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S J Powell

Publications and source records attributed to S J Powell.

At least 19 recordsLinked to original sources
PubMed1993

Human bradykinin B2 receptor: nucleotide sequence analysis and assignment to chromosome 14.

Functional cDNA clones for human bradykinin B2 receptor were isolated from uterus RNA by a polymerase chain reaction (PCR)-based method and by screening a human cosmid library with rat bradykinin B2 receptor probe. We isolated several overlapping clones from the cosmid library, each of which encodes the entire protein coding sequence. The human bradykinin B2 receptor gene codes for a 364-amino-acid protein with a molecular mass of 41,442 Da that is highly homologous to rat bradykinin B2 receptor cDNA (81%). The entire human cDNA sequence was cloned into an expression vector and mRNA was synthesised by in vitro transcription. Applications of bradykinin caused membrane current responses in Xenopus oocytes injected with the in vitro-synthesized mRNA. Preincubation with the potent B2 antagonist, HOE140, prevented this response. The genomic clone is intronless, and we have identified an upstream promoter region and a downstream polyadenylation signal. The human bradykinin B2 receptor gene has been mapped to chromosome 14 using PCR to specifically amplify DNA from somatic cell hybrids.

Amino Acid Sequence↗
PubMed1991

Isolation and characterisation of the human lung NK-1 receptor cDNA.

Functional cDNA clones for human NK-1 receptor were isolated from human lung RNA using the polymerase chain reaction (PCR). We have screened a human cosmid library and isolated a clone which appeared to contain the entire NK-1 receptor gene. From the published rat NK-1 receptor cDNA sequence we designed primers within the protein coding sequence, but outwards towards both the 5' and 3' ends of the putative human protein sequence. By this method we derived DNA sequence from the 3' end of the human gene. In order to determine the 5' end of the gene we used a PCR based method called Rapid Amplification of cDNA Ends (RACE). From the derived human sequences amplimers were designed upstream of the ATG initiation codon and downstream of the stop codon. The entire cDNA was obtained by RNA-PCR from human lung RNA. The sequence obtained was 407 amino acids in length, encoding an open-reading frame that was highly homologous to the rat NK-1 receptor cDNA (89%). The entire human cDNA was then cloned into a mammalian expression vector and mRNA was synthesized by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesized mRNA. The most potent of the three tachykinin peptides tested was Substance P. The human NK-1 receptor gene has been mapped to chromosome 2 using the polymerase chain reaction to specifically amplify the human sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids.

Aged↗
PubMed1991

Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

Aged↗
PubMed1991

Human NK-2 receptor gene maps to chromosome region 10q11-21.

The human NK-2 receptor gene has been mapped to chromosome 10 using the polymerase chain reaction to amplify specifically the human NK-2 receptor sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids. The assignment to chromosome 10 was confirmed by in situ hybridisation to human metaphase chromosomes, giving a regional localisation of 10q11-21.

Base Sequence↗
PubMed1991

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.

Base Sequence↗
PubMed1991

Protection of Nitrosomonas europaea colonizing clay minerals from inhibition by nitrapyrin.

Nitrate production by Nitrosomonas europaea in inorganic liquid medium containing ammonium was limited by reduction in pH. In the presence of montmorillonite and vermiculite, expanding clays with high cation-exchange-capacity (CEC), nitrite yield was increased, ammonia oxidation continued at pH values below those which inhibited growth in the absence of clays and growth was biphasic. The first phase was similar to that in the absence of clays, while the second was characterized by a lower rate of nitrite production. Illite, a non-expanding clay with low CEC, had no significant effect on ammonia oxidation, while oxidation of ammonia-treated vermiculite (ATV) occurred with no significant change in the pH of the medium. ATV, montmorillonite and vermiculite, but not illite, protected cells from inhibition by nitrapyrin at concentrations inhibitory to cells growing in suspended culture. This protection was maintained in ATV homo-ionic to Al3+, but montmorillonite made homo-ionic to Al3+ did not provide protection from inhibition. Attachment of cells to clays with high CEC is therefore advantageous in providing exchange at the clay surface of NH+4 and H+ produced by ammonia oxidation, in reducing pH toxicity, and in protecting cells from inhibition.

