BSci 121M. CYTOGENETICSUNIVERSITY OF SAN AGUSTINCOLLEGE OF PHARMACY AND MEDICAL TECHNOLOGY

Cyto = CellGenes= Elements of heredity (carrying inherited traits) that are transmitted from parents to offspring during reproduction .Genetics= The study of biologically inherited traits./ The study of genes.Genomics= The study of all the genes in an organism to understand their molecular organization, function, interaction and evolutionary history.

Chapter 1.The Genetic Code of Genes & GenomesHistory:1860s- Existence of genes and the rules governing their transmission from generation to generation were discovered by Gregor Mendel. His work with garden peas represents the beginning of what would become the science of Genetics.1869- Friedrich Miescher discovered a new type of weak acid abundant in the nuclei of WBCs that turned out to be the chemical substance of which genes are made. These acid would later be known (until today) as Deoxyribonucleic acid. 1870s- The nuclei of the male and female reproductive cells were observed to fuse in the process of fertilization. This later led to the discovery of thread-like objects with characteristic splitting behavior inside the nucleus that become visible in the light microscope when stained with dyes. These would later be known as Chromosomes.1900s- Chromosomes are indeed carriers of genes.1920s- DNA and other various types of proteins are present in chromosomes.

DNA- Deoxyribonuleic acid. Considered as the Molecule of Heredity. According to studies by James Watson and Francis Crick at Cambridge University (1953), DNA consists of two long chains of subunits twisted around one another to form a double-stranded helix. The subunits of each strand are called nucleotides, each of which contains any one of four chemical constituents called bases (Adenine; Thymine; Guanine and Cytosine). The base pairing between A and T and between G and C is said to be complementary base pairing; the complement of A is T, and the complement of G is C. The complementary pairing in the duplex molecule means that each base along one strand of the DNA is matched with a base in the opposite position on the other strand. Each DNA strand has a polarity or directionality, like a chain of circus elephants linked trunk to tail. In this analogy, each elephant corresponds to one nucleotide along the DNA strand. The trunk end of the strand is called the 5 end of the strand, and the tail end is called the 3 end. In double stranded DNA, the paired strands are oriented in opposite directions: The 5end of one strand is aligned with the 3 end of the other. The oppositely oriented strands are said to be Antiparallel.Replication- The copying process in which a single DNA molecule becomes two identical molecules. In this process, each parental DNA strand directs the synthesis of a new partner strand.For most genes, the genetic information contained in the nucleotide sequence specifies a particular type of protein. Proteins control the chemical and physical processes of cells known as metabolism. Many proteins are enzymes , a term introduced in 1878 to refer to the biological catalysts that accelerate biochemical reactions.

Archibald Garrod- A British physician who studied genetic diseases caused by inherited defects in metabolism. He concluded that an inherited defect in metabolism results from an inherited defect in an enzyme. Example: PKU (Phenylketonuria) results from the absence of (or a defect in) the enzyme Phenylalanine hydroxylase (PAH). When this step in the pathway is blocked, phenylalanine accumulates. The excess phenylalanine is broken down into harmful metabolites that cause defects in myelin formation that damage a childs developing nervous system and lead to severe mental retardation.

The Central Dogma of Molecular Genetics The details of how genes code for proteins were not understood until the 1960s when the Dogma was formulated. Since then, it has become the fundamental principle of molecular genetics because it summarizes how the genetic information in DNA becomes expressed in the amino acid sequence in a polypeptide chain. DNA transcription RNA translation PROTEIN

The main concept in the central dogma is that DNA does not code for protein directly but rather acts through an intermediary molecule of Ribonucleic acid. The structure of RNA is similar to, but not identical with, that of DNA.

