6.MOLECULAR BASIS OF INHERITANCE

DEOXYRIBONUCLEIC ACID (DNA)

• DNA is the long polymer of deoxyribonucleotide.• Its length is defined as the number of nucleotide or base pairs.• The number of base pairs is the characteristics of every organisms. • Ex.Ф 174 ----5386 bp• Lambda phage----48502 bp 6• Escherichia coli ----4.6X1O bp 9• Human being------------3.3x 10 bp(haploid cell)• DNA was discovered by FREDERICH MEISCHER(1869) as an acidic

substance in the nucleus , he called it nuclein.

FREDERICH MEISCHER.

Structure of polynucleotide chain of DNA.• DNA is the largest macromolecule made of helically

twisted, two, antiparallel polydeoxyribonucleotide chains held together by hydrogen bonds.

• X-ray diffraction pattern of DNA by Rosalind Franklin showed DNA a helix.

• Components of DNA are (i) deoxyribose sugar, (ii) a phosphate, and (iii) nitrogen containing organic bases.

• DNA contains four different bases called adenine (A), guanine (G) cytosine (C), and thymine (T).

• These are grouped into two classes on the basis of their chemical structure: (i) Purines (with a double ring structure) and (ii) Pyrimidines (with a single ring structure)

• 1953.James Watson and Francis Crick proposed three dimensional structure of DNA

• DNA double helix with sugar phosphate back bone on outside and paired bases inside.

• Planes of the bases perpendicular to helix axis.• Each turn has ten base pairs.( 34 A0)(3.4nm)• Diameter of helix 20 A0

• Ten base pairs in each turn with 0.34nm between two base pairs.

• The length of DNA in E.coli-----------1.36 mm• The length of DNA in Man -----------2.2m

• Two strands of DNA antiparallel.• DNA found both in nucleus and cytoplasm.• Extranuclear DNA found in mitochondria and chloroplasts.• Two chains complementary• Two chains held together by hydrogen bond.• Adenine-Thymine pair has two hydrogen bonds.• Guanine-Cytosine pair has three hydrogen bonds.• Upon heating at temperature above 80-90 degree two

strands uncoil and separate (Denaturation)• On cooling two strands join together (renaturation

/annealing)• DNA is mostly right handed and B form.• Bacterial nucleoid consists of a single circular DNA molecule .•

PACKAGING OF DNA HELIX• DNA of eukaryotes is wrapped around positively

charged histone proteins to form nucleosome.• # Nucleosome contains 200 base pairs of DNA helix.• # Histone octamer =2(H2a+H2b+H3+H4)• # Linker DNA bears H1 protein• # Chromatin fibres formed by repeated units of

nucleosomes.• # Non histone proteins required for packaging.• # Regions of chromatin, loosely packed and stains

lightly called euchromatin.• # Regions of chromatin, densely packed and stains

darkly is called heterochromatin.

DNA AS THE GENETIC MATERIAL• Transformation experiment or Griffith effect.• • Griffith performed his experiments on Mice using Diplococcus

pneumoniae.• • Two strains of bacteria are S-type and R-type cells.• • Experiments • Living S-strain Injected into mice →Mice killed• Living R-strain Injected into mice → Mice lived• Heat Killed S-strain Injected into mice → Mice lived• Living R-strain + Heat Killed S-strain Injected into mice→ Mice

killed • # Griffith concluded that R type bacteria is transformed into

virulent form.• # Transformation is the change in the genetic constitution of an

organism by picking up genes present in the remains of its relatives.

F.GRIFFITH

• BIOCHEMICAL CHARACTERISATION OF TRANSFORMING PRINCIPLE

• # Proved by Oswarld Avery, Colin Macleod, Maclyn Mc Carty

• From this we conclude that DNA is the genetic material.

Alfred Hershey and Martha Chase Experiment(1952)

• They made two different preparation of bacteriophage , in one the DNA was made radioactive with 32P AND the other the protein coat was made radioactive with 35S.

• These two preparation were allowed to infect the bacterial cell separately.

• Soon after the infection the cultures were gently agitated in a blender to separate the adhering protein coats of the virus from the bacterial cell.

• The culture centrifuged to separate the viral coat and the bacterial cells• It was found that when the phage containing radioactive DNA was used

to infect 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 not proteins.

Characteristics of genetic material

• Genetic material must have the following properties.

• It should be able to generate its own replica.• It should be chemically and structurally stable.• It should provide the scope for slow

changes(mutation) that is necessary for evolution.

