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

                                                        

•       Escherichia coli ----4.6X1O6  bp

                                                          

•Human being------------3.3x 109  bp(haploid cell)

•DNA was  discovered by FREDERICH  MEISCHER(1869) as an acidic substance in the nucleus , he called it  nuclein.

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

•v  Living S-strain Injected into mice  →Mice killed

•v  Living R-strain Injected into mice → Mice lived

•v  Heat Killed S-strain Injected into mice → Mice lived

•v  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.

•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

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

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)

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

               

RNA WORLD .

RNA was the first genetic material.

There are three types of RNA

1.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.

● •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. 

•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 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.

Coming Up Genetic Code   and Translation