FeaturesDNARNA

1. Sugar DeoxyriboseRibose

2. StrandednessDouble strandedSingle stranded

3. BaseThymineUracil

Three Types of RNA:1.) mRNA- carries the genetic information from DNA and is used as a template for polypeptide synthesis.2.) rRNA- the major constituents of the cellular particle Ribosomes on which polypeptide synthesis takes place.3.) tRNA- the carriers of particular amino acids for polypeptide formation. As each tRNA participates in translation, its amino acid becomes the terminal subunit of the growing polypeptide chain.

Transcription- is the production of an RNA strand that is complementary in base sequence to a DNA strand (template).

Translation- the synthesis of a polypeptide under the direction of an mRNA molecule.

Examples of Standard Genetic Code (which comes in groups of 3 bases or CODON): CUA- Leucine GUU- Valine CGG- Arginine

Chapter 2. Transmission Genetics: Heritage from Mendel

Transmission genetics- the study of the patterns of inheritance from generation to generation. In eukaryotic organisms, transmission genetics is often called Mendelian genetics.

Gregor Mendel- Realized that each parent contributed to its progeny a number of separate and distinct elements of heredity (factors as he called them; in modern times genes) - He also realized that each of these parental factors remain unchanged as it was passed from one generation to the next. - He selected peas for his experiments for two reasons:1.) He had access to varieties that differed in observable alternative characteristics (round vs wrinkled seeds, yellow vs green seeds)2.) His preliminary studies had indicated that peas normally reproduce by self- fertilization, in which pollen produced in a flower is used to fertilize the eggs in the same flower. Left alone, pea flowers always self- fertilize . - He did a crossbreeding between two different varieties by opening the keel petal (which encloses the reproductive structures), removing the immature anthers (the pollen- producing structures) before they shed pollen, and dust the stigma(part of the female structure) with mature pollen taken from a flower on a different plant.- He established true- breeding varieties in which the plants produced only progeny like themselves when allowed to self- fertilize. (Ex. One true- breeding variety always yielded round seeds, whereas another true- breeding variety always yielded wrinkled seeds.)-For his experiments, Mendel chose seven pairs of varieties, each of which was true- breeding for a different trait. The contrasting traits affected: seed shape (round vs wrinkled), seed color (yellow vs green), flower color (purple vs white), pod shape (smooth vs constricted), pod color (green vs yellow), flower and pod position (axial vs terminal) and stem length (standard vs dwarf). When two varieties that differ in one or more traits are crossed, the progeny constitute a hybrid between the parental varieties. Crosses in which the parental varieties differ in one, two or three traits of interest are called: Monohybrid, Dihybrid and Trihybrid respectively.Geneticists call the true- breeding parents the P1 generation and the hybrid filial seeds or plants the F1 generation. One pair of traits studied was round vs wrinkled seeds. When pollen from a variety of plants with wrinkled seeds was used to cross- pollinate plants from a variety with round seeds, all of the resulting hybrid seeds were round. When plants from the variety with round seeds were used as the pollen parents and those from the variety with wrinkled seeds as the female parents (reciprocal cross), all of the F1 seeds turned out to be round. Generalizations: 1.) The Traits expressed in the hybrids were called the Dominant traits; while the traits not expressed in the hybrids were called Recessive traits.2.) Two plants with the same outward appearance (for example with round seeds) might nevertheless differ in their hereditary makeup. 3.) The normal gene encodes an enzyme, starch-branching enzyme I (SBEI), required to synthesize a branched chain form of starch known as Amylopectin. As pea seeds dry, they lose water and shrink. Round seeds contain amylopectin and shrink uniformly.Wrinkled seeds lack amylopectin and shrink irregularly. In other words, wrinkled peas have an inborn error in starch metabolism. The molecular basis of the wrinkled mutation is that the SBEI gene has become interrupted by the insertion of aDNA sequence called a transposable element. These are DNA sequences that are capable of moving (transposition) from one location to another within a chromosome or between chromosomes. Many spontaneous mutations result from the insertion of transposable elements into a gene. Terminologies deduced from Mendels experiment:1. Gene- hereditary determinant of a trait2. Alleles- the different forms of a particular gene. (Ex. The alleles of the gene for seed shape are W for round seeds and w for wrinkled seeds. W and w are alleles because they are alternative forms of the gene for seed shape. Alternative alleles are typically represented by the same letter or combination of letters, distinguished either by upper case vs lower case or by means of superscripts or subscripts or some other typographic identifier.)3. Genotype- the genetic constitution of an organism or cell- its molecular makeup. With respect to seed shape in peas, WW, Ww and ww are examples of the possible genotypes for the W and w alleles. Because gametes contain only one allele of each gene, W and w are examples of genotypes of gametes.4. A genotype in which the members of a pair of alleles are different, as in the Ww hybrids, is said to be heterozygous. A genotype in which the two alleles are alike is said to be homozygous. A homozygous organism may be homozygous dominant (WW) or homozygous recessive (ww). The terms homozygous and heterozygous can not apply to gametes because gametes contain only one allele of each gene.5. The observable properties of an organism including its visible traits constitute its phenotype. Round seeds and wrinkled seeds are phenotypes. So are yellow seeds and green seeds. The phenotype of an organism does not necessarily imply anything about its genotype. For example, a seed with the phenotype round could have either the genotype WW or the genotype Ww.6. A dominant trait is that expressed in the phenotype when the genotype is either heterozygous or homozygous. A recessive trait is that expressed in the phenotype when a genotype is homozygous for the alternative allele. The presence of a dominant trait masks a recessive trait.7. Wildtype form- most common form of a trait occurring in a natural population. (Represented as W., ex the round peas.)8. Mutant form- any form that differs from the wildtype.(Ex. The wrinkled peas).

