Molecular Basis of Inheritance

Learning Outcomes:

1. Understand the structure of DNA and its role as genetic material.
2. Explore the central dogma of molecular biology.
3. Comprehend the processes of replication, transcription, and translation.
4. Grasp the regulation of gene expression and the significance of the Human Genome Project.
5. Learn the applications and principles of DNA fingerprinting.

The DNA

DNA, a long polymer of deoxyribonucleotides, serves as the primary genetic material. Its length is typically measured in the number of nucleotides or base pairs (bp). Organisms possess varying lengths of DNA. For instance, bacteriophage φ×174 has 5386 nucleotides, bacteriophage lambda 48502 bp, Escherichia coli 4.6 × 10^6 bp, and human haploid DNA contains 3.3 × 10^9 bp.

Structure of Polynucleotide Chain

A nucleotide comprises three essential components: a nitrogenous base, a pentose sugar (ribose in RNA, deoxyribose in DNA), and a phosphate group. The nitrogenous bases are classified into:

• Purines: Adenine (A) and Guanine (G).
• Pyrimidines: Cytosine (C), Thymine (T) in DNA, and Uracil (U) in RNA.

A nucleoside is formed when a nitrogenous base is linked to the 1’C of a pentose sugar via an N-glycosidic bond. Further, a nucleotide is generated when a phosphate group attaches to the 5’C of the nucleoside. Two nucleotides are linked through a 3′-5′ phosphodiester bond, forming a dinucleotide, which can extend to form a polynucleotide chain.

The DNA’s backbone is made of alternating sugar and phosphate groups, with nitrogenous bases projecting from the backbone. In RNA, an additional –OH group is present at the 2′ position of the ribose.

Discovery of DNA

Friedrich Meischer first identified DNA as an acidic substance in the nucleus in 1869, calling it ‘Nuclein’. However, its structural elucidation remained elusive until 1953, when James Watson and Francis Crick, using X-ray diffraction data from Maurice Wilkins and Rosalind Franklin, proposed the Double Helix Model.

Key Features of DNA Structure:

1. DNA consists of two polynucleotide chains, with the backbone made of sugar-phosphate while the bases project inward.
2. The strands exhibit anti-parallel polarity: one strand is 5’→3′, and the other is 3’→5′.
3. Hydrogen bonds between the bases form base pairs (bp): Adenine pairs with Thymine (2 bonds), and Guanine pairs with Cytosine (3 bonds).
4. The chains are coiled in a right-handed helix with a pitch of 3.4 nm and about 10 bp per turn.
5. The stacking of base pairs stabilizes the helical structure.

Note: The complementarity of strands allows one strand to act as a template for replicating the other, which is crucial for DNA replication.

Packaging of DNA Helix

The length of a typical mammalian DNA, calculated as 2.2 meters, far exceeds the size of the nucleus (about 10^-6 m). In prokaryotes like E. coli, DNA is held with positively charged proteins forming a nucleoid. In eukaryotes, DNA is more complexly organized, wrapped around histones (basic proteins) forming nucleosomes, which constitute chromatin.

The Search for Genetic Material

Early scientists speculated on the nature of the genetic material, whether it was protein or nucleic acid. Various key experiments resolved this question.

Transforming Principle

In 1928, Frederick Griffith discovered the transforming principle in Streptococcus pneumoniae. Griffith observed that heat-killed S strain bacteria could transform live R strain into virulent forms, suggesting a transfer of genetic material.

Biochemical Characterization

Oswald Avery, Colin MacLeod, and Maclyn McCarty determined in the 1940s that DNA is the transforming material. They showed that DNA, not protein or RNA, from S bacteria could transform R bacteria.

Hershey-Chase Experiment

In 1952, Alfred Hershey and Martha Chase conducted experiments using bacteriophages to prove DNA was the genetic material. By labeling viral DNA with radioactive phosphorus and viral protein with radioactive sulfur, they showed that only the DNA entered the bacterial cells and directed viral reproduction.

Properties of Genetic Material (DNA vs RNA)

1. Replication: DNA can generate replicas due to complementary base pairing.
2. Stability: DNA is chemically more stable than RNA due to the absence of a 2′-OH group.
3. Mutation: Both DNA and RNA mutate, but RNA mutates more rapidly, aiding evolution.
4. Expression: DNA expresses itself through proteins.

