The sequence and analysis of duplication-rich human chromosome 16.

Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin.

Identification of two Fas-associated phosphatase-1 (FAP-1) promoters in human cancer cells.

Fas-associated phosphatase-1 (FAP-1) has been reported as a negative regulator of Fas-mediated signal transduction in human cancer cells. To obtain insights into the potential carcinogenesis of the FAP-1 gene, we investigated its transcriptional regulation in normal and cancerous cells. To identify the FAP-1 promoter sequences, we first isolated P1 and cosmid clones that contained the regulatory region upstream from the FAP-1 gene by using the PCR products of 5' rapid amplification of cDNA end (5'-RACE) as probes.

Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization.

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University.

An integrated physical map for the short arm of human chromosome 5.

The short arm of human chromosome 5 contains approximately 48 Mb of DNA and comprises 1.5% of the genome. We have constructed a mega-YAC/ STS map of this region that includes 436 YACs anchored by 216 STSs.

Analysis of a 69-kb contiguous genomic sequence at a putative tumor suppressor gene locus on human chromosome 6q27.

Multiple neoplasias including B-cell non-Hodgkin's lymphoma, breast carcinoma, and ovarian carcinoma, have been associated with frequent deletions of the distal region on the long arm of human chromosome 6, suggesting the presence of one or more tumor suppressor gene(s) at this locus. Loss of heterozygosity analysis of breast and ovarian tumors has further restricted the minimal region of loss within 6q27. To further characterize this genomic region for gene content including putative tumor suppressor genes as well as other elements that may contribute to tumorigenesis, a 68940-bp contiguous sequence, encompassing markers D6S193 and D6S297, was generated by random shotgun sequencing of a cosmid, P1, and PAC contig.

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes.

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family.

New, male-specific microsatellite markers from the human Y chromosome.

Seven novel microsatellite markers have been developed from a new cosmid library constructed from flow-sorted human Y chromosomes. These microsatellites are tetranucleotide GATA repeats and are polymorphic among unrelated individuals. Five of the seven markers are male-specific, with no PCR product being generated from female DNA.

Construction of human chromosome 16- and 5-specific circular YAC/BAC libraries by in vivo recombination in yeast (TAR cloning).

Transformation-associated recombination (TAR) in yeast was exploited for the selective isolation of human DNAs as large circular yeast artificial chromosomes (YACs) from two rodent/human hybrid cell lines containing human chromosomes 5 and 16. TAR cloning vectors containing the F-factor origin of replication were constructed for use in these experiments. Presence of the F-factor origin in TAR vectors provides the capability of transferring the YACs generated by in vivo recombination in yeast into Escherichia coli cells and propagating them as bacterial artificial chromosomes (BACs).

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions.

Cloning of a breakpoint cluster region on chromosome 14 in uterine leiomyoma.

A recurrent reciprocal chromosomal translocation, t(12;14)(q15;q24) is frequently observed in uterine leiomyoma. Chromosome 12 breakpoints have been shown to occur in a region of approximately 150 kb that contains the gene for a high mobility group protein (HMGI-C). The breakpoint region on chromosome 14 has not been precisely defined.

Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine+.cytosine pairs.

Previously, we reported the high resolution NMR structure of the hairpin G-quartet structure formed by the G-rich strand of the insulin minisatellite of repeat sequence, (ACAG4TGTG4/TGTC4ACAC4) located upstream of the human insulin gene. Here, we report structural studies on the C-rich strand of this insulin minisatellite. First, we show by high resolution NMR that (C4TGTC4) forms a hairpin dimer with intercalated C+.

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity.

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago).

Construction of a 1.2-Mb contig surrounding, and molecular analysis of, the human CREB-binding protein (CBP/CREBBP) gene on chromosome 16p13.3.

In the interest of cloning and analyzing the genes responsible for two very different diseases, the Rubinstein-Taybi syndrome (RTS) and acute myeloid leukemia (AML) associated with the somatic translocation t(8;16)(p11;p13.3), we constructed a high-resolution restriction map of contiguous cosmids (contig) covering 1.2 Mb of chromosome 16p13.

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3.

Isolation of a B-cell-specific promoter for the human class II transactivator.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA.

Interactive computer-aided assignment of multiple probes to cytogenetic bands by simultaneous dual color fluorescence in situ hybridization and DAPI banding.

A macro function was developed to run in conjunction with the popular image analysis package NIH Image, to allow simultaneous determination of mapping positions of one or two separate probes with respect to cytogenetic bands by dual color fluorescence in situ hybridization (FISH) and DAPI banding, and by determination of their fractional distance from pter (FLpter). In order to allow maximal flexibility, a user-defined line along the chromosome is used for measurements. Algorithms were developed to detect the ends of the chromosome and the cytogenetic bands.

A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3.

Chromosome 17p13.3 is frequently deleted in human ovarian carcinoma, and the 15 kb critical region of deletion may contain a tumor suppressor gene. A 2.

The generation and regional localization of 303 new chromosome 5 sequence-tagged sites.

With the ultimate goal of creating a sequence-ready physical map of all of chromosome 5, 303 new human chromosome 5-specific STS markers have been systematically generated and regionally ordered. Chromosome 5 DNA prepared from flow-sorted chromosomes was digested with restriction enzymes BamHI and HindIII and cloned in bacteriophage M13mp18. Random clones were sequenced, and appropriate PCR deoxyoligomers were synthesized.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR.

Characterization of a flow-sorted human chromosome 10 cosmid library by FISH.

Fluoresence in situ hybridization (FISH) was used to localize cosmids to regions of human chromosome 10. A total of 301 cosmids were selected randomly from a flow-sorted human chromosome 10 cosmid library constructed from human x hamster cell line 762-8A and arrayed in microtiter storage dishes. Over 70% (211/301) of the cosmids mapped to unique regions of chromosome 10.

Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary.

Selection of chromosomal sublibraries from total human genomic libraries is critical for chromosome-based physical mapping approaches. We have previously reported a method of screening total human genomic library using flow sorted chromosomal DNA as a hybridization probe and selection of a human chromosome 22-enriched sublibrary from a total human bacterial artificial chromosome (BAC) library (Nucleic Acids Res 1995; 23: 1838-39). We describe here further details of the method of construction as well as characterization of the chromosome 22-enriched sublibrary thus constructed.

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis.

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites.

A 500-kilobase region containing the tuberous sclerosis locus (TSC1) in a 1.7-megabase YAC and cosmid contig.

A complete overlapping clone map of a 1.7-Mb region from DBH to D9S67 that includes the TSC1 candidate region has been constructed. The map includes YAC and cosmid clones, contains STS approximately every 50 kb on average, and establishes the order of five previously unordered loci.