A way to identify the W and w forms of gene is the procedure gel electrophoresis. It is used for separating DNA molecules of different sizes. Samples containing relatively small fragments of duplex DNA are placed into slots near one edge of a slab of a jelly-like material (agarose) which is then submerged in a buffer solution and subjected to an electric field. DNA fragments in the samples move in response to the electric field in accordance with their lengths. Shorter fragments move faster and farther than long fragments. The W fragment moves farther than the w fragment because the w fragment is larger owing to the insertion of the transposable element. The separation of the fragments is indicated by the dark rectangles called bands.

9. Morphological trait- one that is manifest, plainly shown, and readily perceived by the senses. Frequently dominant or recessive.10. Molecular trait- one that can be perceived only by means of special methods(such as gel electrophoresis)that enable differences between molecules to be visualized. Often (but not always) codominant.11. Codominant genes- Alternative forms of a gene (W and w) which can both be detected when they are present in a cell or organism using special methods (e.g. gel electrophoresis showing single rapidly migrating band of the true- breeding strain with round seeds; and the single slowly migrating band of the true-breeding strain with wrinkled seeds; and the progeny of the cross which has round seeds BUT exhibit BOTH bands ).

Mendelss hypothesis of genetic transmission:Genes are physical entities that come in pairs, separate in gametes and join randomly in fertilization.In the first generation of hybrids, the recessive visible trait disappeared, only to reappear in the next generation, after the hybrid progeny were allowed to undergo a self fertilization. The progeny seeds produced by self- fertilization of the F1 generation constitute the F2 generation. Mendel found that the dominant and recessive traits appear in the F2 progeny in the proportions 3 round: 1 wrinkled (ratio of dominant: recessive is 3:1).The recessive trait that seemingly disappeared in the F1 generation reappeared again in the F2 generation. Not only did the recessive trait reappear, it was in no way different from the trait present in the recessive P1 plants. Thus, Mendel concluded that the hereditary determinants for the traits in the parental lines were transmitted as two different elements that retain their purity in the hybrids. In other words, the hereditary determinants do not mix or contaminate each other.

