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J M Sikela

Publications and source records attributed to J M Sikela.

At least 19 recordsLinked to original sources
PubMed2006

The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis.

The 19q13 amplicon in pancreatic cancer cells contains a novel pancreatic differentiation 2 (PD2) gene (accession number AJ401156), which was identified by differential screening analysis. PD2 is the human homologue of the RNA polymerase II-associated factor 1 (hPaf1). In yeast, Paf1 is part of the transcription machinery, acting as a docking protein in between the complexes Rad6-Bre1, COMPASS-Dot1p, and the phosphorylated carboxyl terminal domain of the RNA polymerase II. As such, Paf1 is directly involved in transcription elongation via histone H2B ubiquitination and histone H3 methylation. The PD2 sequence is highly conserved from Drosophila to humans with up to 98% identity between rodent and human, suggesting the functional importance of PD2/hPaf1 to maintain cellular homeostasis. PD2 is a modular protein composed of RNA recognition motif, DEAD-boxes, an aspartic/serine (DS)-domain, a regulator of the chromosome condensation domain and myc-type helix-loop-helix domains. Our results further showed that PD2 is a nuclear 80 kDa protein, which interacts with RNA polymerase II. In addition, we have demonstrated that the overexpression of PD2 in the NIH 3T3 cells result in enhanced growth rates in vitro and tumor formation in vivo. Altogether, this paper presents strong evidence that the overexpression of PD2/hPaf1 is involved in cancer development.

Amino Acid Sequence↗
PubMed2001

High-throughput sequence identification of gene coding variants within alcohol-related QTLs.

Low initial response to alcohol has been shown to be among the best predictors of development of alcoholism. A similar phenotypic measure, difference in initial sensitivity to ethanol, has been used for the genetic selection of two mouse strains, the Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice, and for the subsequent identification of four quantitative trait loci (QTLs) for alcohol sensitivity. We now report the application of high throughput comparative gene sequencing in the search for genes underlying these four QTLs. To carry out this search, over 1.7 million bases of comparative DNA sequence were generated from 68 candidate genes within the QTL intervals, corresponding to a survey of over 36,000 amino acids. Eight central nervous system genes, located within these QTLs, were identified that contain a total of 36 changes in protein coding sequence. Some of these coding variants are likely to contribute to the phenotypic variation between ILS/ISS animals, including sensitivity to alcohol, providing specific new genetic targets potentially important to the neuronal actions of alcohol.

Alcoholism↗
PubMed2001

Comparison of global brain gene expression profiles between inbred long-sleep and inbred short-sleep mice by high-density gene array hybridization.

BACKGROUND: Inbred long-sleep (ILS) and short-sleep (ISS) mice show significant central nervous system-mediated differences in sleep time for sedative dose of ethanol and are frequently used as a rodent model for ethanol sensitivity. In this study, we have used complementary DNA (cDNA) array hybridization methodology to identify genes that are differentially expressed between the brains of ILS and ISS mice. METHODS: To carry out this analysis, we used both the gene discovery array (GDA) and the Mouse GEM 1 Microarray. GDA consists of 18,378 nonredundant mouse cDNA clones on a single nylon filter. Complex probes were prepared from total brain mRNA of ILS or ISS mice by using reverse transcription and 33P labeling. The labeled probes were hybridized in parallel to the gene array filters. Data from GDA experiments were analyzed with SQL-Plus and Oracle 8. The GEM microarray includes 8,730 sequence-verified clones on a glass chip. Two fluorescently labeled probes were used to hybridize a microarray simultaneously. Data from GEM experiments were analyzed by using the GEMTools software package (Incyte). Differentially expressed genes identified from each method were confirmed by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A total of 41 genes or expressed sequence tags (ESTs) display significant expression level differences between brains of ILS and ISS mice after GDA, GEM1 hybridization, and quantitative RT-PCR confirmation. Among them, 18 clones were expressed higher in ILS mice, and 23 clones were expressed higher in ISS mice. The individual gene or EST's function and mapping information have been analyzed. CONCLUSIONS: This study identified 41 genes that are differentially expressed between brains of ILS and ISS mice. Some of them may have biological relevance in mediation of phenotypic variation between ILS and ISS mice for ethanol sensitivity. This study also demonstrates that parallel gene expression comparison with high-density cDNA arrays is a rapid and efficient way to discover potential genes and pathways involved in alcoholism and alcohol-related physiologic processes.