• It should be able to express itself in the form of Mendelian characters

• Nucleic acid can replicate but not protein.• Major genetic material is DNA, but viruses

like TMV have RNA as the genetic material.• The 2’ –OH in the nucleotide of RNA is a

reactive group and make the RNA liable and easily degradable, RNA is more reactive and hence DNA has the property to be the genetic material.

Semi conservative nature of DNA Mathew Messelson and Franklin stahl

• E.coli• Grown on 15 NH4Cl culture medium • Both strands of DNA have 15N (N15 N 15)• Shifted to 14NH4Cl culture medium• DNA extracted subjected to CSCl density gradient

centrifugations • After 20 minutes- Hybrid/ Intermediate type of DNA (N15 N14)• After 40 minutes -Equal amount of light DNA (N14 N14) and

hybrid DNA (N15 N14)

Replication of DNA In Eukaryotes• Definition: "Process by which DNA produces daughter DNA

molecules which are exact copies of the original DNA.“• In eukaryotes, DNA is double stranded. The two strands are

complementary to each other because of their base sequences.

• Semi-conservative method of DNA replication Important points:

• i) Most common method of DNA replication. • (ii) Takes place in the nucleus where the DNA is present in the

chromosomes. • (iii) Replication takes place in the S-phase (synthesis phase) of

the interphase nucleus. • (iv) Deoxyribose nucleotides needed for formation of new

DNA strands are present in nucleoplasm

• At the time of replication, the two strands of DNA first separate.

• Each strand then acts as a template for the formation of a new strand.

• A new strand is constructed on each old strand, and two exactly identical double stranded DNA molecules are formed.

• In each new DNA molecule, one strand is old (original) while the other is newly formed. Hence, Watson and Crick described this method as semi-conservative replication.

• An overall process of DNA replication showing replication fork and formation of new strands template and lagging template.

• The various steps involved in this process are summarized as follows:

• i. Mechanism of replication starts at a specific point of the DNA molecule, called origin.

• ii. At origin, DNA strand breaks because of an incision (nick). This is made by an enzyme called incision enzyme (endonuclease).

• iii. The hydrogen bonds joining the two strands are broken by the enzyme.

• iv. The two strands start unwinding. This takes place with the help of a DNA unwinding enzyme Helicases. Two polynucleotide strands are thus separated.

• v. The point where the two strands separate appears like a fork or a Y-shape. This is described as a replicating fork.

• vi. A new strand is constructed on each old strand. This takes place with the help of a small RNA

primer molecule which is complimentary to the DNA at that point.

• vii. Each old DNA strand acts as a template (site) for the construction of new strand.

The RNA primer attaches itself to the old strand and attracts the enzymes(DNA polymerase III) which add new nucleotides through base complementation.

• The deoxyribose nucleotides are present in the surrounding nucleoplasm. New DNA strand is thus constructed opposite to each old strand

• viii. Formation of new complementary strand always begins at the 3' end of the template strand (original strand) and progresses towards the 5' end (i.e in 3' - 5' direction).

Since the new strand is antiparallel to the template strand, it is obvious that the new strand itself is always developed in the, 5'-3' direction.

For this reason when the two original strands separate (then with respect to the origin of separation), one acts as 3'-5' template while the other acts as 5'- 3' template.

• ix. Of the two, the replication of 3'-5' template begins first. • Hence the new strand formed on it is called the leading

strand.• The other template (5'-3') must begin replication at the

fork and progress back toward the previously transcribed fragment.

• The new strand formed on it is called the lagging strand. • x. Replication of the lagging strand takes place in small

fragments called Okazaki fragments.• These are then connected together by the enzyme ligase. • xi. Replication may take place in only one direction on the

DNA helix (unidirectional) or in two directions (bidirectional).

• xii. At the end of the process, two double stranded DNA molecules are formed from the original DNA molecule.

Central dogma of molecular biology Transcription TranslationDNA RNA PROTEIN.RNA WORLD .RNA was the first genetic material.

There are three types of RNA1. Messenger RNA(m RNA ). –It bring the genetic information of DNA transcribed on it

for protein synthesis. It is single stranded.2. Transfer RNA/soluble RNA. It act as an adaptor molecule that reads the code on one

hand and bind to the specific amino acid on the other hand.3. tRNA has a clover leaf like secondary structure It has an amino acid acceptor end at 3’ and an “ anticodon-loop,where the three

bases are complimentary to the bases of the codon of the purticular aminoacid.4. Ribosomal RNA (r RNA ) –It forms the structure of ribosomes.

Clover leaf model of t RNA

• ANTICODON :- The sequence of nitrogenous bases on RNA that is complementary to the codon for particular amino acid.