Mendelss hypothesis of genetic transmission: 1.) Each reproductive cell (or gamete) contains one representative of each kind of hereditary determinant in the plant. 2.) When an F1 plant is self- fertilized, the W and the w determinants separate from one another and are included in the gametes in equal numbers. This separation of the hereditary elements is the heart of Mendelian genetics. The principle is called Segregation. The hereditary determinants are completely unaltered after separation by their having been paired in the previous generation.3.) The gametes produced by segregation come together in pairs at random (subject to chance variation) to yield the progeny of the next generation.

Testcross- A cross between an organism of dominant phenotype and an organism of recessive phenotype (homozygous recessive) which yielded a progeny with Ww and ww ratio of 1:1.

The Human ABO blood groups illustrate both Dominance and codominance.Incomplete dominance- the phenotype of the heterozygous genotype is intermediate between those of the homozygous genotypes. It is more frequent for morphologic traits than for molecular traits. (Ex. The color pink of snapdragon flower that is formed is intermediate between red and white from the parent flowers.)Codominance- the heterozygous genotype exhibits the traits associated with both homozygous genotypes. It is more frequent for molecular traits than for morphologic traits.

ABO blood groups- determined by polysaccharides (polymers of sugars) present on the surface of red blood cells. Both the A and B polysaccharides are formed from a precursor substance that is modifeied by the enzyme product of either the IA or the IB allele. The gene products are transferase enzymes that attach either of two types of sugar units to the precursor. People of genotype IAIA- produce RBCs having only the A polysaccharide and are said to have blood typeA.People of genotype IBIB- produce RBCs having only the B polysaccharide and are said to have blood type B.Heterozygous IAIB people have RBCs with both A and B polysaccharides and are said to have blood type AB. This genotype illustrates codominance because heterozygous genotype has the characteristic of both homozygous genotypes- in this case, the presence of both the A and the B carbohydrate on the RBCs. Although the polypeptides encoded by the IA and the IB differ in only 4 out of 355 amino acids, these differences are at strategic positions in the molecules and change their substrate specificity. Both the IA and the IB are dominant to the recessive allele IO. The IO allele has a single base deletion in codon 86 that shifts the translational reading frame of the mRNA resulting in an incomplete, inactive enzyme. The precursor substrate remains unchanged and neither the A nor the B type of polysaccharide is produced. People of genotype IOIO therefore lack both the A and the B polysaccharide and thus said to have blood type O. In IAIO heterozygotes, presence of the IA allele results in production of the A polysaccharide, and correspondingly in IBIO heterozygotes, presence of the IB allele results in production of the B polysaccharide. Thus persons with IAIO have blood type A and persons with IBIO have blood type B.

Chapter 3. The Chromosomal Basis of Heredity

MitosisMeiosis

The Sex chromosomes an exception to the rule that all chromosomes of diploid organisms are present in pairs of morphologically similar homologs. As early as 1891, microscopic analysis showed that one of the chromosomes in males of some insects (grasshoppers) does not have a homolog. This unpaired chromosome was called the X chromosome and it was present in all somatic cells of the males but in only half the sperm cells. The biological significance of these observations became clear when females of the same species were shown to have two X chromosomes. In other species in which the females have two X chromosomes, the male has one X chromosome along with a morphologically different chromosome, later known to be the Y chromosome. The difference in the chromosomal constitution of males and females is a chromosomal mechanism for determining sex at the time of fertilization. Whereas every egg cell contains an X chromosome, half the sperm cells contain an X chromosome and the rest contain a Y chromosome. Fertilization of an X- bearing sperm results in an XX zygote which normally develops into a female; and fertilization by a Y- bearing sperm results in an XY zygote, which normally develops into a male. The result is a criss- cross pattern of inheritance of the X chromosome in which a male receives his X chromosome from his mother and transmits it only to his daughters.