Alternative Splicing↗
PubMed1998

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

Animals↗
PubMed1998

Discovery of a new Pomc1 allele in the LS x SS recombinant inbred strains: relationship to locomotor behavior.

Various lines of evidence suggest that a polymorphism in the gene for proopiomelanocortin, Pomc1, might account for some portion of the genetic variance for open-field activity in the LS x SS RI strains. To test this hypothesis, approximately 1600 bp of Pomc1 was sequenced from genomic DNA of seven of the LS x SS strains. Two distinct alleles containing a total of five single-base pair differences were detected. A substitution was found in the coding region causing a Pro-to-Ser conversion, two substitutions occurred in the 3' untranslated region of the mRNA, and a substitution and a deletion were detected in the 3' untranscribed flanking region. The fragment containing the coding region substitution was sequenced in an additional 15 of the LS x SS strains. A total of 12 strains contained one form of the allele, while 10 had the other. The strain distribution pattern of open-field activity scores between the two allels suggests that these alleles do not contribute to the genetic variation of open-field activity in the LS x SS RI strains.

Animals↗
PubMed1997

Genomic structure and chromosomal localization of a human myo-inositol monophosphatase gene (IMPA).

Manic-depressive illness is a serious psychiatric disorder that in many, but far from all, patients can be treated with lithium. The main causes for discontinuation of lithium therapy are unpleasant or serious side effects and lack of response. The reason for the striking variation in clinical efficacy of lithium treatment among bipolar patients is not known. The enzyme myo-inositol monophosphatase (IMPase) has been postulated as a target for the mood-stabilizing effects of lithium, but variation in the coding region of the human IMPA gene encoding IMPase activity has not been observed in manic-depressive patients (Steen et al., Pharmacogenetics, 1996, 6, 113-116). It is nevertheless conceivable that polymorphisms or mutations in the noncoding regions of this gene could influence the lithium response in psychiatric patients. As a first step in investigating this possibility, we here report the genomic structure of the human IMPA gene. The gene is composed of at least nine exons and covers more than 20 kb of sequence on chromosome 8q21.13-q21.3. In the 3'-untranslated part of the gene, we observed a polymorphism (a G to A transition) and also two short sequences similar to the inositol/cholin-responsive element consensus. Finally, we postulate that two additional IMPA-like transcripts originate from the human genome, one from a position close to IMPA itself on chromosome 8 and the other from chromosome 18p. Our data may contribute to the identification of genetic factors involved in the pathogenesis and determination of treatment response in manic-depressive illness.

Amino Acid Sequence↗
PubMed1996

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

Amino Acid Sequence↗
PubMed1996

Human chromosome 1 localization of the gene for a prostaglandin F2alpha receptor negative regulatory protein.

A protein that copurifies with the bovine prostaglandin F2alpha (FP) receptor has been isolated and the corresponding rat cDNA has been cloned. Transfection experiments suggest that this protein inhibits binding of [3H]prostaglandin F2alpha ([3H]PGF2alpha) to FP. Histologically, this protein (FP regulatory protein or FPRP) shows a distribution coinciding well with those cells and tissues that respond to PGF2alpha. A portion of the 3' untranslated region of the human homolog to fprp was subcloned, sequenced, and oligonucleotide primers chosen that allow polymerase chain reaction (PCR) amplification specifically of the human fprp sequence. These primers were then used in a PCR-based mapping-protocol. The human fprp gene was first socalized through human/rodent somatic cell hybrids to human chromosome 1 (100% concordance), and further through yeast artificial chromosome (YAC) pools to region 1p13.1-q21.3 (level 1 mapping). In view of the specific histologic localization of this negative regulator, possible pathological conditions are mentioned that may cosegrepate with this chromosomal region.

