The TIGR human cDNA database.
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Amino Acid Sequence↗
Biomedical subjects
E F Kirkness
Publications and source records attributed to E F Kirkness.
Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.
The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.
Alleles↗
Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.
The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.
Amino Acid Sequence↗
Assessment of human gene diversity and expression patterns using expressed sequence tags.
ESTs provide a cheap and effective short-cut for identifying the majority of an organism's genes. Surveys of ESTs can provide a global view of the gene-expression patterns that distinguish different tissues or cell types. The large collection of human ESTs will soon permit the majority of genes to be placed on a chromosomal map.
Amino Acid Sequence↗
Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.
In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.
Adult↗
Comparative expressed-sequence-tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment.
Nerve growth factor-induced differentiation of adrenal chromaffin PC-12 cells to a neuronal phenotype involves alterations in gene expression and represents a model system to study neuronal differentiation. We have used the expressed-sequence-tag approach to identify approximately 600 differentially expressed mRNAs in untreated and nerve growth factor-treated PC-12 cells that encode proteins with diverse structural and biochemical functions. Many of these mRNAs encode proteins belonging to cellular pathways not previously known to be regulated by nerve growth factor. Comparative expressed-sequence-tag analysis provides a basis for surveying global changes in gene-expression patterns in response to biological signals at an unprecedented scale, is a powerful tool for identifying potential interactions between different cellular pathways, and allows the gene-expression profiles of individual genes belonging to a particular pathway to be followed.
Animals↗
Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.
An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.
Bacterial Proteins↗
cDNA sequencing: a means of understanding cellular physiology.
High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.
Animals↗
Site-directed mutagenesis of N-linked glycosylation sites on the gamma-aminobutyric acid type A receptor alpha 1 subunit.
Oligonucleotide-directed mutagenesis was used to mutate the two potential sites for N-linked glycosylation on the rat gamma-aminobutyric acid (GABA)A receptor alpha 1 subunit. Wild-type (WT) or mutant alpha 1 subunits [asparagine to glutamine substitutions at position 10 (alpha 1Q10), 110 (alpha 1Q110), or both 10 and 110 (alpha 1Q10/110)] were coexpressed with beta 1 and gamma 2 subunits in Xenopus oocytes. Removal of either one or both potential sites for N-linked glycosylation resulted in expression, in Xenopus oocytes, of functional GABAA receptors with pharmacological properties similar to those observed for the WT receptor. WT and mutant alpha 1 subunits were co-transfected with beta 1 and gamma 2 subunits in human embryonic kidney 293 cells. WT and mutant alpha 1 subunits expressed in 293 cells were photoaffinity labeled with [3H]flunitrazepam. Co-transfection of alpha 1WT, alpha 1Q10, or alpha 1Q110 subunits in combination with beta 1 and gamma 2 GABAA receptor subunits resulted in the labeling of single bands, with approximate molecular masses of 54, 49, and 50 kDa, respectively. The decrease in molecular mass for both the alpha 1Q10 and alpha 1Q110 mutants suggests that both consensus sequences for N-linked glycosylation are used in 293 cells. Low levels of [3H]flunitrazepam binding prevented visualization of the alpha 1Q10/110 double mutant. The 293 cells transfected with either the alpha 1Q10 or alpha 1Q110 mutant in combination with beta 1 and gamma 2 subunits expressed significantly lower levels of [3H]Ro15-1788 binding, relative to WT levels. In addition, [3H]Ro15-1788 binding was undetectable in 293 cells expressing the alpha 1Q10/110 double mutant. When transfected 293 cells were grown at 30 zero, [3H]Ro15-1788 binding to alpha 1Q10 and alpha 1Q110 GABAA receptors was restored to levels comparable to that for WT receptors. [3H]Ro15-1788 binding to alpha 1Q10/110 was not reliably detected at 30 zero. Similar results were observed using [3H]muscimol. These data suggest that intracellular processing and transport of the glycosylation-deficient GABAA receptor alpha 1 subunit is temperature sensitive. Furthermore, the observed differences between the two expression systems may be accounted for by the typically lower temperature used for maintaining microinjected Xenopus oocytes. Thus, although glycosylation is not an absolute requirement for GABAA receptor expression, it has a profound effect on the processing of at least the alpha 1 receptor and its subsequent assembly into a mature receptor.
Affinity Labels↗
A strong promoter element is located between alternative exons of a gene encoding the human gamma-aminobutyric acid-type A receptor beta 3 subunit (GABRB3).
The gene that encodes the beta 3 subunit of the gamma-aminobutyrate-Type A (GABAA) receptor is widely expressed in brain tissue and has been associated with imprinted genetic disorders. Here, the 5' regions of the human and rat genes were characterized and found to be highly conserved in both coding and non-coding sequences. A novel transcript of the human gene revealed the existence of an alternative exon 1 (exon 1a) that encodes a variant signal sequence. Relative levels of the alternative transcripts were found to vary between fetal and adult brain, and between different brain regions. Endogenous beta 3 subunit transcripts were also detected in several immortalized cell lines, including human kidney 293 cells. Transcription of exon 1 is initiated from multiple sites within a pyrimidine-rich region of the gene. This region of the human gene also exhibits strong promoter activity and binds nuclear factors at a site which overlaps the transcriptional start sites. The promoter element was shown to bind Sp1 and at least one other unidentified nuclear factor.
