Definition of a commonly deleted region in ovarian cancers to a 300-kb segment of chromosome 6q27

Allelic deletions of chromosome 6q that occur frequently in ovarian cancers imply the presence of a putative tumor suppressor gene in this chromosomal vicinity. We analyzed DNA from 32 patients with ovarian carcinomas for loss of heterozygosity at loci on the distal portion of chromosome 6q and constructed a detailed deletion map. The map indicated a commonly deleted region between loci D6S149 (defined by CI6-24) and A2, which are estimated to be 300 kb apart on the basis of our cosmid contig map. By means of exon trapping, we found that the human AF-6 gene, which is disrupted in acute myeloid leukemia cells that carry a (6;11)(q27;q23) translocation, is located within the commonly deleted region. Subsequent screening of the AF-6 gene in ovarian carcinomas revealed no mutations. However, our mapping results, which narrowed the region containing the putative tumor suppressor gene to a 300-kb segment of 6q27, will facilitate further efforts to identify a gene associated with ovarian cancer.     

Advantage of polymerase chain reaction in the diagnosis of herpes simplex encephalitis: presentation of 5 atypical cases

Exon trapping from cosmids mapping to chromosome 19q13.3 yielded 6 exonic sequences that matched the human symplekin gene, which encodes a tight junction-related protein. One exonic sequence identified a 4.0 kb brain cDNA clone, R6E1, which contained 302 bp 5' to the originally reported 3.7 kb symplekin cDNA. A portion of this novel 5' sequence matched an additional trapped exonic sequence which was obtained from the most telomeric cosmid analyzed. The symplekin gene thus lies in a telomeric-to-centromeric direction on 19q13.3. Only three cosmids from a large 19q13.3 contig hybridized with R6E1, thereby assigning the symplekin gene to a 40 kb region immediately telomeric to gene 59 and the DM protein kinase gene. The 5' end of the R6E1 clone has a potential initiation codon with a strong Kozak sequence and Northern blot analysis detected a 4.2 kb signal in most human tissues, indicating that R6E1 may be a complete cDNA sequence. Based on the trapped exonic sequences, twelve exon-intron boundaries were predicted.     

Chromosomal localization of the human and mouse hyaluronan synthase genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17.HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

Decreased release of glutathione into the systemic circulation of patients with HIV infection

The human DDX6 gene (alias RCK) at chromosome 11 band q23 was identified through the study of the breakpoint of t(11;14)(q23;q32) translocation in a B-cell lymphoma cell line, RC-K8. DDX6 encodes a DEAD box protein/RNA helicase. Positive mouse genomic and cDNA recombinant clones were obtained by screening mouse B-cell genomic and cDNA libraries with a human DDX6 cDNA probe. The deduced amino acid sequence of an open reading frame from a cDNA clone revealed a protein with 92.5% identity to human ddx6/p54. All positive mouse genomic recombinant clones, and cDNA clones containing mouse Ddx6 (previous gene symbol: Rck), were localized by fluorescent in situ hybridization to band B of mouse Chromosome 9, a region showing conserved linkage homology to human chromosome 11 band q23. Mouse Ddx6 was localized to the region between Ncam and D9Mit45 by molecular linkage analysis. A 7.5-kb mRNA and a 54-kDa protein were identified as mouse Ddx6 gene products which are similar in size to products of the human DDX6 gene, as shown by Northern and Western blot analyses.     

Mapping the homolog of the human Rb1 gene to chromosome 14 of higher primates

The Rb1 gene has been implicated with retinoblastoma and is located on human Chromosome (Chr) 13q14.2. A unique sequence human Rb1 cosmid DNA probe has been used to localize this region on apes' Chr 14 by the FISH technique. The conservation of the Rb1 gene in higher primates at the corresponding equivalent chromosome locus (14q14) of the human may serve as a phylogenetic marker to further trace the evolutionary pathway of human descent.     

Structure of the m1 muscarinic acetylcholine receptor gene and its promoter

The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.     

Refined chromosomal localization of the putative tumor suppressor gene TP73

We report the identification of a mouse cDNA Tpd52l1 (tumor protein D52-like 1), which represents the first demonstrated orthologue of the human TPD52L1 (alias D53) gene, a member of the breast carcinoma-associated TPD52 (alias D52) gene family. In situ hybridization mapping located the Tpd52l1 gene to chromosome 10A4-10B2. Since the TPD52L1 gene is found at human chromosome 6q22-->q23, the mouse and human TPD52L1 loci are syntenically conserved.     

