The nucleotide sequence of the sheep MHC class II DNA gene.
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Amino Acid Sequence↗
Biomedical subjects
F Wright
Publications and source records attributed to F Wright.
A native plasmid of Pasteurella haemolytica serotype A1: DNA sequence analysis and investigation of its potential as a vector.
The complete nucleotide sequence of a 4.3 kilobase pair plasmid, pAB2, isolated from a bovine strain of Pasteurella haemolytica serotype A1, was determined. It encodes a Rob-1 type beta-lactamase and a region with homology to the mobilisation (mob) region of the Escherichia coli plasmid, ColE1. An insertion mutant of pAB2 (pTC2/81) carrying a copy of Tn5 was transferred to E coli K12 by conjugation. Subsequently pTC2/81 could be transferred by transformation to E coli HB101, but not to P haemolytica serotypes A1 or A2. However, a derivative of this construct containing only a fragment of the Tn5 insertion sequence was able to transform P haemolytica. A further construct containing a fragment of the P haemolytica A1 leucotoxin A gene, was similarly restricted to transforming E coli. These results demonstrate that the pAB2 plasmid is capable of acting as an E coli/P haemolytica shuttle vector. However, the nature of the cloned DNA sequences are important to transformation.
Base Sequence↗
[5-alpha-reductases: physiology and pathology].
In most androgen target tissues, the first step of androgen action is the 5 alpha-reduction of testosterone to DHT which binds to the androgen receptor with an affinity 3 to 4 fold higher than testosterone. Two genes, encoding two isozymes of 5 alpha-reductase (5 alpha-R) have been cloned. The two isoforms, 5 alpha-R1 and 5 alpha-R 2 are located on chromosomes 5 and 2 respectively and differ in optimal pH, substrate and inhibitor affinities and tissue expression. 5 alpha-R 2 is responsible for sexual differentiation. It is the major form expressed in the prostate where it seems necessary for embryonic growth and development. 5 alpha-reductase deficiency results in androgen insensitivity due to abnormal 5 alpha-R 2. Affected patients are XY individuals with a very peculiar form of male pseudohermaphroditism: they have feminine genitalia at birth and masculinize at puberty. 29 mutations, spanning the whole coding portion of the gene, have been described; correlation between mutations and enzyme activity have led to the suggestion that both the N- and the C-terminal end of the gene are involved in substrate binding, whereas the cofactor binding-site is located in the C-terminus. In contrast to androgen insensitivity due to 5 alpha-reductase deficiency, increased 5 alpha-reductase activity can result in androgen hypersensitivity as described in idiopathic hirsutism or benign prostatic hyperplasia. In these case 5 alpha-R 1 could possibly be involved.
Cholestenone 5 alpha-Reductase↗
Asymptotic theory for gene mapping.
In genetic linkage mapping, the precision of the location estimate depends on both the number of individuals and the number of markers. Convergence rates and limit laws are established for several situations in which the number of markers depends on the sample size. Practical implications of the results are discussed.
Chromosome Mapping↗
Cloning of a cDNA encoding ovine interleukin-3.
A cDNA encoding the ovine hematopoietic growth factor interleukin-3 (IL-3), has been cloned. A 560-bp cDNA, containing the entire protein coding region, was amplified from activated mesenteric lymph node cells. The cDNA was found to share 58% nucleotide identity with hIL-3 and 48% identity with mIL-3. The predicted ovine amino-acid sequence shares 36 and 30% identity, respectively, with the corresponding human and murine proteins.
Amino Acid Sequence↗
An algorithm for unwarping multitrack electrophoretic gels.
An algorithm is proposed for removing the effect of "frowns" and "smiles" (i.e. warping) from multitrack electrophoretic gels, so that the bands are horizontal and in alignment between tracks. It has been applied to a DNA sequencing autoradiograph, but could equally be applied to other types of gel separation where warping occurs. The algorithm uses a gradient filter to estimate band orientations. These are smoothed and interpolated by robust regression, and integrated to find the unwarping transform. No manual intervention is required.
