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X chromosome inactivation is a mechanism of dosage compensation that regulates the expression of mammalian X-linked genes between XY males and XX females. This phenomenon is cis-acting, clonally heritable, and requires the presence of an X inactivation center (XIC). In our attempts to characterize this phenomenon, we have focused on the physical organization of the human XIC localized to Xq13. From previous studies, we had determined that the candidate XIC interval contained two loci (DXS128 and XIST) and was bound by the breakpoints of two structurally abnormal inactivated X chromosomes, a t(X;14) and an idic(Xp). Here we present a refined mapping of the XIC-containing region using the breakpoint of a late replicating rearranged X (rea(X)), and the initial characterization of a set of 40 yeast artificial chromosomes (YACs) derived from the XIC-containing region. These YACs form a 2.6 Mb contig which completely covers the XIC, and physically links the RPS4X, PHKA1, XIST, and DXS128E genes, as well as a laminin receptor pseudogene (LAMRP4). Furthermore, we have determined the relative orientations of these four genes, and have derived a restriction map of the region using the rare cutter enzymes BssHII, EagI, MluI, NruI, SalI, SfiI, SstII (or SacII), and NotI. We have identified at least 9 CpG-rich islands within this region, and have discovered a large (approximately 125 kb) inverted duplication proximal to the XIC based on symmetrical restriction patterns and homologous probes. We estimate the maximum size of the XIC-containing interval to be between 680 kb and 1200 kb, based on the localization of the breakpoints of the rearranged X chromosomes mentioned above. This lays the groundwork for the further characterization of the XIC region and the isolation of other expressed sequences therefrom.

The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention. © 2003 Nature Publishing Group.

We hypothesized that individuals who develop fever after smallpox vaccination have genetically determined differences in their immune responses to vaccinia virus. We looked for an association between the development of fever and single-nucleotide polymorphisms (SNPs) in 19 candidate genes in 346 individuals previously assessed for clinical responses to smallpox vaccination. Fever after smallpox vaccination is associated with specific haplotypes in the interleukin (IL)-1 gene complex and in the IL18 gene. A haplotype in the IL4 gene was highly significant for reduced susceptibility to the development of fever after vaccination among vaccinia-naive individuals. Our results indicate that certain haplotypes in the IL-1 gene complex and in IL18 and IL4 predict an altered likelihood of the development of fever after smallpox vaccination. Our findings also raise the possibility that these same haplotypes may identify individuals at risk for the development of fever after receipt of other live virus vaccines, providing information that could be useful in anticipating and preventing more-serious adverse events.

The flaky skin (fsn) mutation in mice causes pleiotropic abnormalities including psoriasiform dermatitis, anemia, hyper-IgE, and anti-dsDNA autoantibodies resembling those detected in systemic lupus erythematosus. The fsn mutation was mapped to an interval of 3.9 kb on chromosome 17 between D17Mit130 and D17Mit162. Resequencing of known and predicted exons and regulatory sequences from this region in fsn/fsn and wild-type mice indicated that the mutation is due to the insertion of an endogenous retrovirus (early transposon class) into intron 14 of the Tetratricopeptide repeat (TPR) domain 7 (Ttc7) gene. The insertion leads to reduced levels of wild-type Ttc7 transcripts in fsn mice and the insertion of an additional exon derived from the retrovirus into the majority of Ttc7 mRNAs. This disrupts one of the TPRs within TTC7 and may affect its interaction with an as-yet unidentified protein partner. The Ttc7 is expressed in multiple types of tissue including skin, kidney, spleen, and thymus, but is most abundant in germinal center B cells and hematopoietic stem cells, suggesting an important role in the development of immune system cells. Its role in immunologic and hematologic disorders should be either investigated.

