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Wednesday, 16 August 2017

Molecular Basis of Inheritance (NOTES)



DEFINITIONS AND TERMS


1.                  Genetic Material        Genetic Material is that substance which controls the inheritance of traits from one generation to next .
2.                  Gene   Gene is a segment of DNA (Deoxyribonucleic Acid), that codes for RNA (Ribonucleic Acid and thereby a polypeptide.The term gene was coined by Johanssen(1909).
3.                  Nucleosome    Nucleosome is a structure formed when the negatively charged DNA is wrapped around the positively charged histone octamer.
4.                  Bacteriophage            A virus that infects a bacterium is called a bacteriophage.
5.                  Replication  Self duplicating property of DNA is called Replication.
6.                  Replication Fork       The Y-shaped structure formed when the double-stranded DNA is unwound upto a point during its replication, is called replication fork.
7.                  Transformation         Transformation is the phenomenon by which the DNA isolated from one type of cell, when introduced into another type, is able to bestow some of the properties of the former to the latter.
8.                  Transcription Transcription is the process of formation of RNA from DNA.
9.                  Translation    Translation is the process of polymerization of amino acids to form a polypeptide dictated by the messenger RNA.Simply it is the biosynthesis of proteins.
10.              Operon           All the genes controlling a metabolic pathway, constitute an operon.
11.              Constitutive Genes    Constitutive genes are those genes which are constantly expressed and whose products are continuously needed for the cellular activity.
12.              Exons  Exons are the regions of a gene, which become part of mRNA and code for the different regions of proteins.
13.              Introns            Introns are the regions of a gene, which do not form part of mRNA and are removed during the processing of mRNA.
14.              Splicing           It is a process in eukaryotic genes, whereby the introns are removed and the exons are joined together to form mRNA.
15.              Codon             Codons a sequence of three nitrogenous bases on mRNA, that codes for a particular amino acid.
16.              Anticodon      Anticodon is a sequence of three nitrogenous bases on tRNA, that is complementary to the codon for the particular amino acid.
17.              Split genes      The genes carrying genetic information in stretches rather than continuously, are called split genes. Eukaryotic genes having coding and non-coding sequences together are called split genes.
18.              Satellite DNA Satellite DNA refers to the repetitive DNA sequences which do not code for any proteins, but form a large portion of human genome; they show high degree of polymorphism.
19.              DNA polymorphism  DNA polymorphism refers to the variation at genetic level, where an inheritable mutation is observed in a population in a frequency  greater than 0.01
20.              Operon system           An operon is a part of genetic material which act as a single single regulated unit having one or more structural genes, an operator gene, a promoter gene and a regulator gene.
21.              Father of DNA fingerprinting         Alec Jaffereys
22.              Father of Indian Dna fingerprinting           Lalji Singh
23.              Actinomycin D           inhibits transcription
24.              Puromycin     inhibits translation
25.              Largest Genome        A very slow growing herb Paris japonica has largest genome (about 50 times the human genome)
26.              Cistron           The term cistron was proposed for the unit of function. Presently a cistron is equivalent to a gene. It is that amount of DNA which can code for one functional polypeptide chain. Both cistron and gene refer to the same entity.
27.              Nucleosides    Nitrogenous base +sugar
28.              Nucleotides     Nitrogenous base +sugar+phosphate
29.              Pribnow box  A sequence of six nucleotides TATATT in prokaryotic promoters.
30.              TATA box     A DNA sequence in promoter region of eukaryotic genes analogous to the prinbox of prokaryotes.








IMPORTANT CONCEPTS

1.                  Deoxyribonucleic Acid (DNA)
·         DNA constitute the genetic material of all the organisms with the exception of riboviruses.
·         DNA is a long polymer of deoxyribonucleotides, whose length is defined as the number of nuleotides or base pairs (bp).
·         The number of base pairs is characteristic of every organism/species, e.g.,
Φ 174 phage                -           5386 bp
Lambda phage                        -           48502 bp
Escherichia coli           -           4.6  106 bp
Human beings             -           3.3  109 bp (haploid number)
·         DNA was discovered by Frederich Meischer (1869) as an acidic substance in the nucleus; he called it  ‘nuclein’.
(A)             Structure of a Polynucleotide Chain of DNA.
-          Each nucleotide has three components,
---- a nitrogenous base,
---- a deoxyribose (pentose) sugar
---- a phosphate group
-          Nitrogenous bases are of two types:
ð  Purines (adenine and guanine) and
ð  Pyrimidines (cytosine and thymine).
-          A nitrogenous base is linked to the pentose deoxyribose sugar through a N-glycosidic linkage to form a nucleoside.
-          When a phosphate group is attached to 5’-OH of a nucleoside through phospho-ester linkage, a corresponding nucleotide is formed.
-          Two nucleotides are linked through 3’-5’ phosphodiester linkage to form a dinucleotide and when many nucleotides are linked in this manner, a polynucleotide is formed
-          The polylnucleotide chain has at the 5’-end of the sugar a free phosphate moiety (it is called 5’-end) and at the 3’-end a OH group (it is called 3’ end).
-          The backbone of the polynucleotide is formed by the sugar and phosphates, while the nitrogen base project from the backbone.
A Polynucleotide Chain

