Molecular Basis of Inheritance

The Dna

In most organisms, Deoxyribonucleic Acid (DNA) is the genetic material that carries hereditary information from one generation to the next. It is a polymer of deoxyribonucleotides.

Structure Of Polynucleotide Chain

DNA is a polynucleotide, meaning it is made up of many repeating units called nucleotides.

Components of a Nucleotide:

Each nucleotide consists of three components:

1. A Nitrogenous Base:
   • Purines: Adenine (A) and Guanine (G). (Double-ring structures)
   • Pyrimidines: Cytosine (C) and Thymine (T). (Single-ring structures)
2. A Pentose Sugar: Deoxyribose sugar (a 5-carbon sugar).
3. A Phosphate group ($PO_4^{3-}$).

A nucleoside is formed when a nitrogenous base is linked to the pentose sugar through an N-glycosidic linkage (at C1' of the sugar). A nucleotide is formed when a phosphate group is attached to the 5' carbon of the nucleoside sugar through a phosphoester linkage.

*(Image shows a diagram illustrating a nitrogenous base, deoxyribose sugar, and a phosphate group, showing how they combine to form a deoxyribonucleotide)*

---

Formation of a Polynucleotide Chain:

• Nucleotides are linked together by phosphodiester bonds to form a polynucleotide chain.
• A phosphodiester bond is a covalent bond formed between the phosphate group of one nucleotide (attached to the 5' carbon of its sugar) and the hydroxyl group on the 3' carbon of the sugar of the next nucleotide.
• This forms a sugar-phosphate backbone.
• A polynucleotide chain has a directionality: a 5' end (with a free phosphate group attached to the 5' carbon of the sugar) and a 3' end (with a free hydroxyl group attached to the 3' carbon of the sugar).

*(Image shows two deoxyribonucleotides linked together by a phosphodiester bond, highlighting the 5' and 3' ends of the growing chain)*

---

The Double Helix Model:

• In 1953, James Watson and Francis Crick proposed the famous double helix model for the structure of DNA, based on X-ray diffraction data provided by Maurice Wilkins and Rosalind Franklin, and Chargaff's rules.
• Key features of the DNA double helix:
  1. DNA is made of two polynucleotide chains.
  2. The two chains run antiparallel to each other, meaning one chain has 5' to 3' directionality, and the other has 3' to 5' directionality.
  3. The sugar-phosphate backbone is on the outside, and the nitrogenous bases are on the inside.
  4. The two chains are held together by hydrogen bonds between the nitrogenous bases.
  5. Bases follow specific pairing rules:
     • Adenine (A) always pairs with Thymine (T) via two hydrogen bonds ($A=T$).
     • Guanine (G) always pairs with Cytosine (C) via three hydrogen bonds ($G \equiv C$).
     This is called complementary base pairing.
  6. The two chains are coiled in a right-handed helix.
  7. Each turn of the helix is approximately 3.4 nm (nanometres) long and contains roughly 10 base pairs.
  8. The distance between two consecutive base pairs is approximately 0.34 nm.
  9. The diameter of the helix is approximately 2 nm.

*(Image shows the DNA double helix, illustrating the key features mentioned above)*

---

Chargaff's Rules:

Erwin Chargaff's quantitative analysis of DNA composition provided crucial data for the Watson-Crick model. Chargaff's rules state that in a double-stranded DNA molecule:

• The amount of Adenine (A) is always equal to the amount of Thymine (T) ($A=T$).
• The amount of Guanine (G) is always equal to the amount of Cytosine (C) ($G=C$).
• Consequently, the ratio of Purines to Pyrimidines is always equal to one (($A+G)/(C+T) = 1$).
• The ratio $(A+T)/(G+C)$ varies among different species but is constant for a given species.

These rules are a direct consequence of complementary base pairing in the double helix.

---

Packaging Of Dna Helix

The length of DNA in a typical human cell is approximately $2.2 \text{ metres}$ ($3.3 \times 10^9 \text{ bp} \times 0.34 \text{ nm/bp}$). Packaging this extremely long molecule into the small nucleus (about $10^{-6} \text{ metres}$ in diameter) is a remarkable feat of organisation.

Packaging in Prokaryotes (e.g., E. coli):

• In prokaryotes, the DNA is circular and located in a region of the cytoplasm called the nucleoid.
• The DNA is not associated with histone proteins.
• The DNA is supercoiled and held together by some non-histone basic proteins. The nucleoid is not a membrane-bound structure.

---

Packaging in Eukaryotes:

• In eukaryotes, DNA is a linear molecule found within the nucleus.
• DNA is associated with positively charged basic proteins called histones. Histones are rich in basic amino acid residues like lysine and arginine.
• There are five types of histone proteins: H1, H2A, H2B, H3, and H4.
• A core particle, called a histone octamer, is formed by eight histone molecules (2 of each H2A, H2B, H3, and H4).
• Negatively charged DNA is wrapped around the positively charged histone octamer, forming a structure called a nucleosome.
• A typical nucleosome contains about 200 base pairs of DNA helix.
• Nucleosomes are the repeating units of a structure called chromatin, which looks like 'beads-on-a-string' under an electron microscope.

*(Image shows diagrams illustrating the levels of DNA packaging from the double helix, wrapping around histone octamers to form nucleosomes ('beads on a string'), further coiling into a chromatin fibre, and finally condensing into a chromosome)*

• The 'beads-on-a-string' structure is further condensed and coiled to form chromatin fibres.
• Chromatin fibres are further coiled and condensed (with the help of additional non-histone chromosomal proteins - NHC proteins) to form the highly condensed structure seen as chromosomes during cell division.
• In a nucleus, some regions of chromatin are lightly stained and less condensed, called euchromatin. Euchromatin is transcriptionally active.
• Other regions are darkly stained and more densely packed, called heterochromatin. Heterochromatin is generally transcriptionally inactive.