Inherited genetic variation has a critical but as yet largely uncharacterized role in human disease. Here we report a public database of common variation in the human genome: more than one million single nucleotide polymorphisms (SNPs) for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations, including ten 500-kilobase regions in which essentially all information about common DNA variation has been extracted. These data document the generality of recombination hotspots, a block-like structure of linkage disequilibrium and low haplotype diversity, leading to substantial correlations of SNPs with many of their neighbours. We show how the HapMap resource can guide the design and analysis of genetic association studies, shed light on structural variation and recombination, and identify loci that may have been subject to natural selection during human evolution. © 2005 Nature Publishing Group., link_to_OA_fulltext

Chromosome 17q25 harbors a susceptibility locus for psoriasis ( PSORS2). This locus may overlap with loci for atopic dermatitis and rheumatoid arthritis. To further refine the location of PSORS2, we genotyped 242 primarily nuclear families for 15 polymorphic microsatellites mapping to chromosome 17q23-q25. Non-parametric linkage analysis revealed a linkage peak lying close to a novel cluster of genes from the immunoglobulin (Ig) superfamily. This cluster spans250 kb and harbors five CMRF35-like genes and a sixth inhibitory receptor ( CMRF35H) with three ITIM motifs that is transcribed in the opposite direction from the rest. The Ig domains encoded by these genes are most similar to those of the TREM (triggering receptor expressed selectively in myeloid cells) molecules, NKp44 and the polymeric immunoglobulin receptor. CMRF35-like genes are only expressed in sub-populations of cells of the myeloid lineage. In order to investigate the association of this region with psoriasis, we genotyped the families for 13 novel microsatellites and 19 SNPs from the region of linkage. A maximum NPL of 1.6 ( P=0.05) was obtained within the interval. Two SNP-based haplotypes revealed some evidence for association with psoriasis. One spanned CMRF35H and includes a non-synonymous polymorphism within CMRF35H (R111Q) (TDT P=0.03). The second was a three-locus haplotype lying within the first intron of CMRF35A2 ( TREM5) (TDT P=0.04). The novel markers described here will facilitate additional linkage and association studies between the CMRF35 family and disease.

Comparison of different metrics, using three large samples of haplotypes from different populations, demonstrates that rho is the most efficient measure of association between pairs of single nucleotide polymorphisms (SNPs). Pairwise data can be modeled, using composite likelihood, to describe the decline in linkage disequilibrium with distance (the Malecot model). The evidence from more isolated populations (Finland, Sardinia) suggests that linkage disequilibrium extends to 427-893 kb but, even in samples representative of large heterogeneous populations, such as CEPH, the extent is 385 kb or greater. This suggests that isolated populations are not essential for linkage disequilibrium mapping of common diseases with SNPs. The in parameter of the Malecot model (recombination and time), evaluated at each SNP, indicates regions of the genome with extensive and less extensive disequilibrium (low and high values of in respectively). When plotted against the physical map, the regions with extensive and less extensive linkage disequilibrium may correspond to recombination cold and hot spots. This is discussed in relation to the Xq25 cytogenetic band and the HFE gene region.

Linkage disequilibrium (LD), or the non-random association of alleles, is poorly understood in the human genome1. Population genetic theory suggests that LD is determined by the age of the markers, population history, recombination rate, selection and genetic drift2. Despite the uncertainties in determining the relative contributions of these factors, some groups have argued that LD is a simple function of distance between markers3,4. Disease-gene mapping studies and a simulation study gave differing predictions on the degree of LD in isolated and general populations5,6. In view of the discrepancies between theory and experimental observations, we constructed a high-density SNP map of the Xq25–Xq28 region7 and analysed the male genotypes and haplotypes across this region for LD in three populations. The populations included an outbred European sample (CEPH males) and isolated population samples from Finland and Sardinia. We found two extended regions of strong LD bracketed by regions with no evidence for LD in all three samples. Haplotype analysis showed a paucity of haplotypes in regions of strong LD. Our results suggest that, in this region of the X chromosome, LD is not a monotonic function of the distance between markers, but is more a property of the particular location in the human genome.

