ABSTRACT
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. Genomic analysis of positive clones revealed that the major FAP-1 mRNA was transcribed from its proximal promoter (pPRM) in all human cancer cell lines tested, but 1 additional large transcript derived from its distal promoter (dPRM) was found in the human colon cancer cell line DLD-1. This suggests that the FAP-1 gene may be aberrantly dysregulated in some types of human cancers, including colon carcinoma. Sequence analysis of the region upstream from the FAP-1 gene strongly suggests that the transcript of the FAP-1 gene may be controlled by a variety of transcriptional regulatory elements, including NF-kappa B, NF-IL6, and p53 in its 2 promoters. These results imply that the FAP-1 gene may be a target gene under the control of important apoptosis-related nuclear factors in human cancers.
Subject(s)
Carrier Proteins/genetics , Promoter Regions, Genetic , Protein Tyrosine Phosphatases/genetics , 5' Untranslated Regions , Base Sequence , Brain/pathology , CCAAT-Enhancer-Binding Protein-beta/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA, Complementary , Gene Expression Regulation , Humans , Interferon-gamma/metabolism , Molecular Sequence Data , NF-kappa B/metabolism , Protein Phosphatase 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 13 , Transcription, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolismABSTRACT
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. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.
Subject(s)
Chromosomes, Bacterial/genetics , Chromosomes, Human, Pair 16/genetics , Contig Mapping/methods , Humans , Nucleic Acid Hybridization/methods , Reproducibility of Results , Restriction Mapping , Sequence Tagged SitesABSTRACT
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. By combining and integrating our map with the 5p maps of other groups using the same recombinant DNA library, a comprehensive map was constructed that includes 552 YACs and 504 markers. The YAC map covers >94% of 5p in four YAC contigs, bridges the centromere, and includes an additional 5 Mb of 5q DNA. The average marker density is 95 kb. This integrated 5p map will serve as a resource for the continuing localization of genes on the short arm of human chromosome 5 and as a framework for both generating and aligning the DNA sequence of this region.
Subject(s)
Chromosomes, Human, Pair 5 , Animals , Expressed Sequence Tags , Genes , Humans , Hybrid Cells , Mice , Physical Chromosome Mapping , Sequence Homology, Nucleic AcidABSTRACT
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. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.
Subject(s)
Chromosomes, Human, Pair 5 , Peptide Termination Factors/chemistry , Peptide Termination Factors/genetics , Pseudogenes , Base Sequence , Chromosomes, Human, Pair 6 , Chromosomes, Human, Pair 7 , Cloning, Molecular , Cosmids , Exons , Gene Library , Humans , Introns , Models, Genetic , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , X ChromosomeABSTRACT
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. One marker produces male-specific, polymorphic PCR products but occasionally produces a much larger, invariant product from female DNA. The remaining marker is polymorphic in both males and females with many shared alleles between the sexes. This report of six new, male-specific markers doubles the number of tetranucleotide markers that are currently available for the human Y chromosome. These new markers will be valuable where nonrecombining, gender-specific DNA markers are desired, including forensic investigations as well as studies of populations and their evolutionary histories.
Subject(s)
Microsatellite Repeats , Y Chromosome , Base Sequence , DNA, Complementary , Female , Humans , Male , Molecular Sequence DataABSTRACT
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 high enrichment of human versus rodent YACs was observed during isolation of human DNA from the rodent/human hybrid cell lines. Although <3% of the DNA content in the hybrid cells was human, as many as 75% of the transformants contained human YACs. In contrast to the standard YAC cloning method based on in vitro ligation, no human/mouse chimeras were observed during TAR cloning. The constructed human chromosome 16 YAC library had approximately 2.6x coverage, represented by 4320 YAC clones with an average insert size of 80 kb. YAC clones generated from chromosome 16 were successfully converted into BACs by electroporation of DNA isolated from yeast transformants into E. coli. The BAC clones represent approximately 0.6x chromosomal coverage. Pilot YAC and BAC libraries of chromosome 5 have been also constructed. The chromosomal distribution of YAC/BACs from chromosome 5 and chromosome 16 was evaluated by fluorescence in situ hybridization (FISH). The distribution of FISH signals appeared random along the length of each chromosome. We conclude that TAR cloning provides an efficient means for generating representative chromosome-specific YAC/BAC libraries.
Subject(s)
Chromosomes, Artificial, Yeast/genetics , Chromosomes, Human, Pair 16/genetics , Chromosomes, Human, Pair 5/genetics , DNA, Circular/genetics , Recombination, Genetic/genetics , Animals , Chromosome Mapping , Cloning, Molecular/methods , Escherichia coli/genetics , Gene Library , Genetic Vectors/genetics , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Rodentia , Saccharomyces cerevisiae/geneticsABSTRACT
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. In addition, exon trapping was performed utilizing a subset of these clones. Sixteen trapped exons, ranging in size from 44 to 399 bp, span this approximately 69-kb region. Many other putative exons have been identified computationally. Further analysis has identified 13 potential promoters and 13 putative polyadenylation sites in the region. Northern analysis identified a transcript mapping within this interval that is expressed in ovarian, breast, and lymphoid-derived tumor cell lines. Consideration of these data, together with the demonstration of several regions of high CpG content, suggests the possibility of several genes at this locus.
