Biomedical subjects

P Taillon-Miller

Publications and source records attributed to P Taillon-Miller.

32 records · Page 2Linked to original sources

PubMed1992

Cloning and physical mapping of the HLA class I region spanning the HLA-E-to-HLA-F interval by using yeast artificial chromosomes.

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.

Chromosomes, Fungal↗

PubMed1992

A yeast artificial chromosome contig encompassing the type 1 neurofibromatosis gene.

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.

Base Sequence↗

PubMed1992

Analysis of the human regulators of complement activation (RCA) gene cluster with yeast artificial chromosomes (YACs).

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.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase 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.

Arylsulfatases↗

PubMed1992

Cloning of the Huntington disease region in yeast artificial chromosomes.

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.

Base Sequence↗

PubMed1992

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.

Base Sequence↗

PubMed1991

A gene deleted in Kallmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules.

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.

Base Sequence↗

PubMed1991

Isolation and characterization of yeast artificial chromosome clones linking the HLA-B and HLA-C loci.

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.

Amino Acid Sequence↗

PubMed1991

Molecular linkage of the HLA-DR, HLA-DQ, and HLA-DO genes in yeast artificial 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.

Base Sequence↗

PubMed1991

Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region.

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.

Cell Line↗

PubMed1991

Yeast artificial chromosome cloning of a two-megabase-size contig within chromosomal band 18q21 establishes physical linkage between BCL2 and plasminogen activator inhibitor type-2.

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.

Animals↗

PubMed1990

Mapping chromosome band 11q23 in human acute leukemia with biotinylated probes: identification of 11q23 translocation breakpoints with a yeast artificial chromosome.

Translocations involving chromosome 11, band q23, are frequent recurring abnormalities in human acute lymphoblastic and acute myeloid leukemia. We used 19 biotin-labeled probes derived from genes and anonymous cosmids for hybridization to metaphase chromosomes from leukemia cells that contained four translocations involving band 11q23: t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13). The location of the cosmid probes relative to the breakpoint in 11q23 was the same in all translocations. Of the cosmid clones containing known genes, CD3D was proximal and PBGD, THY1, SRPR, and ETS1 were distal to the breakpoint on 11q23. Hybridization of genomic DNA from a yeast clone containing yeast artificial chromosomes (YACs), that carry 320 kilobases (kb) of human DNA including CD3D and CD3G genes, showed that the YACs were split in all four translocations. These results indicate that the breakpoint at 11q23 in each of these translocations occurs within the 320 kb encompassed by these YACs; whether the breakpoint within the YACs is precisely the same in the different translocations is presently unknown.

Adult↗

PubMed1984

An improved radioimmunoassay for human ovarian tumor antigen NB/70K.

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.

Antigens, Neoplasm↗

PubMed1984

Selectivity for ovarian cancer of an improved serum radioimmunoassay for human ovarian tumor-associated antigen NB/70K.

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 pretreatment 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 non-ovarian malignant disease, only 4 of 23 patients with non-ovarian cancers had significantly elevated NB/70K levels. However, none of these 4 false positives had levels greater than 20 units.

Animals↗