Molecular Basis of Inheritance — Study Notes

1. Introduction

The molecular basis of inheritance is the study of how genetic information is stored, replicated, transcribed and translated in living organisms. While the previous chapter established that genes are the units of inheritance located on chromosomes, this chapter answers the deeper question: what is the chemical nature of the gene? The answer is deoxyribonucleic acid (DNA), the molecule that carries genetic information in most organisms. The study of DNA and RNA has revolutionised biology, and the discovery of the double helical structure of DNA by James Watson and Francis Crick in 1953 stands as one of the most celebrated achievements in the history of science.

The chapter traces the historical experiments that established DNA as the genetic material, describes the structure of DNA and RNA, explains the processes of replication, transcription, genetic code and translation, and discusses regulation of gene expression. The central dogma of molecular biology, proposed by Francis Crick, states that genetic information flows from DNA to RNA to protein. This chapter links classical genetics with molecular genetics and forms the foundation for the biotechnology chapters that follow.

2. The Search for Genetic Material

The search for the genetic material began with experiments that established that DNA, not protein, is the genetic material.

2.1 Griffith's Transformation Experiment (1928)

Frederick Griffith worked with Streptococcus pneumoniae, which has two strains: the smooth (S) strain, which has a polysaccharide coat and is virulent, and the rough (R) strain, which lacks the coat and is non-virulent. When Griffith injected mice with the live S strain, the mice died. When he injected the live R strain or heat-killed S strain, the mice survived. But when he injected a mixture of heat-killed S and live R strains, the mice died, and live S bacteria were recovered from the dead mice. He concluded that some transforming principle from the heat-killed S strain had converted the R strain into the virulent S strain. This experiment demonstrated transformation but did not identify the chemical nature of the transforming principle.

2.2 Avery, MacLeod and McCarty (1944)

These scientists showed that the transforming principle is DNA. They purified the transforming substance from the heat-killed S strain and found that it was DNA. They demonstrated that if the transforming extract is treated with DNase (which destroys DNA), transformation does not occur, whereas treatment with protease (which destroys proteins) or RNase (which destroys RNA) does not prevent transformation. This proved conclusively that DNA is the genetic material in Streptococcus pneumoniae.

2.3 Hershey and Chase Experiment (1952)

Alfred Hershey and Martha Chase used bacteriophage T2, a virus that infects bacteria, to prove that DNA is the genetic material. They grew the phage in two media, one containing radioactive phosphorus (32P) and the other containing radioactive sulphur (35S). Since DNA contains phosphorus but not sulphur, and protein contains sulphur but not phosphorus, the DNA was labelled with 32P and the protein coat with 35S. After allowing the phages to infect bacteria, they found that the 32P (DNA) entered the bacterial cells, while the 35S (protein) remained outside. This conclusively proved that DNA, not protein, is the genetic material of the phage.

3. Structure of DNA

The double helical model of DNA was proposed by Watson and Crick in 1953. The key features are: - DNA is a polymer of nucleotides. Each nucleotide has a nitrogenous base, a pentose sugar (deoxyribose) and a phosphate group. - The nitrogenous bases are of two types: purines (adenine A and guanine G) and pyrimidines (cytosine C and thymine T). - The two polynucleotide chains are antiparallel; one runs in the 5' to 3' direction and the other in the 3' to 5' direction. - The bases of the two strands are paired by hydrogen bonds: adenine pairs with thymine (A-T, two hydrogen bonds) and guanine pairs with cytosine (G-C, three hydrogen bonds). This is called complementary base pairing. - The two strands are coiled in a right-handed double helix. The sugar-phosphate backbones are on the outside, and the bases are stacked inside. - The distance between the two strands is uniform (20 angstroms or 2 nm), because a purine always pairs with a pyrimidine. - The pitch of the helix is 3.4 nm, with 10 base pairs per turn. Each base pair is 0.34 nm apart.

The double helical model immediately explained Chargaff's rule, which states that in DNA, the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine (A = T and G = C), so the purine:pyrimidine ratio is 1:1.

3.1 Packaging of DNA

In prokaryotes, the DNA is circular and is not associated with histone proteins. In eukaryotes, the DNA is double-stranded, linear and associated with histone proteins. The negatively charged DNA wraps around the positively charged histone proteins (which are rich in basic amino acids like lysine and arginine) to form a nucleosome, the repeating unit of chromatin. Each nucleosome has about 200 base pairs of DNA wrapped around a core of eight histone molecules (H2A, H2B, H3 and H4, two of each). The nucleosomes appear as beads on a string, which are further coiled to form the chromatin fibre. During cell division, the chromatin condenses into chromosomes.

3.2 DNA as the Genetic Material

The genetic material must be able to replicate, store information, express itself and undergo mutation. DNA satisfies all these requirements. In some viruses, however, RNA is the genetic material, for example in tobacco mosaic virus (TMV) and human immunodeficiency virus (HIV).

