Basic Processes Chapter Notes | Biology for Grade 11 PDF Download

DNA as the Genetic Material

• Genes, responsible for trait expression, are located on chromosomes composed of nucleic acids and proteins.
• Deoxyribonucleic acid (DNA) determines traits in all organisms except some viruses, where RNA is the genetic material.
• Johann Friedrich Miescher first isolated an acidic substance from pus cell nuclei, naming it nuclein, which contained DNA and protein.
• DNA and proteins were considered potential genetic materials due to their presence in chromosomes and nuclei.
• Experiments with microorganisms provided evidence supporting DNA as the genetic material.

Discovery of the Transforming Principle

• In 1928, Frederick Griffith observed transformation while developing a pneumonia vaccine using Streptococcus pneumoniae.
• Griffith identified two strains: virulent smooth (S) strain with a polysaccharide capsule and non-virulent rough (R) strain without the capsule.
• Injecting live S bacteria into mice caused pneumonia and death, while R bacteria caused no harm.
• Heat-killed S bacteria did not kill mice, but a mixture of heat-killed S and live R bacteria caused death due to pneumonia.
• Examination revealed live S-type bacteria in dead mice, indicating R bacteria transformed into virulent S bacteria.
• Griffith termed this phenomenon transformation, where genetic material transfer alters the recipient cell’s genetic makeup.
• The nature of the transforming substance remained unidentified at this stage.

Biochemical Characterisation of Transforming Principle

• Oswald T. Avery, Colin Macleod, and Maclyn McCarty conducted experiments in 1944 to identify the transforming principle.
• They prepared an extract from heat-killed S bacteria, removing lipids and carbohydrates, retaining DNA, RNA, and proteins.
• The extract was divided into three parts, each treated with enzymes: ribonuclease (degrades RNA), deoxyribonuclease (degrades DNA), or protease (degrades protein).
• Transformation occurred in cultures with RNase- and protease-treated extracts but not in DNase-treated extracts.
• These results confirmed DNA as the transforming principle.

The Hershey - Chase Experiment

• In 1952, Alfred Hershey and Martha Chase used T2 bacteriophages to confirm DNA as the genetic material.
• T2 bacteriophages, which infect E. coli, consist of DNA encased in a protein coat.
• Phages were grown in media with radioactive phosphorus (³²P) to label DNA or radioactive sulfur (³⁵S) to label proteins.
• ³²P-labeled phages infected E. coli, and after blending and centrifugation, radioactivity was found in the bacterial pellet, indicating DNA entry.
• ³⁵S-labeled phages showed radioactivity in the supernatant, indicating proteins remained outside the bacteria.
• This confirmed that DNA, not protein, is the genetic material responsible for phage multiplication.
• Subsequent studies by Erwin Chargaff, Maurice Wilkins, Rosalind Franklin, James Watson, and Francis Crick elucidated DNA’s structure, explaining its information storage capacity.

Prokaryotic and Eukaryotic Gene Organisation

• Genes, units of inheritance, are carried by DNA in most organisms (RNA in some viruses).
• Gene organization and function differ between prokaryotes and eukaryotes.

Gene Organization in Prokaryotes

• Prokaryotes lack a membrane-bound nucleus; their circular, double-stranded DNA resides in the nucleoid region.
• The large circular DNA is compacted via supercoiling, where the DNA twists into coils and folds into a condensed form.
• Supercoiling can be negative (opposite to the double helix direction) or positive (same direction), with most bacterial genomes negatively supercoiled.
• Prokaryotes may have plasmids, smaller DNA loops not essential for normal growth.
• Proteins like HLU (histone-like protein) aid in condensing prokaryotic DNA.

Gene Organization in Eukaryotes

• Eukaryotes have a membrane-bound nucleus containing linear, double-stranded DNA.
• DNA is packaged by wrapping around histone proteins to form nucleosomes, consisting of a histone octamer (two each of H2A, H2B, H3, H4).
• Nucleosomes appear as beads on a string, connected by linker DNA; H1 histone binds linker DNA.
• Nucleosomes (10 nm) stack into 30 nm fibers, forming loops (300 nm) and condensing into chromosomes (700 nm) during metaphase.
• The genome includes nuclear DNA and organellar DNA (mitochondria and chloroplasts).
• Nuclear DNA contains coding and non-coding regions; only ~2% of the human genome (3 billion base pairs) codes for ~20,000 genes.
• Organellar genomes are circular, double-stranded, and present in multiple copies, encoding organelle-specific functions.
• Most eukaryotic genome is non-coding, with repetitive sequences varying in size and number.
• Early cytological studies revealed chromatin’s bead-on-string structure, later identified as nucleosomes (~200 bp, including 146 bp DNA and linker).
• A gene is a DNA segment with a promoter for RNA polymerase binding, transcribed into mRNA for translation.
• Eukaryotic genes often contain introns (non-coding) and exons (coding), with primary transcripts undergoing splicing to remove introns.