• CODON :- It is a sequence of three nitrogenous bases on m-RNA that code for a particular amino acid.

Transcription unit

• TEMPLATE STRAND • 1. The DNA strand that has the polarity 3‘→5‘ acts as

template during transcription is called as template strand.• 2. It is also called as master strand or (-) or sense strand.• 3. This takes part in transcription.

• CODING STRAND • 1. The strand which has polarity of 5‘→3‘ is called as

codon strand.• 2. It is called (+) because genetic code present in this

strand is similar to genetic code (based on mRNA). Thymine is replaced by uracil in m RNA.

• 3. This does not take part in transcription.

Transcription in Prokaryotes• In prokaryotes the structural genes are polycistronic and continuous.• In prokaryotes there is a single DNAdependent RNA polymerase,that catalyse

the transcription of all the three types of RNA( m RNA, t RNA , r RNA ).Initiation.• RNA polymerase binds to the promoter and initiates the process along with

certain initiating factors.• It uses ribonucleoside triphosphate for polymerisation on a DNA.Elongation.• The enzyme facilitates the opening of the DNA-helix and elongation continues.Termination.• Once the RNA polymerase reaches the terminator , the RNA polymerase falls

off and the nascent RNA separates. It is called termination of transcription.• It is facilitated by certain termination factors• In prokaryotes m RNA SYNTHESISED does not require any processing to become

active.• Both transcription and translation occur in the same cytosol.• Transcription and translation can be coupled(translation can start much before

the m RNA is fully transcribed.)

Transcription in Prokaryotes

Transcription in Eukaryotes• In eukaryote the structural genes are monocistronic and

split.• They have coding sequences called EXONS that forms part of

a m RNA and non coding sequences called INTRONS, that do not form the part of m RNA and are removed during splicing.

• In Eukaryotes three different RNA polymerases.• 1.RNA polymerase-I – Transcribes r RNAs• 2.RNA polymerase-II– Transcribes the precursor of m RNA

(HETEROGENOUS NUCLEAR RNA(hn RNA).• 3.RNA polymerase –III- It catalyses the transcription of t RNA.

• SPLICING :- The process in eukaryotic genes by which the introns are removed and the exons are joined together to form m-RNA.

• The hn RNA undergoes two additional processes called capping and tailing

• In capping ,methyl guanosine triphosphate is added to the 5’ end of hn RNA .

• In tailing ,adenylate residues(200-300) are added at the 3’ end of hn RNA.

• The fully processed hn RNA is called m RNA and is released from the nucleus into the cytoplasm.

Transcription in Eukaryotes

GENETIC CODE• Salient features of genetic code.• Universal –the codons code for any aminoacid in any organism,be it a

bacterium or a human being.• Non ambiguous-each codon codes for only one aminoacid, so the genetic

code is unambiguous and specific.• Comma less-the codons are read in 5’3’ direction and no punctuation.• Degenerate – some amino acids are coded by more than one codon , the

genetic code is said to be degenerate.• Non overlapping-3 successive nitrogen bases code for only one aminoacid.• Nonsense codon (UAA,UAG,UGA)-do not code for any amino acids, but

act as terminating/stop codons of protein synthesis.• Linear-the sequence of amino acids present in a polypeptide chain

corresponds to sequence of nitrogen bases of DNA with 3 successive nitrogen bases forming a single codon.

• Triplet-there are 64(4x4x4) codons,61 codons code for 20 aminoacids.• Initiation Codon AUG

Protein synthesis.• 1.Activation of amino acids AA+ATP+E Mg+2 AA-AMP-E+ PPi AA-AMP-E+tRNA AA-tRNA+AMP+E• 2. Initiation Small subunit (40s) of ribosome binds with mRNA. Charged t RNA specific for initiation codon reaches P site Larger subunit (60s) of ribosome now combines with 40s-m RNA—t RNA met complex in the presence of Mg+2

• 3. Elongation• Second t-RNA charged with amino acid occupies A site of ribosome.• Peptide bond formation between methionine and second amino acids with the

help of enzyme peptide transferase.• Ribosomes moves over m RNA in 5’3’ • 4. Termination• Translation stops when non sense codons (Stop codons) reached.• No t RNA for stop codons (UAA,UAG,UGA)• Synthesized polypeptide is released with the help of release factor.•

• * AA—Amino acid• *ATP—Adenosine Triphosphate• *E—Pyrophosphate• AA—AMP-E-Amino acid adenylate enzyme

complex• AA—t RNA—Amino acyl-t RNA complex•

OPERON

• OPERON :- All the genes controlling a metabolic process constitute an operon.

• Discovered by Jacob and Manod.• *Experimented on E.coli.

