Yeast Artificial Chromosome Contig Research Articles

Mutations in the CNGB3 gene encoding the beta-subunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21.

Achromatopsia is an autosomal recessive disorder featuring total colour blindness, photophobia, reduced visual acuity and nystagmus. While mutations in the CNGA3 gene on chromosome 2q11 are responsible for achromatopsia in a subset of patients, previous linkage studies have localized another achromatopsia locus, ACHM3, on chromosome 8q21. Using achromatopsia families in which CNGA3 mutations have been excluded, we refined the ACHM3 locus to a 3.7 cM region enclosed by markers D8S1838 and D8S273. Two yeast artificial chromosome (YAC) contigs covering nearly the entire ACHM3 interval were constructed. Database searches with YAC content sequences identified two overlapping high throughput genomic sequencing phase (HTGS) entries which contained sequences homologous to the murine cng6 gene encoding the putative beta-subunit of the cone photoreceptor cGMP-gated channel. Using RT-PCR and RACE, we identified and cloned the human cDNA homologue, designated CNGB3, which encodes an 809 amino acid polypeptide. Northern blot analysis revealed a major transcript of approximately 4.4 kb specifically expressed in the retina. The human CNGB3 gene consists of 18 exons distributed over approximately 200 kb of genomic sequence. Analysis of the CNGB3 gene in achromats revealed six different mutations including a missense mutation (S435F), two stop codon mutations (R203X and E336X), a 1 bp and an 8 bp deletion (1148delC and 819-826del) and a putative splice site mutation of intron 13. The 1148delC mutation was identified recurrently in several families, and in total was present on 11 of 22 disease chromosomes segregating in our families.

Construction of a long-range YAC physical map spanning the 10-cM region between the markers D18Mit109 and D18Mit68 on mouse proximal chromosome 18.

Four yeast artificial chromosome (YAC) contigs, physically approximately 8 Mb, have been constructed spanning a 10-cM region on mouse proximal chromosome 18 and include the sites of 21 known genes, including those near the twirler (Tw) locus and the recently isolated Niemann-Pick type C1 (npc1) gene, formerly designated as the spm locus. This physical map consists of 49 YAC clones that cover roughly 15% of the chromosome. The physical order of 38 microsatellite sequence-tagged sites (STSs) could be assembled and confirmed based on their presence or absence in individual YACs, from proximal D18Mit109 through distal D18Mit68. These YACs provide an important resource for the further characterization and identification of known and unknown genes. The physical map has been integrated with our previously published genetic linkage map and showed an average genetic to physical distance of cM/Mb > 1.1.

Physical Map of the Region Surrounding the OTOFERLIN Locus on Chromosome 2p22–p23

The autosomal recessive form of nonsyndromic deafness DFNB9 has been mapped to a 2-cM region on chromosome 2p22–p23, and the responsible gene, OTOF, has been recently identified by positional cloning combined with a candidate gene approach. In the course of this gene cloning, we established a contig of yeast artificial chromosomes, bacterial artificial chromosomes, and P1 phage artificial chromosomes delimited by polymorphic markers D2S2170 and D2S170, i.e., extending over approximately 3500 kb. Sixty expressed sequence tags or genes and 14 sequence-tagged sites, 11 of which are polymorphic, were mapped to this contig and assigned to 21 chromosomal intervals.

No evidence for the involvement of CAG/CTG repeats from within 18q21.33-q23 in bipolar disorder.

We previously identified 18q21.33-q23 as a candidate region in one BP family and constructed a yeast artificial chromosome (YAC) contig map. Here, we mapped eight known CAG/CTG repeats relative to 18q21.33-q23. We also isolated four CAG/CTG repeats from within the region using CAG/CTG YAC fragmentation, one of which is located in the 5' untranslated region of the CAP2 gene coding for a brain-expressed serine proteinase inhibitor. The triplet repeats located in the 18q21.33-q23 BP candidate region showed no expanded alleles in the linked BP family nor in a BP case-control sample. Moreover, only the CAP2 triplet repeat was polymorphic but no genetic association with BP disorder was observed.

