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Biomedical subjects

E F Kirkness

Publications and source records attributed to E F Kirkness.

At least 19 recordsLinked to original sources
PubMed1997

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

Archaeoglobus fulgidus↗
PubMed1997

The complete genome sequence of the gastric pathogen Helicobacter pylori.

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

Antigenic Variation↗
PubMed1997

Identification of a gene within the tandem array of red and green color pigment genes.

The tandem array of color pigment genes on chromosome Xq28 contains nested exons of a distinct gene. This gene (termed TEX28) is composed of five exons that span almost the entire distance between the protein-coding regions of the color pigment genes and a transketolase-related gene. Although most of the TEX28 gene is repeated within the color pigment gene array, the exclusion of exon 1 from the array is predicted to restrict transcription to a single copy of the gene. The TEX28 gene encodes a polypeptide of 410 amino acid residues. This polypeptide does not display significant homology with any known proteins in public databases. Transcripts of the gene (1.8 kb) were detected in testes, but not in any other tissue examined. Color vision disorders that result from the deletion of color pigment genes should be reappraised for associated phenotypes that may derive from disruption of the TEX28 gene.

Amino Acid Sequence↗
PubMed1997

A novel class of GABAA receptor subunit in tissues of the reproductive system.

A novel subunit of the gamma-aminobutyrate, type A (GABAA) receptor family has been identified in human and rat tissues. The subunit displays 30-40% amino acid identity with known family members and represents a distinct subunit class (termed pi). Transcripts of the pi subunit were detected in several human tissues and were particularly abundant in the uterus. The pi subunit protein can assemble with known GABAA receptor subunits and confer unique ligand binding properties to the recombinant receptors in which it combines. Most notably, the presence of the pi subunit alters the sensitivity of recombinant receptors to the endogenous steroid, pregnanolone. Identification of the pi subunit indicates a new target for pharmacological manipulation of GABAA receptors that are located outside of the central nervous system.

Amino Acid Sequence↗
PubMed1997

Human pyridoxal kinase. cDNA cloning, expression, and modulation by ligands of the benzodiazepine receptor.

Peptide fragments of a porcine benzodiazepine-binding protein were used to isolate the cDNA of a related human protein. The cDNA encodes a polypeptide of 312 amino acid residues that is homologous to a bacterial pyridoxal kinase. Transient expression of the cDNA in human embryonic kidney cells confirmed that it encodes human pyridoxal kinase. The recombinant enzyme displayed a Km value of 3.3 microM for pyridoxal and was inhibited competitively by 4-deoxypyridoxine (Ki = 2.8 microM). Benzodiazepine receptor ligands that bound to the purified porcine protein also exerted a potent inhibitory effect on human pyridoxal kinase activity. Transcripts of the pyridoxal kinase gene were detectable in all human tissues examined, and were particularly abundant in the testes. The gene is localized on chromosome 21q22.3 and represents a candidate gene for at least one genetic disorder that has been mapped to this region (autoimmune polyglandular disease type 1).

Amino Acid Sequence↗
PubMed1997

Insensitivity to anaesthetic agents conferred by a class of GABA(A) receptor subunit.

A common feature of general anaesthetic agents is their ability to potentiate neuronal inhibition through GABA(A) (gamma-aminobutyric acid) receptors. At concentrations relevant to clinical anaesthesia, these agents cause a dramatic stimulation of the chloride currents that are evoked by the binding of the natural ligand, GABA. Although there is widespread evidence that the sensitivity of GABA(A) receptors to anaesthetic agents is heterogeneous, the structural basis of these differences is largely unknown. Variations in subunit composition can have profound effects on the sensitivity of GABA(A) receptors to modulatory agents such as benzodiazepines. However, strict subunit specificity has not been demonstrated for the potentiating effects of anaesthetic agents. Here we describe a new class of human GABA(A) receptor subunit (epsilon) that can assemble with alpha- and beta-subunits and confer an insensitivity to the potentiating effects of intravenous anaesthetic agents. The epsilon-subunit also abolishes the normal outward rectification of recombinant receptors in which it assembles. The expression pattern of this subunit in the brain suggests a new target for manipulation of neuronal pathways within the basal ganglia.

Amino Acid Sequence↗
PubMed1997

Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells.

1. Radioligand binding and patch-clamp techniques were used to study the actions of gamma-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5 alpha-pregnan-3 alpha-ol-20-one on rat alpha 1 and beta 3 GABAA receptor subunits, expressed either alone or in combination. 2. Membranes from HEK293 cells after transfection with alpha 1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (< 0.03 pmol mg-1 protein). GABA (100 microM) applied to whole-cells transfected with alpha 1 cDNA and clamped at -60 mV, also failed to activate discernible currents. 3. The membranes of cells expressing beta 3 cDNAs bound [35S]-TBPS (approximately 1 pmol mg-1 protein). However, the binding was not influenced by GABA (10 nM-100 microM). Neither GABA (100 microM) nor picrotoxin (10 microM) affected currents recorded from cells expressing beta 3 cDNA, suggesting that beta 3 subunits do not form functional GABAA receptors or spontaneously active ion channels. 4. GABA (10 nM-100 microM) modulated [35S]-TBPS binding to the membranes of cells transfected with both alpha 1 and beta 3 cDNAs. GABA (0.1 microM-1 mM) also dose-dependently activated inward currents with an EC50 of 9 microM recorded from cells transfected with alpha 1 and beta 3 cDNAs, clamped at -60 mV. 5. Propofol (10 nM-100 microM), pentobarbitone (10 nM-100 microM) and 5 alpha-pregnan-3 alpha-ol-20-one (1 nM-30 microM) modulated [35S]-TBPS binding to the membranes of cells expressing either alpha 1 beta 3 or beta 3 receptors. Propofol (100 microM), pentobarbitone (1 mM) and 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) also activated currents recorded from cells expressing alpha 1 beta 3 receptors. 6. Propofol (1 microM-1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing beta 3 homomers. In contrast, application of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) failed to activate detectable currents. 7. Propofol (100 microM)-activated currents recorded from cells expressing either alpha 1 beta 3 or beta 3 receptors reversed at the Cl- equilibrium potential and were inhibited to 34 +/- 13% and 39 +/- 10% of control, respectively, by picrotoxin (10 microM). 5 alpha-Pregnan-3 alpha-ol-20-one (100 nM) enhanced propofol (100 microM)-evoked currents mediated by alpha 1 beta 3 receptors to 1101 +/- 299% of control. In contrast, even at high concentration 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) caused only a modest facilitation (to 128 +/- 12% of control) of propofol (100 microM)-evoked currents mediated by beta 3 homomers. 8. Propofol (3-100 microM) activated alpha 1 beta 3 and beta 3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 microM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a "surge' current on cessation of propofol (300 microM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) to cells expressing alpha 1 beta 3 receptors. 9. These observations demonstrate that rat beta 3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABAA receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

Anesthetics, General↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1993

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↗
PubMed1991

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↗