DNA Replication

• James Watson and Francis Crick proposed DNA’s double-helix structure in 1953, suggesting semiconservative replication based on base complementarity.
• In semiconservative replication, parental DNA strands separate, each serving as a template for a new complementary strand, forming two identical daughter duplexes.
• Each daughter duplex contains one parental and one newly synthesized strand.

Messelson and Stahl Experiment

• In 1958, Mathew Messelson and Franklin Stahl confirmed semiconservative replication using E. coli and nitrogen isotopes (¹⁴N and ¹⁵N).
• E. coli grown in ¹⁵N medium incorporated heavy nitrogen into DNA, producing a dense band in density gradient centrifugation.
• E. coli grown in ¹⁴N medium produced a lighter DNA band.
• After transferring ¹⁵N-grown E. coli to ¹⁴N medium, DNA after one replication showed an intermediate density band (one ¹⁵N and one ¹⁴N strand).
• After two replications, two bands appeared: one intermediate and one light (¹⁴N only).
• Subsequent replications increased the light band’s intensity, confirming semiconservative replication.
• Density gradient centrifugation used cesium chloride to separate DNA based on density.
• Taylor’s 1958 experiment with Vicia faba using radioactive thymidine also confirmed semiconservative replication in chromosomes.

The Machinery of Replication

• DNA replication involves unwinding the double helix and synthesizing new strands using enzymes and proteins.
• DNA Polymerases: Synthesize new DNA strands in the 5’→3’ direction, forming phosphodiester bonds. In prokaryotes, DNA polymerase III is the main enzyme, with 3’→5’ exonuclease activity for error correction. DNA polymerase I removes RNA primers, and DNA polymerase II repairs DNA. Eukaryotes have multiple polymerases for synthesis and repair.
• Primase: A DNA-dependent RNA polymerase that synthesizes short RNA primers (10-12 nucleotides) to initiate DNA synthesis, as DNA polymerase requires a 3’ OH group.
• Helicases: Unwind the DNA double helix by breaking hydrogen bonds using ATP energy.
• Topoisomerases: Relieve torsional strain by nicking and religating DNA to prevent knotting ahead of the replication fork.
• Single Strand Binding (SSB) Proteins: Stabilize single-stranded DNA to prevent reannealing during replication.
• DNA Ligase: Joins DNA fragments by forming phosphodiester bonds between 3’ OH and 5’ phosphate groups.

Mechanism of DNA Replication

• In prokaryotes, replication begins at a specific origin of replication (oriC in E. coli) on circular, double-stranded DNA.
• Initiator proteins bind and unwind DNA at the origin, followed by helicase separating strands, forming replication forks.
• Replication is bidirectional, with forks moving in opposite directions from the origin.
• SSB proteins stabilize single-stranded DNA, and topoisomerase reduces strain ahead of the fork.
• DNA polymerase III synthesizes new strands in the 5’→3’ direction, antiparallel to the template.
• Primase synthesizes RNA primers to provide a 3’ OH group for DNA polymerase.
• The leading strand is synthesized continuously in the 5’→3’ direction on the 3’→5’ template.
• The lagging strand is synthesized discontinuously on the 5’→3’ template, forming Okazaki fragments (1000-2000 nucleotides) starting with RNA primers.
• DNA polymerase I removes RNA primers and replaces them with DNA nucleotides.
• DNA ligase joins Okazaki fragments by forming phosphodiester bonds.
• Replication terminates at a specific site opposite the origin, where a termination protein blocks helicase, stopping the replication fork.
• Eukaryotic replication is similar but involves multiple origins on linear chromosomes, with simultaneous bidirectional replication from each origin.

Gene Expression

• A gene is a DNA segment carrying information for trait expression, converted into a polypeptide’s amino acid sequence.
• Gene expression follows the central dogma: DNA → RNA (transcription) → polypeptide (translation).
• Transcription transfers genetic information from DNA to mRNA.
• Translation converts mRNA’s nucleotide sequence into a polypeptide’s amino acid sequence in ribosomes.
• mRNA amplifies gene information by producing multiple copies from one DNA gene.
• In eukaryotes, mRNA moves from the nucleus to the cytoplasm for translation, as ribosomes are cytoplasmic.
• In retroviruses, RNA genetic material undergoes reverse transcription to form single-stranded DNA, then double-stranded DNA, followed by transcription to mRNA and translation.