Hemophilia A- a classic example of a human trait with an X-linked pattern of inheritance. A severe disorder of blood clotting determined by a recessive allele. Affected persons lack a blood-clotting protein called factor VIII that is needed for normal clotting, and they suffer excessive, often life threatening bleeding after injury.X-linked inheritance in human pedigrees shows several characteristics that distinguish it from other modes of genetic transmission:1. For any rare trait due to an X-linked recessive allele, the affected individuals are exclusively, or almost exclusively, male. There is an excess of affected males because females carrying the rare X-linked recessive allele are almost exclusively heterozygous and so do not express the mutant phenotype.2. Affected males who reproduce have normal sons. This follows the fact that a male transmits his X chromosome only to his daughters.3. A woman whose father was affected has normal sons and affected sons in the ratio of 1:1. This is true because any daughter of an affected male must be heterozygous for the recessive allele.

Chapter 5. Human Chromosomes and Chromosome Behavior

In most species, organisms with an extra chromosome or a missing chromosome usually have developmental or other types of abnormalities. Some organisms are found to have a variation in chromosome structure (missing segment, duplicated, reversed in orientation or attached to a different chromosome.) Generally speaking, animals are much less tolerant of chromosomal changes than are plants. Human beings have 46 chromosomes in 23 pairs.Chromosome painting - technique in labeling chromosomes. Different colors are painted on each chromosome by hybridization (formation of duplex molecules) with DNA strands labeled with different fluorescent dyes. a. Metaphase spreading- chromosomes are arranged just as they appear in the cytological preparationb. Karyotyping- more conventional representation; autosomes (22 pairs in humans) in the metaphase spread are rearranged systematically in pairs, from longest to shortest and numbered 1 (longest) through 22. The sex chromosomes (X and Y or X and X) are usually set off at the bottom right.Each human chromosome is linear and has a single centromere.Classification of chromosomes according to the relative position of centromeres:1. Metacentric chromosomes ( yield V-shaped daughter chromosomes) - middle location2. Submetacentric chromosome (yield J- shaped daughter chromosomes)- Centromere is off center3. Acrocentric chromosome (yield I- shaped daughter chromosomes)- Centromere is very close to one end Acentric chromosome- A chromosome that lacks a centromere. Genetically unstable because they can not be maneuvered properly during cell division and are lost.Dicentric chromosome- A chromosome with two centromeres. Genetically unstable because it is not transmitted in a predictable fashion.

The Principle of Dosage CompensationFor all the organisms with XX-XY sex determination, there is a problem of the dosage of genes on the X chromosome because females have two copies of this chromosome whereas males have only one. A mechanism of Dosage compensation has evolved in which the unequal dosage in the sexes is corrected either by increasing the activity of genes in the X chromosome in males or by reducing the activity of genes in the X chromosome in females. In the early cleavage divisions of the embryo, one and only one X chromosome in each cell (chosen at random) remains genetically active and any other X chromosomes that may be present in the cell undergo a process of X inactivation (and any inactivated X chromosome remains inactive in all the descendants of that cell). The process of X- chromosome inactivation takes place in all embryos with two or more X chromosomes including normal XX females. The inactivation process is one of chromosome condensation initiated at a site called XIC (X- inactivation Center) near the centromere on the long arm between Xq11.2 and Xq21.1.

Consequences of X-chromosome Inactivation:1. Results in Dosage compensation. It equalizes the number of active copies of X-linked genes in females and males. Single active X- principle (proposed by Mary Lyon).2. A normal female becomes a mosaic for the expression of X-linked genes. (Genetic mosaic= an individual that contains cells of two or more different genotypes.) A normal female is a mosaic for gene expression because the X chromosome that is genetically active can differ from one cell to the next. Approximately 15 % of all recognized pregnancies in human beings terminate in spontaneous abortions, and in about half of all spontaneous abortions, the fetus has a major chromosome abnormality.

