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MOLECULAR BASIS OF INHERITANCE
DEFINITIONS AND TERMS
. 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.
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 X 106 bp
Human beings - 3.3 X 109 bp (haploid number)
Human beings - 6.6 X 109 bp (DIploid 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:
------------- Puri-------------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 or central dogma reverse. 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 negatively charged DNA is wrapped around positively charged histone octamere into a structure called nucleosome.
¾ The nucleosome give chromatin its beads - over - string appearance in electron microscope.
¾ A typical 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
. Transforming Principle
· Transformation is a change in genetic material of an organism by obtaining genes from outside as from remains of its dead relatives.
· Frederick Griffith (1928) conducted as series of experiments with Streptococcus pneumoniae, (Pneumococcus), the bacterium causing pneumonia
· He observed, during the course of his experiment, a living organism (bacteria) had changed in physical form
· He observed two strains of this bacterium on culture plates:
-S strain : forms smooth shiny colonies; have a mucous (polysaccharide) coat;
Virulent/pathogenic
- R strain : forms rough colonies; without a mucous (polysaccharide) coat;
Non-virulent/non-pathogenic
· When live S strains (virulent) were injected into the mice, they produced pneumonia and the mice died
· Mice infected with R strain do not develop pneumonia
· When heat-killed S strains were injected into the mice, it did not kill the mice
· When heat-killed S strains were mixed with live R strains and injected into the mice, the mice died and he recovered live S strain from the dead body of the mice
· He concluded that the R strain bacteria had somehow been transformed by the heat-killed S strain bacteria,
· He further explained that some 'transforming principle' transferred from the heat-killed S strain, had enabled the R strain to synthesise a smooth polysaccharide coat and become virulent;
· This must happened due to the transfer of genetic material; But, the biochemical nature of genetic material was not defined from his experiments
Biochemical Characterisation of Transforming Principle:
· Before the work of Oswald Avery, Colin MacLeod and Maclyn McCarty (1933-44), the genetic material was thought to be a protein.
· They worked to determine the biochemical nature of 'transforming principle' in Griffith's experiment.
· They purified biochemicals (proteins, DNA, RNA, etc.) from the heat-killed S cells and checked which ones could transform live R cells into S cells.
· They found that DNA alone from S bacteria caused R bacteria to become transformed.
· They also observed that protein-digesting enzymes (proteases) and RNA-digesting enzymes (RNases) did not affect transformation, so the transforming substance was not a protein or RNA.
· They observed that Digestion with DNase did inhibit transformation, so the DNA caused the transformation.
· They concluded that DNA is the hereditary material, but not all biologists were convinced.
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 & QB bacteriophage , 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.
(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, on Vicia faba (faba beans) by using radioactive thymidine; they proved that DNA on the chromosomes replicates in a semiconservative manner.