Lecture 2. Biology G4054. Mammalian Cell Genetics. 1/27/00
vs. mutants, continued discussion point: stable
heritable alterations in phenotype that are not due to mutations: already
mentioned the mysterious heritable switches in gene regulation
better understood molecularly: DNA CpG methylation. hemimethylated DNA is a better substrate for methylation than non-methylated DNA
Here frequency ~ rate (if mutations are assumed to be induced over a single generation).
Agents: DNA damaging agents.
Physical = radiation: UV (pyrimidine dimers), ionizing (X-rays, gamma rays --> free radicals --> strand scission; if numerous, then close, ~= DS breaks. )
Chemical: Adduct-formers: e.g., ethyl methane sulfonate (EMS); nitrosoguanidine (MNNG); benzpyrene (BP); other carcinogens
Base analogs e.g., ; bromodeoxyuridine (BrdU)
Biological: Knockout by exogenous DNA insertions: targeted (homologous recomb.) or untargeted (illegitimate recomb.)
Dosage: Kill 9%, 90% or 99%? Answer: 90% (too little effect vs. too few survivors)
Hot spots. Cold spots. Sequence specificity?
Effect of repair. strand specificity: the transcribed strand is often found to host fewer mutations, perhaps preferentially repaired. via PolII?
Expression period: Genotypic fixation Phenotypic lag. For protein degradation, dilution. For mRNA degradation, dilution. HPRT-: 7 days.
Metabolic cooperation: Kiss of death. Cross-feeding: drink of death.
Exploitable metabolic pathways:
Purine and pyrimidine biosynthesis: auxotrophs
Amino acid biosynthesis auxotrophs Lipid auxotrophs (e.g., linoleic acid, cholesterol).
Method= indirect: BUdR (BrdU): Kao and Puck. Penicillin analogy.
Drug resistance: see sheet
A. Mutant lacks toxifying enzyme (e.g., hprt, tk)
B. Enzyme target becomes a better discriminator (E.g., ouabain resistance)
C. Permeation changes (influx blocked (aa analog resistance) or efflux increased (various unrelated ring compounds (P-glycoprotein, MDR)
D. Improved de-toxification via chelation, covalent modification (neo/G408), or overproduction of target
E. Receptor deficiency: E.g., glucocorticoid receptor (Dex-R in lymphoid cells.)
3. Temperature-sensitive mutants: cell cycle mutants. Tritiated amino acid suicide (aa‑tRNA synthetases)
4. Antibodies vs. cell-surface components. Lysis with complement. Targets cell surface constituents mostly (e.g., MHC). Lectin resistance (oligosaccharide components of cell surface glycoprotein)
Visual inspection at colony level:
A. Sib selection
B. Replica plating
C. Secreted product
6. FACS. = Fluorescence-activated cell sorter. 1-D and 2-D fluorescence displays. Used in the Rice et al paper in the reading. 0 or 1 cell per droplet. Charge selected droplets based on fluorescence intensity readout on the fly. Deflect charged droplet to a separate tube. Cell surface proteins via fluorescent antibodies. Internal proteins also (rarer).
7. Brute force (clonal biochemical
analysis, e.g., electrophoretic variants (e.g., Ig)
Molecular (DNA) isolation (via PCR and DGGE [denaturing gradient gel electrophoresis].)
Revertants of mutants: Selection of revertants (or transfectants receiving the wild type gene) is often easy, as it usually involves gain of function and a gain is usually easier to see than a loss. Except for category 4 and some category 2. Ex. HPRT+ from HPRT- in HAT. and APRT= from APRT- in AAT. Prototroph from auxotroph in medium lacking the end-product.