Definition of terms:1. Trisomic- otherwise diploid organism that has an extra copy of an individual chromosome. 2. Monosomic- otherwise diploid organism but having a missing copy of an individual chromosome. Generally, is more frequent and more harmful than chromosome gains . 3. Diploid- Two sets of chromosomes are present (46 chromosomes) 4. Triploids- Three sets of chromosomes are present (69 chromosomes)5. Tetraploids- Four sets of chromosomes are present (92 chromosomes)

Human chromosome abnormalities

A. Deletion/ Deficiency- chromosomes arise in which a segment or segments is/ are missing. Generally harmful to the organism. The larger the deletion, the greater the harm. Very large deletions are usually lethal, even when heterozygous with a normal chromosome. Small deletions are often viable when they are heterozygous with a structurally normal homolog, because the normal homolog supplies gene products that are necessary for survival. However, even small deletions are usually lethal when both members of a pair of homologous chromosomes carry the deletion. Example: ASD (Autism Spectrum Disorders)- characterized by communication deficits, social impairment and repetitive behaviors occurring prior to the age of 3, usually requiring extensive family support and medical intervention. Males: Females ratio 5:1; Identical twin chance of acquiring the defect: 70 90% if the other twin is affected.

Deletions can be formed in 2 major ways:1. Chromosome breakage and reunion- Chromosome breaks result from double- stranded breaks in the DNA backbone. May occur spontaneously at a slow rate or can be induced by xrays and chemicals. 2. Ectopic recombination- pairing and homologous recombination between repeated DNA sequences (direct repeats) present at different sites along the DNA results in deletion of the material between the repeats.

B. Duplication- Abnormal chromosomes having a region that is present twice. Tandem duplication- the duplicated segment is present in the same orientation immediately adjacent to the normal region in the chromosome. This produces even more copies of the duplicated region by means of a process called unequal crossing over.

Human color blindness a result of unequal crossing over Human color vision is mediated by 3 light- sensitive protein pigments present in the cone cells of the retina. Each of the pigments is related to Rhodopsin, the pigment found in he rod cells that mediates vision in dim light. The light sensitivities of the cone pigments are toward blue, red and green (primary colors).Perception of all other colors are brought about by mixtures of these primaries. The gene for the blue- sensitive pigment is in chromosome 7, whereas the genes for the red and green pigments are in the X chromosome (arose from the duplication of a single ancestral pigment gene, are still 96% identical in amino acid sequence and are similar enough that they can pair and undergo unequal crossing over).

Red- green color blindness- one of the most common inherited conditions in humans. 5% of males Preponderance of affected males immediately suggests X-linked inheritance (have normal sons and 50% carrier daughters). Protanopia- inability to perceive red; Protanomaly- impaired ability to perceive red Deuteranopia- inability to perceive green; Deuteranomaly- impaired ability to perceive green

C. Inversion- A chromosome in which the linear order of a group of genes is the reverse of the normal order. Can be formed by 2 methods: 1. Two- break event in a chromosome in which the middle segment is reversed in orientation before the breaks are healed. 2. Ectopic recombination between DNA sequences that are inverted repeats, resulting in a chromosome with an inversion in the order of the gens between the repeats.

In an organism that is heterozygous for an inversion, one chromosome is structurally normal (wildtype) and the other carries an inversion. These chromosomes pass through mitosis without difficulty because each chromosome duplicates and its chromatids are separated into the daughter cells without regard to the other chromosome. However, there could be problems in meiosis. In Prophase I in which there is gene for- gene pairing taking place everywhere along the length of the chromosome, one or the other of the chromosomes must twist into a loop in the region in which the gene order is inverted, forming an inversion loop. When there is crossing over within the inversion loop, the chromatids involved in the crossing over become physically joined and the result is the formation of chromosomes containing large duplications and deletions.

Chapter 7. The Genetics of Bacteria and Their Viruses

Bacteria and their viruses (bacteriophage) have unique and diverse reproductive systems with multiple and novel mechanisms of genetic exchange.

