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Mitosis and Meiosis

We will skip the sections on the regulation of the cell cycle and cell death, and

concentrate on the processes of mitosis and meiosis.

Nomenclature

Each species has a characteristic number of chromosomes; e.g., humans have 46

chromosomes. A sperm and an egg contribute 23 chromosomes each to form a zygote.

A single set of 23 chromosomes is called the haploid. The set is denoted as n.

Gametes are haploid, and are 1n.

The double set of 46 chromosomes is called the diploid. It is denoted as 2n. Zygotes,

and all somatic cells in a human body, are diploid, and are 2n.

Each chromosome of a haploid set is individually identifiable: in humans, they are

designated numbers 1 – 22, plus one sex chromosome (X or Y).

In a diploid nucleus, the two corresponding chromosomes (e.g., the two number 5

chromosomes) are called homologous chromosomes. They form a homologous pair

in the diploid nucleus.

Before replication, a chromosome is a single, double-stranded piece of DNA

associated with proteins. The nucleic acid-protein complex is not compacted, but tends

to be spread out.

After replication, a chromosome consists of two, double-stranded pieces of DNA

associated with proteins.

In preparation for division, the protein-nucleic acid complex is condensed into a

compact structure --- the chromatid. Each chromosome before division consists of two

sister chromatids.

Before replication, a somatic human cell has 46 chromosomes. After replication and

before cell division, each cell has 46 chromosomes, each with 2 chromatids. Note: the

number of distinct chromosomes has not changed, but the DNA content has doubled.

Cell Division

The primary goal of cell division is to produce more cells.

Unicellular organisms use cell division primarily to reproduce.

Multicellular organisms produce more cells for growth and tissue repair in addition to

reproduction.

The process of cell division involves four main events:

A signal to reproduce initiates the process. Signals may include environmental,

nutritional, hormonal, and injury factors, and other cell-cell interactions.

Replication of DNA and other vital cell components.

Karyokinesis: distribution of DNA to the daughter cells and formation of new

nuclei (eukaryotes only).

Cytokinesis: separation of daughter cells.

Cell Division in Prokaryotes

Chromosome replication takes place as the DNA is threaded through a “replication

complex” of proteins.

After replication, the two sister chromosomes separate.

Cytokinesis begins when the plasma membrane pinches in to form a ring composed of

fibers similar to eukaryotic tubulin. As the membrane pinches in, new cell wall

materials are synthesized to separate the two cells.

Cell Division in Eukaryotes

Eukaryotes usually have many chromosomes; newly replicated chromosomes remain

closely associated with each other; mitosis is used to segregate them into two nuclei.

Mitosis ensures that the number and identity of chromosomes in the daughter cells are

exactly identical to those of the parent cell and of each other.

Prior to mitosis, cells destined to divide go through the cell cycle.

The Cell Cycle (sometimes called Mitotic Cycle)

The cell cycle consists of four major phases:

G1 phase, Gap 1, the cell has just completed dividing and begins to grow.

S phase, synthesis phase, chromosome replication occurs. At the end of S phase,

each chromosome consists of two sister chromatids and therefore has twice as much

genetic materials as before.

G2 phase, Gap 2, further growth of the cell in preparation for mitosis.

Mitosis, karyokinesis and cytokinesis occur.

G1, S and G2 phases are collectively called the interphase of the cell cycle.

Cells not destined to divide are usually arrested at the G1 phase of the cell cycle. These

cells are sometime considered to be in the G0 phase of the cycle.

Mitosis

Mitosis is designed to produce genetically identical cells.

Mitosis is subdivided into five major phases defined by the disposition and behavior of

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chromosomes:

Prophase: the beginning of mitosis, at which time individual chromatids become

visible under the microscope.

Sister chromatids are held together by a small amount of the protein cohesin at

the centromere.

The kinetochore forms late in prophase.

The centrosomes migrate to opposite poles of the nucleus. These serve as

mitotic centers toward which the chromosomes will move during anaphase.

Polar microtubules form between the two centrosomes and make up the

developing spindle.

Prometaphase: the beginning of chromosome movement.

The nuclear envelope breaks down.

Kinetochore microtubules form and connect each chromosome with both

centrosomes.

The kinetochore microtubules move the chromosomes towards the equatorial

(metaphase) plate.

Metaphase: all chromosomes are aligned at the equatorial plate.

Sister chromatids are of each chromosome are connected through the

kinetochore to opposite mitotic centers.

Anaphase: separation of sister chromatids and migration towards opposite poles.

The separated chromatids are sometimes called daughter chromatids.

Power for movement is provided by “molecular motors,” or cytoplasmic

dynein located at the kinetochores. The dynein molecules move along the

mitotic spindles, dragging the chromosomes toward the centrosomes.

Kinetochore microtubules shorten from the poles, drawing chromosomes toward

them.

Telophase: chromosome movement ceases.

The daughter chromatids have reached the poles and stopped moving.

Nuclear envelopes coalesce and re-form around the chromosomes.

The Centrosome

During S phase, the centrosome replicates to form two centrosomes.

Each centrosome consists of a pair of centrioles, each a hollow tube lined with nine

microtubules. The centrioles are oriented perpendicular to each other.

During prophase, the two centrosomes move to opposite ends of the nuclear envelope.

The orientation of the centrosomes determines the plane of cell division. The positions

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of the centrosomes are called the mitotic center.

Microtubules are organized at and grow from the centrosome.

Cytokinesis: Division of the Cytoplasm

Cytokinesis in animal cells begins with a furrowing of the plasma membrane.

Filaments of actin and myosin, located in a contractile ring, interact to produce a

contraction to pinch the cell into two halves.

In plant cells, vesicles from the Golgi apparatus appear in the equatorial region after the

breakdown of mitotic spindles.

The vesicles fuse to form a new plasma membrane and the contents of the vesicles

combine to form the cell plate, which is the beginning of the new cell wall.

Organelles and other cytoplasmic resources are not necessarily distributed equally in

daughter cells.

Meiosis

Meiosis is a specialized cell division used for sexual reproduction. The genetic

information in the chromosomes is shuffled, and the resultant cells, the gametes,

typically receive one-half of the full complement of chromosomes.

Shuffling of the genetic material results in new gene combinations and diversity.

Sexual reproduction results in an organism that is not identical to either parent

organism.

Unlike in mitosis, the resultant cells of meiosis are not genetically identical to one

another.

Meiosis involves one round of DNA replication, and two rounds of cell division. Each

diploid parent cell gives rise to four haploid daughter cells.

Meiosis I: Reduction of Chromosome Number

DNA replication occurs during the S phase of the cell cycle.

Meiosis can be divided into the same phases as mitosis.

Meoisis I is characterized by two important features:

Homologous chromosomes come together and synapse along their entire length.

During anaphase I, the homologous chromosomes separate; sister chromatids stay

together. Individual chromosomes remain intact until the end of metaphase II.

The kinetochores of both sister chromatids in each chromosome become attached to the

same half-spindle; the entire chromosome migrates to one pole. The homologous

chromosome migrates to the opposite pole.

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After anaphase I, each of the two daughter nuclei contains only a haploid set of

chromosomes, but each chromosome consists of two chromatids.

The pairs of homologous chromosomes are sorted randomly into each of the two

daughter nuclei. The two daughter cells are therefore not identical to each other.

Genetic Recombination

Genetic recombination, or cross-over, occurs during meiosis I only.

During prophase I, synapsis between homologous chromosomes occurs: The two

chromosomes are joined together by a synaptonemal complex of proteins, forming a

four-chromatid tetrad, or bivalent.

Adjacent chromatids of the homologs attach to each other at points called chiasmata ( shaped).

Segments of homologous chromosomes are exchanged after these chiasmata form and

break away again.

Meiosis II: Separation of Sister Chromatids

The mechanics of meiosis II is similar to mitosis.

There are three major differences between meiosis II and mitosis:

There is no DNA replication before meiosis II.

The number of chromosomes is haploid during meiosis II.

The sister chromatids of each chromosome are not identical (as a result of

recombination during meiosis I).

The four daughter cells (gametes) produced at the end of meiosis are genetically

distinct from each other, and are all haploid.

Chapter 12: Inheritance, genes and chromosomes

What Are the Mendelian Laws of Inheritance?

• Before the acceptance of Mendel’s research, the concept of blending was favored.

• Kölreuter and others believed that hereditary determinants existed in the egg and sperm

cells, which came together in a single cell after mating and blended together. According to

the theory, once heritable elements were combined, they could not be separated.

• According to this theory, it was thought, for example, that a cross between red and blue

flowers, resulting in purple flowers, cannot be separated back into the parental types.

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Mendel brought new methods to experiments on inheritance

• Gregor Mendel, a monk with training in mathematics, physics, and biology, crossed 24,034

plants and presented the results of his seven-year-long project orally in 1865 and in writing in

1866, but he was ahead of his time, and his findings were ignored.

• His challenge to the blending concept was ignored, perhaps because biologists were not

accustomed to reviewing mathematical data. Even Darwin, whose evolutionary theory rests

on genetic variation among individuals, failed to understand Mendel and relied on the

concept of blending.

Mendel devised a careful research plan

• Mendel chose garden peas as his subjects because they are easily grown and their pollination

is easily controlled, either by manually moving pollen between plants or by allowing plants

to self-pollinate.

• Mendel selected varieties of peas that could be studied for their heritable characters

(observable features passed from parent to offspring) and traits (the particular forms the

features take).

• Mendel looked for characters that had well-defined alternative traits and that were true-

breeding.

• A trait is true-breeding when it is the only trait that occurs through many generations of

breeding individuals.

• A true-breeding white-flowered plant would have only white-flowered progeny when

crossed with others in its strain.

• True-breeding plants, when crossed with other true-breeding plants, are called the parental

generation, designated P.

• The progeny from the cross of the P parents are called the first filial generation, designated

F1.

• When F1 individuals are crossed with each other or self-fertilized, their progeny are

designated F2.

Mendel’s first experiments involved monohybrid crosses

• Mendel crossed true-breeding plants that differed in a given character.

• A monohybrid cross involves one (mono) character and different (hybrid) traits.

• Pollen from true-breeding pea plants with wrinkled seeds (one trait) was placed on stigmas

of true-breeding plants with spherical seeds (another trait).

• The F1 seeds were all spherical; the wrinkled trait failed to appear at all.

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• Because the spherical trait completely masks the wrinkled trait when true-breeding plants

are crossed, the spherical trait is considered dominant and the wrinkled trait recessive.

• The F1 plants were then allowed to self-pollinate.

• The progeny, called F2, were examined: 5,474 were spherical and 1,850 were wrinkled.

• From the results of his experiments, Mendel reached a number of conclusions.

• Rejection of the blending theory; inheritance cannot be the result of blending.

• The units responsible for inheritance are discrete particles that exist within an organism in

pairs, separate during gamete formation, and retain their integrity; this is called the

particulate theory.

• Each pea has two units of inheritance for each character.

• During production of gametes, only one of the pair for a given character passes to the

gamete.

• When fertilization occurs, the zygote gets one unit from each parent, restoring the pair.

• Mendel’s units of inheritance are now called genes.

Alleles are different forms of a gene

• Alleles are different forms of a gene.

• Each allele is given a symbol (e.g., S to represent smooth seeds and s to represent

wrinkled).

• True-breeding individuals have two copies of the same allele (i.e., they are homozygous ss or

homozygous SS).

• Individuals that have two different alleles of the same gene are heterozygous.

• The physical appearance of an organism is its phenotype; the actual composition of the

organism’s alleles for a gene is its genotype.

Mendel’s first law says that the two copies of a gene segregate

• Mendel’s first law (stating that each gamete receives one member of the pair of alleles) is

called the law of segregation.

• The Punnett square is a simple grid device that shows the expected frequencies of

genotypes.

• The S and s symbols represent the single allele each gamete receives.

• The Punnett square shows that the genotypes

and associated ratios for a monohybrid cross are

1 SS:2 Ss:1 ss.

• Any progeny with an S would have the dominant (smooth) phenotype, so the phenotypic

ratio is 3 smooth to 1 wrinkled.

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Mendel verified his hypothesis by performing a test cross

• A test cross of an individual with a dominant trait with a true-breeding recessive

(homozygous recessive) can determine the first individual’s genotype (heterozygous or

homozygous.)

• If the unknown is homozygous dominant, all the progeny will have the dominant trait.

• If the unknown is heterozygous, approximately half the progeny will have the dominant

trait and half will have the recessive trait.

Mendel’s second law says that copies of different genes assort independently

• Mendel’s second law describes the outcome of dihybrid (two-character) crosses, or hybrid

crosses involving additional characters (e.g., smooth or wrinkled shape, yellow or green

color).

• Called the law of independent assortment, the second law states that alleles of different genes

(e.g., Ss and Yy ) assort into gametes independently of each other.

• Segregation of S from s is independent of segregation of Y from y.

• In this case, four different gametes are possible and will be produced in equal proportions:

SY, Sy, sY, and sy.

Mendel’s laws can be observed in human pedigrees

• Because humans cannot be studied using planned crosses, human geneticists rely on

pedigrees, which show phenotype segregation in several generations of related individuals.

• Because humans have such small numbers of offspring, human pedigrees do not show clear

proportions. In other words, outcomes for small samples fail to follow the expected outcomes

closely.

• If neither parent has a given phenotype, but it shows up in their progeny, the trait is recessive,

and the parents are heterozygous.

• Half of the children from such a cross will be carriers (heterozygous for the trait).

• The chance of any one child’s getting the trait is 1⁄4.

• See Figure 12.10A for a pedigree analysis of the dominant allele for Huntington’s disease.

• Every affected person has an affected parent.

• About half the offspring of an affected person are also affected (assuming only one parent

is affected).

• The phenotype occurs equally in both sexes.

• See Figure 12.10B for a pedigree analysis of a recessive allele for a rare trait.

• Affected people usually have parents who are both unaffected.

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• On average, one-quarter of the children of unaffected parents would be affected.

• The phenotype occurs equally in both sexes.

• Marriage between close relatives results in a higher likelihood that both parents will be

carriers of a rare allele and produce affected children. Pedigree analysis is used mostly in

clinical evaluation and counseling of patients with inherited abnormalities.

How Do Alleles Interact?

• Differences in alleles of genes consist of slight differences in the DNA sequence at the same

locus, resulting in slightly different protein products.

• Some alleles are not simply dominant or recessive. There may be many alleles for a single

character or a single allele may have multiple phenotypic effects.

New alleles arise by mutation

• Different alleles exist because any gene is subject to mutation into a stable, heritable new

form.

• Alleles can mutate randomly to become a different allele depending on DNA sequence

changes.

• The most common allele in the population is called the wild type.

• Other alleles, often called mutant alleles.

Dominance is not always complete

• Heterozygotes may show an intermediate phenotype.

• For example, red-flowered snapdragons when crossed with white will generate pink-flowered

plants.

• This phenotype might seem to support the blending theory.

• The F2 progeny, however, demonstrate Mendelian genetics. For self-fertilizing F1 pink

individuals the blending theory would predict all pink F2 progeny, whereas the F2 progeny

actually have a phenotypic ratio of 1 red:2 pink:1 white.

• This mode of inheritance is called incomplete dominance.

In codominance, both alleles at a locus are expressed

• In codominance, two different alleles for a gene produce two different phenotypes in the

heterozygotes. The AB of the human ABO blood group system is an example.

• Around 1900, Karl Landstein found that only certain combinations of blood types are

compatible.

• The alleles for blood type are IA, IB, and iO, all occupying one locus.

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Some alleles have multiple phenotypic effects

• Pleiotropic alleles are single alleles that have more than one distinguishable phenotypic

effect.

How Do Genes Interact?

• Epistasis occurs when the alleles of one gene cover up or alter the expression of alleles of

another gene; that is, the phenotypic expression of one gene is affected by another gene.

• An example is coat color in Labrador retrievers. (See Figure 12.14.)

• The B allele (black pigment) is dominant to b allele (brown).

• Another locus determines if pigment deposition occurs. Gene e determines the expression of

B/b.

• Allele E (pigment deposition in hair) is dominant to e (yellow hair).

• The F2 phenotypic ratio (from BbEe x BbEe) is 9 black:3 brown:4 yellow.

The environment affects gene action

• Genotype and environment interact to determine the phenotype of an organism.

• Variables such as light, temperature, and nutrition can affect the translation of genotype into

phenotype.

What Is the Relationship between Genes and Chromosomes?

• How do we determine the order and distance between genes that are located on the same

chromosome?

• In 1909, Thomas Hunt Morgan’s lab began its pioneering studies in Drosophila melanogaster,

an organism that is still used in chromosomal studies today.

Genes on the same chromosome are linked

• One early exception to Mendel’s second law (the law of independent assortment) was found

in a test cross between flies that were hybrids for two particular alleles (body color and wing

size) and flies that were recessive for both alleles.

• The results were not the expected 1:1:1:1; instead, two of the four possible genotypes

occurred at a higher frequency.

• These results make sense if the two loci are on the same chromosome, and thus their

inheritance is linked. All the loci on a given chromosome make up a linkage group.

• Many genes exist together on the same chromosome, although the linkage is not absolute.

