Crispr-Cas9 Mode Of Action – Ru
7.1 Molecular classes #
7.1.1 From base substitution to splice site #
The chemistry of damage and repair is Molecular Biology. Genetics classes the outcome. A transition swaps purine for purine or pyrimidine for pyrimidine; a transversion crosses families. Either can be silent, missense, or nonsense.
| Class | Text | Usual product |
|---|---|---|
| Silent | Codon changes, amino acid does not | Sequence unchanged; splicing may still change |
| Missense | Different amino acid | Mild to null |
| Nonsense | Stop (UAA, UAG, UGA) | Truncation; mRNA may be destroyed |
| Frameshift | Indel not a multiple of three | Downstream frame wrong |
| In-frame indel | Multiple of three | A local insertion or deletion |
| Splice-site | Intron boundary damaged | Exon skip, intron retention, or a cryptic site |
Sickle cell is a missense in HBB: GAG to GTG, glutamic acid to valine at beta position 6. A synonymous codon change can still alter a splice signal, so “silent” is not a synonym for neutral. Splice-site alleles are Mendelian losses whose target is an RNA signal; the spliceosome is Molecular Biology. Muller showed in 1927 that X-rays raise the Drosophila mutation rate. Per locus per generation, rates are often about 10^−6 to 10^−5 and vary widely. That scale is enough for Chapter 9.
7.2 What the allele does to the organism #
7.2.1 Loss, gain, and haploinsufficiency #
Molecular class does not fix dominance. An amorph is a null and a hypomorph a partial loss. If one wild-type copy suffices, as for many metabolic enzymes, the loss is recessive.
Haploinsufficiency is a loss that is dominant because half dose is not enough: a loss-of-function LDL-receptor allele raises cholesterol in the heterozygote. A hypermorph or neomorph does too much or something new. Achondroplasia is a gain-of-function missense in FGFR3, dominant, and many cases are new mutants because transmission is reduced. A dominant negative, or antimorph, poisons a multimer, so the heterozygote is worse than a heterozygote for a null; some severe collagen alleles do this. An added wild-type copy rescues a loss and does not rescue a neomorph. Human pedigrees often cannot include that test.
7.3 Finding mutagens, and unstable repeats #
7.3.1 The Ames test as a principle #
The Ames test asks whether a chemical raises mutation in a bacterium. Histidine-requiring cells are exposed and then asked to grow without histidine. Only revertants form colonies. A rise above the spontaneous background means the compound was mutagenic under those conditions.
Strains differ so that substitutions and frameshifts are separately visible. A liver extract supplies mammalian metabolism that can turn a precursor into the active compound. A negative test is not proof of safety, and a positive test is not a diagnosis of human cancer. The endpoint is heritable reversion. No protocol or dose is given here.
7.3.2 Trinucleotide-repeat expansion #
A run of three bases can change length when copied, usually toward expansion once it is already long. The enzymology is Molecular Biology. The genetic signs are anticipation, earlier or more severe disease in later generations, and a parent-of-origin bias.
| Huntington | Fragile X | |
|---|---|---|
| Gene and repeat | HTT, CAG in the coding sequence, a polyglutamine tract | FMR1, CGG in the 5′ untranslated region |
| Pathogenic effect | Longer tract in the protein | Methylation and silencing once the repeat is a full mutation |
| Pattern | Autosomal dominant; expansion often paternal | X-linked; a maternal premutation expands to a full mutation |
Teaching boundaries, not clinical cuts: for HTT, about 26 or fewer repeats is the usual normal range, about 27–35 can expand without itself causing disease, about 36–39 is reduced penetrance, and about 40 or more is fully penetrant. For FMR1, up to about 44 is normal, about 45–54 is intermediate, a premutation is about 55–200, and a full mutation is above about 200. The Sherman paradox was this maternal expansion. Both are unstable triplets with anticipation. They are not the same kind of allele.
7.4 Where variation comes from #
7.4.1 Mutation, recombination, and independent assortment #
Mutation is the only source of new sequence: substitutions, indels, copy-number changes, expanding repeats, new junctions. Recombination by crossing over makes new combinations along a chromosome without new sequence.
Independent assortment makes new packages of whole chromosomes: 2^n gametes from n pairs even with no crossover, more than eight million in humans before crossing over multiplies them. Segregation only samples which allele of a heterozygote enters a gamete. Fertilisation joins two samples. Polyploidy and rearrangement are further mutational routes, not a fourth shuffle. Chapter 9 then moves the frequencies of whatever mutation has supplied.
7.5 Key terms, summary, and self-check #
Key terms. Transition; transversion; missense; nonsense; frameshift; splice-site mutation; haploinsufficiency; dominant negative; Ames test; anticipation; HTT; FMR1.
Summary. Substitutions are transitions or transversions and silent, missense, or nonsense by the codon; frameshifts are indels not divisible by three. Loss of function is recessive when one copy suffices and dominant when dose is insufficient or the product is a poison. The Ames test counts bacterial revertants, with host metabolism supplied as an extract. Huntington lengthens a polyglutamine tract; a full fragile-X mutation silences FMR1. Mutation creates alleles; crossing over and independent assortment only recombine them.
Self-check.
1. Why is a synonymous change not automatically neutral?
2. A nonsense heterozygote is affected, and an added wild-type copy restores wild type. Which functional class is this?
3. What does a positive Ames test measure, and what does it not measure?
4. Where do the Huntington and fragile-X repeats sit in the gene, and what does each expansion do to the product?
5. With no new mutation for a generation, which kinds of difference among offspring can still arise?
