Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas.

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Positioning of 72 potentially full size LTRs of human endogenous retroviruses HERV-K on the human chromosome 19 map. Occurrences of the LTRs in human gene sites.

Seventy-two near full size long terminal repeats (LTRs) of human endogenous retrovirus of K-family (HERV-K) have been precisely located on the metric map of human chromosome 19. The LTR-related sequences were identified and assigned to cosmids by hybridization with two independent chromosome 19 specific cDNA clones corresponding to different parts of U3 region of LTR of HERV-K. The presence of full-size LTR sequences in a cosmid was further verified by PCR assay with a pair of primers complementary to the termini of the LTR. Coincidences of the LTR and the known genes positions are discussed.

Mapping of transcribed sequences on human chromosome 19.

30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a source of transcribed sequences. In addition more than 50 sites constituting 19 families of closely related sequences containing at least one transcribed member each were mapped across the chromosome. Chromosome-19 specific hncDNA clones were hybridized to chromosome 19 cosmids that were previously assembled into contigs covering about 80% of Chr19. The hybridization results were verified by PCR. Such an approach to EST mapping provides information on possible locations of genes as transcribed units of genome and on location of repeated elements used for the priming the hncDNA synthesis. Mapped hncDNA sequences may serve as good starting points for the systematic sequencing of transcribed genomic regions.

[Isolation and characteristics of an ordered library of transcribed sequences of human chromosome 19 from hybrid human-hamster cells]. / Poluchenie i kharakteristiki uporiadochennoi biblioteki transkribiruemykh posledovatel'nostei khromosomy 19 cheloveka iz gibridnykh kletok chelovek-khomiak.

A new technique based on Alu-PCR amplification of hn-RNA is described for the extraction of human-specific transcribed sequences from a hybrid cell line. Arrayed library of hn-cDNA was constructed and characterized by sequencing about 80 individual clones. A high enrichment by human-specific sequences (about 95%) was demonstrated.

Genome sequence and splice site analysis of low-fidelity DNA polymerases H and I involved in replication of damaged DNA.

POLH and POLI are paralogs encoding low-fidelity, class Y, DNA polymerases involved in replication of damaged DNA in the human disease xeroderma pigmentosum variant. Analysis of genomic regions for human and mouse homologs, employing the analytic tool Genome Cryptographer, detected low-repetitive or unique regions at exons and other potential control regions, especially within intron I of human POLH. The human and mouse homologs are structurally similar, but the paralogs have undergone evolutionary divergence. The information content of splice sites for human POLH, the probability that a base would contribute to splicing, was low only for the acceptor site of exon II, which is preceded by a region of high information content that could contain sequences controlling splicing. This analysis explains previous observations of tissue-specific skipping during mRNA processing, resulting in the loss of the transcription start site in exon II, in human tissues.

A comparative analysis of the putative regulatory regions in human genes for the alpha-subunit family of Na(+)-K+ ATPase.

The sequences of a 1.5 kb long stretch of the 5' flanking region of the gene for the alpha 3 isoform of the catalytic subunit of human Na(+)-K+ ATPase (located on chromosome 19) and of more than a 2 kb stretch of the 5' flanking region of the gene for the alpha 2 isoform (located on chromosome 1) have been determined. Transcription start sites for the gene for the alpha 3 isoform have been mapped at positions -152 and -155 relative to the translation initiation codon by primer extension analysis and S1-nuclease mapping of mRNA from human brain. The 5' flanking region of the gene for isoform alpha 3 contains a CCAAT box on the noncoding chain and six putative Sp1 binding sites. Absence of a conventional TATA box and a high GC content are other features of the region. The 5' upstream region of the gene for the alpha 2 isoform contains potential TATA and CCAAT boxes and one potential Sp1 binding site. Upstream of the putative TATA box there is an octanucleotide repeat, GGGGGAGA, which is also found in several eukaryotic genes in analogous positions. Pairwise comparison of the putative 5' regulatory regions of the genes coding for the different isoforms of the Na(+)-K(+)-ATPase catalytic subunit shows the existence of conserved elements, as well as of oligonucleotide blocks with very different structures. It is suggested that the differences in the primary structure of the 5' upstream regions may provide the basis for tissue-specific expression of the Na(+)-K(+)-ATPase isoforms.

