Gene Expression, Human Genome Project and DNA Fingerprinting — Notes

Transcription copies the information in one strand of DNA into RNA. A transcription unit has three regions: a promoter (upstream; RNA polymerase binds here), the structural gene, and a terminator (downstream). Of the two DNA strands, the one with polarity 3′→5′ acts as the template strand; the other (5′→3′) is the coding strand, whose sequence is the same as the RNA except that thymine is replaced by uracil. RNA is always made in the 5′→3′ direction. Both strands are not copied because the two RNAs would be complementary, pair up, and prevent translation.

Stages: initiation, elongation and termination. In bacteria a single DNA-dependent RNA polymerase makes all RNAs; the sigma (σ) factor helps initiation and the rho (ρ) factor helps termination. Transcription and translation can be coupled in the cytoplasm.

In eukaryotes there are three RNA polymerases in the nucleus: RNA polymerase I (rRNAs: 28S, 18S, 5.8S), II (hnRNA, the precursor of mRNA) and III (tRNA, 5S rRNA, snRNAs).

Processing of hnRNA (in the nucleus):

• Splicing: introns (non-coding intervening sequences) are removed and exons (coding sequences) are joined.
• Capping: methyl guanosine triphosphate is added to the 5′ end.
• Tailing: 200–300 adenylate residues (poly-A tail) are added to the 3′ end. The fully processed hnRNA, now mRNA, is transported to the cytoplasm. Genes with introns are split genes.

Terms: exon — coding sequence present in mature mRNA; intron — intervening sequence removed by splicing; cistron — segment of DNA coding for one polypeptide; recon — smallest unit capable of recombination; muton — smallest unit capable of mutation; monocistronic — one structural gene in a transcription unit (mostly eukaryotes); polycistronic — several structural genes in one unit (mostly bacteria).

The sequence of bases in mRNA determines the sequence of amino acids in a protein. George Gamow argued that with 4 bases and 20 amino acids, the code must be a triplet: 4³ = 64 combinations (a doublet gives only 4² = 16). Har Gobind Khorana developed chemical methods to synthesise RNA with defined base combinations; Marshall Nirenberg used a cell-free system for protein synthesis to decode codons; Severo Ochoa’s enzyme polynucleotide phosphorylase made RNA with defined sequences without a template.

Features:

1. The codon is a triplet; 61 codons code for amino acids and 3 (UAA, UAG, UGA) are stop codons.
2. One codon codes for only one amino acid: the code is unambiguous and specific.
3. Some amino acids have more than one codon: the code is degenerate.
4. The code is read in a continuous manner: it is comma-less.
5. The code is nearly universal: UUU codes for phenylalanine in bacteria and in humans (exceptions in mitochondria and some protozoans).
6. AUG has dual functions: it codes for methionine and acts as the initiator codon.

Example (computed). Template 3′–TACAAGCCTACCATT–5′ gives mRNA 5′–AUGUUCGGAUGGUAA–3′, which codes for Met–Phe–Gly–Trp, then stop.

Translation is the polymerisation of amino acids into a polypeptide in the order set by mRNA codons, on ribosomes in the cytoplasm.

tRNA — the adapter molecule. tRNA has an anticodon loop with bases complementary to the codon and an amino-acid acceptor end (3′ end) to which the amino acid binds. Its secondary structure is a clover leaf; its 3-D structure is an inverted L. There are no tRNAs for stop codons.

Steps:

1. Charging (aminoacylation): each amino acid is activated in the presence of ATP and linked to its tRNA.
2. Initiation: the small ribosomal subunit binds mRNA; the initiator tRNA carrying methionine pairs with the start codon AUG; the large subunit joins.
3. Elongation: the ribosome has two sites for tRNAs. A charged tRNA whose anticodon matches the codon enters the A site; a peptide bond forms between the growing chain (on the tRNA in the P site) and the new amino acid; the rRNA of the large subunit (23S rRNA in bacteria) acts as the enzyme — a ribozyme. The ribosome then moves one codon towards the 3′ end (translocation).
4. Termination: when a stop codon reaches the A site, a release factor binds and the polypeptide is released; the ribosome dissociates.

mRNA has untranslated regions (UTRs) before the start codon (5′ UTR) and after the stop codon (3′ UTR); they are needed for efficient translation.