Genes can be exchanged between chromatids

• Absolute or total linkage of all loci is extremely rare.

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• Genes at different loci on the same chromosome do sometimes separate during meiosis.

• Sometimes two homologous chromosomes physically exchange corresponding segments

during prophase I of meiosis (crossing over).

• If just a few exchanges occur, genes that are closer together tend to stay together.

• The progeny resulting from crossing over appear in repeatable proportions, called the

recombinant frequency.

• Greater recombination frequencies are observed for genes that are farther apart on the

chromosomes because an exchange event is more likely to cut between genes that are far

apart than between genes that are closer together.

Geneticists can make maps of chromosomes

• The farther apart on the same chromosome genes are, the more likely they are to separate

during recombination.

• The two extremes are independent assortment and complete or absolute linkage.

• Alfred Sturtevant, an undergraduate student working in Morgan’s fly room, realized that

determining recombinant frequencies for many pairs of linked genes could be used to create

genetic maps showing the arrangement of genes along the chromosome.

• Scientists now measure distances between genes in map units. One map unit corresponds to a

recombination frequency of 0.01 (or a 1 percent recombination). It also is referred to as a

centimorgan (cM).

Linkage is revealed by studies of the sex chromosomes

• In some cases, parental origin of a chromosome is important.

• Sex is determined in different ways in different species.

• In corn (and peas), which are monoecious (“one house”), every diploid adult has both male

and female reproductive structures.

• Other plants and most animals are dioecious (“two houses”), meaning that some individuals

produce only male gametes, and others produce only female gametes.

• In most dioecious organisms, sex is determined by differences in the chromosomes, but

such determination operates in different ways in different organisms.

• Many organisms have homologous pairs of all chromosomes except for those that

determine sex. The homologous pairs are called autosomes; the unpaired X and Y

chromosomes are called sex chromosomes.

• Female mammals have two X chromosomes, whereas male mammals have two different

sex chromosomes, X and Y.

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• Sex of the offspring is determined by the sperm: If a sperm with an X chromosome reaches

the egg, the resulting offspring will be female (XX); if a sperm with a Y chromosome

reaches the egg, the resulting offspring will be male (XY).

• In birds, males are XX (ZZ) and females are XY (ZW). The bird egg, Z or W, determines

the sex of the offspring.

• Drosophila chromosomes follow the same pattern as humans, but the mechanism is

different.

• The males are XY, and females are XX.

• The ratio of X chromosomes to the autosomal sets determines sex.

• One X chromosome for each set of autosomes yields females.

• One X for the two sets of autosomes yields males. (XO is sterile; XY is fertile).

• Y chromosome is needed for male fertility. It is not a sex-determinant.

Genes on sex chromosomes are inherited in special ways

• The Y carries very few genes, whereas the X carries a great variety of characters.

• Females with XX are diploid for X-linked genes; males with XY are haploid. Therefore,

females may be heterozygous for genes on the X chromosome, whereas males are always

hemizygous.

• This difference generates a special type of inheritance called sex-linked inheritance.

Humans display many sex-linked characters

• The probability of a male having an X-linked genetic disease caused by a mutant recessive

allele is much higher than it is for a female.

• Pedigree analysis of X-linked recessive phenotypes reveals certain patterns.

• The phenotype appears much more often in males than in females.

• A male with the mutation can pass it on only to his daughters, through an X-bearing sperm;

his sons get his Y chromosome, which does not carry the trait.

• Daughters who receive one mutant X are heterozygous carriers. They pass the allele to

approximately half of their sons and daughters.

• The mutant phenotype can skip a generation if the mutation is passed from a male to his

daughter and then to her son.

Chapter 13: DNA and Its Role in Heredity

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What Is the Evidence that the Gene Is DNA?

• By the early twentieth century, geneticists had associated the presence of genes with chromosomes and

had begun researching which chemical component of chromosomes comprised this genetic material.

• Circumstantial evidence pointed to DNA as the genetic material.

• It was in the right place. DNA was found in the nucleus and chromosomes, which were already

known to carry genes.

• DNA varied among species. DNA-binding dyes revealed each species appeared to have its own

specific nuclear DNA content.

• It was present in the right amounts. The quantity of DNA in somatic cells was twice that in eggs or

sperm, as would be expected from Mendel’s discoveries.

DNA from one type of bacterium genetically transforms another type

• In the 1920s, the English physician Frederick Griffith made a landmark discovery about heredity

while looking for a vaccine against Streptococcus pneumoniae, one of the bacteria that cause

pneumonia in humans.

• Griffith worked with two different strains of the bacterium.

• The S strain produced shiny, smooth colonies when grown in the laboratory. This strain was

virulent (mice injected with the S strain died within a day); a capsule around the S strain bacteria

protected them from the host’s immune system.

• The R strain produced colonies that looked rough, lacked this capsule, and were nonvirulent.

• Griffith heated some S strain bacteria to kill them and then injected the bacteria into mice; the heat-

killed bacteria did not kill the mice.

• A mixture of heat-killed S strain bacteria and living R strain bacteria did kill the mice, however, and

Griffith found living S strain bacteria in the hearts of the mice killed in this way.

• He concluded that some of the living R strain bacteria had been transformed by the presence of the

heat-killed S strain bacteria.

• Further tests demonstrated that some substance from the dead S strain bacteria could cause a heritable

change in the R strain bacteria.

• Results showed that this transforming principle carried heritable information.

The transforming principle is DNA

• Oswald T. Avery and colleagues spent several years identifying the transforming principle by a

process of elimination.

• They treated the extract from heat-killed S strain bacteria in various ways to destroy different types of

substances but retain others.

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• Invariably, when DNA was destroyed, the transforming activity was lost, but when DNA was left

intact, the transforming activity survived.

• This work, published in 1944, was not immediately appreciated for two reasons:

• Most scientists did not believe that DNA was chemically complex enough to be the genetic

material.

• Little was known about bacterial genetics, and it was not yet obvious that bacteria even had genes.

Viral replication experiments confirmed that DNA is the genetic material

• In 1952, Alfred Hershey and Martha Chase performed experiments using a virus that infects bacteria

to confirm that DNA is the genetic material.

• The T2 bacteriophage, a virus that attacks E. coli, consists almost entirely of a DNA core packed in a

protein coat.

• When a T2 bacteriophage attacks a bacterium, part, but not all, of the virus enters the bacterial cell.

• The Hershey–Chase experiment determined which part of the virus (protein or DNA) entered the

bacterium.

• To trace the two components of the virus over its life cycle, Hershey and Chase labeled each with a

specific radioactive tracer.

• Some viruses were labeled with radioactive sulfur. Sulfur is present in proteins but not in DNA.

• Other viruses were labeled with radioactive phosphorus. Phosphorus is present in DNA but absent

from most proteins.

• In separate experiments, viruses with labeled sulfur and labeled phosphorus were combined with

bacteria.

• Blending the resulting bacteria removed the viral material that had not entered the bacteria.

• Centrifuging revealed that the labeled sulfur (and thus the viral protein) had separated from the

bacteria, but the labeled phosphorus (and thus the viral DNA) remained with the bacteria.

• Experiments on later generations of bacteria confirmed that the labeled phosphorus remained with

subsequent generations, whereas the labeled sulfur was quickly lost.

Eukaryotic cells can also be genetically transformed by DNA

• The question arose as to whether DNA could be the same genetic material found in complex

eukaryotes.

• Genetic transformation of eukaryotic cells (transfection) can be demonstrated using a genetic marker,

a gene whose presence in the recipient cell results in an observable phenotype.

• In the absence of a gene that codes for thymidine kinase, an enzyme that is needed to use thymidine,

mammal cells do not grow. However, when the DNA containing the thymidine kinase marker gene is

added to a culture of mammalian cells lacking this gene, some cells will grow.

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• Any cell can be transfected this way; the result is a whole new genetically transformed organism.

What Is the Structure of DNA?

• Scientists set out to determine the structure of DNA hoping to find the answers to two questions:

• How is DNA replicated between nuclear divisions?

• How does DNA cause the synthesis of specific proteins?

• X-ray crystallography provided clues to DNA structure.

• The positions of atoms in a crystalline substance can be inferred from the pattern of diffraction of X

rays passed through it.

• In the 1950s, the English chemist Rosalind Franklin, building on previous work by Maurice Wilkins,

was able to provide key information about the structure of DNA based on X-ray crystallography.

• The crystallographs suggested a spiral or helical molecule.

The chemical composition of DNA was known

• Biochemists knew that DNA was a polymer of nucleotides.

• The four nucleotides that make up DNA differ only in their nitrogenous bases.

• There are two purines (adenine and guanine) and two pyrimidines (cytosine and thymine).

• In 1950, Erwin Chargaff noted that in DNA from all species tested, the amount of adenine equals the

amount of thymine, and the amount of guanine equals the amount of cytosine.

• In other words, the total abundance of purines equals the total abundance of pyrimidines, even though

the actual proportions of each base vary in different species. This is now known as “Chargaff’s rule.”

Watson and Crick described the double helix

• The English physicist Francis Crick and the American geneticist James D. Watson used the technique

of model building to establish the general structure of DNA.

• The results of X-ray crystallography convinced them that the DNA molecule was helical.

• X-ray crystallography also provided the values of certain distances within the helix.

• Density measurements and earlier models pointed to a structure with two polynucleotide chains

running antiparallel to each other.

• Although there have been modifications, the principle features of the model they built in 1953 have

remained unchanged.

Four key features define DNA structure

• Four features summarize the molecular architecture of DNA

• The DNA molecule is a double-stranded helix of uniform diameter.

• The twist in DNA is right-handed.

• The two strands run in different directions (they are antiparallel).

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• The outer edges of the nitrogenous bases are exposed in the major and minor grooves.

• The helix:

• The sugar–phosphate backbones of each strand coil around the outside of the helix, and the

nitrogenous bases point toward the center.

• Hydrogen bonds between complementary bases hold the two strands together.

• A always pairs with T (two hydrogen bonds).

• G always pairs with C (three hydrogen bonds).

• Each base pair has one purine and one pyrimidine, so the diameter of the double helix remains

constant. This is called complementary base pairing.

• Antiparallel strands:

• The direction of a polynucleotide is defined by the linkages between adjacent nucleotides.

• The phosphate groups link the 3ʹ carbon of one deoxyribose molecule to the 5ʹ carbon of the next.

• Thus a single strand of DNA has a 5ʹ phosphate group at one end (the 5ʹ end) and a free 3ʹ hydroxyl

group at the other end (the 3ʹ end).

• In a double helix, the 5ʹ end of one polypeptide is hydrogen-bonded to the 3ʹ end of the other, and

vice-versa.

• Base exposure at the grooves:

• The exposed outer edges of the flat, hydrogen-bonded base pairs are accessible for potential

hydrogen bonding.

• The surfaces of the AT and GC base pairs have different, chemically distinct surfaces due to the

different number of hydrogen bonds.

• Access to exposed base-pair sequences is essential to protein–DNA interactions in replication and

expression of genetic information.

The double-helical structure of DNA is essential to its function

• The genetic material must perform four important functions:

• It must be able to store all of an organism’s genetic information.

• It must be susceptible to mutation.

• It must be precisely replicated in the cell division cycle.

• It must be expressible as the phenotype.

How Is DNA Replicated?

Three modes of DNA replication appeared possible

• Three years after Watson and Crick published their structure of DNA, the American biochemist

Arthur Kornberg demonstrated that the DNA molecule contains the information needed for its own

replication.

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• Kornberg showed that DNA can replicate in a test tube with only DNA as a template, a specific

enzyme (DNA polymerase), and a mixture of four deoxyribonucleoside triphosphates (dNTPs):

dATP, dCTP, dGTP, and dTTP.

• Theoretically, DNA could serve as its own template in one of three different ways:

• Semiconservative replication would use each parent strand as a template for a new strand. Each new

DNA double helix would then have one parent strand and one new strand.

• Conservative replication would build an entirely new double helix based on the template of the old

double helix. The new strand would contain none of the original DNA.

• Dispersive replication would use fragments of the original DNA molecule as templates for

assembling two molecules. All the resulting strands would be mixtures of old and new material.

An elegant experiment demonstrated that DNA replication is semiconservative

• Matthew Meselson and Franklin Stahl demonstrated in 1957 that DNA replication is

semiconservative, by using a technique they devised called density labeling.

• Centrifuging can separate DNA labeled with “heavy” nitrogen (15N) from unlabeled DNA.

• Meselson and Stahl grew a culture of E. coli for many generations in a medium with 15N instead of 14N.

• As a result, all the DNA in the bacteria was “heavy.”

• They then transferred bacteria grown on the heavy medium to a normal medium and allowed the

bacteria to continue growing.

• They sampled the DNA at each generation time, starting with the parental, all-heavy generation.

• Centrifuging the DNA after the first cell division (20 minutes) yielded a single band of DNA

intermediate in density between the heavy and light forms.

• Centrifuging the DNA after the second cell division (40 minutes) yielded an intermediate band and a

light band, the result predicted by the semiconservative replication hypothesis.

• If one of the other models was correct, the results would have been different:

• The conservative replication hypothesis would have yielded two bands, one heavy and one light.

• Dispersive replication would have yielded a single band with a density less than heavy DNA, but

greater than light DNA.

There are two steps in DNA replication

• DNA replication takes place in two steps:

• The double helix is unwound to separate the two template strands.

• The new nucleotides are joined by phosphodiester linkages in a sequence determined by

complementary base pairing.

• Nucleotides are added to the 3ʹ end of the growing polynucleotide.

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• One of the phosphate groups of the deoxyribonucleoside triphosphate is attached to the 5ʹ position of

the sugar.

• The bonds linking the other two phosphate groups to the nucleotide are broken, releasing energy used

in the reaction.

DNA polymerases add nucleotides to the growing chain

• DNA is replicated through the interaction of the template strand and the replication complex, which

catalyzes the reactions of DNA replication.

• This replication complex recognizes an origin of replication (ori) on a chromosome.

• DNA replication begins with a starter primer of RNA using a primase enzyme.

• A polymerase then continues adding nucleotides to the 3ʹ end of the strand.

• The RNA primer is later degraded and replaced with DNA, so the final DNA molecule has no RNA.

• DNA polymerase is a large complex with a groove that the DNA attaches to and can slide through.

• Cells have more than one kind of DNA polymerase.

• Many other proteins assist with DNA polymerization.

• DNA replicates in both directions from the origin, forming two replication forks.

• Both strands of DNA act as templates.

• The process:

• Localized unwinding (denaturation) of DNA takes place at the origin of replication, via DNA

helicase.

• Single-strand binding proteins bind to the unwound strands to keep them apart.

• The two DNA strands grow differently; one continuously through 3ʹ extension (leading strand) and

one discontinuous resulting in short Okazaki fragments (lagging strand).

• DNA ligase fills in gaps between adjacent Okazaki fragments.

• A sliding clamp resembling a donut clamps around the DNA strand and attaches to DNA

polymerase dramatically increasing the efficiency of the rate of DNA replication.

• Small, circular DNAs replicate from a single origin.

• Small chromosomes, such as those found in bacteria, have a single origin of replication, and

replication forks grow around the circle.

• Two interlocking circular DNAs, that are separated by the enzyme DNA topoisomerase, are formed.

• Large, linear DNAs have many origins.

• Large chromosomes can have hundreds of origins of replication, and replication occurs at many

different sites simultaneously.

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Telomeres are not fully replicated and are prone to repair

• Beyond the very end of a linear DNA molecule there is no place for a primer to bind, resulting in new

chromosomes formed after DNA replication that have single-stranded DNA at each end.

• This single-stranded region is cut off, along with some of the intact double-stranded end, slightly

shortening the chromosome after each cell division.

• Many eukaryotic chromosomes have repetitive sequences called telomeres at their ends, which

shorten after each round of cell division. These repeats bind to special proteins that maintain the

stability of the chromosome ends.

• After a given number of cell divisions (20 to 30 in some human cells), the telomeres have shortened

to the extent that they are no longer able to stabilize the ends of the chromosomes, and the cell dies.

This explains in part why cells do not last the entire lifetime of the organism.

• Constantly dividing cells, such as bone marrow, germ line, and more than 90 percent of cancer cells,

produce an enzyme called telomerase that catalyzes the addition of any lost telomeric sequences.

13.4 How Are Errors in DNA Repaired?

• To minimize the number of errors, our cells normally have at least three DNA repair mechanisms at

their disposal:

• A proofreading mechanism corrects errors during the replication process.

• A mismatch repair mechanism scans and repairs errors in DNA shortly after replication.

• An excision repair mechanism operates over the life of the cell to repair errors that result from

chemical or radiation damage.

• As they add new bases to a growing strand, DNA polymerases make a proofreading check to make sure

they have added the correct base. When a DNA polymerase recognizes an error, it removes the wrong

nucleotide and tries again.

• The error rate of DNA polymerase on each attempt is only about 1 in 10,000, so the second attempt at

matching the template is very likely to be successful.

• After DNA has been replicated, the mismatch repair mechanism looks for mismatched base pairs that

were missed in proofreading.

