14.7 Regulation of Gene Expression, Mutation and Mutagens, DNA Repair, Carcinogenesis, and cDNA Cloning

14.7 Regulation of Gene Expression, Mutation and Mutagens, DNA Repair, Carcinogenesis, and cDNA Cloning

Quick Answer: The nucleic-acid area of the Biochemistry TOS (12 items) goes beyond replication, transcription, and translation (Section 14.6). It adds regulation of gene expression, mutations and mutagens, DNA repair and carcinogenesis, cloning, and cDNA. Expect operon logic, classifying mutations, matching each kind of DNA damage to its repair pathway, and explaining why cDNA is used to express eukaryotic genes in bacteria.

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Regulation in Prokaryotes: Operons

An operon is a cluster of genes transcribed together from one promoter and controlled by an operator.

The lac Operon (Inducible)

The lacZ, lacY, and lacA genes encode beta-galactosidase, lactose permease, and transacetylase.

• Negative control: the LacI repressor binds the operator and blocks transcription. Allolactose, made from lactose, binds the repressor and releases it.
• Positive control: when glucose is low, cAMP rises and the cAMP-CAP (catabolite activator protein) complex binds near the promoter, recruiting RNA polymerase.

Glucose suppressing the use of other sugars is called catabolite repression.

The trp Operon (Repressible)

When tryptophan is abundant, it acts as a corepressor: it binds the inactive trp repressor so that the repressor binds the operator. Attenuation fine-tunes transcription: when tryptophan is plentiful, the ribosome moves quickly through the leader peptide's Trp codons, and the leader RNA forms a terminator hairpin that ends transcription early.

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Regulation in Eukaryotes

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Mutations

Mutagens

The Ames test detects mutagens by their ability to revert histidine-requiring Salmonella strains to growth without histidine. A liver S9 extract is added to activate pro-mutagens, as the body would. Most carcinogens are mutagens.

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DNA Repair Pathways

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Carcinogenesis

Cancer usually needs several mutations in one cell lineage:

• Oncogenes are gain-of-function mutations of proto-oncogenes, such as constitutively active RAS. One mutant allele is enough.
• Tumour suppressor genes, such as TP53 and RB1, need loss of both copies (the "two-hit" hypothesis). p53 halts the cell cycle or triggers apoptosis after DNA damage, and it is mutated in about half of human cancers.
• Defects in DNA repair speed the accumulation of mutations.

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Cloning, cDNA, and Modern Tools

Cloning vectors (plasmids) need an origin of replication, a selectable marker (such as ampicillin resistance), and a multiple cloning site. In blue-white screening, the insert disrupts lacZ-alpha, so recombinant colonies stay white on X-gal plates while non-recombinant colonies turn blue.

Complementary DNA (cDNA):

1. Isolate mRNA; an oligo(dT) primer anneals to the poly(A) tail.
2. Reverse transcriptase, a retroviral enzyme, copies the mRNA into DNA.
3. The RNA is removed and the second DNA strand is synthesized.

cDNA lacks introns, so bacteria, which cannot splice, can express it. Recombinant human insulin was the first approved recombinant-DNA drug, in 1982. A cDNA library contains only genes expressed in the source tissue; a genomic library contains all DNA, including introns and non-coding regions.

RT-qPCR measures mRNA levels: each PCR cycle doubles the product, so a sample that crosses the threshold 3.3 cycles earlier ($\Delta C_t = 3.3$) has about 10 times more template, since $2^{3.3} \approx 10$. CRISPR-Cas9 uses a guide RNA to direct Cas9 to cut a chosen DNA sequence for gene editing (Nobel Prize in Chemistry 2020, Charpentier and Doudna).