Lecture 1.  Biology G4054. Mammalian Cell Genetics.    1/20/00

Mutation.

Outline:
Mammalian cells lines
The problem of diploidy and polyploidy
Mutation rates. Rates vs. frequency. Measurements of rates. Fluctuation analysis.
Mutagenesis. Chemical and physical agents.  Dosage considerations. Expression period. Metabolic cooperation.
Dominant vs. recessive mutations.
Mutagen specificity.  Mutational spectra.  Repair.  Strand specificity. 

Introduction:
Genetics as a subject and genetics as a tool.  As a subject: Understanding what genes do. What do genes do, in somatic cells?
    Answer: Replicate, get transmitted, recombine, express their information.
Genetics as a tool: Most advantageous, the less you know about a process: mutations can illuminate
(Genetics vs. biochemistry: E. coli PolI of Kornberg vs. PolIII of Cairns)

Basic genetic manipulation using genetics as a tool:
1) Mutation.  The primary tool. Absence of function lets you pinpoint the WT function.   Random in vivo + selection vs. site-directed.
2) mapping How: mating (with whole organism, but not mammalian cells); cell fusion = surrogate sex :-) ; FISH
3) juxtaposition (complementation): heterokaryons= 2 nuclei in one cell; hybrid cells= 2 sets of chromosomes in one nucleus
Advantages of cultured cells for using genetic tools: numbers (10^8); homogeneity (cell clones)
Disadvantages: limited phenotypes; limited differentiation in culture (although can maintain a differentiated phenotype); no sex

Mammalian cell lines.
Primary, secondary cultures; passages, senescence
Crisis, rare establishment = immortalization.  Transformation (neoplastic transformation) vs. immortalization. 
Mechanism of immortalization, or rather mortalization: telomere shortening due to a relative telomerase deficiency in WT cells
  (See reading)
Heteroploidy common in established lines. Diploidy. Haploidy not achievable (except very partial monosomies) 

Mutation:
Problems of  diploid or heteroploid cells: Recessive mutations masked. Dominant mutations are expressed but are less common.
    Example: thymidine kinase (TK) recessive (loss of enzyme) ; alpha amanitin resistance dominant (altered RNA Pol II).
Problem of epigenicity: epigenetic change = "outside" of genetics = non-mutational (mutation being a heritable change in 1 the 4 bases).
Example: almost all of differentiation. How to distinguish an epigenetic variant (heritable change in phenotype) from true mutants? 
    Caskey and CRM, Capecchi and heat sensitive enzymes; Chasin and ploidy dependence. Not predicted by epigenetic silencing.
Clone mRNA. See sequence change directly.
Measurement of spontaneous mutation rates. Frequency is not rate, for spontaneous mutations.
Fluctuation analysis.  Luria & Delbruck: Random mutations, not induced by selection (anti-Lamarkianism). High variance/mean.
Po method.  Variance method (use of more data). Also median method (not discussed). Other methods: rate of increase of frequency.