HUMAN GENOME PROJECT (HGP)

• STEPS• Sequence annotation (Sequence the whole set of genome)• -Isolation of total DNA from the cell• -Fragmentation by restriction endonuclease• Fragments cloned in suitable host BAC/YAC • Fragments sequenced using automated DNA sequences.• Sequences arranged on the basis of overlapping regions.• Alignment of the sequences by specialized computer

based programmes• Expressed sequence Tags (EST) (Identifying all the genes

Expressed as RNA)

Salient features of Human Genome

1. Functions of 50% discoursed genes unknown 2.Repetitive sequences contribute large portion 3.Largest gene dystrophic 4.3164.7 millions Nucleotides5.Average gene consists of 3000 bases 6.Total genes 30,000 7.<2% gene codes protein 8.Chromosome-1 has 2968 genes 9.Y chromosome has 231 genes

Application of Human genome project

• Identification of defective genes.• -: Opportunity to offer early treatment.• -: Identification of genes that confer

susceptibility to certain disease.• -: Prediction of protein that the genes

produce.• -: Drug designing to enhance or inhibit the

activities of the proteins.

TECHNIQUE FOR DNA FINGER PRINTING

• Technique developed by Dr.Alec Jeffreys.• Process is also known as DNA typing/DNA profiling.• DNA extraction from the cells in high speed refrigerated centrifuge. .DNA extraction from the cells in high speed refrigerated centrifuge• Amplification of DNA content by PCR (Polymerase chain reactions)• DNA fragmentation by Restriction endonuclease• Gel electrophoresis• Double stranded DNA split into single stranded• Southern blotting (Transferring separated DNA to nylon or

nitrocellulose sheet)• Nylon sheet immerse in a bath having probes/marker* (Hybridisation)• Nylon membrane pressed on X-ray film (Autoradiography)• Dark band develops at probe site• *Probes/ Markers are radioactive synthetic DNA complementary to

VNTR

DEFINITIONS• ANTICODON :- The sequence of nitrogenous bases

on RNA that is complementary to the codon for particular amino acid.

• BACTERIOPHAGE :- A virus that infects a bacterium. • CODON :- It is a sequence of three nitrogenous

bases on m-RNA that code for a particular amino acid.

• CONSTITUTIVE GENES :- Constitutive genes are those genes which are constantly expressed & whose products are continuously needed for cellular activity.

• DNA POLYMORPHORISM :- Refers to the variations at genetic level where an inheritable mutation is observed in a population in a frequency greater than 0.01.

• EXON :- The regions of a gene which become part of m-RNA & code for the different regions of proteins.

• FRAME SHIFT MUTATION :- A type of mutation where addition 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.

• GENE :- Segment of DNA that code for RNA/functional unit of heredity.

• INTRONS :- The regions of a gene which do not form part of m-RNA and are removed.

• NUCLEOSOME :- Structure formed when negatively charged DNA is wrapped around the positively charged histone octamer.

• OPERON :- All the genes controlling a metabolic process constitute an operon.

• ORIGIN OF REPLICATION :- It is the definite region of DNA where replication originates/ starts.

• REPLICATION FORK :- The Y- shaped structure formed when the double standard DNA is unwound up to a point during its replication

• SATELLITE DNA :- The repetitive DNA sequences which do not code for any protein, but form a large portion of human genome; and show high degree of polymorphorism.

• SILENT MUTATION :- Mutation which do not cause any change in protein.

• SPLICING :- The process in eukaryotic genes by which the introns are removed are the exons are joined together to form m-RNA.

• TRANSCRIPTION :- It is the process of formation of RNA from DNA.

• • TRANSFORMATION :- It 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 later.

• TRANSLATION :- It is the process of polymerization of amino acids to form a polypeptide dictated by mRNA

Differences • DNA • 1. It usually occurs inside nucleus and some cell organelles. • 2. DNA is a genetic material. • 3. It is a double stranded with the exception of some viruses (rabies, AIDS etc.) • 4. DNA contains over a million nucleotides. • 5. It contains deoxyribose sugar. • 6. Nitrogen bases thymine occurs in DNA along with three others- adenine,

cytosine and three guanine. • RNA • 1. Very little RNA occurs inside nucleus. Most it is found in the cytoplasm. • 2. RNA is not a genetic material except in certain viruses, e.g., Reovirus. • 3. It is a single stranded with the exception of some viruses(e.g., double

stranded in Reovirus) • 4. Depending upon the type, RNA contains 70-12000 nucleotides. • 5. It contains ribose sugar. • 6. Thymine is replaced by uracil in RNA. The other are similar – adenine, cytosine

