molecular biology-reading material.docx

7/26/2019 Molecular Biology-Reading Material.docx

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Ministry of Health of the Republic of Moldova

State Medical and Pharmaceutical University

Nicolae Testemitanu

Molecular Biology

Chisinau, 2012


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CONTENT

I. INTRODUCTION TO CELL AND MOLECULAR BIOLOGY. GENERAL PRINCIPLES OF

CELLULAR ORGANIZATION ..................................................................................................... 4

Common characteristics of living cells ....................................................................................... 4

Levels of organization of biological systems .............................................................................. 4

Cellmorphological and structural unity of life ........................................................................ 5

Comparative characterization of prokaryotes and eukaryotes ..................................................... 5

Viruses ......................................................................................................................................... 6

II. CHEMICAL ORGANIZATION OF CELLS. MACROMOLECULES .................................... 7

CARBOHYDRATES .................................................................................................................. 7

LIPIDS ......................................................................................................................................... 7

PROTEINS .................................................................................................................................. 7

NUCLEIC ACIDS ....................................................................................................................... 9

DNA .......................................................................................................................................... 11

RNA ........................................................................................................................................... 14

III. BIOLOGICAL MEMBRANES .............................................................................................. 16

Functions of biological membranes ........................................................................................... 16

Membrane Structure .................................................................................................................. 16

Fluid mosaic model ................................................................................................................... 19

Membrane biogenesis ................................................................................................................ 19

Transport through the membrane .............................................................................................. 19

DIVERSITY OF BIOLOGICAL MEMBRANES .................................................................... 24

Plasma membrane ...................................................................................................................... 25

Intercellular connections and communication ........................................................................... 26

IV. COMPARTMENTALIZATION OF EUKARYOTIC CELL ................................................. 27

ER - endoplasmic reticulum ...................................................................................................... 28

Golgi complex (Golgi apparatus) .............................................................................................. 29

Lysosomes ................................................................................................................................. 30

Peroxisomes ............................................................................................................................... 32

Nucleus ...................................................................................................................................... 33

Mitochondrion ........................................................................................................................... 34

Ribosomes ................................................................................................................................. 34

Cytoskeleton .............................................................................................................................. 35

V. NUCLEUS ................................................................................................................................ 37

Chromatin .................................................................................................................................. 37

Levels of DNA condensation .................................................................................................... 38

Nuclear Envelope ...................................................................................................................... 40

Karyoplasm ................................................................................................................................ 41


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Nucleolus ................................................................................................................................... 41

VI. THE GENE ............................................................................................................................. 43

Peculiarities of organization of the structural (II-ndclass) genes in eukaryotic cells ................. 44

Organization Peculiarities of genes Encoding rRNA and tRNA ............................................... 45

Mitochondrial Genome .............................................................................................................. 45

THE PECULIARITIES OF PROKARYOTES GENES ORGANIZATION ........................... 46

VII. TRANSCRIPTION AND RNA PROCESSING ................................................................... 48

Components required for transcription: ..................................................................................... 48

Particularities of Transcription of Eukaryotic Structural Genes ............................................... 49

RNA PROCESSING ................................................................................................................. 52

PARTICULARITIES OF I-STAND IIIrd CLASS GENES TRANSCRIPTION ....................... 53

Transcription in Mitochondria ................................................................................................... 54

Transcription in prokaryotes ...................................................................................................... 54

VIII. TRANSLATION .................................................................................................................. 55

THE GENETIC CODE ............................................................................................................. 55

The Components Required for Translation ............................................................................... 56

THE MECHANISM OF TRANSLATION ............................................................................... 58

POST-TRANSLATIONAL EVENTS ...................................................................................... 60

IX. DNA REPLICATION AND REPAIR .................................................................................... 61

Components required for replication: ........................................................................................ 61

MECHANISMS OF REPLICATION ....................................................................................... 61

DNA REPAIR ........................................................................................................................... 64

MECHANISMS OF DNA REPAIR ......................................................................................... 65

X. THE CELL CYCLE ................................................................................................................. 67

Interphase................................................................................................................................... 67

Mitosis ....................................................................................................................................... 68

Regulation of the cell cycle ....................................................................................................... 70

Apoptosis ................................................................................................................................... 71

XI. RECOMBINATION OF GENETIC MATERIAL.................................................................. 73

STAGES OF MEIOSIS ............................................................................................................. 73

Fertilization ................................................................................................................................ 76

XII. METHODS OF MOLECULAR GENETICS ........................................................................ 77

ISOLATION OF NUCLEIC ACIDs ......................................................................................... 77

DNA CLONING ....................................................................................................................... 77

POLYMERASE CHAIN REACTION (PCR) .......................................................................... 79

DNA SEQUENCING ................................................................................................................ 82

HYBRIDIZATION OF NUCLEIC ACIDS .............................................................................. 83


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I. INTRODUCTION TO CELL AND MOLECULAR BIOLOGY. GENERAL

PRINCIPLES OF CELLULAR ORGANIZATION

Biology is a science that studies life. It is based on the fundamental laws of nature

embodied in chemistry and physics. The field of biology today is so wide, that it has been

divided into some separate disciplines. Molecular biology is one of these disciplines. The termmolecular biology was first used in 1945 by William Astbury and was referred to the study of the

chemical and physical structure of biological macromolecules (biopolymers). By that time

biochemists had discovered many fundamental intracellular chemical reactions and explained the

importance of proteins in cell activity. In 1953, scientists identified that DNA was the

macromolecule containing the genetic information of a cell. Following this discovery, the new

field of molecular genetics appeared. In the late 1970, a new method recombinant DNAtechnologywas elaborated. This provided new tools and, in time, information about all cells

became available at an extraordinary rate. As the molecular mechanisms of life have become

clearer, the underlying similarities became more impressive than the differences. Biologists are

confident that a limited number of general principles, summarizing common molecular

mechanisms, will eventually explain even the most complex life processes in terms of chemistryand physics.

Common characteristics of living cellsThere are certain common characteristics of all living organisms: growth, reproduction,

homeostasis, metabolism, sensitivity, and energy acquisition.

1.

Growth and development. Even single-celled (unicellular) organisms grow. When first

formed by cell division, the cells are small, and must grow and develop into mature cells.

Multicellular organisms pass through a more complicated process of differentiation and

organogenesis.

2.Reproduction. All living things must be able to reproduce. Through reproduction the

species continues to survive. All their hereditary characteristics that determine a species and

make it suitable for a particular environment are transmitted to their offspring.

3.

Homeostasis. It is the maintenance of a constant internal environment in terms of

temperature, pH, water concentrations etc. An organism adjusts its metabolism to maintain stable

internal conditions for an effective functioning of the organism.

4.Metabolism. All living beings must have a metabolism, the ability to carry out chemical

reactions and exchange substances.

5.Sensitivity is the ability to respond to stimuli (both internal and external).

6.Energy acquisition and release. One view of life is that it is a struggle to acquire energy

(from sunlight, inorganic chemicals or another organisms), and release it in the process of

forming ATP.

Levels of organization of biological systems

Life on Earth is incredibly extensive and, to make it easier to study, biologists have broken

living systems up into generalized hierarchical levels:

molecules

cells

tissues

organisms

populations

communities

ecosystems

biosphere


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ribosomes

Fig. 1.Bacterium

Structure

The focus of this course is the fundamentals of life, the properties that are held in common

among all living things. We will concentrate almost exclusively on molecular and cellular levels.

Cellmorphological and structural unity of lifeCells are the structural units of all living beings (with the possible exception of viruses).

All cells have the following characteristics in common. CELL MEMBRANE that separates the chaos outside a cell from the high degree of

organization within the cell. A cell without a cell membrane is not a cell.

CONTAINS DNA as its genetic material. All cells contain several varieties of RNAmolecules and proteins; most of them are enzymes.

All cells are composed of the same BASIC CHEMICALS: carbohydrates, proteins,

nucleic acids, minerals, fats and vitamins.

All cells REGULATE the flow of nutrients and wastes that enter and leave the cell.

All cells REPRODUCE and are the result of reproduction.

All cells require a SUPPLY OF ENERGY.

All cells are HIGHLY REGULATED by ELABORATING SENSING SYSTEMS

(chemical noses) that allow them to be aware of every reaction and many of theenvironmentalconditions around them; this information is continually PROCESSED to make metabolic

decisions.