(B)              Double Helix – Model of DNA
-          Watson and Francis Crick (1953) proposed a double-helix model of DNA, based on the X-ray diffraction data, produced by Maurice Wilkins and Rosalind Franklin.
-          One of the important features of this model is the complementary base-pairing.
-          Later Erwin Chargaff observed that in a double-stranded DNA, the ratios between adenine and thymine and that between guanine and cytosine are constant, i.e., A : T = 1 and G :C = 1
-          The salient features of the double helical model are given below:
(I)        DNA is made of two polynucleotide chains, where the backbone is constituted by sugar-phosphate and the nitrogen bases project inside.
(II)       The base in the two strands are held together by hydrogen bonds forming base pairs, Adenine pairs with thymine through two hydrogen bonds and guanine with cytosine through three hydrogen bonds.
(III)     The two chains have an anti-parallel polarity, i.e., one  chain has the polarity 5’à  3’, the other has 3’ à 5’polarity.
(IV)     The two chains are coiled in a right-handed fashion and the pitch of the helix is 3.4 nm; there are about 10 base pairs in each turn with 0.34 nm between two base pairs.
(V)       The plane of one base pair stacks over the other in the double helix. This, in addition to H-bonds,confers the stability of the helical structure.The length of DNA in E. coli is approximately 1.36 mm, while that of humans 2.2 m.
                      
Double stranded polynucleotide chain                        
DNA double helix
CENTRAL DOGMA OF MOLECULAR BIOLOGY
Francis crick proposed the central dogma of molecular biology, which states that the genetic information flows from DNAà RNAà Protein.
                                 
In some viruses the flow of information is in reverse direction ,that is, from RNA to DNA, This is called reverse transcription or teminism. It was discovered by TEMIN AND BALTIMORE in 1970
(C)       Packaging of DNA
¾    In prokaryotes, with no well-defined nucleus, the DNA is organised in large loops held by certain positively charged proteins, in a region called nucleoid.
¾    In eukaryotes, the DNA is wrapped around positively charged histone octamere into a structure called nucleosome.
¾    A typicl nucleosome  consists of 200 bp of DNA helix
¾    The nucleosomes are the repeating unit that form chromatin fibres.
¾    The chromatin fibres condense at metaphase stage of cell division to form chromosomes.
¾    The packaging of chromatin at higher level requires additional set of proteins called non-histone chromosomal (NHC) proteins.
¾    In a nucleus, certain regions of the chromatin are loosely packed and they stain lighter than the other regions; these are called euchromatin.
¾    The other regions are tightly packed and they stain darker and are called heterochromatin.
¾    Euchromatin is transcriptionally more active than heterochromatin.

                                 Nucleosome                                           EM picture-Beads on string



2.         Transformation and the Transforming Principle.
·         Frederick Griffith (1928) conducted as series of experiments with Streptococcus pneumoniae, (Pneumococcus), the bacterium causing pneumonia.
·         He observed two strains of this bacterium; one forming smooth colonies with capsule (S-type) and the other forming rough colonies without capsule (R-type).
·         When live S-type cells were injected into the mice, they produced pneumonia (pathogenic/virulent) and the mice died.
·         When heat-killed S-type cells were injected into the mice, the disease did not appear.
·         When heat-killed S-strain cells were mixed with live R-strain cells and injected into the mice, the mice died and he could isolate live S-strain cells from the body of the mice.
·         He concluded that the R-strain bacteria had somehow been transformed by the heat-killed S-strain bacteria, which must be due to the transfer of the genetic material (transforming principle).
·         They also discovered that proteases and RNase did not affect transformation, while DNase inhibited the process; this proves that DNA is the transforming principle.
3.         DNA is the Genetic Material
·         The proof for DNA as the genetic material came from the experiments of Alfred Hershey and Martha Chase (1952), who worked with bacteriophages.
·         The bacteriophage on infection injects only the DNA into the bacterial cell and not the protein coat; the bacterial cell treats the viral DNA as its own and subsequently manufactures more virus particles.
·         They made two different preparations of the phage; in one, the DNA was made radioactive with 32p and in the other, the protein coat was made radioactive with 35S.
·         These two phage preparations were gently agitated in a blender to separate the adhering protein coat of the virus from the bacterial cells.
·         The culture was also centrifuged to separate the viral coat and the bacterial cells.
·         It was found that when the phage containing radioactive DNA was used to infect the bacteria, its radioactivity was found in the bacterial cells (in the sediment) indicating that the DNA has been injected into the bacterial cell.
·         So, DNA is the genetic material and not proteins.

