Evolutionary and genetic implications of sequence variation in two nonallelic HLA-DR beta-chain cDNA sequences.

Most HLA haplotypes carry two expressed DR beta-chain genes; in the DR4 haplotype, the polymorphic locus has been called DR beta 1 and the apparently nonpolymorphic locus has been called DR beta 2. We have isolated nearly full-length DR beta-chain cDNA clones representing each of these two loci from a cell line homozygous for DR4 and Dw4. The clones have been sequenced and the sequences compared with published DR beta cDNA sequences derived from other haplotypes. A comparison of our sequences with other published cDNA sequences did not allow assignment of these other sequences to either the beta 1 or beta 2 locus. Comparison of our DR4 beta 1 sequence with DR beta 1 sequences isolated from other DR4-positive cells suggests that the alleles of DR4 beta 1 may have recently diverged from a common ancestor. The apparent lack of polymorphism of DR beta 2 may in part be a reflection of this recent divergence.     

Site-specific genetic engineering of the Anopheles gambiae Y chromosome

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line–specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.Mosquito species of the Anopheles gambiae complex represent the principal vectors of human malaria, and they pose an enormous burden on global health and economies. Every year, 300–500 million people are infected by malaria and more than 1 million people die as a consequence of Plasmodium parasite infections (1). The malaria mosquito A. gambiae has two pairs of autosomes, termed 2 and 3, and a pair of heteromorphic sex chromosomes X and Y, XX in females and XY in males (2). Extensive nonpairing regions exist between the X and the degenerate Y chromosome. and evidence points to a factor located on the Y chromosome that primarily determines the sex in Anopheles (3). Current models suggest that the evolutionary differentiation of Y chromosomes begins with the acquisition of a male determining factor on a proto-Y chromosome (4, 5). This event is followed by a progressive suppression of recombination between the still largely homomorphic proto-sex chromosomes, a process attributed to the acquisition of sexually antagonistic mutations, which are beneficial to the heterogametic sex but detrimental to the homogametic sex (6–8). The lack of recombination, together with the male-limited transmission, leads to the degeneration of the Y chromosome, which involves accumulation of deleterious mutations, spread of transposable elements, and silencing of all or most of the genes present on the proto-Y (9–11). As a result, Y chromosomes of many species appear to be strongly heterochromatic and harbor only few genes often involved in male fertility (12–17). The accumulation of repetitive sequences, many of which are also present on other chromosomes, hampers the assembly of Y chromosome contigs following shotgun sequencing. Indeed, despite the completion of the A. gambiae genome project (18), and the knowledge that the primary signal is likely to be associated with the inheritance of the Y (3, 19), no assembly of the Anopheles Y chromosome has been achieved. At present, public databases host only a few hundred kilobases of A. gambiae sequences attributed to the Y, a chromosome that is estimated to comprise 10% of the genome and to be at least 20 Mb in size. None of these Y-specific scaffolds have been physically mapped, because the Y chromosome does not polytenize. The exploration of the Y chromosome will improve our understanding of the evolutionary forces involved in driving chromosome evolution and may enable the manipulation of the molecular pathways that control sex determination and sexual differentiation in mosquitoes. In a number of organisms, Y chromosome genes have been found to be essential for male fertility or sex determination. Recently, a number of excellent candidate genes potentially involved in these processes have been identified on the Y chromosome of anopheline mosquitoes (20, 21). Because interfering with male fertility is an essential part of vector control strategies such as the sterile insect technique, the identification of such genes is of particular interest to mosquito biologists. In this paper, we demonstrate the targeted molecular manipulation of the Y chromosome in A. gambiae, thus opening up a number of ways to explore one of the most fascinating of evolution’s upshots and to harness this genetic tool for vector control.     

Structural genes on the Y chromosome of Drosophila melanogaster.

Testis proteins of Drosophila melanogaster deficient for six different Y-chromosome regions were fractionated by means of a sodium dodecyl sulfate/polyacrylamide gel system designed to separate high molecular weight polypeptides (Mr, greater than 200,000). Analysis of the banding patterns indicates that the three regions containing fertility genes kl-2, kl-3, and kl-5 are responsible for three different high molecular weight polypeptides. Several observations indicate that these polypeptides are structural components of the sperm axoneme. They are present in seminal vesicles, which are highly enriched for mature sperm. They are first detected during development at a time when the first spermatids are elongating. Finally, deletion of either kl-5 or kl-3 leads to the absence of the outer dynein arm of the peripheral doublets of the axoneme. Although absence of the kl-2 region eliminates the third polypeptide, an associated structural defect in the axoneme has yet to be identified. The three polypeptides are in the Mr 300,000-350,000 range, and their mobilities are similar to those of dynein polypeptides from Chlamydomonas axonemes. Experiments using dosage variation and a temperature-sensitive sterile mutation in kl-5 suggest that the Y-chromosome regions contain the coding sequences for the polypeptides.     