A high-density single-nucleotide polymorphism (SNP) map was developed for Xq25–q28 using a targeted approach to SNP discovery. This high-density map includes 217 new SNP markers, and 117 are informative in the CEPH parent population with >20% minor allele frequency. The average distance between SNP markers is 100 kb in the targeted regions. This is the densest genetic map of Xq25–q28 to date. The SNP markers are presented in order by their distance in megabases along the X chromosome, and the markers from the current genetic map are placed using the same scale to produce an integrated map of the region.

An efficient strategy to develop a dense set of single-nucleotide polymorphism (SNP) markers is to take advantage of the human genome sequencing effort currently under way. Our approach is based on the fact that bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) used in long-range sequencing projects come from diploid libraries. If the overlapping clones sequenced are from different lineages, one is comparing the sequences from 2 homologous chromosomes in the overlapping region. We have analyzed in detail every SNP identified while sequencing three sets of overlapping clones found on chromosome 5p15.2, 7q21–7q22, and 13q12–13q13. In the 200.6 kb of DNA sequence analyzed in these overlaps, 153 SNPs were identified. Computer analysis for repetitive elements and suitability for STS development yielded 44 STSs containing 68 SNPs for further study. All 68 SNPs were confirmed to be present in at least one of the three (Caucasian, African-American, Hispanic) populations studied. Furthermore, 42 of the SNPs tested (62%) were informative in at least one population, 32 (47%) were informative in two or more populations, and 23 (34%) were informative in all three populations. These results clearly indicate that developing SNP markers from overlapping genomic sequence is highly efficient and cost effective, requiring only the two simple steps of developing STSs around the known SNPs and characterizing them in the appropriate populations.[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AC003015 (for GS113423),AC002380 (GS330J10), AC000066 (RG293F11), AC003086 (RG104F04), AC002525(257C22A), and U73331 (96A18A).]

The region surrounding the ZNF35 zinc finger protein gene on 3p21 is of particular interest, as this region of chromosome 3 is frequently involved in rearrangements and/or deletions associated with various human tumors including lung and renal carcinoma. We have analyzed yeast artificial chromosomes (YACs), identified by PCR screening, using oligonucleotides derived from the ZNF35 gene. PFGE and Southern blot/hybridization analysis revealed that the clones cover 750-kb including the ZNF35 gene. The use of specific somatic cell hybrids have allowed us to locate the YAC contig telomeric to the D3F15S2 locus, in a region which is frequently deleted in lung carcinomas. In addition, we have developed a novel cDNA hybridization protocol allowing the isolation of transcribed sequences present in the overlapping YAC clones. Using the cDNA hybridization selection, we have isolated and characterized one transcribed sequence (D3S1362E) from the 3p21 YAC contig and the corresponding cDNA has been isolated. DNA sequencing analysis indicated that the D3S1363E cDNA codes for a putative transcription factor. Northern blot analysis indicated that the D3S1362E sequence hybridized to multiple transcripts in skeletal muscle, and weakly hybridizing transcripts of similar sizes were detected in other tissues.

The human regulators of complement activation gene cluster (RCA cluster) have been partially characterized with yeast artificial chromosomes (YACs). While the data confirm many points previously elucidated, the finer resolution of YAC mapping has allowed the discovery and/or localization of partial gene duplications, the determination of gene orientations, and the measurement of gaps between known genes. Here nine overlapping YACs that encompass a genomic region of 800 kb, encoding four RCA genes and three gene-like elements, are described. The encoded genes and two of the gene-like elements share the same orientation and are ordered (5′ to 3′) DAF, CR2, CR1, MCP-like, CR1-like, and MCP. A C4bp-like region lies upstream from DAF and is likely to correspond to one recently observed by F. Pardo-manuel, J.Rey-Campos, A. Hillarp, B. Dahlback, and S. Rodriguez de Cordoba (1990, Proc. Natl. Acad. Sci. USA 87: 4529–4533). MCP-like, a new genetic element, was discovered and found to be homologous to the 5′ portion of the MCP gene. Two large gaps of 85 kb (between CR2 and DAF) and 110 kb (between DAF and the C4bp-like element) could carry additional RCA genes. The arrangement of CR1, MCP-like, CR1-like, and MCP, in that order, strongly suggests that this region was generated by a single duplication of neighboring CR1 CR1 -like and MCP MCP -like forerunners. The RCA YACs will now serve as convenient DNA sources for the subcloning and further characterization of this region.