The above criteria are the minimal requirements of life. Two general cell types have

evolved: prokaryotic and eukaryotic cells. Current data supports the theory that prokaryotes

represent the initial or primitive (the simplest) cell type on earth and that eukaryotic cell types

evolved from them. There is strong data to support the idea that eukaryotes evolved from

aggregates of prokaryotic cells that became interdependent upon one another and eventually

merged (fused) into a single larger cell. Eukaryotic cells are structurally and biochemically more

complex than prokaryotes. They contain many membrane-bound organelles(cell structures withspecific molecular organization and distinct functions), whereas prokaryotic cells contain no

organelles.

Comparative characterization of prokaryotes and eukaryotesThe both cell types do have DNA as genetic material (thus also possessing different RNAs,

ribosomes and proteins), have an exterior membrane (plasma membrane), and are very diverse.

Prokaryotes are bacteria and blue green algae (Kingdom Monera). Prokaryotes are cells

without a nucleus and membrane-bound organelles. They have

genetic material, which is however not enclosed

within a membrane. The genetic material is a

single circular DNA situated in the cytoplasm.The prokaryotic DNA is associated with

proteins, which can be easily separated. The

reproduction of prokaryotes is through binary

fission (no sexual process takes place).

Nevertheless, there is a way for the exchange of

genetic information through transfer of

plasmids (short circles of DNA that pass from

one bacterium to another). Often plasmids carry

genes of resistance to antibiotics. The prokaryotes do not engulf

solids nor do they have centrioles. The membrane of prokaryotes

lacks cholesterol. Prokaryotes have a cell wall made up of

peptidoglycan. The cytoplasm of prokaryotes is motionless. Any internal membranes are


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elaborations of the plasma membrane and are known as mesosomes. The mesosome is believed

to play the role of ATP synthesis or energy center of prokaryotic cell.

Eukaryotes form the remaining four kingdoms: Protista (ex.: Protozoa like Amoeba or

Trypanosoma; Algal Protists such as Euglena or Chlamydomonas; and Fungus-like Protists,

which include species of Myxomycota or slime molds), Fungi (yeasts, rusts, smuts, puffballs,

truffles, molds),Plantae (plants), and Animalia (animals and humans). These are cells with a

nucleus an organelle where the genetic material is surrounded by an envelope (doublemembrane). The genetic material is encoded by linear DNA molecules that in complex withproteins form multiple chromosomes. Eukaryotes also contain membrane-bound organelles

(Endoplasmic Reticulum (ER), Golgi apparatus (GA), lysosomes, peroxisomes, mitochondria,

vesicles, endosomes) and some non-membranous organelles (ribosomes, nucleolus, cenrioles).

The most complex eukaryotes are composed of plant and animal cells. Plants vary from

animal cells in that they have large vacuoles, cell wall, chloroplasts, and a lack of lysosomes,

centrioles, pseudopods, and flagella or cilia. Animal cells do not have the chloroplasts, and may

or may not have cilia, pseudopods or flagella, depending on the type of cell.

Viruses

A virus is a submicroscopic infection particle composed of a protein coat and a nucleicacid core. The diameter of viral particles is 20-30 nm. Thus, they are much smaller than any

prokaryotic cell. Viruses, like cells, carry genetic information encoded in their nucleic acid, and

can undergo mutation and reproduce; however they cannot carry out metabolism.

Viruses are obligated intracellular parasites, meaning that they require host cells to

reproduce. In the viral life cycle, a virus infects a cell, allowing the viral genetic information to

direct the synthesis of new virus particle by the cell. Outside the cell they can only be in non-

replicative state, as they lack enzymes necessary for complete reproduction of virus particle.

There are many kinds of viruses. Viruses are classified by the type of nucleic acid they

contain and the shape of their protein capsule. They can be: DNA containing and RNA-containing. DNA-containing viruses can be spiral, octahedral, complex without envelope,

complex with envelope. RNA containing viruses have RNA instead of DNA and the enzymereverse transcriptase. Once inside the host cell, reverse transcription (making DNA from RNA)

is accomplished by the reverse transcriptase. This new DNA is incorporated into the host DNA,

where it transcribes new viral RNA genomes, as well as the RNA to synthesize new reverse

transcriptase and protein capsules.

Viruses cause a variety of diseases among all groups of living organisms. Viral diseases

include the flu, common cold, herpes, measles, chicken pox and encephalitis.

Bacteriophages (viruses that infect bacteria) invade the host cell and begin replicating

viruses, eventually lysing or bursting the host cell, realizing the new viruses.


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II. CHEMICAL ORGANIZATION OF CELLS. MACROMOLECULESThe most frequent chemical elements of living cells are H, C, N, O, P, S, representing 99%

of cell mass. These elements form the organic molecules of the cells - biomolecules: nucleic

acids, proteins, carbohydrates, and lipids.

Organic substances are represented by simple substancesand polymers(biopolymers). The

polymers are macromolecules made of monomers. Monomers are linked together through

covalent bonds, which occur when electrons are shared between atoms; this form of bond isstrongest and is found in both energy-rich molecules and molecules essential to life.

Some polymers the homopolymers are made from only one type of monomers (ex:cellulose, starch), otherthe copolymers (heteropolymers)are composed from different typesof monomers (ex: DNA, RNA, proteins). Proteins and nucleic acids are responsible for

conveying genetic information. Nucleic acids carry the information, while proteins provide the

means for executing it. The sequence in which the individual building blocks are joined is the

critical feature that determines the property of the resulting macromolecule.

A polymer has:

-a backboneconsisting of a regularly repeating series of bonds;

-side-groupsof characteristic diversity that stick out from the backbone.

The process of polymers synthesis is called polymerization (or polycondensation). Theprocess of polymers destruction is named hydrolysis.

CARBOHYDRATESThe general formula of carbohydrates is Cx(H2O)y, where x and y can be grater then 3.

There are several types of carbohydrates: monosaccharides (glucose, fructose, ribose,

deoxyribose), disaccharides (sucrose), oligosaccharides (different types of cellular receptors)

and polysaccharides (cellulose, starch, glycogen). The monomers are linked by glycosidic

bonds.

The main functions of carbohydrates are structural, energetic and depositary.

LIPIDS

Lipids are organic substances insoluble in water, but soluble in nonpolar solvents

(chloroform, ether). The simplest lipids are the fatty acids with the general formula R-COOH,

where R is the hydrocarbon tail. Fatty acids can also be constituents of more complex lipids such

as triacylglycerols, glycerophospholipids, and sphingolipids, as well as waxes and

eicosanoids. On the other hand there are structurally distinct lipids like steroids and lipid

vitamins which are derived from a five carbon molecule called isoprene.

Lipids can be divided in structural lipids(lipids from membranes) and storage lipids.

Functions of l ipids: energetic; structural; mechanical; hormonal; vitamins.

PROTEINSEach protein consists of a unique sequence of amino acids. A

free amino acid contains an amino group, a carboxyl group(constant

part) and a side group (variable part) (fig. 2). Depending on the

structure of side group there are four types of amino acids: basic,

acidic, neutral and hydrophobic. There are about 150 types of amino

acids; only 20 however participate in protein synthesis.

Amino acids are joined into a chain by peptide bonds (fig. 3),

which are created by the condensation of carboxyl (COOH) group of

one amino acid with the amino (NH2) group of the next one. A longer

chain of amino acids joined in this manner is called a polypeptide.

The proteinrepresents the functional unit, which may consist of oneor more polypeptide chains.

Fig. 2. The general

structure of amino acids


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A critical feature of protein is its ability to fold into a three dimensional conformation. A major

force underlying the acquisition of conformation in

all proteins is the formation of non-covalent bonds:

- Hydrogen bondsare weak electrostatic bonds

that arise between NH and C=O groups of the

peptide backbone. Although these bonds are weak,

the number of hydrogen bonds formed in amacromolecule may be significant and their overall

contribution to the stability of the conformation is

substantial. Despite of this, physical (high

temperature) and physiological (action of enzymes)

factors are often causes of hydrogen bonds disruption.

-Ionic bonds occur between groups

that have opposite charges (usually between

acidic and basic amino acids).

-Hydrophobic bonds occur between

amino acids with apolar side chains, which

aggregate together to exclude water.-Van der Waals attractionvery

weak interactions between atoms.

There is also a covalent bond -

disulphide bondformed between the sulphur

atoms of cysteine.

The conformation can be described in

terms of several levels of structure.

