YAC Clones Research Articles

Localization of the Gene (LAMA4) to Chromosome 6q21 and Isolation of a Partial cDNA Encoding a Variant Laminin A Chain

Laminin is a basement membrane glycoprotein composed of three nonidentical chains, A, B1, and B2. Variant chains such as merosin and S-laminin have been found in different tissues. We have isolated a cDNA encoding a novel laminin A variant that hybridizes to a 6.45-kb mRNA. Using amplification of genomic DNA and flow-sorted chromosomes we have assigned the gene (LAMA4) for this new laminin A variant to chromosome 6. Fluorescence in situ hybridization of a YAC clone further localized the gene to 6q21.

Analysis of Mnk, the Murine Homologue of the Locus for Menkes Disease, in Normal and Mottled (Mo) Mice

Menkes disease (MNK) lies immediately proximal to phosphoglycerate kinase (PGK1) in Xq13 in human. Phenotypic similarities between MNK patients and murine mottled (Mo) mutants strongly suggest that both defects are caused by mutations at the same locus. Human MNK cDNA clones and a genomic subclone derived from a 40-kb YAC clone that includes Pgk1 have been used to position the murine homologue of Menkes disease (MNK, Mnk) immediately proximal to, and within 150-200 kb of, phosphoglycerate kinase (Pgk1) on the mouse X chromosome using interspecific backcross analysis and pulsed-field gel electrophoresis. A related autosomal locus has been mapped to mouse chromosome 18. RFLVs at Mnk between inbred strains of mice that show a strong association with the presence of the Mo phenotype have been detected. Hybridization of 4.1 kb of the 4.5-kb MNK coding sequence failed to reveal any deletions or alterations to restriction fragments containing exons of the Mnk locus in 9 Mo mutants. Furthermore, no genomic deletions or alterations > 20 kb were detected in 10 independently derived Mo mutants using pulsed-field gel electrophoresis. As no deletions or alterations at the Mnk gene were found, we suggest that any mutations in Mnk that cause the Mo phenotype are likely to be due to small changes at the nucleotide level and/or small deletions (<20 kb) that lie outside the coding sequence.

Refined mapping of a gene (NPH1) causing familial juvenile nephronophthisis and evidence for genetic heterogeneity.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive progressive tubulo-interstitial kidney disorder, responsible for 6-10% of end-stage renal failure in children, and is frequently associated with Leber amaurosis (termed Senior-Løken syndrome). The biochemical basis of NPH is unknown. We recently reported linkage of the purely renal form of NPH to three markers on chromosome 2. Our results also suggested the existence of genetic heterogeneity between NPH and SLS. To map this NPH gene more precisely, we have now tested the segregation of six new microsatellite markers and five additional families. Haplotype analyses show unequivocally that four NPH families are not linked to the chromosome 2 markers, although there are no clinical or pathological features discernible in these families that could separate them from the families linked to the chromosome 2 NPH locus (NPH1). This reveals genetic heterogeneity in the purely renal form of NPH. In situ hybridization of YAC clones isolated with two closely linked markers assigned the NPH1 region to 2q13. Furthermore, based on haplotype analysis and specific recombination events, the NPH1 gene has been placed between D2S293/D2S340 and D2S121, a genetic interval of about 5-7 cM.

MHCDB--database of the human MHC.

Genetic and physical data relating to the human major histocompatibility complex (MHC) were compiled and analyzed, using a genome analysis program. The current contents of the database include: 1) location of over 100 genes and other markers; 2) location of over 250 YAC and cosmid clones; 3) 150 kilobases of genomic DNA sequence including full annotation (exon/intron boundaries, repeats, promoters, etc.); 4) cDNA sequences of currently-known class I and class II alleles; and 5) accompanying descriptive data--references, comments, laboratory addresses, and so on. The database allows rapid access, retrieval, and display of all these data, which should make it a useful tool for the study of the human MHC. MHCDB is publicly available and will be updated as new data become available.

Efficient identification and regional positioning of YAC and cosmid clones to human chromosome 21 by radiation fusion hybrids.

