Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.
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PURPOSE: To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. METHODS: A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. RESULTS: The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. CONCLUSIONS: The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases. (+info)
Structure, chromosomal location, and analysis of the canine Cu/Zn superoxide dismutase (SOD1) gene.
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Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA. (+info)
Prostate cancer aggressiveness locus on chromosome 7q32-q33 identified by linkage and allelic imbalance studies.
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The biologic aggressiveness of prostate tumors is an important indicator of prognosis. Chromosome 7q32-q33 was recently reported to show linkage to more aggressive prostate cancer, based on Gleason score, in a large sibling pair study. We report confirmation and narrowing of the linked region using finer-scale genotyping. We also report a high frequency of allelic imbalance (AI) defined within this locus in a series of 48 primary prostate tumors from men unselected for family history or disease status. The highest frequency of AI was observed with adjacent markers D7S2531 (52%) and D7S1804 (36%). These two markers delineated a common region of AI, with 24 tumors exhibiting interstitial AI involving one or both markers. The 1.1-Mb candidate region contains relatively few transcripts. Additionally, we observed positive associations between interstitial AI at D7S1804 and early age at diagnosis (P=.03) as well as a high combined Gleason score and tumor stage (P=.06). Interstitial AI at D7S2531 was associated with a positive family history of prostate cancer (P=.05). These data imply that we have localized a prostate cancer tumor aggressiveness loci to chromosome 7q32-q33 that is involved in familial and nonfamilial forms of prostate cancer. (+info)
ChickRH6: a chicken whole-genome radiation hybrid panel.
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As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6,000 rads with HPRT-deficient hamster Wg3hCl2 cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs. (+info)
Parametric and non-parametric linkage analysis of several candidate regions for genes for human handedness.
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The frequency of left-handedness in the general population is around 11%. Both environmental and genetic models have been proposed to explain the aetiology of human handedness. The majority of genetic models, such as those of Annett, McManus and Klar, propose a single gene determinant with a non-Mendelian inheritance pattern. As left-handedness is correlated with cerebral asymmetry and is a feature of left-right asymmetry, genes involved in the development of left-right asymmetry can be considered as candidate genes. Candidate gene analysis was performed using an informative extended pedigree, and also using nuclear families of right-handed parents with left-handed children. Segregation analysis in the extended pedigree identified allele sharing in the NODAL and DNAHC13 candidate regions on chromosome 10 and 1. Linkage analysis using the models of Klar and McManus, and non-parametric analysis on nuclear families, subsequently excluded all candidate regions tested. This demonstrates the power to identify the genes specifying handedness by the conduct of extended genetic studies on these and similar cohorts. (+info)
Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences.
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BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA. (+info)
Functional second genes generated by retrotransposition of the X-linked ribosomal protein genes.
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We have identified a new class of ribosomal protein (RP) genes that appear to have been retrotransposed from X-linked RP genes. Mammalian ribosomes are composed of four RNA species and 79 different proteins. Unlike RNA constituents, each protein is typically encoded by a single intron- containing gene. Here we describe functional autosomal copies of the X-linked human RP genes, which we designated RPL10L (ribosomal protein L10-like gene), RPL36AL and RPL39L after their progenitors. Because these genes lack introns in their coding regions, they were likely retrotransposed from X-linked genes. The identities between the retrotransposed genes and the original X-linked genes are 89-95% in their nucleotide sequences and 92-99% in their amino acid sequences, respectively. Northern blot and PCR analyses revealed that RPL10L and RPL39L are expressed only in testis, whereas RPL36AL is ubiquitously expressed. Although the role of the autosomal RP genes remains unclear, they may have evolved to compensate for the reduced dosage of X-linked RP genes. (+info)
The comprehensive mouse radiation hybrid map densely cross-referenced to the recombination map: a tool to support the sequence assemblies.
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We have developed a unique comprehensive mouse radiation hybrid (RH) map of nearly 23,000 markers integrating data from three international genome centers and over 400 independent laboratories. We have cross-referenced this map to the 0.5-cM resolution recombination-based Jackson Laboratory (TJL) backcross panel map, building a complete set of RH framework chromosome maps based on a high density of known-ordered anchor markers. We have systematically typed markers to improve coverage and resolve discrepancies, and have reanalyzed data sets as needed. The cross-linking of the RH and recombination maps has resulted in a highly accurate genome-wide map with consistent marker order. We have compared these linked framework maps to the Ensemble mouse genome sequence assembly, and show that they are a useful medium resolution tool for both validating sequence assembly and elucidating chromosome biology. (+info)