Monday, 23 September 2019

Molecular Basis of Inheritance

Molecular Basis of Inheritance

Molecular Basis of Inheritance

• The genetic material must be able to serve as a repository of genetic information, whether or not it is expressed. It should be able to accurately pass its copies into the progeny. Replication allows the genetic material to precisely duplicate itself forming the exact copies.
• Expression of stored genetic information is the basis for the concept of information flow within the cell.
• The genetic material must also serve as the basis of newly arising variability among organisms through the process of mutation.

DNA AS GENETIC MATERIAL

• The concept of DNA as the genetic material of most organisms has been developed and supported by following direct and indirect evidence.

Direct Evidence
• The most convincing evidence in support of DNA as the genetic material came from the three approaches on microorganisms – transformation in bacteria, mode of infection of bacteriophages and conjugation in bacteria.

TransformationGriffith's experiment
• Transformation is the mode of exchange or transfer of genetic information from one strain of bacterium to another strain of bacterium without involving any direct contact between them.
• Griffith found that extracts of dead pathogenic strains of the bacterium Streptococcus pneumonia can transform live harmless strains into live pathogenic strains. Later, Avery, McLeod, and McCarty analyzed the extract and demonstrated that the material was DNA.
• The bacterium has two strains — virulent and non-virulent. The virulent strain causes pneumonia and its bacteria are S-type (forming smooth colony), surrounded by polysaccharide sheath. The non-virulent bacteria are R-type (forming irregular or rough colonies).

Griffith's experiment
Step-I
Live cells of S-III type were injected into mice, all the mice died due to pneumonia, live S-III type cells were recovered from the serum of dead mice.

Step-II
Live cells of R-II type was injected into mice, mice remained healthy, R-II type cells were recovered from the serum of mice.

Step-III
Heat killed S-III type were injected into mice, none of the mice died, showing that virulence
is lost after heat killing.

Step-IV
Heat killed S-III type and live R-II type injected together into mice, mice died due to pneumonia. Autopsy of dead mice showed the presence of
both S-type and R-type bacteria.

• Griffith concluded that there was something in heat-killed S-III strains that transformed live R-II strains into live S-III strains. The R-II strains that were transformed into S-III strains continued thereafter producing S-III strain only.

The Avery, McLeod and McCarty experiment

• Three scientists Avery, McLeod, and McCarty fractionated the killed S-type bacteria into three components-DNA, carbohydrate and protein and performed the following experiment.

Steps in the experiment

(i) Removed the polysaccharide capsule from heat-killed S-III
type and R-II type 

(ii) Removed protein fraction from heat-killed S-III type + R-II type Mice died Mice died

(iii) Added deoxyribonuclease enzyme into heat-killed S-III type + R-II type Mice survived

(iv) Added proteases into heat-killed S-III type + R-II type Mice died

• In experiments (i), (ii) and (iv), DNA of heat-killed S-III type was intact and so it transformed live R-II type into S-III types, but in the experiment (iii) the enzyme disintegrated the DNA and so R-II type was not transformed.
• It clearly showed that the DNA component of heat-killed S-III type transformed live R-II type into live S-III type and thus, DNA forms the molecular basis of heredity.

Transduction

• It is the process in which bacterium infecting virus (bacteriophage) serves as a vector transferring DNA from one bacterium cell to another, e.g., T 2 bacteriophage. Hershey and Martha Chase performed an experiment to confirm that DNA of bacteriophage (virus infecting bacteria) enters into host (bacterial) cell and carries the necessary information for the formation of new phages. Their experiment was based on the fact that DNA contains phosphorus but no sulphur whereas proteins contain sulphur but no phosphorus. 

The Hershey-Chase experiment

Step-I
T 2 bacteriophages are labelled with radioactive isotopes. Protein coats of phages are labelled with 35 S and DNA of phages are labelled with 32 P. Hershey and Chase’s Experimental Steps

Step-II
Two cultures of E.coli were grown. Bacteriophages infect bacterial cells. In culture, infected with radioactive 35 S, radioactive sulphur gets incorporated into sulphur-containing amino acids and becomes part of bacterial proteins. In culture, infected with radioactive 32 P, radioactive phosphorus gets incorporated into nucleotides.

Step-III
Bacterial cells are agitated to remove protein coats and centrifuged. 35 S radioactivity was found in the supernatant, which contains only empty phage capsids or ghosts. 32 P radioactivity was found in the bacterial cells, proving only DNA of the phage entered the bacteria.