• In eukaryotes, methyl groups are added some time after replication to some cytosines. The mismatch

repair mechanism operates before the new DNA strand is chemically modified (methylated).

• This mechanism can distinguish between the methylated template strand and the unmethylated new

strand.

• Thus, this mechanism can determine which base is correct (the base on the template strand) and which

base needs to be replaced.

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• Some cells live for many years, during which time their DNA is subject to damage by chemicals,

radiation, and random spontaneous chemical reactions.

• Excision repair proteins operate over the life of a cell.

• Excision repair enzymes “inspect” the cell’s DNA for mispaired bases and chemically modified bases

and points where one strand has more bases than the other.

• These enzymes cut the damaged strand and remove the modified base and a few bases on either side

of it.

• DNA polymerase and DNA ligase fill in and seal up the resulting gap.

Chapter 14: From DNA to Protein: Gene Expression

Experiments on bread mold established that genes determine enzymes

• Because of life’s basis in the cell theory, scientists can assume that what is found in one

organism can apply to others, and they often search for a model organism.

• The common bread mold Neurospora crassa is one such model organism. This mold is

haploid for most of its life, making its genetics straightforward (because there are no

dominant–recessive relationships).

• In the 1940s, Beadle and Tatum undertook studies to chemically define the phenotype of

Neurospora.

• They hypothesized that the expression of a gene as a phenotype could occur through an

enzyme.

• They grew Neurospora on a minimal nutritional medium consisting of sucrose, minerals, and

a vitamin.

• Using this medium, the enzymes of wild-type Neurospora (called prototrophs; “original

eaters”) could catalyze the metabolic reactions needed to make all the chemical

constituents.

• Beadle and Tatum treated wild-type Neurospora with X rays, which act as a mutagen. They

found that some of the mutant strains could no longer grow on the minimal medium; they

grew only if supplied with additional nutrients.

• Beadle and Tatum hypothesized that these auxotrophs (“increased eaters”) must have

suffered mutations in genes that coded for the enzymes used to synthesize the nutrients

they now needed to obtain from their environment.

• For each auxotrophic strain, Beadle and Tatum were able to find a single compound that

could support its growth.

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• This result suggested that mutations have simple effects, and that each mutation causes a

defect in only one enzyme in a metabolic pathway, the one-gene, one-enzyme hypothesis of

Garrod (see textbook) was confirmed.

• One group of auxotrophs could grow only if the minimal medium was supplemented with

arginine (arg mutants).

• Beadle and Tatum found several genetically different arg mutant strains with the same

phenotype. They proposed two alternative hypotheses to explain why these different strains

had the same phenotype:

• The different arg mutants could have mutations in the same gene, in this case the gene

might code for an enzyme involved in arginine synthesis.

• The different arg mutants could have mutations in different genes, each coding for a

separate function that leads to arginine production. These independent functions might be

different enzymes along the same biochemical pathway.

• Genetic crosses showed that some of these arg mutants had mutations at the same

chromosomal locus and were different alleles of the same gene; other mutations were at

different loci or on different chromosomes and were thus in different genes.

• Beadle and Tatum concluded that these different genes participated in the same

biochemical pathway—the pathway leading to arginine synthesis.

• By growing different arg mutants in the presence of various compounds suspected to be

intermediates in the arginine pathway, Beadle and Tatum were able to classify each

mutation as affecting one enzyme or another and to order the compounds along the

pathway. They then broke open the wild-type and mutant cells and, by examining their

enzymatic activities, confirmed their hypothesis that each mutant strain was indeed missing

a single active enzyme in the pathway.

One gene determines one polypeptide

• The gene–enzyme connection has undergone several modifications. It was learned, for

example, that some enzymes are composed of different subunits coded for by separate

genes, thus suggesting, instead of the one-gene, one-enzyme hypothesis, a one-gene, one-

polypeptide relationship.

• In other words, the function of a gene is to control the production of a single, specific

polypeptide. This statement remains true, even though we know that some genes code for

forms of RNA that are not translated into polypeptides and have discovered that still other

gene sequences do not produce polypeptides but instead control which other gene

sequences are expressed.

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How Does Information Flow from Genes to Proteins?

• The expression of a gene to form a polypeptide occurs in two steps:

• Transcription copies the information of a DNA sequence (a gene) into corresponding

information in an RNA sequence.

• Translation converts this RNA sequence into the amino acid sequence of a polypeptide.

RNA differs from DNA and plays a vital role in gene expression

• RNA (ribonucleic acid) is a polynucleotide and a key intermediary between DNA and

polypeptide. It differs from DNA in three ways:

• RNA consists of only one polynucleotide strand.

• The sugar in RNA is ribose, not deoxyribose.

• Although three of the nitrogenous bases (adenine, guanine, and cytosine) are identical, the

fourth base in DNA is thymine, whereas in RNA it is uracil (similar to thymine, but lacking

the methyl group).

• RNA can base pair with single-stranded DNA (with adenine pairing with uracil instead of

thymine, as in complementary base-pairing of DNA) and also can fold over and base pair

with itself.

Two hypotheses were proposed to explain information flow from DNA to protein

• Central dogma suggested information flows from DNA to RNA to protein, raising two

questions:

• How does genetic information get from the nucleus to the cytoplasm?

• What is the relationship between DNA sequence and amino acid sequence?

• Francis Crick’s central dogma stated that DNA codes for RNA, RNA codes for protein (more

correctly polypeptide), and protein does not code for the production of protein, RNA, or DNA.

• Crick proposed two hypotheses to answer two questions.

• The messenger hypothesis and transcription:

• To answer the question of how information gets from the nucleus into the cytoplasm,

Crick proposed the messenger hypothesis.

• He proposed that the RNA molecule forms as a complementary copy of one DNA strand

of a particular gene. This messenger RNA (mRNA) travels from the nucleus to the

cytoplasm, where it serves as a template for the synthesis of protein.

• The process by which RNA forms is called transcription.

• The adapter hypothesis and translation:

• To answer the question of how a DNA sequence gets transformed into the specific

amino acid sequence of a polypeptide, Crick proposed the adapter hypothesis.

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• There must be an adapter molecule that can both bind a specific amino acid and

recognize a sequence of nucleotides.

• Eventually, adapter molecules (transfer RNA, or tRNA) were found. Because tRNA

recognizes the genetic message of mRNA and carries specific amino acids, tRNA can

translate the language of DNA into the language of proteins. The tRNA adapters,

carrying bound amino acids, line up on the mRNA sequence so that the amino acids are

in the proper sequence for a growing polypeptide chain. This process is called

translation.

RNA viruses are exceptions to the central dogma

• Many viruses (e.g., influenza, poliovirus) use RNA rather than DNA as their genetic material.

• These viruses transcribe from RNA to RNA; they make an RNA strand that is

complementary to their genome and then use this “opposite” strand to make multiple copies

of the viral genome by transcription.

• HIV and certain tumor viruses have RNA as their genome; they convert it to a DNA copy

inside the host cell and then use it to make more RNA.

How Is the Information Content in DNA Transcribed to Produce RNA?

• In normal prokaryotic and eukaryotic cells, transcription (formation of a specific RNA from a

specific DNA) requires the following:

• A DNA template for complementary base pairing.

• The appropriate ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) to act as

substrates.

• The enzyme RNA polymerase.

• Just one DNA strand (the template strand) is used to make the RNA; the complementary non-

template strand remains untranscribed.

• For different genes in the same DNA molecule, however, different strands may be transcribed.

• The same process, transcription, is responsible for the synthesis of tRNA and ribosomal RNA.

Like polypeptides, these RNAs are coded by specific genes.

RNA polymerases share common features

• RNA polymerases from both prokaryotes and eukaryotes all share a common structure that

resembles a crab claw. Catalysis occurs in several steps:

• The enzyme recognizes certain bases within the DNA double helix and binds to them.

• Once the template DNA has bound to the enzyme, the “pincers” close, keeping DNA in a

double-stranded form called a closed complex.

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• A conformational change in the RNA polymerase occurs, denaturing a short (10 base

pairs) stretch of DNA and forming an open complex.

• The open complex makes the unpaired bases within DNA available to pair with

ribonucleotides, and RNA synthesis begins.

• Like DNA polymerases, RNA polymerases are processive; a single enzyme–template

binding event results in the polymerization of hundreds of RNA bases. Unlike DNA

polymerases, they do not require a primer.

Transcription occurs in three steps

• Initiation:

• The first step of transcription, initiation, requires a promoter, a special sequence of DNA to

which RNA polymerase binds very tightly. It orients the RNA polymerase to the correct

strand to use as a template and “tells” it where to start transcription and the direction to

take from the start.

• Promoters function like punctuation marks that determine how a sequence of words is to

be read as a sentence.

• Part of each promoter is the initiation site where transcription begins. Groups of

nucleotides lying upstream form the initiation site (5′ on the non-template strand and 3′ on

the template strand) help the RNA polymerase bind.

• Not all promoters are identical. Some are more effective at transcription than others.

Furthermore, there are differences between transcription initiation in prokaryotes and in

eukaryotes.

• Elongation:

• After binding, RNA polymerase begins the process of elongation and unwinds the DNA

about 10 base pairs at a time and reads the template in the 3′-to-5′ direction.

• The new RNA elongates from its 5′ end to its 3′ end; thus the RNA transcript is antiparallel

to the DNA template strand.

• Unlike DNA polymerases, RNA polymerases do not proofread and correct their work.

Transcription errors occur at a rate of every 104 to 105 bases. Because many copies of the

RNA are made and they have a relatively very short life span, these errors are not as

potentially harmful as mutations in DNA.

• Termination:

• Particular base sequences in the DNA specify termination.

• Gene mechanisms for termination vary.

• For some genes, the newly formed transcript simply falls away from the DNA template

and the RNA polymerase.

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• For other genes, a helper protein pulls the transcript away.

• In prokaryotes, translation of the mRNA often begins before transcription is complete.

• In eukaryotes, the process is more complicated and involves spatial separation of

transcription (in the nucleus) and translation (in the cytoplasm). In addition, there is further

processing of the first product of transcription, pre-mRNA (which is longer than the final

mRNA), before translation.

The information for protein synthesis lies in the genetic code

• A genetic code relates genes (DNA) to mRNA and mRNA to the amino acids of proteins,

specifying which amino acids will be used to build a protein by the transcription process.

• The genetic information in an mRNA molecule can be thought of as sequential

nonoverlapping three-letter “words.”

• Each sequence of three nucleotide bases (the three “letters”) specifies a particular amino

acid.

• Each three-letter “word” is called a codon.

• Characteristics of the code:

• In the early 1960s, molecular biologists broke the genetic code determining how only four

bases (A, U, G, and C) could code for 20 different amino acids.

• A triplet code was considered likely because only a triplet code could contain up to 64 (4 x

4 x 4) codons.

• Nirenberg prepared an artificial mRNA in which all bases were uracil (poly U).

• When added to a test tube with the required additional ingredients for bacterial protein

synthesis, the poly U mRNA led to synthesis of a polypeptide chain consisting only of

phenylalanine amino acids.

• UUU appeared to be the codon for phenylalanine.

• Two other codons were deciphered from this starting point (CCC for proline, AAA for

lysine).

• Other scientists discovered that artificial mRNAs only three nucleotides long could bind to

ribosomes.

• The resulting complex then caused the tRNA to bind with its specific amino acid.

• After this discovery, Nirenberg repeated his experiments, and the code was fully

deciphered.

• The number of different codons possible is 64 (43), because each position in the codon can

be occupied by one of four different bases.

• The 64 possible codons code for only 20 amino acids and the start and stop signals found

in all mRNA molecules.

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• AUG, which codes for methionine, is called the start codon, the initiation signal for

translation.

• Three codons (UAA, UAG, and UGA) are stop codons, which direct the ribosomes to end

translation.

• The genetic code is redundant but not ambiguous:

• After subtracting start and stop codons, the remaining 60 codons code for 19 different

amino acids.

• For almost all amino acids there is more than one codon. Thus, the code is redundant.

• But the code is not ambiguous. Each codon codes for only one amino acid.

• The genetic code is (nearly) universal:

• The genetic code applies to all species on our planet.

• Minor variations are found within mitochondria and chloroplasts; other exceptions are few

and slight.

• The common genetic code means there is also a common language for evolution.

• The common genetic code also has profound implications in genetic engineering.

How Is Eukaryotic DNA Transcribed and the RNA Processed?

• Even though they share the genetic code, several unique things separate eukaryotic from

prokaryotic gene expression.

• Eukaryotic genes have noncoding sequences=

• Preceding the coding region of a eukaryotic gene is a promoter, to which RNA polymerase

binds to begin transcription.

• At the other end of the gene is a terminator, which signals the end of transcription.

• The terminator sequence is usually after the stop codon.

• The stop codon is in the coding region and, when transcribed into mRNA, signals the end

of translation at the ribosome.

• Eukaryotic protein-coding genes also contain noncoding sequences called introns.

• Introns are interspersed with the coding regions, called exons.

• Transcripts of the introns appear in the pre-mRNA but are removed before the final mRNA is

translated. Pre-mRNA processing involves cutting out introns and splicing together exons.

• Introns interrupt, but do not scramble, the DNA sequence that codes for a polypeptide chain.

Eukaryotic gene transcripts are processed before translation

• The pre-mRNA gets modified at the 5′ and 3′ ends.

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• A G cap, a modified guanosine triphosphate (GTP), is added to the 5′ end. It facilitates the

binding of mRNA to the ribosome and protects the mRNA from being digested by

ribonucleases.

• A poly A tail is added to the 3′ end of pre-mRNA. It is 100 to 300 residues of adenine (poly

A) in length and may assist the export of mRNA from the nucleus.

• Splicing removes introns from the primary transcript. If introns were not deleted, a

nonfunctional protein would result.

• RNA splicing removes the introns and splices the exons together.

• As soon as the mRNA is transcribed, it is bound by several small ribonucleoprotein particles

(snRNPs). The RNA in one of the snRNPs binds by complementary base pairing to the

consensus sequence (sequences with little variation) at the 5′ exon–intron boundary.

Another snRNP binds near the 3′ exon–intron boundary.

• Next, other proteins bind, forming a large RNA–protein complex called a spliceosome.

• The spliceosomes cut out the introns and join the exons together to make mature mRNA.

How Is RNA Translated into Proteins?

• As proposed by Crick’s adaptor hypothesis, translation requires tRNA to bridge mRNA to

specific amino acids. To accomplish this, two things are key:

• tRNAs must read mRNA correctly.

• tRNAs must deliver the correct amino acid.

Transfer RNAs carry specific amino acids and bind to specific codons

• The codon in mRNA and the amino acid in a protein are related by way of an adapter—a

specific tRNA molecule that carries (is “charged with”) an amino acid, associates with mRNA

molecules, and interacts with ribosomes.

• A tRNA molecule has 75 to 80 nucleotides and a three-dimensional shape (conformation)

maintained by complementary base pairing and hydrogen bonding.

• The three-dimensional shape of the tRNAs allows them to combine with the binding sites of

the ribosome.

• At the 3′ end of every tRNA molecule is a site to which its specific amino acid binds

covalently (the amino acid attachment site).

• Midpoint in the sequence are three bases called the anticodon.

• Each tRNA species has a unique anticodon, which is complementary to the mRNA codon for

that tRNA’s amino acid.

• At contact, the codon and the anticodon are antiparallel to each other. As an example,

consider arginine:

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• The DNA sequence that codes for arginine is 3′-GCC-5′, which is transcribed by

complementary base paring to produce the mRNA codon 5′-CGG-3′.

• That mRNA codon binds by complementary base pairing to a tRNA with the anticodon 3′-

GCC-5′, which is charged with arginine.

• The cell does not need 61 different tRNA species, each with a different anticodon; in fact, it

gets by with about two-thirds of that number because the specificity for the base at the 3′

end of the codon is not always strictly observed. This phenomenon is called wobble.

• Wobble is allowed in some matches but not in others. It does not allow the genetic code to

be ambiguous.

Activating enzymes link the right tRNAs and amino acids

• The charging of each tRNA with its correct amino acid is achieved by a family of activating

enzymes called aminoacyl-tRNA synthetases.

• Each activating enzyme is specific for one amino acid and its tRNA.

• The enzyme has a three-part active site that recognizes a specific amino acid, ATP, and a

specific tRNA.

• The process of tRNA charging is sometimes called the second genetic code.

• A clever experiment by Seymour Benzer demonstrated the importance of the specificity of

the attachment of tRNA to its amino acid. The amino acid cysteine, already attached to its

tRNA, was chemically modified to become a different amino acid, alanine. The tRNA, not the

amino acid, was recognized when this hybrid charged tRNA was put into a protein

synthesizing system.

• This experiment showed that the protein synthesis machinery recognizes the anticodon of

the charged tRNA, not the amino acid attached to it.

The ribosome is the workbench for translation

• The structure of a ribosome allows it to hold mRNA and charged tRNAs in the right

positions.

• A ribosome can use any mRNA and all species of charged tRNAs and thus can be used to

make many different polypeptide products. The mRNA, as a linear sequence of codons,

specifies the polypeptide sequences to be made.

• Each ribosome consists of two subunits, one large and one small.

