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.

Yeast artificial chromosome cloning of 3.2 megabases within chromosomal band 11q24 closely linking c-ets 1 and Fli-1 and encompassing the Ewing sarcoma breakpoint.

Human chromosome 11 harbors many genes of medical significance and cancer-related rearrangements. The availability of cloned DNA in cosmids and in yeast artificial chromosomes (YACs), combined with fluorescence in situ hybridization analysis, has led to the cloning of genes at sites of chromosomal breakpoints in acute leukemias in 11q23 and in Ewing tumors in 11q24. YAC cloning has facilitated the construction of contigs covering large portions of chromosomes for the detailed analysis of disease gene regions. Here we have cloned in YACs approximately 3.2 Mb of DNA within band 11q24, spanning the Ewing sarcoma breakpoint. Landmark cosmids 23.2 (D11S374) and 5.8 (D11S372), shown by FISH to flank the breakpoint within a 1.5- to 1.8-Mb segment, were used to seed two YAC "walks" both centromeric and telomeric to the breakpoint by YAC-end cloning and screening of two total genomic YAC libraries. The centromeric YAC contig, which consists of 23 overlapping YACs and orders 19 sequence-tagged sites (STSs), covers a minimum of 2.2 Mb and spans the Ewing sarcoma breakpoint. c-ets 1 and Fli-1, two members of the ets family, have been linked within 400 kb of intervening DNA within this contig, which also comprises a polymorphic microsatellite, D11S912 (CA)n, which we have localized within the Fli-1 gene. The telomeric YAC contig, which consists of 11 overlapping YACs, comprises 5 STSs and covers a minimum of 1 Mb distal to the breakpoint. Taken together, the two contigs, which consist of a total of 34 YACs and comprise 24 STSs, are separated by a maximum gap of 200-400 kb and cover as a whole 3.2 Mb of DNA. This represents about 70% of human chromosomal band 11q24, which extends over approximately 4.4 Mb of DNA.

Alternatively spliced pp52 mRNA in nonlymphoid stromal cells.

The 52-kDa phosphoprotein, also reported as lymphocyte-specific gene 1 and WP34, is transcribed as a 1.6-kb mRNA in B lymphocytes, B cell lines, and untransformed T cells. This gene encodes a cytoplasmic and plasma membrane-associated protein that is phosphorylated at a casein kinase II site and reportedly binds calcium. Based on these properties, it has been hypothesized that lymphoid form of the 52-kDa phosphoprotein protein may play a role in lymphocyte signal transduction. We show that alternatively spliced mRNA are expressed from this gene in nonlymphoid cell lines (myocytes, stromal cells, fibroblasts). These cell lines do not express the 1.6-kb lymphoid cell-specific transcript. Instead, mRNA of 2.0 and 2.8 kb are detected in varying abundance. A full-length 2.0-kb cDNA has been cloned and sequenced from the BMS2 stromal cell line by conventional screening and polymerase chain reaction-based methods. This cDNA clone, designated S37, has a single open reading frame encoding a 328 amino acid peptide. The nucleotide sequence of the S37 stromal cell cDNA is identical to that of the lymphocyte derived pp52 cDNA from the 3' poly(A) tail to the codon encoding the amino acid at residue 24. This region of the S37 cDNA clone encodes a protein that is identical to that encoded by the lymphoid pp52 cDNA and includes a casein kinase II phosphorylation site. However, the two clones differ in their 5' nucleotide sequence and their NH3 terminal amino acid sequence. This organization is consistent with alternative exon utilization. These results suggest that tissue-specific control mechanisms are used to generate different forms of lymphoid form of the 52-kDa phosphoprotein mRNA in lymphoid cells versus mesoderm-derived, nonlymphoid cell lineages.

Localization of the human oncostatin M gene (OSM) to chromosome 22q12, distal to the Ewing's sarcoma breakpoint.

Using fluorescence in situ hybridization, a cosmid clone containing the gene for oncostatin M (OSM) was mapped to human chromosome 22q12, placing the OSM gene in the same chromosome band as the leukemia-inhibitory factor gene (LIF). The location of the OSM gene was determined relative to the t(11;22)(q24;q12) of Ewing's sarcoma and found to be distal to the translocation breakpoint on chromosome 22. Analysis of physical distances by pulsed-field gel electrophoresis demonstrated further that the two genes lie within 500 kb of each other.

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.

B29 gene products complex with immunoglobulins on B lymphocytes.