•The viruses derived from parents having labelled DNA possessed radioactivity. This experiment demonstrated clearly that genetic material is DNA and not the protein.

Indirect Evidence

DNA is capable of controlling the cell structure and cell functions through transcription and translation. DNA replicates prior to cell division and is equitably distributed in the daughter cells. DNA is capable of replication. DNA copies are similar to the original DNA. DNA has a system of repair. DNA can show infinite variations due to changes in its nucleotide type, sequence and length.

DEOXYRIBONUCLEIC ACID

• DNA is the largest macromolecule which consists of two complementary strands of deoxyribonucleotides that run antiparallel.
• It is composed of small monomeric units called nucleotides. Nucleotides (deoxyribonucleotides) are made up of a pentose sugar (deoxyribose type), a phosphate group and a nitrogenous base.
• The pentose sugar and the nitrogenous base constitute nucleosides. Nitrogen base is linked
to pentose sugar through N-glycosidic linkage to form a nucleoside. There are four nucleotides in DNA, differ from each other in the type of the nitrogenous base which could be adenine (A), guanine (G), thymine (T) or cytosine (C).


DEOXYRIBONUCLEIC ACID


Structure and Function of DNA
• The linkage between the two nucleotides consists of a phosphate group linked to two sugars. A phosphodiester bond is formed between C-3 and C-5 of different deoxyribose sugars of two adjacent nucleotides.
• One end of the polynucleotide chain has a sugar residue with C-3, not linked to another nucleotide having free 3'-OH group and the other end has sugar residue with C-5, linked to a phosphate group thus, showing polarity. These are named as 3' and 5' ends respectively.
• The two polynucleotide chains are antiparallel to each other. One has phosphodiester linkage in 3'→5' direction while the other has phosphodiester linkage in 5'– 3'direction. The two polynucleotide chains are held together by hydrogen bonding between specific pairs of purines and pyrimidines.
• The pairing is always between A and T and G and C.
• There are two hydrogen bonds between A and T and three hydrogen bonds between G and C.
• The stacking of bases creates two types of grooves — major and minor grooves.

Chargaf’s Rules
• The purines and pyrimidines are always in equal amounts i.e., A + G = T + C.
• In human DNA, A = 30.9%, T = 29.4%, G = 19.9% and C = 19.8%.
• DNA carries all the hereditary information coded in the arrangement of its nitrogen bases i.e., genetic code.
• Changes in the sequence and number of nucleotides produce mutations which are responsible for variations and formation of new species.
• DNA produces RNAs through transcription.
• DNA controls the metabolic reactions of cells through RNAs and synthesis of proteins, enzymes and other biochemicals.

Types of DNA
• There are five types of DNA: B, Z, A, C and D. B, A (widest diameter of the helix), C and D are right-handed helix while Z-DNA (thinnest diameter of the helix) is left-handed.

PACKAGING OF DNA HELIX

• Long sized DNA molecules are compacted in small areas (about 1μm in E.coli and 5μm nucleus in human beings) only through packing.

DNA Packaging in Prokaryotes
• Bacterial DNA is relatively simpler in form, double-stranded molecule compacted into a structure referred to as nucleoid.
• DNA in a bacterial chromosome is found to be associated with DNA and nonhistone basic proteins like polyamines.

DNA Packaging in Eukaryotes
• DNA packaging in eukaryotes followsμμ the nucleosome-solenoid model. RNA is complexed with lysine and arginine-rich basic proteins called histones to form nucleosomes.
• Each nucleosome consists of eight histone proteins (2 molecules of each H 2 A, H 2 B, H 3 and H 4 ) around which the DNA wraps 1.65 times.
• Histone H1 is a linker protein which binds DNA of two adjacent nucleosomes.
• 10 nm fibre of nucleosomes gets coiled upon itself to form 30 nm wide helix with five or six nucleosomes per helix. This 30 nm structure is called a solenoid. The packing of DNA has the ‘beads on a string’ appearance.
• Solenoid further condenses to form loops averaging 300 nm in length.
• Two types of chromatin material form chromosomes — 
(i) euchromatin, that is loosely packed and lightly stained and
transcriptionally active; 
(ii) heterochromatin, that is densely packed, darkly stained and transcriptionally inactive.