The use of integrated mapping strategies involving bacterial, yeast, and rodent cells as hosts simplifies the construction of maps, which combine long-range order, high resolution, and easy access to the cloned DNA. Radiation-fusion hybrids offer a specially powerful long-range mapping system for human chromosomes. We describe here techniques for establishing a radiation-fusion hybrid map of Chromosome (Chr) 21q and its integration with local information on YAC and cosmid positions.

YAC clones targeting gene-rich regions of human Chromosome 3

YAC clones targeting gene-rich regions of human Chromosome 3

Analysis of clones carrying repeated DNA sequences in two YAC libraries of Arabidopsis thaliana DNA.

YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library. Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries. The coordinates of YAC clones hybridizing to these sequences are given. A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome. None of the EW YAC clones analysed were chimaeric in this way. YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability. These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments.

Deletion of a common region on the long arm of chromosome 6 in acute lymphoblastic leukaemia.

We have characterised a region of deletion on the long arm of chromosome 6 (6q) in six cases of acute lymphoblastic leukaemia, by fluorescence in situ hybridisation, using a series of YAC clones which map to 6q. Conventional cytogenetic analysis of four of these cases had been interpreted as showing terminal deletions of 6q. We demonstrated by FISH that in all cases the deletions were interstitial. D6S246 (6q16.3) was the only marker which was missing in all six cases, indicating a common region of deletion between the markers M6P1 at 6q14-15 and FYN at 6q21. Our results suggest the presence of a tumour suppressor gene within this interval.

A YAC Contig Spanning the Hypophosphatemic Rickets Disease Gene (HYP) Candidate Region

Dominant X-linked hypophosphatemic rickets (HYP) is the most common form of familial rickets. Linkage studies have localized the gene for this disorder to Xp22.1 between the markers DXS365 and DXS274, a region estimated to be approximately 3.5 cM. We have constructed a 1.5-Mb YAC contig encompassing this region by hybridization screening of high-density YAC clone filters. Rapid chromosome walking was achieved by direct hybridization of a pool of Alu -PCR products derived from a YAC containing DXS365 to the filter grids. Overlaps between YACs in the contig were estimated by hybridization of end probes to YAC digest blots and by analysis of cosmid fingerprints obtained by hybridization of YAC inserts to a flow-sorted chromosome X cosmid library. All YACs in the contig have been verified by fluorescence in situ hybridization. Several YACs spanning the HYP gene candidate region were selected for further analysis by rare-cutter enzyme digestion and pulsed-field gel electrophoresis. We estimate that the markers flanking the disease region, DXS365 and DXS274, are less than 1 Mb apart. This clone contig map provides an essential resource for the isolation of the HYP gene.

A 2-Mb YAC Contig Encompassing Three Loci (DXF34, DXS14, and DXS390) That Lie between Xp11.2 Translocation Breakpoints Associated with Incontinentia Pigmenti Type 1

We demonstrate that all the repeat elements representing the conserved loci DXF34 and DXS390 lie between the X;9 and the X;17 translocation breakpoints associated with incontinentia pigmenti type 1 (IP1). Sequence-tagged sites (STSs) at DXF34S1, DXS14, and DXS390 have been used to isolate YAC clones containing these loci, and a contig of approximately 2 Mb has been constructed. Patterns of hybridization observed in the YAC clones indicate that DXS390 comprises two distinct regions (A and B). The STS at DXS390 detects the A region and includes a polymorphic CA repeat (PIC = 0.25). This expansion of the cloned region around DXF34 and DXS390 will enable the isolation of additional conserved sequences that will help in understanding both the lesions underlying the pathogenesis of IP1 and the size and extent of the man-mouse homologous block defined by DXF34.