The primary structurethe sequenceof amino acids linked into polypeptide chain. This linear sequence

is the essential information encoded by genetic material (DNA)

(fig. 3). The primary structure folds into secondary structure,

which describes the path that the polypeptide backbone of the

protein follows in the space. Hydrogen bondingbetween groups

on the same polypeptide chain causes the backbone to twist into a

helix (-helix; ex: keratin, myosin) or sheet-like structure (-

sheet; ex: fibroin, collagen) (fig. 4).

The tertiary structure describes the three dimensional

organization of all the atoms in the polypeptide chain. This

involves side chain interactions and packing of secondary

structure motifs (a combination of -helixes and -sheets; ex:enzymes, myoglobin, globulins) (Fig. 5).

The highest level of organizationquaternary structureoccurs in mul timeric proteins, which consist of aggregates of two

or more polypeptide chains. The individual chains that make up a

multimeric protein are called protein subuni ts. Fig. 6 represents

the structure of hemoglobin, which consists of two chains andtwo chains.

Among the major bio-organic molecules, only proteins

require up to four levels of structure in order to be functional.Fig. 6. Quaternary structure of

hemoglobin

Fi . 4. A, B - -helix; C, D - -seets

Fig. 3. Primary structure of proteins

Fig. 5. Tertiary structure ofroteins


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Fig. 10 Polynucleotide Chain

Table 1. Bases, nucleosides and nucleotides

Base Nucleoside Nucleotide

Abbreviation

(1P)

Abbreviation

(2P)

Abbreviation

(3P)

RNA DNA RNA DNA RNA DNA

Adenine AdenosineAdenilic

acid

AMP dAMP ADP dADP ATP dATP

Guanine GuanosineGuanilic

acidGMP dGMP GDP dGDP GTP dGTP

Cytosine CytidineCytidilic

acidCMP dCMP CDP dCDP CTP dCTP

Thymine ThymidineThymidilic

aciddTMP dTDP dTTP

Uracil UridineUridylic

acidUMP UDP UTP

Nucleotides are building blocks from

which nucleic acids are constructed.

The nucleotides are linked into a

polynucleotide chain. The 5 positionof one pentose ring is connected to the

3 position of the next pentose ring via

a phosphate group. The bond that linknucleotides is called phosphodiester

bond(fig. 10)

Number, type and sequence of

nucleotides in polynucleotide chain

represent the primary structure of

nucleic acids.

Fig. 9. Nucleoside TriphosphatesMonomers of DNA (left) and RNA (right)


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Fig. 12. The double helix of DNA2

antiparallel chains bounded by hydrogen bonds

DNADNA represents the molecule of heredity and

variability, which contains the genetic program of

an organism, ensuring the passage of characteristic

traits from one generation to another.

The molecule of DNA represents a double

helix, which consists of two polynucleotide chainsassociated by hydrogen bounding between the

nitrogenous bases. There are 3 H-bonds between G

and C and 2 H-bonds between A and T (fig. 11).

These reactions are described as base

pairing, and the paired bases are said to be

complementary. The polynucleotide chains

run in opposite directions are antiparallel(fig. 12).

The double helix of antiparallel

polynucleotide chains linked by hydrogen

bonds between purines and pyrimidinesrepresents the secondary structur e of DNA.

This structure was established in 1953 by

James Watson and Francis Crick. Each base

pair (bp) is rotated ~360around the axis of the

helix relative to the next pair. Thus ~10 base

pairs make a complete turn of 3600. The

twisting of the two strands around each other

forms a double helix with narrow groove

(~12 across) and a wide groove (22 across). The double helix is right-handed(fig.

12).The complementarity of DNA bases assure:

DNA stability;

Replication;

Transcription;

Recombination;

DNA repair.

Different conditions (heating in laboratory experiences or in vivoaction of enzymes, such

as DNA-helicases) determine the conversion of the molecule from

double-stranded to the single-stranded state. This process is called DNA

denaturation. Slow cooling of DNA or action of enzymes may determine

the DNA renaturation reassociation of denatured complementarysingle strands of a DNA into a double helix.

DNA can exist in more than one type of double-helical structure.

Each family represents a characteristic type of double helix described by

parameters n (number of nucleotides per turn) and h (the distance

between adjacent repeating units) (Tab. 2). The B-form represents the

general structure of DNA under conditions of a living cell (fig. 13). The

A-form is probably characteristic for hybrid duplex with one strand of

DNA and one strand of RNA. This may occur during transcription. The

Z-formis the only left-handed helix. Its name arises from the zigzag paththat the sugar-phosphate backbone follows along the helix.

Fig. 13. B-DNA

Fig. 11. Complementary Base Pairing


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Table 2. Parameters of double-helical DNA structures

Parameters of helix ADNA BDNA ZDNA

Direction Right-handed Right-handed Left-handed

Base pair in turn 11 10,4 12

Distance between bases () 2,9 3,4 3,7

Helical diameter () 25,5 23,7 18,4The total length of DNA contained within the genome of

an organism varies depending on its genetic complexity, and

may fluctuate from micrometers (viruses) or millimeters

(bacteria), to several centimeters (in higher eukaryotes).

Therefore, the DNA must be organized and packed into higher

order forms within the cell, and DNA molecules in vivo may

acquire a compact shape by existing in circular forms (ex: in

prokaryotes), where the two ends of a linear DNA are

covalently bound to each other. These circular

DNA molecules can be twisted into supercoiledmolecules to adopt an even more condensed

configuration than the relaxed circularequivalent (fig.14). In prokaryotic cells, this

supercoiling of the DNA is critical for DNA

packaging into the cells. In prokaryotes the

superhelical state of the DNA plays an

important part in various biological processes

including replication, transcription and

recombination, by altering the accessibility of

the DNA to proteins (fig. 15).

In eukaryotes the DNA is packed (condensed)in the nucleus (0.5mm in diameter) with the help of

basic polypeptides called histones(fig. 16). There are

5 classes oh histones: H1, H2A, H2B, H3, H4. Each

DNA molecule is packed in the form of a

chromosome. Chromosomes are composed of

chromatin, a densely staining material initially

recognized in two different forms: highly condensed

heterochromatin and more diffuse euchromatin.

Decondensed chromatin resembles beads on astring. Each bead, called nucleosome, contains about

two supercoils of DNA wrapped around a corehistone octamer (made of 8 proteins). Interaction of

DNA with proteins represents the terti ary structure of DNA.

Properties of DNA MoleculesFrom the perspective of its function in replicating itself and being expressed as protein, the

central property of the double helix is the ability to separate the two strands without disrupting

covalent bonds. This allows the strands to get separated and reunited under physiological

conditions. The specificity of the process is determined by complementary base pairing.

Chemical structure and negative charging permit the interactions of DNA with other molecules.

The main properties of DNA are the following:

Replicationsynthesis of new molecules identical with initial copy, which is possible dueto the double stranded state of the molecule.

Fig. 16. Interaction of DNA with Histones in

Eukaryotes

Fig. 14. Supercoiling of Circular

DNA

Fig. 15. DNA Packing in Prokaryotes


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Repair represents a range of cellular responses associated with restoration of primarystructure of DNA, based on complementarity of bases.

Denaturation- the conversion of the molecule from double-stranded to the single-stranded

state.

Renaturationreassociation of denatured complementary single strands of a DNA into adouble helix.

Coiling, supercoiling and unwindingare properties of double helix to change its functionalstates.

Chargaff rule the quantity of purines is equal with quantity of pyrimidines ([G] = [C]and [A] = [T]).

Flexibility DNA ability to change the type (ex.: from B-DNA to A-DNA duringtranscription).

Variability the state of being variable, changeable. The tendency of individual geneticcharacteristics in a population to vary from one another; is assured mainly by mutations and

recombination.

Recombination- the process of exchanges of fragments between different DNA molecules,

resulting in a different genetic combination.

Hybridization - the process of forming a double stranded nucleic acid from joining twocomplementary strands of DNA (or RNA).

DNA FunctionsDNA is the main genetic molecule of life that carries the hereditary information, which

determines the structure of proteins in all eukaryotic and prokaryotic organisms. It contains the

instructions by which cells grow, divide and differentiate, and has provided a basis for the

evolutionary process both within and between related species. DNA is the information-carrying

material that comprises the genes, or units of inheritance, which are arranged in linear arrays

along the chromosomes of the cell.