The Hershey-Chase experiment

4.         Characteristics of Genetic Material.
·         A molecule that can act as genetic material must have the following properties:
(I)                It should be able to generate its replica.
(II)             It should be chemically and structurally stable.
(III)          It should provide the scope for slow changes (mutation) that are necessary for evolution.
(IV)           It should be able to express itself in the form of Mendelian characters.
·         Nucleic acids ie. DNA and RNA can replicate, but not protein.
·         The predominant genetic material is DNA, while few viruses like tobacco mosaic virus have RNA as the genetic material.
·         The 2’-OH group in the nucleotides of RNA is a reactive group and makes RNA labile and easily degradable; RNA as a catalyst is also more reactive and hence DNA has the property to be the genetic material.
5.         RNA-World
·         Ribonucleic acid (RNA) was the first genetic material.
·         The 2’-OH group of ribonucleotides is a reactive group that makes RNA a catalyst.
·         It is evident that essential life processes such as metabolism, translation, splicing etc, have evolved around RNA, even before DNA has evolved as a genetic material.
·         Structure of a Polynucleotide Chain (RNA)
¾    In RNA also, each nucleotide has three components as in DNA
¾    The nitrogen bases are of two types:
Þ    Purines – (Adenine and Guanine)
Þ    Pyrimidines – (Cytosine and Uracil).
·         There are three types of RNA:
(A)             Messenger RNA (mRNA)
¾    About 3-5% of total RNA of the cell is m-RNA.
¾    It brings the genetic information of DNA transcribed on it for protein synthesis.
¾    It is single-stranded.
(B)       Transfer/Soluble RNA (tRNA/sRNA)
¾    It makes about 15% of the total RNA of the cell and having on average 80 nucleotides per molecule. It is the smallest of all the RNAs.
¾    It acts as an adapter molecule that reads the code on one hand and binds to the specific amino acid on the other hand.
¾    tRNA has a clover leaf like secondary structure but actually it is an inverted L-shaped  compact molecule.
¾    It has an ‘amino acid acceptor end’ (3’-end) and an ‘anticodon-loop’, where the three bases are complementary to the bases of the codon of the particular amino acid.
¾     
(C)       Ribosomal RNA (rRNA)
¾    Ribosomal RNA was tha first RNA to be identified, and it constitutes approximately 80% of the total RNA of the cell.
¾    It forms the structure of ribosomes.
¾    It also plays a catalytic role during translation.                                 
6.         Replication of DNA
·         Watson and Crick had proposed a scheme for replication of DNA, when they proposed the double helical structure for DNA.
·         The scheme suggested that the two strands would separate and each acts as a template for the synthesis of a new complementary strand.
·         After complete replication, each DNA molecule would have one parental and one newly synthesized strand.
                                 
                          Watson-Crick model for semiconservative DNA replication
(A)       Proof for Semiconservative Replication of DNA
¾    Mathew Meselson and Franklin Stahl have performed an experiment using Escherichia coli to prove that DNA replication is semiconservative.
¾    They grew E.coli in a medium containing15NH4Cl unitil 15N was incorporated in the two strands of newly synthesised DNA; this heavy DNA can be separated from the normal (14N) DNA by centrifugation in Cesium Chloride (CsCl) density gradient.
¾    Then they transferred the cells into a medium with normal 14NH4Cl and took out samples at various time intervals and extracted DNAs and centrifuged them to measure their densities.
¾    The DNA extracted from the cells after one generation of transfer from the 15N medium to 14N medium (i.e., after 20 minutes) had an intermediate/hybrid density.
¾    Similar experiments were conducted by Taylor et al in 1958, by using radioactive ethymidine; they proved that DNA on the chromosomes replicates in a semiconservative manner.
                                                      Messelson and Stahl’s Experiment
(B)       The Process of Replication of DNA
¾    The process involves a number of enzymes/catalysts, of which the main enzyme is DNA-dependent DNA-polymerase, that catalyses of the polymerization of the deoxynucleotides at a rate of approximately 200 bp per second.
¾    The process is also an energy-expensive process; deoxyribonucleoside triphosphates serve the dual purpose of (I) acting as substrate and (II) providing energy (from the two terminal phosphates).
¾    The intertwined strands of DNA separate from a particular point called origin of replication.
¾    Since the two strands cannot be separated in its entire length, replication occurs with small opening of the DNA- helix; the Y-shaped structure formed, is called replication fork.
¾    The DNA-dependent DNA-polymerases catalyse polymerisation of the nucleotides only in 5’-3’ direction.
¾    Consequently, one of the template strands (with 3’-5’polarity) the synthesis of DNA is continuous, while on the other template strand (with polarity 5’-3’), the synthesis of DNA is discontinuous, i.e., short stretches of DNA are synthesised.
¾    The discontinuously synthesisted strands are later joined together by the enzyme DNA-ligase.
                                 