Rescuing YAC‐Insert Ends as E. coli Plasmids

End clones from YACs (terminal fragments of the YAC-insert DNA cloned into a plasmid vector) are essential ingredients for contig building using chromosome walking strategies, for fluorescent in situ hybridization experiments, and for generating repeat-free probes for pulsed-field gel electrophoresis or genetic linkage analysis. The basic protocol describes a method for rescuing the CEN (centromere) ends of YACs constructed in the vector pYAC4. However, because this method relies on XhoI and SalI sites, which are relatively rare in the mammalian genome, it is not always possible to obtain a subclone in this manner. An alternate protocol presents a method utilizing integrative plasmid-rescue vectors to facilitate the isolation of both ends of any YAC clone even in the absence of convenient restriction enzyme sites.

Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma.

We report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, we first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). We then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and established long-range physical maps of these. Cosmid clones LLNL12NCO1-98C10 and LLNL12NCO1-113D12 were isolated using STSs within the CRI-C86- and the p7G11-derived YAC contigs, respectively, and a mixture of them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, our studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells.

The human NFKB3 gene encoding the p65 subunit of transcription factor NF-kappa B is located on chromosome 11q12.

A YAC clone that contains the human gene NFKB3, encoding the p65 subunit of transcription factor nuclear factor kappa B (NF-kappa B), was isolated. The YAC contains the entire NFKB3 gene, which is smaller than 15 kb and present in a single copy in the genome. Fluorescence in situ hybridization with metaphase chromosomes showed two different chromosomal locations (11q12 and Xp11.4) for sequences present in the YAC. The NFKB3 gene was assigned to chromosome 11q12 by PCR analysis of a panel of relevant hybrid cell lines. Thus, no linkage exists between NFKB3 and genes encoding other known members of the NF-kappa B family.

The DCC Gene: Structural Analysis and Mutations in Colorectal Carcinomas

DCC is a candidate tumor-suppressor gene encoding a protein with sequence similarity to cell adhesion molecules such as N-CAM. A set of overlapping YAC clones that contains the entire DCC coding region was isolated. Studies of this YAC contig showed that the DCC gene spans approximately 1.4 Mb. For elucidation of exon-intron structure, lambda phage clones containing all known coding sequences were isolated from a genomic library. These clones were used to demonstrate the existence of 29 DCC exons, and the sequences of the exon-intron boundaries were determined for each. Twenty-three polymorphic markers from chromosome 18 were then studied in a panel of primary colorectal tumors that had lost some, but not all, of chromosome 18. In most of these tumors, the region that was lost included DCC. Finally, Southern blot and PCR-based approaches were used to search for subtle mutations in several DCC exons. One tumor that had a point mutation in exon 28 was found, resulting in a proline to histidine substitution. A second tumor with a point mutation in intron 13 was also found. The regional map and genomic structure of DCC should provide the means to more extensively study DCC gene alterations and protein function in normal and neoplastic cells.

Targeted development of microsatellite markers from inter-Alu amplification of YAC clones.

Primary genetic maps based on highly informative markers are now available. The local density of these markers may, however, not be sufficient. There is thus a need for new means to generate polymorphic markers from targeted regions of the genome. This can be achieved by selectively cloning and sequencing (CA)n-positive human inter-Alu sequences from targeted YAC clones. This method was tested on 21 YACs and led to the development of seven new polymorphic microsatellite markers.

A genomic search for the gene conferring resistance to fusarium wilt in tomato

Fusarium wilt is an economically important disease of tomatoes, caused by the soil-born fungus Fusarium oxysporum f. sp. lycopersici. There are three host-specific races of this pathogen. The dominant tomato gene I-2 confers resistance to race 2. The I-2 fusarium resistance gene was mapped genetically to chromosome 11 of tomato, between the RFLP markers TG105 and TG36, 0.4 centiMorgan (cM) from TG105. A mean value of 43 kb for each cM was assigned in the vicinity of I-2. We have generated new RFLP markers in the region by chromosome walking from TG105 towards I-2 on lambda clones, and by subcloning a 350 kb long YAC clone (YAC 8) that contains TG105. These RFLP markers were mapped physically on YAC 8 by PFGE. The location of I-2 relative to these markers was genetically estimated using a recombinant inbred (RI) segregating population. The order of the markers according to the RI population is inconsistent with their order on the physical map. A cDNA clone, D14, that was isolated by YAC 8, turned out to be 53% similar to xanthine dehydrogenase from mammals and flies. Antibodies raised against a part of the protein encoded by D14 recognize cross reacting material of MW 80 kD, that is highly enriched in nodules of legumes, and seems to be induced by various environmental and pathogenic stress conditions.