4. DNA Replication

DNA replication is the process by which a DNA molecule makes copies of itself. It occurs during the S phase of the cell cycle. The model of replication is the semi-conservative model, proposed by Watson and Crick, which states that each daughter DNA molecule consists of one parental strand and one newly synthesised strand. This was experimentally proven by Matthew Meselson and Franklin Stahl in 1958 using Escherichia coli and the isotopes of nitrogen, 15N (heavy) and 14N (light).

4.1 Steps of Replication

• The DNA double helix unwinds at the origin of replication, and the hydrogen bonds between the base pairs are broken by the enzyme helicase, forming a replication fork.
• The enzyme DNA polymerase adds nucleotides to the growing strand in the 5' to 3' direction, using the parental strands as templates. Since the strands are antiparallel, one strand (the leading strand) is synthesised continuously, while the other (the lagging strand) is synthesised discontinuously in short fragments called Okazaki fragments, which are later joined by DNA ligase.
• DNA polymerase requires a primer (a small RNA or DNA segment) to begin synthesis.
• Replication is initiated at specific sequences called origins of replication.
• The E. coli replication origin is called oriC, and replication is bidirectional.

4.2 Enzymes in Replication

• Helicase: Unwinds the DNA double helix.
• DNA polymerase: Synthesises new DNA strands; it can only add nucleotides in the 5' to 3' direction and requires a primer.
• DNA ligase: Joins the Okazaki fragments on the lagging strand.
• Primase: Synthesises the RNA primers.

5. Transcription

Transcription is the process of copying genetic information from DNA to RNA. It occurs in the nucleus of eukaryotic cells. The segment of DNA that is copied is called the gene. During transcription, the enzyme RNA polymerase binds to the promoter region and synthesises RNA in the 5' to 3' direction, using one of the two DNA strands as a template.

5.1 Template and Coding Strands

In transcription, only one of the two DNA strands is used as a template for RNA synthesis. The strand that is copied is called the template strand (or antisense strand), and the other strand, which has the same sequence as the RNA (with T replaced by U), is called the coding strand (or sense strand). The coding strand runs in the 5' to 3' direction, and the template strand in the 3' to 5' direction. The promoter is the sequence where RNA polymerase binds to initiate transcription, and the terminator sequence signals the end of transcription.

5.2 Transcription in Prokaryotes and Eukaryotes

• In prokaryotes, there is a single RNA polymerase enzyme that synthesises all types of RNA. Transcription and translation can occur simultaneously in the cytoplasm because there is no nuclear membrane.
• In eukaryotes, there are three RNA polymerases: RNA polymerase I (synthesises rRNA), RNA polymerase II (synthesises mRNA, the precursor of hnRNA) and RNA polymerase III (synthesises tRNA). Transcription occurs in the nucleus, and the primary transcript is processed by splicing, capping and polyadenylation to form the mature mRNA. The split gene arrangement, where coding sequences (exons) are interrupted by non-coding sequences (introns), requires the removal of introns by splicing.

6. Genetic Code

The genetic code is the set of rules by which the sequence of nucleotides in mRNA is translated into the sequence of amino acids in a protein. Each group of three consecutive nucleotides in mRNA is called a codon, and it codes for one amino acid. The genetic code was deciphered by Har Gobind Khorana and Marshall Nirenberg.

6.1 Salient Features of the Genetic Code

• The code is a triplet code: three nucleotides (codon) code for one amino acid.
• The code is universal: the same codons code for the same amino acids in all organisms, from bacteria to humans.
• The code is degenerate: more than one codon can code for the same amino acid (e.g., both GGU and GGC code for glycine), because there are 64 codons but only 20 amino acids.
• The code is unambiguous: a particular codon codes for only one specific amino acid.
• The code is non-overlapping: codons are read sequentially, one after another.
• There are start and stop codons. AUG codes for methionine and is the initiator codon. The stop or termination codons are UAA, UAG and UGA, which do not code for any amino acid.

6.2 tRNA and the Adaptor Molecule

Transfer RNA (tRNA) acts as the adaptor molecule that reads the codon on mRNA and brings the appropriate amino acid. The tRNA has an anticodon, a sequence of three nucleotides complementary to the codon on mRNA, and it carries a specific amino acid at its 3' end.

7. Translation

Translation is the process of synthesising a polypeptide from the mRNA sequence. It occurs on ribosomes in the cytoplasm and involves three steps: - Initiation: The ribosome binds to the mRNA. The small ribosomal subunit recognises the initiation codon (AUG) with the help of the initiator tRNA carrying methionine. The large subunit then joins. - Elongation: Amino acids are added one by one to the growing polypeptide chain. The tRNA anticodons pair with the mRNA codons, and peptide bonds are formed between adjacent amino acids. - Termination: When a stop codon (UAA, UAG or UGA) is reached, the ribosome detaches and the completed polypeptide is released.

The polypeptide then undergoes folding and post-translational modifications to become a functional protein. In eukaryotes, the process is more complex, and many ribosomes can translate a single mRNA molecule simultaneously, forming a polysome.