Replicating Fork
7.         Transcription
·         Transcription is also governed by the complementarity of bases as in DNA. (Except that uracil in place of thymine is complementary to ademine).
·         Only one of the strands of the DNA acts as the template for RNA synthesis for the following reasons:
(I)        If both the strands code for RNA, two different (complementary) RNA molecules and two different proteins would be formed; hence the genetic information-transfer machinery would become complicated.
(II)       Since the two RNA molecules produced would be complementary to each other, they would wind together to form a double-stranded RNA without carrying out translation; that means the process of transcription would become futile.
·         A transcription unit in DNA has three regions:
(I)        A Promoter     (II)       Structural and            (III)     A terminator.
Schematic structure of transcriptional unit

·         The process is catalysed by DNA-dependent RNA-polymerase, which catalyses the polymerisation of nucleotides only in 5’-3’ direction.
·         The DNA strand with 3’-5’ polarity is called ‘template strand’, while the other strand with 5’-3’ polarity is called ‘coding strand’.
·         The coding strand is displaced and does not code for RNA, but reference points regarding transcription are made in relation to it.
·         The promoter refers to a particular sequence of DNA located towards the 5’ end (upstream) of the coding strand, where the RNA polymerase becomes bound for transcription.
·         The terminator is a sequence of DNA located towards the 3’ end (downstream) of the coding strand, where the process of transcription would stop.
·         There are additional regulatory sequences that may be present upstream or downstream to the promoter.
(A)       Transcription in prokaryotes
¾    In prokaryotes, the structural genes are polycistronic and continuous.
¾    In prokaryotes, there is a single DNA-dependent RNA polymerase, that catalyses the transcription of all the three types of RNA (mRNA, tRNA, rRNA).
¾    RNA polymerase binds to the promoter and initiates the process along with certain initiation factors () (sigma).
¾    It uses ribonucleoside triphosphates (also called ribonucleotides) for polymerisation on a DNA template following complementarity of bases.
¾    The enzyme facilitates the opening of the DNA-helix and elongation continues.
¾    Once the RNA polymerase reaches the terminator, the nascent RNA falls off and the RNA polymerase also separates; it is called termination of transcription and is facilitated by certain termination factors () (Rho).
¾    In prokaryotes, the mRNA synthesised does not required any processing to become active and both transcription and translation occur in the same cytosol; translation can start much before the mRNA is fully transcribed, i.e., transcription and translation can be  coupled.
                                                Process of transcription in Bacteria

(B)       Transcription in Eukaryotes.
¾    In eukaryotes, the structural genes are monocistronic and ‘split’.
¾    They have coding sequences called exons that form part of mRNA and non-coding sequences, called introns, that do not form part of the mRNA and are removed during splicing.
¾    In eukaryotes, there are atleast three different RNA polymerases in the nucleus, apart from the RNA polymerase in the organelles, which function as follows:
                                             Process of transcription in Eukaryotes


Þ    RNA polymerase I transcribes rRNAs (26S, 18S and 5.8S),
Þ    RNA polymerase II transcribes the precursor of mRNA (called as heterogenous nuclear RNA (hnRNA) and
Þ    RNA-polymerase III catalyses transcription of tRNA.
¾    The primary transcript contains both exons and introns and it is subjected to a process, called splicing, where the introns are removed and the exons are joined in a definite order to form mRNA.
¾    The hnRNA undergoes two additional processes called ‘capping’ and ‘tailing’.
¾    In capping, methyl guanosine triphosphate is added to the 5’ end of hnRNA.
¾    In tailing, adenylate residues (about 200-300) are added at the 3’-end of hnRNA.
¾    The fully processed hnRNA is called mRNA and is released from the nucleus into the cytoplasm.