Physical mapping of sequences homologous to an endogenous retrovirus LTR on human chromosome 19.

The human genome contains multiple copies of sequences related to the HERV-K family of endogenous retroviruses, homologous to the B-type mouse mammary tumour virus. A DNA fragment closely resembling an HERV-K long tandem repeat (LTR) was detected in a library of hncDNA clones enriched for sequences from human chromosome 19. Sites showing homology to the sequence of this fragment have been identified on human chromosome 19 by hybridization to previously mapped chromosome 19 cosmids. Thus the distribution of LTR sequences on a specific human chromosome has been mapped for the first time. We estimate the total number of such sites on human chromosome 19 to be at least 110. Many of these sites are located in the vicinity of known genes. The precise localizations (to specific cosmids) of LTR-homologous sequences on chromosome 19 can serve as a reference source and will automatically provide further insight into LTR-gene relationships as new genes are mapped onto the chromosome.

An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis.

A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.

Characterization of the mouse collectin gene locus.

Three of the four known mouse collectin genes have been mapped to chromosome 14. To further characterize the spatial relationship of these genes, a bacterial artificial chromosome (BAC) library of mouse chromosome 14 was screened using mouse surfactant protein (SP)-A and -D complementary DNAs (cDNAs). One large clone hybridized to both SP-A and SP-D cDNAs and was found by polymerase chain reaction (PCR) to contain sequences from one of the mouse mannose-binding lectin genes (Mbl1). We used Southern mapping and subcloning of overlapping restriction fragments to characterize the gene locus. Mapping was confirmed by fluorescent in situ hybridization of fiber-stretched BAC DNA and by Southern hybridization of restriction endonuclease-digested and PCR-amplified genomic DNA. We found that the SP-A, Mbl1, and SP-D genes reside contiguously within a 55-kb region. The SP-A and Mbl1 genes are in the same 5' to 3' orientation and 16 kb apart. The SP-D gene is in the opposite orientation to the two other collectin genes, 13 kb away from the 3' end of the Mbl1 gene. The mouse SP-D gene had not previously been characterized. We found its size (13 kb) and organization to be similar to that of human SP-D. Exon I is untranslated. The second exon is a hybrid exon that contains signal for initiation of translation, signal peptide, N-terminal domain, and the first seven collagen triplets of the collagen-like domain of the protein. Four short exons (III through VI) encode the collagen-like domain of the protein, and exons VII and VIII the linking and the carbohydrate-recognition domains, respectively.

Comprehensive genome sequence analysis of a breast cancer amplicon.

Gene amplification occurs in most solid tumors and is associated with poor prognosis. Amplification of 20q13.2 is common to several tumor types including breast cancer. The 1 Mb of sequence spanning the 20q13.2 breast cancer amplicon is one of the most exhaustively studied segments of the human genome. These studies have included amplicon mapping by comparative genomic hybridization (CGH), fluorescent in-situ hybridization (FISH), array-CGH, quantitative microsatellite analysis (QUMA), and functional genomic studies. Together these studies revealed a complex amplicon structure suggesting the presence of at least two driver genes in some tumors. One of these, ZNF217, is capable of immortalizing human mammary epithelial cells (HMEC) when overexpressed. In addition, we now report the sequencing of this region in human and mouse, and on quantitative expression studies in tumors. Amplicon localization now is straightforward and the availability of human and mouse genomic sequence facilitates their functional analysis. However, comprehensive annotation of megabase-scale regions requires integration of vast amounts of information. We present a system for integrative analysis and demonstrate its utility on 1.2 Mb of sequence spanning the 20q13.2 breast cancer amplicon and 865 kb of syntenic murine sequence. We integrate tumor genome copy number measurements with exhaustive genome landscape mapping, showing that amplicon boundaries are associated with maxima in repetitive element density and a region of evolutionary instability. This integration of comprehensive sequence annotation, quantitative expression analysis, and tumor amplicon boundaries provide evidence for an additional driver gene prefoldin 4 (PFDN4), coregulated genes, conserved noncoding regions, and associate repetitive elements with regions of genomic instability at this locus.