• In eukaryotes, the large subunit has three different associated rRNA molecules and 45

different proteins.

• The smaller subunit has one rRNA and 33 different protein molecules.

• When they are not translating, the two subunits are separate.

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• The different proteins and rRNAs are held together by ionic bonds and hydrophobic forces,

not covalent bonds.

• The structure can self-assemble if disassembled by detergents.

• On the large subunit of the ribosome are three sites to which tRNA can bind. A charged

tRNA traverses these three sites in order:

• The A (amino acid) site is where the charged tRNA anticodon binds to the mRNA codon,

lining up the correct amino acid to be added to the polypeptide chain.

• The P (polypeptide) site is where the tRNA adds its amino acid to the polypeptide chain.

• The E (exit) site is where the tRNA, having given up its amino acid, resides before being

released to pick up another amino acid to begin the process again.

Translation takes place in three steps

• Initiation:

• The translation of mRNA begins with the formation of an initiation complex, which consists

of a charged tRNA bearing the first amino acid of the polypeptide chain and a small

subunit of the ribosome, both bound to the mRNA.

• This complex first binds to a complementary ribosomal binding site (the Shine–Dalgarno

sequence) on the mRNA upstream (toward the 5′ end) of the actual start codon that begins

translation.

• The anticodon of a methionine-charged tRNA binds to this start codon (AUG) by

complementary base pairing to complete the translation. Thus, the first amino acid in a

polypeptide chain is always methionine. (However, in many cases the initiator methionine

is removed by an enzyme after translation.)

• The large subunit then joins the complex.

• The mRNA, two ribosomal subunits, and methionine-charged tRNA are put together by

initiation factors.

• Elongation:

• A charged tRNA whose anticodon is complementary to the second codon of the mRNA

now enters the open A site of the large ribosomal subunit. The large subunit then catalyzes

two reactions:

• Breakage of the bond between the tRNA in the P site and its amino acid (on the

polypeptide).

• Peptide bond formation between that amino acid and the one attached to the tRNA in the

A site.

• Because the large subunit performs these actions, it said to have peptidyl transferase

activity.

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• After the first tRNA releases its methionine, it moves to the E site and is dissociated from

the ribosome, returning to the cytosol. The second tRNA, now bearing a dipeptide, is

shifted to the P site as the ribosome moves one codon along the mRNA (5′-to-3′).

• The elongation process continues and the steps are repeated:

• The next charged tRNA enters the open A site, where its anticodon binds with the mRNA

codon.

• Its amino acid forms a peptide bond with the amino acid chain in the P site, so that it

picks up the growing polypeptide chain from the tRNA in the P site.

• The tRNA in the P site is transferred to the E site and released. The ribosome shifts one

codon, so the entire tRNA polypeptide complex moves to the newly vacated P site.

• All these steps are assisted by proteins called elongation factors.

• Termination:

• When a stop codon—UAA, UAG, or UGA—enters the A site, it binds a protein release

factor, which allows hydrolysis of the bond between the polypeptide chain and the tRNA in

the P site.

• The newly completed protein then separates from the ribosome. Its C terminus is the last

amino acid in the chain; its N terminus, at least initially, is methionine (as a consequence

of the AUG start codon); and in its amino acid sequence, it contains information specifying

its conformation and ultimate cellular destination.

Polysome formation increases the rate of protein synthesis

• Several ribosomes work simultaneously at translating a single mRNA molecule, producing

multiple molecules of the protein at the same time. As soon as the first ribosome moves far

enough away from the Shine–Dalgarno sequence, a second initiation complex can form,

then a third, etc.

• An assemblage consisting of a strand of mRNA with its beadlike ribosomes and their

growing polypeptide chains is called a polysome.

• A polysome is like a cafeteria line, where one patron follows another, all adding items to their

trays. The person at the start of the line has little food (a newly initiated protein); the person

at the end has a complete meal (a completed protein).

What Happens to Polypeptides after Translation?

• Especially in eukaryotic cells, the site of a polypeptide’s function may be far away from its

point of synthesis in the cytoplasm. In addition, polypeptides are often modified by the addition

of new chemical groups that have a functional significance.

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Signal sequences in proteins direct them to their cellular destinations

• As the polypeptide emerges from the ribosome, it folds into its three-dimensional shape.

• The amino acid sequence also contains a signal sequence, its “address label,” indicating

where in the cell the polypeptide belongs.

• All protein synthesis begins on free ribosomes in the cytoplasm.

• As the polypeptide chain is made, information contained in its amino acid sequence gives it

one of two sets of instructions:

• Finish translation and be released to the cytoplasm. Such proteins are sent to the nucleus,

mitochondria, plastids, or peroxisomes or, lacking such specific instructions, remain in the

cytosol.

• Stop translation, go to the endoplasmic reticulum, and finish synthesis there. After protein

synthesis is completed, such proteins may be retained in the ER and sent to the Golgi

apparatus from where they may be sent to the lysosomes, to the plasma membrane, or,

lacking such specific instructions, be secreted from the cell.

• Destination: nucleus, mitochondrion, or chloroplast:

• After translation some polypeptides have a short exposed (signal) sequence of amino

acids, usually at the N terminus or in the interior part of the chain, that directs them to an

organelle.

• Dilworth et al. in England demonstrated that the peptide sequence of Pro–Pro–Lys–Lys–

Lys–Arg–Lys–Val would target newly synthesized proteins to the nucleus.

• Signal sequences have a conformation that allows them to bind to the docking protein at

the outer membrane of the appropriate organelle.

• A channel then opens in the membrane, allowing the protein to pass into the organelle.

• In the process, the protein usually is unfolded by a chaperonin so that it can pass through

the channel, after which it refolds to its normal conformation.

• Destination: endoplasmic reticulum:

• Those with specific 15–30 amino acid-long hydrophobic sequences at the N terminus part

of the chain are sent initially to the ER and then to the Golgi.

• In the cytoplasm, before translation is finished, the signal sequence binds to the signal

receptor particle composed of protein and RNA.

• The binding blocks further protein synthesis until the ribosome attaches to a specific

receptor protein on the surface of the ER.

• Once again the receptor protein is converted into a channel through which the growing

peptide passes.

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• The elongating polypeptide may be retained in the ER or enter the lumen of the ER. In

either case, an enzyme in the lumen of the ER removes the signal sequence.

• Then, protein synthesis resumes and the chain grows longer until its sequence is

completed.

• If the finished protein enters the lumen, it can be transported to other cellular

compartments outside the cell via the ER and the Golgi apparatus.

• Additional signals are needed to further direct the protein. These are of two kinds:

• Sequences of amino acids that allow the protein’s retention in the ER.

• Sugars, added in the Golgi apparatus. The resulting glycoproteins end up either at the

plasma membrane or in the lysosome (or plant vacuole).

• Proteins with no additional signals pass from the ER through the Golgi apparatus and are

secreted from the cell.

Many proteins are modified after translation

• Most finished proteins are not identical to the translation from the mRNA code. Most

polypeptides are modified in a number of ways after translation.

• These modifications are essential to the final functioning of the protein.

• Proteolysis is the cutting of the polypeptide chain.

• Cleavage of the signal sequence from the growing polypeptide chain is an example of

proteolysis.

• Some proteins are made from polyproteins (long polypeptides) that are cut into final

products by enzymes called proteases.

• Glycosylation involves the addition of sugars to the protein to form glycoproteins.

• In both the Golgi apparatus and the ER, resident enzymes directly catalyze the addition of

various sugars or sugar chains.

• One such type of “sugar coating” is essential for addressing proteins to lysosomes. Others

are important in the conformation and recognition functions of proteins at the cell surface.

Still other attached sugars help to stabilize proteins stored in vacuoles or plant seeds.

• Phosphorylation is the addition of phosphate groups to proteins. The charged phosphate

groups change the conformation of a protein, often exposing the active site of an enzyme or

a binding site for another protein.

Lecture #14 — Animal Development

Information germane to animal development is divided between chapters 19 and 44 in the

textbook. We shall not be able to cover both chapters in their entirety. We will instead sample a

32

few salient features of animal development, but not necessarily in the order presented in the two

chapters. We believe that the order at which topics are presented here paints a more coherent

picture of development. We shall elide the techniques involved in studying development, and

ignore details and names of genes and members of signaling pathways that mediate

developmental processes, establishment of polarity, segmentation, and homeosis. We will also

bypass the growth of the embryo. Be mindful that the study of development is an enormous

field; we hardly doing it justice in highlighting a handful of landmark events.

Development is the Progressive Restriction in Potential (pp. 408, 410 – 412)

the zygote is a totipotent cell; it has the potential to give rise to all cell types and an

entire individual.

as development proceeds, cell become gradually multipotent, or pluripotent cells (see

fig. 42.2), their ability to produce sundry cell types become gradually more restricted.

[Note: totipotency, pluripotency, and multipotency are not necessarily well-defined terms.

The boundaries among them may be fuzzy.]

Major Developmental Processes (p. 406)

determination confers cells with a particular fate, although this fate may yet to be

expressed. For example, at the end of cleavage, cells in different parts of the blastula are

determined to be ectoderm, mesoderm, or endoderm.

differentiation occurs when cells express traits of the tissue to which they have been

determined. For example, skin cells (ectodermal) become squamous, or muscle cells

(mesodermal) become fusiform as they develop.

morphogenesis is the process of shape and pattern formation. Formation of the neural

tube and the heart, for example, are early morphogenetic events.

growth is the process of size increase. Differential growth leads to further changes in

shape and pattern.

[Note: the transition between determination and differentiation is not necessarily a sharp

one. It is not always easy to determine when determination ends and

differentiation begins.]

Fertilization and the Establishment of the Cardinal Axes in Amphibian Egg (pp 905 – 906;

924 – 925)

the unfertilized egg is radially symmetrical. The amphibian egg contains a yolk-poor,

pigmented, animal hemisphere, and a yolk-rich, non-pigmented, vegetal hemisphere.

Cytoplasmic constituents are largely homogeneously distributed.

33

fertilization usually occurs in the animal pole. The sperm entry point defines the midline

of the egg and future embryo, bisecting the left and the right sides.

the sperm centriole, along with the acrosome body, introduced during fertilization

organizes local tubulins into parallel arrays of spindles, which generates tension which

pulls and rotates the cortex of the egg towards the sperm entry point.

rotation of the pigmented cortex towards the sperm entry point exposes the grey crescent

in the egg opposite to the sperm entry point. The grey crescent marks the future dorsal

side of the embryo; the sperm entry point becomes the ventral side. The position of the

grey crescent will come to contain cytoplasmic materials critical for the organizer.

the animal and vegetal poles become roughly the anterior and posterior ends,

respectively, of the future embryo.

Cortical Rotation and the Rearrangement of the Egg Cytoplasm (pp 924 – 925)

the egg cytoplasm contain maternal materials, proteins and mRNAs, needed for the early

development of the egg prior to transcription of the embryonic genome.

some of the materials are structural proteins and enzymes, some are transcription factors

and signaling molecules, some are evenly distributed within the cytoplasm, some are

uneven.

The uneven distribution of these cytoplasmic determinants will help to guide the

development of the embryo.

cortical rotation is one of the processes that trigger the establishment of gradients of

certain materials in the dorsal-ventral axis in the egg cytoplasm.

in the amphibian egg, the sequence of event following cortical rotation results in an

uneven distribution of -catenin in the grey crescent (future dorsal) side of the embryo. -catenin is a transcription factor, a “genetic master switch” that coordinates the

expression of a host of “dorsal” gene products. [cf. coordinated expression of stress

response genes (pp. 355-356).]

Cleavage (pp 925 – 926)

fertilization activates the egg and initiates mitosis. The first plane of division forms at the

midline, dividing the egg into a left and a right cell. Additional divisions occur in the

orthogonal planes.

in complete cleavage, the cleavage furrows cut through the entire egg. Incomplete or

discoidal cleavage occurs in eggs with a large amount of yolk. The embryo forms as a

disc on one side of the egg. Insect eggs exhibit superficial cleavage. Nuclear division is

not accompanied by cell division. Nuclei migrate to the surface of the egg before they

34

are partitioned into cells.

during cleavage, DNA replication and mitosis are not accompanied by cell growth. The

G1 and G2 phases of the cell cycle are bypassed.

at the end of cleavage, the egg becomes a blastula, and the cells that make up the blastula

are called blastomeres.

blastomeres compartmentalize the cytoplasm of the initial zygote. Cytoplasmic

constituents that are non-homogeneously distributed initially will be unevenly distributed

among blastomeres. Blastomeres are different from each other by virtue of the

cytoplasmic constituents that they have acquired.

The Fate Map (pp. 927 – 928)

blastomeres located at different parts of the blastula are determined: they have acquired a

particular fate, depending on their position.

as a result, a fate map of the three germ layers can be charted on the blastula.

cells located in the animal hemisphere are destined to become ectodermal tissues; cells

located in the vegetal hemisphere are destined to become endodermal tissues; cells that

occupy an equatorial belt in between the two hemispheres are destined to become

mesodermal tissues.

Gastrulation (pp. 928 – 930, 933 – 934)

gastrulation involves cell and tissue movement that rearranges the blastula into a

gastrula, in which the three germ layers are positioned with the ectoderm on the outside,

the endoderm in the inside, and the mesoderm in between.

there are many modes of gastrulation:

sea urchin eggs gastrulate by invagination: internalization of the vegetal pole.

amphibian eggs gastrulate by involution: movement of presumptive mesodermal cells

located in the equator into the interior of the blastula through the blastopore.

Involuted mesoderm cells will position themselves between ectoderm and endoderm.

Ectodermal tissues from the animal pole move ventrad to encircle the entire embryo.

in discoidal eggs that contain a large amount of yolk, gastrulation occurs by

involution of external cells — epiblasts — into the blastoceol through the primitive

groove. The primitive groove is analogous to the blastopore, and Henson’s node

analogous to the dorsal lip of the frog egg.

Determination and Lability of Cell Fate (pp. 407 – 408, 413 – 415, 930 - 932)

determination of cell fate is progressive; fate slowly becomes irreversible as development

35

proceeds. The timing of determination and its reversibility are species- and tissue-

dependent.

a fate map is demonstrable in the blastula at the end of cleavage. At this time, fate may

or may not be fixed.

dorsal and ventral fate is determined as early as the 8-cell stage in the sea urchin embryo.

If separated from their counterpart, dorsal and ventral cells can form dorsal and ventral,

respectively, embryonic structures only. Cytoplasmic determinant missing from dorsal or

ventral cells are irreplaceable. This is an example of mosaic development, in which a

complete embryo cannot be reconstituted from the remaining parts.

in the frog embryo, part of the egg or early blastula can form a normal embryo as long as

it contains at least a part of the grey crescent. This is an example of regulative

development. Parts of egg that do not contain the grey crescent will develop into a

“belly piece”, which contains mostly endoderm covered by ectoderm.

in the frog embryo, determination of germ layer fate is labile before gastrulation, and

fixed after gastrulation. Before gastrulation, if cells destined to become ectoderm

(according to the fate map) are transplanted to a position where cells are destined to

become endoderm, the transplanted cells will become endodermal cells, viz. cells can

take on new fates depending on the new environment in which they find themselves.

The Organizer and Organization of the Embryonic Axis (p. 930 – 932)

before gastrulation, transplanted blastular tissues will have their fate re-determined by

their new position. This is true of all parts of the blastula except the dorsal lip of the

blastopore.

the blastopore appears in the dorsal side of the embryo where the grey crescent was

immediately after fertilization.

cells involuting into the interior during gastrulation pass through the blastopore.

cells located at the dorsal lip of the blastopore at the onset of gastrulation are the first to

go through the blastopore, and form the leading edge of the involution. They are

mesodermal cells destined to form part of the notochord.

transplantation of the dorsal lip to anywhere on the embryo results in the formation of a

secondary blastopore and a new focus of involution. The newly involuted cells form a

secondary notochord, and organize surround tissues to form a supernumerary (secondary)

embryo. The dorsal lip of the blastopore was thus christened the organizer.

the supernumerary embryo is made up of tissue from the host, not from the transplanted

organizer. Not only does the organizer retain its own fate regardless of the environment

in which it finds itself, it changes the fate of the surrounding tissues.

36

we now know that cells of the dorsal lip are unique because they have acquired -catenin

during early cleavage.

Neurulation (p. 935) [We shall revisit this when we talk about the nervous system.]

neurulation is the first of the morphogenetic events that follow gastrulation.

the notochord induces the overlying ectoderm to become the neural plate and take on

neural fate.

the neural plate folds upwards at the neural folds, which eventually meet at the midline.

the neural tube pinches off from the overlying epidermis.

neurulation does not occur if the notochord is removed after gastrulation.

transplanting the notochord underneath the ectoderm anywhere on the body will result in

neural induction and formation of a neural tube out of the overlying ectoderm.