8.         Genetic Code
·         Genetic code refers to the relationship between the sequence of nucleotides on mRNA and the sequence of amino acids in the polypeptide.
·         It was George Gamow, who suggested that the code must be made up of three bases, in order to code for the twenty different amino acids; with only four bases (A, T, G, C), there would be (43) or              (4  4  4) = 64 triplet condons.
·         Har Gobind Khorana Could synthesise RNA molecules with definite combinations of bases. (homopolymers and copolymers).
·         Marshal Nirenberg made a cell-free system for protein synthesis, that helped in deciphering the genetic code.
·         Severo Ochoa discovered enzyme polynucleotide phosphorylase, that could polymerise RNA with a definite sequence in template-independent manner.
·         The checkerboard pattern of genetic code was prepared as given below:
                                              
                                                     The Codons for various amino acids

·         This salient features of genetic code are given below:
(I)        The codons are triplets and there are sixty-four codons; sixty-one codons code for twenty amino acids and three codons (UAA, UAG, UGA) do not code for any amino acid, but function as stop/termination codons. UAA, UAG and UGA are also known as ochre, amber and opal.
(II)       Each codon codes for only one/particular amino acid and so the genetic code ‘unambiguous’ and ‘specific’.
(III)     Since some amino acids are coded by more than one codon, the genetic code is said to be ‘degenerate’.
(IV)     The codons are read in a contiguous manner in the 5’-3’ direction and have no punctuations (commaless).
(V)       The genetic code is universal, i.e., the codons code for the same amino acid in any organism, be it a bacterium or a human being.
(VI)     AUG has dual functions of coding for methionine and acting as initiation codon.
(VII)    Wobble hypothesis : It is believed that third base of a codon is not very important and that the specificity of a codon is particularly determined by first two bases. Same tRNA can recognize more than one codons differing only at third position.
9.         Mutations
(A)       Point Mutation
¾    This type of mutation involves a change in single base pair.
¾    An example of point mutation is a change of single base pair in the gene for beta globin chain (of haemoglobin) that results in substitution of glutamate by valine; it causes a disease called sickle-cell anaemia.
(B)       Frameshift mutation
¾    It is the type of mutation where addition/insertion or deletion of one or two bases changes the reading frame from the site of mutation, resulting in a protein with a different set of amino acids.
¾    This also forms the basis of proof that codon is a triplet and codons are read in a contiguous manner.
¾    Insertion or deletion of three or its multiples of bases does not alter the reading frame, but one/more amino acids are added or deleted in the protein translated.
(C)       Silent mutation  If a base change in a codon does not alter the amino acid coded, the    mutation is said to be silent mutation.
10.       Translation
·         In this process, amino acids become joined together by peptide bonds, to form polypeptides.
·         The formation of peptide bonds requires energy and hence in the first phase, the amino acids are activated.
(A)       Activation of amino acids
¾    In this process, a particular amino acid becomes activated and attached to the 3’ end of a specific tRNA molecule.
¾    The reaction is catalysed by the enzyme amino –acyl-tRNA synthetase.
Amino acid (AA) + ATP    Enzyme            AA – AmP – ENZ + PPi      
AA-AMP – ENZ + tRNA                  AA –tRNA + AMP + PPi      
(B)       Initiation of polypeptide synthesis
¾    The ribosome, in its inactive state exits as two subunits – a large subunit and a small subunit.
¾    When the small subunit encounters the mRNA, translation begins.
¾    The mRNA binds to the small subunit of ribosome, following base pair rule between the bases of mRNA and those on rRNA; it is catalysed by ‘initiation factors’.
¾    There are two sites on the larger subunit, the P-site and the A-site
¾    The small subunit (with the mRNA) attaches to the large subunit in such a way that the initiation codon (AUG) comes on the P-site.
¾    The anticodon UAC of initiator tRNA (methionyl tRNA) carrying amino acid methionine(AUG) undergoes pairing with the initiation codon AUG of mRNA through hydrogen bonds at P site .

                                               tRNA- the adapter molecule
(C)       Elongation of polypeptide chain
¾    A second tRNA charged with an appropriate amino acid binds to the A-site of the ribosome.