The role of human and mouse Y chromosome genes in male infertility

It was suggested by Ronald Fisher in 1931 that genes involved in benefit to the male (including spermatogenesis genes) would accumulate on the Y chromosome. The analysis of mouse Y chromosome deletions and the discovery of microdeletions of the human Y chromosome associated with diverse defective spermatogenic phenotypes has revealed the presence of intervals containing one or more genes controlling male germ cell differentiation. These intervals have been mapped, cloned and examined in detail for functional genes. This review discusses the genes mapping to critical spermatogenesis intervals and the evidence indicating which are the most likely candidates underlying Y-linked male infertility.     

Polynesian origins: insights from the Y chromosome

The question surrounding the colonization of Polynesia has remained controversial. Two hypotheses, one postulating Taiwan as the putative homeland and the other asserting a Melanesian origin of the Polynesian people, have received considerable attention. In this work, we present haplotype data based on the distribution of 19 biallelic polymorphisms on the Y chromosome in a sample of 551 male individuals from 36 populations living in Southeast Asia, Taiwan, Micronesia, Melanesia, and Polynesia. Surprisingly, nearly none of the Taiwanese Y haplotypes were found in Micronesia and Polynesia. Likewise, a Melanesian-specific haplotype was not found among the Polynesians. However, all of the Polynesian, Micronesian, and Taiwanese haplotypes are present in the extant Southeast Asian populations. Evidently, the Y-chromosome data do not lend support to either of the prevailing hypotheses. Rather, we postulate that Southeast Asia provided a genetic source for two independent migrations, one toward Taiwan and the other toward Polynesia through island Southeast Asia.     

Worldwide DNA sequence variation in a 10-kilobase noncoding region on human chromosome 22

Human DNA sequence variation data are useful for studying the origin, evolution, and demographic history of modern humans and the mechanisms of maintenance of genetic variability in human populations, and for detecting linkage association of disease. Here, we report worldwide variation data from a approximately 10-kilobase noncoding autosomal region. We identified 75 variant sites in 64 humans (128 sequences) and 463 variant sites among the human, chimpanzee, and orangutan sequences. Statistical tests suggested that the region is selectively neutral. The average nucleotide diversity (pi) across the region was 0.088% among all of the human sequences obtained, 0.085% among African sequences, and 0.082% among non-African sequences, supporting the view of a low nucleotide diversity ( approximately 0.1%) in humans. The comparable pi value in non-Africans to that in Africans indicates no severe bottleneck during the evolution of modern non-Africans; however, the possibility of a mild bottleneck cannot be excluded because non-Africans showed considerably fewer variants than Africans. The present and two previous large data sets all show a strong excess of low frequency variants in comparison to that expected from an equilibrium population, indicating a relatively recent population expansion. The mutation rate was estimated to be 1.15 x 10(-9) per nucleotide per year. Estimates of the long-term effective population size N(e) by various statistical methods were similar to those in other studies. The age of the most recent common ancestor was estimated to be approximately 1.29 million years ago among all of the sequences obtained and approximately 634,000 years ago among the non-African sequences, providing the first evidence from a noncoding autosomal region for ancient human histories, even among non-Africans.     