Heterogeneity of DNA in Eukaryotes

The total amount of DNA in the genome is characteristic for each living species and is

known as its C-value. There is enormous variation in the range of C-values (106-1011). There is a

discrepancy between genome size and genetic complexity, called C-value paradox:

there is an excess of DNA compared with the amount that could be expected to code for

proteins;

there are large variations in C-values between certain species whose apparent complexity

does not vary much.

There is a rough correlation between the DNA content and the number of genes in a cell or

virus. However, this correlation breaks down in several cases of closely related organisms where

the DNA content per haploid cell (C-value) varies widely. This C-value paradox is probably

explained, not by extra genes, but by extra noncoding DNA in some organisms.

Eukaryotic DNA may be divided in some groups depending on complexity: nonrepetitive

DNA45-56%; moderately repetitive DNA8-30%; highly repetitive DNA12-25%.Nonrepetiti ve DNA consists of sequences existing in genome in one or a few copies and

usually encodes information about proteins.

Moderately repetit ive DNA consists of families of sequences that are not exactly the same,

but are related. In human genome such sequences are represented by genes encoding for

histones, rRNA, actins. Another example is Alu familyshort fragments (~300 bp (base pairs))distributed among the nonrepetitive sequences.

Highl y repetiti ve DNArepresents very short sequences repeated many times in tandem in

large clusters. They are also called simple sequencesor satel li te DNA. The simple sequences arelocated mainly in the heterocromatine, especially around the centromere. The tandem clusters of

satellite DNA are highly polymorphic, with wide variations between individuals. Such


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Fig. 17. The structure of palindrome andcruciform

Palindrome

sequences, called minisatellites, can be used to characterize individual genomes in the technique

of DNA fingerprinting.Additional classes of highly repeated

DNA are palindromes, which consist of

inverted repeatsa region of dyad symmetry(fig. 17). In a double-strand DNA, the

complementary sequences on one strand havethe opportunity to base pair only if the strand

separates from its partner. As a result a

hairpin could be formed. The formation of

two apposed hairpins creates a cruciform.

Palindromes are very important for DNA

interactions with proteins; they serve as

indicators in the processes of initiation and

termination of transcription, replication etc.

RNAMolecules of RNA represent single-stranded polymers and consist of nucleotides linked by

phosphodiester bonds 3-5 (some viral RNA may be double-stranded). The primary structure ofRNA represents a sequence of nucleotides covalently linked through 3,5-phosphodiester bonds

into an unbranched single-stranded chain. The four major bases in RNA are the purines adenine

and guanine, and the pyrimidines cytosine and uracil (A, G, C, and U, respectively). The thymine

nucleoside (T) in DNA is transcribed into U in RNA.

Regions of complementarity within a single-stranded RNA sequence can base-pair with

one another to generate double-stranded regions of secondary structu re (hairpin loops). U base-

pairs with A, and G with C, via hydrogen bonding. Further folding can take place to form

complex terti ary structures. The cloverleaf structure of the tRNA molecule is possibly the best-

defined RNA secondary structure (fig. 18).

Both prokaryotic and eukaryotic cells contain messenger RNA (mRNA), transfer RNA

(tRNA) and r ibosomal RNA(rRNA), all of which are involved in various ways in the accurateconversion of genetic information (in the form of DNA) into protein. This process in eukaryotic

cells also involves two additional classes of RNA - heterogeneous nuclear (or pre-messenger)

Fig. 18. A - Secondary structure of tRNA (cloverleaf); BTertiary structure oftRNA


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RNA (hNRNA or pre-mRNA) and small nuclear RNAs (snRNA). All cellular RNAs are

synthesized through the process of transcription, in which a complementary RNA copy of a

DNA template is made.

Messenger RNA is a temporary complementary copy of the sense str and (anticoding

strand) of protein-coding DNA. Messenger RNAs range in length from a few hundred to many

thousands of nucleotides depending largely upon the size of the protein they encode. An mRNA

contains a region that specifies the protein sequence (the protein-coding region), flanked oneither sides by untranslated regions (5 and 3 untranslated regions). The coding regionrepresents a sequence of codons, each codon being made of three nucleotides. The sequence of

codons specifies the amino-acid sequence of the polypeptide chain, with each codon representing

a particular amino acid. The mature mRNA molecule is transported to the cytoplasm where it is

translated into protein on the ribosome.

Ribosomal RNA is a major structural

component of the ribosome. It participates in

the process of protein biosynthesis. Depending

on sedimentation coefficient, there are several

classes of rRNAs in prokaryotic and eukaryotic

cells (Table 3). Sedimentation coefficientcharacterizes the speed of sedimentation of

particle during centrifugation. As the

sedimentation coefficient is dependent on the

mass of the particle it can be used to estimate

molecular mass. Larger molecules have higher

values of sedimentation coefficient.

Sedimentation coefficients are generally expressed as Svedberg units [S].

Transfer RNAsare small RNAs (70-80 nucleotides), which have a key role in translation

(fig. 17). They act as adaptor molecules, each tRNA molecule having a 3-base anticodoncomplementary to one of the codons, and also having a site at which the appropriate amino acid

is attached. During translation, the anticodon on the charged tRNA pairs with the codon on themRNA within the ribosome, and the amino acid is then transferred from the tRNA to the

growing polypeptide chain. Accuracy in the mRNA codon:tRNA anticodon interaction, and in

the recognition of amino acids by tRNAs is central to the insertion of the appropriate amino acid

into the polypeptide chain during translation. There are at least 20 types of tRNAs in the cell

(according to the number of amino acids participating in translation). The maximal number of

tRNA types is 61.

Small nuclear RNAs (snRNA) are present only in eukaryotes. They are parts of some

nuclear enzymatic complexes (primase, telomerase) and participate in the post-transcriptional

modification of RNA.

Heterogeneous nuclear RNAs (hnRNA) represent the primary RNA transcripts, which

are found in the nucleus. It is, as the name implies, a heterogeneous collection of RNA molecules

which are on average some four to five times larger than matured mRNAs and in some cases

more unstable.

Short interfering RNAs (siRNA), as well as microRNA (miRNAs) are 2 classes of

double-stranded RNAs, which are the effector molecules of RNA interference - an endogenous

post-transcriptional gene-silencing mechanism with a great clinical potential. In other words,

siRNAs and miRNAs can knockdown the homologous mRNAs associated with different

diseases.

Small cytoplasmic RNA (scRNA) aresmall (7S; 129 nucleotides) RNA molecules found

in the cytosol and rough endoplasmic reticulum associated with proteins that are involved in

specific selection and transport of other proteins.

Table 3. Types of rRNAs

Type of cell

Sedimentati

on

coefficient

Length

(bases)

Prokaryotes 5S 12016S 1540

23S 2900

Eukaryotes

5S 120

5,8S 160

18S 1900

28S 4700


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Fig. 19.Diagram of the

arrangement of amphipathic lipid

moleculesto form a lipid bilayer.

Individual phospholipids can rotate

and move laterally within a bilayer.

III. BIOLOGICAL MEMBRANESA biological membrane(biomembrane) is an enclosing or separating layer (possessing

both hydrophilic and hydrophobic properties) that acts as a selective barrier within or around

a cell.Cell membranes define and compartmentalize space, regulate the flow of materials, detect

external signals, and mediate interactions between cells.

Functions of biological membranes Biological barrier;

Selective transportation of ions, micromolecules and macromolecules;

Receiving and delivering of intracellular and extracellular signals;

Support for a number of enzymes;

Enclosing of compartments (organelles) in eukaryotic cells;

Control of intracellular and intercellular homeostasis.

Membrane StructureBiological membranes are made of three main components: lipids, proteins and

carbohydrates. All membranes have common general structures, which is highly fluid and most

of the lipid and protein molecules can move about in the plane of the membrane. The lipid and

protein molecules are held together mainly by non-covalent interactions. Sugars are attached by

covalent bonds to some of the lipid and protein molecules.

Lipids

The cell membrane consists of several classes of lipids: phospholipids, glycolipids,

and steroids. The amount of each depends upon the type of cell, but in the majority of cases

phospholipids are the most abundant.

Lipids constitute approximately 50% of the mass of

most biological membranes, although this proportion varies

depending on the type of membrane. Plasma membranes, for

example, are approximately 50% lipid and 50% protein. Theinner membrane of mitochondria, on the other hand, contains

an unusually high fraction (about 75%) of protein, reflecting

the abundance of protein complexes involved in electron

transport and oxidative phosphorylation. The lipid

composition of different cell membranes also varies.