The packaging of DNA in eukaryotes is complex and hierarchical, allowing a very long DNA molecule to fit within the nucleus and also playing a role in regulating gene expression.

The Search For Genetic Material

For many years, scientists debated whether protein or DNA was the molecule that carried hereditary information. Several experiments were conducted to identify the genetic material.

Transforming Principle

In 1928, Frederick Griffith conducted experiments using Streptococcus pneumoniae bacteria, which cause pneumonia.

• He used two strains of the bacteria:
  • S strain (Smooth): Virulent (cause pneumonia), have a smooth polysaccharide coat.
  • R strain (Rough): Non-virulent, lack the polysaccharide coat.

Griffith's Experiment:

1. Injected R strain bacteria into mice: Mice survived.
2. Injected S strain bacteria into mice: Mice died (due to pneumonia).
3. Injected heat-killed S strain bacteria into mice: Mice survived. (Heat killed the bacteria, so they were harmless).
4. Injected a mixture of heat-killed S strain and live R strain bacteria into mice: Mice died. Living S strain bacteria were recovered from the dead mice.

*(Image shows the four steps of Griffith's experiment with diagrams showing what was injected, the mouse's outcome, and what was recovered, highlighting the result of the fourth step)*

Conclusion:

• Griffith concluded that some factor from the heat-killed S strain bacteria had 'transformed' the live R strain bacteria into virulent S strain bacteria. This unknown substance was called the 'Transforming Principle'.
• The nature of this transforming principle was not clear from this experiment. It could be protein, DNA, RNA, or other substances.

---

Biochemical Characterisation Of Transforming Principle

Building on Griffith's work, Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-1944) conducted experiments to identify the chemical nature of the transforming principle.

Avery, MacLeod, and McCarty's Experiment:

• They purified biochemicals (proteins, DNA, RNA, etc.) from heat-killed S strain bacteria.
• They mixed each purified component separately with live R strain bacteria in culture medium.
• They tested which component could transform the R strain into the S strain.
• Results:
  • Adding purified proteins from S strain to R strain: No transformation.
  • Adding purified RNA from S strain to R strain: No transformation.
  • Adding purified DNA from S strain to R strain: Transformation occurred; live S strain bacteria were produced.
• They further confirmed this by treating the purified DNA with enzymes:
  • DNA + Protease (enzyme that digests protein): Transformation occurred.
  • DNA + RNase (enzyme that digests RNA): Transformation occurred.
  • DNA + DNase (enzyme that digests DNA): No transformation occurred.

*(Image shows a diagram illustrating the steps of isolating DNA from S strain, adding it to R strain culture, and showing that this results in S strain bacteria, and the result of treating the S strain extract with DNase preventing transformation)*

Conclusion:

• Avery, MacLeod, and McCarty concluded that DNA is the transforming principle and therefore the genetic material responsible for carrying hereditary information.
• Their work was initially met with scepticism by many scientists who still believed protein was the genetic material.

---

The Genetic Material Is Dna

The definitive proof that DNA is the genetic material came from the experiments of Alfred Hershey and Martha Chase in 1952. They worked with bacteriophages (viruses that infect bacteria).

Hershey and Chase Experiment:

• Bacteriophages consist of a protein coat and DNA inside. They inject their genetic material into the bacterium, and the bacterial cell then produces new virus particles.
• Hershey and Chase used radioactive isotopes to label the viral DNA and protein separately.
  • DNA contains phosphorus (P) but no sulfur (S). They labelled the DNA with radioactive phosphorus ($^{32}P$).
  • Protein contains sulfur (S) but usually no phosphorus (P). They labelled the protein coat with radioactive sulfur ($^{35}S$).
• They infected *E. coli* bacteria with these labelled bacteriophages.
• After infection, they agitated the mixture in a blender to separate the viral coats from the bacterial cells.
• They centrifuged the mixture. The heavier bacterial cells settled at the bottom (pellet), while the lighter viral coats remained in the supernatant.
• They then checked for the presence of radioactivity in the pellet (bacteria) and the supernatant (viral coats).
• Results:
  • The pellet containing the bacteria was radioactive when the phages were labelled with $^{32}P$ (DNA).
  • The supernatant containing the viral coats was radioactive when the phages were labelled with $^{35}S$ (protein).

*(Image shows diagrams of the Hershey-Chase experiment steps: labelling phages with 32P and 35S, infection of bacteria, blending, centrifugation, and finding 32P in the bacterial pellet but 35S in the supernatant)*

Conclusion:

• Since $^{32}P$ (representing DNA) was found in the bacteria (pellet), it meant that DNA was injected into the bacteria during infection.
• Since $^{35}S$ (representing protein) was found in the viral coats (supernatant), it meant that the protein coat did not enter the bacteria.
• The genetic material responsible for directing the synthesis of new viral particles must be the substance that entered the bacterium.
• Therefore, DNA is the genetic material.

This experiment provided clear and strong evidence confirming DNA as the genetic material.