A 290-kilobase-pair chromosomal segment containing the genes encoding the human class I major histocompatibility complex molecules HLA-B and HLA-C as well as a class I pseudogene has been isolated on three overlapping yeast artificial chromosome (YAC) clones. One YAC clone contains both the HLA-B and HLA-C genes. These loci are located approximately 85 kilobase pairs apart, each in close association with a CpG island. Southern blotting and nucleotide sequencing showed no evidence of alteration of the structure of the cloned DNA in the YACs. End fragments from the YAC inserts have been isolated and used to confirm the overlaps between clones. These fragments can also serve as polymorphic markers for structural analysis of the major histocompatibility complex. Our data show that YAC cloning offers an attractive alternative for analysis of the structures of large gene complexes such as HLA.

The construction of large-scale physical maps requires efficient approaches to generate new probes and link informative markers. The mapping of a human chromosomal segment was initiated by using the 18q21.3 probes, plasminogen activator inhibitor type-2 (PLANH2) and BCL2, to screen a yeast artificial chromosome (YAC) library. An inverse polymerase chain reaction technique rescued genomic ends of the YAC inserts. These new probes were used in a chromosomal walking strategy which established that the PLANH2 gene was 600 kb telomeric and in the opposite transcriptional orientation to that of BCL2. Overall, 16 YACs with a mean size of approximately 300 kb were analyzed using rare-cutting restriction endonucleases and 10 end-rescued probes. A contiguous map within 18q21.3 that spans approximately 2 Mb was assembled. This establishes the feasibility of using YACs in the efficient cloning and physical surveying of expanses of the human genome lacking closely spaced, genetic landmarks.

Psoriasis is a complex inflammatory disease of the skin affecting 1–2% of the Caucasian population. Associations with alleles from the HLA class I region (now known as PSORS1), particularly HLA-Cw*0602, were described over 20 years ago. However, extensive linkage disequilibrium (LD) within this region has made it difficult to identify the true susceptibility allele from this region. A variety of genes and regions from a 238-kb interval extending from HLA-B to corneodesmosin (CDSN) have been proposed to harbor PSORS1. In order to identify the minimum block of LD in the MHC class I region associated with psoriasis we performed a comprehensive case/control and family-based association study on 242 Northern European psoriasis families and two separate European control populations. High resolution HLA typing of HLA-A, -B and -C alleles was performed, in addition to the genotyping of 18 polymorphic microsatellites and 36 SNPs from a 772-kb segment of the HLA class I region harboring the previously described interval. This corresponded on average to one SNP every 7 kb in the candidate 238 kb region. With all tests, the association was the strongest with single markers and haplotypes from a block of LD harboring HLA-C and SNP n.9. Logistic regression analyses indicated that association seen with candidate genes from the interval such as CDSN and HCR was entirely dependent on association with HLA-Cw*0602 and SNP n.9-G alleles. The previously reported association with CDSN and HCR was observed to be due to the existence of the associated alleles lying on the most commonly over-transmitted haplotype. Rare over-transmitted haplotypes also harbored HLA-Cw*12 alleles. HLA-Cw*12 family members are closely related to HLA Cw*0602, sharing identical sequences in their alpha-2 domains, peptide-binding pockets A, D and E and all 3′ introns. The introduction of a potential binding site for the RUNX/AML family of transcription factors in intron 7, is also specific to these HLA-C alleles. These variants need to be investigated further for their role as PSORS1.