Algorithms↗
Characterisation of the cDNA clones of two beta-tubulin genes and their expression in the potato plant (Solanum tuberosum L.).
The cDNA clones of two potato beta-tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar beta-tubulin genes, designated TUBST1 and TUBST2. The expression pattern of beta-tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding beta-tubulin probes revealed that there are multiple beta-tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3' non-coding sequences. Phylogenetic analysis of plant beta-tubulin genes is described.
Amino Acid Sequence↗
Genetic diversity and phylogenetic relationships among strains of Prevotella (Bacteroides) ruminicola from the rumen.
A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length polymorphisms in 16S rRNA genes, sodium dodecyl sulfate-polyacrylamide gel profiles of total-cell proteins, and G + C contents of chromosomal DNAs. In order to obtain information on phylogenetic relationships, the sequences of a 389-bp region of the 16S rRNA gene, including variable regions 4 and 5, were compared for 10 strains. These 10 strains formed a single group when their sequences were compared with 16S ribosomal DNA sequences from other species, including Bacteroides spp. from the human colon. On the other hand, the great genetic distances between many P. ruminicola strains, including P. ruminicola subsp. brevis B(1)4 and GA33 and P. ruminicola 23T (T = type strain), support the hypothesis that these organisms should be reclassified into new species. We identified signature oligonucleotides based on 16S ribosomal DNA sequences that distinguished strains related to strains 23T, B(1)4, GA33, and M384, as well as an oligonucleotide that specifically recognized all but one of the Bacteroides and Prevotella strains tested. On the basis of the priming activities of these signature oligonucleotides in PCR reactions and on other criteria, we concluded that 12 of the original 29 strains were related to strain 23T, 4 were related to strain B(1)4, and 4 were related to strain GA33. While there are clear grounds for subdividing the species P. ruminicola on the basis of genotypic differences, it is appropriate to delay formal reclassification until further work on the phenotypic differentiation of the new groups is completed.
Animals↗
Performance and metabolic effects of benzodiazepine during submaximal exercise.
The present study examined whether benzodiazepine (BZ) intake alters performance and selected hormonal and metabolic variables during submaximal exercise. Seven triathletes completed two cycling trials at 85% maximum O2 uptake starting 3 h after an ingestion of either a placebo (PLA) of gelatin or BZ (1.5 mg lorazepam) and continuing until exhaustion, according to a double-blind randomized protocol. Blood samples were collected at rest; 5, 10, and 15 min; and exhaustion for dopamine (DA), norepinephrine (NE), epinephrine (Epi), adrenocorticotropic hormone (ACTH), cortisol (CORT), insulin (INS), free fatty acid, blood glucose, and lactate (La) determinations. Time of cycling was not significantly changed after BZ or PLA administration (22.9 +/- 2.5 vs. 23.5 +/- 3.8 min, respectively). A decrease in CORT and an increase in INS (P < 0.05) were observed with BZ before cycling. In comparison with rest, exercise resulted in a decrease in INS and an increase in all the other variables investigated (P < 0.001), but DA, NE, Epi, ACTH, CORT, La, and free fatty acid were significantly less elevated under BZ (P < 0.05). No change was found in glucose and INS levels between the two treatments at the end of the test. There was a strong correlation under both PLA and BZ conditions between DA, NE, Epi, and ACTH and also between Epi and La levels. From these data, BZ intake did appear to alter metabolism but did not influence performance during intense submaximal exercise.
Adrenocorticotropic Hormone↗
E2A/HLF fusion cDNAs and the use of RT-PCR for the detection of minimal residual disease in t(17;19)(q22;p13) acute lymphoblastic leukemia.