---

Properties Of Genetic Material (Dna Versus Rna)

Once DNA was established as the genetic material, scientists began to consider the properties that a molecule must possess to function as genetic material. These include:

1. Ability to Replicate (Self-duplicate): The genetic material must be able to make copies of itself, ensuring that hereditary information is passed on to daughter cells and offspring.
2. Chemical and Structural Stability: The genetic material should be stable enough not to change drastically during the life cycle, providing a reliable template for information transfer. However, some degree of change (mutation) is necessary for evolution.
3. Ability to Mutate: It must have the potential for mutation (change) to provide variation necessary for evolution.
4. Ability to Express itself as Phenotypic Characters: The genetic material must be able to direct the synthesis of proteins and other molecules that determine the observable traits of an organism.

Comparison of DNA and RNA as Genetic Material:

• Replication: Both DNA and RNA have the ability to replicate (using specific enzymes and mechanisms).
• Stability:
  • DNA is chemically more stable than RNA. The presence of the 2'-OH group in ribose sugar of RNA makes it more reactive and easily hydrolysed. Deoxyribose sugar in DNA lacks this 2'-OH group, making DNA more stable.
  • Thymine (T) is present in DNA instead of Uracil (U) in RNA. Thymine provides additional stability to DNA.
  • DNA is double-stranded, while RNA is generally single-stranded (though it can fold). Double-stranded structure provides more stability to DNA, protecting it from degradation.
  • Due to its stability, DNA is better suited for storing genetic information over long periods.
• Mutation: Both DNA and RNA can mutate. However, RNA is less stable and mutates at a faster rate. Viruses with RNA genomes (e.g., HIV, influenza virus) evolve faster than viruses with DNA genomes.
• Expression: RNA can directly code for protein synthesis (mRNA). DNA requires transcription into mRNA for protein synthesis. So, RNA can directly express traits, while DNA requires an intermediate step.

---

Based on these properties, DNA is considered a better genetic material for most organisms because of its greater stability, which ensures accurate transmission of information over generations. RNA, being less stable, is more suited for dynamic functions like acting as a messenger (mRNA) or a catalyst (ribozyme).

Some viruses have RNA as their genetic material (e.g., Tobacco Mosaic Virus, HIV). In these viruses, RNA performs both the function of genetic material and acts as a template for protein synthesis (via reverse transcription in some cases like HIV).

Rna World

The "RNA world" hypothesis is a theoretical concept that proposes that RNA was the primary genetic material and performed catalytic roles in the early forms of life, before the evolution of DNA and proteins.

Evidence Supporting the RNA World Hypothesis:

• RNA as genetic material: In some viruses, RNA serves as the genetic material.
• RNA as a catalyst (Ribozymes): Some RNA molecules have catalytic activity (like enzymes). These are called ribozymes. Examples include ribosomal RNA (rRNA) that catalyses peptide bond formation during protein synthesis. This suggests that RNA could have catalysed early biochemical reactions.
• RNA involved in key biological processes: RNA plays crucial roles in transcription, translation, and gene regulation in all living organisms, suggesting its ancient origin.
• DNA synthesis requires RNA primer: The process of DNA replication relies on RNA primers synthesised by RNA primase, suggesting RNA's role predates DNA.

Transition from RNA to DNA:

It is hypothesised that DNA evolved from RNA. DNA's greater stability (due to deoxyribose sugar and thymine) made it a better molecule for long-term storage of genetic information. Proteins, with their diverse structures and catalytic capabilities, evolved to perform most enzymatic functions more efficiently than ribozymes.

The transition to DNA as the primary genetic material and protein as the primary catalyst represents a major evolutionary step, leading to more stable and efficient biological systems.

Replication

Replication is the process by which a DNA molecule makes an exact copy of itself. This is essential for passing on genetic information during cell division and reproduction.

Semi-Conservative Nature of Replication:

The Watson-Crick model of DNA structure suggested a mechanism for replication. They proposed that when DNA replicates, the two strands of the double helix separate, and each strand serves as a template for the synthesis of a new complementary strand. Thus, each new DNA molecule consists of one original (parental) strand and one newly synthesised (daughter) strand. This is called semi-conservative replication.

*(Image shows a diagram illustrating the semi-conservative nature of replication: a double helix separating, each strand serving as a template, resulting in two new double helices each containing one original and one new strand)*

The Experimental Proof

The semi-conservative nature of DNA replication was experimentally proven by Matthew Meselson and Franklin Stahl in 1958 using *E. coli* bacteria.

Meselson and Stahl's Experiment:

• They grew *E. coli* for several generations in a medium containing a heavy isotope of nitrogen, $^{15}N$ (instead of the normal $^{14}N$). The DNA of these bacteria became heavy ($^{15}N$-DNA).
• They then transferred the bacteria to a medium containing normal nitrogen, $^{14}N$, and allowed them to divide.
• They extracted DNA at different generation times and analysed its density using cesium chloride (CsCl) density gradient centrifugation. Heavy DNA ($^{15}N$-DNA) would sediment lower than light DNA ($^{14}N$-DNA$).
• Results:
  • Generation 0 (before transfer): DNA was heavy ($^{15}N$-DNA), forming a single band at the bottom of the tube.
  • Generation 1 (after one generation in $^{14}N$ medium): All DNA was of intermediate density (hybrid DNA, containing one $^{15}N$ strand and one $^{14}N$ strand), forming a single band in the middle of the tube.
  • Generation 2 (after two generations in $^{14}N$ medium): Two bands were observed: one at the intermediate density (hybrid DNA) and one at the light density ($^{14}N$-DNA$).
  • Subsequent generations: The light DNA band became progressively stronger, while the intermediate band persisted but its relative amount decreased.