Cervical cancer suppressor gene is within 1 cM on 6p23

We previously showed loss of heterozygosity at 6p to be a common genetic alteration in cervical cancer and cervical intraepithelial neoplasia. To characterize this critical area of deletion in chromosome 6, we evaluated 107 invasive cervical cancers, using 23 polymorphic markers. Genomic DNA from microdissected frozen or paraffin-embedded cervical tumors and corresponding normal tissue was analyzed. Fifty-three percent (57/107) of the cervical tumors showed loss in 6p. Deletions were found in all stages and histologic types. Ninety-one percent (52/57) of these tumors had a loss at 6p23. One tumor defined the distal area of deletion at marker D6S429. Two tumors defined the proximal area of deletion at marker D6S1578. Genotyping of parental DNA was done on 16 cases to evaluate the origin of chromosomal loss. The deletion occurred in the paternal chromosome in 10 tumors and in the maternal in six. Within each tumor, the same parental chromosome was lost at all tested heterozygous 6p markers. The order of the polymorphic markers and estimate of distances in the critical region were confirmed by generation of a yeast artificial chromosome (YAC) contig and pulse-field gel electrophoresis. Our data strongly suggest that a gene important in cervical cancer tumorigenesis is located within a 1-cM region of 6p23, and it is not imprinted.

Mapping of target regions of allelic loss in primary breast cancers to 1-cM intervals on genomic contigs at 6q21 and 6q25.3.

Allelic losses on the long arm of human chromosome 6 are frequently observed in cancers of the ovary, prostate, and breast. To identify the locations of putative tumor suppressor genes on 6q, we examined 192 primary breast cancers for patterns of allelic loss at 16 polymorphic microsatellite loci distributed along this chromosome arm. Allelic losses at one or more loci were observed in 105 (55%) of the tumors examined. Detailed deletion mapping with appropriate yeast artificial chromosome (YAC) contigs identified two distinct commonly deleted regions; one was confined to a 1‐cM interval at 6q21 flanked by D6S1040 and D6S262 and the other to a 1‐cM interval at 6q25.3 flanked by D6S305 and D6S411. Allelic losses at 6q21 were more frequent in invasive solid tubular and scirrhous carcinomas than in tumors of less aggressive histologic types (P=0.0006). Allelic loss at 6q25.3 was associated with loss of progesterone receptor (P=0.0256). Our results suggest the presence of two tumor suppressor genes for breast cancer on 6q that are likely to be associated with tumor progression and/or loss of hormonal dependency.

Somatic cell hybrids for high‐density mapping of chromosome 2 breakpoints in radiation‐induced myeloid leukemia cell lines from inbred mice

Chromosome 2 (chr 2) deletions are recurrent abnormalities in acute myeloid leukemia (AML) induced by ionizing radiation in the mouse. The localization of deletion sites has proven extremely useful in providing information on the molecular mechanisms of leukemogenesis. The models available for the study of AML are mostly represented by inbred mouse strains, in which the molecular resolution of breakpoints is problematic. In this study, we have examined five leukemic cell lines exhibiting hemizygous chr 2 loss, derived from CBA, C3H, or (C57BL×CBA/H) F1 mice in which AML had been induced by a whole-body dose of radiation. By application of a somatic cell hybridization technique, we have generated interspecific cell hybrids retaining the deleted murine chr 2 homologue. This strategy permitted a very detailed genetic analysis allowing the utilization of any genetic marker on chr 2 without a requirement for polymorphism. Somatic cell hybrid clones were subjected to a high-density polymerase chain reaction–based microsatellite screening using 62–106 informative markers for each cell line. Detailed maps accurately defining chr 2 breakpoints were obtained. The identification of critical breakpoint markers allowed the construction of partial yeast artificial chromosome contigs across chr 2 breakpoints. These maps represent an essential resource for cloning of the breakpoint regions. Mol. Carcinog. 27:219–228, 2000. Published 2000 Wiley-Liss, Inc.

An Integrated Map of Human 6q22.3–q24 Including a 3-Mb High-Resolution BAC/PAC Contig Encompassing a QTL for Fetal Hemoglobin

Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of ∼4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1–2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig.

Somatic cell hybrids for high-density mapping of chromosome 2 breakpoints in radiation-induced myeloid leukemia cell lines from inbred mice

Chromosome 2 (chr 2) deletions are recurrent abnormalities in acute myeloid leukemia (AML) induced by ionizing radiation in the mouse. The localization of deletion sites has proven extremely useful in providing information on the molecular mechanisms of leukemogenesis. The models available for the study of AML are mostly represented by inbred mouse strains, in which the molecular resolution of breakpoints is problematic. In this study, we have examined five leukemic cell lines exhibiting hemizygous chr 2 loss, derived from CBA, C3H, or (C57BLxCBA/H) F1 mice in which AML had been induced by a whole-body dose of radiation. By application of a somatic cell hybridization technique, we have generated interspecific cell hybrids retaining the deleted murine chr 2 homologue. This strategy permitted a very detailed genetic analysis allowing the utilization of any genetic marker on chr 2 without a requirement for polymorphism. Somatic cell hybrid clones were subjected to a high-density polymerase chain reaction-based microsatellite screening using 62-106 informative markers for each cell line. Detailed maps accurately defining chr 2 breakpoints were obtained. The identification of critical breakpoint markers allowed the construction of partial yeast artificial chromosome contigs across chr 2 breakpoints. These maps represent an essential resource for cloning of the breakpoint regions.