To develop an efficient strategy for mapping genetic factors associated with common diseases, we constructed linkage disequilibrium (LD) maps of human chromosomes 5, 7, 17, and X. These maps consist of common single nucleotide polymorphisms at an average intermarker distance of 100 kb. The genotype data from these markers in a panel of American samples of European descent were analyzed to produce blocks of markers in strong pair-wise LD. Power calculations were used to guide block definitions and predicted that high-level LD maps would be useful in initial genome scans for susceptibility alleles in case-control association studies of complex diseases. As anticipated, LD blocks on the X chromosome were larger and covered more of the chromosome than those found on the autosomes.

Complete hydatidiform moles (CHMs) are diploid tumors that result from fertilization of an empty ovum by a haploid 23,X sperm. In most cases, the resulting duplication of the genome gives rise to a 46,XX genotype and is thought to be androgenetic in origin. If this hypothesis is correct, then the genotypes of all polymorphic markers in CHMs should be homozygous. We used a dense set of single-nucleotide polymorphism (SNP) markers, evenly spaced throughout the genome, to definitively test this hypothesis. We genotyped genomic DNA samples from five CHMs and their corresponding maternal samples with 1494 SNP markers using high-density microarrays (HuSNP). As predicted, the maternal samples were heterozygous at >25% of the markers, which is consistent with the expected average heterozygosity of this panel of SNPs. In contrast, the five CHM samples were heterozygous at

While scanning for single-nucleotide polymorphisms (SNPs) in the human Xq25–q28 region of CEPH families, we found six long “deserts” of low SNP incidence representing 28% of the investigated genome. One was 1.66 Mb in length. To determine whether these SNP deserts were due to reduced input of mutations or to recent coalescent events such as bottlenecks or selective sweeps, comparative sequence was determined from a female orangutan. The mean divergence was 2.9% and was not reduced in deserts compared with nondesert regions. Thus, the best explanation for the SNP deserts is recent coalescent events in humans. These events are the cause of substantial variation in human noncoding SNP incidence. In addition, the mutational spectrum in humans and orangutans was estimated as 63% AG (and CT), 17% AC (and GT), 8% CG, 4% AT, and 8% insertion/deletions. The average lifetime of a SNP destined to become fixed for a new allele between these species was estimated as 284,000 years.