*(Image shows diagrams illustrating Meselson and Stahl's experiment steps: growing bacteria in N15, transferring to N14, extracting DNA at different generations, and showing the banding pattern in density gradient centrifugation tubes (heavy, hybrid, light bands))

Conclusion:

• The results clearly showed that in the first generation, all new DNA molecules were hybrids (one old $^{15}N$ strand, one new $^{14}N$ strand). This is consistent with semi-conservative replication.
• In subsequent generations, the hybrid DNA persisted, and new light DNA molecules ($^{14}N$ double helix) were formed, further supporting the semi-conservative model.

This experiment provided conclusive evidence that DNA replication is semi-conservative.

J. Herbert Taylor conducted similar experiments in 1958 on root tips of *Vicia faba* (faba bean) using radioactive thymidine to detect newly synthesised DNA. His results also supported the semi-conservative mode of replication in eukaryotes.

---

The Machinery And The Enzymes

DNA replication is a complex process involving several enzymes and proteins.

Key Enzymes and Proteins:

• DNA-dependent DNA polymerase: The main enzyme. It catalyses the polymerisation of deoxyribonucleotides to form new DNA strands using the parental DNA strand as a template. It polymerises nucleotides only in the 5' $\rightarrow$ 3' direction. It also has proofreading activity.
• Helicase: Unwinds the DNA double helix by breaking hydrogen bonds between base pairs. This creates a replication fork.
• Topoisomerase (or DNA gyrase in bacteria): Relieves the tension (supercoiling) that builds up in the DNA ahead of the replication fork.
• Single-strand binding proteins (SSBs): Bind to the separated DNA strands to prevent them from re-annealing (coming back together).
• Primase (RNA polymerase): Synthesises a short RNA primer on the template strand. DNA polymerase cannot initiate synthesis on its own; it requires a primer.
• DNA ligase: Joins the Okazaki fragments (short DNA segments synthesised on the lagging strand) together by forming phosphodiester bonds.

---

The Process of Replication:

1. Origin of Replication (Ori): Replication begins at specific sites on the DNA molecule called origins of replication. Eukaryotic DNA has multiple origins, while prokaryotic DNA typically has a single origin.
2. Unwinding of DNA Helix: Helicase unwinds the double helix at the origin, forming a replication bubble with two replication forks moving in opposite directions. Topoisomerase relieves supercoiling.
3. Primer Synthesis: Primase synthesises short RNA primers (complementary to the template strand) on both template strands at the replication fork.
4. Synthesis of New DNA Strands: DNA polymerase adds deoxyribonucleotides to the 3' end of the RNA primer, using the parental strand as a template.
   • Since DNA polymerase works only in the 5' $\rightarrow$ 3' direction, and the two parental strands are antiparallel:
   • One strand (the leading strand) is synthesised continuously in the 5' $\rightarrow$ 3' direction (towards the replication fork).
   • The other strand (the lagging strand) is synthesised discontinuously in short segments called Okazaki fragments. Each Okazaki fragment requires a new RNA primer. Synthesis proceeds in the 5' $\rightarrow$ 3' direction (away from the replication fork).
5. Primer Removal and Fragment Joining: RNA primers are removed by an enzyme (e.g., DNA polymerase I in prokaryotes), and the gaps are filled with DNA by DNA polymerase. DNA ligase joins the Okazaki fragments of the lagging strand together.
6. Proofreading: DNA polymerase has a 3' $\rightarrow$ 5' exonuclease activity that allows it to remove incorrectly paired nucleotides, reducing the error rate.
7. Termination: Replication stops when the replication forks meet or at specific termination sites.

Replication is a highly accurate process due to the specificity of base pairing, the action of DNA polymerase, and its proofreading ability.

*(Image shows a diagram of a replication fork illustrating the unwinding helix, leading strand synthesis (continuous), lagging strand synthesis (discontinuous with Okazaki fragments and primers), and indicating the direction of synthesis and some enzymes)*

Transcription

Transcription is the process of synthesising a complementary RNA molecule from a DNA template. It is the first step in gene expression, where the genetic information encoded in DNA is copied into RNA.

Transcription Unit

A transcription unit is a segment of DNA that is transcribed into RNA. It is defined by three regions:

1. Promoter:
   • A DNA sequence located upstream (towards the 5' end of the coding strand, typically) of the structural gene.
   • It is the binding site for RNA polymerase.
   • It dictates which strand of the DNA helix will be transcribed and where transcription starts.
2. Structural gene:
   • The segment of DNA that contains the genetic information to be transcribed into RNA.
   • In prokaryotes, a structural gene can be polycistronic, meaning it codes for multiple polypeptide chains (proteins).
   • In eukaryotes, a structural gene is usually monocistronic, meaning it codes for a single polypeptide chain. Eukaryotic structural genes also contain coding sequences called exons and non-coding intervening sequences called introns. Introns are removed during RNA processing.
3. Terminator:
   • A DNA sequence located downstream (towards the 3' end of the coding strand) of the structural gene.
   • It signals the end of transcription and causes RNA polymerase to detach from the DNA.

The structural gene's sequence is transcribed. One DNA strand serves as the template strand (or antisense strand, 3' $\rightarrow$ 5' direction), from which the RNA is synthesised in the 5' $\rightarrow$ 3' direction. The other DNA strand is the coding strand (or sense strand, 5' $\rightarrow$ 3' direction). The coding strand sequence is identical to the RNA sequence, except that DNA has Thymine (T) while RNA has Uracil (U).

*(Image shows a diagram of a double-stranded DNA segment illustrating the promoter, structural gene, and terminator regions, indicating the coding and template strands and the direction of transcription)*

Transcription Unit And The Gene

The concept of a gene is broader than just the DNA sequence coding for a polypeptide. The DNA sequence coding for a tRNA or rRNA molecule is also considered a gene.