Mammalian plasma membranes, for example, are complex,

containing four major phospholipids: phosphatidylcholine,

phosphatidylserine, phosphatidylethanolamine, and

sphingomyelin, which together constitute 50 to 60% of total

membrane lipid.

The membrane (fig. 19) consists primarily of a thin layerof amphipathic phospholipids,consisting of two hydrophobic fatty acid chains linked to a

phosphate-containing hydrophilic head group, which spontaneously arrange so that the

hydrophobic "tail" regions are shielded from the surrounding polar fluid, causing the more

hydrophilic "head" regions to associate with the cytosolic and extracellular faces of the resulting

bilayer. This forms a continuous, spherical lipid bilayer. The arrangement of hydrophilic and

hydrophobic heads of the lipid bilayer prevent polar solutes (e.g. amino acids, nucleic acids,

carbohydrates, proteins, and ions) from diffusing across the membrane, but generally allows for

the passive diffusion of hydrophobic molecules. This affords the cell the ability to control the

movement of these substances via transmembrane protein complexes such as pores and gates.


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Fig. 20. Insertion of

cholesterol in a membrane.Cholesterol inserts into the

membrane with its polar

hydroxyl group close to the

polar head groups of the

phospholipids.

The entire membrane is held together via non-

covalent interaction of hydrophobic tails; however the structure is

quite fluid and not fixed rigidly in place. Phospholipid molecules in

the cell membrane are "fluid" in the sense that they are free to diffuse

and exhibit rapid lateral diffusion along the layer in which they are

present. However, movement of phospholipid molecules between

layers is not energetically favorable and does not occur to anappreciable extent. So, phospholipids freely move via rotational

movement (around the longitudinal axis of the molecule), lateral

movement (in the same layer of the membrane) and rarely flip to the

other layer transversionor flip-flop. Flipping of phospholipidsfrom one layer to the other rarely occurs because flipping requires

the hydrophilic head to pass through the hydrophobic region of the

bilayer.

In animal cells cholesterol(a lipid-based molecule - steroid),

is normally found dispersed in varying degrees throughout cell

membranes, in the irregular spaces between the hydrophobic tails of

the membrane lipids (fig. 20). Cholesterol actually has twofunctions: I) To help stabilize the membrane. 2) To maintain

membrane flexibility as temperature changes. Normally the human

body is capable of producing all the cholesterol it needs. Dietary ingestion of excess saturated

fats and cholesterol is currently thought to be the source of the plaque that builds up in arteries

and can cause heart attacks and strokes.

Other membrane lipids are: glycolipids that contain sugar residues attached to a

phospholipid or sphingolipid, common in myelin; cardiolipids, which are diphosphatidyl

glycerols, found in heart mitochondria.

Carbohydrates

About 5%- 10% of the membrane weight is carbohydrate: glycoprotein and glycolipid.They are involved in cell recognition.

In many eukaryotic and prokaryotic cells, carbohydrates-containing molecules from the

outer part of plasma membrane with their sugars exposed at the cell surface form the glycocalyx,

which is an important feature in all cells, especially epithelia with microvilli. Recent data suggest

the glycocalyx participates in cell adhesion, lymphocyte homing, and many others.

ProteinsProteins are the other major constituent of membranes, constituting 25 to 75% of the mass

of the various membranes of the cell. While phospholipids provide the basic structural

organization of membranes, membrane proteins carry out the specific functions of the different

membranes of the cell. These proteins are divided into Integral proteins, lipid anchored proteins,

peripheral proteins (Table 4).

Most membrane proteins must be inserted in some way into the membrane. For this to

occur, an N-terminus "signal sequence" of amino acids directs proteins to the endoplasmic

reticulum, which inserts the proteins into a lipid bilayer. Once inserted, the protein is then

transported to its final destination in vesicles, where the vesicle fuses with the target membrane.

Many integral membrane proteins (called transmembrane proteins)span the lipid bilayer,

with portions exposed on both sides of the membrane. The membrane-spanning portions of these

proteins are usually -helical regions of 20 to 25 nonpolar amino acids. The hydrophobic side

chains of these amino acids interact with the fatty acid chains of membrane lipids, and the

formation of an helix neutralizes the polar character of the peptide bonds. Like thephospholipids, transmembrane proteins are amphipathic molecules, with their hydrophilic

portions exposed to the aqueous environment on both sides of the membrane. Some
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886#3400http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886#3400


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transmembrane proteins span the membrane only once; others have multiple membrane-spanning

regions. Most transmembrane proteins of eukaryotic plasma membranes have been modified by

the addition of carbohydrates, which are exposed on the surface of the cell and may participate in

cell-cell interactions.

Table 4. Types of Membrane Proteins

Type Description Examples

Integral proteins

or transmembrane

proteins

Span the membrane and have a hydrophilic

cytosolic domain, which interacts with internal

molecules, a hydrophobic membrane-spanning

domain that anchors it within the cell membrane, and

a hydrophilic extracellular domain that interacts with

external molecules. The hydrophobic domain

consists of one, multiple, or a combination of -helices and sheetprotein motifs.

Ion channels, proton

pumps, G-protein-

coupled receptor

Lipid anchored

proteins

Covalently-bound to single or multiple lipid

molecules; are firmly attached to membrane and can

only be removed by treatments disrupting the

membrane (using detergents or organic solvents).

G-proteins (or GTP-binding proteins)involved in sendingmessages (visual, taste,smell, some hormones)across membranes

Peripheral proteins

Attached to integral membrane proteins, or

associated with peripheral regions of the lipid

bilayer. These proteins are weakly bound to

membrane, tending to have only temporary

interactions with biological membranes, and, once

reacted the molecule, dissociates to carry on its workin the cytoplasm.

Some enzymes, some

hormones

Proteins can also be anchored in membranes by lipids that are covalently attached to the

polypeptide chain. Distinct lipid modifications anchor proteins to the cytosolic and extracellular

faces of the plasma membrane.

By other classification, there are:

Intrinsicmembrane proteins that are embedded within the membrane. They may extend

through the membrane and project on both surfaces (transmembrane proteins) or just one

(anchored proteins). Ex.: glycophorin,

which is a glycoprotein with branched

oligosaccharide residues on the part of

the protein projecting on the outer

surface of the plasma membrane. It is

responsible for the MN blood group on

red blood cells.

Extrinsic membrane proteins

are situated on the surface and are

loosely bound to the membrane

(peripheral proteins). They are easily

removed by mild treatments (altering

the pH or ionic strength). E.g.cytochromes are found only on the


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Fig. 22. Fluid Mosaic Model of

Membrane Structure

inner surface of the inner mitochondrial membrane (fig. 21).

Fluid mosaic modelThe current model of membrane structure, frequently referred to as thefluid mosaic model,

is based on work completed by Seymour J. Singer and Garth L. Nicholson in 1972. Their

research revealed that the plasma membrane is a mosaic of integral proteins bobbing in a fluid

bilayer of phospholipids. This pattern is not staticbecause the positions of the proteins are constantly

changing, moving about like icebergs in a sea of lipids.

Peripheral proteins are not embedded in the lipid

bilyaer but are appendages loosely bound to the

membrane surface. Membrane carbohydrates on the

surface function as cell markers to distinguish one cell

from another. This model has been tested repeatedly

and has been shown to accurately predict the

properties of many kinds of cellular membranes; this

structure has also been confirmed using a technique known

as freeze-fracture electron microscopy.

Membrane biogenesis

Membranes grow by the expansion of pre-existing membranes. The membranes are

renewed by synthesis of its components on ER, processing and assembling in Golgi apparatus

and transportation in vesicles to plasma membrane or other organelles (Fig. 23).

Transport through the membrane

Macrotransport Microtransport

Endocytosis active transport passive transport co-transport Exocytosis carrier proteins diffusion, osmosis Transcytosis ionic pumps

Passive Transport (is performedin the energetically favorable direction, as determined byconcentration and electrochemical gradient)

Fig. 23. Scheme of

Membrane

Biogenesis

in Eukaryotic Cell


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Fig. 24. Permeability of

phospholipid bilayers.Small uncharged molecules

can diffuse freely through a

phospholipid bilayer.

However, the bilayer is

impermeable to larger polarmolecules (such as glucose

and amino acids) and to ions.