The Neural Crest Cells (p. 935) [We shall revisit this when we talk about the nervous system.]

neural crest cells originate at the boundary between the neural plate and adjacent

epidermis.

as the neural tube forms, neural crest cells separate from both the neural tube and the

overlying epidermis, and migrate away from the crest of the neural tube.

these cells migrate throughout the entire body, and give rise to all the pigment cells under

the skin, all the neurons and supportive cells of the peripheral nervous system and the

autonomic nervous system, all the neurons in the digestive tract (enteric nervous system),

adrenaline producing (chromaffin) cells in the adrenal medulla, some cells in the inner

lining of the heart, and cartilage in the head.

due to the their versatility, many embryologists consider the neural crest cells the fourth

germ layer.

Body Segmentation and Somitogenesis (pp. 935 – 936)

for segmental animals such as vertebrates, the body is composed of homologous, repeated

segments that are modified regionally into the various body parts.

formation of the somites is the earliest sign of segmentation.

somites form from partitioning of the mesodermal tissues that lie on both sides of the

neural tube. Somitogenesis progresses from anterior to posterior.

the somites soon break up; its cells migrates out to form the dermis under the skin, as

well as all the bones and muscles of the body (except the head, where cells from the

neural crest contribute to much of the skeletal and muscular systems).

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Induction and Morphogenesis (pp. 414 – 415)

do not confuse induction as a morphogenetic mechanism with induction in regulation of

gene expression.

induction is a important developmental mechanism. Groups of cells interact with each

other, either by direct contact or via diffusible signals, and induce each other to take on

new fates.

neural induction – the induction of the formation of the neural tube by the notochord, is a

good example.

another good example is the sequence of inductive events in the formation of the

vertebrate eye.

at the onset of eye development, an anterior region of the neural tube evaginates to

form the optic vesicle.

the optic vesicle induces the epidermis to thicken to form the optic or lens placode.

the optic vesicle involutes to form the optic cup.

the optic cup induces the involution of the lens placode, which pinches off from the

overlying epidermis to form the lens.

the lens induces the overlying epidermis to become transparent and form the cornea.

transplantation of the optic vesicle under the epidermis anywhere on the body induces

the formation of an eye via the same stepwise inductive process.

removal of the optic vesicle prevents the formation of the optic placode and a lens.

removal of the lens results in an opaque epidermis covering the optic cup.

Apoptosis and Morphogenesis (p. 417)

apoptosis is coordinated elimination of cells; it is often called programmed cell death.

strategic removal of cells and tissues is an important morphogenetic mechanism.

the vertebrate limb forms from a bud of solid tissue; digits are formed when connective

tissues in between are removed by apoptosis.

apoptosis of the overlying epidermal tissues allows the eye to “open”.

Lecture #15 — Homeostasis and Thermoregulation

The section in Chapter 40 on the microscopic structures of cells and tissues is important.

We have covered some aspects this in the laboratory, and will continue to visit tissue

microscopic anatomy in conjunction with our excursions through the organ systems both in the

laboratory and in lecture.

Homeostasis (from the Greek homoios, same, and stasis, stance, posture) is the

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establishment and maintenance of a constant, stable internal environment in metazoans.

Constancy of the internal environmental condition is maintained by dynamic equilibrium through

interactive and integrated regulatory mechanisms. For an organism, the immediate surrounding

is the external environment, and the body cavity is the internal environment. For an organ or an

organ system, the body cavity is the external environment. For a cell, the extracellular fluid is

the external environment, and the cytoplasm is the internal environment. In all cases,

homeostasis is exercised to maintain a stable internal environment and to keep biochemical and

cellular activities constant.

All organ systems participate in the maintenance of a constant internal environment. Most

organ systems contribute to homeostasis of more than one physiological parameter; the

homeostasis of each parameter involves the coordinated action of multiple organ systems.

The nervous system and the endocrine systems are the chief homeostatic integrators — they

coordinate the activities of other organ systems.

Main Features of Homeostatic Systems

Stability — homeostatic systems must be intrinsically stable.

Set-point — a reference point to which internal condition is brought.

Adjustability — internal conditions are adjustable to accommodate changes in the external

environment and to bring conditions back to the set-point.

Negative feedback loop — prevents deviation from the set-point.

Feed-forward mechanism — adjusts or changes set-point.

Participate in coordinating multiple organ systems.

Body Mass, Body Surface, Surface-to-Volume ratio, and the Heat Budget

Organisms produce heat through internal metabolic activity, and gain heat from radiation and

conduction from the environment.

Organisms lose heat through conduction and convection to the environment, and evaporation

of fluid.

Generation of metabolic heat is proportional to body mass.

Heat loss is proportional to body surface area.

Animals with low surface-to-volume ratios tend to have low metabolic rates and vice versa.

Animals can increase heat loss by increasing body surface relative to body mass.

Thermobiology

The freezing of water and the thermal denaturation of proteins (disruption of hydrogen

bonds) set the lower and upper temperature limits, respectively, of biological processes.

39

[Animals and plants that have evolved anti-freezing mechanisms can survive below the

freezing point of water. Animals with modified proteins that can function at high

temperature can survive at temperature close to (or sometimes higher than) the boiling point

of water.]

Temperature-dependence of chemical reactions is the most important impact of temperature.

The temperature quotient, Q10, is a measure of temperature sensitivity of physiochemical

parameters. The majority of biological reactions have Q10’s between 2 and 3.

Thermoregulation exemplifies many of the salient features of homeostatic systems. Vertebrates

employ two primary mechanisms for generating heat:

Endothermy — metabolic heat is generated internally.

Ectothermy — heat is gained from the environment.

Based on body temperature relative to ambient temperature, animals can be classified into three

main types:

Homeotherms — temperature regulators.

maintain a stable body temperature in spite of changes in ambient temperature, i.e.

complete temperature homeostasis.

homeotherms tend to be endothermic.

the challenges for homeotherms are in generating heat when ambient temperature is low,

and dissipating heat when ambient temperature is high.

Poikilotherms — temperature conformers.

body temperature fluctuates along with ambient temperature, usually (but not necessarily)

matches ambient temperature.

poikilotherms tend to be ectotherms.

the challenge for poikilotherms is in maintaining constancy in physiological functions

over a wide range of body temperature.

Heterotherms — partial regulators.

body temperature conforms to ambient temperature within a set range, but regulates when

temperature is above or below this range.

heterotherms tend to be ectothermic (poikilothermic) when ambient temperature is

relatively high, and become endothermic (homeothermic) when ambient temperature falls

below a set point.

Maintenance of Body Temperature in Poikilotherms

Behavioral adaptation is used to regulate body temperature in response to changes in

40

diurnal or seasonal ambient temperature.

Acclimatization — biochemical adaptation — is used to compensate for seasonal changes in

ambient temperature. Metabolic rates remain basically constant in spite of seasonal

temperature changes. Isozymes (cf. chapter 8) with different temperature coefficients are

expressed during different seasons.

Physiological or anatomical adaptation, e.g. modulation of heart rate or pattern of blood

flow, allows poikilotherms to conserve metabolic heat and maintain a body temperature

higher than ambient temperature.

Maintenance of Body Temperature in Homeotherms

Body temperature is preset and regulated by the hypothalamus, which acts as a biological

“thermostat”. Direct warming of the hypothalamus decreases body metabolic rate; direct

cooling of the hypothalamus increases metabolic rate.

Deviations from the set-temperature — the set-point — activate behavioral and physiological

compensatory mechanisms.

When environmental temperature is within the thermoneutral zone, which is usually

slightly lower then the desired body temperature (37ºC for mammals), basal metabolic

activity generates sufficient heat for maintaining a constant body temperature. Non-

metabolic heat conservation or dissipation mechanisms: peripheral vasoconstriction,

piloerection, vasodilatation, are employed for thermoregulation

The thermoneutral zone is bounded by a lower critical temperature and an upper

critical temperature. Outside of the thermoneutral zone, feedback regulatory

mechanisms initiate metabolic activities that are used to generate or dissipate heat.

Metabolic heat-generating mechanisms include: shivering (in birds and mammals), and

increasing brown fat mitochondrial thermogenin production of metabolic heat instead of

ATP (in mammals).

Metabolic cooling mechanisms include: sweating, salivating, and panting. Metabolic

cooling mechanisms also generate heat.

The cellular correlates of the set point, and how the set point is set and reset, are poorly

understood.

Febrile Response — Resetting the Thermostat Upwards

Homeostatic systems change equilibrium set-points by feedforward control. [Feedback

control prevents change; feedforward control executes change.]

To increase body temperature, resetting is required. After resetting, the body uses the same

thermoregulatory mechanisms to maintain temperature at the new set-point.

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Note: fever, pyrexia, is not hyperthermia. Fever is a new equilibrium; the thermoregulatory

machinery is functioning as it should. Hyperthermia, sometimes commonly called heat

stroke, is an increase in body temperature above the set-point; it is the result of a breaking of

the thermoregulation machinery — metabolic cooling mechanisms generate heat that cancel

and overwhelm its own ability to cool the body.

Hibernation — Resetting the Thermostat Downwards

During hibernation, the feedforward mechanism resets the set-point at a lower temperature.

This creates a new dynamic equilibrium. The same thermoregulatory mechanisms are kicked

into action when body temperature deviates in either direction from the new set-point.

Arousal at the end of hibernation is preceded by a new resetting of the set-point to a higher

temperature. Once this is established, the thermoregulatory machinery will warm up the

body.

Lecture #16 — Endocrine Systems and Hormones

There are hundreds of animal hormones. The textbook presents several interesting

examples. I suggest that you read about all of them. To illustrate some fundamentals of

endocrine systems, we will focus on insect ecdysone, calcitonin, parathyroid hormone and

calcitriol, and hormones of the cortisol pathway. We will skip the section on strategies involved

in studying hormones. We will discuss sex steroids and a few other hypothalamic-pituitary

hormones when we examine the reproduction system.

Hormones (from the Greek homoios, I excite) are chemical messengers, released by

secretory cells of endocrine organs, and act to influence the physiology of other organ systems

by transport through the systemic circulation.

The Vertebrate Endocrine System

consists of endocrine glands, including two pairs of gonads. The brain, sometimes called the

Master Gland, is generally considered to be a part of the endocrine system.

serves as the interface between the external and internal environment: sensory signals from

the environment are translated into chemical signals via the nervous and endocrine systems.

coordinates organ systems through chemical messengers in both homeostatic and non-

homeostatic functions.

Major Classes of Hormones

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Peptide hormones

act through integral membrane receptors, most commonly G-protein coupled or kinase

receptors.

usually involve “second” messenger systems (commonly G-proteins, cAMP, kinases,

calcium, et cetera).

cellular responses include protein synthesis, anabolic and catabolic changes, membrane

channel opening or closing, membrane pump activation, cytoskeletal protein assembly or

disassembly, shape changes, motility changes, gene regulation (c.f. chapter 7).

the hypothalamus and pituitary release peptide hormones only, a few other glands also

release peptide hormones.

Steroid hormones

cholesterol derivatives; lipid soluble.

act through cytoplasmic nuclear receptors, also called nuclear hormone receptors.

ligand-receptor complex acts as transcription factors that bind to consensus sequences at

gene promoter or repressor sites; ligands alone or receptors alone are not active factors.

cellular responses include positive or negative transcription regulation.

mostly released by peripheral glands in response to central tropic hormone stimulation.

Others

monoamines: amino acid derivatives: tyroxine, epinephrine, norepinephrine, melatonin.

These hormones act through membrane receptors and second messengers.

non-steroidal lipid-soluble hormones: prostaglandins, vitamin D, juvenile hormone. These

hormones act through cytoplasmic nuclear receptors.

Hormone Release and Control Mechanisms

Simple on/off switch

e.g. ecdysone or juvenile hormone release in insects.

usually triggered by environmental or sensory cues, e.g. changes in light-dark cycle, inflation

of abdomen after a blood meal, presence of a reproductively receptive member of the

opposite sex, or under neural control, e.g. cyclical release of juvenile hormone in insects.

generally lack feedback regulation; when cue or trigger is removed, release ends.

Simple feedback control

e.g. calcium control of parathyroid hormone release by parathyroid gland.

high plasma Ca++ concentration inhibits parathyroid hormone release, low plasma Ca++

43

concentration removes inhibition.

Dual (antagonistic) hormone feedback control

e.g. calcitonin-parathyroid hormone maintenance of Ca++ homeostasis.

high plasma Ca++ level inhibits parathyroid hormone release and stimulates calcitonin release.

low plasma Ca++ level removes stimulus for calcitonin release and permits parathyroid

hormone release.

Multimodal negative feedback control

e.g. regulation of the release of hypothalamic corticotropin releasing hormone and pituitary

adrenocorticotropic hormone (ACTH).

corticotropin releasing hormone stimulates the release of ACTH, which in turn stimulates the

release of cortisol by the adrenal cortex.

two feedback inhibitors acting at two sites:

ACTH feedback inhibits corticotropin releasing hormone secretion.

cortisol feedback inhibits corticotripin releasing hormone and ACTH secretion.

Sir Vincent Wigglesworth’s Rhodnius Experiment

molting is triggered by a blood meal (inflation of the abdomen).

stretching of the abdominal wall sends sensory signal to the brain to initiate the molting

process (which takes about two weeks).

if head is removed one hour after a blood meal, the body does not molt.

if head is removed one week after a blood meal, the body molts into an adult.

if a body with head removed one hour after a blood meal, which would not normally molt, is

connected to a body with head removed one week after a blood meal, both bodies molt into

an adult.

conclusions:

signal from the brain initiates the molting process.

the signal is a diffusible substance that diffuses from the brain to the body, and to the

body of the parabiotic Rhodnius.

there is a lag time between a blood meal and the release and delivery of the signal from

the brain.

we now know that this substance is the steroidal molting hormone ecdysone.

the brain also delivers a substance that prevents metamorphosis at the time of molting. In

the absence of this substance, the insect molts into an adult. We now know that this

substance is juvenile hormone.

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Parathyroid Hormone, Calcitonin, Vitamin D, Calcitriol, and Calcium Homeostasis

parathyroid hormone and calcitonin are released by the parathyroid gland and the thyroid

gland, respectively, vitamin D is produced in the skin in the presence of ultraviolet radiation,

and calcitriol, the active form of vitamin D, is produced in the liver from vitamin D.

plasma calcium is maintained at between 9 and 11 mg/100 ml. High plasma Ca++ triggers the

release of calcitonin and inhibits the release of parathyroid hormone.

calcitonin promotes the deposition of calcium in bone, thereby removing Ca++ from

circulation.

parathyroid hormone maintains Ca++ homeostasis by orchestrating the activity of three organ

systems: the digestive, skeletal, and renal systems:

increases Ca++ uptake by stimulating absorption of Ca++ in the gut.

stimulates the re-absorption of Ca++ from bone.

helps to conserve Ca++ by stimulating the re-uptake of Ca++ in the kidney.

the vitamin D derivative, calcitriol, works synergistically with parathyroid hormone in

effecting calcium homeostasis.

Organization of the Anterior Pituitary System

consists of two groups of cells: hypothalamic cells and pituitary cells, and a vascular delivery

system: the hypothalamic portal in the pituitary stalk.

hypothalamic cells have short axons that ends in the pituitary stalk. There terminals secrete

releasing hormones (or inhibiting hormones) from their terminals in the pituitary stalk,

which are delivered to the anterior pituitary via the portal veins.

in the anterior pituitary, releasing hormones stimulate cells to release tropic hormones,

which are delivered to the body via the vasculature.

there are many cell types in the hypothalamus and anterior pituitary. Different hypothalamic

cells release specific releasing or inhibiting hormones, each one of these hormones acts on

corresponding pituitary cells that release specific tropic hormones. There are more than a

dozen releasing hormone-tropic hormone pairs.

Functional Organization of the Hypothalamic-Pituitary-Adrenal Axis in Response to Stress

the adrenal gland consists of an adrenal medulla, which releases epinephrine and

norepinephrine under neural control, and an adrenal cortex, which releases cortisol

(glucocorticoid) under pituitary control.

in response to stressful stimuli, rapid release of epinephrine and norepinephrine increases

heart rate, blood pressure, and glucose delivery to the nervous system and muscles, and

45

decreases glucose utilization and movement of the digestive tract. This rapid response starts

within seconds of the stress stimulus, and may last for minutes.

in response to stressful stimuli, hypothalamus releases corticotropin releasing hormone

(CRH), which stimulates corticotropes in the anterior pituitary to release

adrenocorticotropic hormone (ACTH), which in turn stimulates gland cells in the adrenal

cortex to secrete cortisol.

the hypothalamic-pituitary-adrenal axis is organized in two superimposing pathways:

a direct, stimulatory pathway involving corticotrophin releasing hormone,

adrenocorticotropic hormone, and cortisol, each hormone stimulates the release of the

next hormone in the hierarchy.

a feedback, inhibitory pathway in which each hormone lower in the hierarchy inhibits

release of hormones higher up in the hierarchy ― cortisol inhibits the release of CRH and

ACTH; ACTH inhibits the release of CRH.

the feedback inhibition is a self-regulating mechanism ensuring a steady-state level of

circulating cortisol that is appropriate for a particular environmental (stressful) stimuli.

the stress response is a coordinated homeostatic response. Epinephrine and cortisol

coordinate the physiological activity of multiple organ systems. The concentration and

duration of availability of epinephrine and cortisol are appropriate for the stress level.

46

47

Human body has ~3 x 1013 (30 trillion cells)

~10 billion mitosis per second; much division in bone marrow (RBC live 4

months) and epithelial lining of gut (cells in harsh environment replaced

every 2-3 days)

human feces is 20-30% dead epithelial cells (and ~50% bacteria)

Why do cells divide?

Reproduction

Growth

Regeneration

Unicellular organisms use cell division for reproduction.

In multicellular organisms, cell division is also important in growth and repair

of tissues.

Four events must occur for cell division to occur:

Reproductive Signal: to initiate cell division

Replication: of DNA

Segregation: distribution of the DNA into the two new cells

Cytokinesis: separation of the two new cells

DNA replication and segregation are perhaps the most complicated process

in cell division, especially in eukaryotes.

Most prokaryotes have one chromosome: a single, usually circular, molecule

of DNA.

Eukaryotes have multiple chromosomes, all of which are replicated before

cell division.

Ensuring that each daughter cell contains a complete set of chromosomes is

a critical part of cell division.

Prokaryote Cell division

In prokaryotes, binary fission results in two new cells.

External factors such as nutrient concentration and

environmental conditions are the reproductive signals

that initiate cell division

For many bacteria, abundant food supplies speed up

the division cycle

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The Cell Cycle- Eukaryotic cell cycle

A dividing cell cycles between interphase and mitosis.

Interphase is divided into three subphases:

G1: Gap 1 — between end of mitosis and S phase. Cell completed division

and starts growing.

S phase: Synthesis — DNA replicates; each chromosome becomes two sister

chromatids.

G2: Gap 2 — DNA replication completed, cell grows and prepares for mitosis.

If a cell is not destined to divide further, it

usually leaves the cell cycle at this point ---

before DNA replication. Cells are arrested in

what is sometimes called the G0 phase.

A chromosome before replication

consists of a single double-helix.

In a non-dividing human cell, there are 2 sets of 23, or 46 of these

chromosomes.

One set originates from the father, one set from the mother.

Each of the two members of a corresponding pairs of chromosomes are call

homologous chromosomes. Each pair contains information for same

biological functions and same genes at same loci or order

49

Karyotype: the number, shapes, and sizes of the chromosomes in a cell.

Individual chromosomes can be recognized by length, position of

centromere, and banding patterns. They are numbered 1 – 22, plus 2 sex

chromosomes.

A nucleus that contains the full set of 46 chromosomes is called diploid, or

2n.

A nucleus that contains one set of 23 chromosomes is called haploid, or 1n.

Gametes, sperms or eggs, are haploid (1n). Upon fertilization of an egg by a

sperm, the resulting zygote is diploid (2n).

During DNA replication --- in the S-phase of the cell cycle --- a single

chromosome is duplicated into a chromosome containing two sister

chromatids.

Since all chromosomes replicate at the

same time during S-phase, the

homologous chromosome is also

replicated. A diploid nucleus will

therefore contain 23 pairs of sister

chromatids.

50

DNA molecules are “packed” even during interphase.

Chromosomes have many histones—proteins with positive charges that

attract negative phosphate groups of DNA.

Interactions result in the formation of beadlike units, nucleosomes

Spindle has two types of microtubules:

Polar microtubules—form spindle;

overlap in center

Kinetochore microtubules—attach

to kinetochores on the chromatids.

Sister chromatids attach to opposite

halves of the spindle

Mitosis can be divided into phases:

Prophase— chromosomes condense

Prometaphase— nuclear envelope breaks down

Metaphase— chromosomes align at equatorial plate

Anaphase— chromosomes begin moving to spindle poles

Telophase— chromosomes decondense, nuclear envelope reforms

51

52

Meiosis consists of two nuclear divisions (meiosis I & II) but DNA is

replicated only once. The function of meiosis is to:

Reduce the chromosome number from diploid to haploid

Ensure that each haploid has a complete set of chromosomes

Generate diversity among the products—products are different from the

parent cell, and from each other

Unlike mitosis:

In meiosis I, homologous pairs

of chromosomes come

together and pair along their

entire lengths.

After metaphase I, the

homologous pairs segregate;

the sister chromatids remain

together until after meoisis II.

53

Comparing itosis and Meiosis.

Prophase I may last a long time.

Human males: about one week for prophase I and

one month for entire meiotic cycle.

Human females: prophase I begins before birth, and

ends up to decades later during the monthly ovarian

cycle.

Prometaphase I: nuclear envelope and nucleoli

disappear.

Spindle forms; kinetochores of both chromatids of a

chromosome attach to the same half-spindle.

Which member of each homologous pair goes to

which pole is random (called independent

assortment).

Metaphase I: chromosomes are at the equatorial

plate; homologous pairs held together by

chiasmata.

Anaphase I: homologous chromosomes separate;

daughter nuclei contain only one set of

chromosomes. Each chromosome consists of two

chromatids.

Which members of each homologous pair goes to

which pole is random, called independent

assortment.

Telophase I: occurs in some organisms.

54

Nuclear envelope reaggregates; followed by an interphase called

interkinesis.

In other organisms, meiosis II begins immediately after anaphase I.

55

Inheritance, Genes and Chromosomes 2/26/12 12:06 AM

Hereditary materials from each parent is blended in an offspring.

Offspring in an intermediate form of the parent (e.g., red + blue gives

purple).

Once blended, the hereditary material is inseparable; new genetic materials

form and the original materials are lost.

Gregor Mendel’s new theory of inheritance was published in 1866, and

refuted these ideas

This theory was mostly ignored until 1900, when meiosis had been observed.

Three plant geneticists realized that chromosomes and meiosis provided a

physical explanation for Mendel’s results

Rapid growing; produces a lot of seeds; requires

little space.

Naturally self-pollinating, but pollination and

fertilization can be controlled (done by Mendel)

to be sure of offspring and parents.

Has distinct characters: observable physical

feature (e.g., flower color) and traits: form of a

character (e.g., purple flowers or white flowers,

wrinkled). Heritable traits are passed from parents to offspring.

Mendel looked for well-defined, true-breeding traits—the observed trait is

the only one present for many generations. Are homozygous.

True-breeding strains were isolated by inbreeding and selection.

He concentrated on seven traits.

Pollen from one parent was transferred to the stigma of the other parent.

Parental generation = P.

Resulting offspring = first filial generation or F1.

If F1 plants self-pollinate, produces second filial generation or F2.

He crossed plants differing in just one trait, the F1 generations are called

monohybrids.

57

The monohybrids were then allowed to self pollinate to form the F2

generation: A monohybrid cross.

Mendel repeated this for all seven characters.

One trait of each pair disappeared in the F1 generation and reappeared in the

F2—these traits are recessive.

The trait that appears in the F1 is the dominant trait.

The ratio of dominant to recessive in the F2 was about 3:1.

Mendel proposed:

the heritable units were discrete particles—the particulate theory.

each plant has two particles for each character, one from each parent (today

we call this diploid).

During gamete formation only one of these paired units is given to the

gamete (today we call this haploid)

The true-breeding plants in the P generation had two identical copies of the

particle (gene) for each character.

Example: Spherical SS; wrinkled ss

gametes from SS will have one S

gametes from ss will have one s

offspring (F1) will be Ss

S is dominant; s is not expressed in F1.

Alleles: different forms of a gene

Homozygous: an individual having two copies of the same allele of a gene

in the genome. These individuals are true-breeding.

SS ss

Heterozygous: an individual carrying two different alleles of the same

gene.

Ss

Dominant allele: allele that is preferentially expressed in an organism that

also carry another allele of the same gene.

Recessive allele: allele that is not expressed or weakly expressed.

S s

58

Phenotype: Physical appearance of an organism (e.g., spherical seeds).

Genotype: The genetic makeup (e.g., Ss).

Spherical seeds can be the result of two different genotypes—SS or Ss.

A true-breeding spherical seed plant and

true-breeding wrinkled seed plant were

crossed

Wrinkled seeds disappeared in the F1

generation.

Wrinkled seeds reappeared in 1/4 of the F2

generation.

The true-breeding plants of the P generation have two identical copies of the

allele (particle in Mendel’s terminology) for each trait: SS for spherical and

ss for wrinkle seeds.

SS parent produces S gametes, ss parent produces s gametes

All members of the F1 generation have Ss genotype, and spherical

phenotype.

Monohybrid cross produces F2 generation with 3

genotypes and 2 phenotypes.

The Law of Segregation: two copies of a gene

separate when an individual makes gametes.

59

When the F1 self-pollinates, there are three ways to get the dominant trait

(e.g., spherical), but only one way to get the recessive (wrinkled)—resulting

in the 3:1 ratio.

Different alleles of a gene are located on

the same locus in homologous

chromosomes.

The alleles separate during meiosis.

Mendel’s next experiment:

Crossing peas that differed in two characters—seed shape and seed color. He

wanted to see how two characters behaved in the same crosses

True-breeding parents:

SSYY—spherical yellow seeds

60

ssyy—wrinkled green seeds

SSYY ´ ssyy4 SsYy

F1 generation is SsYy—all spherical yellow.

Crossing the F1 generation (all identical double heterozygotes) is a dihybrid

cross.

Mendel asked whether, in the gametes produced by SsYy, the traits would be

linked (i.e., will SY of maternal origin and sy of paternal origin always go into

same gamete), or segregate independently (one paternal and one maternal

allele).

If linked, gametes would be SY or sy; F2 would have three times more

spherical yellow than wrinkled green (3:1).

If independent, gametes could be SY, sy, Sy, or sY. F2 would have nine

different genotypes; phenotypes would be in 9:3:3:1 ratio.

Results indicated new combinations called recombinant phenotypes.

61

F1 all identical double

heterozygotes

Mendel asked

whether, in the

gametes produced by

SsYy, the traits would

be linked, or

segregate

independently.

Result was 9:3:3:1,

therefore independent

segregation

Alleles of different genes sort independently during gamete formation.

Note: this is not true for genes that are on the same chromosome

(sometimes called genes that belong to the same linkage group), but

chromosomes do segregate independently.

62

Human pedigrees can

show Mendel’s laws.

Humans have few

offspring; pedigrees do

not show the clear

proportions that the

pea plants showed.

Geneticists use

pedigrees to determine

whether a rare allele is

dominant or recessive.

Every affected person

has an affected parent

~ half of the offspring

of an affected parent

are affected

Sexes equal

63

If a recessive allele is rare

in the general population, it

is unlikely that two people

that marry will both carry it

unless they are related

(e.g., cousins).

affected person has neither

parent affected

~ a quarter of the offspring

of an unaffected parent are

affected

Sexes equal

Genes do interact, and are not always independent, and dominance and

recessiveness are not always absolute.

Polymorphism -- genes can have more than 2 alleles

Incomplete dominance (semi-dominance) -- some alleles are neither

dominant nor recessive

Epigenesis -- non-genetic affects on phenotype

Not all genes are discrete and qualitative. Many phenotypes are continuous

over a range of variations. Multiple genes that make up quantitative trait

loci determine complex characters (e.g., height).

Different alleles arise through mutation: rare, stable, inherited changes in

the genetic material.

Because of random mutations, more than 2 alleles of a gene may exist in a

population (any one individual has only 2 alleles)

Wild type: allele present in most of the population. Other alleles are

mutant alleles.

Multiple alleles increase the number of possible phenotypes. This is

especially true if dominance of one allele over another is not complete.

64

A given gene may have more than two alleles.

Example: Coat color in rabbits

Multiple alleles increase the number of possible phenotypes.

C = color (dark gray)

cch = chinchilla (gray)

ch = himalayan (point restricted, temperature-dependent)

c = albino (white)

Dominance hierarchy: C > cch > ch > c. Depending on the combination of

alleles, there is a graded series of coat color phenotype.

Some alleles are neither dominant nor

recessive—a heterozygote has an

intermediate phenotype: Incomplete

dominance.

Example: Snapdragons.

Environment factors --- light, temperature,

nutrition, parasites, etc. affect phenotype.

65

DNA and Its Role in Heredity 2/26/12 12:06 AM

Determining that DNA is the Hereditary molecule

In the 1920’s, Frederick Griffith transformed a benign strain of

pneumococcus bacteria into a virulent strain by allowing the former to

interact with dead cells of the latter. He hypothesized that the benign

strained acquired some “transforming principle” from the virulent strain.

To identify the transforming principle:

Avery (1940s) treated samples to destroy different

molecules; if DNA was destroyed, the transforming

activity was lost.

There was no loss of activity with destruction of

proteins, carbohydrates, or lipids.

-When DNA was destroyed, the bacteria

remained virulent.

Deoxoribose Nucleic Acid

Phosphate nucleotide, and base

Carbon 3 and carbon 5 are important.

DNA is a polymer of nucleotides.

The four nucleotides that make up DNA differ only in their nitrogenous bases.

There are two purines (adenine and guanine) and two pyrimidines

(cytosine and thymine).

The ratio of adenine:thymine and of guanine:cytosine is 1:1 (Chargaff’s

Rule).

Rosalind Franklin and Maurice Wilkins studied DNA structure by X-ray

crystallography in the early 1950’s.

James Watson and Francis Crick studied DNA structure by model building at

exactly the same time.

DNA is a double stranded, right-handed helix with a constant diameter.

The strands are antiparallel, held together by hydrogen bonds between

complementary base pairs on opposite strands:

A to T with 2 hydrogen bonds

G to C with 3 hydrogen bonds

A purine pairs with a pyrimidine, thus the

polymer has a constant diameter.

The large numbers of hydrogen bonds produce a

highly stable double helix.

Carbon 3 has the hydroxyl group

Carbon 5 has the Phosphate free

DNA Backbone has a negative charge

How is DNA replicated so that genetic

information is preserved and transmitted from

generation to generation?

How does DNA mediate the synthesis of specific proteins?

Complementary base pairing suggests an obvious mechanism by which

exact copies of DNA can be produced.

67

Kornberg showed that DNA contains information for its own replication.

He combined in a test tube: DNA, the four deoxyribonucleoside

triphosphates, DNA polymerase, salts, and pH buffer.

Each strand of the double helix serves as a template for the synthesis of the

complementary strand.

Semiconservative replication: the new double

helix consists of one parent strand and one new

strand.

Conservative replication: the parent helix is

unchanged, an entirely new helix is synthesized.

Dispersive replication: fragments of the parent

helix serve as templates, two new helices are

synthesized.

Meselson and Stahl demonstrated in 1957 that DNA replication is

semiconservative by using

a technique called density

labeling.

They used DNA labeled

with “heavy” nitrogen (15N).

In a cesium gradient, heavy

DNA moves near the

bottom and light DNA stays

near the top after

centrifugation.

Intermediate DNA moves to

an intermediate position.

Observations:

-- All heavy DNA at time 0.

-- After one generation, all DNA is intermediate.

68

-- After two generations, light DNA appears.

The parental helix is not conserved. This eliminates the conservative model.

The intermediate helix is retained after two generations eliminating the

dispersive model.

The original parental strands are never destroyed. This eliminates the

dispersive model.

DNA replication takes place in two steps:

The hydrogen bonds between the two

strands are broken by the enzyme

helicase. The double helix opens up,

exposing each strand for base pairing.

The new nucleotides are covalently bonded

to each growing strand by DNA

polymerase. In DNA replication,

nucleotides are added to the 3’ end of

each growing strand --- elongation

proceeds from 5’ to 3’.

The triphosphate of a nucleotide is bonded

to the 5’ carbon. A new phosphodiester bond is made with the 3’ carbon.

Energy for synthesis is provided by the hydrolysis of the phosphate bonds in

the nucleotide triphosphates.

New DNA Strand grows from 5’ end to the 3’ end.

A huge protein complex catalyzes DNA replication.

This replication complex recognizes an origin of replication (ori) on a

chromosome. Prokaryote chromosomes usually have one origin. Eukaryote

chromosomes have many origins.

DNA replicates simultaneously in both directions from the origin, forming two

replication forks.

69

Both strands of DNA act as templates.

Recent evidence suggests that the replication complex is stationary, and

DNA threads through it.

The two interlocking DNA rings are

separated by the enzyme DNA

topoisomerase.

In bacteria, DNA Polymerase III

performs most of the synthesis.

DNA polymerases can only build from an existing strand 3’ end.

An RNA primer (a “starter” strand), made by the enzyme primase provides

the needed 3’-OH.

The primer strand is complementary to the DNA template strand.

Since the two template strands are antiparallel, the leading strand and

the lagging strand are synthesized in different ways.

As the helix passes through the replication complex, the leading strand is in

the correct orientation (5’ 3’) for addition of nucleotides.

The lagging strand has the reverse orientation (3’ 5’), and must be

synthesized in fragments.

70

Helicase “unzips” the double helix.

Primase adds RNA primer.

DNA polymerase elongates new strand from primer.

Formation of Okazaki fragments is a mechanism for overcoming the “wrong”

orientation of the lagging strand.

Elongation of the leading strand is continuous.

Elongation of the lagging strand occurs in small, non-continuous, Okazaki

fragments.

Helicase unwinds the helix.

Primase adds RNA primer.

71

DNA polymerase III synthesizes Okazaki fragment.

DNA polymerase I removes RNA

primer and replaces it with DNA.