Three cases of acute lymphoblastic leukemia (ALL) with the rare t(17;19)(q22;p13) translocation were investigated for E2A/HLF fusion genes using reverse transcription coupled with polymerase chain reaction (RT-PCR). The patients had C-ALL, F/17 years (case 1) or pre-B ALL, M/11 years (case 2) and M/13 years (case 3). Case 1 had an event-free survival (EFS) of 42 months. Case 2 was ultimately refractory to treatment. Case 3 presented following EFS of 16 months in morphological remission (1% blasts), but with immunological and cytogenetic evidence of active disease, then relapsed, remitted and relapsed. Type II E2A/HLF fusion cDNA was found at diagnosis (cases 1, 2), at presentation (case 3) and in all samples tested, whether with active disease or in complete remission (CR). Case 3 showed, in addition, type I fusion E2A/HLF cDNA at presentation, through induction therapy when there was evidence of active disease, but not in CR. Cases 1 and 3 had bone marrow transplantation while in CR but with residual disease detectable by RT-PCR. All patients have died of ALL. Two cases (2 and 3) had hypercalcemia with bone lesions. No case had any evidence of disseminated intravascular coagulation. This is the first demonstration of the value of RT-PCR for the detection of minimal residual disease in t(17;19) ALL.
Adolescent↗
Serotyping and distribution of Listeria isolates from cases of ovine listeriosis.
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Animals↗
MK 886, an antagonist of leukotriene generation, inhibits DNA synthesis in a subset of acute myeloid leukaemia cells.
We have studied the actions of inhibitors of leukotriene generation on DNA synthesis (measured by 3H-thymidine incorporation) in blast cells from patients with acute myeloid leukaemia (AML). Cells from a subset only of these patients were sensitive to MK 886, a potent selective inhibitor of leukotriene synthesis. By contrast, DNA replication in cells from all of the patients was inhibited by nordihydroguiaretic acid (NDGA), a leukotriene synthesis inhibitor of lower selectivity. DNA synthesis in normal bone marrow cells and phytohaemagglutinin-stimulated lymphocytes was sensitive to NDGA but not to MK 886. The data suggest that NDGA inhibits DNA replication by a mechanism other than the abolition of leukotriene biosynthesis, but that DNA synthesis in a subset of AML cells may be dependent on the generation of lipoxygenase products, as indicated by sensitivity to MK 886.
Adult↗
Evolution of complex chromosomal rearrangements in a case of biphenotypic pre-B/myeloid acute leukemia.
A case of acute biphenotypic leukemia, in which blast cells coexpress antigens of both lymphoid and myeloid lineages, was seen in a 24-year-old female. The clonal karyotype was complex but did not include any of the abnormalities previously found in biphenotypic leukemia. Chromosome abnormalities included breakpoints seen in both acute lymphoblastic and acute myeloid leukemias as well as a number of known fragile sites, namely the rare heritable fragile site at 10q24, and the common, aphidicolin-induced sites on chromosome 7.
Adult↗
Minimal residual disease in acute lymphoblastic leukaemia--PCR analysis of immunoglobulin gene rearrangements.
Rearrangement of the immunoglobulin heavy chain (IgH) gene can be utilized as a marker of clonality in a number of B-lineage lymphoproliferative disorders including acute lymphoblastic leukaemia (ALL). We have used a PCR technique involving a panel of amplimers for the 6 different Variable (VH) region families and for a consensus sequence of the Joining (JH) segment to detect clonal IgH rearrangements in the peripheral blood (PB) and/or bone marrow (BM) of 28 patients (17 children and 11 adults) with B-lineage ALL at presentation (20 patients) or with overt relapse (8 patients). The age range of the patients was 2-65 years (mean 15.7 years). Follow up remission BM samples were analysed in 22 patients during and after therapy (2-7 samples per patient), 1-50 months after presentation or relapse. In 1 relapsed case, previously stored complete remission (CR) samples were analysed retrospectively. Clonal IgH chain rearrangements were detected by PCR in 90% of patients studied initially. The 2 VH region families most commonly used were the large VH3 family (65%) and the smaller more JH-proximal VH4 family (22%). More than one VH clone was detectable in 25% of the cases. A gene "fingerprinting" modification of a previously described method was applied to the detection of minimal residual disease (MRD) in follow up BM samples with a sensitivity of 10(-3) to 10(-4). In 8 of 14 patients remaining in complete remission (CR) during the time of study, all PCR analyses on BM samples in the first 6 months were negative, in some cases as early as 2 weeks post-induction therapy, and a further patient reverted from being PCR positive in the first month after the commencement of therapy to sustained PCR negativity. One adult remains in CR at 50 months after presentation and has been PCR negative at 2 time points after cessation of maintenance therapy (30 and 50 months). Eight patients relapsed in the study period comprising 6 BM and 2 isolated CNS relapses. In 4 cases of BM relapse occurring within 7 months of the start of therapy, all BM remission samples tested in this period were PCR positive. In 2 other patients BM samples tested 7 and 2 months respectively prior to relapse were PCR negative. In the 2 patients with isolated CNS relapse, PCR of BM samples from 2 and 10 months before the relapse were negative.(ABSTRACT TRUNCATED AT 400 WORDS)
Adolescent↗
Codon usage in the G+C-rich Streptomyces genome.