A 6-Mb High-Resolution Physical and Transcription Map Encompassing the Hereditary Prostate Cancer 1 (HPC1) Region

Several hereditary disease loci have been genetically mapped to the chromosome 1q24–q31 interval, including the hereditary prostate cancer 1 (HPC1) locus. Here, we report the construction of a 20-Mb yeast artificial chromosome contig and a high-resolution 6-Mb sequence-ready bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) contig of 1q25 by sequence and computational analysis, STS content mapping, and chromosome walking. One hundred thirty-six new STSs, including 10 novel simple sequence repeat polymorphisms that are being used for genetic refinement of multiple disease loci, have been generated from this contig and are shown to map to the 1q25 interval. The integrity of the 6-Mb BAC/PAC contig has been confirmed by restriction fingerprinting, and this contig is being used as a template for human chromosome 1 genome sequencing. A transcription mapping effort has resulted in the precise localization of 18 known genes and 31 ESTs by database searching, exon trapping, direct cDNA hybridization, and sample sequencing of BACs from the 1q25 contig. An additional 11 known genes and ESTs have been placed within the larger 1q24–q31 interval. These transcription units represent candidate genes for multiple hereditary diseases, including HPC1.

High resolution genetic and physical mapping of the mouse asebia locus: A key gene locus for sebaceous gland differentiation

Summary The asebia (ab) mutation in the mouse is an autosomal recessive trait with hypoplastic sebaceous glands. As a first step toward cloning the ab gene, we report here the genetic mapping of the ab locus with respect to Chromosome 19 microsatellite markers. 644 backcross progeny were generated by mating (CAST/EiJ × DBA/1LacJ- ab 2J /ab 2J ) F 1 heterozygous females and parental ab 2J /ab 2J mutant males. Our results located the ab gene to an interval of 1.6 cM on mouse Chromosome 19 defined by flanking markers D19Mit11 and D19Mit53/D19Mit27 , and identified a tightly linked polymorphic marker, D19Mit67, that co-segregates with the mutation in the backcross progeny examined. This places ab locus 4 cM distal to its present position as indicated in Mouse Genome Database at The Jackson Laboratory. We have also mapped a yeast artificial chromosome (YAC) contig in this locus interval which suggests the ab interval to be less than one megabase of DNA.

A Sequence-Ready BAC/PAC Contig and Partial Transcript Map of Approximately 1.5 Mb in Human Chromosome 17q25 Comprising Multiple Disease Genes

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1.5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.

A high-resolution physical map of human chromosome 21p using yeast artificial chromosomes.

The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes. Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region. We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs). To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs). Thirty YACs were selected from the St. Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries. These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12. The total size of the region covered by YACs is estimated between 2.9 and 5 Mb. The integrity of the YAC contig was confirmed by restriction enzyme fingerprinting and fluorescence in situ hybridization (FISH). One gap with an estimated size of 400 kb remained near the telomeric end of the contig. This YAC contig map of the short arm of human chromosome 21 constitutes a basic framework for further structural and functional studies of chromosome 21p.

Distal Hereditary Motor Neuropathy Type II (Distal HMN Type II): Phenotype and Molecular Genetics.

The distal hereditary motor neuropathies (distal HMN) are clinically and genetically heterogeneous and are subdivided in seven subtypes according to the mode of inheritance, age at onset and clinical evolution. We studied a multigenerational Belgian pedigree with autosomal dominant distal HMN type II. The clinical phenotype closely resembles classical Charcot-Marie-Tooth (CMT) disease with an age at onset between 15 and 25 years. Linkage studies have shown that distal HMN II is not linked to the known CMT1 and CMT2 loci. A genome-wide search was performed and significant linkage was obtained between markers D12S86 and D12S340, suggesting that a gene causing distal HMN II is located on chromosome 12q24.3. The gene encoding the human pancreatic phospholipase A2 (PLA2A), which is expressed in peripheral nerves during degeneration, is a positional candidate gene. Because no disease-specific mutations were detected in the coding region, however, PLA2A is most likely not the disease causing gene. A yeast artificial chromosome (YAC) contig map spanning the candidate region has been constructed to isolate the gene responsible for distal HMN II. Positional and functional candidate genes are currently being screened for the presence of mutations in distal HMN II patients.

