Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands.

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.     

Molecular evolution of duplicated amylase gene regions in Drosophila melanogaster: evidence of positive selection in the coding regions and selective constraints in the cis-regulatory regions

In this study, we randomly sampled Drosophila melanogaster from Japanese and Kenyan natural populations. We sequenced duplicated (proximal and distal) Amy gene regions to test whether the patterns of polymorphism were consistent with neutral molecular evolution. F(st) between the two geographically distant populations, estimated from Amy gene regions, was 0.084, smaller than reported values for other loci, comparing African and Asian populations. Furthermore, little genetic differentiation was found at a microsatellite locus (DROYANETSB) in these samples (G'st = -0.018). The results of several tests (Tajima's, Fu and Li's, and Wall's tests) were not significantly different from neutrality. However, a significantly higher level of fixed replacement substitutions was detected by a modified McDonald and Kreitman test for both populations. This indicates that positive selection occurred during or immediately after the speciation of D. melanogaster. Sliding-window analysis showed that the proximal region 1, a part of the proximal 5' flanking region, was conserved between D. melanogaster and its sibling species, D. simulans. An HKA test was significant when the proximal region 1 was compared with the 5' flanking region of Alcohol dehydrogenase (Adh), indicating a severe selective constraint on the Amy proximal region 1. These results suggest that natural selection has played an important role in the molecular evolution of Amy gene regions in D. melanogaster.     

The Tarsius gamma-globin gene: pseudogene or active gene?

We sequenced the approximately 5-kb long gamma-globin gene locus from Tarsius bancanus and compared it to the published gamma-globin gene sequence from the related species Tarsius syrichta. The T. syrichta gene's promoter lacks the distal CCAAT box and has a point mutation in the functionally important proximal CCAAT box (CCgAT). This previous finding had suggested that in tarsiers the gamma-globin gene might be a nonexpressed pseudogene. The two tarsier species show the same point mutation at the third nucleotide of the proximal CCAAT element and absence of the distal CCAAT element. Nevertheless, our results indicate that in tarsiers the gamma-globin gene is active, since all three coding regions show only synonymous substitutions and a much lower level of divergence than the noncoding regions. This is significantly different from what would be expected for a silent gene evading stabilizing selection. Thus, we hypothesize that the tarsier's gamma-globin gene locus is expressed even with the mutation in the proximal CCAAT box.     

Restriction fragment length polymorphism and divergence in the genomic regions of high and low recombination in self-fertilizing and cross-fertilizing aegilops species.

RFLP was investigated at 52 single-copy gene loci among six species of Aegilops, including both cross-fertilizing and self-fertilizing species. Average gene diversity (H) was found to correlate with the level of outcrossing. No relationship was found between H and the phylogenetic status of a species. In all six species, the level of RFLP at a locus was a function of the position of the locus on the chromosome and the recombination rate in the neighborhood of the locus. Loci in the proximal chromosome regions, which show greatly reduced recombination rates relative to the distal regions, were significantly less variable than loci in the distal chromosome regions in all six species. Variation in recombination rates was also reflected in the haplotype divergence between closely related species; loci in the chromosome regions with low recombination rates were found to be diverged less than those in the chromosome regions with high recombination rates. This relationship was not found among the more distantly related species.     

Isolation and characterization of a human locus homologous to the transforming gene (v-fes) of feline sarcoma virus

A single locus (designated c-fes) in the human genome which exhibits homology to the transformation-specific onc gene (v-fes) of Snyder-Theilen feline sarcoma virus was identified by the Southern blot technique. Recombinant clones containing 16- to 18-kilobase inserts of human DNA including the c-fes locus were constructed. Restriction endonuclease mapping of these clones verified their identity with native human c-fes and demonstrated the presence of at least two sequences in human c-fes interrupting v-fes-homologous regions. The v-fes-homologous locus in the human genome spans about 4 kilobases. The 5'-3' orientation of the c-fes clones with respect to feline sarcoma virus proviral DNA was determined. The region of the human genome that is homologous to v-fes is proximal to the highly reiterated human Alu sequence but not to the highly reiterated human alphoid sequence.     

