Biomedical subjects

K Gregg

Publications and source records attributed to K Gregg.

At least 19 recordsLinked to original sources

PubMed2000

Construction of a promoter-rescue plasmid for Butyrivibrio fibrisolvens and its use in characterization of a flagellin promoter.

The Butyrivibrio fibrisolvens/Escherichia coli shuttle vector pBHerm has been modified to produce a plasmid (pBHE) that can be used for the identification and characterization of promoters in B. fibrisolvens. pBHE allows the insertion of a test promoter immediately upstream of a promoterless erythromycin resistance gene (ermAM). The efficacy of the pBHE plasmid in isolating and characterizing promoters was tested by inserting the flagellin gene (flaA) promoter from B. fibrisolvens OR77. Transcription of the ermAM gene from the flaA promoter was significantly higher than that observed when the ermAM gene was under the control of its own promoter. The flagelling gene of OR77 appears to be transcribed from two different promoters that produce transcripts initiating approximately 130 bp apart. Two mutant flaA promoter constructs, containing mutations in the -10 and -35 regions of either of the two putative promoter regions, showed drastic alterations in both the origin and amounts of the two transcripts produced. Mutations in either promoter affected transcription from both promoters, indicating that both regions contribute to gene expression.

DNA, Bacterial↗

PubMed2000

CDP is a repressor of mouse mammary tumor virus expression in the mammary gland.

Mouse mammary tumor virus (MMTV) transcription is highest in the lactating mammary gland but is detectable in a variety of other tissues. Previous results have shown that MMTV expression is suppressed in lymphoid and other tissues through the binding of the homeodomain-containing repressor special AT-rich binding protein 1 to a negative regulatory element (NRE) in the MMTV long terminal repeat (LTR). Another homeoprotein repressor, CCAAT displacement protein (CDP), also binds to the MMTV NRE, but a role for CDP in MMTV transcriptional suppression has not yet been demonstrated. In this paper, we show that the level of CDP decreases during development of the mammary gland and that this decline in CDP level correlates with the known increase in MMTV expression observed during mammary gland differentiation. Moreover, CDP overexpression was able to suppress MMTV LTR-reporter gene activity up to 20-fold in transient-transfection assays of mouse mammary cells. To determine if this effect was due to direct binding of CDP to the promoter-proximal NRE, we performed DNase I protection assays to map two CDP-binding sites from +835 to +845 and +920 to +931 relative to the first base of the LTR. Mutations engineered into each of these sites decreased CDP binding to the proximal NRE, whereas a combination of these mutations further reduced binding. Subsequently, each of these mutations was introduced into the full-length MMTV LTR upstream of the luciferase reporter gene. Analysis of stable transfectants of LTR constructs showed that CDP binding site mutations in the proximal NRE elevated reporter gene expression two- to sixfold compared to wild-type LTR constructs. Thus, MMTV expression increases during mammary gland development, in part due to decreased CDP levels and CDP binding to the LTR. Together, these experiments provide the first evidence that CDP acts as a repressor of MMTV transcription in the mammary gland.

Animals↗

PubMed1998

Genetically modified ruminal bacteria protect sheep from fluoroacetate poisoning.

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10(6) to 10(7) cells ml-1 in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.

Animals↗

PubMed1997

Improvement of expression and secretion of a fungal xylanase in the rumen bacterium Butyrivibrio fibrisolvens OB156 by manipulation of promoter and signal sequences.

Promoters and signal sequences for expression and secretion of a fungal xylanase encoded by a modified Neocallimastix patriciarum xynA cDNA in the rumen bacterium, Butyrivibrio fibrisolvens OB156, were investigated. Successful expression of the fungal xylanase in OB156 was obtained using the putative xylanase promoter from B. fibrisolvens strain 49. Replacing the putative -35 region sequence (TTGCAC) of the xylanase promoter with the sequence TTGACA by mutagenesis reduced the fungal xylanase expression level 4-fold in OB156, indicating that this B. fibrisolvens strain did not efficiently recognise the E. coli consensus -35 sequence. Reduction of the spacer length between the -35 and -10 regions of the xylanase promoter from 18 to 17 base-pairs (bp) considerably increased the expression levels of the fungal enzyme in both E. coli and OB156. Insertion of a pUB110 mob promoter upstream of the xylanase promoter also significantly improved the fungal xylanase expression. Secretion of the fungal xylanase mediated by the alpha-amylase signal peptide from B. fibrisolvens strain H17c was efficient in E. coli, but very poor in OB156. An increase in the hydrophobicity of the signal sequence resulted in a 4-fold increase in the extracellular portion of the fungal xylanase in OB156, indicating marked improvement in xylanase secretion efficiency. The recombinant plasmids and xylanase expression/secretion cassettes were found to be stable in OB156 after prolonged cultivation (100 generations) in the absence of antibiotic selection. These results suggest that the rumen bacterium B. fibrisolvens can be manipulated to produce and secrete a eukaryotic extracellular protein with stable maintenance of the expression cassette in plasmid form.

