Regular ArticleDevelopment of 124 Sequence-Tagged Sites and Cytogenetic Localization of 217 Cosmids for Human Chromosome 10
Abstract
A total of 124 new chromosome 10-specific sequence-tagged sites (STSs) were derived from two sources: (1) DNA sequences obtained from anonymous clones in new libraries enriched for human chromosome 10 inserts, and (2) published sequences of genes and other loci already known to map to chromosome 10. Libraries were constructed from a somatic cell hybrid carrying human chromosomes 10 and Y. A cosmid library was made from total DNA of the hybrid and probed with labeled total human DNA to identify clones with human DNA inserts. Two hundred seventeen cosmids were mapped to regions of human chromosome 10 by fluorescence in situ hybridization. Twenty-five cosmids represent probes that have been placed on the genetic map previously. One hundred ninety-two cosmids represent new probes that have not been mapped previously. Cosmids carrying inserts with CA repeats were identified by hybridization with a labeled poly(dC-dA)-poly(dG-dT) probe and subcloned to yield microsatellite STS markers. Two small insert plasmid libraries were made, the first by subcloning inserts from a chromosome 10-enriched λ phage library (LL10NS01) and the second by cloning Alu element-mediated PCR products amplified from hybrid DNA. STSs were generated from the DNA sequences of clone inserts. Chromosome 10-specific STSs were distinguished from Y chromosome STSs by one or both of the following criteria: (1) successful PCR amplification from a template consisting of DNA from another chromosome 10-containing cell line, NA10926B, or (2) FISH localization to chromosome 10 of the source cosmid or of YACs isolated by PCR screening with the STS. These libraries were the source of 90 new chromosome 10-specific STSs, 42 of which contain CA repeats.
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A DNA fragment containing 1.5 kb of the 5′-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5′ flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.
Identification of the promoter region of human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene
2000, Biochemical and Biophysical Research CommunicationsThe placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) is expressed in several tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, and primary blood mononuclear cells. To better understand the regulation of HP2K gene expression, we isolated and characterized its genomic DNA, which includes the promoter region. The results of oligo-capping analysis indicate that the transcription start point (tsp) is an adenine residue 329 bp upstream of the translational start codon. DNA sequence analysis of this gene shows that the promoter region that contains the TATA box sequence and the 5′-UTR is different from the other known PFK-2/F2,6BPase genes. In addition, its 5′-flanking and 5′-UTR both have G + C-rich sequences containing Sp1 binding sites. To identify the promoter/enhancer region of HP2K gene, we performed transfection analyses of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. These experiments identified a promoter region 164 bp upstream from the tsp and an enhancer region between −1265 and −1329 on the 5′-flanking sequences. We also showed that Sp1 sites were not essential for HP2K transcription. Following transfection, stimulation experiments with serum, progesterone and phorbol 12-myristate 13-acetate showed that only the construct with the enhancer containing putative early growth response-1 binding motif was responsive to serum. We propose that the transcription of HP2K is strictly controlled by tissue-specific factors even though its genomic DNA contains several transcriptional elements.
Frequent loss of chromosome 10q sequences in endometrial cancers suggests the involvement of a tumorsuppressor gene. Previous loss-of-heterozygosity (LOH)studies have pointed to the 10q25–q26 region as the likely site of a tumor suppressor involved in endometrial tumorigenesis (S. L. Peifferet al.,1995,Cancer Res.55: 1922–1926; S. Nagaseet al.,1996,Br. J. Cancer74: 1979–1983; S. Nagaseet al.,1997,Cancer Res.57: 1630–1633). In an attempt to define further the localization of a tumor suppressor gene at 10q25, we screened a panel of 123 endometrioid adenocarcinomas for loss of heterozygosity of 10q25.3 sequences. Forty-three (35%) revealed LOH at one or more loci. The observed patterns of allelic loss define a minimum consensus region of deletion between D10S221 and D10S610. A sequence-ready bacterial clone contig and a long-range restriction map for a 1-Mb interval spanning the deletion region were developed as the first step in experiments directed toward the discovery the 10q25 tumor suppressor.
We present a yeast artificial chromosome (YAC) and pulsed-field gel electrophoresis (PFGE) map across the centromere of human chromosome 10 that links expressed sequences in 10p11 to expressed sequences in 10q11.2. This map is the first of its kind to link genes across a human centromere. It consists of a 2.5-Mb YAC contig extending from 10p11 to our previously published 5.35-Mb PFGE map of the centromeric satellite arrays, and a 2.65-Mb YAC contig extending from these satellite arrays to 10q11.2. This map covers approximately 6.5–7% of the total DNA of chromosome 10. Two Généthon genetic markers,D10S578andD10S604,are included. These markers are only 1 cM apart but are separated by a physical distance of more than 9.2 Mb, including the centromere. This gives a ratio of genetic to physical distance of 0.11 cM/Mb, 9–11 times lower than average estimates for the human genome and chromosome 10. Markers linked to the centromere include the duplicated zinc finger genesZNF11A, ZNF33A,andZNF37A(which map to 10p11) andZNF11B, ZNF33B,andZNF37B(which map to 10q11.2). Restriction mapping confirms that the genes on each arm lie in opposite orientation with respect to the centromere, consistent with the hypothesis that a pericentric inversion has occurred in this region during primate evolution.
Integration of the cytogenetic, genetic, and physical maps of the human genome by FISH mapping of CEPH YAC clones
1996, GenomicsThe physical locations on human metaphase chromosomes of over 950 yeast artificial chromosome (YAC) clones from the CEPH library have been determined by fluorescencein situhybridization and described as fractional chromosome length relative to the terminus of the short arm. Collectively, these clones contain approximately 1 billion basepairs of human DNA, about one-third of the human genome. In addition, the locations of 337 of these clones were established in terms of cytogenetic bands for chromosomes 1–18, 20, and X. Since most clones are positive for one or more of the Généthon polymorphic STS markers with defined genetic linkage distances corresponding to their physical locations, these data facilitate the integration of the cytogenetic, genetic, and physical maps of the human genome. Use of these mapping data in conjunction with public database information on CEPH YACs greatly facilitates the identification of YACs or polymorphic markers at specific locations in the genome.
Genetic aberrations in glioblastoma multiforme: Translocation of chromosome 10 in an O-2A-like cell line
1999, British Journal of Cancer