                                                  Translation
¾    A peptide (CO-NH) bond is formed between the carboxyl group of methionine and the amino group of the second amino acid; this reaction is catalysed by the enzyme peptidyl transferase.
¾    The ribosome moves from codon to codon along the RNA in the 5’-3’ direction.
¾    Amino acids are added one by one in the sequence of the codons and become joined together to form a polypeptide.
(D)       Regulation of Gene Expression
¾    When one of the termination codons comes at the A-site, it does not code for any amino acid and there is no tRNA molecule for it.
¾    As a result, the polypeptide synthesis (or elongation of polypeptide) stops.
¾    The polypeptide synthesised is released from the ribosome, catalysed by a ‘release factor’.
11.       Regulation of polypeptide synthesis
·         In prokaryotes, the gene expression is controlled by the initiation of transcription.
·         In eukaryotes, the regulation can be exerted at four levels:
(I)        Transcriptional level (formation of primary transcript)
(II)       Processing level (splicing)
(III)     Transport of mRNA from nucleus to cytoplasm and
(IV)     Translational level
·         The regulation at transcriptional level was elucidated by Jacob and Monod.
·         All the genes controlling one metabolic pathway constitute an operon.
·         A few examples are lac-operon, try-operon, val-operon, his-operon, etc. in E.coli.
·         An operon consists of the following components:
(A)       Structural Gene (s)
¾    They transcribe the mRNA for the amino acid sequence of proteins (enzymes).
(B)       Promoter Gene
¾    The promoter gene is a sequence of DNA, where the RNA polymerase binds and initiates transcription
(C)       Operator
¾    The operator is a sequence of bases on DNA adjacent to the promoter.
¾    The accessibility of promoter gene for RNA polymerase is regulated by proteins, called repressor.
¾    In most cases a specific repressor protein binds to the operator.
(D)       Regulator Gene
¾    This codes for the repressor protein that binds to the operator and ‘switches off the transcription unit.
¾    So the regulatory gene is also represented as ‘i’ gene (inhibitory gene).
(E)       Inducer
¾    The substance/substrate that prevents the repressor from binding to the operator, is called an inducer; it keeps the switch ‘on’ and transcription continues.
12.       Lac-operon in Escherichia coli
¾    It is an inducible operon, where Lactose is the inducer; it is the substrate for the enzyme -galactosidase.
¾    The components of lac-operon and their functions are as follows:
(A)       Structural Genes
¾    There are three structural genes (z, y, a) which transcribe a polycistronic mRNA.
¾    Gene z codes for enzyme -galactosidase (b-gal), that catalyses the hydrolysis of lactose into galactose and glucose.
¾    Gene y codes for permease, which increases the permeability of the cell to enzyme -galactosidase (lactose).
¾    Gene a codes for transacetylase, that catalyses the transacetylation of lactose into its active form.
(B)       Promoter
¾    It is a sequence of bases near to the structural genes; it is the site where RNA-polymerase binds for transcription.
(C)       Operator
¾    It is a sequence of base of DNA near the promoter, where a repressor always binds.
¾    It functions as a switch for the operon.
(D)       Repressor
¾    It is a protein coded by i gene, sythesised all the time constitutively.
¾    It binds to the operator and prevents the RNA polymerase from transcribing.
¾    Lactose is the inducer that inactivates the repressor and prevents it from binding to the operator.
¾    This allows an access for the RNA polymerase to the promoter and transcription continues.
                                                             The lac Operon



13.       Human Genome Project (HGP)
·         Human Genome Project was a 13-year project, that was launched in the year 1990 and completed in 2003.
·         This project was coordinated by the U.S. Department of Energy and the National Institute of Health.
·         During the early years of the project, the Wellcome Trust (U.K) became a major partner; other countries like Japan, Germany, China and France contributed significantly.
·         Its aim was to find out the complete DNA sequences for the human genome.
·         The two factors that made this possible are:
(I)        Genetic engineering techniques, with which it was possible to isolate and clone any segment of DNA
(II)       Availability of simple and fast techniques, for determining the DNA sequences.
·         Human Genome Project was called a mega project for the following facts:
(I)        The human genome has approximately 3.3  109 bp; if the cost of sequencing is US $3 per bp, the approximate cost is about US $9 billions.
(II)       If the sequence obtained were to be stored in typed form in books and if each page contained 1000 letters and each book contained 1000 pages, then 3300 such books would be needed to store the complete information.
(III)     The enormous quantity of data expected to be generated also necessitates the use of high speed computational devices for data storage, retrieval and analysis.
·         The project was closely associated with a new branch of biology, called bioinformatics.
                             