Amino Acid Sequence↗

PubMed1996

Positional information and whorl morphogenesis in Polysphondylium.

Cellular slime molds of the genus Polysphondylium periodically release cell masses from the base of culminating fruiting bodies. These masses quickly undergo a change in symmetry from spherical to radial as they differentiate into distinctive arrays of secondary fruiting bodies arranged about a primary axis of stalk cells. Here we show that a major event in whorl morphogenesis is the activation of a prestalk-specific promoter early and globally in newly forming whorls. With time, transcript synthesis and amplification become restricted to the equator of the whorl and then to patches which define where secondary tip morphogenesis will occur. The localization of early prestalk message synthesis depends on positional information, in contrast to the establishment of early prestalk/prespore patterns in both Polysphondylium and Dictyostelium.

Animals↗

PubMed1996

Role of serotonergic neurotransmission in the hypnotic response to dexmedetomidine, an alpha 2-adrenoceptor agonist.

The role of serotonergic pathways in the hypnotic response to dexmedetomidine was examined in neurochemical and behavioral studies. Following acute administration of dexmedetomidine, loss of righting reflex and changes in serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine turnover in different brain regions (locus coeruleus and hippocampus) were assessed. In separate experiments, the effect of dexmedetomidine on 5-HT turnover was measured in rats rendered tolerant to the hypnotic effects of dexmedetomidine. These neurochemical data were complemented by a study of dexmedetomidine-induced hypnotic response in the presence of a 5-HT2 receptor agonist and antagonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and ritanserin, respectively. Dexmedetomidine (1-500 micrograms.kg-1) dose dependently reduced 5-HT and norepinephrine turnover in both the locus coeruleus and hippocampus. The decrease in 5-HT turnover more closely correlated with the dose-response curve for loss of righting reflex, a behavioral measure of hypnosis, than did the norepinephrine turnover. In previous studies with chronic administration of dexmedetomidine (3 micrograms.kg-1.h-1 for 7 days), the norepinephrine turnover effect of acute dexmedetomidine (30 micrograms.kg-1) persisted while the hypnotic effect was blunted. Following the same regimen, the drug's ability to diminish 5-HT turnover was also blunted. This biochemical evidence for the role of 5-HT in sleep was supported by the behavioral evidence that dexmedetomidine (100 micrograms.kg-1 i.p. or 7 micrograms.0.2 microliter-1 locus coeruleus)-induced hypnosis was dose dependently blocked by DOI (0.08-0.32 mg.kg-1 i.p.). The selectivity of this effect was demonstrated by the finding that ritanserin (0.16 mg.kg-1 i.p.) pretreatment blocked the effects of DOI (0.16 mg.kg-1 i.p.) on dexmedetomidine (100 micrograms.kg-1 i.p. or 7 micrograms.0.2 microliter-1 locus coeruleus)-induced loss of righting reflex. In conclusion, these findings suggest that the hypnotic effect of the alpha 2-adrenoceptor agonist, dexmedetomidine, is not mediated solely by changes in noradrenergic neurtransmission, but instead is strongly associated with a decrease in serotonergic neurotransmission and correspondingly diminished by stimulation of 5-HT2 receptors.

Adrenergic alpha-Agonists↗

PubMed1995

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156.

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM beta 1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 micrograms erythromycin/ml. Maximum efficiency was 1.1 x 10(5) transformants per micrograms plasmid DNA (average 3 x 10(4)), and restriction mechanisms reduced efficiency by a factor of 2 x 10(2). Nonselective growth for 200 generations gave no measurable loss of plasmid.

Escherichia coli↗

PubMed1995

Engineering gut flora of ruminant livestock to reduce forage toxicity: progress and problems.

The rumen bacterium Butyrivibrio fibrosolvens has been genetically modified to detoxify fluoroacetate (a poisonous component of trees and shrubs in Australia, Africa and Central America) and has been shown to persist when it is returned to the rumen. Such bacteria may save animals from poisoning and, therefore, reduce economic losses for livestock industries in those countries. The ability to make genetic changes to rumen bacteria raises important questions about their practicality, and about the environmental factors that must be considered before releasing modified strains. The fluoroacetate-detoxifying bacterium provides an important model by which these issues can be examined.

Animals↗

PubMed1994

Detoxification of the plant toxin fluoroacetate by a genetically modified rumen bacterium.