Gene expression in eukaryotes can be regulated at the levels of (i) transcription, (ii) processing (splicing), (iii) transport of mRNA from nucleus to cytoplasm and (iv) translation. In bacteria, control of the rate of transcription initiation is the main level.

An operon is a group of genes that are transcribed together and regulated as a unit. François Jacob and Jacques Monod first described the lac operon of E. coli:

• i — the regulatory gene, makes the repressor protein (constitutively; it has its own promoter);
• p — promoter; o — operator, where the repressor binds;
• z — β-galactosidase, which hydrolyses lactose into galactose and glucose; y — permease, which increases the permeability of the cell to lactose; a — transacetylase.

No lactose: the repressor binds the operator and prevents RNA polymerase from transcribing the structural genes. Lactose present: a few lactose molecules enter (low permease level); lactose (strictly allolactose) acts as the inducer, binds the repressor and inactivates it, so it cannot bind the operator; RNA polymerase transcribes z, y and a as one polycistronic mRNA. When lactose is used up, the repressor binds again. This is negative regulation, since the repressor switches the operon off.

The Human Genome Project (HGP), launched in 1990 and completed in 2003, was coordinated by the US Department of Energy and the National Institutes of Health, with the UK, Japan, France, Germany, China and others.

Goals: identify all the genes (about 20 000–25 000) in human DNA; determine the sequence of the 3 billion chemical base pairs; store the information in databases; improve tools for data analysis; transfer related technologies to other sectors; and address the ethical, legal and social issues (ELSI) that may arise.

Methodologies: (i) Expressed Sequence Tags (ESTs) — identifying all the genes that are expressed as RNA; (ii) sequence annotation — sequencing the whole set of the genome, coding and non-coding, and later assigning functions to different regions. Total DNA was broken into fragments, cloned in hosts using BAC (bacterial artificial chromosome) and YAC (yeast artificial chromosome) vectors, sequenced with automated DNA sequencers (Frederick Sanger’s method) and arranged by specialised computer programs using overlapping regions.

Salient features:

• The genome has 3164.7 million nucleotide bases.
• The total number of genes was estimated at about 30 000 (later estimates nearer 20 000).
• The average gene has 3000 bases; the largest known gene, dystrophin, has 2.4 million bases.
• Almost 99.9 percent of bases are the same in all people.
• The functions of over half the discovered genes were unknown.
• Less than 2 percent of the genome codes for proteins — at most about 63.3 million bp.
• Repeated sequences make up a very large portion of the genome.
• Chromosome 1 has the most genes (2968) and Y the fewest (231).
• About 1.4 million locations of single-base DNA differences, single nucleotide polymorphisms (SNPs), were identified.

Applications: finding the genes behind disorders, designing new diagnosis and treatment (including gene therapy), understanding human biology and evolution; the sequences of other organisms (bacteria, yeast, Caenorhabditis elegans, Drosophila, rice, Arabidopsis) were also completed.

About 99.9 percent of the human sequence is the same in everyone; DNA fingerprinting compares the differences in repetitive DNA. Repetitive DNA separates from the bulk DNA as satellite DNA peaks in density-gradient centrifugation. Satellite DNA is classified as micro-satellites and mini-satellites by length and base composition; it does not code for proteins and shows high polymorphism.

Variable number tandem repeats (VNTRs) — mini-satellites in which a short sequence is repeated many times — differ in copy number from person to person, so the size of the DNA fragment containing them differs. The technique was developed by Alec Jeffreys, who used a VNTR as a probe.

Steps: (i) isolation of DNA; (ii) digestion with restriction endonucleases; (iii) separation of fragments by gel electrophoresis; (iv) Southern blotting — transfer to a nylon or nitrocellulose membrane; (v) hybridisation with a labelled VNTR probe; (vi) detection by autoradiography. The pattern of bands is the DNA fingerprint; it is unique to each individual except identical twins. Small samples can be amplified by PCR first.

Applications: forensic science (identifying suspects from blood, hair, semen or skin), paternity and maternity disputes, identification of disaster victims, studies of population and genetic diversity and evolution.

Ethical issues: genetic data reveal private information about a person and relatives; samples need informed consent; databases need secure storage and controlled access; misuse could lead to discrimination (for example by employers or insurers); results must be handled carefully in court.