Search PubMed⌕ Search

Biomedical subjects

M K Trower

Publications and source records attributed to M K Trower.

At least 19 recordsLinked to original sources
PubMed1999

Identification of two novel diurnal genes by screening of a rat brain cDNA library.

While the hypothalamus is fundamental for sleep and circadian regulation, the molecular mechanism involved are poorly understood. We have used a differential gene expression technique to identify hypothalamic genes which have altered expression in rat sleep periods. Complex cDNA probes from rat hypothalami removed at Zeitgeber times 4 and 15 were hybridised to rat brain cDNA library girds. From 30 differentially expressed clones, six were further analysed and two were confirmed to exhibit increased expression at Zeitgeber time 4. A Northern blot hybridization of brain, heart, kidney, lung, testis and skin mRNA showed that both clones were brain specific. Therefore, we have identified two novel brain specific diurnally expressed hypothalamic genes. Both genes may have roles in sleep or circadian regulation.

Animals↗
PubMed1999

CCR7 (EBI1) receptor down-regulation in asthma: differential gene expression in human CD4+ T lymphocytes.

Asthma is an inflammatory disorder, and the CD4+ T lymphocyte plays a key role in mediating the inflammatory response. We used a high-density grid, hybridization-based, differential gene expression technology to analyse molecular mechanisms underlying in vivo CD4+ T-cell activation in both steroid-resistant asthma (SRA) and steroid-sensitive asthma (SSA). Hybridization of radioactively-labelled first-strand cDNAs prepared from different biological samples, to identical high-density gridded arrays of PCR amplicons derived from cDNA clone inserts immobilized on nylon membranes, was compared by phosphorimaging. Hybridization data were captured and processed using image analysis software that can identify the location and signal intensity of each hybridized cDNA. This produces a hierarchy of signals of differing intensities between the two grids, representing differential gene expression in the two different RNA samples. CCR7 (EBI1), a lymphocyte-specific G-protein-coupled receptor, was down-regulated in the CD4+ T cells of SRA and SSA non-atopic, compared to non-asthmatic non-atopic individuals. This observation is intriguing given that CCR7 and its ligand EBI1-Ligand Chemokine (ELC), may play a role in the migration and homing of normal lymphocytes. Also, TNFR2 is up-regulated in both SSA non-atopic and SRA atopic as compared to non-asthmatic controls. LAMR1 is down-regulated in CD4+ T cells of SRA compared to non-asthmatic individuals, irrespective of their atopic status. These could be general phenomena resulting from cytokine release.

Adult↗
PubMed1998

Identification of two novel diurnal genes by screening of a rat brain cDNA library.

While the hypothalamus is fundamental for sleep and circadian regulation, the molecular mechanisms involved are poorly understood. We have used a differential gene expression technique to identify hypothalamic genes which have altered expression in rat sleep periods. Complex cDNA probes from rat hypothalami removed at Zeitgeber times 4 and 15 were hybridised to rat brain cDNA library girds. From 30 differentially expressed clones, six were further analysed and two were confirmed to exhibit increased expression at Zeitgeber time 4. A Northern blot hybridization of brain, heart, kidney, lung, testis and skin mRNA showed that both clones were brain specific. Therefore, we have identified two novel brain specific diurnally expressed hypothalamic genes. Both genes may have roles in sleep or circadian regulation.

Animals↗
PubMed1996

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.

Acyltransferases↗
PubMed1993

PCR cloning, sequence analysis and expression of the cybC genes encoding soluble cytochrome b-562 from Escherichia coli B strain OP7 and K strain NM522.

The cybC genes encoding cytochrome b-562 from B and K strains of Escherichia coli were shown to differ in size following their isolation by in vitro amplification using the polymerase chain reaction. Nucleotide sequencing of the genes and flanking regions revealed the discrepancy was due to a 26 bp deletion at the 5' end of the K strain sequence that included the initiation codon. Expression studies confirmed that the K strain gene was untranslated. These data indicate that the cybC gene product is non-essential in E. coli.

Amino Acid Sequence↗
PubMed1992

Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy (soyC) and ferredoxinsoy (soyB) from Streptomyces griseus.

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P-450, designated cytochrome P-450soy. A DNA segment carrying the structural gene encoding P-450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P-450soy was compared with that for P-450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe-4S] ferredoxin (soyB), named ferredoxinsoy, was identified.

Amino Acid Sequence↗
PubMed1992

A rapid PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis.

A simple and rapid method for identifying DNA sequences altered by site-directed mutagenesis is described. The procedure requires that a restriction endonuclease recognition sequence is either created or removed from the target DNA sequence by the site-directed mutagenesis reaction. In the screening method presented, transformant colonies or M13 plaques are directly subjected to PCR using universal primers that flank the region containing the site-directed changes. The double stranded DNA products generated are then digested, with no purification, by the restriction enzyme whose site is modified, allowing mutant clones to be differentiated from those carrying wild type sequences. The protocol also provides for recovery of the bacterial cultures harbouring the mutated plasmid or M13 for further characterisation. All the procedures are carried out in small volumes in microtitre plates, thereby lowering costs and enabling the investigation of large numbers of clones. The technique allows a considerable amount of effort to be saved compared to conventional screening practices by circumventing time consuming DNA template preparations.

Base Sequence↗
PubMed1990

Lack of regio- and stereospecificity in oxidation of (+) camphor by Streptomyces griseus enriched in cytochrome P-450soy.

Oxidation of (+) camphor by cytochrome P-450soy-enriched intact cells of Streptomyces griseus resulted in the formation of one major and several minor metabolites. The minor metabolites were identified as 3-endo-hydroxycamphor (2%), 5-endo-hydroxycamphor (7%), 5-exo-hydroxycamphor (9%), 2,5-diketobornane (2%), and camphorquinone (3%). The major metabolite was isolated and conclusively identified as 6-endo-hydroxycamphor (60%). When supplemented with NADPH, spinach ferredoxin:NADP oxidoreductase and spinach ferredoxin, homogeneous preparations of cytochrome P-450soy oxidized camphor to a mixture of 3-endo-, 5-endo-, 5-exo-and 6-endo-hydroxycamphor. The data presented indicates that cytochrome P-450soy resembles its mammalian counterparts in its lack of regio- and stereospecificity in camphor oxidation.

Camphor↗