Single-nucleotide polymorphisms (SNPs) are the most frequently found DNA sequence variations in the human genome (Taillon-Miller et al. 1998). It has been argued that a dense set of SNP markers can be used to identify genetic factors associated with complex disease traits (Risch and Merikangas 1996; Collins et al. 1997). Advocates of these approaches suggest that some 100,000 or more SNP markers (at 30-kb intervals or up to five markers per gene) will be needed in population studies to detect genetic factors with moderate effects in the complex traits being investigated (Collins et al. 1997). Several efforts, sponsored by both the National Human Genome Research Institute and private industry, have been launched to develop SNP markers with the goal of achieving the numbers needed for association studies within the next 3 years (Marshall 1997, 1998; Wang et al. 1998). Because all high-throughput genotyping methods capable of handling large numbers of markers and samples require precise knowledge of the DNA sequence surrounding the SNP markers, and the usefulness of the markers is determined by their heterozygosity in the population, any SNP discovery approach must involve the determination of DNA sequence and allele frequencies. Furthermore, most high-throughput genotyping methods also require a genomic DNA amplification step, making it necessary to develop sequence-tagged sites (STSs) that amplify only the DNA fragments containing the SNPs and nothing else from the rest of the genome. This is not a trivial concern because there are many repetitive elements and duplicated regions in the genome in which near identical sequences are found on different chromosomal regions. If the DNA fragments amplified by the PCR (Saiki et al. 1988) came from different parts of the genome but were near identical, the DNA sequence differences might be erroneously considered alleles of an SNP, leading to highly confusing results when the genotyping experiments were performed. The use of computer programs such as REPEAT MASKER (A.F.A. Smit and P. Green, unpubl.) has made it a simple task to avoid developing SNP markers from common repetitive regions such as those containing Alu or L1 elements. What is more difficult to detect is the presence of a putative SNP in a duplicated region of the genome. In this report, we demonstrate the utility of a SNP screening approach that yields the DNA sequence and allele frequency information while screening out duplications with minimal cost and effort. The approach combines the use of a complete hydatidiform mole (CHM) as a homozygous DNA reference sample and a pooled DNA sequencing strategy for SNP identification and allele frequency estimation (Kwok et al. 1994). A CHM is usually a 46, XX homozygote formed by the fertilization of an empty ovum by a single haploid sperm, which later duplicates its chromosomes to give a diploid tumor (Lawler et al. 1991). The worldwide incidence of hydatidiform moles is 1/1000 pregnancies (Grimes 1984). We have reported previously that the CHM can be used as a homozygous DNA reference in SNP marker development (Taillon-Miller et al. 1997). In the course of screening anonymous STSs for SNPs, we have noticed that the DNA from this CHM1 can be used to identify false-positive SNPs that are the result of amplification of duplicated regions of the genome as in the case of multigene families or low-frequency repeats. In this study we show that in every case in which the CHM sequence contains a heterozygous base, it is the result of duplication, and the sequence differences are not in fact allelic. In regions of the genome in which high-quality, large-scale sequencing is being performed, we have shown that the most efficient and cost-effective approach to SNP identification is comparison of the consensus sequences of the overlapping regions of the large-insert clones being sequenced (Taillon-Miller et al. 1998). In regions in which no such overlapping sequences are available, one has to develop STSs and screen the DNA fragments amplified from multiple individuals for DNA sequence variations (Kwok et al. 1996; Wang et al. 1998). We have advocated a sequence comparison approach consisting of obtaining the DNA sequences from four individuals (eight chromosomes) plus a pooled DNA sample for allele frequency estimation (Kwok et al. 1994, 1996). This strategy allows one to identify, with >85% probability all the SNPs with >20% allele frequency for the minor allele (Kwok et al. 1994). SNPs developed by use of the population pool method of estimating frequencies have been confirmed by subsequent genotyping of the markers in every individual present in the pool and the frequencies have been shown to be accurate (±5%) (Kwok et al. 1994). With the advent of two new classes of dye-labeled dideoxy chain terminators (the dRhodamine and the energy transfer, BigDye terminator) that have improved spectral properties and give more even peaks in cycle sequencing (Zakeri et al. 1998), we show in this study that one can reduce the number of samples used in each screening experiment from five to just two (CHM and pooled sample) and still identify all the SNPs found with the previous approach. Reducing the number of sequencing reactions required to identify SNPs from anonymous STSs and screening out duplications undetected by computer filters greatly lowers the reagent and labor cost of SNP development.

The most frequent type of complete hydatidiform mole is a 46, XX homozygote formed by the fertilization of an empty ovum by a single haploid sperm that later duplicates its chromosomes to give a diploid tumor. The homozygous nature of these complete hydatidiform moles makes them unique resources for human genome studies. They can serve as homozygous controls in the development of single nucleotide polymorphism (SNP) markers and provide a way to obtain long-range haplotypes that are useful in population studies. The use of a homozygous control makes it possible to estimate the allele frequencies of the SNP markers in any population by sequencing pooled DNA samples. In this report, we present evidence of homozygosity of a complete hydatidiform mole using 20 diallelic markers distributed across the genome. Furthermore, its usefulness as a homozygous control in SNP development and as a resource for long-range haplotype determination is demonstrated using 11 newly discovered loci in the BRCA2 region on chromosome 13q12-q13.

A cDNA clone coding for human protein kinase CK1 (casein kinase 1) has been isolated and sequenced demonstrating that it corresponds to a homolog of the CK1 alpha form found in bovine brain. The derived amino acid sequence of the human CK1 alpha is identical to the bovine counterpart except that it contains 12 extra amino acids at the carboxyl end. Using this cDNA sequence and PCR amplification, YAC genomic clones that contain this human CK1 alpha sequence have been isolated. These YACs have been used for fluorescent in situ hybridization in order to localize the human CK1 alpha gene to chromosome 13q13.

The yeast artificial chromosome (YAC) system (Burke et al. , 1987, Science 236: 806–812) allows the direct cloning of large regions of the genome. A YAC contig map of approximately 700 kb encompassing the region surrounding the type 1 neurofibromatosis ( NF1 ) locus on 17q11.2 has been constructed. A single YAC containing the entire NF1 locus has been constructed by homologous recombination in yeast. In the process of contig construction a novel method of YAC end rescue has been developed by YAC circularization in yeast and plasmid rescue in bacteria. YACs containing homology to the NF1 region but mapping to another chromosome have also been discovered. Sequences of portions of the homologous locus indicate that this other locus is a nonprocessed pseudogene.

The gene responsible for Huntington disease has been localized to a 2.5 million base pair (Mb) region between the loci D4S10 and D4S168 on the short arm of chromosome 4. As part of a strategy to clone the HD gene on the basis of its chromosomal location, we isolated genomic DNA from the HD region as a set of overlapping yeast artificial chromosome (YAC) clones. Twenty-eight YAC clones were identified by screening human YAC libraries with twelve PCR-based sequence-tagged sites (STSs) from the region. We assembled the YAC clones into overlapping sets by hybridizing them to a large number of DNA probes from the HD region, including the STSs. In addition, we isolated the ends of the human DNA inserts of most of the YAC clones to assist in the construction of the contig. Although almost half of the YACs appear to contain chimeric inserts and several contain internal deletions or other rearrangements, we were able to obtain over 2.2 Mb of the HD region in YACs, including one continuous segment of 2.0 Mb covering the region that most likely contains the HD gene. Ten of the twenty eight YAC clones comprise a minimal set spanning the 2.2 Mb. These clones provide reagents for the complete characterization of this region of the genome and for the eventual isolation of the HD gene.

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.

Eight major histocompatibility complex (MHC) class II loci and the newly defined Y3/Ring 4 locus were isolated in overlapping yeast artificial chromosome (YAC) clones defining a 420-kb segment of human chromosome 6p21.3. YAC B1D12 spanning 320 kb contained seven of these loci from HLA-DRA to HLA-DQB2. A 330-kb YAC, A148A7, spanned from the HLA-DQA1 locus through the Y3/Ring 4 locus and extended at least 130 kb centromeric of YAC B1D12. Southern blotting demonstrated that YAC B1D12 derived from the HLA-DR3 haplotype and that YAC A148A7 derived from the HLA-DR7 haplotype of the heterozygous library donor. A third 150-kb YAC, A95C5, lay within this contig and contained only the HLA-DRA locus. A fourth 300-kb YAC, A76F11, was isolated by chromosome walking from the telomeric end of YAC B1D12. Probes isolated from the ends of the YAC genomic inserts have been used to confirm overlaps between the clones. These analyses demonstrated that the centromeric end of YAC A76F11 used the same genomic EcoRI cloning site as the telomeric end of YAC A95C5. YAC B1D12 used an EcoRI site only 2.1 kb telomeric of the aforementioned EcoRI site. These data suggest that certain EcoRI sites are used preferentially during construction of the library. These YACs complete the linkage of the DR and DQ subregions of the HLA complex in cloned DNA and provide the substrate for precise analysis of this portion of the class II region.

Kallmann's syndrome (clinically characterized by hypogonadotropic hypogonadism and inability to smell) is caused by a defect in the migration of olfactory neurons, and neurons producing hypothalamic gonadotropin-releasing hormone. A gene has now been isolated from the critical region on Xp22.3 to which the syndrome locus has been assigned: this gene escapes X inactivation, has a homologue on the Y chromosome, and shows an unusual pattern of conservation across species. The predicted protein has significant similarities with proteins involved in neural cell adhesion and axonal pathfinding, as well as with protein kinases and phosphatases, which suggests that this gene could have a specific role in neuronal migration.

