Molecular Basis of Inheritance

The DNA

Deoxyribonucleic acid (DNA) is the long polymer of deoxyribonucleotides. The length of DNA is usually defined as the number of nucleotides or a pair of nucleotides referred to as base pairs present in it.

Structure of Polynucleotide Chain

A nucleotide has three components: a nitrogenous base, a pentose sugar (deoxyribose), and a phosphate group. Nitrogenous bases are divided into two types: Purines (Adenine and Guanine) and Pyrimidines (Cytosine and Thymine). A nitrogenous base is linked to the pentose sugar through an N-glycosidic linkage to form a nucleoside. When a phosphate group is linked to 5'-OH of a nucleoside through phosphoester linkage, a corresponding nucleotide is formed. Two nucleotides are linked through 3'-5' phosphodiester linkage to form a dinucleotide. More nucleotides can join to form a polynucleotide chain.

A nucleotide is formed when a nucleoside is linked to a phosphate group.

Exam-Oriented Note: The backbone of a polynucleotide chain is formed due to sugar and phosphate. The nitrogenous bases project from the backbone.

Double Helix Model for Structure of DNA

James Watson and Francis Crick proposed the double helix model for DNA based on X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin.

• DNA consists of two polynucleotide chains where the backbone is constituted by sugar-phosphate, and the bases project inside.
• The two chains have anti-parallel polarity. It means, if one chain has the polarity 5'-3', the other has 3'-5'.
• The bases in two strands are paired through hydrogen bond (H-bonds) forming base pairs (bp). Adenine forms two hydrogen bonds with Thymine from the opposite strand, and Guanine is bonded with Cytosine by three hydrogen bonds. This generates complementarity between the two chains.
• The two chains are coiled in a right-handed fashion. The pitch of the helix is 3.4 nm and there are roughly 10 bp in each turn. The distance between a base pair in a helix is approximately 0.34 nm.

Common Mistake: Confusing the number of hydrogen bonds between A-T and G-C. Remember: A=T (two bonds) and G≡C (three bonds).

Central Dogma of Molecular Biology

Francis Crick proposed the central dogma of molecular biology, which states that genetic information flows in one direction: DNA to mRNA via Transcription, and mRNA to Protein via Translation.

DNA -> Transcription -> mRNA -> Translation -> Protein

The Search for Genetic Material

The quest to determine whether protein, RNA, or DNA acts as the genetic material involved key landmark experiments.

Griffith's Experiment (Transforming Principle)

Frederick Griffith (1928) used Streptococcus pneumoniae (bacterium) infecting mice. He observed two strains: smooth shiny colonies (S strain) with a mucous coat (virulent, causes pneumonia) and rough colonies (R strain) without the coat (non-virulent).

• S strain -> Inject into mice -> Mice die
• R strain -> Inject into mice -> Mice live
• S strain (heat-killed) -> Inject into mice -> Mice live
• S strain (heat-killed) + R strain (live) -> Inject into mice -> Mice die

Griffith concluded that the R strain bacterium had somehow been transformed by the heat-killed S strain bacterium, enabling the R strain to synthesize a smooth polysaccharide coat and become virulent. This was termed the transforming principle.

Avery, MacLeod, and McCarty Experiment

Oswald Avery, Colin MacLeod, and Maclyn McCarty worked to determine the biochemical nature of the transforming principle in Griffith's experiment. They purified biochemicals (proteins, DNA, RNA) from heat-killed S cells to see which ones transformed live R cells into S cells.

• Protease digestion: Transformation occurred (Protein is not genetic material).
• RNase digestion: Transformation occurred (RNA is not genetic material).
• DNase digestion: Transformation did not occur (DNA is the genetic material).

They concluded that DNA is the hereditary material, though not all biologists were convinced at first.

Hershey-Chase Experiment

Alfred Hershey and Martha Chase (1952) conclusively proved that DNA is the genetic material by working with bacteriophages (viruses that infect bacteria).

• They grew some viruses on a medium that contained radioactive phosphorus (P-32) and some others on medium containing radioactive sulfur (S-35).
• Viruses grown in P-32 contained radioactive DNA because DNA contains phosphorus, but proteins do not. Viruses grown in S-35 contained radioactive protein because proteins contain sulfur, but DNA does not.
• Radiolabeled phages were allowed to infect Escherichia coli bacteria.
• As the infection progressed, the viral coats were removed from the bacteria by agitating them in a blender. The virus particles were separated from the bacterial cell by spinning them in a centrifuge.
• Bacteria infected with viruses that had radioactive DNA were radioactive, indicating that DNA was injected into the bacteria. Bacteria infected with viruses that had radioactive proteins were not radioactive, indicating that proteins did not enter the bacteria.

DNA is the genetic material that is passed from virus to bacteria.