Radiation Hybrid and Cytogenetic Mapping of SOCS1 and SOCS2 to Chromosomes 16p13 and 12q, Respectively

Suppressor of cytokine signaling (SOCS) proteins are involved in the negative regulation of cytokine-induced STAT (signal transducers and activators of transcription) factor signaling. We cloned genomic regions of SOCS1 and SOCS2 genes and mapped these genes to chromosome 16p12–p13.1 and chromosome 12q21.3–q23 regions, respectively, by cytogenetic and radiation hybrid mapping. In addition, we mapped SOCS2 by yeast artificial chromosome (YAC) pool screening to YAC contig WC 12.5 on chromosome 12 with an unambiguous hit to CHLC.ATA19H12 and WI-5940, which is 461.5 cR from the top of the map.

Localization on a Physical Map of the NKC-Linked Cmv1 Locus Between Ly49b and the Prp Gene Cluster on Mouse Chromosome 6

Abstract The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of ∼390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.

High-Resolution Landmark Framework for the Sequence-Ready Mapping of Xq23–q26.1

We have established a landmark framework map over 20-25 Mb of the long arm of the human X chromosome using yeast artificial chromosome (YAC) clones. The map has approximately one landmark per 45 kb of DNA and stretches from DXS7531 in proximal Xq23 to DXS895 in proximal Xq26, connecting to published framework maps on its proximal and distal sides. There are three gaps in the framework map resulting from the failure to obtain clone coverage from the YAC resources available. Estimates of the maximum sizes of these gaps have been obtained. The four YAC contigs have been positioned and oriented using somatic-cell hybrids and fluorescence in situ hybridization, and the largest is estimated to cover approximately 15 Mb of DNA. The framework map is being used to assemble a sequence-ready map in large-insert bacterial clones, as part of an international effort to complete the sequence of the X chromosome. PAC and BAC contigs currently cover 18 Mb of the region, and from these, 12 Mb of finished sequence is available.

Deletion mapping at 12p12-13 in metastatic prostate cancer

The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN. We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits. We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion. A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse. Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted. Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest. We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358. We then used the same primers to construct a YAC contig across the homozygous deletion. PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion. Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft. Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.

Identification and mapping of novel tumor suppressor loci on 6p in diffuse large B-cell non-Hodgkin's lymphoma

Allelic deletions have been thought to be indicators of the presence of tumor suppressor genes (TSGs). As indicated by this allelotype study using 39 highly informative microsatellite markers distributed among all autosomal chromosomes, frequent loss of heterozygosity (LOH) has been found at 6p in B-cell non-Hodgkin's lymphoma. To identify the commonly deleted regions (CDRs), we performed fine deletion mapping using 26 highly polymorphic microsatellite markers on 6p. The most frequent LOH occurred at D651721, where 9 of 18 of the informative cases (50%) had allelic losses. Seventeen of 32 cases (53%) exhibited LOH at least at one locus on 6p. Ten of these 17 cases showed interstitial deletions, and their LOH patterns indicated two CDRs on 6p; one between D6S1721 and D6S260 (at 6p23-24), and the other between D6S265 and D6S291 (at 6p21). The genetic distance of both CDRs was 6 cM. The CDKN1A (p21) gene is reported to be located within the interval of the CDR at 6p21, but no mutation of the gene was found in these 32 patients. These data suggested that these two loci might harbor novel putative TSGs responsible for the pathogenesis of malignant lymphoma. We have constructed a contig of yeast artificial chromosome (YAC) clones spanning the most frequent CDR at 6p23-24. This YAC contig can be used for fine physical mapping of the region and cloning of candidate TSGs.

Isochromosome 17q in Blast Crisis of Chronic Myeloid Leukemia and in Other Hematologic Malignancies Is the Result of Clustered Breakpoints in 17p11 and Is Not Associated With Coding TP53 Mutations

An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome-positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies. The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression. This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail. We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes [MDS], 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH). Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p. In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable. In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone. Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region. Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11). Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region. In 16 cases, no TP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53. Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia.