Molecular Basis of Inheritance — Notes

Griffith (1928), Streptococcus pneumoniae: S strain (capsule, virulent) and R strain (no capsule). Live S → mice die; live R → live; heat-killed S → live; heat-killed S + live R → mice die and live S recovered. Some "transforming principle" from dead S cells transformed R cells.

Avery, MacLeod and McCarty (1933–44): purified DNA, RNA and proteins from heat-killed S cells; only DNA transformed R cells. Proteases and RNase did not stop transformation; DNase did.

Hershey and Chase (1952): bacteriophages grown with radioactive ³²P (DNA contains P, protein does not) or ³⁵S (protein contains S, DNA does not). After infection, blending and centrifugation, bacteria infected with ³²P phages were radioactive, those with ³⁵S phages were not: DNA enters the bacterium and is the genetic material.

Criteria for genetic material: replicates; chemically and structurally stable; allows slow changes (mutation) for evolution; expresses itself as Mendelian characters. DNA vs RNA: RNA's 2′-OH makes it reactive and less stable, and it mutates faster; uracil in RNA is replaced by the more stable thymine in DNA. RNA was probably the first genetic material ("RNA world"); some viruses (TMV, QB bacteriophage) still use RNA.

Polynucleotide: nitrogenous base + pentose (deoxyribose in DNA, ribose in RNA) + phosphate. Base + sugar (N-glycosidic linkage) = nucleoside; + phosphate (phosphoester at 5′-OH) = nucleotide; nucleotides joined by 3′–5′ phosphodiester linkages. Purines: adenine, guanine; pyrimidines: cytosine, thymine (DNA), uracil (RNA). DNA was first identified by Friedrich Miescher (1869) as "nuclein".

Double helix (Watson and Crick, 1953; X-ray data of Wilkins and Franklin; Chargaff: A = T, G = C in double-stranded DNA):

• two antiparallel polynucleotide chains, sugar-phosphate backbone outside, bases inside;
• A=T (two H-bonds), G≡C (three H-bonds); a purine always faces a pyrimidine (uniform width);
• right-handed; pitch 3.4 nm; about 10 bp per turn; 0.34 nm between base pairs. Length = number of bp × 0.34 nm: human diploid DNA (6.6 × 10⁹ bp) ≈ 2.2 m; E. coli (4.6 × 10⁶ bp) ≈ 1.56 mm.

Packaging: prokaryotes — DNA in large loops held by positively charged proteins in the nucleoid. Eukaryotes — histones (rich in lysine and arginine; positive) form an octamer; DNA wraps around it as a nucleosome (about 200 bp); nucleosomes form the "beads-on-string" chromatin fibre, which coils further; non-histone chromosomal (NHC) proteins help higher packing. Euchromatin: loosely packed, light-staining, transcriptionally active. Heterochromatin: densely packed, dark-staining, inactive.

Semi-conservative (Watson and Crick): strands separate and each templates a new strand, so each daughter has one old and one new strand.

Meselson and Stahl (1958): E. coli grown in ¹⁵NH₄Cl, then in ¹⁴N; DNA separated in a CsCl density gradient. Generation 1: all hybrid (¹⁵N/¹⁴N); generation 2: hybrid and light in equal amounts. After n generations, hybrid fraction = 2/2ⁿ. (Taylor and colleagues showed the same in Vicia faba chromosomes using radioactive thymidine.)

Machinery: DNA-dependent DNA polymerase (polymerises only 5′ → 3′; E. coli copies 4.6 × 10⁶ bp in about 18 minutes, roughly 2000 bp/s); deoxyribonucleoside triphosphates are substrates and energy source; replication starts at an origin of replication; at the replication fork the leading strand is continuous and the lagging strand is made as Okazaki fragments joined by DNA ligase. In eukaryotes replication occurs in the S phase.

Central dogma (Crick): DNA → RNA → protein; reverse transcription (RNA → DNA) in some viruses (retroviruses).

Transcription unit: promoter (upstream, binding site of RNA polymerase), structural gene, terminator. Template strand 3′→5′; coding strand 5′→3′ has the RNA sequence (T for U). Structural genes may be monocistronic (mostly eukaryotes) or polycistronic (mostly bacteria).

• Bacteria: one DNA-dependent RNA polymerase; σ (initiation) and ρ (termination) factors; transcription and translation can be coupled.
• Eukaryotes: RNA pol I (rRNAs 28S, 18S, 5.8S), pol II (hnRNA → mRNA), pol III (tRNA, 5S rRNA, snRNAs). Split genes: exons (coding) and introns. Processing: splicing (remove introns, join exons), capping (methyl guanosine triphosphate at the 5′ end), tailing (200–300 adenylates at the 3′ end).

Genetic code: proposed as a triplet by Gamow (4³ = 64); RNAs of defined sequence made by Khorana; cell-free decoding by Nirenberg. Features: triplet; 61 sense codons, 3 stop (UAA, UAG, UGA); unambiguous and specific; degenerate; read contiguously (no punctuation); nearly universal; AUG codes for methionine and is the start codon. Mutations: a point mutation changes one codon (sickle-cell GAG → GUG); insertion or deletion of 1–2 bases causes a frameshift; of 3 bases adds or removes one amino acid.

tRNA (adapter): clover-leaf secondary structure, inverted-L 3D shape; anticodon loop pairs with the codon; amino acid acceptor end carries the amino acid; a specific tRNA for each amino acid; an initiator tRNA for methionine; no tRNA for stop codons.

Translation:

1. Charging (aminoacylation): amino acids activated with ATP and linked to their tRNA.
2. Initiation: the small ribosomal subunit meets the mRNA at the start codon AUG; the large subunit joins.
3. Elongation: charged tRNAs enter codon by codon; peptide bonds form. In bacteria the 23S rRNA catalyses peptide-bond formation, acting as a ribozyme.
4. Termination: a release factor binds the stop codon; the polypeptide is released.

UTRs (untranslated regions) at the 5′ and 3′ ends of mRNA help efficient translation.

Regulation can occur at transcription, processing, transport of mRNA or translation; in prokaryotes it is mainly at the initiation of transcription. An operon is a set of genes controlled together.

lac operon (Jacob and Monod): regulatory gene i (makes the repressor), promoter, operator, structural genes z (β-galactosidase: lactose → galactose + glucose), y (permease: lets lactose in), a (transacetylase).

• No lactose: the repressor binds the operator and prevents RNA polymerase from transcribing the operon.
• Lactose present (inducer): lactose binds the repressor, inactivating it; RNA polymerase transcribes z, y and a.
• When lactose is used up, the repressor binds again. This is negative regulation.

HGP (1990–2003). Goals: identify all the approximately 20,000–25,000 human genes; determine the sequence of about 3 billion base pairs; store the information in databases; improve analysis tools; transfer technologies; address ethical, legal and social issues. Methods: Expressed Sequence Tags (ESTs) and sequence annotation; DNA fragments cloned in BAC and YAC vectors; automated sequencers based on Sanger's method; computer alignment of overlaps.

Salient observations: 3164.7 million bases; average gene about 3000 bases (largest, dystrophin, 2.4 million); about 30,000 genes; 99.9 % of bases identical in all people; functions of over half the genes unknown; less than 2 % of the genome codes for proteins; repeated sequences make up a large part; chromosome 1 has most genes (2968), Y fewest (231); about 1.4 million single-base differences (SNPs).

DNA fingerprinting (Alec Jeffreys): compares repetitive DNA; satellite DNA forms minor peaks in density gradients; VNTRs (variable number tandem repeats, about 0.1–20 kb) vary between individuals (polymorphism) and are inherited, half from each parent. Steps: isolate DNA → digest with restriction endonucleases → separate by gel electrophoresis → Southern blot onto nylon or nitrocellulose → hybridise with a labelled VNTR probe → detect by autoradiography. Uses: forensic identification, paternity testing, population and genetic diversity studies.