Animals↗
PubMed1995

The gamma-aminobutyric acid receptor gamma 3 subunit gene (GABRG3) is tightly linked to the alpha 5 subunit gene (GABRA5) on human chromosome 15q11-q13 and is transcribed in the same orientation.

GABAA receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter gamma-aminobutyric acid. The GABAA receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies (alpha, beta, gamma, delta, rho) on the basis of sequence similarity. Chromosomal mapping studies have revealed that several of the GABAA receptor subunit genes appear to be organized as clusters. One such cluster, which consists of the GABAA receptor beta 3 (GABRB3) and alpha 5 (GABRA5) subunit genes, is located in chromosome 15q11-q13. It is shown here that the GABAA receptor gamma 3 subunit gene (GABRG3) also maps to this region. Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5' end of GABRG3 were linked to an existing phage contig spanning the 3' end of GABRA5. The two genes are located within 35 kb of each other and are transcribed in the same orientation.

Animals↗
PubMed1995

Localization of the gene for a novel human adenylyl cyclase (ADCY7) to chromosome 16.

A novel form of human adenylyl cyclase (ADCY7) has been discovered in the human erythroleukemia cell line (HEL). This cell line has been widely used as a model for studies of the characteristics of human platelets. Data from HEL cells suggests that ADCY7 may be the major AC form in human platelets. In the current study polymerase chain reaction (PCR) techniques coupled with use of human/rodent somatic hybrid panels and a yeast artificial chromosome (YAC) library were used to determine the chromosomal localization of the gene (adcy7) for ADCY7 enzyme. A 251-bp product from the 3' untranslated region of human adcy7 was amplified for PCR mapping and the results localize the adcy7 gene to region 16q12-16q13 of the human genome. The AC enzyme family is characterized by the presence of 12 membrane-spanning domains in its sequences, and this chromosomal region is known to contain other genes coding for proteins characterized by 12 membrane-spanning domains.

Adenylyl Cyclases↗
PubMed1995

Gene-based sequence-tagged-sites (STSs) as the basis for a human gene map.

Using our data set of 3,143 single pass sequences from human brain cDNA libraries, we have developed a strategy in which gene-based sequence-tagged-sites (STSs), derived from 3'untranslated regions of human cDNAs, are rapidly assigned to megabase-insert yeast artificial chromosomes and somatic cell hybrids to generate regional gene mapping data. Employing this approach, we have mapped 318 cDNAs, representing 308 human genes. Ninety-two of these mapped to regions implicated in human genetic diseases, identifying them as candidate genes. Extension of this strategy has the potential to result in virtually every human gene having, at its 3' end, its own associated STS, with each STS in turn specifying both a corresponding genomic clone and a specific regional location in the genome.

Animals↗
PubMed1995

A strategy for the identification of candidate genes for alcohol-related phenotypes and other human disorders using rapid polymerase chain reaction mapping of gene-based sequence-tagged sites.

We describe a method for the rapid identification and mapping of human genes, including those possibly contributing to disease and alcohol-related phenotypes. New human genes are identified from cDNA libraries through single-pass sequencing into the 3' untranslated (3'UT) regions of human brain cDNAs. Primers derived from the 3'UT region sequences [representing gene-based, sequence-tagged sites (STSs)] are used for polymerase chain reaction (PCR) analyses of the CEPH megabase insert yeast artificial chromosome (YAC) DNA pools. With this approach, approximately 18,000 megabase YACs can be screened and a single YAC identified using only 52 PCR reactions. The YAC localization in conjunction with other mapping techniques, such as PCR mapping to human chromosomes using somatic cell hybrids, allows identification of chromosomal band locations. In this manner, each gene can be associated with its own STS, which in turn specifies both a corresponding genomic clone and specific location in the genome. These locations can be compared with the purported locations of disease genes. The locations of the STSs can also be compared with those of Quantitative Trait Loci implicated for quantitative traits (e.g., alcohol-related phenotypes) on the basis of synteny between the mouse and human genes. Using this strategy, we found candidates for 78 human disease/syndrome genes among the first 220 genes mapped.