Fig. 25. Aquaporin

The selective permeability of biological membranes to small molecules allows the cell to

control and maintain its internal composition. Only small uncharged molecules can diffusefreely

through phospholipid bilayers. Small nonpolar molecules, such as O2and CO2, are soluble in the

lipid bilayer and therefore can readily cross cell membranes. Small uncharged polar molecules,

such as H2O, also can diffuse through membranes (process called osmosis), but larger uncharged

polar molecules, such as glucose, cannot. So, the movement of substances from an area of higher

concentration to an area of lower concentration, independent from the motion of other moleculeswith no energy requirement, is called simple diffusion.

Charged molecules, such as ions, are unable to diffuse through a phospholipid bilayer

regardless of size; even H+ions cannot cross a lipid bilayer by free diffusion.

Although ions and most polar molecules cannot diffuse across a lipid bilayer, many such

molecules (such as glucose) are able to cross cell membranes. These molecules pass across

membranes through facilitated diffusion - via the action of specific transmembrane proteins,

which act as transporters. Such transport proteins determine the selective permeability of cell

membranes and thus play a critical role in membrane function. They contain multiple membrane-spanning regions that form a passage through the lipid bilayer, allowing polar or charged

molecules to cross the membrane through a protein pore without interacting with the

hydrophobic fatty acid chains of the membrane phospholipids.

There are two general classes of membrane transport proteins:

channel proteinsform open pores through the membrane, allowing the free passage of

any molecule of the appropriate size. I on channels (or ionophores), for example, allow the

passage of inorganic ions such as Na+ (carried by Monesin), K+ (carried by Valinomycin), H+

(transported byProton carriers), Ca2+, and Cl-across the plasma membrane. Once open, channel

proteins form small pores through which ions of the appropriate size and charge can cross the

membrane by free diffusion. Not all pores formed by these channel proteins are permanently

open; rather, they can be selectively opened and closed in response to extracellular signals (gated

channels, which are specific ion channels that open only in response to a particular stimulus; ex.:

electrically gated channels, chemically gated channels), allowing the cell to control the

movement of ions across the membrane. Such regulated ion channels have been particularly well

studied in nerve and muscle cells, where they mediate the transmission of electrochemical

signals. Another example of channel proteins is aquaporin - water channel protein - that

accomplishes the water-transporting task. Water permeation through aquaporins is a passive

process that follows the direction of osmotic pressure

across the membrane (fig. 25). Aquaporins are crucial

for life and they are found in all organisms, from

bacteria to man. Aquaporins facilitate rapid, highlyselective water transport, thus allowing the cell to

regulate its volume and internal osmotic pressure


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according to hydrostatic and/or osmotic pressure differences across the cell membrane. The

physiological importance of the aquaporin in human is perhaps most obvious in the kidney,

where ~150-200 liters of water need to be reabsorbed from the primary urine each day. In

kidneys, this is made possible mainly by two aquaporins denoted AQP1and AQP2(11 different

aquaporins are known in humans). Studies of water transport in various organisms and tissues

suggested that aquaporins have a narrow pore preventing any large molecule and ions flow while

maintaining an extremely high water permeation rate (~ 109 molecules H2O per channel persecond). Several diseases, such as congenital cataractsand nephrogenic diabetes insipidus, are

connected to the impaired function of these channels.

In contrast to channel proteins, carrier proteins (fig. 26) selectively bind and transport

specific small molecules, such as glucose. Rather than forming open channels, carrier proteins

act like enzymes to facilitate the passage of specific molecules across membranes. In particular,

carrier proteins bind specific molecules and then undergo conformational changes that open

channels, through which the molecule to be transported can pass across the membrane and be

released on the other side. Carriers operate in the presence of a concentration gradient.

Fig. 26.Channel and carrier proteins(A) Channel proteins form open pores through which moleculesof the appropriate size (e.g., ions) can cross the membrane. (B) Carrier proteins selectively bind the smallmolecule to be transported and then undergo a conformational change to release the molecule on the otherside of the membrane.

Active Transport is energetically consumable, usually is coupled to ATP hydrolysis as a

source of energy; it is performed against the gradient(Fig. 27).

As described so far, molecules may be transported by either channel or carrier proteins

cross membranes in the energetically favorable direction through passive transport. However,

carrier proteins, called pumps, provide a mechanism through which the energy changes are

associated with transporting molecules (such as small ions (Na+, K+, Cl-, H+), amino acids, and

monosaccharides) across a membrane. Molecules are transported in an energetically unfavorable

direction across a membrane (e.g., against a concentration gradient) if their transport in thatdirection is coupled to ATP hydrolysis as a source of energy a process called active transport.The free energy stored as ATP can thus be used to control the internal composition of the cell, as

well as to drive the biosynthesis of cell constituents.

The protein pump binds to a molecule of the substance to be transported on one side of the

membrane, then it uses the released energy (ATP) to change its shape, and releases it on the

other side. Protein pumps are catalysts in the splitting of ATP ADP + phosphate, so they arecalled ATPase enzymes. Most of these are ion pumps. They are specific: there is a different

pump for each molecule to be transported. Ex.: the sodium-potassium pump (also called the

Na+/K

+-ATPase enzyme) actively moves sodium out of the cell and potassium into the cell.

These pumps are found in the membrane of virtually every cell, and are essential in transmission

of nerve impulses and in muscular contractions.


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Fig. 28. Scheme of Protein-

mediated Transport

Fig. 29. Co-transport of

Sodium and glucose

(antiport)

Cystic fibrosisis a genetic disorder that results in a mutated chloride ion channel (mutated

Cl--ATPase). By not regulating chloride secretion properly, water flow across the airway surface

is reduced and the mucus becomes dehydrated and thick.

Fig. 27. Model of active transport. H+-ATPase. Energy derived from the hydrolysis of ATP is used to

transport H+against the electrochemical gradient (from low to high H+concentration). Binding of H+is

accompanied by phosphorylation of the carrier protein, which induces a conformational change thatdrives H

+ transport against the electrochemical gradient. Release of H

+ and hydrolysis of the bound

phosphate group then restore the carrier to its original conformation.

Protein-mediated TransportThere are three main types of proteins

that shuttle molecules across the membrane

(fig. 28):

Uniport: transport a single

molecule across the membrane. Ex.: glucose

transporter.

Symport: transport two different

molecules in the same direction. Ex.: amino

acids and Na+.

Antiport: transport two different

molecules in opposite directions. Ex.: the

mitochondrial ADP/ATP translocase (AAT)

catalyses the exchange, across the inner membrane ofmitochondrion, of cytosolic ADP for ATP, produced in the

matrix by ATP-synthase. The AAT undergoes substantial

conformational changes during transport. An example of active

antiport transporter that uses the energy of ATP to transport 3

Na+ ions outside of the cell in exchange for the influx of 2 K+

ions into the cell is Na+/K+-ATPase.

Symport and antiport are examples of co-transport (the

transport of more than one molecule at the same time; fig. 29).

This process couples an energy-utilizing transport to an

energy-releasing transport. Example: Co-transport of glucose.


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Fig. 30. Receptor-

mediated Endocytosis

Glucose may be transported into a cell against a concentration gradient utilizing the energy

generated by the flow of Na+ions along a concentration gradient.

Macrotransport (vesicular transport - transport of large molecules via vesicles)

The main purpose of the plasma membrane is to provide a barrier that keeps cellular

components inside the cell while simultaneously keeping unwanted substances from entering the

cell. The membrane allows essential nutrients to be transported into the cell and aids in the

removal of waste products from the cell.Cells constantly transport substances across their cell membranes. Endocytosis (from the

Greek endo meaning "in" and cytosis meaning cell") is the process by which cells bringmolecules into their structure.

There are different types of endocytosis:

Pinocytosis (from the Greek pino meaning "to drink") - during pinocytosis the cellmembrane folds inward, forming a small pocket (vesicle) around fluid that is directly outside

the cell membrane. Fluid consumed by cells may contain small molecules, such as lipids.

Endothelial cells, which line capillaries, are constantly undergoing the process of

pinocytosis, "drinking" from the blood within the capillaries.

Phagocytosis (from the Greek phago meaning "to eat"). It also plays a critical role in

defense systems by eliminating microorganisms or damaged cells in mammals. Once aparticle (or microorganism) is ingested, it is wrapped within a vesicle, and then the vesicle

fuses with a lysosome. The digestive enzymes of the lysosome then digest the contents of the

vesicles.