Ligase joins two adjacent

Okazaki fragments.

72

Many eukaryotic chromosomes have repetitive sequences (which do not

contain genes) called telomeres at their ends that shorten after each round

of cell division.

Eventually the telomeres shorten to the extent that they no longer stabilize

the ends of the chromosomes, and cell division stops.

Constantly dividing cells, such as bone marrow, germ line, and greater than

90% of cancer cells, produce an enzyme called telomerase that catalyzes

the addition of any lost telomeric sequences. Telomerase contains an RNA

sequence—acts as template for telomeric DNA sequences.

When the RNA primer is removed from the end of the chromosome from the

lagging strand, there is no 3’-OH to extend

There is not enough room to build a primer at the lagging strand.

73

Solution: build an over-hanging primer, use it as a template to extend the

parent DNA.

• DNA polymerases make mistakes in replication, and DNA can be

damaged in living cells.

• Cells have three repair mechanisms:

• Proofreading

• Mismatch repair

74

• Excision repair

• As they add new bases to a growing strand, DNA polymerases make a

proofreading check.

• When an error is detected, it is removed and corrected.

• The error rate of DNA polymerase on each attempt is about 1 base in

105.

• This proofreading function reduces the error rate to about 1 base in

1010. (< 1 mistake per cell division.)

• The mismatch repair mechanism using a different enzyme scans new

DNA for mismatched base pairs.

• This mechanism can distinguish between the methylated template

strand and the unmethylated new strand. This helps to ascertain that

the error is in the new strand, not the template strand.

• The mismatch repair mechanism in E. coli operates before the new

DNA strand is methylated.

• DNA is subject to damage by chemicals, radiation, and random

spontaneous chemical reactions.

• Excision repair operates over the life of a cell to inspect the DNA for

damage, then cut the damaged strand and remove it.

• DNA polymerase and DNA ligase fill in and seal up the resulting gap.

75

76

2/26/12 12:06 AM

The molecular basis of phenotypes (specific proteins) was discovered before

it was known that DNA is the genetic material.

Identification of a gene product as a protein began with a mutation.

Beadle and Tatum used Neurospora (haploid for most of its life cycle—all

alleles are expressed as phenotypes) to propose that each mutation caused

a defect in only one enzyme in a metabolic pathway

They showed that

an altered gene an altered enzyme

an altered enzyme an altered phenotype

This confirmed Garrod’s one-gene, one-enzyme hypothesis.

Beadle and Tatum formulated the one-gene, one-protein hypothesis.

However, some proteins are composed of multiple subunits coded for by

separate genes. Therefore one-gene, one-polypeptide is a more accurate

statement.

Other genes code for RNA are not translated to polypeptides; some genes

are involved in controlling other genes.

Gene expression to form a specific polypeptide occurs in two steps:

Transcription—copies information from a DNA sequence (a gene) to a

complementary RNA sequence. DNa is copied in the nucleus.

Translation—converts RNA sequence to amino acid sequence of a

polypeptide. Done in the cytoplasm

Genetic information is contained in DNA. How do we produce proteins from

DNA?

Francis Crick proposed the following scheme, which had come to be called

the Central Dogma!

The information in DNA is first transcribed into RNA.

It is then translated into protein.

Transcription: conversion of a language from one medium to another, e.g.,

converting speech sound in phonetic symbols, or tone into musical notations.

Translation: rendering representation of one language into another.

RNA differs from DNA in three major ways:

RNA consists of only one polynucleotide strand.

78

The sugar in RNA contains a hydroxyl group at the 2’ carbon (it is not a

deoxyribose).

RNA contains uracil instead of thymine.

RNA can base-pair with single-stranded DNA (adenine pairs with uracil

instead of thymine) and also can fold over and base-pair with itself.

The following RNAs are produced in the

nucleus by transcription. All function in the

cytoplasm.

Messenger RNA, mRNA, carries copy of a

DNA sequence to site of protein synthesis at

the ribosome

Transfer RNA, tRNA, is the link between the

code of the mRNA and the amino acids of the

polypeptide, specifying the correct amino acid

sequence in a protein.

Ribosomal RNA, rRNA, in ribosome catalyzes

(i.e., it is an enzyme) peptide bonds and

provides structure; carries out key steps in

ensuring the correct translation of mRNA.

Transcription has three stages

Initiation: Opening DNA and bringing in RNA polymerase

Elongation: adding nucleotides to lengthen the mRNA

Termination: releasing the completed mRNA

79

Genes may be located anywhere on a chromosome, and on either strand of a

double helix.

Both strands can encode genes (but not in same spot on DNA)

How does the transcription machinery know where to go, where to start, and

where to end?

Initiation, begins with binding of RNA polymerase to the promoter, of a

gene.

The promoter is a special sequence of nucleotides on the DNA.

The promoter sequence tells the RNA polymerase:

where the gene begins

which strand is the template for transcription,

which direction the RNA polymerase should move.

- genes get copied from the 3’ end to the 5’ end.

-RNA grows from 5’ to 3’

- it starts with the promoter, which is not copied. The promoter just tells it

where to start.

RNA polymerase unwinds the double helix and slides down the DNA (in the 3’

5’ direction of the template strand).

A primer is not required for RNA elongation.

80

Nucleotides are added to the 3’ end of the growing RNA polymer: RNA

elongation proceeds from 5’ 3’

No proofreading and no error correction.

The control of transcription termination is not well understood.

A termination sequence on the DNA signals the end of a gene.

Information encoded in groups of 3 nucleic acid bases is translated into

amino acid sequence by a universal genetic code

81

Deciphering the Genetic Code

Artificial polynucleotides were synthesized and translated in a cell-free

system.

The resulting polypeptides were sequenced.

For most Amino acids, there is more than one codon. The genetic code is

redundant. But the genetic code is not ambiguous. Each codon specifies

only one amino acid.

61 three-nucleotide sequences code for 20 amino acids.

Each 3-nucleotide sequence is called a codon.

Three codons do not code for amino acids; they are the stop codons.

There is only one codon for methionine; it is also the initiation codon, or

start codon.

Many codons are redundant. In many cases, the first two bases of a codon

are sufficient for coding. In these cases, the third codon is called the

wobble base, because it can be any one of two [usually U and C

(pyrimidines), or A and G (purines)] or four nucleotides.

Eukaryotic genes may have noncoding sequences—introns.

82

The coding sequences are exons.

Introns and exons appear in the primary mRNA transcript—pre-mRNA;

introns are removed from the final mRNA.

Pre-mRNA processing

5’ G cap added

3’ Poly (A) tail added

Introns spliced out

83

• tRNA speaks the language of both nucleic acid and amino acid.

• tRNA serves three major functions:

• it carries an amino acid.

• it has an anti-codon complementary to the RNA codon.

• it interacts with mRNA and ribosomes.

• Each tRNA binds covalently to a specific amino acid. When an amino

acid is bound, the tRNA is said to be charged.

• The anti-codon is the contact point between tRNA and mRNA.

• An anti-codon is complementary --- and antiparallel --- to a codon.

The anti codon is what is on the TRNA and it base pairs to the specific codon

that is required for the amino acid.

Conformation of TRNA results from base pairing within the molecule.

Three end is the amino acid attachment site-binds covalently

Anticodon- at the midpoint of the trna sequence site of base pairing with

mrana. Unique for each species of trna.

DNA codon is the similar to the anticodon.

Wobble- specificity for the base at the 3’ end of the codon is not always

observed.

84

Example- codons for Leucine- CUA and CUG are recognized by the same

tRNA

Wobble allows cells to produce fewer tRNA species, but does not allow the

genetic code to be ambiguous.

• Each ribosome consists of a large and a small subunit.

• The subunits are made up of ribosomal RNAs and proteins.

• The rRNAs and proteins are held together by ionic bonds and

hydrophobic interactions.

• When not translating, the two subunits are separate.

• A: Amino acid site, tRNA anticodon binds to mRNA codon.

• P: Polypeptide site, tRNA adds its amino acid to polypeptide chain.

• E: Exit site, tRNA remains until released.

Three steps of translation

• Initiation: mRNA, ribosome and tRNA are brought together.

• Elongation: amino acids are added (via tRNA-mRNA interaction) to

lengthen the peptide chain.

• Termination: releasing the completed polypeptide and the mRNA

from the ribosomal complex.

85

• rRNA binds to

complementary mRNA-

ribosome recognition

sequence upstream of the

start codon

• Initiator complex

• charged (methionine) tRNA

binds to codon on mRNA

• rRNA validates mRNA - tRNA

match, and removes tRNA if

incorrect

• Large ribosomal subunit

binds, along with other

initiation factors

• rRNA of large subunit has peptidyl transferase activity. It is the rRNA

that catalyzes the peptide chain.

• additional elongation factors are involved

86

No tRNA has anticodon recognizing stop codon (UAA,

UGA, UAG).

• Release factor recognizes stop codon.

• Binding of release factor helps to hydrolyzes

bond between polypeptide and tRNA at P site

• Ribosomal complex disassembles.

• DNA 5’-ATG- - - - -CGG- - - - -AGT-3’ gene

• 3’-TAC- - - - -GCC- - - - -TCA-5’ template

• mRNA 5’-AUG- - - - -CGG- - - - -AGU-3’ same as gene

• tRNA3’-UAC- - - - -GCC- - - - -UCA-5’ anticodon

complementary

• to mRNA

• Peptide Methionine- - -Arginine- - -Serine

87

• Proteins may be destined for the nucleus, mitochondria or other

organelles

88

• Proteins destined for secretion or packaged into vesicles are directed

to rough endoplasmic reticulum, Golgi apparatus, and plasma

membrane.

• Amino acid sequence provides a signal for targeting in cell. Called

signal sequence.

Regulation of Gene Expression

-not every gene in every cell is copied into RNA

• Eukaryotic gene expression:

• Must be regulated to ensure proper timing and location of protein

production.

• Regulation can occur at multiple points in transcription and translation.

89

Eukaryotic Gene Transcription is regulated

• Gene expression begins at the promoter—regions of DNA where RNA

polymerase binds and initiates transcription

• Transcription factors (regulatory proteins) must assemble on the

chromosome before RNA polymerase can bind to the promoter.

• Besides the promoter, other sequences bind regulatory proteins that

interact with RNA polymerase and regulate rate of transcription.

• Some are positive regulators—enhancers; others are negative—

repressors.

• The combination of factors present determines the rate of

transcription.

90

• Functionally related genes are often on different chromosomes in

eukaryotes.

• These genes share the same regulatory sequences in the regulatory

region and are therefore activated by the same regulator protein.

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2/26/12 12:06 AM

Animal Development

Development is primarily a process of gene regulation.

- starts with a zygote and ends up with an organism.

The egg is one cell. It differentiates into an organism.

How do we build something

Cookie cutter- prepare raw materials ad cut products out of molds.

o This is how membranes are made.

o Organisms are not made out of this method

Lego approach- prefabricated, standardized building blocks that

create an end product.

o Organism are not built this way

Origami- Construct products from gradual modification.

o Organisms are formed similar to this.

Development is the gradual restriction of potential.

Loss of potentiality

The egg has a lot of potential to form any cell or structure within the

organisms potential

o Over the course of development, it will lose its ability to form

various structures.

Totipotent- an egg cell with total potential to form any cell

Pluripotent- the organism has lost the ability to form some aspects

Multipotent- the organisms have fewer options to form into.

No distinct point between either of the toti-multi-pluri

Example of gradual restriction of potential in hematopoietic progenitor cells.

Once the cell has chosen a pathway, it cannot switch. The myeloid

progenitor cell cannot become a lymphoid progenitor cell.

Development is the progressive restriction of potential. These stages are not

easily distinct

93

determination — defining the fate of cells. Once they take on one fate,

they cannot change.

differentiation —expressing the fate of cells. Bone cells become bone cells.

morphogenesis — organization and arrangement of cells. Requires

movement of cells.

growth — increase in size. Adding more cells and more proteins.

Establishment of axis: bilaterality, dorsal-ventral, anterior-posterior.

Production of large numbers of pluripotent cells: cleavage.

Arrangement of pluripotent cells into tissue layers that resemble an

organism: gastrulation.

Organogenesis: neurulation, cardiogenesis.

Establishment of body segments: somitogenesis.

Morphogenesis: induction, apoptosis, cell shape changes, cell migration.

Establishment of axes

Egg is a radial structure. It is symmetrical. Has two hemispheres

o Animal Hemisphere-

o Vegetal Hemisphere- Contains the nutrients

Axese are determined. This is extremely important.

o Sperm plays a huge role in this.

o The sperm contributes the nucleus, a centriole, and the

acrosomal (actin) Microtubule production process to the egg.

o Egg already has a centriole, but for cell division, two are

required, so the sperm contributes the second centriole.

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Point of sperm entry is usually in the animal pole.

The sperm entry point defines the midline of the zygote.

The centriole organizes rearrangement of the cortex and the cytoplasm.

This determines the left and right axeses.

The sperm entry point is going ot be the ventral side of the egg, and the

opposite side will be the dorsal side.

This is true because of the rotation of the egg.

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When the sperm enters, it causes a shift in the cortical cytoplasm. The

microtubules create tension and shift towards the sperm causing the cortical

rotation

It exposes the grey crescent.

Grey side will turn into the dorsal side.

GSK will decay Beta Catenin. Upon shift in the cortical cytoplasm, the sperm

shifts the egg so that the stuff that was located at the vegetal pole, which

happens to be an inhibitor to GSK, moves over to the dorsal side. The dorsal

side will have more Beta Catenin than the rest of the egg.

The rotation turns the homogenous distribution in the egg into a

heterogeneously distributed egg. When distribution is uneven, axeses form.

Centriole organizes microtubules at the sperm entry point, creating

tension that rotates the cortex.

Cortical rotation brings ventral vesicles opposite sperm entry point,

establishing the dorsal pole of the future embryo.

Inhibitor released from vesicles in dorsal pole inhibits GSK-3.

GSK-3 breaks down b-catenin in ventral hemisphere.

β-catenin is enriched in dorsal hemisphere.

The dorsal cytoplasm becomes distinct from the ventral cytoplasm.

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Cleavage is to cut.

Cleavage is cell division without cell growth.

Complete Cleavage

Cleavage is cell division without cell growth.

In relatively small eggs, or eggs with a relatively small amount of

yolk, the entire egg is divided.

The egg becomes a blastula.

At the end of cleavage, the blastula contains the same type of cells as its

neighbor, except it has different types of cytoplasm

Incomplete Cleavage

in eggs with relatively large amounts of yolk, the early cleavage

furrows do not cut through the entire egg

called the Blastodisc

Superficial cleavage

In insect eggs, nuclei divide in the middle of the egg without cell division.

The nuclei then migrate to the periphery of the egg to form the blastula

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Cleavage is cell division without cell growth.

G1 and G2 phases of the cell cycle are much reduced relatively to the S and M

phases.

The end result is the partition of the egg cytoplasm among thousands of

cells, forming a blastula.

Because the egg cytoplasm is heterogeneous, cells of the blastula have

different cytoplasmic contents.

Cells of the blastula have taken on different fate.

The Fate Map. Cells located in different parts of the blastula are destined to

become different types of tissues.

They are Determined.

At this stage, the cells are not expressing their phenotype.

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Three germ layers

Ectoderm — outermost layer, gives rise to epidermis and nervous system.

Mesoderm — middle layer, gives rise to muscles, bones, heart, blood,

kidney and gonads.

Endoderm — innermost layer, gives rise to visceral organs and lung.

Gastrulation and the restriction of cell fate.

Gastrulation is a rearrangement of the blastula into an arrangement that

resembles the future embryo.

Gastrulation is the rearrangement of cells of the blastula to bring them

to positions consistent with their fates.

At the end of gastrulation, endoderms are in the core of the embryo,

mesoderm in the middle, and ectoderm on the outside.

Sea urchin eggs gastrulate by invagination.

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Frog eggs gastrulate by involution.

Shape changes in cells at the grey crescent

produces a blastopore.

Bottle-shaped cells at the dorsal lip of the

blastopore enter the interior of the blastula

along the inner surface of the overlying

cells.

Ectoderm on the outside, Mesoderm and

endoderm in the inside.

Cells move from outside to inside the embryo.

The movement begins where the grey crescent

formed, on the opposite side of sperm entry. The

point of starting is called the blastopore.

The process brings cells destined to

become mesodermal tissue to the

inside.

Internalized mesodermal cells surround

endodermal cells.

Ectodermal cells are left on the

outside.

Gastrulation- technically means the

formation of the stomach.

Most important event in life is gastrulation

-rearrangement of cells into what looks like

an embryo

Gastrulation in Discoidal Eggs

The embryonic disc (blastodisc) forms on the surface of the yolk mass.

Gastrulation occurs by migration of surface cells inwards through the

primitive streak.

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The embryonic shield forms in the posterior end of it. It is called the

primitive streak, which extends from the postierior end to the anterior end.

The streak forms Hensen’s node, and the groove that is left is called the

promative groove.

This part is similar to the blastopore in the frog egg.

Fomrs two layers.

Epiblast, and hypoblast.

Internalized cells become mesoderms.

