Lecture 2.  Biology G4054. Mammalian Cell Genetics.    1/27/00

Variants 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:  

   1. Auxotrophs:  
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. 

2. 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)

5. 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.