Receptor-mediated

endocytosisoccurs when specific

molecules in the fluid

surrounding the cell bind to

specialized receptors in the

plasma membrane. As in

pinocytosis, the plasma

membrane folds inward and theformation of a vesicle follows.

Certain hormones are able to

target specific cells by receptor-

mediated endocytosis. Receptor-

hormone complexes cluster

together in special regions of

plasma membrane (coated pits)

that have a clathrin coating (fig.

30).

The release of material

from a cell is known as exocytosis(from the Greek exo meaning "out). First the cell formsthe cell product and then packages it. The package, or vesicle, is comprised of the same material

that makes up the cell membrane. When the vesicle reaches the membrane, the two structures

merge together much like air bubbles do in liquid. The contents of the vesicle are then expelled

from the cell. For example, secretory cells that manufacture specific proteins, such as the

pancreatic cells that manufacture insulin, use the process of exocytosis to secrete insulin into the

blood.

Another type of vesicular transport is transcytosis - a mechanism for transcellulartransport in which a cell encloses extracellular material in an invagination of the cell membrane


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Fig. 31. Erythrocyte

Plasma Membrane

to form a vesicle, then moves the vesicle across the cell to eject the material through the opposite

cell membrane by the reverse process.

DIVERSITY OF BIOLOGICAL MEMBRANES

Different types of biological membranes have diverse lipid and protein compositions. The

content of membranes defines their physical and biological properties. Some components of

membranes play a key role in medicine, such as the eff lux pumpsthat pump drugs out of a cell.

Natural membranes from different cell types exhibit a variety of shapes, which complement a

cells function. The smoothflexible surface of the erythrocyte

plasma membrane (the shape of

which is maintained by spectrin)

allows the cell to squeeze through

narrow blood capillaries (Fig.

31). Some cells have a long,

slender extension of the plasma

membrane, called a cilium orflagellum, which beats in a whip

like manner. This motion causes

fluid to flow across the surface of

an epithelium or a sperm cell to

swim through the medium. The

axons of many neurons are encased by multiple layers of modified

plasma membrane called the myelin sheath. This membranous

structure is elaborated by an adjacent supportive cell and facilitates the conduction of nerve

impulses over long distances.

TYPES OF BIOLOGICAL MEMBRANES

Within a eukaryotic cell, there are membranous compartments, as well as other structures

(such as ribosomes) that lack membranes but possess distinctive shapes and functions, which are

called organelles. Each of these organelles has specific roles in its particular cell. These roles are

defined by chemical reactions:

The nucleus contains most of the cells genetic material (DNA). The duplication of thegenetic material and the first steps in decoding genetic information take place in the nucleus.

The mitochondrion is a power plant and industrial park, where energy stored in the bonds of

carbohydrates is converted to a form more useful to the cell (ATP) and certain essential

biochemical conversions of amino acids and fatty acids occur.

The endoplasmic reticulum and Golgi apparatus are compartments in which proteins arepackaged and sent to appropriate locations in the cell.

Lysosomes and vacuoles are cellular digestive systems in which large molecules arehydrolyzed into usable monomers.

The membrane surrounding each organelle does two essential things: (1) it keeps the

organelles molecules away from other molecules in the cell with which they might reactinappropriately; (2) it acts as a traffic regulator, letting important raw materials into the organelle

and releasing its products to the cytoplasm. The evolution of compartmentalization was an

important development in the ability of eukaryotic cells to specialize, forming the organs and

tissues of a complex body.

According to the structure it surrounds, the membranes are:

Plasma membrane= plasmalemma


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Fig. 32. Structure of a Plasma Membrane

Inner membranes: -- nuclear envelope, membranes of smooth and rough ER,

membranes of Golgi apparatus, membranes of mitochondrion (inner and outer), membrane of

lysosome, membrane of peroxisome, membranes of different vesicles.

There are two classes of internal membrane-bound structures in eukaryotic cells. There are

discrete organelles such as mitochondria and peroxisomes; then there is the dynamic

endomembrane system: nuclear membrane, endoplasmic reticulum, Golgi apparatus, lysosomes,and vesicles.

The membranes could also be classified in:

Single Membrane Double Membrane Special Membrane1. plasma membrane 1. nuclear membrane 1. human myelin

2. Golgi body membrane 2. mitochondrial memb. 2. light sensitive

membrane

3. Endoplasmatic reticulum memb. 3. chloroplast membrane

4. Lysosome membrane

5. Vesicle membrane

Plasma membrane

The plasma membrane is a thin membrane that surrounds and defines the boundaries of all

living cells. It consists of a double layer (bilayer) of phospholipids with various proteins attached

to or embedded in it. In many cases, these proteins protrude into the cytoplasm and into the

extracellular environment.

Functions of the plasma membrane:

it allows the cell to maintain a more or less constant internal environment, which is a key

characteristic of life.

it acts as a selectively permeable barrier, preventing some substances from crossing while

permitting other substances to enter and leave the cell.

as the cells boundary with the outside environment, the plasma membrane is important incommunicating with adjacent cells and receiving extracellular signals.

the plasma membrane often has molecules protruding from it that are responsible for binding

and adhering to adjacent cells.

The specific functionsof a membrane depend on the kinds of phospholipids and proteins

present in the plasma membrane.

The main componentsof the plasma membrane are:

Component Function

Cell surface markers "Self'-recognition; tissue recognition

Interior protein network Determines shape of cell

Phospholipid molecules Provide permeability barrier, matrix for proteins

Transmembrane proteins Transport molecules across membrane and against gradient.

The cell membrane, being exposed to the outside environment, is an important site of cell-

cell communication. As such, a large variety of protein receptors and identification proteins,

such as antigens, are present on the surface of the membrane. Functions of membrane proteins

can also include cell-cell contact, surface recognition, cytoskeleton contact, signaling, enzymatic

activity, or transporting substances across the membrane.


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Intercellular connections and communicationPlasma membranes can form different types of "supramembrane" structures such as cell

junctions, desmosomes, hemidesmosomes, etc.

Cell junctions are the specialized connections between the plasma membranes of

adjoining cells. The three general types of cell junctions are tight junctions, anchoring junctions,

and communicating junctions. Tight junctionsbind cells together, forming a barrier that is leak-

proof. For example, tight junctions form the lining of the digestive tract, preventing the contentsof the intestine from entering the body. Anchoring (or adhering) junctions, such as

desmosomes (protein attachments between adjacent cells,bearing a disk shaped structure from

which protein fibers extend into the cytoplasm), link cells together, enabling them to function as

a unit and forming tissues that undergo considerable stress, such as heart muscle or the

epithelium that comprises skin. Communicating (or gap) junctions allow rapid chemical and

electrical communication between cells. They consist of channels that connect the cytoplasm of

adjacent cells.

Cells communicate with each other via small, signaling molecules that are produced by

specific cells and received by target cells. This communication systemoperates on both a local

and long-distance level. The signaling molecules can be proteins, fatty acid derivatives, or gases.

Nitric oxide is an example of a gas that is part of a locally based signaling system and is able to

signal for a human's blood pressure to be lowered. Hormones are long-distance signaling

molecules that must be transported via the circulatory system from their production site to their

target cells.

In order to respond to a signal, a cell needs a receptor molecule that recognizes the signal.

A cell's response to a specific signal varies according to the signal. Some signals are local signals

(e.g., growth factors), while others act as distance signals (e.g., hormones). There are two basic

types of hormones, those that bind to receptors on the cell surface and those whose receptors are

found within cytoplasm. Both types cause the cellular machinery to change its activities.


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IV. COMPARTMENTALIZATION OF EUKARYOTIC CELLIn eukaryotes (which are approximately a thousand times the volume of bacteria) the rates

of chemical reactions would be limited by the diffusion of small molecules if a cell were not

partitioned into smaller compartments termed organelles. Each organelle is surrounded by one

or more biomembranes, and each type of organelle contains a unique complement of proteinssome embedded in its membrane(s), others in its aqueous interior space, or lumen. These

proteins enable each organelle to carry out its characteristic cellular functions.The table below provides a short description of the main organelles of an animal cell

(Table 5, fig. 33).

Table 5. Main Organelles of an Animal Cell.