The human HNRPD locus maps to 4q21 and encodes a highly conserved protein

The hnRNP D protein interacts with nucleic acids both in vivo and in vitro. Like many other proteins that interact with RNA, it contains RBD (or "RRM") domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the human HNRPD gene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3' UTR. Use of two of the coding exons is determined by alternative splicing of the HNRPD mRNA. The human HNRPD gene maps to 4q21. The mouse Hnrpd gene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.     

Human PEG1/MEST, an imprinted gene on chromosome 7

The mouse Peg1/Mest gene is an imprinted gene that is expressed particularly in mesodermal tissues in early embryonic stages. It was the most abundant imprinted gene among eight paternally expressed genes (Peg 1-8) isolated by a subtraction-hybridization method from a mouse embryonal cDNA library. It has been mapped to proximal mouse chromosome 6, maternal duplication of which causes early embryonic lethality. The human chromosomal region that shares syntenic homology with this is 7q21-qter, and human maternal uniparental disomy 7 (UPD 7) causes apparent growth deficiency and slight morphological abnormalities. Therefore, at least one paternally expressed imprinted gene seems to be present in this region. In this report, we demonstrate that human PEG1/MEST is an imprinted gene expressed from a paternal allele and located on chromosome 7q31-34, near D7S649. It is the first imprinted gene mapped to human chromosome 7 and a candidate for a gene responsible for primordial growth retardation including Silver-Russell syndrome (SRS).     

The structure and organization of the human NPAT gene

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, and ATM, a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene, NPAT, is located upstream of ATM on human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5' flanking sequence. The structure and organization of NPAT were determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22-q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22-q24. For investigation of the role of NPAT in AT and these tumors with allelic loss of 11q22-q24, appropriate primer sequences and PCR conditions for amplification of all the NPAT exons from genomic DNA were determined. We previously reported that no recombinations are found among Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis using NPAT- and ACAT-specific primers and human genomic DNA allowed us to map ACAT 12 kb centromeric to NPAT.     

Simplified guide for precise implant placement: a technical note

Loss of heterozygosity (LOH) at chromosome band 10q23 occurs frequently in a wide variety of human tumors. A recently identified candidate tumor suppressor gene, PTEN located on 10q23, is mutated in multiple advanced cancers. To explore whether PTEN is associated with human squamous cell carcinoma of the head and neck (SCCHN), DNAs from both normal muscle and tumor tissue in 19 SCCHN were used for detecting LOH at chromosome 10q23 and mutational analysis of PTEN by direct polymerase chain reaction (PCR)-DNA sequencing. LOH at 10q23 was identified in 6/15 SCCHN. Mutation of PTEN was identified in 3/19 SCCHN. Of these 3 patients, 2 had stage IV disease; the third patient, with recurrent, metastatic and stage III disease, showed a 36 bp germline heterozygous deletion within intron 7. Furthermore, a missense mutation at codon 501 (TCT --> TTT: Ser --> Phe) in exon 8 was also found in tumor from the same patient. Our results suggest that PTEN may play a role in the genesis of some SCCHNs.     

Gene and locus structure and chromosomal localization of the protease-activated receptor gene family

Protease-activate receptors (PARs) mediate activation of platelets and other cells by thrombin and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis, and other normal and pathological processes. We report here the structure of the mouse and human PAR3 genes as well as the organization of a PAR gene cluster encompassing the genes encoding PARs 1, 2, and 3. We also report the structure of the mouse and human PAR4 genes, which map to distinct chromosomal locations and encode a new thrombin receptor. PARs 1-4 are all encoded by genes with the same two exon structure. In each case, exon 1 encodes a signal peptide, and exon 2 encodes the mature receptor protein. These are separated by an intron of variable size. The genes encoding PARs 1-3 all map to chromosome 13D2 in mouse and chromosome 5q13 in human. In mouse, all three genes are located within 80 kilobases of each other. The PAR1 gene is located centrally and is flanked upstream by the PAR3 gene and downstream by the PAR2 gene in both species. The proximity of the PAR1 and PAR3 genes suggests the possibility that these genes might share regulatory elements. A comparison of the structures of the PAR amino acid sequences, gene structures, locus organization, and chromosomal locations suggests a working model for PAR gene evolution.     

Isolation and chromosomal mapping of a mouse homolog of the Batten disease gene CLN3

We describe the isolation and chromosomal mapping of a mouse homolog of the Batten disease gene, CLN3. Like its human counterpart, the mouse cDNA contains an open reading frame of 1314 bp encoding a predicted protein product of 438 amino acids. The mouse and human coding regions are 82 and 85% identical at the nucleic acid and amino acid levels, respectively. The mouse gene maps to distal Chromosome 7, in a region containing genes whose homologs are on human chromosome 16p12, where CLN3 maps. Isolation of a mouse CLN3 homolog will facilitate the creation of a mouse model of Batten disease.