Subject(s)
Chromosomes, Human, Pair 6/genetics , Genes, Tumor Suppressor , Alu Elements , Base Sequence , Breast Neoplasms/genetics , Chromosome Deletion , Chromosome Mapping , Cloning, Molecular , DNA/genetics , Exons , Female , GC Rich Sequence , Genetic Variation , Genome, Human , Humans , Lymphoma, B-Cell/genetics , Molecular Sequence Data , Ovarian Neoplasms/genetics , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid , Tumor Cells, CulturedABSTRACT
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+.C pairs (referred to as the hairpin i-motif); 340 NOE distance constraints uniquely define the nature of hairpin folding and the pattern of C+.C intercalation. Second, we show by one-dimensional NMR spectroscopy and molecular modeling studies that (C4TGTC4ACA4TGTC4) forms an intramolecularly folded hairpin with intercalated C+.C pairs. Third, we demonstrate by in vitro replication studies that several such hairpin i-motifs are present in long (C4TGTC4ACA)n (n>/=6) sequences, even in the presence of their complementary strands. Finally, we discuss structural and biological significance of the hairpin i-motifs formed by the C-rich strands of the insulin minisatellite.
Subject(s)
Cytosine/chemistry , Insulin/genetics , Minisatellite Repeats , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Base Sequence , Computer Simulation , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Dimerization , Humans , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Sequence DataABSTRACT
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). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric-directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non-homologous interchromosomal exchange.
Subject(s)
Adrenoleukodystrophy/genetics , Centromere/genetics , Repetitive Sequences, Nucleic Acid , X Chromosome , Amino Acid Sequence , Base Sequence , Biological Evolution , Chromosome Breakage , Chromosome Mapping , Chromosomes, Human , Humans , Male , Molecular Sequence Data , Phylogeny , Sequence Analysis/methods , Sequence Homology, Amino AcidABSTRACT
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. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.
Subject(s)
Chromosomes, Human, Pair 16/genetics , Familial Mediterranean Fever/genetics , Base Sequence , Chromosomes, Artificial, Yeast/genetics , Cloning, Molecular , Cosmids , DNA Primers/genetics , Genetic Markers , Haplotypes , Humans , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Restriction Mapping , Sequence Tagged SitesABSTRACT
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 16p13.3. By fluorescence in situ hybridization and Southern blot analysis, we assigned all tested RTS and t(8;16) translocation breakpoints to a 100-kb region. We have previously reported exact physical locations of these 16p breakpoints, which all disrupt one gene we mapped to this interval: the CREB-binding protein (CBP or CREBBP) gene. Intriguingly, mutations in the CBP gene are responsible for RTS as well as the t(8;16)-associated AML. CBP functions as an integrator in the assembly of various multiprotein regulatory complexes and is thus necessary for transcription in a broad range of transduction pathways. We report here the cloning, physical mapping, characterization, and full cDNA nucleotide sequence of the human CBP gene.
Subject(s)
Chromosomes, Human, Pair 16/genetics , Nuclear Proteins/genetics , Trans-Activators , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , CREB-Binding Protein , Chromosome Mapping , Chromosomes, Artificial, Yeast/genetics , Chromosomes, Human, Pair 8 , Cloning, Molecular , Cosmids , DNA Primers/genetics , DNA, Complementary/genetics , Humans , In Situ Hybridization, Fluorescence , Leukemia, Myeloid, Acute/genetics , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Rubinstein-Taybi Syndrome/genetics , Translocation, GeneticABSTRACT
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. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA.
Subject(s)
B-Lymphocytes , Genes, MHC Class II , Nuclear Proteins , Promoter Regions, Genetic , Trans-Activators/genetics , Base Sequence , Cells, Cultured , Cosmids , DNA , Epithelium , Humans , Luciferases/genetics , Macrophages , Molecular Sequence Data , Transcriptional ActivationABSTRACT
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. Results of the analysis are presented in graphical form, comprising a display of the DAPI intensity along the chromosome, the positions of the probe(s), the locations of bands as determined by analysis of the second derivative of the DAPI intensity profile, and a standard ideogram of the chromosome for comparison. The approach was validated and compared to visual assignment of probes to DAPI bands using the cosmid clone PYGM, which has been previously mapped to chromosome 11q13, and has been used as a landmark for mapping for other probes.