RNA World

RNA was the first genetic material. Essential biochemical processes such as metabolism, translation, and splicing evolved around RNA. RNA used to act as a genetic material as well as a catalyst (ribozymes).

• DNA being double-stranded and having complementary strands resists changes by evolving a process of repair. Therefore, DNA is a better genetic material than RNA.
• RNA is labile and easily degradable. Thus, RNA is catalytic, but DNA is chemically less reactive and structurally more stable.

Replication

Watson and Crick proposed the semi-conservative scheme for DNA replication, suggesting that the two strands would separate and act as a template for the synthesis of new complementary strands.

The Experimental Proof (Meselson and Stahl Experiment)

Matthew Meselson and Franklin Stahl performed an experiment in 1958 on Escherichia coli to prove that DNA replication is semi-conservative.

• They grew E. coli in a medium containing heavy isotope of nitrogen (N-15) as the sole nitrogen source for many generations. The resulting N-15 DNA could be distinguished from N-14 DNA by centrifugation in a cesium chloride (CsCl) density gradient.
• Then they transferred the cells into a normal N-14 medium. Samples were taken at various time intervals and extracted DNA was centrifuged to measure densities.
• The DNA extracted from the culture after one generation (20 minutes) had a hybrid or intermediate density. The DNA extracted after two generations (40 minutes) consisted of equal amounts of light DNA and hybrid DNA.

The Machinery and Enzymes

Replication requires a set of catalysts (enzymes). The main enzyme is DNA-dependent DNA polymerase. It uses DNA template to catalyze the polymerization of deoxynucleotides with high degree of accuracy and very fast speed.

• Replication occurs within a small opening of the DNA helix known as the replication fork.
• DNA polymerase can catalyze polymerization only in one direction, that is 5'-3'.
• Consequently, on one template strand with polarity 3'-5', replication is continuous (leading strand), while on the other template strand with polarity 5'-3', replication is discontinuous (lagging strand). The discontinuously synthesized fragments are joined by DNA ligase.

Transcription

Transcription is the process of copying genetic information from one strand of the DNA into RNA. Here, only a segment of DNA is transcribed and only one of the strands is copied into RNA.

Transcription Unit

A transcription unit in DNA has primarily three regions:

• A Promoter
• The Structural Gene
• A Terminator

The promoter is located towards 5'-end (upstream) of the coding strand and provides binding site for RNA polymerase. The terminator is located towards 3'-end (downstream) and defines the end of the transcription process.

Transcription Unit and the Gene

A gene is defined as the functional unit of inheritance. DNA sequence coding for tRNA or rRNA molecule also define a gene. The structural gene in a transcription unit could be monocistronic (mostly in eukaryotes) or polycistronic (mostly in bacteria).

Types of RNA and the Process of Transcription

In bacteria, there is a single DNA-dependent RNA polymerase that catalyzes transcription of all types of RNA (mRNA, tRNA, rRNA). Transcription has three steps:

• Initiation: RNA polymerase binds to promoter and initiates transcription with the help of a factor called sigma (σ) factor.
• Elongation: RNA polymerase uses nucleotides as substrate and polymerizes them in a template-dependent fashion following the rule of complementarity.
• Termination: Once the polymerase reaches the terminator region, it uses a termination factor called rho (ρ) factor to disengage the RNA polymerase and newly synthesized RNA.

In eukaryotes, there are two additional complexities:

• There are at least three types of RNA polymerases (RNA Polymerase I, II, and III). RNA Pol I transcribes rRNAs, RNA Pol II transcribes precursors of mRNA (heterogeneous nuclear RNA or hnRNA), and RNA Pol III transcribes tRNA, 5s rRNA, and snRNAs.
• The primary transcripts (hnRNA) contain both exons (expressed regions) and introns (non-coding regions) and are subjected to a process called splicing where introns are removed and exons are joined in a defined order.

Capping and Tailing: In hnRNA, cap is added at the 5'-end (an unusual nucleotide, methyl guanosine triphosphate) and poly-A tail (adenylate residues) is added at the 3'-end in a template-independent manner. Fully processed hnRNA, now called mRNA, is transported out of the nucleus for translation.

Genetic Code

Genetic code directs the sequence of amino acids during protein synthesis. George Gamow argued that since there are 20 amino acids and only 4 bases (A, U, C, G), a combination of bases must code for amino acids. A code of 3 nucleotides (triplet code) yields 64 codons (4 cubed), which is sufficient to code for 20 amino acids.

Salient Features of Genetic Code

Translation

Translation refers to the process of polymerization of amino acids to form a polypeptide. The order and sequence of amino acids are defined by the sequence of bases in the mRNA.

• Amino acids are joined by a peptide bond.
• The first phase involves charging of tRNA (aminoacylation of tRNA) where amino acids are activated in the presence of ATP and linked to their specific tRNA.
• The cellular factory for protein synthesis is the ribosome. Ribosome consists of structural RNAs and about 80 different proteins. In its inactive state, it exists as two subunits: a large and a small subunit.
• When the small subunit encounters an mRNA, the process of translation of the mRNA begins. mRNA also has some untranslated regions (UTR) that are not translated but are required for efficient translation process.
• The ribosome binds to the mRNA at the start codon (AUG) recognized by the initiator tRNA.
• The ribosome proceeds from codon to codon along the mRNA. Amino acids are added one by one translating the polypeptide sequence dictated by DNA and represented by mRNA.
• At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.

Regulation of Gene Expression

Regulation of gene expression refers to a broad term that could control at various levels. In eukaryotes, regulation can be exerted at:

1. Transcriptional level (formation of primary transcript)
2. Processing level (regulation of splicing)
3. Transport of mRNA from nucleus to the cytoplasm
4. Translational level

In prokaryotes, control of the rate of transcriptional initiation is the predominant site for control of gene expression.

The Lac Operon

The elucidation of the lac operon was a milestone in the understanding of transcriptional regulation, pioneered by François Jacob and Jacques Monod. An operon is a set of genes regulated together, such as the lac operon (lac stands for lactose).

• The lac operon consists of one regulatory gene (the i gene, which codes for the repressor of the lac operon) and three structural genes (z, y, and a).
• The z gene codes for beta-galactosidase, which hydrolyzes lactose into galactose and glucose. The y gene codes for permease, which increases permeability of the cell to beta-galactosides. The a gene encodes a transacetylase.
• Lactose acts as the inducer that switches the operon on and off.
• In the absence of inducer (lactose), the repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the structural genes.
• In the presence of an inducer such as lactose or allolactose, the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the promoter and transcription proceeds. Regulation of lac operon by repressor is referred to as negative regulation.

Human Genome Project

Launched in 1990, the Human Genome Project (HGP) was a mega project aimed at sequencing the complete human genome of approximately 3 billion base pairs.

Goals of HGP

• Identify all the approximately 20,000 to 25,000 genes in human DNA.
• Determine the sequences of the 3 billion chemical base pairs that make up human DNA.
• Store this information in databases.
• Improve tools for data analysis.
• Address the ethical, legal, and social issues (ELSI) that may arise from the project.

Salient Features of Human Genome

• The human genome contains 3164.7 million nucleotide bases.
• The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases.
• Total number of genes is estimated at 30,000—much lower than previous estimates of 80,000 to 140,000 genes. Almost 50 percent of the discovered proteins are unknown function.
• Less than 2 percent of the genome codes for proteins.
• Repeated sequences make up a very large portion of the human genome. Repetitive sequences are stretches of DNA repeated many times, sometimes hundred to thousand times. They have no direct coding functions, but they shed light on chromosome structure, dynamics, and evolution.
• Chromosome 1 has most genes (2968), and the Y has the fewest (231).
• Scientists have identified about 1.4 million locations where single base DNA differences (SNPs - single nucleotide polymorphism) occur in humans.

DNA Fingerprinting

DNA fingerprinting is a technique to determine genetic identity by examining individual nucleotide sequences. It was developed by Alec Jeffreys.

Basis of DNA Fingerprinting

Short Tandem Repeats (STRs) or Variable Number of Tandem Repeats (VNTRs) form the basis of DNA fingerprinting.

• In genomic DNA, most of the sequence is non-coding and appears as stretches of repeated DNA sequences. These sequence repetitions do not code for proteins but make up a large portion of human genome.
• In some DNA regions, a short nucleotide sequence is repeated many times in tandem. These are called Variable Number of Tandem Repeats (VNTRs).
• The size of VNTR varies from size 0.1 to 20 kb. Any human genome exhibits polymorphism in the size of VNTR. This polymorphism is inheritable from parents to offspring, making DNA fingerprinting the foundational tool for paternity testing and forensic science.

Steps of DNA Fingerprinting Technique

1. Isolation of DNA: DNA is extracted from biological samples like blood, hair follicles, skin, or saliva.
2. Digestion of DNA by Restriction Endonucleases: Restriction enzymes cut the DNA molecules into smaller fragments.
3. Separation of DNA Fragments by Electrophoresis: The fragmented DNA is separated according to size through agarose gel electrophoresis.
4. Southern Blotting: The separated DNA fragments are transferred to a synthetic membrane such as nitrocellulose or nylon.
5. Hybridization using labeled VNTR probe: A radioactive or fluorescently labeled VNTR probe (known single-stranded DNA sequence) is added, which binds to complementary sequences on the membrane.
6. Autoradiography: The hybridized membrane is exposed to an X-ray film to detect the radioactive probe spots, producing distinctive autoradiograms (DNA fingerprints) unique to each individual.