DNA REPLICATION

• In replication, DNA acts as its own template and produce exact copies of itself which is an autocatalytic function of DNA.
• DNA replicates by the semiconservative method in which one strand of the daughter duplex is derived from the parent while
the other strand is newly formed.
• Semiconservative replication of DNA was proved by the work of Meselson and Stahl. In their experiment, E.coli was grown
in 15 N medium having heavy isotope of nitrogen, for many generations. After that, the labelled bacteria were transferred to
a fresh 14 N medium and they were allowed to grow in that medium. DNA samples were tested for the heavy isotope of nitrogen.
• DNA from bacteria that had been grown on medium with 15 N, contained only 15 N isotopes. DNA of the first generation grew
on 14 N medium, was hybrid or intermediate ( 15 N and 14 N). The second generation of bacteria after 40 minutes contained two
types of DNA, 50% light ( 14 N 14 N) and 50% intermediate ( 15 N 14 N). It proved the DNA replication in E.coli is semiconservative.

Steps of DNA Replication
• Enzyme helicase unwinds the parental double helix.
• Topoisomerase releases tension of the DNA strand.
• Replication over the two templates proceeds in opposite directions.
• One strand with polarity 3'→5'forms its complementary strand continuously
because 3' end of the latter is always open for elongation. It is called
the leading strand.

• As RNA primer is also required every time a new Okazaki fragment is to be built.
• Okazaki fragments are joined together by means of enzyme DNA ligase.

DNA polymerases in prokaryotes
• DNA polymerase I have 5'→ 3' polymerising activity (due to which it removes or excises RNA primers from Okazaki
fragments and fills it with DNA) as well as 5'→3'and 3'→5' exonuclease activity due to which mispaired nucleotide is
removed. This is called proofreading function.
• DNA polymerase II has 5'→ 3' polymerising activity as well as 3' →5'exonuclease activity but lacks 5'→3'
exonuclease activity.
• DNA polymerase III has an essential role in DNA replication. It has both 5'→3 polymerising activity as well as 3' →5'
exonuclease activity (proofreading activity).

DNA polymerases in eukaryotes
• DNA polymerase α- is the largest and main enzyme of DNA replication, synthesise DNA on lagging strand.
• DNA polymerase β- nuclear polymerase found only invertebrates.
• DNA polymerase γ- mitochondrial polymerase.
• DNA polymerase δ- synthesises DNA on the leading strand.
• DNA polymerase ε- help in elongation of lagging strand.

Proof-reading and DNA repair
• DNA polymerase III sense a wrong base introduced during replication. It goes back, removes the wrong base, allows the addition of proper base and then proceeds forward.
• There is a separate repair mechanism for any damage caused to DNA due to mutation, UV exposure or mismatching that escapes proof-reading mechanism.
• A nick or break is caused by an endonuclease near the region of repair. DNA polymerase I remove the mismatched or wrong nucleotides if present and synthesises a correct replacement by using the intact strand as a template. The newly formed segment is sealed by DNA ligase.

TRANSCRIPTION

• The process of transferring genetic information from the template strand of the DNA to RNA is called transcription. It takes place in the following three steps :
(i) Initiation: The σ subunit of RNA polymerase recognises specific DNA sequences called promoters at –35 region. As initiation continues, RNA polymerase binds more tightly to the promoter at –10 region (TATA box), accompanied by a local untwisting of DNA.

(ii) Elongation: Subsequent ribonucleotide complements are inserted and linked together by phosphodiester bonds, chain elongation continues in 5'⟶ 3' direction creating a temporary DNA/RNA hybrid, the σ subunit dissociates from the RNA polymerase.

(iii) Termination: As polymerase transcribes away from the promoter, rho factor binds to RNA and follows the polymerase hen polymerase reaches some sort of pause site, rho factor catches up with polymerase and unwinds the DNA-RNA hybrid, resulting in the release of the polymerase.

Post-transcriptional processing
• Transcription in eukaryotes occurs within the nucleus and this primary transcript, called heterogeneous nuclear RNA (hnRNA), moves out of the nucleus into the cytoplasm for translation. The functional mRNA is processed from hnRNA and the process is called maturation.
• Initially, a cap (consisting of 7-methyl guanosine or 7 mG) at the 5' end and a tail of poly-A at the 3' end is added. It is known as capping and tailing respectively.

RIBONUCLEIC ACID (RNA)

• RNA is a single-stranded polyribonucleotide which functions as a carrier of coded genetic information from DNA to the cytoplasm for taking part in protein and enzyme synthesis. In some viruses, they may appear partially double-stranded due to folding or coiling of a single strand. dsRNA can be seen in viruses only.
• The axis or backbone of RNA is formed of alternate residues of phosphate and ribose sugar. Phosphate combines with 5' carbon of its sugar and 3'carbon of next sugar similar to the arrangement found in the DNA strand. Nitrogen bases are attached to sugars at 1' carbon of the latter and are of four types — adenine (A), guanine (G), cytosine (C) and uracil (U).