Aluminum Silicates↗
PubMed1990

The epsilon-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence, expression in bovine tissues and evidence of homologous sequences in man and rat.

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3' non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.

Amino Acid Sequence↗
PubMed1989

ATP synthase from bovine mitochondria: complementary DNA sequence of the import precursor of a heart isoform of the alpha subunit.

The alpha-subunit of ATP synthase from mitochondria is a major component of the extrinsic membrane sector of the enzyme. It is encoded in nuclear DNA. A family of overlapping complementary DNA clones encoding its precursor has been isolated from a bovine library by using in the first instance a mixture of 128 synthetic oligonucleotides designed on the basis of the known protein sequence, and the sequence of the full-length cDNA has been determined. The deduced protein sequence shows that the alpha-subunit of ATP synthase has a presequence of 43 amino acids that is not present in the mature protein. Presumably it directs the protein into the mitochondrial matrix and is removed during the import process. The encoded protein sequence is also longer by one amino acid at its C-terminal end than the protein isolated from F1-ATPase, but this alanine residue may have been removed artifactually during release of the F1-ATPase particle from the inner mitochondrial membrane. With the exception of one uncertainty caused by an ambiguity at one position in the nucleotide sequence, the mature protein sequence encoded in the cDNA is exactly the same as the sequence determined previously by direct analysis of the protein isolated from bovine heart mitochondria [Walker et al. (1985) J. Mol. Biol. 184, 677-701]. The cDNA sequence differs in 158 nucleotides over a region of alignment of 1097 nucleotides from a partial cDNA for the alpha-subunit that has been isolated from a bovine cDNA derived from liver RNA [Breen (1988) Biochem. Biophys. Res. Commun. 152, 264-269].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

ATP synthase from bovine mitochondria: complementary DNA sequence of the mitochondrial import precursor of the gamma-subunit and the genomic sequence of the mature protein.

The gamma-subunit of mitochondrial ATP synthase is part of the extrinsic membrane sector of the enzyme F1-ATPase. It is a nuclear gene product. Complementary DNA clones encoding a precursor of the protein have been isolated from a bovine library. The initial partial clone was identified with a mixture of 32 synthetic oligonucleotides designed from the known protein sequence (Walker et al., 1985), and this isolate was then used to screen the library again in order to find a complete cDNA. The DNA sequence of a clone that encodes the entire mature protein has been established, and the deduced protein sequence agrees exactly with that determined by direct sequence analysis of protein isolated from bovine hearts (Walker et al., 1985). At the 3' ends of two independently isolated clones, alternative polyadenylation sites have been observed; otherwise, the DNA sequences of the clones are concordant. In common with many other mitochondrial proteins encoded in nuclear genes, the deduced protein sequence has an N-terminal extension that is absent from the mature protein. These presequences direct the protein to its appropriate mitochondrial compartment and are removed during the import process. The cDNA clone has been employed to isolate bovine genomic clones containing the gene for the gamma-subunit. From them, the DNA sequence has been established of a region encoding the mature protein and six amino acids in the presequence, but not the remainder of the proposed import sequence. This sequence extends over almost 10 kb and is divided into eight exons. Intron B between exons I and II contains a sequence that is related to long interspersed repetitive elements (LINEs) that have been described in other mammals. Human LINEs are usually flanked by directly repeated sequences with a poly(A) tract at their 3' ends, and these features are present in the bovine LINE which is truncated. This sequence contains an open reading frame encoding part of a protein that is closely related to a protein encoded in mouse LINEs, to reverse transcriptase, and to DNA binding proteins. We have also made a preliminary investigation by DNA hybridization of the number of sequences related to the bovine gene in both the bovine and human genomes. Under the experimental conditions employed, one fragment hybridized in digests of bovine DNA, and two to four bands were detected in digests of human DNA; these latter fragments have originated from either expressed genes or pseudogenes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS).

We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.

Base Sequence↗
PubMed1989

Two bovine genes for mitochondrial ADP/ATP translocase expressed differences in various tissues.

