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J M Sikela

Publications and source records attributed to J M Sikela.

At least 37 records · Page 2Linked to original sources
PubMed1993

Chromosomal distribution of 320 genes from a brain cDNA library.

We have determined the chromosomal assignment of 320 brain expressed genes by studying the segregation of polymerase chain reaction (PCR) products in human rodent somatic cell hybrids and by genetically mapping polymorphic cDNAs using the CEPH (Centre d'Etude du Polymophisme Humaine) reference pedigrees and database. These mapped genes can function as markers on the physical map of the human genome, as well as serve as candidate disease gene loci. Distribution of these genes to the human chromosomes correlates well with the GC content of the chromosomes. However, the distribution of these genes does not correlate well with the cytogenetic length of each chromosome.

Animals↗
PubMed1993

FISH ordering of reference markers and of the gene for the alpha 5 subunit of the gamma-aminobutyric acid receptor (GABRA5) within the Angelman and Prader-Willi syndrome chromosomal regions.

We have established a probe order within the Angelman/Prader-Willi chromosomal regions by multicolor fluorescence in situ hybridization (FISH). The probe [locus] order extending distally from the centromere is 34[D15S9]-IR4-3R[D15S11]-189-1[D15S13]-PW71++ + [D15S63]-3-21[D15S10]-28 beta 3-H3[GABRB3]-IR10-1 [D15S12]. This order agrees with that recently reported (1) with the exception of PW71 [D15S63]. In addition, a second gamma-aminobutyric acid (GABAA) receptor, the alpha 5 subunit, has been localized within the reference map to between GABRB3 and D15S12. The locus order was further confirmed by DNA hybridization analysis of two patients, one with Angelman syndrome and one with Prader-Willi syndrome, with different unbalanced translocations and molecular extents of deletion. Our results provide a framework map of chromosome 15q11-q13 into which additional markers can be oriented and allow a further differentiation of the critical genetic regions of the two syndromes.

Amino Acid Sequence↗
PubMed1993

Role of the beta subunit in determining the pharmacology of human gamma-aminobutyric acid type A receptors.

A cDNA encoding the human gamma-aminobutyric acid (GABA)A receptor beta 2 subunit has been cloned and sequenced. The deduced amino acid sequence of this cDNA shows only a single amino acid change from the rat sequence (Asn-347 in rat, serine in human). Using polymerase chain reaction amplification of human-specific products from human x rodent somatic cell hybrid DNAs, the gene has been assigned to human chromosome 6. By expressing recombinant human GABAA receptors containing different beta subunits (beta 1, beta 2 or beta 3) in both transfected cells and Xenopus oocytes, we have been able to determine the influence of the beta subunit on the pharmacology of the receptor. For a number of benzodiazepine binding site compounds, a barbiturate, and several neurosteroids, neither the affinity nor the efficacy of the compounds is influenced by the type of beta subunit present in the receptor molecule. These data suggest that the beta subunit does not significantly influence the benzodiazepine, barbiturate, or steriod site pharmacologies of human GABAA receptor subtypes.

Amino Acid Sequence↗
PubMed1992

Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome.

The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol. The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism. We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs. Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2. These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5. To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2. A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes. Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other. The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.

Base Sequence↗
PubMed1992

GABAA receptor function and regional analysis of subunit mRNAs in long-sleep and short-sleep mouse brain.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.

Animals↗
PubMed1992

A radiation hybrid map of 18 growth factor, growth factor receptor, hormone receptor, or neurotransmitter receptor genes on the distal region of the long arm of chromosome 5.

The distal portion of the long arm of human chromosome 5 contains an impressive number of genes encoding growth factors, growth factor receptors, and hormone/neurotransmitter receptors. The order of and relative distance between 18 of these genes was determined by radiation hybrid mapping. There is only a single gap in a contiguous radiation map from 5q22-5q35. For this set of radiation hybrids, one map unit (centiray) corresponds to 20-50 kb of DNA. Close physical proximity for several pairs of loci was predicted by the map. Two sets of these were found to be contained in single YAC clones. The physical map produced by radiation hybrid mapping should prove useful in efforts to identify four disease genes that have been assigned to distal 5q by linkage studies.

Base Sequence↗
PubMed1992

Single pass sequencing and physical and genetic mapping of human brain cDNAs.

We have performed single pass sequencing of 1,024 human brain cDNAs, over 900 of which seem to represent new human genes. Library prescreening with total brain cDNA significantly reduced repeated sequencing of highly represented cDNAs. A subset of sequenced cDNAs were physically mapped to their chromosomal locations using gene-specific STS primers derived from 3' untranslated regions. We have also determined that human brain cDNAs represent a rich source of gene-associated polymorphic markers. Microsatellite-containing cDNAs can be physically mapped and converted to highly informative genetic markers, thus facilitating integration of the human physical, expression and genetic maps.