Types of RNA
• There are two types of RNA :
(a) Genetic RNA: It takes part in genetic transmission. It is further divided into two types: double-stranded (mammalian reovirus) and single-stranded (TMV, LTS virus).

(b) Non-genetic RNA: It is of three types :
(i) mRNA (messenger RNA): Brings coded information from DNA and takes part in its translation by bringing amino acids in a particular sequence while synthesising polypeptide.

(ii) tRNA (transfer RNA) - Transfers an amino acid from cytoplasm to the site of protein synthesis. It has a clover leaf-shaped structure with four recognisable sites - amino acid binding site at 3', anticodon loop (codon recognition sites), DHU loop (amino acid recognition site) and TΨCloop (ribosome recognition site).

(iii) rRNA (ribosomal RNA) - It is involved in the translation of the message of DNA. rRNA forms the structural benchwork on which a polypeptide is formed, during protein synthesis.
• Other non-genetic RNA: snRNA, siRNA, hnRNA and antisense RNA.

CENTRAL DOGMA OF MOLECULAR BIOLOGY

• Crick (1958) proposed the central dogma of molecular biology.
• Central dogma says there is the unidirectional flow of information from DNA to RNA and from RNA to polypeptide.
• Many tumour viruses contain RNA as genetic material and replicate by first synthesising a complementary DNA. This process is called reverse transcription.
• It is carried out by an RNA-dependent DNA polymerase called reverse transcriptase. RNA of these viruses first synthesises DNA through reverse transcription, Now, DNA transfers information to RNA which takes part in the translation of coded information to form a polypeptide.


Reverse flow of information

GENETIC CODE

• The genetic code is made up of codons through which information in RNA is decoded in a polypeptide chain. A codon is a three-letter sequence.
• The codons which initiate the protein synthesis are called initiation codons. They are AUG (methionine) and GUG (valine).
• The codons which do not code for any amino acid are called non-sense codons or termination codons. They are UAG, UAA and UGA.

Characteristics of the Genetic Code

• Universal: A codon specifies the same amino acid in all organisms ranging from a bacterium to human beings.

• Non-overlapping: One letter or base cannot be used for two different adjacent codons while reading a polynucleotide chain.

• Degenerate: 61 out of 64 codons code for only 20 amino acids. 

• Non-ambiguous: A particular codon will always code for the same amino acid.

• Triplet codon: Three adjacent nitrogen bases constitute a codon which specifies the placement of one amino acid in a polypeptide.

• Commaless: No punctuations are needed between any two words i.e. after one amino acid is coded, the second amino acid will be automatically coded by the next three letters. One gene-one enzyme hypothesis given by Beadle and Tatum states that a gene controls a structural or functional trait through controlling the synthesis of a specific protein or enzyme. Later, it has been proved that a single protein may have a number of polypeptides and each polypeptide is controlled by a separate gene. As a result, one gene-one enzyme hypothesis was replaced by one gene-one polypeptide hypothesis. Further, when the gene was identified as a functional unit or cistron, the same was termed as one cistron-one polypeptide hypothesis.

TRANSLATION

• This activation process, called charging, occurs under the influence of aminoacyl tRNA synthetases. It takes place in three steps:

Initiation
• The translation of mRNA begins with the formation of the initiation complex. The 30S ribosomal subunit binds with two initiation factors (IF-1 and IF-3) then with the mRNA. The initiating codon (5') AUG is guided to its correct position by the Shine-Dalgarno sequence in the mRNA.
• In the second step, both GTP-bound IF-2 and the initiating fMet-tRNA bind with this complex.
• In the third step, this large complex combines with the 50S ribosomal subunit.
• Functional 70S ribosome called the initiation complex is formed and it includes the mRNA and the initiating fMet-tRNA.

Elongation
• The GTP is hydrolysed and an EF-Tu–GDP complex is released from the 70S ribosome. The EF-Tu–GTP complex is regenerated in a process involving EF-Ts and GTP.
• In the next step, a peptide bond is formed between the two amino acids bound by their tRNAs to the A and P sites on the ribosome. This occurs by the transfer of the initiating N-formyl methionyl group from its tRNA to the amino group of the second amino acid, now in the A site.
• This reaction produces a dipeptidyl-tRNA in the A site and the now uncharged (deacylated) tRNA feet remains bound to the P site. The enzymatic activity that catalyses peptide bond formation has been referred to as peptidyl transferase.
• In the final step of the elongation cycle (translocation), the ribosome moves one codon towards the 3' ends of the mRNA.
This movement shifts the anticodon of the dipeptidyl-tRNA from the A site to the P site and shifts the deacylated tRNA from the P site to the E site, from where the deacylated t RNA is released into the cytosol.