We isolated the fluoroacetate dehalogenase gene (H1), from Moraxella species strain B, and placed it under the transcriptional control of a 154 bp fragment of the erm gene promoter. The promoter/gene construct was attached to the Butyrivibrio fibrisolvens shuttle vector pBHerm, and the resulting dehalogenase expression plasmid (pBHf) was transferred to B. fibrisolvens OB156 by electroporation. The erm gene promoter directed expression of dehalogenase activity in both E. coli and B. fibrisolvens OB156. Cell-free lysates of the genetically modified OB156 defluorinated 10.6 nmol fluoroacetate/min/mg protein. Growing cultures of OB156 were able to detoxify fluoroacetate in the culture medium, at the rate of 9.9 nmol/min/mg. Plasmid pBHf was retained by 100% of OB156 cells after 500 generations of non-selective culture. The restriction pattern of pBHf remained unchanged after extensive non-selective growth and host bacteria continued to produce active dehalogenase. The construction of rumen bacteria that are able to detoxify an important natural poison supports the feasibility of using genetically modified rumen bacteria to aid animal production.

Animals↗

PubMed1994

Cloning and DNA sequence analysis of the region containing attP of the temperate phage phi AR29 of Prevotella ruminicola AR29.

Phage phi AR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (attP) was located on a 4.5 kb EcoRV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRV/HindIII fragment was isolated, cloned in Escherichia coli and sequenced. DNA hybridization localized the attP site to the vicinity of an internal DraI site. Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage lambda. Two open reading frames are present adjacent to attP (ORF1 and ORF2). The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to attP. The proposed phi AR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.

Amino Acid Sequence↗

PubMed1992

DNA sequence and transcription of an endoglucanase gene from Prevotella (Bacteroides) ruminicola AR20.

The endoglucanase gene was sequenced from Prevotella ruminicola AR20, isolated as clone pJW4. The endoglucanase (BrEND) is encoded by an open reading frame (ORF1) of 501 codons, corresponding to a protein of calculated molecular weight 55.7 kDa. Analysis of proteins on SDS-PAGE revealed a protein corresponding to the calculated molecular weight of the processed BrEND. The protein showed substantial homology to members of the A4 sub-family cellulases. Primer extension studies revealed that transcription of celA is initiated at different sites in Escherichia coli and Prevotella ruminicola. E. coli sigma 70 recognition sequences were identified, which were located upstream from the transcription initiation site (TIS) functional in E. coli. A longer extension product was identified using RNA from P. ruminicola, indicating that the gene may normally be transcribed as part of a polycistronic message. The end of the primer extension product corresponded to a site beyond the 5' boundary of the cloned fragment, thus preventing identification of native promoter sequences. A second ORF of 110 codons (ORF2) was identified on the antisense strand, and primer extension indicated that transcription through ORF2 was initiated at an identical site in both E. coli and P. ruminicola. E. coli-like consensus sequences were located at positions -10 and -35 upstream from this site, suggesting that some promoter sequences in P. ruminicola are similar to E. coli consensus sequences, although others recognized by E. coli are non-functional in P. ruminicola.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

The structure and expression of a gene encoding chick claw keratin.

A cDNA library was constructed from embryonic chick claw mRNA and a claw keratin (cKer)-encoding clone was isolated and sequenced. Subsequently, a genomic clone, containing four cKer-encoding genes (cKer) was isolated and one of the genes (cKer1) was completely sequenced. The cKerl gene appears to be differentially expressed in the keratinizing tissue appendages of the embryonic chick, being abundantly expressed in the claw and at a low level in feather tissue. Comparison of the deduced amino acid (aa) sequence of the cKer to those of feather (fKer) and scale keratins (sKer) showed that the regions conserved between fKer and sKer are also found in the cKer. The glycine-rich as repeat region characteristic of sKer is also present in a shortened form in the cKer sequence. Like the fKer genes (fKer) and the feather histidine-rich protein-encoding gene (HRP), the cKer1 gene also contains one intron which interrupts the 5'-noncoding region at an equivalent position to that found in the fKer and HRP genes. Genomic Southern analysis using the cKer cDNA as a probe indicated the presence of several related genes in the chick genome.

Amino Acid Sequence↗

PubMed1990

Mode of action and substrate specificity of a purified exo-1,4-beta-D-glucosidase cloned from the cellulolytic bacterium Ruminococcus albus AR67.

A gene encoding exo-1,4-beta-D-glucosidase, from Ruminococcus albus AR67, was cloned in Escherichia coli, restriction mapped, and shown to be expressed from sequences within the insert that function as a promoter in E. coli. The cloned enzyme was located predominantly in the cytoplasm (40%) and attached to insoluble cell components (48%). After purification to homogeneity, the enzyme (Mr = 64,000, monomeric) was specific for substrates with beta-D-glucopyranosyl configuration and was inactive against alpha-glucosides, lactosides and xylosides. Km values of the enzyme decreased with increasing chain length (G2-G5). Glucose was the major product of hydrolysis from cellodextrins. Preference for longer chain cellodextrins is consistent with exo-1,4-beta-D-glucan glucohydrolase mode of action [E.C. 3.2.1.74].