The primitive streak extends from posterior to anterior, lead by

Hensen’s node. Hensen’s node is the organizer in discoidal eggs.

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At what point does fate become irreversible.

Hans Driesch bisected sea urchin embryos

horizontally or vertically at the 8-cell stage.

Dorsal and ventral cell

fates have already been

established and appear to

be irreversible by the 8-cell

stage, i.e. after 3 rounds of

cell division.

Cell fate is established by cytoplasmic determinants distributed in the egg

soon after fertilization.

Fate is gradually determined and certain axes are

determined before others.

Cytoplasmic determinants become unevenly distributed

in the egg cytoplasm in the egg.

As cleavage proceeds, cytoplasmic determinants are

segregated into subsets of cells.

Cells that have inherited different cytoplasmic determinants

take on different fates.

Chemicals in the vegetal pole are not present in the animal

pole.

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Hans Spemann’s Newt Splitting Experiment

The midline of the embryo bisects the grey crescent.

Splitting the egg bilaterally produces 2 normal

embryos.

Splitting the egg perpendicular to the midline

produces one normal embryo.

As long as a piece contains the grey crescent, it

produces a normal embryo.

Cells destined to be skin are transplanted to

positions in which cells are destined to become skin,

brain or notochord.

If skin fate is irreversible, the

transplanted cells will become skin.

If skin fate is not fixed, the transplanted

cells will take on a new fate according to

their new position.

The transplanted cells take on new fate

according to their position in the host.

Therefore, cell fate is not determined

before gastrulation.

He took the different parts of the embryo

that is destined to become something,

and placed it in a host but in another

location. If the fate was already

determined, no matter where it was

placed in the host it would become

whatever it was already intended to

become.

The part of tissue that was meant to

be skin, but was placed in the brain

region of the host, became a brain.

Before gastrulation, the fate is

determined. Beforehand, the cell can

change fates.

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This is true except for the dorsal of the blastopore.

Dorsal lip cannot be changed no

matter what. The Dorsal lip will induce other cells to take on new fates.

Dorsal lip is called the organizer. If you remove the dorsal lip, no

gastrulation will occur. The grey crescent makes the dorsal lip.

• Not only does the transplanted dorsal lip of the blastopore retain its

own fate when transplanted to a new position, it changes the fate of

the adjacent cells in the recipient.

• Spemann called the dorsal lip the organizer, because it organizes the

cells around it to form a new axes, and a new embryo.

• The organizer itself has a fate: it is mesodermal tissue; it becomes a

part of the notochord.

Beta Cetinin is the master transcription factor that turns on many of the

events of gastrulation.

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Dorasl lips come in and forms the notochord. It is what is going to induce the

ectoderm to become neural tissue.

If the notochord is removed, it will just become skin. The organism will no

have a nervous system

The organizer becomes part

of the notochord.

• The notochord induces

the overlying ectoderm

to become neural tissue.

• During neurulation, the

neural plate folds

upwards to become the

neural tube.

• The neural tube pinches

off from the ectoderm

and becomes

internalized.

• The overlying ectoderm

become the dorsal epidermis.

• As the neural tube folds, 2 bands of cells (dark green) that originate at

the border between the neural plate and epidermis remain at the crest

of the neural tube.

• These cells migrate away from the crest of the neural tube after

neurulation.

• Neural crest cells give rise to all neurons and supportive cells of the

peripheral nervous system and the gut, all pigment cells of the body,

the adrenal medulla, some cells of the heart, facial cartilage, and

dentine of teeth.

Somite formation and the segmentation of the body.

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• Somites are mesodermal tissues. They give rise to all the muscles and

bones of the body (except those of the head), and the dermis.

Cell-cell interaction. What can change the fate of cells.? Induction

• The neural component of the eye originates from the neural tube.

• The neural tube evaginates to form the optic vesicle.

• The optic vesicle induces the overlying ectoderm to thicken to form the

lens placode.

• The optic vesicle invaginates to form the optic cup.

• The optic cup induces invagination of the optic placode, which pinches

off, clears up, and form the lens.

• The lens induces the overlying epidermis to clear up and form the

cornea.

• Lens is ectodermal tissue coming from the skin.

• Induction is better than parceling out cytoplasm because the organism

is made up a lot of cells. Would be hard to organize.

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• Scaling and matching of size. A large lens will induce a large cornea

• Apoptosis is coordinated, programmed death of cells.

• Orchestrated apoptosis are an important morphogenetic events.

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2/26/12 12:06 AM

Homeostasis is the maintenance of balance in the organisms

External environments are variable, sources of energy and repository of

waste

Internal environments are stable,

Goal of homeostasis is responses to changes in both the external and the

internal environment. Maintain constant environment so that functions can

occur.

The amount of neurons dedicated to maintaining the internal environment is

10,000 times more than the amount of neurons dedicated to detecting the

external environment.

Ideal Levels

temperature (thermoregulation) — 37ºC

pH (buffering) — plasma pH 7.35 – 7.40; H2CO3: 1.35 mM; HCO3-: 27 mM;

H2CO3/HCO3-: 1/20

fluid volume, electrolyte concentration (osmoregulation) — plasma

osmolarity: 300 mOsm; Na+: 140 mM; Cl-: 100 mM; K+: 3.5 – 5.0 mM; Ca++:

4.5 – 5.5 mM

blood flow, blood pressure (haemodynamics) — systolic/diastolic pressure:

120/80 mm Hg

glucose, other fuel (energetics) — plasma glucose: 0.1%

oxygen, carbon dioxide (gas exchange)

Homeostatic regulation

nutrient, storage, waste

cell number (proliferation, growth, death)

organ size (hypertrophy, atrophy)

behavior — stress response

Cells, tissues, and organ systems are the effectors of homeostasis.

Each organ system contributes to a part of the overall homeostasis, and

each organ system is balanced by more than one system

Most organ systems are involved in homeostasis of more than one

parameter:

respiratory system — O2, CO2, pH

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cardiovascular system — fluid pressure, fluid volume, electrolyte balance

renal system — fluid volume, fluid pressure, electrolyte balance, pH,

erythrocyte concentration, nitrogen level

Properties that homeostatic systems must have

Stable — a homeostatic system must itself be stable.

Sensors — must be able to monitor conditions. Sensors are essential.

Set-point — reference point against which homeostatic adjustments are

made. Goal is always to maintain set-point

Set-point adjustable — change set-point to accommodate various

contingencies.

Up- and down-regulation — adjustment can be made on both sides of set

point

Integrative and interactive — coordinate control of other systems.

Feed-back loops

— negative feedback loops: return activity to set-point, prevent

changes, stabilizing. Homeostatic systems rely on negative feedbacks. With

no negative feedback, the system is not homeostatic.

— positive feedback loops: Not part of homeostatic systems. amplify

changes, they are destabilizing (e.g. nerve conduction, blood clot,

childbirth), not a common feature of homeostatic systems.

Feed-forward loops — change set-point.

Positive feedback

There is a stimulus that will

stimulate A which will then

stimulate B.

B then becomes the stimulus

for A so the original stimulus is

not necessary.

This will go until either A or B

is exhausted.

always goes until completion

Negative feedback

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B inhibits A, so the system regulates

itself.

Does not go until completion

Thermoregulation

Body maintains specific temperature

Temperature and life

Temperature sets the limits for life.

The freezing point of water sets the lower limit.

The denaturation temperature of protein sets the upper limit.

The major impact of temperature is on the rate of chemical reactions:

Metabolic rates varies with temperature.

Low temperatures reduce sensory and motor efficacy and affect behavior.

Q10 ― Temperature Quotient of Reaction

Rate

Q10 is a ratio; it has no unit.

Most biological processes have Q10

between 2 and 3.

The Q10 is characteristic of a reaction.

The value varies with different reactions.

Q10 of 1 means no change when

temperature changes.

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Because different components have different Q10, a change in temperature

can upsets chemical/physiological balance in may ways.

Organisms either keep temperature constant, or compensate for disparate

changes.

Poikilotherms (G. poikilos = varied, irregular) — temperature conformers —

body temperature follows ambient temperature

mechanism: ectothermy

Homeotherms (G. homoio = similar) — temperature regulators — body

temperature maintained at preset temperature

mechanism: endothermy

Heterotherms (G. heteros = other, different) — partial regulators — body

temperature maintained at preset temperature under some conditions, and

follows ambient temperature under other conditions

mechanism: partial endothermy

As the temperature increases, the lizard’s body temperature increases and

the metabolic rate of the lizard also increases. Very little metabolic rate is

necessary to maintain body function.

As the temperature increases, the mouse’s body temperature remains the

same. When the environment is within the thermoneutral zone, the

metabolic rate is constant. Around that, the metabolic rate increases. Takes

a lot of energy to maintain the body temperature.

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Heterotherms

Body temperature is allowed to drop with ambient temperature up to a set

level.

Below this ambient temperature, body temperature is kept constant.

The overall use of energy is much reduced.

The Heat Budget

Heat in = Heat out

Heat in- metabolic heat, environmental heat, (radiation, conduction,

conection)

Heat out- radiation, conduction, convection, and evaporation.

Evaporation is a great way to lose heat.

Cellular respiration and metabolism produce heat as a by-product.

(Remember: ATP production and expenditure are not 100% efficient.)

Total metabolic heat production is directly proportional to body mass.

Heat loss is directly proportional to surface area.

Heat loss can be reduced by reducing surface area.

Heat loss can be increased by increasing surface area.

Heat loss has to be through the skin. By reducing the surface area of the

body, heat loss is therefore reduced.

Relative heat loss is a

function of surface area-

to-volume ratio.

Larger animals have low

surface area-to-volume

ratio and relatively low

heat loss.

Smaller animals have

high surface area-to-

volume ratio and relatively high heat loss.

Small animals have a hard time maintaining heat

Big animals have a hard time dissipating heat

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Small animals increase

metabolic rate to

overcome relatively

high heat loss.

Large animals decrease

metabolic rate to

balance relatively low

heat loss.

This is adjusted to the

surface to volume raito

An elephant with the

metabolic rate of a mouse with boil.

A mouse with the metabolic rate of an elephant will freeze.

Morphological adaptation- adjusting surface to volume ratio

Hotter temperatures- animals will attempt to increase total surface

area

Colder temperatures- animals will have smaller appendages

Vasomotor Modulation of blood flow to

the skin

When ambient temperature is

low, blood flow to skin is

restricted; heat loss is

reduced

When ambient temperature is

high, or during high metabolic

activity, blood flow to skin is

enhanced to increase heat

loss.

Shunt system will open or

close depending on whether it

is hot or cold.

Open shunt will decrease heat loss

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Thermoregulation in homeotherms

37 degrees is roughly the body temperatures of all mammals.

Only the body core is kept at 37. The appendages are not

Mammalian thermoregulatory systems

• Heat production (metabolic):

• muscle shivering

• brown fat activation (by epinephrine)

• Heat conservation (non-metabolic):

• peripheral vasoconstriction

• piloerection

• behavioral/postural changes

• Heat dissipation (non-metabolic and metabolic):

• peripheral vasodilatation

• sweating, salivating

• increase ventilation ¾ panting

• behavioral/postural changes

Metabolic thermoregulation in mammals

• Basal metabolic rate is the rate required to maintain minimal body

function.

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• The thermoneutral zone is

the ambient temperature at

which the basal metabolic

rate generates sufficient heat

to maintain body

temperature.

• Below the lower critical

temperature, increase in

metabolic rate is necessary

to produce heat.

• Above the upper critical

temperature, increase in

metabolic rate is necessary

to increase heat loss.

• Shivering ¾ rapid contraction of muscles generates heat.

• Brown fat ¾ mitochondrial metabolic heat production. Brown Fat does

not store lipid droplets. Called brown fat because these cells have

many mitochondrion which look brown.

• It generates heat the same way the ETC creates ATP

• The proton is pumped into the space during ET which generates

a proton gradient. Instead of going through the ATP synthase, it

goes through thermogenin, which is a different proton channel

that allows the proton to drain through the thermogenin.

Metabolic Cooling- cooling is much harder than

heating.

• Heat removal by vaporization ¾ sweating, salivating.

• Increase ventilation ¾ panting.

• Cooling is more difficult than heating —

activities that cool the body also

produce heat.

• A lot of these ativities themselves

generate heat. Causes heat stroke.

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Thermostat is located in the hypothalamus- temperature sensors are located

in the skin, the hypothalamus and elsewhere

Can locally change the temperature near the hypothalamus, and

the hypothalamus will react to that temperature.

Must be able to adjust the thermostat

When someone has a fever, the

body resets the temperature to

40. The only difference is the

set point.

This is the only way to change

the body’s temperature.

So why do we shiver when the

body has temperature. We do

this to generate heat. As long as

the body temperature is below

40, the body will attempt to get

to 40.

• At the onset of

hibernation, the

thermoregulatory set

point is reset to a low

temperature. The same

homeostatic mechanisms

maintain the low body

temperature.

• At the end of hibernation,

set point is reset to 37°C.

The same homeostatic

mechanisms bring body

temperature up.

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Endocrine system. It integrates things and communication is essential Done by systesm.

o Circulatory system.o Lymphatic system.o Immune system: Cellular communication; relies on

circulatory and lymphatic systems for distribution.o Endocrine system: Molecular communication; relies on

circulatory system for distribution.o Nervous system: Electrical and molecular communications;

relies on intrinsic network and circulatory system.

Endocrine System relies on circulatory system for distribution. Located throughout the body.

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Hormone-secreting cells are endocrine cellsHormone-receiving cells are target cellsTarget cells must have appropriate hormone receptors to respond

Autocrine is a feedback regulatory system

Hormone only affects cells that have the receptor for the hormone

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Secreted by gland cells located in endocrine organs.Affect metabolism and activity of distant target cells and tissues.Integrated with the nervous system. Top-down, hierarchical regulatory chain — brain-pituitary-gland axis.Homeostatic functions: coordinates anatomical, physiological and behavioral stability.

Hormones can be classified by their structuresPolypeptides — most common, interact with integral membrane, G protein-associated or kinase receptors.Steroids — derivatives of cholesterol, interact with cytoplasmic receptors.Monoamines — epinephrine, norepinephrine, melatonin, thyroxine, interact with integral membrane receptors.Lipid soluble molecules — vitamin D, juvenile hormone, prostaglandins, interact with cytoplasmic receptors.

Single hormone on/off switch, e.g. ecdysone in arthropods

Cyclic/pulsatile release, e.g. ecdysone and juvenile hormone in insects, adrenocorticotropin releasing hormone in hypothalamus

Dual, reciprocal hormones, e.g. calcitonin and parathyroid hormone, insulin and glucagon

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Negative feedbackSimple feedbackPolynodal hierarchical feedback — adrenocorticotropin releasing hormone and cortisol

Endocrine regulation of metamorphosis in Insects. Insects are good to study because they are full of hormones.

Vincent Wigglesworth

The Kissing bug circulates the disease of Chagas Disease- Chagas disease is slowly spreading- Chagas disease will circulate through the body and slowly decay brain cells.

Insects have rigid exoskeletons; they must molt periodically and therefore have episodic growth.The growth stage between each molt is an instar.The adult has wings, where the larval instarts do not have.

Wiggleworth’s experiment Observations:Molting is stimulated by a blood meal.Intact bugs molt into the next instar.If decapitated immediately after feeding, no molting.If decapitated 1 week after feeding, bugs molt into adults.

Observations:If a bug decapitated 1 hour after blood meal is connected to a bug decapitated 1 week after a blood meal, both molt into an adult.Conclusion:Molting is triggered by a diffusible substance that originates in the head.

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Wiggleworth’s ConclusionsBlood meal initiates molting by stimulating the brain to release a molting factor.The release of molting factor requires about a week.The molting factor is a diffusible substance; it circulates around the body to orchestrate tissue and behavior in preparation for molting.We now know that ecdysone is the molting hormone.

Why does the bugs molt into an adultWithout the head, the bug molts into an adult. Therefore, the larval brain must release a substance that prevents molting into an adult.In the presence of this substance, larvae molt into the next instar. In the absence of this substance, larvae molt into an adult.The substance is release by the brain just before molting.We now know that juvenile hormone prevents adult development.

When Ecdysone is present, the body goes on to molt- example of hormone being an on off switch.

Calcium Homeostasis- Reciprocal Hormonal Control Need at least two- one does one thing and one does the opposite Calcitonin that is released by the thyroid gland Parathyroid hormone that is released by the parathyroid glands The skin uptakes vitamin D which results in the release of Calcitriol

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Calcium Budget

Involve the Digestive, Renal and Circulatory system

Brain-Pituitary Axis of the Vertebrate Endocrine SystemHypothalamus-Anterior Pituitary SystemAnterior contains a lot of cellsPosterior pituitary does not contain a lot of cells, it stores the hormones produced by the hypothalamus

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• Feedback Loops of the Hypothalamus-Anterior Pituitary-Endocrine Gland Axis

Two hierarchical, superimposing pathways:

• (a) a stimulatory brain-pituitary-gland pathway; and

• (b) a feedback inhibitory pathway.

• Hormones lower in the stimulatory hierarchy feedback inhibit hormones higher in the hierarchy.

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