In terms of a transcription unit, a gene can be defined as the functional unit of inheritance that is transcribed into an RNA molecule. This transcribed RNA can be mRNA (coding for a protein), tRNA, or rRNA.

As mentioned, eukaryotic genes are typically monocistronic (coding for one polypeptide) and interrupted by introns. Prokaryotic genes are often polycistronic (coding for multiple polypeptides from a single mRNA).

Types Of Rna And The Process Of Transcription

There are different types of RNA molecules, each serving a specific function in the cell:

1. Messenger RNA (mRNA): Carries the genetic information from DNA in the nucleus to the ribosomes in the cytoplasm, where it serves as a template for protein synthesis.
2. Ribosomal RNA (rRNA): A structural component of ribosomes and also has catalytic activity (ribozyme) during peptide bond formation in protein synthesis.
3. Transfer RNA (tRNA): Acts as an adapter molecule. It brings specific amino acids to the ribosome during protein synthesis and reads the genetic code on mRNA (anticodon).

The Process of Transcription:

Transcription is catalysed by the enzyme RNA polymerase. The process has three main steps:

1. Initiation:
   • RNA polymerase binds to the promoter region of the DNA.
   • The DNA double helix unwinds briefly at the promoter.
   • RNA polymerase starts synthesising a new RNA strand complementary to the template strand, using ribonucleotide triphosphates as substrates.
2. Elongation:
   • RNA polymerase moves along the DNA template strand, unwinding the helix ahead and synthesising the RNA strand in the 5' $\rightarrow$ 3' direction.
   • The RNA polymerase adds ribonucleotides one by one based on complementary base pairing with the DNA template strand (A pairs with U, T pairs with A, G pairs with C, C pairs with G).
   • The synthesised RNA strand peels away from the DNA template.
3. Termination:
   • RNA polymerase reaches the terminator region on the DNA.
   • The terminator sequence signals the enzyme to stop transcription.
   • RNA polymerase detaches from the DNA, and the newly synthesised RNA molecule is released.

Transcription in Prokaryotes vs. Eukaryotes:

• Prokaryotes:
  • Have a single type of RNA polymerase that transcribes all types of RNA (mRNA, tRNA, rRNA).
  • Transcription occurs in the cytoplasm.
  • Transcription and translation can occur simultaneously (coupled) because there is no nucleus.
  • mRNA is usually polycistronic and does not require much processing.
• Eukaryotes:
  • Have three types of RNA polymerases:
    • RNA polymerase I: Transcribes rRNA (except 5S rRNA).
    • RNA polymerase II: Transcribes mRNA precursors (hnRNA) and some small nuclear RNAs (snRNAs).
    • RNA polymerase III: Transcribes tRNA, 5S rRNA, and some other small RNAs.
  • Transcription occurs in the nucleus.
  • Transcription and translation are separated in space and time.
  • mRNA precursor (hnRNA) is monocistronic and contains both exons and introns. It undergoes post-transcriptional modifications (RNA processing).

---

Post-Transcriptional Modifications in Eukaryotes:

The primary transcript (hnRNA) in eukaryotes is non-functional and needs to be processed before it can be translated. This involves:

• Splicing: Introns (non-coding regions) are removed from the hnRNA, and exons (coding regions) are joined together. This process is catalysed by a complex called the spliceosome.
• Capping: An unusual nucleotide, methylguanosine triphosphate, is added to the 5' end of the hnRNA. This cap is important for ribosome binding and protection from degradation.
• Tailing: A tail of about 200-300 adenine nucleotides (poly-A tail) is added to the 3' end of the hnRNA. This tail is important for mRNA stability and export from the nucleus.

After these modifications, the hnRNA becomes mature mRNA, which is then transported out of the nucleus to the cytoplasm for translation.

*(Image shows a diagram illustrating a hnRNA molecule with exons and introns, showing the removal of introns (splicing), addition of a 5' cap and a 3' poly-A tail to form mature mRNA)*

Genetic Code

The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequence) is translated into proteins (amino acid sequence). It defines the correspondence between nucleotide triplets (codons) and amino acids.

Salient Features of the Genetic Code:

1. Triplet Codon: The code is read in units of three consecutive nucleotides called codons. A sequence of three nucleotides specifies one amino acid.
2. 64 Codons: Since there are 4 different nucleotide bases (A, U, G, C) and codons are triplets, there are $4^3 = 64$ possible codons.
3. Amino Acids: These 64 codons code for 20 different amino acids.
4. Degeneracy: Most amino acids are coded by more than one codon. This is called degeneracy of the code. For example, both UUU and UUC code for Phenylalanine.
5. Non-overlapping: The code is read sequentially in groups of three nucleotides without any overlap. Each nucleotide is part of only one codon.
6. Commaless: There are no punctuation marks or gaps between codons. The code is read continuously.
7. Universal: The genetic code is largely universal across almost all organisms, from bacteria to humans. A codon specifies the same amino acid in almost all species. (Some minor exceptions exist, e.g., in mitochondria and some protozoans).
8. Start Codon: AUG serves as the start codon, initiating protein synthesis. It also codes for the amino acid Methionine (Met) in eukaryotes and formyl-Methionine in prokaryotes.
9. Stop Codons (Termination Codons): Three codons (UAA, UAG, UGA) do not code for any amino acid. They signal the termination of protein synthesis. They are also called non-sense codons.

*(Image shows a standard genetic code table listing the 64 codons and the amino acids they specify, including start and stop codons)*

Mutations And Genetic Code

Changes in the genetic material (mutations) can affect the genetic code and the protein synthesised.