and guanine. •

• PROCARYOTIC TRANSCRIPTION

• 1. It occurs in contact with cytoplasm. • 2. Products of transcription become effective in situ. 3. There is only one RNA polymerase. 4. RNA polymerase does not have separate transcription factors. 5. mRNA is generally polycistronic. 6. Splicing is generally not required. • EUKARYOTIC TRANSCRIPTION • 1. It occurs inside the cytoplasm. • 2. Products of transcription come out of the nucleus for functioning in

cytoplasm. • 3. There are three types of RNA polymerase. • 4. Transcription factors are involved in recognition of promoter site. • 5. mRNA is generally monocistronic. • 6. In most of the cases splicing required for removing intervening

sequences •

• LEADING STRAND • 1. It is a replicated strand of DNA which grows continuously without any gap.• 2. It does not require DNA ligase for its growth.• 3. The direction of growth of the leading strand is 5‘→3‘• 4. Only a single RNA primer is required.• 5. Its template opens in 3‘→5‘ direction.• 6. Formation of leading strand begins immediately at the beginning of

replication.• LAGGING STRAND • 1. Lagging strand is a replicated strand of DNA which is formed in short segment

called discontinuous. • 2. DNA ligase is required for joining Okazaki fragments. • 3. The direction of growth of the lagging strand is 3‘→5‘though in each Okazaki

fragment it is 5‘→3‘. • 4. Starting of each Okazaki fragment requires a new RNA. • 5. Its template opens in 5‘→3‘ direction • 6. Formation of lagging strand begins a bit later than that of leading strand. •

QUESTIONS• 1. There are proteins which are positively charged and

there are also negatively charged proteins. What makes the protein get its charge

• 2. What is ESTs? 3. A particular human gene has the largest number of bases. Identify it

• . 4. Why is mRNA of eukaryotic cells said to monocistronic, while that of prokaryotic cell is polycistronic?

• 5. A point mutation leads to adverse change in the function of hemoglobin (B-globin chain). Identify the disease that may occur due to this mutation. Mention the change of amino acids in the polypeptide due to mutation

• 6. Two persons filed a case against a lady claiming to be the father of her only daughter. How to find the real biological father

• 7. If a nucleosome contains 200bps, how many nucleosome are there in a mammalian cell? What changes occur to beads of strings of DNA during metaphase?

• 8. Write the mRNA transcribed from the DNA segment with the base sequence TAC TAG TCG ACT. How many amino acids will there be in the oligopeptide translated by the mRNA? Why?

• 9. Lac operon is negatively regulated. What is meant by this? Why is lactose called the inducer of lac operon in E.coli?

• 10. (i) Describe the two major approaches to sequencing of genomes? (ii) Expand SNPs. What are they? (iii) Explain VNTR as the basis of DNA fingerprinting?

• 1. The two strands of DNA have antiparallel polarity. What does it mean?

• 2. DNA fingerprinting is a technique to find out variations at DNA level among individuals of population. What is the principle on which it works?

• 3. What term is given to the flow of information from RNA to DNA in certain viruses?

• 4. A criminal case is 10 years old was registered for investigation. What samples they might have tested?

• 5. Pick out the untransalated regions from the given mRNA. 5‘ ACG UCG AUG GCG CCC UUU UAG GAG GAA 3‘ Where are they normally located

only ONE MARK QUESTION• 1.Name the genetic material in TMV.• 2.Write the scientific name of the plant on which

Taylor et al performed their experiment.• 3.What would be the proportion of light and hybrid

density DNA molecules after 80 minutes of a single cell of E. coli growth?

• 4.When does DNA replicate in the cell cycle ?• 5.Name the amino acids having one codon.

.

TWO MARK QUESTION1.What is meant by semiconservative nature of DNA replication?2. What are the functions of DNA polymerase?3. What is frame shift mutation ?Name the type of mutation that does not affect protein synthesis .4.What are the untranslated regions (UTRs) ?5.Briefly describe polymorphism

THREE MARK QUESTIONS1.Describe the discontinuous synthesis of DNA.2. How is Lac operon “switched on” in an E.coli cell ?3.Name the three RNA Polymerases found in eukaryotes and mention their functions.4.Explain the two major approaches involved in the sequencing of genomes.FIVE MARKS QUESTIONS 1.Describe the salient features of the double helical model of DNA.2. Bring out the salient features of genetic code .3.Describe in detail the steps in the technique of DNA finger printing.4.Describe the process of replication of DNA.5. What is satellite DNA ?Name their types. Mention their basis for the classification of satellite DNA