Human heterozygosity: a new estimate.

Abstract

Several hundred polypeptides from four human diploid fibroblast cell lines were compared by high-resolution two-dimensional polyacrylamide gel electrophoresis and double-label autoradiography under conditions where allelic products that differ by a single charged amino acid would be distinguished. The average heterozygosity represented by this set of gene products appears to be less than 1% for changes involving charged amino acids.The nature and extent of genetic diversity among humans is a matter of great theoretical and practical significance. Our current views on this subject depend heavily on the summaries by Harris and colleagues (1, 2). Of the 87 (6)(7)(8). These facts suggested to one of us several years ago that an attempt to measure heterozygosity by comparison of O'Farrell gel patterns of polypeptides from cells of different individuals would be fruitful. Side-by-side comparison of complex patterns, however, is fatiguing and inaccurate. The problem of finding a few spots unique to one two-dimensional gel pattern among hundreds of spots common to both patterns in any pairwise comparison has been solved by the development of double-label autoradiography (9). This method permits the simultaneous electrophoresis of 3H-labeled and '4C-labeled protein mixtures on the same gel, followed by a two-step autoradiographic procedure that identifies the polypeptides that are unique to the 3H-labeled preparation. We have used this technique to compare approximately 400 fibroblast polypeptides from several individuals. We find that the heterozygosity of the genes represented by this set of polypeptides is significantly less than one would have expected from the enzyme surveys (1, 2). (2.5 ,Ci/ml, 300 Ci/mol) (1 Ci = 3.7 X 1010 becquerels). Cells were harvested by scraping with a rubber policeman, rinsed with 0.14 M NaCl/0.01 M sodium phosphate, pH 7.0, and pelleted in a conical tube which was then carefully wiped dry. Fresh or frozen cell pellets were lysed in freshly made 10 M urea, with vigorous mixing on a Vortex. The protein concentration of a 5-to 10-,ul sample of homogenate was measured with the Bio-Rad Reagent (bulletin 1069), and 1% sodium dodecyl sulfate/10 M urea was added so that the final mass ratio of detergent to protein was 1.5:1. If necessary, 10 M urea was added so that the final protein concentration did not exceed 1 mg/ml. The homogenate was then made 0.1 M in acetic acid (final pH 4.0-4.2) and 5 mM in ZnSO4; SI nuclease (Sigma or P-L Biochemicals) was added at 2 units of enzyme per 1ug of homogenate protein, and the mixture was incubated at 450C for 5 min, then chilled. An equal volume of 1 M acetic acid/1% 2-mercaptoethanol was added, followed by 10 homogenate volumes of cold acetone, and the proteins were allowed to precipitate at -20°C overnight. The precipitate was collected by centrifugation at 10,000 X g for 15 min. MATERIALS AND METHODS CellSamples were dissolved in O'Farrell's buffer (5) and centrifuged for 10 min at 10,000 X g at 25°C just before electrofocusing gels were lo...

Biochemical Population Genetics and Speciation

“…The rule that emerges is that loci that are highly variable under one condition of detection McConkey et al, 1979;Racine and Langley, 1980). A similar result was obtained when several groups of water-soluble proteins of non-enzymatic nature were analyzed electrophoretically .…”

Protein complexity of central nervous system cell lines

“…In addition, the frequency of transcript data from whole brain could be misleading because the extreme cellular heterogeneity in the CNS would tend to reduce the copy number for each cell that is actually synthesizing the protein. Finally, it is unlikely that polymorphic differences between individual rats could significantly alter the above data, for the majority of the cell lines were from the BDIX inbred strain, and the average heterozygosity between human fibroblasts is less than 1% as defined by 2D gel analysis (McConkey et al, 1979;Walton et al, 1979).…”

The estimate of protein polymorphism in human populations: Lack of evidence for overestimation due to post-translational modification.

“…Virtually no information exists concerning levels of genetic variation of membrane bound proteins. Some data has been collected using two dimensional electrophoresis (Comings 1979;McConkey, Taylor and Phan 1979;Smith, Racine and Langley 1980), although the interpretation of the resolving power of 2-D electrophoresis remains somewhat questionable (Edwards and Hopkinson 1980;Warner, Neel and Meisler 1982;McLellan, Ames and Nikaido 1983). We can say nothing concerning possible effects of GMPTM on levels of variation estimated using these techniques.…”

“…Some of these methods may increase the estimated average heterozygosity in a population, compared to the values obtained by more traditional screening methods, because they separate previously undifferentiated alleles (Ayala 1982), while others may have an unknown effect because they are increasing the number and type of loci which are being sampled, but with an undetermined sensitivity to allelic differences (McConkey, Taylor and Phan 1979;McLellan, Ames and Nikaido 1983). Nei and Roychoudhury (1982) reanalyzed the data on genetic variation in major racial groups and reported that average heterozygosity levels for protein loci were increased to the range 12-16% when they included loci for which new screening methods (excluding two-dimensional electrophoresis) had been used during the collection of gene frequency data.…”