                            A representative diagram of human genome project
(A)       Goals of HGP
·         Some major/important goals of HGP are to:
(I)        Identity all the genes (approximately 20000-25000) in human DNA.
(II)       Determine the sequences of the three billion base pairs present in human DNA.
(III)     Store this information in data bases.
(IV)     Improve the tools for data analysis.
(V)       Transfer the technologies to other sectors (like industries).
(VI)     Address the ethical, legal and social issues (ELSI), that may arise from this project.
(B)       Advantages/Uses of HGP
(I)        Knowledge of the effects of variations of DNA among individuals can revolutionise the ways to diagnose, treat and even prevent a number of diseases/disorders that affect human beings.
(II)       It provides clues to the understanding of human biology.
(C)       Methodologies of HGP
¾    The methods involved two major approaches:
(I)        One approach, called Expressed Sequence Tags (ESTs), focused on identifying all the genes that expressed as RNA.
(II)       Second approach, called Sequence Annotation, was to simply sequence the whole set of genome, that included all the coding and non-coding sequence and later assigning functions to different regions in the sequence.
¾    The total DNA from the cells is isolated and converted into random fragments of relatively smaller used hosts are bacteria and yeast and the vectors are bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
¾    The fragments are then sequenced using automated DNA sequences, which work on the principle developed by Frederick Sanger.
¾    The sequences were them arranged on the basis of certain overlapping regions present in them; this required the generation of overlapping fragments for sequencing.
¾    Specialised computer based programmes were developed for alignment of the sequences.
¾    These sequences were annotated and assigned to the respective chromosomes.
¾    The next task was to assign the genetic and physical maps on the genome; this was generated using the information on polymorphism of restriction endonuclease recognition sites and certain repetitive DNA sequences, called microsatellites.
(D)       Salient features of Human Genome
¾    Following are some of the salient observations derived from HGP.
(I)        The human genome contains 3164.7 million nucleotides (base pairs).
(II)       The size of the genes varies; an average gene consists of 3000 bases, while the largest gene, dystrophin consists of 2.4 million bases.
(III)     The total number of genes is estimated as 30000 and 99.9% of the nucleotides are the same in all humans.
(IV)     The functions of over 50% of the discovered genes are not known.
(V)       Only less than 2% of the genome codes for proteins.
(VI)     Repetitive sequences make up a large portion of the human genome.
(VII)    Repetitive sequences throw light on chromosome structure and dynamics and evolution, thought they are thought to have no direct coding functions.
(VIII)  Chromosome 1 has 2968 genes (the maximum) and the Y-chromosome has 231 genes (the least).
(IX)     Scientists have identified about 1.4 million locations, where DNA differs in single base in human beings; these are called single nucleotide polymorphisms (SNPs).
(E)       Applications/Future challenges of HGP
(I)        Having the complete sequence of human genome, will enable a radically new approach to biological research, i.e., a systematic approach on a much broader scale.
(II)       All the genes in a genome or all the transcripts in a particular tissue/organ/tumor can be studied.
(III)     It will be possible to understand how the enormous number of genes and projects work together in interconnected networks in the chemistry of life.
14.       DNA-Fingerprinting
·         The technique of DNA-fingerprinting was developed by Dr. Alec Jaffreys, in an attempt to find out markers for inherited disease; the process is also known as DNA-typing or DNA-profiling.
·         DNA-fingerprinting involves identifying differences in some specific short nucleotide repeats, called Variable Number Tandem Repeats (VNTRs), that vary in number from person to person and are inherited.
·         The VNTRs of two persons may be of same length and sequence at certain sites, but very at others.
·         The procedure of DNA-fingerprinting includes the following major steps:
(I)        Extraction:-    DNA is extracted from the cells in a high-speed, refrigerated centrifuge.
(II)       Amplification:-          Many copies of the extracted DNA are made by polymerase chain reaction (PCR).
(III)     Restriction Digestion:-          DNA is cut into fragments with restriction enzymes into precise reproducible sequences.
(IV)     Separation of DNA sequences/restriction fragments:-      The cut DNA fragments are introduced and passed through electrophoresis set-up containing agarose polymer gel; the separated fragments can be visualized by straining them with a dry that show fluorescence under ultraviolet radiation.
(V)       Southern Blotting:-   The separated DNA sequences are transferred onto a nitrocellulose or nylon membrane or sheet placed over the gel.
(VI)     Hybridisation:-          The nylon membrane is immersed in a bath and radioactive probes (DNA segments of known sequence) are added; these probes target a specific nucleotide sequence that is complementary to them.
(VII)    Autoradiography:-    The nylon membrane is pressed on to an x-ray film and dark bands develop at the probe sites which resemble the bar-codes.
·         DNA-fingerprinting technique is use for the following purposes:
(I)        To identify criminals in the forensic laboratories.
(II)       To determine the real parents, i.e., to identify the true biological father or mother in cause of disputes.
(III)     To verify whether an immigrant is really a close relative (as he/she claims) of mentioned resident.
(IV)     To identify racial groups to rewrite the biological evolution.