Base Sequence↗
PubMed1991

cDNA sequence and differential expression of the mouse Ca2+/calmodulin-dependent protein kinase IV gene.

We have isolated and sequenced a mouse brain cDNA encoding Ca2+/calmodulin-dependent protein kinase IV. The sequence predicts an acidic protein (pI = 4.56) of 469 amino acids (Mr = 52,627) that contains kinase catalytic and calmodulin-binding domains. The carboxy region has several primary structural features that suggest it may be a readily cleaved attachment domain. This region is highly charged and hydrophilic and contains several PEST sequences, motifs associated with high turnover proteins. Of the tissues examined, expression of the CaM kinase IV gene is restricted to brain and testis, where transcripts are differentially expressed to produce a kinase in both tissues and a calmodulin-binding protein, calspermin, in testis that lacks a kinase catalytic domain.

Amino Acid Sequence↗
PubMed1991

A novel gamma subunit of the GABAA receptor identified using the polymerase chain reaction.

We have utilized a polymerase chain reaction (PCR) strategy to identify a novel subunit, gamma 3, of the GABAA receptor. The gamma 3 cDNA encodes a mature protein of 450 amino acids that contains structural features typically conserved among subunits of the GABAA receptor family. The gamma 3 subunit shares approximately 66% sequence identity with the gamma 2 subunit but only 38% and 29% with alpha 1 and beta 1 subunits, respectively. Localization of the gamma 3 mRNA indicates that it is widely distributed throughout the mouse brain in a pattern similar to that observed for mRNAs encoding the gamma 2 subunits.

Amino Acid Sequence↗
PubMed1991

Use of 3' untranslated sequences of human cDNAs for rapid chromosome assignment and conversion to STSs: implications for an expression map of the genome.

A general mapping strategy is described in which the 3'untranslated regions of human cDNAs are used to design PCR primers which will selectively amplify human genomic sequences in a rodent background. When applied to panels of human x hamster somatic cell hybrid DNAs, this approach provides a PCR-based method for rapidly assigning genes to specific chromosomes and chromosomal regions. In addition, it follows from the virtual absence of introns in the 3'untranslated region of vertebrate genes that within this region the cDNA sequences almost always will be identical to those of the genomic DNA and can therefore be used to automatically generate gene-specific sequence-tagged sites (STSs). We have applied this strategy to six human cDNAs and demonstrate that 1) the primers selectively amplify human genomic DNA and 2) the PCR product is of the size predicted from the cDNA. To test this approach further we have utilized it to confirm the known chromosomal location of the retinoblastoma gene. Lastly, we describe how this strategy can readily be applied to unknown human cDNAs, and thereby be integrated into efforts to generate a human STS expression map of the genome. A strategy for production of such a map, using human brain cDNAs as a model, is described.

Animals↗
PubMed1991

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.

Amino Acid Sequence↗
PubMed1991

Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Amino Acid Sequence↗
PubMed1991

A general method for quantitative PCR analysis of mRNA levels for members of gene families: application to GABAA receptor subunits.

We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits.

Animals↗
PubMed1990

Genetic mapping of the gene for Ca2+/calmodulin-dependent protein kinase IV (Camk-4) to mouse chromosome 18.

Southern blot analysis of Chinese hamster x mouse somatic cell hybrids was used to map the gene for a serine/threonine protein kinase expressed in brain and testis. This locus, termed Camk-4, encodes Ca2+/calmodulin-dependent protein kinase IV. Progeny of an interspecific backcross were analyzed to position Camk-4 in the centromeric region of chromosome 18 near two mutations known to affect neurological function and fertility. This raises the possibility that a defect in Camk-4 may be responsible for one of these mutant phenotypes.

Animals↗
PubMed1989

Sequence and expression of the murine diazepam binding inhibitor.

Previous studies suggest that a diazepam binding inhibitor (DBI, also referred to as endozepine) present in the brain may function anxiogenically as a modulator of the gamma-aminobutyric acid receptor complex (GABAA). An expression library representing mouse brain mRNA was screened using antisera that recognizes the 11 kDa DBI protein. A cDNA clone was isolated and sequenced. Comparison of the amino acid sequence of mouse DBI to that for human, rat and bovine DBI shows that the size of DBI is conserved at 87 amino acids in all of these mammals. DBI cDNA hybridizes to an mRNA of about 600 nucleotides. This mRNA is not restricted to the brain, being prevalent in other organs such as the liver and kidney. Its moderate to high abundance, as judged from mRNA levels, in several organs suggests that DBI might have functions other than, or in addition to, the possible regulation of benzodiazepine binding sites.

Amino Acid Sequence↗