Termination

• Termination is signalled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA), immediately following the final coded amino acid.

OPERON

•  Operator, promoter and regulatory genes constitute the regulatory region. Operon system is common in prokaryotes.


OPERON


Inducible Operon

• An inducible operon system is a regulated unit of genetic material which is switched on in response to the presence of an inducer. It is usually found in catabolic pathways.
• The lac operon consists of the regulator (I), promoter (P), operator (O) sites and structural genes (lac Z, Y and A) that code for the protein (enzymes).
• In the absence of lactose, the regulator protein (repressor) binds to the operator and inhibits transcription.
The structural genes are transcribed, ultimately resulting in the production of the enzymes (b-galactosidase, permease and transacetylase) needed for lactose catabolism.

Repressible Operon
• Repression is blocking off the operator gene of operon through a complex repressor that is formed by the union of corepressor formed by regulator gene and corepressor, a product of the anabolic pathway.
• Tryptophan (Trp) operon consists of regulator (R), promoter (P), operator (O) and structural (trp - E, D, C, B, A) genes.
• In the absence of corepressor, the repressor becomes inactive, the structural genes are transcribed and tryptophan is produced ultimately.
• In the presence of corepressor, repressor becomes activated which binds to the operator and blocked the same thus, the structural genes stop transcription. This is called feedback repressor and it also exerts negative control.

HUMAN GENOME PROJECT

• It is called International Human Genome Sequencing Consortium and is aimed at finding out all the genes in each of the human chromosomes determining their function and hopefully understanding how they together form the complete organism. The two factors that made this possible are :
(i) Genetic engineering techniques, which made it possible to isolate
and clone any segment of DNA, 
(ii) Availability of simple and fast techniques, for determining the DNA sequences.
• The goals of the human genome project are as follows : 
(i) To develop a genetic linkage map of the human genome by identifying
thousands of genetic markers and mapping them in the genome. 
(ii) To obtain a physical map of the human genome by cloning
genomic DNA into YACs and cosmids.
(iii) To sequence the entire human genome.
• The human genome consists of about 30,000 genes. It has 3.1647 billion nucleotide base pairs. The average size is 8000 base pairs of which Duchenne Muscular Dystrophy on the X chromosome is the largest gene.

DNA FINGERPRINTING

• DNA fingerprinting is a technique of determining certain nucleotide sequences, generally repeated sequences (Satellite DNA) in the human genome that produce a pattern of bands which is unique for every individual. The basis of DNA fingerprinting is short nucleotide repeats in DNA called the Variable Number of Tandem Repeats or VNTRs that vary in number from person to person but are inherited.

The technique for DNA Fingerprinting

• DNA fingerprints can be prepared from extremely minute amounts of blood, semen, hair bulb or any other cells of the body.
The major steps are as follows :
• DNA is extracted from the cells in a high-speed refrigerated centrifuge.to restriction fragment length analysis.
• These DNA fragments are separated through gel electrophoresis. The separated fragments can be visualized by staining them with a dye that fluoresces under ultraviolet radiation.
• Double-stranded DNA is then split into single-stranded DNA using alkaline chemicals.
Separated DNA sequences are transferred from the gel onto a nitrocellulose or nylon membrane (Southern blotting).
• The nylon sheet is then exposed to probes or markers that are radioactive, synthetic DNA segments and complementary to known sequences. 
• Lastly, X-ray film is exposed to the nylon sheet containing radioactive probes. Dark bands develop at the probe bound DNA sites. Thus, hybridised fragments are detected by autoradiography and the film developed represents DNA fingerprint.

Applications of DNA Fingerprinting

• For identifying the true (biological) father/mother, DNA samples of the child, mother and father are taken. 
• DNA fingerprints of suspects from blood or hair or semen picked up from the scene of the crime are prepared and compared.
The DNA fingerprint of the person matching the one obtained from the sample obtained from the scene of a crime can give a clue to the actual criminal.
• It is used to determine and study human lineages.
• It can be used to identify genes associated with hereditary disorders.
• It is useful in determining population and genetic diversities.

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