• Point Mutations: Changes in a single nucleotide.
  • Substitution: Replacing one nucleotide with another.
    • Silent mutation: If the new codon codes for the same amino acid (due to degeneracy). No effect on protein sequence.
    • Missense mutation: If the new codon codes for a different amino acid. Can change protein function (e.g., Sickle cell anaemia - a single base substitution changes a codon from GAG (Glutamic acid) to GUG (Valine), altering the beta-globin protein).
    • Nonsense mutation: If the new codon is a stop codon. Leads to premature termination of protein synthesis, usually resulting in a non-functional protein.
• Frameshift Mutations: Insertions or deletions of nucleotides (that are not in multiples of three).
  • These mutations shift the reading frame of the codons downstream of the mutation.
  • This results in a completely different sequence of amino acids being incorporated, usually leading to a non-functional protein.
  • Frameshift mutations can be very detrimental.

The effect of mutations on the phenotype depends on the type of mutation and its location in the gene.

Trna– The Adapter Molecule

tRNA (transfer RNA) plays a crucial role in translating the genetic code. It acts as an adapter molecule that links the genetic code (codon on mRNA) to the corresponding amino acid.

Structure of tRNA:

• tRNA is a small RNA molecule.
• In its 2-dimensional structure, it resembles a cloverleaf model with several loops:
  • Acceptor stem (at the 3' end, where the amino acid binds).
  • Anticodon loop (contains the anticodon, which pairs with the mRNA codon).
  • D loop (contains dihydrouridine).
  • T$\Psi$C loop (contains pseudouridine $\Psi$).
  • Variable loop.
• In its 3-dimensional structure, tRNA has an inverted L-shape.

*(Image shows diagrams of the cloverleaf 2D structure and the inverted L 3D structure of tRNA)*

---

Function of tRNA:

• Reads the codon: The anticodon loop contains an anticodon sequence of three nucleotides that is complementary to a specific codon on the mRNA. This ensures the correct amino acid is matched to the codon.
• Carries the amino acid: The 3' end of the tRNA molecule has an amino acid acceptor site, to which the corresponding amino acid is attached.
• The attachment of the amino acid to the tRNA is catalysed by a specific enzyme called aminoacyl-tRNA synthetase (also called amino acid activating enzyme). This process is called charging of tRNA or aminoacylation. The enzyme is specific for both the amino acid and its corresponding tRNA.

---

tRNA molecules are essential intermediaries in the process of translation, ensuring the accurate synthesis of proteins according to the genetic code.

Translation

Translation is the process of synthesising a polypeptide chain (protein) using the information encoded in a messenger RNA (mRNA) molecule. This process occurs in the ribosomes, which serve as the cellular machinery for protein synthesis.

Components Involved in Translation:

• mRNA: Carries the codons (genetic information) from DNA.
• Ribosomes: Provide the site for protein synthesis. Consist of rRNA and ribosomal proteins. Have two subunits (small and large) that come together during translation. Ribosomes have binding sites for mRNA and tRNA.
• tRNA: Adapter molecules that bring specific amino acids to the ribosome and recognise codons on mRNA via their anticodons.
• Amino acids: The building blocks of proteins.
• Aminoacyl-tRNA synthetases: Enzymes that catalyse the attachment of amino acids to their corresponding tRNAs (charging of tRNA).
• Energy sources: ATP (for charging tRNA) and GTP (for initiation and elongation steps on the ribosome).
• Initiation factors, elongation factors, termination factors: Proteins that regulate the different stages of translation.

Steps of Translation:

Translation proceeds in three main stages:

1. Initiation:
   • The small ribosomal subunit binds to the mRNA molecule. In prokaryotes, this binding occurs at a specific sequence near the start codon (Shine-Dalgarno sequence). In eukaryotes, it binds to the 5' cap.
   • The initiator tRNA (carrying Methionine, or formyl-Methionine in prokaryotes) binds to the start codon (AUG) on the mRNA. This occurs at the P site (peptidyl site) on the ribosome.
   • The large ribosomal subunit then joins the complex, forming the complete ribosome with the mRNA positioned in the groove between the subunits.
2. Elongation:
   • A new charged tRNA (carrying the next amino acid) enters the A site (aminoacyl site) on the ribosome, binding to the next codon on the mRNA.
   • A peptide bond is formed between the amino acid carried by the tRNA at the A site and the growing polypeptide chain attached to the tRNA at the P site. This reaction is catalysed by peptidyl transferase, a ribozyme (part of the large rRNA).
   • The ribosome moves along the mRNA in the 5' $\rightarrow$ 3' direction by one codon. This movement is called translocation and requires GTP and elongation factors.
   • The tRNA that was at the P site (now carrying the growing polypeptide) moves to the E site (exit site) and leaves the ribosome. The tRNA that was at the A site (now holding the polypeptide chain) moves to the P site. The A site is now free to receive the next charged tRNA.
   • This cycle of charged tRNA binding to A site, peptide bond formation, and translocation continues, adding amino acids sequentially to the polypeptide chain.
3. Termination:
   • Elongation continues until the ribosome reaches a stop codon (UAA, UAG, or UGA) on the mRNA.
   • Stop codons are not recognised by any tRNA.
   • Release factors bind to the stop codon, which causes the release of the completed polypeptide chain from the ribosome.
   • The ribosome then dissociates into its small and large subunits, and the mRNA is released.