Structure and evolution of human sub-telomeric regions

Recent progress in the field of human genome analysis has led to the development of new concepts in the definition of subtelomeric domains. Analysis of DNA sequences from human and yeast chromosome ends have shown that short stretches of degenerate TTAGGG are found at a distance from the telomeric repeats. These stretches define a boundary between two structurally different regions. The distal domain is characterised by numerous, short segments of interrupted homology to many other human telomeric regions and to a number of ESTs. The proximal domain shows much longer uninterrupted homology to a few chromosome ends. This domain evolved quickly within primates at least, as demonstrated by the detailed study of locus DNF92 which spread very recently in humans from 17 qter to at least ten other chromosome ends. At the different sites, presence-absence polymorphisms are observed within humans. The region remained single locus at the paralogous site in higher primates. Conversely, a human and orangutan single locus telomeric domain occupies multiple chromosome ends in chimpanzee. Balanced translocation is the likely mechanism through which the spreading occurred. Some members of the olfactory receptor gene family show a similar behaviour: multiple telomeric locations, and presence-absence polymorphism. Strikingly, the set of chromosome ends occupied by the two regions is identical, except for the two ancestral sites. Moreover, the relative frequency of detection of the region at the different sites indicates some kind of competition between the two regions. Consequently, these two regions represent major new tools to investigate recent human genome evolution and human genome diversity in different populations.     

The distribution of RFLP markers on chromosome 2(2H) of barley in relation to the physical and genetic location of 5S rDNA

The 5S rDNA locus on the long arm of barley chromosome 2(2H) was genetically mapped in two crosses in relation to 30 other RFLP loci. Comparison of the genetic maps with the previously published physical position of the 5S rDNA, determined by in-situ hybridization, showed that there was a marked discrepancy between physical and genetic distance in both crosses, with recombination being less frequent in the proximal part of the arm. Pooled information from the present study and other published genetic maps showed that at least 26 of the 44 (59%) RFLPs that have been mapped on 2(2H)L lie distal to the 5S rDNA locus even though this region is only 27% of the physical length of the arm. The distribution of RFLP markers is significantly different from expected (P < 0.01), implying that the low-copy sequences used for RFLP analysis occur more frequently in distal regions of the arm and, or, that sequences in distal regions are more polymorphic.     

Assessing genetic diversity of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) germplasm using microsatellite markers

A set of 24 wheat microsatellite markers, representing at least one marker from each chromosome, was used for the assessment of genetic diversity in 998 accessions of hexaploid bread wheat (Triticum aestivum L.) which originated from 68 countries of five continents. A total of 470 alleles were detected with an average allele number of 18.1 per locus. The highest number of alleles per locus was detected in the B genome with 19.9, compared to 17.4 and 16.5 for genomes A and D, respectively. The lowest allele number per locus among the seven homoeologous groups was observed in group 4. Greater genetic variation exists in the non-centromeric regions than in the centromeric regions of chromosomes. Allele numbers increased with the repeat number of the microsatellites used and their relative distance from the centromere, and was not dependent on the motif of microsatellites. Gene diversity was correlated with the number of alleles. Gene diversity according to Nei for the 26 microsatellite loci varied from 0.43 to 0.94 with an average of 0.77, and was 0.78, 0.81 and 0.73 for three genomes A, B and D, respectively. Alleles for each locus were present in regular two or three base-pair steps, indicating that the genetic variation during the wheat evolution occurred step by step in a continuous manner. In most cases, allele frequencies showed a normal distribution. Comparative analysis of microsatellite diversity among the eight geographical regions revealed that the accessions from the Near East and the Middle East exhibited more genetic diversity than those from the other regions. Greater diversity was found in Southeast Europe than in North and Southwest Europe. The present study also indicates that microsatellite markers permit the fast and high throughput fingerprinting of large numbers of accessions from a germplasm collection in order to assess genetic diversity.     