B29 is a B-lineage-specific gene predicted from sequence information to be a transmembrane member of the immunoglobulin (Ig) superfamily, with a single extracellular Ig-like domain. Its presumptive cytoplasmic region contains a peptide motif present in CD3 and other molecules involved in lymphocyte activation. Affinity-purified goat antibodies were prepared to a TrpE fusion protein of B29 and used to study B29 expression on lymphoid cells. The antiserum precipitated surface-labeled heterodimers from B lymphoma cells. One was 65-88 kDa (unreduced) or 36-47 plus 32-34 kDa (reduced) by SDS/PAGE analysis, regardless of detergent. A smaller heterodimer was detected only with Triton detergent extraction. IgM molecules were coprecipitated by the B29 antiserum when the weak detergent digitonin was used. In addition, cocapping experiments revealed that most B29 molecules codistribute with Ig on the cell surface. Although early B-lineage cells and plasma cells contain B29 mRNA, surface expression was detectable only on B cells that had significant amounts of surface Ig. The surface expression was B-lineage-specific and included cells from mutant xid mice and B-cell lines representing mu, delta, gamma, and alpha heavy-chain isotypes and both kappa and lambda light-chain types. The density of surface B29 protein correlated directly with surface mu heavy-chain density on subclones of a B-cell lymphoma and lipopolysaccharide-stimulated pre-B cells. These findings show that B29 is covalently linked in a heterodimer and are consistent with a recently proposed model of surface Ig complexes.

Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.

Chromosomal in situ hybridization using yeast artificial chromosomes.

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.

Molecular localization of the t(11;22)(q24;q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization.

Chromosome translocations are associated with a variety of human leukemias, lymphomas, and solid tumors. To localize molecular markers flanking the t(11;22) (q24;q12) breakpoint that occurs in virtually all cases of Ewing sarcoma and peripheral neuroepithelioma, high-resolution chromosomal in situ suppression hybridization was carried out using a panel of cosmid clones localized and ordered on chromosome 11q. The location of the Ewing sarcoma translocation breakpoint was determined relative to the nearest two cosmid markers on 11q, clones 23.2 and 5.8, through the analysis of metaphase chromosome hybridization. By in situ hybridization to interphase nuclei, the approximate physical separation of these two markers was determined. In both Ewing sarcoma and peripheral neuroepithelioma, cosmid clone 5.8 is translocated from chromosome 11q24 to the derivative chromosome 22 and a portion of chromosome 22q12 carrying the leukemia inhibitory factor gene is translocated to the derivative chromosome 11. The physical distance between the flanking cosmid markers on chromosome 11 was determined to be in the range of 1000 kilobases, and genomic analysis using pulsed-field gel electrophoresis showed no abnormalities over a region of 650 kilobases in the vicinity of the leukemia inhibitory factor gene on chromosome 22. This approach localizes the Ewing sarcoma breakpoint to a small region on chromosome 11q24 and provides a rapid and precise technique for the molecular characterization of chromosomal aberrations.

Immunoglobulin enhancer and promoter motifs 5' of the B29 B-cell-specific gene.

B29 is a B-cell-specific member of the immunoglobulin gene superfamily that is expressed throughout B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy-chain gene segment rearrangements. We have analyzed the region upstream of the B29 gene to identify DNA sequences involved in transcriptional regulation of this gene. The B29 gene lacks a TATA box and transcription is initiated at multiple sites. The B29 gene sequence 5' of these transcription start sites contains six promoter and enhancer motifs known to control immunoglobulin gene transcription. The most notable is a perfect octamer (5'-ATTTGCAT-3'), which binds the Oct-2 B-cell-specific transcription factor and thereby can account for the tissue-specific expression of this gene.

B29: a member of the immunoglobulin gene superfamily exclusively expressed on beta-lineage cells.

A number of the glycoproteins identified on the surfaces of cells of the immune response belong to the immunoglobulin superfamily. We have isolated and characterized cDNA clones and the complete genomic gene encoding a B-cell-specific member of the immunoglobulin superfamily called "B29." This isolate is expressed at all stages in B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy chain gene diversity region----joining region gene (DH----JH) rearrangements. The protein sequence predicted by the B29 coding region contains a leader sequence and a single extracellular immunoglobulin-like domain, followed by a hydrophobic transmembrane segment and a charged intracytoplasmic domain. The immunoglobulin-like domain contains cysteines and other conserved amino acids characteristic of light chain variable and joining regions, but overall the sequence is only distantly related to immunoglobulins. Each of these domains is encoded in separate exons in the B29 gene, in analogy to other members of the immunoglobulin superfamily. The conserved structural features of the immunoglobulin-like domain in the B29 gene product resemble those of other members of the immunoglobulin superfamily involved in cell recognition and adhesion.