Amino Acid Sequence↗
Isolation, characterization, and localization of human genomic DNA encoding the beta 1 subunit of the GABAA receptor (GABRB1).
Genomic DNA that encodes the beta 1 subunit of the human gamma-aminobutyric acidA (GABAA) receptor was cloned and mapped. Exons and flanking introns (greater than 14 kb) were sequenced to determine the structural organization of the gene. The gene was localized on human chromosome 4, in bands p12-13. The beta 1 subunit is encoded by a relatively large gene (greater than 65 kb) on nine exons. In contrast to other conserved regions of the subunit polypeptide, the proposed channel-forming domain (M2) is derived from more than one exon. The organization of exons was compared with that of the genes that code for subunits of nicotinic acetylcholine receptors. There is no evidence for conservation of gene structure between these two members of the proposed gene superfamily. However, intron-exon junctions were found to be conserved precisely between subtypes of GABAA receptor subunits.
Amino Acid Sequence↗
Probing the GABAA receptor with antibodies.
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Amino Acid Sequence↗
Genetic mapping of the beta 1 GABA receptor gene to human chromosome 4, using a tetranucleotide repeat polymorphism.
As more coding loci for functional human genes are described, there is a growing need to identify DNA polymorphisms in specific genes. By examining DNA sequences within the introns of the beta 1 subunit of the gamma-aminobutyric acid receptor gene, GABARB1, we found a tetranucleotide repeat sequence (GATA). Amplification of this region by using PCR revealed seven alleles and a high degree of polymorphism (PIC = .75) in human populations. DNAs from the CEPH families were typed for the GABARB1 intron polymorphism and were analyzed with respect to 20 linked markers on chromosome 4. The results permit placement of GABARB1 on the linkage map of chromosome 4, between D4S104 and ALB. These results affirm that sequence analysis of noncoding segments included within or adjacent to functional genes has value as a strategy to detect highly informative polymorphisms.
Alleles↗
Localization of the gene encoding the GABAA receptor beta 3 subunit to the Angelman/Prader-Willi region of human chromosome 15.
Deletions of the proximal long arm of chromosome 15 (bands 15q11q13) are found in the majority of patients with two distinct genetic disorders, Angelman syndrome (AS) and Prader-Willi syndrome (PWS). The deleted regions in the two syndromes, defined cytogenetically and by using cloned DNA probes, are similar. However, deletions in AS occur on the maternally inherited chromosome 15, and deletions in PWS occur on the paternally derived chromosome 15. This observation has led to the suggestion that one or more genes in this region show differential expression dependent on parental origin (genetic imprinting). No genes of known function have previously been mapped to this region. We show here that the gene encoding the GABAA (gamma-aminobutyric acid) receptor beta 3 subunit maps to the AS/PWS region. Deletion of this gene (GABRB3) was found in AS and PWS patients with interstitial cytogenetic deletions. Evidence of beta 3 gene deletion was also found in an AS patient with an unbalanced 13;15 translocation but not in a PWS patient with an unbalanced 9;15 translocation. The localization of this receptor gene to the AS/PWS region suggests a possible role of the inhibitory neurotransmitter GABA in the pathogenesis of one or both of these syndromes.
Amino Acid Sequence↗
Phosphorylation of gamma-aminobutyrate (GABA)/benzodiazepine receptors by cyclic AMP-dependent protein kinase.
Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.
Animals↗
Steroid modulation reveals further complexity of GABAA receptors.
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Humans↗
Antibodies directed against a nonapeptide sequence of the gamma-aminobutyrate (GABA)/benzodiazepine receptor alpha-subunit. Detection of a distinct alpha-like subunit in pig cerebral cortex but not cerebellum.
A synthetic peptide, corresponding to amino acid residues 101-109 of the bovine gamma-aminobutyrate/benzodiazepine receptor alpha-subunit, was used to raise a polyclonal antiserum. The reactivity of this antiserum towards polypeptides of both bovine and pig receptor preparations was established by immunoprecipitation and immunoblotting. Anti-peptide antibodies recognized the alpha-subunit (51 kDa) of receptor prepared from pig cerebellum or cerebral cortex. However, a polypeptide of 57 kDa was additionally recognized in cortical, but not cerebellar, preparations. This alpha-like polypeptide appeared larger than the band of polypeptides labelled irreversibly with [3H]muscimol (beta-subunit, 55-57 kDa) and corresponds to a polypeptide detected only in cortex after silver-staining or irreversible labelling with [3H]flunitrazepam. These results support the idea that the distinct regional patterns of polypeptides labelled irreversibly with [3H]flunitrazepam reflect the existence of heterologous distributions of distinct alpha-like subunits.
Affinity Labels↗