*(Image shows diagrams illustrating the steps of translation: ribosome binding to mRNA and initiator tRNA (initiation), sequential addition of amino acids and movement of ribosome (elongation), and release of polypeptide at stop codon (termination))*

Polysome:

A single mRNA molecule can be simultaneously translated by multiple ribosomes. A complex formed by an mRNA molecule and several ribosomes translating it is called a polysome or polyribosome. Polysomes allow for the rapid synthesis of multiple copies of the same protein.

Regulation Of Gene Expression

Not all genes are expressed in all cells at all times. The process by which the expression of genes is controlled to produce the necessary proteins at the right time and in the right amount is called regulation of gene expression.

Gene expression can be regulated at various levels:

• Transcriptional level: Controlling the rate of transcription (formation of primary transcript).
• Processing level (in eukaryotes): Regulating splicing, capping, tailing.
• Transport level (in eukaryotes): Regulating mRNA export from the nucleus to the cytoplasm.
• Translational level: Controlling the rate of translation (protein synthesis).
• Post-translational level: Modifying the protein after synthesis.

Regulation of gene expression is crucial for differentiation, development, and adaptation to the environment.

The Lac Operon

The lac operon is a classic example of transcriptional regulation in prokaryotes (bacteria), studied by Francois Jacob and Jacques Monod. It regulates the expression of genes involved in the metabolism of lactose in *E. coli*.

Components of the Lac Operon:

The lac operon consists of:

• Regulatory gene (i gene): Located upstream of the operon. It codes for the repressor protein.
• Promoter (p): The binding site for RNA polymerase to initiate transcription.
• Operator (o): The binding site for the repressor protein. It is located adjacent to the promoter.
• Structural genes: Genes that are transcribed together to produce enzymes for lactose metabolism.
  • lacZ: Codes for $\beta$-galactosidase, which hydrolyses lactose into glucose and galactose.
  • lacY: Codes for permease, which increases the permeability of the bacterial cell to lactose.
  • lacA: Codes for transacetylase (function less clear in lactose metabolism).

*(Image shows a diagram illustrating the arrangement of the i gene, promoter, operator, lacZ, lacY, and lacA genes in the lac operon)*

---

Regulation of the Lac Operon (Negative Regulation):

The lac operon is under negative control by the repressor protein.

• In the absence of an inducer (lactose):
  • The regulatory gene (i gene) constitutively (continuously) produces the repressor protein.
  • The repressor protein binds to the operator region of the operon.
  • Binding of the repressor to the operator blocks the binding of RNA polymerase to the promoter, preventing transcription of the structural genes. The operon is switched OFF.
• In the presence of an inducer (lactose or allolactose):
  • Lactose (or its isomer allolactose) acts as an inducer. It binds to the repressor protein.
  • Binding of the inducer causes a conformational change in the repressor protein, making it unable to bind to the operator.
  • RNA polymerase can now bind to the promoter and initiate transcription of the structural genes (lacZ, lacY, lacA). The operon is switched ON.
  • This leads to the synthesis of $\beta$-galactosidase, permease, and transacetylase, allowing the cell to metabolise lactose.

*(Image shows two diagrams side-by-side: one showing the repressor binding to the operator when lactose is absent, blocking transcription; the other showing lactose binding to the repressor, releasing it from the operator, allowing transcription)*

---

Positive Regulation (Catabolite Repression):

• The lac operon is also regulated positively by CAP (Catabolite Activator Protein) in the presence of glucose.
• When glucose is absent, cAMP levels are high. cAMP binds to CAP, forming a complex that binds to the promoter and enhances the binding of RNA polymerase, promoting transcription.
• When glucose is present, cAMP levels are low. CAP does not bind to the promoter effectively, and transcription of the lac operon is low even if lactose is present. This is known as catabolite repression, ensuring the cell preferentially uses glucose.

The lac operon demonstrates a finely tuned regulatory mechanism that allows bacteria to utilise lactose efficiently only when glucose is absent and lactose is available.

Human Genome Project

The Human Genome Project (HGP) was an international research project that aimed to sequence and map all of the genes within the entire human genome (all the DNA in a human cell). It was a landmark project in the history of biology, often referred to as a 'mega project'. It was launched in 1990 and completed in 2003.

Goals Of HGP

The major goals of the Human Genome Project were:

• To sequence the entire 3.16 billion base pairs of the human genome.
• To identify and map all the human genes (estimated 20,000-25,000 initially, later refined).
• To store this information in databases.
• To develop tools and techniques for data analysis.
• To transfer related technologies to other sectors, such as industries.
• To address the ethical, legal, and social issues (ELSI) that arose from the project.

Methodologies

Two major approaches were involved in the HGP:

• Expressed Sequence Tags (ESTs): Identifying genes that are expressed as RNA. This approach focused only on sequencing the coding regions of the genome.
• Sequence Annotation: Sequencing the entire genome (both coding and non-coding regions). This involved:
  1. Isolating the total DNA from a cell.
  2. Fragmenting the DNA into smaller, manageable pieces.
  3. Cloning the fragments in suitable vectors (e.g., Bacterial Artificial Chromosomes - BACs, Yeast Artificial Chromosomes - YACs) in host organisms (bacteria or yeast). This amplified the DNA fragments.
  4. Sequencing the cloned fragments using automated DNA sequencers (based on the principle developed by Frederick Sanger).
  5. Arranging the sequences based on overlapping regions (like solving a puzzle).
  6. Annotating the sequences by assigning functions to the identified genes.