STRUCTURE DESCRIPTION BIOLOGICAL ROLE

STRUCTURAL ELEMENTS

Cytosketeton Network of protein filaments Structural support; cell movement

Flagella(cilia,

microvilli)

Cellular extensions Motility or moving fluids over

surfaces

Centrioles Hollow microtubules Moving chromosomes during cell

divisionENDOMEMBRANE SYSTEM

Plasma membrane Lipid bilayer in which proteins are

embedded

Regulates what passes into and out of

cell; cell-to-cell communication

Endoplasmic

reticulum

Network of internal membranes;

forms compartments and vesicles

Rough type processes proteins for

secretion and synthesizes

phospholipids; smooth type synthesize

fats and steroids

Nucleus Structure bounded by double

membrane; contains chromosomes

Control center of cell; directs protein

synthesis and cell reproduction

Golgi apparatus Stacks of flattened vesicles Modifies and packages proteins for

export from cell; forms secretory

vesicles

Lysosomes Vesicles derived from Golgi

complex that contain hydrolytic

digestive enzymes

Digest worn-out mitochondria and cell

debris; play role in cell death

ENERGY-PRODUCTING ORGANELLES

Mitochondria Bacteria-like elements with double

membrane

Power plant of the cell; site of

oxidative metabolism; synthesis of

ATP

ORGANELLES OF GENE EXPRESSION

Chromosomes Long threads of DNA that form acomplex with protein

Contain hereditary information

Nucleolus Site of rRNA synthesis Assembles ribosomes

Ribosomes Small, complex assemblies of

protein, often bound to ER

Site of protein synthesis

The cytoplasm is the part of the cell outside the largest organelle, the nucleus. It is

composed of hyaloplasm and cell organelles dispersed in it. The cytosol, the aqueous part of the

hyaloplasm also contains its own distinctive proteins.

Cytoplasm

Hyaloplasm

Cell organellesCytosol Cytoskeleton


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The cytoplasm is a colloidal solution. It is viscous, semifluid and precipitates if placed into

water. It is constantly active (rotation and streaming), allowing the cell to be in a continuous

dynamic flux.

Fig. 33. An Animal Cell

ER - endoplasmic reticulumGenerally, the largest membrane in a eukaryotic cell encloses the endoplasmic reticulum

(ER)an extensive network of closed, flattened membrane-bounded sacs called cisternae. Theendoplasmic reticulum has a number of functions in the cell but is particularly important in the

synthesis of lipids, membrane proteins, and secreted proteins.

The Smooth Endoplasmic Reticulum - it lacks ribosomes and is the place for synthesis of

fatty acids and phospholipids. Although many cells have very little smooth ER, this organelle is

abundant in hepatocytes. Enzymes in the smooth ER of the liver also modify or detoxify

hydrophobic chemicals such as pesticides and carcinogens by chemically converting them into


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Fig. 34. Golgi Apparatus

more water-soluble, conjugated products that can be excreted from the body. High doses of such

compounds result in a large proliferation of the smooth ER in liver cells.

The Rough Endoplasmic Reticulum its cytosolic face is studded with ribosomes.

Ribosomes bound to the rough ER synthesize certain membrane and organelle proteins and

virtually all proteins to be secreted from the cell. A ribosome that fabricates such a protein is

bound to the rough ER by the nascent polypeptide chain of the protein and is attached by

ribophorin (an integral membrane protein). As the growing polypeptide emerges from theribosome, it passes through the rough ER membrane, with the help of translocases - specific

proteins in the membrane. Newly made membrane proteins remain associated with the rough ER

membrane, and proteins to be secreted accumulate in the lumen of the organelle. All eukaryotic

cells contain a noticeable amount of rough ER because it is needed for the synthesis of plasma

membrane proteins and proteins of the extracellular matrix. Rough ER is particularly abundant in

specialized cells that produce an abundance of specific proteins to be secreted. For example,

plasma cells produce antibodies, pancreatic acinar cells synthesize digestive enzymes, and cells

in the pancreatic islets of Langerhans produce the polypeptide hormones insulin and glucagon. In

secretory cells a large part of the cytosol is filled with rough ER and secretory vesicles.

Several minutes after proteins are synthesized in the rough ER, most of them leave the

organelle within small membrane-bounded transport vesicles. These vesicles, which bud fromregions of the rough ER not coated with ribosomes, carry the proteins to another membrane-

limited organelle, the Golgi complex.

Golgi complex (Golgi apparatus)

This organelle is a series of flattened

membrane vesicles or sacs (cisternae),

surrounded by a number of more or less

spherical membrane-limited vesicles. The stack

of Golgi cisternae has three defined regions

the cis, the medial, and the trans. Transportvesicles from the rough ER fuse with the cis

region of the Golgi complex, where they deposit

their protein contents. These proteins then

progress from the cis to the medial and to the

trans region (fig. 34). Within each region are

different enzymes that modify proteins to be

secreted and membrane proteins differently,

depending on their structures and their final

destinations.

After proteins to be secreted and

membrane proteins are modified in the Golgicomplex, they are transported out of the

complex by a second set of vesicles, which

seem to bud from the trans side of the Golgi

complex. Some vesicles carry membrane proteins destined for the plasma membrane or soluble

proteins to be released from the cell surface; others carry soluble or membrane proteins to

lysosomes or other organelles.

So, Golgi complex functions as a factory in which proteins received from the ER are

further processed and sorted for transport to their eventual destinations: lysosomes, the plasma

membrane, or secretion. In addition, glycolipids and sphingomyelin are synthesized within the

Golgi. Proteins, as well as lipids and polysaccharides, are transported from the Golgi apparatus

to their final destinations through the secretory pathway. This involves the sorting of proteins

into different kinds of transport vesicles, which bud from the transGolgi network and deliver


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Fig. 35. Transport from the Golgi apparatus.

Proteins are sorted in the transGolgi network and

transported in vesicles to their final destinations. In

the absence of specific targeting signals, proteins are

carried to the plasma membrane by constitutive

secretion. Alternatively, proteins can be diverted

from the constitutive secretion pathway and targeted

to other destinations, such as lysosomes or regulated

secretion from the cells.

Fig. 36. Content of aL sosome

their contents to the appropriate cellular

locations (Fig. 35). Proteins that function

within the Golgi apparatus must be retained

within that organelle, rather than being

transported along the secretory pathway. In

contrast to the ER, all of the proteins retained

within the Golgi complex are associated withthe Golgi membrane rather than being soluble

proteins within the lumen. The signals

responsible for retention of some proteins

within the Golgi have been localized to their

transmembrane domains, which retain proteins

within the Golgi apparatus by preventing them

from being packaged in the transport vesicles

that leave the transGolgi network.

Functions of Golgi apparatus:

1) biogenesis of membranes, the formation of

the plasma membrane;2) the synthesis of polysaccharides,

glycoproteins, glycolipids and sphingomyelin

(the only nonglycerol phospholipid in cell

membranes);

3) the modification of products through the

addition of fatty acids, sulfation and

glycosylation;

4) the concentration and packaging of

synthesized material into secretory versicles;

5) the biogenesis of lysosomes.

Biogenesis: Large membrane-bounded

organelles, such as the Golgi apparatus and the endoplasmic reticulum, break up into many

smaller fragments during M phase, which ensures their even distribution into daughter cells

during cytokinesis. In such a way these organelles are developed from the preexisting organelles

(ER or GA).

LysosomesLysosomesprovide an excellent example of the ability of

intracellular membranes to form closed compartments in which

the composition of the lumen differs substantially from that of the

surrounding cytosol. Found exclusively in animal cells,lysosomes are responsible for degrading certain components that

have become obsolete for the cell or organism. The process by

which an aged organelle is degraded in a lysosome is called

autophagy (eating oneself). A process in which excessiveamounts of secreted material (ex.: proteins, hormones, etc.) are

fused with lysosomes for hydrolysis is called crinophagy.

Materials taken into a cell by endocytosis or phagocytosis also

may be degraded in lysosomes. In phagocytosis, large, insoluble

particles (e.g., bacteria) are enveloped by the plasma membrane

and internalized.

Lysosomes contain a group of enzymes that degrade

polymers into their monomeric subunits (fig. 36). For example,


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Fig. 37

Fi . 38. Functions of L sosomes

nucleasesdegrade RNA and DNA into nucleotides; proteasesdegrade a variety of proteins and

peptides; phosphatases remove phosphate groups from nucleotides, phospholipids, and other

compounds, etc. All the lysosomal enzymes work most efficiently at acid pH values and

collectively are termed acid hydrol ases.The enzymes are synthesized by ribosomes on the ER

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