Large-scale isolation of human 1p36-specific P1 clones and their use for fluorescence in situ hybridization

A series of 80 microclone probes derived from the chromosomal region 1p36 was used to isolate corresponding clones from the ICRF human P1 library (see Francis et al., this issue). Hybridization screenings were performed using probe pools on high-density filter grids. A total of 87 P1 clones specific for 1p36 were isolated. This large-scale approach allowed a detailed evaluation of the complexity, quality, and utility of this library. The isolated P1 clones were used both for size determination by pulsed-field gel electrophoresis and as probes for fluorescence in situ hybridization (FISH) analysis. FISH of P1 clones is shown to be both easy and efficient to perform on metaphase chromosomes and interphase nuclei. This observation is expected to reveal new avenues for diagnosis of disease-related chromosomal changes. The use of P1 clones as a tool in clinical and tumor interphase cytogenetics is discussed and compared with FISH data of other long insert clones such as cosmids and YAC clones.

A high resolution physical map of 2.5 Mbp of the Down syndrome region on chromosome 21.

The region surrounding D21S55 in band 21q22 of human chromosome 21 has been implicated in the etiology of Down syndrome (DS). In this paper, we report the construction of a high resolution map of a 2.5 Mb region around the marker D21S55. Characterization of YAC clones by accurate size determination, end isolation and marker assignment was used to build a refined YAC-based map. The YAC clones were then used to isolate 284 cosmid clones, covering 2.3 Mb, from a chromosome 21-specific cosmid library. The cosmid clones were ordered into overlapping groups and a restriction map of each group was determined. The groups of cosmids were then ordered and oriented with respect to the YAC-based map. This high resolution map provides the framework for further analysis of the region by transcribed sequence mapping and sequence determination.

Construction of a YAC contig and a STS map spanning at least seven megabasepairs in chromosome 5q34-35.

We have constructed a YAC contig containing 54 clones and a minimum of 7 Mbp of human DNA, that maps to bands q34-35 on chromosome 5. The contig was nucleated using FISH mapped cosmid clones shown to flank the t(2;5)(p23;q35) translocation breakpoint in a CD30-positive large cell lymphoma cell line. Thirty of the 54 YAC clones are non-chimeric and six span the translocation breakpoint, as determined by FISH analysis. A total of 28 YAC clone end fragments, 14 non-polymorphic YAC end STS probes and 13 polymorphic microsatellite STS markers have been used to order clones within the contig. The most distal genetic markers (D5S498 and D5S619) are separated by 15 cM based on multipoint linkage analysis. This map of overlapping clones and the set of densely spaced physical markers will promote our understanding of the 5q34-35 region and its associated genes.

Localization of the gene (RSN) coding for restin, a marker for Reed-Sternberg cells in Hodgkin's disease, to human chromosome band 12q24.3 and YAC cloning of the locus.

A novel 160-kDa intermediate filament associated protein, named restin (Reed-Sternberg intermediate filament associated protein), is specifically expressed in the malignant cells of Hodgkin's disease and anaplastic large cell lymphoma (Ki-1 lymphoma). The combination of chromosomal R-banding and fluorescence in situ hybridization (FISH) with the use of two fluorescent dyes, fluorescein isothiocyanate and propidium iodide, allowed simultaneous detection of the hybridized DNA sequence and chromosomal R-banding. By this technique, the gene coding for restin (RSN) was assigned to chromosome region 12q24.31-->q24.33, while localization of the alpha-2-macroglobulin receptor (A2MR) was refined to 12q13.1-->q13.3. To further analyze the restin gene, a 500-kb YAC clone containing the gene was isolated and analyzed. A restriction map of this area is presented.