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.

Spontaneous and UV induced unequal mitotic sister chromatid recombination was examined in RAD+ and rad52-1 strains carrying the LEU2 gene inserted in the rDNA locus. The rad52-1 mutation does not affect spontaneous sister chromatid recombination but greatly reduces the frequency of UV induced sister chromatid recombination.

An improved assay for the measurement of human ovarian tumor antigen NB/70K (triton NB/70K assay) has been developed. This assay makes the accurate and reproducible estimation of serum NB/70K possible. Freezing and thawing of serum up to three times or refrigeration of diluted serum up to 7 days does not significantly alter the NB/70K levels detected by the triton NB/70K assay. NB/70K levels in different volumes of the same sample showed a linear relationship to volume, with a correlation coefficient usually above 0.98.

An improved NB/70K assay which can be used to reproducibly and accurately measure the NB/70K content of unextracted serum samples has been developed. Analysis of pre-treatment serum samples has indicated that the NB/70K assay shows selectivity for ovarian cancer with respect to nonmalignant ovarian cysts and tumors as well as controls; if one considers 10 units of NB/70K as a significant level of antigen, then there are no false positives in the benign and control groups, while 10 of 21 in the ovarian cancer group have significant NB/70K levels. When NB/70K levels of ovarian cancer patients are compared to those of patients with nonovarian malignant disease, only 4 of 23 patients with nonovarian cancers had significantly elevated NB/70K levels. However, none of these 4 false positives had levels greater than 20 units.

Allelic association between pairs of loci is derived in terms of the association probability ρ as a function of recombination θ, effective population size N , linear systematic pressure v , and time t , predicting both ρ rt , the decrease of association from founders and ρ ct , the increase by genetic drift, with ρ t = ρ rt + ρ ct . These results conform to the Malecot equation, with time replaced by distance on the genetic map, or on the physical map if recombination in the region is uniform. Earlier evidence suggested that ρ is less sensitive to variations in marker allele frequencies than alternative metrics for which there is no probability theory. This robustness is confirmed for six alternatives in eight samples. In none of these 48 tests was the residual variance as small as for ρ. Overall, efficiency was less than 80% for all alternatives, and less than 30% for two of them. Efficiency of alternatives did not increase when information was estimated simultaneously. The swept radius within which substantial values of ρ are conserved lies between 385 and 893 kb, but deviation of parameters between measures is enormously significant. The large effort now being devoted to allelic association has little value unless the ρ metric with the strongest theoretical basis and least sensitivity to marker allele frequencies is used for mapping of marker association and localization of disease loci.

The HLA class I genes are located within a 2-million-base pair (2-Mbp) region constituting the telomeric half of the human major histocompatibility complex. The large majority of the class I sequences, including the HLA-A, -E, -F, and -G genes, is found within the telomeric 1 Mbp. We report here the isolation and characterization of yeast artificial chromosome (YAC) clones that span a contiguous region of greater than 1.2 Mbp and include 14 of the 18 characterized class I sequences. Restriction enzyme mapping and the use of locus-specific probes have allowed all of the class I genes and sequences to be ordered and positioned within the region. In addition, the transcriptional orientation of the four class I genes has been determined. Using probes derived from the ends of YAC inserts and from class I pseudogenes, we describe a highly polymorphic region between the HLA-A and HLA-G genes. This region appears to be deleted in certain HLA haplotypes, shortening the distance between HLA-A and HLA-G by greater than 50 kilobase pairs (kbp). As part of the characterization of the YAC clones, unique sequence probes derived from the ends of each YAC insert were identified. When combined with probes derived from HLA genes and pseudogenes, 25 locus-specific probes spanning the 1.2-Mbp region have been identified for an average of 1 probe every 48 kbp.