The codon usage (CU) patterns of 64 genes from the Gram+ prokaryotic genus Streptomyces were analysed. Despite the extremely high overall G+C content of the Streptomyces genome (estimated at 0.74), individual genes varied in G+C content from 0.610 to 0.797, and had third codon position G+C contents (GC3s) that varied from 0.764 to 0.983. The variation in GC3s explains a significant proportion of the variation in CU patterns. This is consistent with an evolutionary model of the Streptomyces genome where biased mutation pressure has led to a high average G+C content with random variation about the mean, although the variation observed is greater than that expected from a simple binomial model. The only gene in the sample that can be confidently predicted to be highly expressed, EF-Tu of Streptomyces coelicolor A3(2) (GC3s = 0.927), shows a preference for a third position C in several of the four codon families, and for CGY and GGY for Arg and Gly codons, respectively (Y = pyrimidine); similar CU patterns are found in highly expressed genes of the G+C-rich Micrococcus luteus genome. It thus appears that codon usage in Streptomyces is determined predominantly by mutation bias, with weak translational selection operating only in highly expressed genes. We discuss the possible consequences of the extreme codon bias of Streptomyces and consider how it may have evolved. A set of CU tables is provided for use with computer programs that locate protein-coding regions.
Base Composition↗
Isochromosome 1q in acute monocytic leukemia: a new nonrandom association.
We report a case of de novo acute monocytic leukemia in a 25-year-old man in whom the sole clonal cytogenetic abnormality was an additional isochromosome 1q. Cytogenetic polymorphisms indicate that, in addition to the formation of the isochromosome, a duplication of the remaining normal chromosome 1 had taken place. Other reported cases suggest an association between acute monocytic leukemia and i(1q).
Adult↗
A 2359-base pair DNA fragment from Cryptosporidium parvum encoding a repetitive oocyst protein.
A Cryptosporidium parvum lambda gt11 expression library was constructed using EcoRI-digested genomic DNA extracted from in vitro-excysted oocysts. Screening of this library with rat anti-Cryptosporidium antiserum led to the isolation of a clone containing a 2359-bp EcoRI fragment. When this fragment was ligated into the EcoRI site of plasmid vector pMS1S, the resulting clone expressed a 200-kDa beta-galactosidase fusion protein. Western blot analysis using serum raised against this fusion protein indicated that the EcoRI fragment represented part of a gene encoding a 190-kDa oocyst wall protein of C. parvum. Sequencing of the fragment revealed a continuous open reading frame encoding 786 amino acids. The DNA sequence is relatively low in G+C (39.1%), and the third codon position contains only 17.9% G+C. The deduced peptide sequence has unusually high proportions of cysteine, proline, glutamine and histidine. Another striking feature of the amino acid sequence is the presence of distinctly repetitive regions based on conserved cysteine residues.
Amino Acid Sequence↗
Isoelectric focusing and 2D electrophoresis of the human androgen receptor.
Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polyacrylamide gels before and after photoaffinity labeling with [3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 +/- 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated in IEF sample buffer containing no salt, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 +/- 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with Mw 90-95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with Mw 90-95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc+) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and Mw 90-95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc+) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.
Autoradiography↗