Detection of nonrandom association of alleles from the distribution of the number of heterozygous loci in a sample.

The distribution of the number of heterozygous loci in two randomly chosen gametes or in a random diploid zygote provides information regarding the nonrandom association of alleles among different genetic loci. Two alternative statistics may be employed for detection of nonrandom association of genes of different loci when observations are made on these distributions: observed variance of the number of heterozygous loci (s2k) and a goodness-of-fit criterion (X2) to contrast the observed distribution with that expected under the hypothesis of random association of genes. It is shown, by simulation, that s2k is statistically more efficient than X2 to detect a given extent of nonrandom association. Asymptotic normality of s2k is justified, and X2 is shown to follow a chi-square (chi 2) distribution with partial loss of degrees of freedom arising because of estimation of parameters from the marginal gene frequency data. Whenever direct evaluations of linkage disequilibrium values are possible, tests based on maximum likelihood estimators of linkage disequilibria require a smaller sample size (number of zygotes or gametes) to detect a given level of nonrandom association in comparison with that required if such tests are conducted on the basis of s2k. Summarization of multilocus genotype (or haplotype) data, into the different number of heterozygous loci classes, thus, amounts to appreciable loss of information.

Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

Estimation of the incidence of a rare genetic disease through a two-tier mutation survey.

Recent attempts to detect mutations involving single base changes or small deletions that are specific to genetic diseases provide an opportunity to develop a two-tier mutation-screening program through which incidence of rare genetic disorders and gene carriers may be precisely estimated. A two-tier survey consists of mutation screening in a sample of patients with specific genetic disorders and in a second sample of newborns from the same population in which mutation frequency is evaluated. We provide the statistical basis for evaluating the incidence of affected and gene carriers in such two-tier mutation-screening surveys, from which the precision of the estimates is derived. Sample-size requirements of such two-tier mutation-screening surveys are evaluated. Considering examples of cystic fibrosis (CF) and medium-chain acyl-CoA dehydrogenase deficiency (MCAD), the two most frequent autosomal recessive disease in Caucasian populations and the two most frequent mutations (delta F508 and G985) that occur on these disease allele-bearing chromosomes, we show that, with 50-100 patients and a 20-fold larger sample of newborns screened for these mutations, the incidence of such diseases and their gene carriers in a population may be quite reliably estimated. The theory developed here is also applicable to rare autosomal dominant diseases for which disease-specific mutations are found.

Population amalgamation and genetic variation: observations on artificially agglomerated tribal populations of Central and South America.

The interpretation of data on genetic variation with regard to the relative roles of different evolutionary factors that produce and maintain genetic variation depends critically on our assumptions concerning effective population size and the level of migration between neighboring populations. In humans, recent population growth and movements of specific ethnic groups across wide geographic areas mean that any theory based on assumptions of constant population size and absence of substructure is generally untenable. We examine the effects of population subdivision on the pattern of protein genetic variation in a total sample drawn from an artificial agglomerate of 12 tribal populations of Central and South America, analyzing the pooled sample as though it were a single population. Several striking findings emerge. (1) Mean heterozygosity is not sensitive to agglomeration, but the number of different alleles (allele count) is inflated, relative to neutral mutation/drift/equilibrium expectation. (2) The inflation is most serious for rare alleles, especially those which originally occurred as tribally restricted "private" polymorphisms. (3) The degree of inflation is an increasing function of both the number of populations encompassed by the sample and of the genetic divergence among them. (4) Treating an agglomerated population as though it were a panmictic unit of long standing can lead to serious biases in estimates of mutation rates, selection pressures, and effective population sizes. Current DNA studies indicate the presence of numerous genetic variants in human populations. The findings and conclusions of this paper are all fully applicable to the study of genetic variation at the DNA level as well.

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

BIOCHEMICAL CHARACTERIZATION AND GENETIC MAPPING OF WILD TYPE AND MUTANT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ALLELES IN CHINESE HAMSTER OVARY CELLS

A UV-induced mutation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) was characterized in the CHO clone A24. The asymmetric 4-banded zymogram and an in vitro GAPD activity equal to that of wild type cells were not consistent with models of a mutant heterozygote producing equal amounts of wild type and either catalytically active or inactive mutant subunits that interacted randomly. Cumulative evidence indicated that the site of the mutation was the GAPD structural locus expressed in CHO wild type cells, and that the mutant allele coded for a subunit that differed from the wild type subunit in stability and kinetics. The evidence included the appearance of a fifth band, the putative mutant homotetramer, after addition of the substrate glyceraldehyde-3-phosphate (GAP) to the gel matrix; dilution experiments indicating stability differences between the subunits; experiments with subsaturating levels of GAP indicating differences in affinity for the substrate; GAPD zymograms of A24 x mouse hybrids that were consistent with the presence of two distinct A24 subunits; independent segregation of A24 wild type and mutant electrophoretic bands from the hybrids, which was inconsistent with models of mutation of a locus involved in posttranslational modification; the mapping of both wild type and mutant forms of GAPD to chromosome 8; and the failure to detect any evidence of posttranslational modification (of other A24 isozymes, or through mixing of homogenates of A24 and mouse).^ The extent of skewing of the zymogram toward the wild type band, and the unreduced in vitro activity were inconsistent with models based solely on differences in activity of the two subunits. Comparison of wild type homotetramer bands in wild type cells and A24 suggested the latter had a preponderance of wild type subunits over mutant subunits, and had more GAPD tetramers than did CHO controls.^ Two CHO linkages, GAPD-triose phosphate isomerase, and acid phosphatase 2-adenosine deaminase were reported provisionally, and several others were confirmed. ^

Cell survival in early embryogenesis requires proper dosage of GCN5 and p300

Histone acetylation is a central event in transcriptional activation. The importance of this modification in mammalian development is highlighted by knockout studies that revealed loss of the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality. Furthermore, early embryogenesis is sensitive to the dosage of p300 and CBP since double p300 +/−CBP+/− heterozygotes die in utero, although either single heterozygote survives. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of HATs interact functionally in vivo, we created mice lacking one or more allele of p300, GCN5 or PCAF. As expected, we found that mice heterozygous for any one of these null alleles are viable. The majority of GCN5 p300 double heterozygotes also survive to adulthood with no apparent abnormalities. However, a portion of these mice die prior to birth. These embryos are developmentally stunted and exhibit increased apoptosis compared to wild type or single GCN5 or p300 heterozygous littermates at E8.5. Tissue specification is unaffected in these embryos but organ formation is compromised. In contrast, no abnormalities were observed in mice harboring mutations in both PCAF and p300 , emphasizing the specificity of HAT functions in mammalian development. ^ Since GCN5 null embryos die early in embryogenesis because of a marked increase in apoptosis, studies of its function and mechanism in late development and in tissue specific differentiation are precluded. Here, we also report the establishment of a GCN5 null embryonic stem cell line and a conditional floxGCN5 mouse line, which will serve as powerful genetic tools to examine in depth the function of GCN5 in mammalian development and in adult tissues. ^