Transvection at the vestigial locus of Drosophila melanogaster

Transvection is a phenomenon wherein gene expression is effected by the interaction of alleles in trans and often results in partial complementation between mutant alleles. Transvection is dependent upon somatic pairing between homologous chromosome regions and is a form of interallelic complementation that does not occur at the polypeptide level. In this study we demonstrated that transvection could occur at the vestigial (vg) locus by revealing that partial complementation between two vg mutant alleles could be disrupted by changing the genomic location of the alleles through chromosome rearrangement. If chromosome rearrangements affect transvection by disrupting somatic pairing, then combining chromosome rearrangements that restore somatic pairing should restore transvection. We were able to restore partial complementation in numerous rearrangement trans-heterozygotes, thus providing substantial evidence that the observed complementation at vg results from a transvection effect. Cytological analyses revealed this transvection effect to have a large proximal critical region, a feature common to other transvection effects. In the Drosophila interphase nucleus, paired chromosome arms are separated into distinct, nonoverlapping domains. We propose that if the relative position of each arm in the nucleus is determined by the centromere as a relic of chromosome positions after the last mitotic division, then a locus will be displaced to a different territory of the interphase nucleus relative to its nonrearranged homolog by any rearrangement that links that locus to a different centromere. This physical displacement in the nucleus hinders transvection by disrupting the somatic pairing of homologous chromosomes and gives rise to proximal critical regions.     

Genetic mapping and assignment of a long-range repeat cluster to band D of chromosome 1 in Mus musculus and M. spretus.

A cluster (D1Lub1) of a long-range repeat family was mapped to the proximal part of the Giemsa-negative band D in Chromosome 1 of Mus musculus and M. spretus by in situ hybridization with cloned probes of the long-range repeat family. By making use of restriction fragment length polymorphisms in DNAs from interspecific backcross mice, the cluster could be mapped to a position 5.3 +/- 2.1 cM distal to the Inha locus and the same distance proximal to the Bcl-2 locus. D1Lub1 was inseparable in 114 meiotic events from Acrg, Sag, and Akp-3. Taken together, the data may serve as a reference for coordinating the genetic and cytogenetic maps of Mus Chromosome 1. High-copy-number variants of the cluster, which appear cytogenetically as homogeneously staining regions at the same chromosome location, presumably arose by amplification of the long-range repeat family in situ.     

Analysis of two additional loci in Neurospora crassa related to Spore killer-2

Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2).     

Regional localization of the autosomal dominant polycystic kidney disease locus

The localization of the autosomal dominant polycystic kidney disease locus (PKD1) within an array of anonymous polymorphic DNA sequences on chromosome 16 band p13 was determined by multipoint mapping. Nine polymorphic DNA markers, including two hypervariable sequences, were used to study 19 PKD1 and 21 reference families. PKD1 was found to lie proximal to the 3' and 5' hypervariable regions of alpha-globin and distal to the anonymous sequence CRI-0327. Somatic cell hybrid mapping places PKD1 within the region 16p13.11-16pter. The availability of an array of linked markers which bracket the PKD1 locus provides a framework for further attempts to identify the PKD1 gene and offers an improved method of presymptomatic diagnosis of the disease.     