*(Image shows a simplified diagram illustrating the process: isolating DNA, breaking into pieces, inserting into vectors, cloning in bacteria, sequencing fragments, and assembling the sequences based on overlaps)*

Salient Features Of Human Genome

Some key findings from the Human Genome Project:

• The human genome contains approximately 3.1647 billion base pairs.
• The average gene consists of 3000 bases, but sizes vary greatly (largest known human gene is Dystrophin, over 2.4 million bases).
• The total number of genes is estimated to be approximately 20,000 - 25,000, much lower than earlier estimates.
• More than 50% of the human genome consists of repetitive sequences (sequences that are repeated many times). These sequences do not directly code for proteins but have various functions, including structural roles and contributing to variation. They were initially referred to as 'junk DNA' but are now known to have important regulatory roles.
• Less than 2% of the genome codes for proteins.
• About 99.9% of the nucleotide bases are exactly the same in all people. The variations at single nucleotide positions (Single Nucleotide Polymorphisms or SNPs, pronounced 'snips') are responsible for individual differences.
• Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231).

Applications And Future Challenges

The HGP has opened up new avenues in biology and medicine.

• Applications:
  • Understanding human biology: Provides insights into gene function, regulation, and interactions.
  • Disease identification and diagnosis: Helps identify genes associated with various diseases, improving diagnostic tools.
  • Developing new therapies: Leads to the development of targeted therapies, including gene therapy and personalised medicine.
  • Risk assessment: Helps assess an individual's risk of developing certain diseases.
  • Evolutionary studies: Provides data to study human evolution and migration patterns.
• Future Challenges:
  • Understanding gene function: Identifying all genes is only the first step; understanding what they do (functional genomics).
  • Proteomics: Studying the complete set of proteins produced by an organism.
  • Interpreting complex interactions: Understanding how genes interact with each other and the environment.
  • Ethical, Legal, and Social Implications (ELSI): Issues related to privacy of genetic information, potential for discrimination, use of genetic data in healthcare and society.

The HGP was a monumental achievement that has transformed biological research and holds immense promise for improving human health.

Dna Fingerprinting

DNA fingerprinting is a technique used to identify individuals at the molecular level based on the unique patterns in their DNA. It exploits the fact that the DNA sequence is largely the same among individuals, but there are certain regions that show a high degree of variation.

Basis of DNA Fingerprinting:

• The technique was developed by Alec Jeffreys. In India, Dr. Lalji Singh is considered the father of DNA fingerprinting.
• The basis is the presence of repetitive DNA sequences in the genome. These sequences are repeated many times and do not code for proteins.
• A type of repetitive DNA is satellite DNA, which can be classified into mini-satellites and micro-satellites based on the length and number of repetitions.
• Mini-satellites often contain sequences called Variable Number Tandem Repeats (VNTRs). VNTRs are short DNA sequences that are repeated tandemly (one after another). The number of repetitions of a particular VNTR sequence varies from person to person (except identical twins), making these regions highly polymorphic.
• The VNTRs are inherited from parents, but their specific pattern (allele) at different loci in an individual is unique.

*(Image shows diagrams of DNA from two different individuals, illustrating different loci and showing varying numbers of tandem repeats (VNTRs) at those loci)*

Steps in DNA Fingerprinting:

The technique involves several steps:

1. Isolation of DNA: DNA is extracted from any tissue or cell (e.g., blood, hair follicle, skin, semen).
2. Digestion of DNA: The DNA is cut into smaller fragments using restriction enzymes. Restriction enzymes cut DNA at specific recognition sequences. The fragments will vary in size depending on the location of these sites relative to the VNTRs.
3. Separation by Gel Electrophoresis: The DNA fragments are separated based on their size by electrophoresis through an agarose gel. Smaller fragments move faster and further than larger fragments.
4. Blotting (Southern Blotting): The separated DNA fragments are transferred from the gel onto a nylon or nitrocellulose membrane (a solid support). This process is called Southern blotting.
5. Hybridisation: The membrane is incubated with a labelled VNTR probe. A probe is a single-stranded DNA sequence complementary to the VNTR sequence, labelled with a radioactive isotope (e.g., $^{32}P$) or a fluorescent dye. The probe hybridises (binds) only to the complementary VNTR fragments on the membrane.
6. Detection (Autoradiography): The membrane is exposed to X-ray film (if radioactive label) or viewed under UV light (if fluorescent label). The bound probe reveals the positions of the VNTR fragments, creating a pattern of bands called a DNA fingerprint.

The pattern of bands is unique for each individual (except identical twins).

*(Image shows a step-by-step diagram illustrating the DNA fingerprinting process)*

Applications of DNA Fingerprinting:

• Forensic Science: Identifying suspects or victims in criminal investigations by comparing DNA from crime scene samples (blood, semen, hair) with suspect DNA.
• Paternity Testing: Determining biological parentage by comparing the DNA fingerprint of a child with that of the alleged father and mother.
• Identification of individuals: In cases of mass casualties, disasters, or identifying unknown remains.
• Conservation Biology: Studying genetic diversity within populations, identifying endangered species, wildlife forensics (identifying source of poached animal products).
• Evolutionary Biology: Studying evolutionary relationships and migration patterns.

Example 4. A crime scene sample (S) is collected. Three suspects (A, B, C) are apprehended. DNA fingerprinting is performed on all four samples. The resulting band patterns are compared. Based on the following hypothetical result, which suspect is most likely linked to the crime scene?

*(Image shows four vertical lanes labelled S, A, B, C, with hypothetical bands in each lane. Assume Suspect B's bands match the Crime Scene Sample bands)*

Answer:

DNA fingerprinting is a powerful and reliable technique for identification due to the high variability of repetitive DNA sequences among individuals.