Transcription

• Transcription involves synthesizing RNA from a DNA template, occurring in prokaryotes and eukaryotes with differences.
• Initiation: RNA polymerase binds to the promoter, unwinds DNA, and forms a transcription bubble.
• Elongation: RNA polymerase adds nucleotides to the 3’ OH end of the growing RNA chain at ~40 nucleotides/second in bacteria.
• Termination: At the terminator sequence, RNA polymerase dissociates, releasing the RNA and allowing DNA to rewind. Prokaryotic termination may be rho-dependent or rho-independent.
• In prokaryotes, multiple genes can be transcribed into polycistronic RNA, with translation often starting before transcription completes.
• In eukaryotes, transcription requires transcription factors for RNA polymerase I, II, and III to initiate at rRNA, mRNA, and tRNA promoters, respectively.
• Eukaryotic genes often contain introns and exons, with primary transcripts (hnRNA) undergoing post-transcriptional modifications:
  • Capping: Addition of a 5’ methyl G cap (GTP with a methyl group) to protect mRNA from exonuclease degradation.
  • Splicing: Removal of introns and joining of exons to form mature mRNA.
  • Poly-A Tail: Addition of adenine nucleotides at the 3’ end to enhance mRNA stability.

Genetic Code

• The genetic code translates mRNA’s nucleotide sequence into a polypeptide’s amino acid sequence.
• Unlike replication and transcription, translation lacks complementarity between nucleotides and amino acids.
• Francis Crick hypothesized in 1961 that base sequences carry genetic information.
• George Gamow proposed that three nucleotides (codons) code for one amino acid, as four bases yield 64 codons (4³), sufficient for 20 amino acids.
• Marshall Nirenberg and Johann Matthaei deciphered the first codon in 1961, showing UUU codes for phenylalanine using poly(U) RNA.
• Similar experiments showed CCC codes for proline and AAA for lysine.
• Nirenberg, Matthaei, Leder, Ochoa, and Khorana deciphered all 64 codons.
• Features of the Genetic Code:
  • Triplet codons: 64 codons, with 61 coding for 20 amino acids.
  • Stop codons: UAA, UAG, UGA signal translation termination.
  • AUG: Codes for methionine and serves as the initiator codon.
  • Unambiguous: Each codon codes for one amino acid.
  • Degenerate: Most amino acids are coded by multiple codons, except methionine and tryptophan (single codon each).
  • Non-overlapping: Each base is part of only one codon.
  • Universal: Same code applies from bacteria to humans, with exceptions in mitochondrial and some protozoan codons.

Translation

• Charging of tRNA: tRNA’s anticodon base-pairs with mRNA’s codon, carrying a specific amino acid (e.g., tRNA^met for methionine). Aminoacyl-tRNA synthetase activates amino acids with ATP and attaches them to tRNA’s 3’ CCA end. Wobble pairing allows one tRNA to recognize multiple codons due to flexible base pairing at the codon’s third position.
• Initiation: In prokaryotes, the 30S ribosomal subunit binds the Shine-Dalgarno sequence, positioning AUG in the P site. fMet-tRNA^fmet (formylated methionine) pairs with AUG, and the 50S subunit forms the 70S complex with initiation factors and GTP. In eukaryotes, the 40S subunit binds the 5’ cap, scans for AUG, and uses Met-tRNA^met.
• Elongation: A second aminoacyl-tRNA enters the A site, and peptidyl transferase forms a peptide bond between P and A site amino acids. The ribosome translocates, moving the uncharged tRNA to the E site, peptidyl-tRNA to the P site, and a new codon to the A site. Elongation factors and GTP facilitate this process.
• Termination: Stop codons (UAA, UAG, UGA) enter the A site, recognized by release factors, which release the polypeptide and dissociate the ribosome, mRNA, and tRNA.

Post-translational modifications (PTMs) by enzymes produce mature proteins.
Polyribosomes form when multiple ribosomes translate a single mRNA, increasing polypeptide production.

Gene Mutation

• Mutations are sudden changes in genetic material (DNA or RNA in some viruses) during processes like replication or cell division.
• Mutations in germinal cells are heritable, while somatic mutations are not.
• Chromosomal Mutations: Include aberrations (loss, addition, or rearrangement of chromosome segments) or ploidy changes (aneuploidy or polyploidy). Mutagens like ionizing radiation or chemicals induce aberrations, detectable by banding or FISH.
• Gene (Point) Mutations: Alterations in DNA’s nucleotide sequence, affecting gene expression (e.g., sickle cell anemia due to a single nucleotide substitution).

