Meiotic recombination in human oocytes.

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

The synaptonemal complex and meiotic recombination in humans: new approaches to old questions.

Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633-638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363-365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405-408; Pathak and Elder (1980) Hum Genet 54:171-175; Solari (1980) Chromosoma 81:315-337; Speed (1984) Hum Genet 66:176-180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215-226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833-848; Vidal et al. (1982) Hum Genet 60:301-304; Bojko (1983) Carlsberg Res Commun 48:285-305; Bojko (1985) Carlsberg Res Commun 50:43-72; Templado et al. (1984) Hum Genet 67:162-165; Navarro et al. (1986) Hum Reprod 1:523-527; Garcia et al. (1989) Hum Genet 2:147-53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.

Punctuated duplication seeding events during the evolution of human chromosome 2p11.

Primate genomic sequence comparisons are becoming increasingly useful for elucidating the evolutionary history and organization of our own genome. Such studies are particularly informative within human pericentromeric regions--areas of particularly rapid change in genomic structure. Here, we present a systematic analysis of the evolutionary history of one approximately 700-kb region of 2p11, including the first autosomal transition from pericentromeric sequence to higher-order alpha-satellite DNA. We show that this region is composed of segmental duplications corresponding to 14 ancestral segments ranging in size from 4 kb to approximately 115 kb. These duplicons show 94%-98.5% sequence identity to their ancestral loci. Comparative FISH and phylogenetic analysis indicate that these duplicons are differentially distributed in human, chimpanzee, and gorilla genomes, whereas baboon has a single putative ancestral locus for all but one of the duplications. Our analysis supports a model where duplicative transposition events occurred during a narrow window of evolution after the separation of the human/ape lineage from the Old World monkeys (10-20 million years ago). Although dramatic secondary dispersal events occurred during the radiation of the human, chimpanzee, and gorilla lineages, duplicative transposition seeding events of new material to this particular pericentromeric region abruptly ceased after this time period. The multiplicity of initial duplicative transpositions prior to the separation of humans and great-apes suggests a punctuated model for the formation of highly duplicated pericentromeric regions within the human genome. The data further indicate that factors other than sequence are important determinants for such bursts of duplicative transposition from the euchromatin to pericentromeric regions.

Segmental duplications and copy-number variation in the human genome.

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders.

Comparison of helium leak test and vacuum leak test using canned foods: collaborative study.

Two can leak tests were compared by 7 collaborators. In the helium leak test, pressurized helium is applied to the outside of the container, and a headspace gas sample from the can is then analyzed for the presence of helium. The vacuum test is described in the Bacteriological Analytical Manual. Ninety No. 303 cans of creamed-style corn, green beans, carrots, fruit cocktail, and whole-kernel corn were shipped in 3 groups. Two groups of 30 cans had 10 dented flat cans, 5 flat controls (nondented), 10 dented swollen cans, and 5 swollen control cans (nondented). The third group had 10 dented swollen cans and 5 swollen control cans. Of 600 cans analyzed, 37 (6.2%) were deleted from the analysis because results were not available for both tests. One laboratory was constrained by scheduling to analyze 15 of 45 swollen cans. The helium leak test found 12 (13%) positives of 92 nondented swollen cans. One pressurization test yielded 7 of those 12 positives. Of the 400 dented cans sent as possible leakers, the helium test found 267 positives, and the vacuum test found 181. Five of the 7 analysts had significantly (alpha = 0.05) higher percent positive helium results. One analyst found more leakers by the vacuum leak test. Both tests found fewer positives in the swollen dented cans than in the flat dented cans. After exposure to pressurized helium, all cans with greater than 8 psi headspace pressure were positive helium leakers. The method was adopted official first action.

Rapid Colorimetric Test for Alkaline Phosphatase in Dairy Products.

The Scharer rapid test for measuring alkaline phosphatase activity in milk and milk products was modified to include a photoelectric colorimeter in place of visual observation of color. Dairy products containing various amounts of added enzyme (3-15 µg phenol/ml or g) were prepared for analysis as per standard methods and analyzed by the rapid colorimetric test. A linear relationship was found between the percent of raw milk added and the enzyme activity with a correlation coefficient (r) range of 0.963 to 1.000. Hydrolysis of the substrate (disodium phenyl phosphate) by the surviving enzyme in different dairy products was similar. Recovery of the added enzyme varied, depending on the nature of the product. The method is quantitative, reproducible, and can be used as a rapid confirmatory procedure. Similarly, this method was applied to differentiating residual and reactivated enzyme in milk, cream and buttermilk. Compared to the Scharer rapid and the Rutgers visual methods, this test was more reliable in borderline cases because it eliminated the bias encountered in visual examination. A method was developed to analyze alkaline phosphatase in casein.