Replication timing of the human X-inactivation center (XIC) region: correlation with chromosome bands

The human genome is composed of long-range G+C% mosaic structures, which are thought to be related to chromosome bands. Replication timing during S phase is associated with chromosomal band zones; thus, band boundaries are thought to correspond to regions where replication timing switches. The proximal limit of the human X-inactivation center (XIC) has been localized cytologically to the junction zone between Xq13.1 and Xq13.2. Using PCR-based quantification of the newly replicated DNA from cell-cycle fractionated THP-1 cells, the replication timing in and around the XIC was determined at the genome sequence level. We found two regions where replication timing changes from the early to late period during S phase. One is located near a large inverted duplication proximal to the XIC, and the other is near the XIST locus. We propose that the 1Mb late-replicated zone (from the large inverted duplication to XIST) corresponds to a G-band Xq13.2. Several common characteristics were observed in the XIST region and the MHC class II-III junction which was previously defined as a band boundary. These characteristics included differential high-density clustering of Alu and LINE repeats, and the presence of polypurine/polypyrimidine tracts, MER41A, MER57 and MER58B.     

西藏和埃塞俄比亚大麦6个同工酶位点遗传变异的对比分析

我们对比分析了埃塞俄比亚(简称埃)和西藏共777份栽培大麦材料在6个同工酶位点(Est1、Est2、Kst3、Est4、Acp1和Got1)的遗传变异。结果表明,无论是从单个位点上分析还是在多位点基因组合形式上评价,埃大麦与西藏大麦群体在遗传组成和多位点基因结构上都有着很大的差异。遗传多样性对比分析表明,从单个位点基因类型看,西藏大麦与埃大麦遗传变异程度大致相当,但从多位点基因组合形式看,西藏大麦遗传多样性程度极显著地高于埃大麦。     

Molecular characterization of two proximal deletion breakpoint regions in both Prader-Willi and Angelman syndrome patients.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation syndromes caused by paternal and maternal deficiencies, respectively, in chromosome 15q11-q13. Approximately 70% of these patients have a large deletion of approximately 4 Mb extending from D15S9 (ML34) through D15S12 (IR10). To further characterize the deletion breakpoints proximal to D15S9, three new polymorphic microsatellite markers were developed that showed observed heterozygosities of 60%-87%. D15S541 and D15S542 were isolated from YAC A124A3 containing the D15S18 (IR39) locus. D15S543 was isolated from a cosmid cloned from the proximal right end of YAC 254B5 containing the D15S9 (ML34) locus. Gene-centromere mapping of these markers, using a panel of ovarian teratomas of known meiotic origin, extended the genetic map of chromosome 15 by 2-3 cM toward the centromere. Analysis of the more proximal S541/S542 markers on 53 Prader-Willi and 33 Angelman deletion patients indicated two classes of patients: 44% (35/80) of the informative patients were deleted for these markers (class I), while 56% (45/80) were not deleted (class II), with no difference between PWS and AS. In contrast, D15S543 was deleted in all informative patients (13/48) or showed the presence of a single allele (in 35/48 patients), suggesting that this marker is deleted in the majority of PWS and AS cases. These results confirm the presence of two common proximal deletion breakpoint regions in both Prader-Willi and Angelman syndromes and are consistent with the same deletion mechanism being responsible for paternal and maternal deletions. One breakpoint region lies between D15S541/S542 and D15S543, with an additional breakpoint region being proximal to D15S541/S542.     

Forum domain in Drosophila melanogaster cut locus possesses looped domains inside.

We have studied the relationship between chromosomal forum domains and looped domains in the cut locus of Drosophila melanogaster . Forum domains were earlier detected by separation in pulsed-field gels of 50-150 kb chromosomal DNA fragments obtained after spontaneous non-random degradation of chromosomes. We have localized the boundary region where cleavage sites are scattered between two forum domains in the regulatory region of the cut locus. We have sequenced a 13 kb region spanning few kilobases from distal domain, the boundary region and part of the proximal forum domain where several scaffold associated regions (SARs) were observed. We conclude that forum domains and looped domains are physically different types of domains and belong to different levels of organization in eukaryotic chromosomes. The boundary region between the neighboring forum domains in the cut locus possesses the Doc element insertion and a micro-satellite stretch and thus might remind a small island of heterochromatin and correspond to so-called intercalary heterochromatin that is known to be located in the 7B1-2 band where the major part of the cut locus is reside.