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1.
Neuroreport ; 10(5): 1155-61, 1999 Apr 06.
Article in English | MEDLINE | ID: mdl-10321501

ABSTRACT

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.


Subject(s)
Circadian Rhythm/physiology , Hypothalamus/physiology , Animals , Biological Clocks , Blotting, Northern , DNA, Complementary/genetics , Gene Expression/physiology , Gene Library , Genetic Testing , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Sleep/physiology
2.
QJM ; 92(8): 463-71, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10627863

ABSTRACT

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.


Subject(s)
Asthma/metabolism , CD4-Positive T-Lymphocytes/metabolism , Chemokines, CC/genetics , Gene Expression Regulation , Integrin beta Chains , Adult , Asthma/drug therapy , Asthma/genetics , Chemokine CCL19 , Chemokines, CC/metabolism , Genes, fos , Genes, jun , Glucocorticoids/therapeutic use , HLA-DR Antigens/genetics , Humans , Immunoblotting , In Situ Hybridization , Integrins/genetics , Middle Aged , RNA, Messenger/analysis , Receptors, Laminin/genetics , Receptors, Tumor Necrosis Factor/genetics , Reverse Transcriptase Polymerase Chain Reaction
3.
Neuroreport ; 9(17): 3935-41, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9875732

ABSTRACT

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.


Subject(s)
Circadian Rhythm/physiology , DNA, Complementary/genetics , Gene Expression Regulation/physiology , Gene Library , Genetic Testing , Hypothalamus/metabolism , Animals , Blotting, Northern , Cloning, Molecular , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley
5.
Proc Natl Acad Sci U S A ; 93(4): 1366-9, 1996 Feb 20.
Article in English | MEDLINE | ID: mdl-8643637

ABSTRACT

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.


Subject(s)
Alzheimer Disease/genetics , Chromosome Mapping/methods , Chromosomes, Human, Pair 14/genetics , Fishes, Poisonous/genetics , Genes, fos , Genome , Acyltransferases/genetics , Amino Acid Sequence , Animals , Base Sequence , Evolution, Molecular , Genes , Genetic Linkage , Humans , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
13.
Biochim Biophys Acta ; 1143(1): 109-11, 1993 Jun 10.
Article in English | MEDLINE | ID: mdl-8499452

ABSTRACT

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.


Subject(s)
Cytochrome b Group/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genes, Bacterial , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/enzymology , Gene Expression , Molecular Sequence Data , Polymerase Chain Reaction
15.
Mol Microbiol ; 6(15): 2125-34, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1406253

ABSTRACT

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.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Ferredoxins/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Glycine max/genetics , Streptomyces griseus/genetics , Amino Acid Sequence , Base Sequence , Biodegradation, Environmental , DNA, Bacterial/isolation & purification , Ferredoxins/biosynthesis , Genetic Vectors , Molecular Sequence Data , Oligonucleotide Probes , Peptide Fragments/isolation & purification , Plant Proteins, Dietary/genetics , Sequence Homology , Glycine max/enzymology , Streptomyces griseus/enzymology
16.
DNA Seq ; 3(4): 233-5, 1992.
Article in English | MEDLINE | ID: mdl-1338368

ABSTRACT

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.


Subject(s)
DNA Mutational Analysis/methods , Mutagenesis, Site-Directed , Polymerase Chain Reaction/methods , Base Sequence , DNA Transposable Elements , DNA, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Evaluation Studies as Topic , Genes, Bacterial , Kanamycin Kinase , Molecular Sequence Data , Phosphotransferases/genetics
18.
Biochem Biophys Res Commun ; 170(2): 456-61, 1990 Jul 31.
Article in English | MEDLINE | ID: mdl-2116789

ABSTRACT

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.


Subject(s)
Camphor/metabolism , Streptomyces griseus/metabolism , Cytochrome P-450 Enzyme System/metabolism , Flour , Oxidation-Reduction , Oxidoreductases/metabolism , Glycine max/enzymology , Stereoisomerism , Streptomyces griseus/enzymology , Streptomyces griseus/growth & development
19.
Biochim Biophys Acta ; 1037(3): 281-9, 1990 Mar 01.
Article in English | MEDLINE | ID: mdl-2155656

ABSTRACT

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14,000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6-7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g = 2.01 which is typical of [3Fe-4S]1+ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g = 1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]1+ clusters at much higher yields (0.4-0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. griseus ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.


Subject(s)
Ferredoxins/isolation & purification , Iron/analysis , Streptomyces griseus/analysis , Chemical Phenomena , Chemistry, Physical , Chromatography, Gel , Cytochrome P-450 Enzyme System/metabolism , Dithionite , Electron Spin Resonance Spectroscopy , Electron Transport , Electrophoresis, Polyacrylamide Gel , Ferredoxins/metabolism , Molecular Weight , Oxidation-Reduction , Photochemistry , Sulfur/analysis
20.
Biochim Biophys Acta ; 1037(3): 290-6, 1990 Mar 01.
Article in English | MEDLINE | ID: mdl-2106913

ABSTRACT

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12,291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the Fe-S clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two Fe-S clusters are conserved.


Subject(s)
Ferredoxins/analysis , Iron/analysis , Streptomyces griseus/analysis , Amino Acid Sequence , Amino Acids/analysis , Cytochrome P-450 Enzyme System/metabolism , Ferredoxins/metabolism , Molecular Sequence Data , Molecular Weight , Mycobacterium/analysis , Peptide Fragments , Sequence Homology, Nucleic Acid , Serine Endopeptidases , Sulfur/analysis , Trypsin
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