MOLECULAR BASIS OF INHERITANCE

Nucleotide = Base + Sugar + Phosphate; Nucleoside = Base + Sugar (NO phosphate).

Purines = A & G (double ring); Pyrimidines = C, T, U (single ring).

Nuclein first discovered by Friedrich Miescher (1869).

Chargaff's Rule — A=T, G≡C; ratio constant & equals one for dsDNA only.

A=T (2 H-bonds), G≡C (3 H-bonds); purine always opposite pyrimidine.

DNA is negatively charged (acidic); histones are positively charged (basic).

Central Dogma — Francis Crick; DNA → RNA → Protein. Reverse flow in some viruses.

φ×174 = 5386 nucleotides; λ = 48502 bp; E. coli = 4.6×10⁶ bp; Human haploid = 3.3×10⁹ bp.

Mammalian cell DNA length = ~2.2 metres; Nucleus = ~10⁻⁶ m (DNA far greater than nucleus!).

In E. coli, negatively charged DNA held with positively charged non-histone proteins in nucleoid.

Histones = positively charged, basic, rich in Lysine & Arginine.

Histones are enriched with Lysine & Arginine.

Histones are organised to form a unit of eight molecules called histone octamer.

Histones are positively charged basic proteins rich in basic amino acid residues.

Nucleosome = DNA wrapped around histone octamer; 200 bp per nucleosome; beads-on-string.

Negatively charged DNA is wrapped around the positively charged histone octamer to form the nucleosome.

Euchromatin = active, light staining; Heterochromatin = inactive, dark staining.

Packaging of chromatin at higher levels requires an additional set of proteins called non-histone chromosomal (NHC) proteins.

Euchromatin = loosely packed and light-stained; Heterochromatin = densely packed and dark-stained.

Griffith — Streptococcus pneumoniae; transformation experiment. Limitation: biochemical nature NOT defined.

Frederick Griffith ↔ Streptococcus pneumoniae.

Hershey-Chase — unequivocal proof; ³²P labels DNA, ³⁵S labels protein.

If DNA had S and protein had P, then bacterial cells would contain radioactive Sulphur after infection.

Alfred Hershey & Martha Chase ↔ confirmation of DNA as the genetic material.

RNA mutates faster; viruses with RNA genome evolve faster.

Ribozyme = catalytic RNA; 23S rRNA in bacteria acts as ribozyme.

RNA was the first genetic material — it acts as genetic material as well as catalyst, and being reactive it is unstable.

DNA evolved from RNA and is more stable; its complementary double-helical strands resist changes by evolving a repair mechanism.

TMV, QB bacteriophage — RNA is genetic material; mutate & evolve faster.

Meselson-Stahl (E. coli, ¹⁵N heavy isotope); Taylor (Vicia faba, radioactive thymidine).

¹⁵N is NOT radioactive — separated by density (CsCl gradient), not radioactivity.

dNTPs serve dual purpose — substrate for polymerisation + energy source.

Continuous (3′→5′ template) vs Discontinuous (5′→3′ template); Okazaki fragments elongate lagging strand AWAY from replication fork.

In transcription, A pairs with U (not T).

Transcription unit = Promoter (5′-end) + Structural gene + Terminator (3′-end).

A transcription unit is defined primarily by three regions — promoter, structural gene and terminator.

The promoter is located towards the 5′-end of the structural gene and provides the binding site for RNA polymerase.

The promoter defines the template strand and the coding strand.

The terminator is located towards the 3′-end of the coding strand and defines the end of transcription.

Template strand = 3′→5′; Coding strand = 5′→3′ = same sequence as mRNA.

Cistron = segment of DNA coding for polypeptide.

Split-gene arrangement in eukaryotes (exons + introns).

σ factor (initiation), ρ factor (termination) — transient association with RNA polymerase.

RNA polymerase facilitates opening of DNA helix.

ρ (rho) factor is required for termination of transcription; σ (sigma) factor for initiation.

RNA Pol I = rRNA; RNA Pol II = hnRNA/mRNA; RNA Pol III = tRNA, 5S rRNA, snRNA.

RNA polymerase II synthesises the precursor of mRNA (hnRNA).

Splicing = removal of introns + joining of exons in defined order.

Capping = methyl guanosine triphosphate at 5′-end (NOT 3′-end!).

Spliceosomes (snRNPs) are NOT found in bacteria — prokaryotes lack split genes.

The post-transcriptional events are — removal of introns & joining of exons (splicing), addition of methyl group at 5′-end of hnRNA (capping), and addition of adenine residues at 3′-end of hnRNA (tailing).

Splicing occurs inside the nucleus — hnRNA is transported to the cytoplasm only after complete processing.

Base pairing of two complementary RNAs is RNA interference, not post-transcriptional processing.

Khorana — chemical method for RNA synthesis (homopolymers & copolymers).

George Gamow proposed that the genetic code for amino acids should be made up of three nucleotides.

AUG = Methionine + initiator codon (dual function).

Genetic code is nearly universal → bacteria can produce human insulin.

Salient features of genetic code — triplet, degenerate, unambiguous, universal.

Frameshift mutations; insertion/deletion of 3 bases → no frame change.

Sickle cell anaemia — point mutation in β-globin gene; Glu → Val.

Substitution of Glutamic acid by Valine at the 6th position of the β-globin chain causes sickle-cell anaemia.

The sixth mutant codon of the β-globin gene causing Hb polymerisation and change in RBC shape is GUG.

Peptide bond formation requires energy (ATP).

Ribosomes = structural RNAs + ~80 different proteins.

Translation begins when SMALL subunit encounters mRNA.

Eukaryotic ribosome = 80S (60S + 40S); Prokaryotic ribosome = 70S (50S + 30S); each ribosome has two sub-units.

Jacob & Monod — Lac operon; Lactose = inducer.

z = β-galactosidase; y = permease; a = transacetylase.

In the lac operon, the z gene codes for β-galactosidase.

The i gene is expressed constitutively (repressor is synthesised all the time).

The i gene has its own separate promoter — only z, y and a share the common operon promoter.

Regulation by repressor = negative regulation.

Glucose/Galactose CANNOT induce lac operon.

Lactose = inducer for lac operon.

Lactose inactivates the repressor; the repressor binds the operator only when lactose is absent.

Galactose cannot act as an inducer of the lac operon.

ESTs = genes expressed as RNA.

Sequence Annotation = whole genome + function assignment (blind approach).

BAC & YAC vectors; Chromosome 1 sequenced last (May 2006).

BAC & YAC = Bacterial & Yeast Artificial Chromosomes.

Chromosome 1 has the highest number of genes (2968); Y chromosome the fewest (231).

DNA fingerprinting basis = DNA polymorphism in repetitive/satellite DNA.

Polymorphisms are inheritable from parents to children.

VNTR = Variable Number of Tandem Repeats; mini-satellite DNA.

Zinc finger analysis is NOT used in DNA fingerprinting.

Correct sequence = Isolation of DNA & digestion by restriction endonucleases → Separation of fragments by electrophoresis → Transfer of fragments to synthetic membranes → Hybridisation using labelled VNTR probe → Detection by autoradiography.

RNAi takes place in all eukaryotic organisms as a method of cellular defence.

Silencing of a specific mRNA through RNAi is possible because of complementary dsRNA.

tRNA and rRNA both interact with mRNA — tRNA reads the codon through its anticodon, rRNA aligns mRNA and catalyses peptide bond formation.