Note: DNA is preferred as the genetic material due to its stability, while RNA serves better for genetic transmission.

RNA World

RNA is believed to be the first genetic material, evolving essential life processes such as metabolism and translation. RNA also acted as a catalyst, but due to its reactivity and instability, DNA evolved as the stable genetic material.

Replication

Watson and Crick proposed semi-conservative replication, where each strand of the DNA double helix serves as a template for a new strand.

Meselson-Stahl Experiment

In 1958, Meselson and Stahl proved semi-conservative replication by growing E. coli in 15N medium and then shifting it to 14N medium. After DNA replication, they observed that each new DNA molecule consisted of one parental and one newly synthesized strand.

Concept: Semi-conservative replication ensures genetic fidelity across generations.

Transcription

Transcription is the process of copying genetic information from DNA to RNA. It differs from replication as only a segment of DNA is copied, and only one strand serves as a template.

Transcription Unit

A transcription unit consists of:

1. Promoter: The binding site for RNA polymerase.
2. Structural gene: The gene that gets transcribed.
3. Terminator: Defines the end of transcription.

Types of RNA

There are three major types of RNA:

1. mRNA (messenger RNA): Provides the template for protein synthesis.
2. tRNA (transfer RNA): Brings amino acids and reads the genetic code.
3. rRNA (ribosomal RNA): Plays a structural and catalytic role during translation.

Transcription in Eukaryotes

Eukaryotic transcription is more complex, involving:

1. Splicing: Removal of introns and joining of exons to produce mature mRNA.
2. Capping and Tailing: Adds stability to mRNA.

Genetic Code

The genetic code translates nucleotide sequences into amino acids. It consists of triplet codons, each coding for one amino acid.

Features of the Genetic Code:

1. Triplet Code: Each codon consists of three nucleotides.
2. Degenerate: Some amino acids are coded by multiple codons.
3. Universal: The code is nearly the same across all organisms.
4. Non-overlapping: Codons are read sequentially without overlap.

Mutations and Genetic Code

Point mutations, like the one that causes sickle cell anemia, alter a single nucleotide. Frameshift mutations result from insertion or deletion of nucleotides, changing the reading frame of the genetic code.

tRNA: The Adapter Molecule

tRNA acts as an adapter by carrying specific amino acids and recognizing codons on mRNA through its anticodon loop.

DNA	RNA
Double-stranded	Single-stranded
More stable	Less stable
Thymine as a base	Uracil as a base
Genetic material in most	Genetic material in some viruses

Translation

Translation is the process of synthesizing proteins from amino acids as directed by mRNA. The ribosome serves as the site of protein synthesis, facilitating peptide bond formation between amino acids.

Steps in Translation:

1. Initiation: The ribosome assembles at the start codon (AUG) on the mRNA.
2. Elongation: Amino acids are added sequentially to form the polypeptide.
3. Termination: The process stops at a stop codon (UAA, UAG, or UGA).

Regulation of Gene Expression

Gene expression can be regulated at various levels, including:

1. **

Transcriptional level**: Regulation of RNA synthesis.

2. Post-transcriptional level: Processing of RNA.
3. Translational level: Control over protein synthesis.

Lac Operon

The lac operon in E. coli is a classic example of gene regulation. It controls the expression of genes required for lactose metabolism.

Important Concept: The lac operon is activated by the presence of lactose, which inactivates the repressor, allowing transcription.

Human Genome Project

The Human Genome Project (HGP) was an ambitious initiative to sequence the entire human genome, which consists of 3.3 billion base pairs. The project was completed in 2003, revealing that humans have approximately 25,000 genes.

Key Findings:

1. The human genome contains 3164.7 million base pairs.
2. Less than 2% of the genome encodes proteins.
3. The genome includes many repeated sequences with unknown functions.

DNA Fingerprinting

DNA fingerprinting is a technique used to identify individuals based on variations in their DNA. It relies on polymorphisms in repetitive DNA sequences and has applications in forensic science and paternity testing.

MCQ: What is the significance of the lac operon in E. coli?
Answer: It regulates the metabolism of lactose by controlling the expression of specific enzymes.