Two different bovine cDNAs have been characterized that encode closely related homologues of the mitochondrial membrane carrier protein ADP/ATP translocase. One of them codes for the protein that has been characterized previously from bovine heart mitochondria, and the other codes for a protein that differs from it in 33 amino acids out of 297. Including the base substitutions required to bring about these changes in amino acid sequence, the coding regions of the cDNAs differ at 184 positions. In addition, they are extensively diverged in their 3' noncoding sequences, which differ greatly in both length and sequence, and these segments of the cDNAs have been used as hybridization probes to demonstrate that the expression of the two genes giving rise to the two proteins is very different in various bovine tissues. Expression of one gene predominates in heart muscle and that of the other in intestine. Hybridization experiments with digests of genomic DNA have shown the presence of numerous sequences related to the two cDNAs in both the bovine and human genomes. Some of these probably arise from pseudogenes, but three expressed genes have been detected in the human genome. The study of the regulation of the expression of these genes may help to illuminate the basis of tissue-specific human mitochondrial diseases which arise because of defects in mitochondrial enzymes only in the affected tissue and not in other tissues of the same individual.

Amino Acid Sequence↗
PubMed1989

Molecular characterisation of three alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) nullcardiff (Asp256----Val); PiMmalton (Phe51----deletion) and PiI (Arg39----Cys).

Three mutations causing alpha-1-antitrypsin deficiency have been identified by gene amplification and direct DNA sequencing. In the Pi (proteinase-inhibitor) nullcardiff gene, the codon for aspartate at position 256 has mutated to encode valine. In PiMmalton and Pi I, the respective mutations are the deletion of the codon for a phenylalanine residue at position 51 or 52, and a single base substitution resulting in arginine being replaced by cysteine at position 39. Examination of the protein tertiary structure suggests that all of these mutations are likely to result in folding abnormalities that may explain the deficiency states.

Amino Acid Sequence↗
PubMed1987

ATP synthase from bovine mitochondria: sequences of imported precursors of oligomycin sensitivity conferral protein, factor 6, and adenosinetriphosphatase inhibitor protein.

Oligomycin sensitivity conferral protein (OSCP), factor 6 (F6), and ATPase inhibitor protein are all components of the ATP synthase complex of bovine mitochondria. They are encoded in nuclear DNA. Complementary DNA clones encoding the precursors of these proteins have been isolated from a bovine library by using mixtures of synthetic oligonucleotides as hybridization probes, and their DNA sequences have been determined. The deduced protein sequences show that the OSCP, F6, and inhibitor proteins have N-terminal presequences of 23, 32, and 25 amino acids, respectively. These presequences are not present in the mature proteins. It is assumed that they serve to direct the proteins into the mitochondrial matrix. The cDNA clones have also been employed as hybridization probes to investigate the genetic complexity of the three proteins in cows and humans. These experiments indicate that the bovine and human inhibitor and bovine F6 proteins are encoded by single genes but suggest the possibility of the presence in both species of more than one gene (or pseudogenes) for the OSCP.

Adenosine Triphosphatases↗
PubMed1987

Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and the brown fat mitochondria uncoupling protein.

A cDNA encoding the precursor of the bovine mitochondrial phosphate carrier protein has been cloned from a bovine cDNA library using a mixture of 128 different 17-mer oligonucleotides as hybridisation probe. The protein has an N-terminal extension of 49 amino acids not present in the mature protein. This extension has a net positive charge and is presumed to direct the import of the protein from the cytoplasm to the mitochondrion. Comparison of the protein sequence of the mature phosphate carrier with itself, with ADP/ATP translocase and with the uncoupling protein from brown fat mitochondria shows that all three proteins contain a 3-fold repeated sequence approximately 100 amino acids in length, and that the repeats in the three proteins are related to each other. This implies that the three proteins have related three-dimensional structures and mechanisms and that they share a common evolutionary origin. The distribution of hydrophobic residues in the phosphate carrier protein suggests that each repeated 100 amino acid element is composed of two membrane-spanning alpha-helices linked by an extensive hydrophilic domain. This model is similar to that first proposed for the ADP/ATP translocase and later for the brown fat mitochondria uncoupling protein.

Adipose Tissue, Brown↗