Schematic representation of DNA fingrerprinting. It is clear that the banding pattern of DNA from crime scene matches with individual B, and not with A.

·         GENE BANK : With a view  towards rapidly proceeding genetic erosion, it is important to conserve germplasm because it is difficult to say which gene may prove to be lifesaving in future. Also, plant breeders require a variety of genes for developing new varieties. To serve these purposes, the plant species are searched for variations and are stored safely in a gene bank, in the form of seeds, other propagules, or even tissues.
GENE BANKS         are included in ex-situ conservation strategies and aim at static conservation, that is to retain, as far as possible, the genetic structure of theoriginal population in the same pattern. In this type, conservation is carried out by reducing the life processes to a low level and is the safest and cheapest method for conserving genetic information.

Multiple Choice Questions
1.         Formation of RNA from DNA is known as
(A)       Transcription                                       (B)       Translation
(C)       Replication                                          (D)       Recombination
2.         Transcription is catalyzed by
(A)       RNA polymerase                                (B)       DNA polymerase
(C)       Endonucleases                                                (D)       None of these
3.         During RNA transcription, unwinding of DNA is performed by
(A)       Topoisomerase                                                (B)       Helicase
(C)       RNA polymerase                                            (D)       Ligase
4.         mRNA is synthesized on DNA template in which direction
(A)       5’-3’                                                    (B)       3’-5
(C)       Bothe A and B                                                (D)       None of these
5.         Which of the following reunites the exon segments after RNA splicing?
(A)       RNA polymerase                                (B)       RNA primase
(C)       RNA ligase                                         (D)       RNA proteases
6.         If the sequence of bases in DNA is ATTCGATG, then the sequence of bases in its transcript will be
(A)       CAUCGAAU                                     (B)       GAUGCUUA
(C)       UAAGCUAC                                     (D)       AUUCGAUG
7.         In transcription the terminating factor is
(A)        factor                                               (B)       p factor
(C)        factor                                                          (D)       TATA box
8.         During transcription, the DNA site at which RNA polymerase bind is called
(A)       Promoter                                             (B)       Regulator
(C)       Receptor                                              (D)       Enhancer
9.         Segments of mRNA removed during splicing are
(A)       Introns                                                 (B)       Exons
(C)       Promotor regions                                 (D)       Integrator regions
10.       Removal of introns and joining the exons in a defined order in a transcription unit is called
(A)       tailing                                                  (B)       transcription
(C)       capping                                                (D)       splicing
11.       The DNA strand that is transcribed to mRNA is
(A)       antisense strand                                   (B)       sense strand
(C)       both sense and antisense strand          (D)       parts of the sense and antisense strand
12.       Transcription in case of eukaryotes takes place in
(A)       nucleus                                                (B)       nucleolus
(C)       cytoplasm                                            (D)       ribosome
13.       Which of the following type of RNA molecule is essential for protein synthesis?
(A)       mRNA                                                (B)       tRNA
(C)       rRNA                                                  (D)       All of these
14.       Central dogma is directly connected with synthesis of
(A)       mRNA                                                            (B)       poltpeptide
(C)       both A and B                                      (D)       lipids
15.       The anticodon of tRNA binds with
(A)       nucleic bases of mRNA                      (B)       nucleic bases of rRNA
(C)       codons  of  tRNA                               (D)       amino acids
16.       The function of tRNA is
(A)       selection of amino acids                      (B)       production of mRNA
(C)       production of ribosomes                     (D)       production of microsomes
17.       Polysome is formed by
(A)       a ribosome with several subunits       
(B)       ribosomes attached to each other in a linear arrangement
(C)       several ribosomes attached to a single mRNA
(D)       many ribosomes attached to a strand of endoplasmic reticulum
18.       During protein synthesis AUG function as a initiator codon in mRNA. What should be the anticodon on the tRNA molecule that picks up and brings the amino acids specified by this codon?
(A)       UAC                                                   (B)       TAC
(C)       CAU                                                   (D)       GUA
19.       mRNA directs the building of proteins through a sequence of
(A)       exons                                                   (B)       introns
(C)       codons                                                 (D)       anticodons
20.       Degeneration of a genetic code is attributed to the
(A)       first member of a codon                      (B)       second member of a codon
(C)       entire codon                                        (D)       third member of codon










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