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J J Murtagh

Publications and source records attributed to J J Murtagh.

31 records · Page 2Linked to original sources
PubMed1992

Guanine nucleotide-binding proteins in the intestinal parasite Giardia lamblia. Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor.

Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.

ADP-Ribosylation Factors↗
PubMed1991

Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium.

The predicted protein domains coded by exons 9-12 and 19-23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide-binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9- mRNA transcripts). In most (9 of 12), the exon 9- transcripts represented less than or equal to 25% of the total CFTR transcripts. However, in three individuals, the exon 9- transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re-evaluation of the same individuals 2-4 months later showed the same proportions of exon 9- transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon-intron junctions relevant to exon 9 deletion (exon 8-intron 8, intron 8-exon 9, exon 9-intron 9, and intron 9-exon 10) were identical except for the intron 8-exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9- transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype.

Base Sequence↗
PubMed1991

Different forms of Go alpha mRNA arise by alternative splicing of transcripts from a single gene on human chromosome 16.

Go alpha, (gene symbol GNA01), a member of the signal-transducing guanine nucleotide-binding (G) protein family, has been implicated in ion channel regulation. Some tissues contain multiple Go alpha mRNAs of different sizes that differ in the 3' untranslated regions (UTRs). Using sequence-specific 48-base oligonucleotides, two complementary to the different 3' UTRs and one complementary to the coding region, we investigated the origin of the multiple Go alpha transcripts, the organization of the Go alpha gene, the interspecies conservation of 3' UTRs, and the chromosomal localization of Go alpha. Oligonucleotides labeled to high specific activity by using terminal deoxynucleotidyltransferase each hybridized with a single band of restriction enzyme-digested mouse and human DNAs. In three of four digests of human DNA, the two probes specific for the different 3' UTRs hybridized with the same restriction fragment. Thus, these nucleotide sequences are in close proximity in the human genome. The order of the UTRs in the bovine, human, and mouse genomes was confirmed directly by polymerase chain reaction (PCR) amplification and sequencing. Hybridization of bovine oligonucleotide sequence with mouse and human genomic DNA indicated a high degree of interspecies sequence conservation: conservation was confirmed by PCR amplification and sequencing. Bands detected by both UTR probes, as well as the predominant bands detected by a bovine Go alpha cDNA, segregated with human chromosome 16 on Southern blot analysis of human-mouse somatic cell hybrids. We conclude that Go alpha mRNAs with different 3' UTRs arise by alternative splicing of transcripts from a single gene. The UTRs, which exhibit a high degree of interspecies conservation, may play a role in regulation of Go alpha expression during differentiation or in specific tissues. The use of oligonucleotide probes of the type described here represents a new strategy, potentially widely applicable for mapping and elucidating structural features of genes.

Animals↗
PubMed1990

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions.

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.

Animals↗
PubMed1990

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin.

ADP-ribosylation factors (ARFs) are approximately 20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known ARFs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone psi ARF 4, a putative ARF pseudogene, from a human genomic library in lambda phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A)+ RNA the cDNA corresponding to the expressed homolog of psi ARF 4, referred to as human ARF 4. It appears that psi ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

ADP-Ribosylation Factors↗
PubMed1989

Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin.

ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFs also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.

ADP-Ribosylation Factors↗
PubMed1989

Isolation and immunological properties of adenosine kinase.

Bovine liver adenosine kinase is a 43 kDa protein that catalyzes the transfer of phosphate from GTP or ATP to adenosine. Its immunological properties were compared to other GTP-binding proteins of approximately 40 kDa, in particular those involved in signal transduction, such as Gs and Gi, the stimulatory and inhibitory regulatory proteins of adenylyl cyclase, Gt, from the visual excitation system, and Go, a similar protein of unknown function. Antibodies elicited in rabbits against adenosine kinase did not significantly cross-react with other guanyl nucleotide-binding proteins. Antibodies against the other GTP-binding proteins did not react with adenosine kinase. Thus these GTP-binding proteins do not exhibit immunological cross-reactivity.

Adenosine Kinase↗
PubMed1984

Beneficial effects of nafazatrom on ischemic reperfused myocardium.

The effect of nafazatrom, a new antithrombotic agent, was studied in a canine model of regional myocardial ischemia. Nafazatrom was administered 1 mg/kg intravenously every 6 h for 48 h. After 24 h of drug or placebo administration, animals underwent 90 min of occlusion of the proximal left circumflex coronary artery followed by gradual reperfusion over a period of 30 min. Twenty-four hours later, the animals were sacrificed and infarct size was determined by histochemical staining with triphenyltetrazolium chloride. Nafazatrom-treated animals had a significant reduction in infarct size expressed as a percent of the anatomical area at risk for infarction: 21 +/- 5% in the treated group vs. 41 +/- 5% in the control group (X +/- S.E.M., P less than 0.05). Histological examination confirmed the gross results of postmortem histochemical staining. Salvage of ischemically jeopardized tissue appeared to be unrelated to myocardial oxygen demand as there were no hemodynamic differences between groups. The beneficial effects of nafazatrom are presumably related to a limitation of autolytic processes on the heart during and after ischemia as a result of the drug's ability to inhibit lipoxygenase and to prevent the enzymatic degradation of prostacyclin.

Animals↗
PubMedDate not supplied

Hypertrophic cardiomyopathy: failure to demonstrate mutations in exon 13 of the cardiac beta myosin heavy-chain gene.

Familial hypertrophic cardiomyopathy (FHCM) has been linked to the cardiac beta-myosin heavy-chain (MHC) genes on chromosome 14 (14q1), and a missense mutation within exon 13 of the beta MHC gene has been implicated in the pathogenesis of the disease. To test whether this constitutional mutation occurs somatically in the myocardium of the sporadic form of the disease, we studied seven patients with familial (n = 3) or sporadic (n = 4) hypertrophic cardiomyopathy (HCM). Amplification of exon 13 of the beta MHC from paraffin-embedded myocardium using the polymerase chain reaction (PCR) was performed and analysis of the amplified product for migration abnormalities using denaturing gradient gel electrophoresis (DGGE) and direct sequencing of the PCR product were used. Neither patients with HCM nor subjects with dilated cardiomyopathy (DCM) (n = 2) exhibited an aberration within exon 13 of the myocardial beta MHC. It is concluded that a specific beta MHC gene mutation is displayed only in a subset of patients with familial disease, thus further emphasizing the notion of genetic heterogeneity. In addition, in the sporadic form of the disease, somatically occurring mutations in this particular exon could not be demonstrated.

Base Sequence↗