8. Regulation of Gene Expression and the Operon

Gene expression is the process by which a gene's information is used to produce a functional product. Regulation of gene expression ensures that genes are expressed only when needed, saving energy and resources. In prokaryotes, gene expression is regulated mainly at the level of transcription through operons. The operon model was proposed by Francois Jacob and Jacques Monod.

8.1 The Lac Operon

The lac operon of Escherichia coli consists of the structural genes (Z, Y and A), a promoter (P) and an operator (O). The structural genes code for enzymes that metabolise lactose. The regulator gene (I) codes for the repressor protein, which binds to the operator and prevents transcription. - When lactose is absent: The repressor binds to the operator, blocking RNA polymerase, so the lac genes are not transcribed. This is called the switched off state. - When lactose is present: Lactose acts as an inducer and binds to the repressor, inactivating it. The repressor cannot bind to the operator, so RNA polymerase can transcribe the genes. This is the switched on state.

The lac operon is thus an inducible operon, regulated by the inducer lactose. This is an example of negative regulation, where the repressor is the control element.

9. Human Genome Project and DNA Fingerprinting

The Human Genome Project (HGP) was a mega project launched in 1990, coordinated by the US Department of Energy and the National Institutes of Health, with India among the six countries that contributed. The goal was to determine the complete nucleotide sequence of the human genome. The HGP was completed in 2003 and revealed that the human genome contains about 3 billion base pairs and approximately 20,500 genes. The project used two approaches: expressed sequence tags (ESTs) and sequence annotation.

DNA fingerprinting is a technique used to identify individuals by analysing their DNA sequences, based on the presence of variable number of tandem repeats (VNTRs) and short tandem repeats (STRs). It was developed by Alec Jeffreys and has applications in forensic science (identifying criminals and victims), parentage testing and determining genetic relationships. The technique involves DNA isolation, restriction digestion, separation by gel electrophoresis, transfer to a membrane (Southern blotting), hybridisation with radioactive probes and autoradiography.

Quick Revision Tables

Table 1: Experiments Establishing DNA as Genetic Material

Table 2: Differences Between DNA and RNA

Table 3: Features of the Genetic Code

Mind Map

Important Diagrams (SVG)

Common Mistakes

1. Students think the 3:1 ratio belongs to molecular biology; the molecular chapter focuses on DNA structure, replication, transcription and translation, not Mendelian ratios.
2. The template strand is confused with the coding strand; the template strand is copied by RNA polymerase, while the coding strand has the same sequence as the RNA (with T as U).
3. DNA polymerase is said to synthesise in the 3' to 5' direction; DNA polymerase always synthesises new strands in the 5' to 3' direction, so the lagging strand is made in fragments.
4. A-T pairing is said to have three hydrogen bonds; A-T has two hydrogen bonds, while G-C has three.
5. Students think all genes have introns; introns are a feature of eukaryotic split genes, not typical of prokaryotic genes.
6. The genetic code is said to be overlapping; it is non-overlapping, with codons read sequentially without sharing bases.
7. UAA is said to code for an amino acid; UAA, UAG and UGA are stop codons that code for no amino acid and terminate translation.
8. DNA fingerprinting is said to compare proteins; it compares specific DNA sequences like VNTRs and STRs after gel electrophoresis and hybridisation.

Exam Tips

1. Memorise the key historical experiments: Griffith (transformation), Avery-MacLeod-McCarty (DNA is the transforming principle), Hershey-Chase (DNA is genetic material using 32P and 35S).
2. Learn the Watson-Crick model facts: antiparallel strands, A-T (2 H-bonds), G-C (3 H-bonds), 10 bp per turn, pitch 3.4 nm, diameter 2 nm.
3. For replication, remember the enzymes: helicase (unwinds), DNA polymerase (synthesis 5' to 3'), DNA ligase (joins Okazaki fragments), and the Meselson-Stahl experiment.
4. In transcription, distinguish template and coding strands, promoter and terminator, and the three eukaryotic RNA polymerases (I: rRNA, II: mRNA, III: tRNA).
5. The genetic code features, especially triplet, universal, degenerate, non-overlapping, with start (AUG) and stop (UAA, UAG, UGA) codons, are high-frequency questions.
6. The lac operon is a must-know: inducible, structural genes Z, Y, A, and the role of the repressor and inducer (lactose).
7. For the HGP, remember 3 billion base pairs, about 20,500 genes, and that India contributed to the project.

Conclusion

The molecular basis of inheritance reveals the chemical machinery behind the hereditary phenomena described by Mendel. Through the landmark experiments of Griffith, Avery and the decisive work of Hershey and Chase, DNA was established as the genetic material, and the elegant double helical structure proposed by Watson and Crick explained how genetic information could be stored in a stable, replicable form. The processes of semi-conservative replication, transcription and translation, governed by the triplet, universal and degenerate genetic code, convert the genetic information stored in DNA into the proteins that build and maintain organisms. Regulation of gene expression through operons such as the lac operon shows how cells economise and respond to their environment, while the Human Genome Project and DNA fingerprinting demonstrate the applied power of this knowledge. Together these concepts form the foundation for the biotechnology chapters, where these molecular tools are harnessed for human welfare.