Chromatography, Gel↗

PubMed1989

Avian keratin genes. I. A molecular analysis of the structure and expression of a group of feather keratin genes.

The nucleotide sequence of the four complete chicken feather keratin genes A to D contained in the previously isolated recombinant lambda CFK1 has been determined. All four genes have a very similar structure; each gene encodes a polypeptide of 97 amino acid residues and contains an intron in the 5' non-coding region, 37 base-pairs from the cap site. Comparison of the previously determined feather keratin gene C sequence to genes A, B and D indicates that a high level of gene correction has occurred in the protein coding and 5' non-coding regions, which show more than 90% homology, whereas the intron and 3' non-coding regions are by contrast poorly conserved with one or two exceptions. The dramatic conservation of the 5' non-coding region between the feather keratin sequences and an unrelated but co-expressed gene encoding a histidine-rich protein suggests that this segment may play an important role in transcriptional regulation. In addition, both gene types contain an identically positioned intron in the 5' non-coding region. Northern blots performed using gene-specific probes show that the four characterized genes A to D plus gene E, which is partially contained in the recombinant lambda CFK1, are all expressed in feather tissue from 14-day old chick embryos. In addition, we report that a scale keratin gene (originally isolated from a scale complementary DNA library) is expressed at a low level in the embryonic feather.

Animals↗

PubMed1989

The isolation and comparison of cellulase genes from two strains of Ruminococcus albus.

Endo-1,4-beta-glucanase genes have been cloned from two strains of Ruminococcus albus recently isolated in this laboratory. Although the strains were phenotypically similar, cross-hybridization studies between them showed significant genetic differences, with only 20% of the genome forming DNA heteroduplexes. Heteroduplexes displayed an average dissociation temperature 9 degrees C lower than that of the homoduplex. Consistent with this, restriction maps of the two endoglucanase genes showed no similarity, and hybridization work using the endoglucanase genes as probes revealed that neither gene was present in the genome of the other isolate of R. albus. Comparative enzyme characterization showed differences between the enzymes in their response to temperature, pH and substrate preference.

Animals↗

PubMed1989

Isolation of an endoglucanase gene from Bacteroides ruminicola subsp. brevis.

A gene coding for endo-1, 4-beta-glucanase activity has been isolated from Bacteroides ruminicola subsp. brevis by cloning in Escherichia coli. After restriction mapping of a 6.4 kb insert, a 2.2 kb DNA fragment was sub-cloned in pUC19 to produce the enzymically active clone pJW3. Recloning of the gene fragment in the reverse orientation in pUC18 (clone pJW4) indicated that a gene promoter was present in the cloned fragment and was able to function in E. coli. The clone pJW4 displayed increased activity which was attributed to expression from the lac promoter of pUC18. The enzyme encoded by pJW4 was optimally active at pH 5.5-6.0, and in the temperature range 37-42 degrees C. The preferred substrate was carboxymethylcellulose, but the enzyme displayed 50-60% of maximal activity on both acid-swollen cellulose and soluble xylan. No significant activity was detected on ball-milled filter paper or particulate xylan. Deletion experiments confirmed that both cellulase and xylanase activities were altered to a similar extent by deletion of DNA from the 3' end of the gene, suggesting that both are a function of the same polypeptide product.

Bacteroides↗

PubMed1987

Promoter efficiency depends upon intragenic sequences.

Experiments concerning gene transcription in Xenopus oocytes have revealed that the efficiency of the HSV-TK promoter is dependent upon the nature of the attached gene sequences. Transcriptional efficiency of the TK promoter when attached to its own gene was 30-fold higher than that observed when the promoter was attached either to an avian keratin gene or to the chicken histone H2B gene. Furthermore, attachment of the keratin gene promoter to the TK gene resulted in a 20-fold increase in keratin promoter efficiency. It was found by subsequent experiments that the TK gene is able to exert a stimulatory influence upon attached promoter sequences. This cis effect was shown to be independent of orientation but dependent upon the distance between the DNA sequences involved.

Animals↗

PubMed1986

Intron sequences modulate feather keratin gene transcription in Xenopus oocytes.

We have shown that chicken feather keratin genes are transcribed at extremely low levels in Xenopus oocytes. Primer extension reactions using carefully selected primers have shown that none of the detectable transcripts are spliced. When the single intron, located in the 5' untranslated region, was removed from the gene, we observed a 5-fold increase in transcript initiation from the in vivo "cap" site. Significantly, this 5-fold increase was also observed when the feather keratin intron was replaced with a similar sized fragment from pBR322. We conclude that the efficiency of accurate transcriptional initiation of chicken feather keratin genes in the oocyte is affected by DNA sequences within the intron.

Animals↗