Subject(s)
Chromosome Banding , Fluorescent Dyes/chemistry , Image Processing, Computer-Assisted , In Situ Hybridization, Fluorescence , Indoles/chemistry , Cells, Cultured , Cosmids , Fibroblasts/cytology , Humans , SkinABSTRACT
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 2.3 kb cDNA has been identified which spans 17 kb of genomic DNA, including 8.1 kb within the critical region, and thus is a candidate tumor suppressor gene. This highly conserved gene has significant sequence similarity to a yeast gene of unknown function and to one of the yeast enzymes in the diphthamide synthetic pathway, DPH2, that has a role in global protein synthesis regulation. This gene, named DPH2L (diphthamide biosynthesis protein 2-like), is expressed in multiple tissues and stages of development.
Subject(s)
Chromosomes, Human, Pair 17 , DNA, Neoplasm/genetics , Gene Deletion , Ovarian Neoplasms/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Exons , Female , Gene Amplification , Genes, Tumor Suppressor , Humans , Molecular Sequence Data , Nucleic Acid Hybridization , Sequence Homology, Amino AcidABSTRACT
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. An acceptable sequence-tagged site (STS)-PCR assay yielded the appropriate size amplification product from both total human DNA and hybrid cell line DNA containing only human chromosome 5. Each STS has been regionally localized by breakpoint analysis using a set of hybrid cell panels consisting of natural deletions or translocations of human chromosome 5. This hybrid cell panel was able to localize the STSs to 1 of 51 bins on the short arm and 1 of 15 bins on the long arm. The STS markers appear to be randomly distributed along the length of this 194-Mb chromosome. The current overall density of these markers (approximately 1 STS/640 kb), combined with the numerous PCR-based physical and genetic markers generated by other groups, will provide sufficient "nucleation points" for YAC contig assembly and verification in any region of human chromosome 5.
Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 5/genetics , Sequence Tagged Sites , Animals , Chromosome Banding , Chromosomes, Artificial, Yeast/genetics , Chromosomes, Human, Pair 5/ultrastructure , Cloning, Molecular , Cricetinae , Genetic Markers , Humans , Hybrid Cells , Polymerase Chain ReactionABSTRACT
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. About 7% (22/301) produced multiple hybridization signals indicative of chimeric clones or sequences repeated at low copy number. Three cosmids (3/301, or 1%) hybridized to the centromeric regions of chromosome 10 and one or more other human chromosomes. About 19% (59/301) consisted mostly or entirely of hamster DNA inserts, and about 2% (6/301) appeared to be nonrecombinants.
Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 10 , Cosmids , Gene Library , In Situ Hybridization, Fluorescence , Animals , Cell Line , Cricetinae , DNA Probes , Flow Cytometry , Humans , Hybrid Cells , Molecular Sequence DataABSTRACT
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. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.
Subject(s)
Breast Neoplasms/genetics , Chromosome Mapping/methods , Chromosomes, Human, Pair 13/genetics , Neoplasm Proteins/genetics , Software , Transcription Factors/genetics , BRCA2 Protein , Base Sequence , Chromosomes, Artificial, Yeast/genetics , Chromosomes, Human, Pair 13/ultrastructure , Cosmids/genetics , DNA, Complementary/genetics , Disease Susceptibility , Female , Gene Expression , Humans , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction , Repetitive Sequences, Nucleic Acid , Selection, GeneticSubject(s)
Adenine Phosphoribosyltransferase/genetics , Chromosome Mapping , Chromosomes/ultrastructure , Mice, Inbred AKR/genetics , Mice, Inbred C57BL/genetics , Animals , Cricetinae , Crosses, Genetic , DNA Probes , Hybridization, Genetic , In Situ Hybridization, Fluorescence , Mice , Polymerase Chain ReactionABSTRACT
A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17,952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494,487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1,536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.
Subject(s)
Chromosomes, Human, Pair 11 , Cosmids , Gene Library , Animals , Chromosomes, Human , Cricetinae , DNA Probes , Flow Cytometry/methods , Humans , Hybrid Cells , Molecular Sequence Data , Nucleic Acid HybridizationABSTRACT
A cDNA encoding human glutamine phosphoribosylpyrophosphate amidotransferase for step one in de novo purine nucleotide synthesis was cloned, sequenced, and expressed in Chinese hamster ovary cells to yield functional enzyme. Enzyme function was dependent upon removal of an 11-amino-acid propeptide. A mutant enzyme having three propeptide amino acid replacements was not processed and was not active. The human genes GPAT, encoding the amidotransferase, and AIRC, encoding a bifunctional enzyme for steps six and seven in the pathway, were cloned and characterized. GPAT and AIRC are closely linked and divergently transcribed from an intergenic region of approximately 625 base pairs. Expression of a luciferase reporter from the GPAT promoter was approximately 3-4-fold higher than from the AIRC promoter. The GPAT gene was mapped to the q12 region of chromosome 4.