A high percentage of bacteria isolated from clinical infections are resistant to one or more antibiotics. The widespread antibiotic- resistance genes almost never originate from new mutations in the bacterial genome, but they are actually acquired, usually several at a time, in various forms of Mobile DNA (sequences in bacteria which are mobile and can be transferred between DNA molecules and from one cell to another, between individuals and among species).

Plasmids- nonessential DNA molecules that exist inside bacterial cells. They replicate independently of the bacterial genome and segregate to the progeny when a bacterial cell divides, so they can be maintained indefinitely in a bacterial lineage. Many are circular DNA molecules but others are linear. They range in size from a few kilobases to a few hundred kilobases.

The presence of plasmids can be detected physically by electron microscopy or by gel electrophoresis of DNA samples. Some can be detected because of phenotypic characteristics that they confer on the host cell (like antibiotic resistance).(Text book: Fig. 7.1)

Plasmids rely on the DNA- replication enzymes of the host cell for their reproduction, but the initiation of replication is controlled by plasmid genes.

High- copy-number plasmids- found in as many as 50 copies per host cell. Replication is initiated multiple times during replication of the host genome.Low-copy-number plasmids- present in 1 to 2 copies per cell. Replication is initiated only once per round of replication of the host genome.

Pilus (plural: pili)- a tube-like structure formed between the cells through which the plasmid DNA passes as they are being transferred between cells.

Conjugation- the joining of bacterial cells in the transfer process. Plasmids that can be transferred in this manner are called conjugative plasmids. Not all plasmids are conjugative. Most small plasmids are nonconjugative. They can be maintained in a bacterial lineage as the cells divide, but they do not contain the approximately 20 genes necessary for pilus assembly or those for DNA transfer. Hence they are unable to be transferred on their own.

(Text book: Fig. 7.2)

The pilus between the E. coli cells above is an F pilus whose synthesis results from the presence of a conjugative plasmid called the F factor (F stands for fertility). Cells that contain the F plasmid (low-copy number plasmid) are donors and are designated the F+ cells (F plus); those lacking F are recipients and are designated the F- cells (F minus.) Conjugation begins with physical contact between a donor cell and a recipient cell. Once the pilus contacts the F- cell, the pilus retracts and the cell membranes of the donor and recipient are brought into close proximity. Then the donor DNA moves through a pore in the membrane from the donor to the recipient. The transfer is always accompanied by replication of the plasmid. Contact between an F+ and an F- cell initiates rolling circle replication which results in the transfer of a single- stranded linear branch of the rolling circle to the recipient cell. During transfer, DNA is synthesized in both donor and recipient. When transfer is complete, the linear F strand becomes circular again in the recipient cell. After the transfer, both cells contain F and can function as donors. The F- cell has been converted into an F+ cell. (Text book: Fig. 7.3)

Transposable elements are DNA sequences that can jump from one position to another or from one DNA molecule to another. Bacteria contain a wide variety of transposable elements. The smallest and simplest are insertion sequences or IS elements, which are typically 1-3 kb in length and usually encode only the transposase protein required for transposition and one or more additional proteins that regulate the rate of transposition.

Other transposable elements in bacteria contain one or more genes unrelated to transposition that can be mobilized along with the transposable element; this type of element is called a transposon. Much of the widespread antibiotic resistance among bacteria is due to the spread of transposons that include one or more antibiotic- resistance genes. When a transposon mobilizes and inserts into a conjugative plasmid, it can be widely disseminated among different bacterial hosts by means of conjugation.

Some transposons have composite structures with antibiotic resistance sandwiched between insertion sequences, as is the case with the Tn5 element (illustration below), which terminates in two IS 50 elements in inverted orientation. Transposons are designated by the abbreviation Tn followed by an italicized number (ex. Tn5). For example, Tn5( neo- r ble-r str-r) contains genes for resistance to three different antibiotics: Neomycin, Bleomycin and Streptomycin.