Alcoholism↗
PubMed1994

Cloning, functional coexpression, and pharmacological characterisation of human cDNAs encoding NMDA receptor NR1 and NR2A subunits.

Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of rat N-methyl-D-aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C-terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N-terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C-terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors in Xenopus oocytes. These receptors had EC50 values of 2.14 and 2.05 microM for glutamate and glycine, respectively, and an IC50 of 46.8 microM for Mg2+. Responses were antagonised by D-2-amino-5-phosphonovalerate, L-689,560, pH 6.3, zinc, and MK-801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.

Amino Acid Sequence↗
PubMed1993

A cluster of three GABAA receptor subunit genes is deleted in a neurological mutant of the mouse p locus.

The mouse pink-eyed cleft-palate (p(cp)) mutation is characterized by hypopigmentation associated with cleft palate, neurological disorders and runting. Most p(cp) homozygotes are born with cleft palate and die shortly after birth, presumably as a result of feeding problems. A few exceptional p(cp) mutants live beyond this stage but display tremor and jerky gait. We report here that the genes encoding the gamma-aminobutyric acid type A (GABAA) receptor subunits alpha 5 (originally described as alpha 4; ref. 4), beta 3 and gamma 3 are disrupted by a deletion in p(cp) mice. We also show that the alpha 5 and gamma 3 genes are located between the p and beta 3 genes on mouse chromosome 7. The p(cp) deletion leads to alterations of binding properties of the GABAA receptors in the brain, providing an in vivo model system for studying GABAA receptor function. The human homologue of the region deleted in p(cp) mice is associated with Angelman syndrome. Thus, p(cp) mice may be useful in defining the region containing the gene(s) for this syndrome.

Animals↗
PubMed1993

Isolation and sequence of a mouse brain cDNA coding for protein kinase C-gamma isozyme.

The regulatory enzyme, protein kinase C (PKC), is characterized by a family of related isozymes. Currently, nucleotide (nt) sequences for seven members of this family have been reported from the bovine, human and rat genomes. Only four of these seven PKC isoforms have been isolated in mouse: alpha, beta II, delta and epsilon. Here, we report the cDNA sequence encoding mouse PKC-gamma isolated from a C57BL/6 brain cDNA library. The mouse and rat PKC-gamma nt and deduced amino acid sequences share 97 and 100% identity, respectively.

Amino Acid Sequence↗
PubMed1993

Prodynorphin and vasopressin mRNA levels are differentially affected by chronic ethanol ingestion in the mouse.

Opioid peptides derived from the precursor, prodynorphin, are co-localized with vasopressin in the hypothalamus and posterior pituitary, and vasopressin and prodynorphin synthesis are coordinately regulated during salt-loading. We had previously found that chronic ethanol ingestion resulted in decreased levels of hypothalamic and extrahypothalamic vasopressin mRNA, and the current study investigated the effect of ethanol ingestion on prodynorphin mRNA levels. A cRNA probe was constructed from a PCR product amplified from mouse genomic DNA. Cloning and sequencing of the PCR product revealed that the sequence of the mouse prodynorphin gene used to synthesize the probe is highly conserved, with high sequence similarity to corresponding regions of the gene in other mammalian species. In situ hybridization using the cRNA probe showed a widespread distribution of prodynorphin mRNA in mouse brain. In dehydrated mice, prodynorphin mRNA was significantly increased in the hypothalamus and nearly all other brain areas examined. In ethanol-fed mice, prodynorphin mRNA was also significantly increased in hypothalamus (50-60%) and in most brain areas. In the same mice, measurement of hypothalamic vasopressin mRNA confirmed a significant (approximately 60%) decrease. These results indicate that hypothalamic vasopressin and prodynorphin mRNA can be differentially regulated in certain situations.

Alcoholism↗