Types of Gene Mutations:

• Addition: Insertion of nucleotides, shifting the reading frame.
• Deletion: Removal of nucleotides, altering the reading frame.
• Substitution: Replacement of one nucleotide, either transition (purine to purine or pyrimidine to pyrimidine) or transversion (purine to pyrimidine or vice versa).

Addition and deletion cause frameshift mutations, altering RNA transcription and polypeptide synthesis, potentially producing non-functional proteins.

Molecular Mechanism of Mutation

• Spontaneous Mutations: Tautomeric shifts in nitrogenous bases (keto to enol or amino to imino forms) alter base-pairing properties. For example, guanine’s imino form pairs with thymine instead of cytosine, leading to a G=C to A=T substitution after replication (e.g., sickle cell anemia).
• Induced Mutations: Caused by:
  • Physical Mutagens: X-rays break phosphodiester bonds, causing deletions and frameshift mutations. UV rays excite electrons, inducing deletions or substitutions.
  • Chemical Mutagens: Base analogs like 5-bromouracil pair with adenine in keto form but guanine in enol form, causing A=T to G=C substitutions. Alkylating agents like EMS ethylate nucleotides, altering pairing (e.g., guanine pairs with thymine, mutating G=C to A=T).

DNA Repair

• Base Excision Repair (BER): DNA glycosylase removes damaged bases (caused by oxidation, alkylation, or deamination), creating an AP site. Endonuclease cleaves the site, DNA polymerase adds correct bases, and DNA ligase seals the gap.
• Nucleotide Excision Repair (NER): Repairs larger DNA damage, like UV-induced pyrimidine (T-T) dimers. The UVr complex scans DNA, cleaves phosphodiester bonds around the damage, removes the fragment, and DNA polymerase I and ligase restore the correct sequence.

Mismatch Repair (MMR): Corrects replication errors using MutH, MutL, MutS, and MutT proteins to cleave mismatched segments (~1,000 nucleotides), followed by DNA polymerase and ligase to fill and seal the gap.
Other repair mechanisms exist in prokaryotes and eukaryotes, ensuring genetic information stability.

Regulation of Gene Expression

• In multicellular organisms, all cells have identical genes, but different cell types express specific genes based on function.
• Gene expression is regulated by switching genes on or off, controlling protein production.
• Bacteria like E. coli regulate gene expression based on environmental nutrients (e.g., expressing glucose or lactose metabolism genes).
• Regulation occurs at chromatin, transcription, mRNA processing, mRNA transport, or translation levels.
• Constitutive Genes: Expressed constantly (e.g., citric acid cycle enzymes), called housekeeping genes.
• Regulated Genes: Expression varies based on cellular needs, controlled by molecular signals.

Regulation of Gene Expression in Bacteria

• Bacterial genes with related functions are clustered in operons, transcribed into a single mRNA for coordinated control.
• An operon includes structural genes (encoding metabolic proteins), a promoter (RNA polymerase binding site), an operator (repressor binding site), and a regulator gene (encoding a repressor protein).
• The repressor protein binds the operator, blocking RNA polymerase and preventing transcription.
• Francois Jacob and Jacques Monod proposed the operon model in 1961 for lactose metabolism in E. coli.

Inducible Operons: Activated by inducers (substrates) that bind and inactivate the repressor, allowing transcription (e.g., lactose induces enzyme production).
Repressible Operons: Inhibited by corepressors (end products) that activate the repressor, blocking transcription (e.g., histidine represses its synthesis enzymes).
Transcriptional Control: Negative control (repressor inhibits transcription) or positive control (activator stimulates transcription).

The Lac Operon - an Inducible Operon

• The lac operon in E. coli regulates lactose metabolism, producing enzymes when lactose is present.
• It includes three structural genes: lacZ (encodes β-galactosidase, breaks lactose into glucose and galactose, converts lactose to allolactose), lacY (encodes β-galactoside permease for lactose transport), and lacA (encodes β-galactoside transacetylase, function unclear).
• The lacI regulator gene, upstream of the promoter, encodes a repressor protein with binding sites for the operator and allolactose (inducer).
• In the absence of lactose, the active repressor binds the operator, blocking RNA polymerase and preventing transcription (negative control).
• When lactose is present, β-galactosidase converts some to allolactose, which binds the repressor, inactivating it and allowing RNA polymerase to transcribe the genes.
• Positive control occurs when an activator, activated by an inducer, binds DNA to enhance RNA polymerase binding and transcription.