Association of genetic variation on chromosome 9p21.3 and arterial stiffness

Objectives. Genome wide association studies have consistently reported associations between a region on chromosome 9p21.3 and a broad range of vascular diseases, such as coronary artery disease (CAD), aortic and intracranial aneurysms and type‐2 diabetes (T2D). However, clear associations with intermediate phenotypes have not been described so far. To shed light on a possible influence of this chromosomal region on arterial wall integrity, we analysed associations between single nucleotide polymorphisms (SNPs) and degree of stiffness of the abdominal aorta in elderly individuals. Methods and results. A total of 400 subjects, 212 men and 188 women, aged 70–88 years were included. Arterial stiffness was examined at the midpoint between the renal arteries and the aortic bifurcation. Two CAD‐ and aneurysm‐associated SNPs (rs10757274 and rs2891168) and one T2D‐associated SNP (rs1081161) within the 9p21.3 region were genotyped. Aortic compliance and distensibility coefficients were higher in carriers of the rs10757274G and rs2891168G alleles in men reflecting a decrease in aortic stiffness. Adjustment for age and mean arterial pressure had no effect on these associations. The two SNPs were not associated with intima‐media thickness or lumen diameter of the abdominal aorta. There were no associations between the rs10811661 SNP and any measure of aortic stiffness. Conclusions. Impaired mechanical properties of the arterial wall may explain the association between chromosome 9p21.3 polymorphisms and vascular disease.     

Quantitative trait loci define genes and pathways underlying genetic variation in longevity

Quantitative trait locus (QTL) mapping provides a means to discover and roughly position regions of the genome that harbor genes responsible for natural variation in a complex trait. QTL mapping has been utilized extensively in the pursuit of genes contributing to longevity, chiefly in two animal models, the nematode Caenorhabditis elegans and the dipteran insect Drosophila melanogaster. Research on both species has demonstrated that a relatively small set of loci accounts for most of their genetic variance in lifespan. QTL mapping complements the discovery of longevity genes by mutagenesis screens, because the two procedures are predicted to unveil overlapping but distinct types of genes. We argue that information gained from animal models, even invertebrates, can greatly facilitate the process of gene identification and testing of homologous genes in humans.     

Y chromosome microdeletions and alterations of spermatogenesis,patient approach and genetic counseling

Infertility affects 15% of couples at reproductive age and human male infertility appears frequently idiopathic. The main genetic causes of spermatogenesis defect responsible for non-obstructive azoospermia and severe oligozoospermia are constitutional chromosomal abnormalities and microdeletions in the azoospermia factor region of the Y chromosome. The improvement of the Yq microdeletion screening method gave new insights in the mechanism responsible for the genesis of Yq microdeletions and for the consequences of the management of male infertility and genetic counselling in case of assisted reproductive technology.     

Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage.

We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis. One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome. Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb. We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions.     

Significant population variation in adult male height associated with the Y chromosome and the aromatase gene

The determination of human adult height is dependent on both environmental and genetic factors. Rare causes of abnormal stature have been identified, including mutations in the gene encoding aromatase (CYP19) and regions on the Y chromosome. However, the possible role of these loci in the genetic control of normal adult height is unknown. We have performed an association study using common biallelic polymorphisms within CYP19 and the Y chromosome to determine whether these loci are associated with variation in height in 413 adult males and 335 females drawn at random from a large population sample. An association between CYP19 and height was found (difference, 2.0 cm; 95% confidence interval, 0.16-3.8; P = 0.003), but this was more evident in men (difference, 2.3 cm; 95% confidence interval, 0.38-4.4; P = 0.05) than women (difference, 0.2 cm; 95% confidence interval, -2.1 to 1.6; P = 0.94). An association was also found with the Y chromosome (P = 0.009; difference of 1.9 cm; 95% confidence interval, 0.5-3.4). Additionally, when men were grouped according to haplotypes of the CYP19 and Y chromosome polymorphisms, a difference of 4.2 cm (95% confidence interval, 0.67-7.3) was detected (P = 0.004). These results suggest that in men, genetic variation in CYP19 and on the Y chromosome are involved in determining normal adult height, and that these loci may interact in an additive fashion.     

Tandemly repeated genomic sequence demonstrates inter- and intra-strain genetic variation in Schistosoma japonicum*

Genetic variability within and among four geographical strains of Schistosoma japonicum was examined using a novel repetitive element. The element, termed Sjrh1.0, was isolated from genomic DNA of a Philippine strain of S. japonicum using a combination of restriction fragment PCR and band-stab PCR. Sjrh1.0 is a tandemly repeated element, the sequence of which appears to be species-specific, in that it hybridized to DNA from S. japonicum but not to DNA from S. mansoni . Its sequence does not match previously deposited sequences in GenBank. When employed as a probe in Southern hybridization analysis, radiolabelled Sjrh1.0 revealed sex-specific and strain-specific differences in genomic DNA of individual worms. We also found individual genetic variation within geographical isolates of the Asian schistosome.