(Text book: Fig. 7.4)

Nonconjugative and conjugative plasmids typically coexist in the same cell along with host genomic DNA, and when a transposable element is mobilized, all of the DNA molecules present are potential targets for insertion. Many nonconjugative and conjugative plasmids present in a bacterial cell come to carry one or more copies of the same transposable element. Because these copies are homologous DNA sequences, they can serve as substrates for recombination. When 2 plasmids undergo recombination in a region of homology, the result is as shown below. The recombination forms a composite plasmid called a cointegrate. By this mechanism, nonconjugative plasmids can temporarily ride along with conjugative plasmids and be transferred from cell to cell.

(Text book: Fig. 7.5)

In the evolution of multiple antibiotic resistance, bacteria have also made liberal use of a set of enzymes known as Site- specific recombinases which were present in bacterial populations and functioned in the evolution of other traits long before the antibiotic era. Each type of site specific recombinase binds with a specific nucleotide sequence in duplex DNA. When the site is present in each of two duplex DNA molecules, the recombinase brings the sites together and catalyzes a reciprocal exchange between the duplexes. Site specific recombinases are used in the assembly of multiple antibiotic-resistance units called integrons. An integron is a DNA element that encodes a site- specific recombinase as well as a recognition region that allows other sequences with similar recognition regions to be incorporated into the integron by recombination. The elements that integrons acquire are known as cassettes. In the context of integrons, a cassette is a circular antibiotic- resistance- coding region flanked by a recognition region for an integron. Because the site specific recombinase integrates cassettes, the integron recombinase is usually called an integrase.

(Text book: Fig. 7.7)

In nature, a conjugative plasmid can accumulate different transposons containing multiple independent antibiotic- resistance genes, or transposons containing integrons that have acquired multiple antibiotic- resistance cassettes with the result that the plasmid confers resistance to a large number of completely unrelated antibiotics. These multiple resistance plasmids are called R plasmids. The evolution of R plasmids is promoted by the use of and overuse of antibiotics which selects for resistant cells because in the presence of antibiotics, resistant cells have a growth advantage over the sensitive cells. The presence of multiple antibiotics in the environment selects for multiple drug resistance. Serious complications result when plasmids resistant to multiple drugs are transferred to bacterial pathogens or agents of disease.

Transduction- bacterial DNA is transferred from one bacterial cell to another by a phage particle containing the DNA. Such a particle is called a transducing phage. Two types of transducing phages are known:1.) Generalized transducing phage- produces some particles that contain only DNA obtained from the host bacterium, rather than phage DNA; the bacterial DNA fragments can be derived from any part of the bacterial chromosome.2.) Specialized transducing phage- produces particles that contain both phage and bacterial genes linked in a single DNA molecule, but the bacterial genes are obtained from a particular region of the bacterial chromosome.

Bacteriophage life cycles:

Lytic cycle- the reproductive cycle of a phage. Phage DNA enters a cell and replicates repeatedly, bacterial ribosomes are used to produce phage protein components, the newly synthesized phage DNA molecules are packaged into protein shells to form progeny phage, and the bacterium is split open (lysis), releasing the progeny phages from the cell.

Lysogenic cycle- The alternative to the lytic cycle. No progeny particles are produced, the infected bacterium survives, and a phage DNA molecule is transmitted to each bacterial progeny cell when the cell divides. All phage species can undergo a lytic cycle. Those phages that are also capable of the lysogenic cycle are called temperate phage , and those capable of only the lytic cycle are called virulent phage. In the lysogenic cycle, a replica of the infecting phage DNA becomes inserted, or integrated into the bacterial chromosome. The inserted DNA is called a prophage, and the surviving bacterial cell is called a lysogen.

(Text book: Fig. 7.22)

A mutation is any heritable change in the genetic material.

(Text book: Table 12.1)

1