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Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 235 records · Page 13Linked to original sources
PubMed1989

A 10- rather than 9-bp duplication associated with insertion of Tn5 in Escherichia coli K-12.

The composite transposable element Tn5, which is made up of two inverted IS50 elements surrounding genes encoding drug resistance, generally generates 9-bp duplications at the site of insertion. In our studies of three Tn5 insertion mutants at one location in the Escherichia coli chromosome, we have observed that one contains a duplication of 10 bp, while the other two have the usual 9-bp duplication. Three other insertion elements, IS1, IS4, and IS186, give variable-sized target site sequence duplications. We observed a similarity of amino acid sequence in a small region of the putative transposases among IS4, IS186, and Tn5 suggesting a conservation of function in this group of transposases.

Amino Acid Sequence↗
PubMed1989

Haematology of the haemolytic uraemic syndrome.

The Haemolytic Uraemic Syndrome (HUS), characterised by a microangiopathic haemolytic anaemia, thrombocytopenic purpura and renal failure, is the commonest cause of acute renal failure in children. Most cases of HUS are associated with enteropathogenic Escherichia coli which elaborate an exotoxin called verotoxin. Verotoxin has been shown to produce red cell abnormalities and induce a platelet aggregating activity. It also exerts a cytopathic effect on cultured endothelial cells. These findings are discussed in relation to the pathogenesis of HUS. Recent studies demonstrating homology between the verotoxin glycolipid receptor and the red cell P group antigens have led to the hypothesis that variability in the expression of these red cell antigens may account for the marked individual variations in the degree of organ damage in children with HUS. This hypothesis could provide a criterion for rational immunisation of at risk children.

Hemolytic-Uremic Syndrome↗
PubMed1989

Suppression of a frameshift mutation in the recE gene of Escherichia coli K-12 occurs by gene fusion.

The nucleotide sequences of a small gene, racC, and the adjacent N-terminal half of the wild-type recE gene are presented. A frameshift mutation, recE939, inactivating recE and preventing synthesis of the active recE enzyme, exonuclease VIII, was identified. The endpoints of five deletion mutations suppressing recE939 were sequenced. All five delete the frameshift site. Two are intra-recE deletions and fuse the N- and C-terminal portions of recE in frame. Three of the deletions remove the entire N-terminal portion of recE, fusing the C-terminal portion to N-terminal portions of racC in frame. These data indicate that about 70% of the N-terminal half of recE is not required to encode a hypothesized protein domain with exonuclease VIII activity.

Amino Acid Sequence↗
PubMed1989

The molecular manipulation of milk composition.

The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep beta-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising beta-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.

Animal Husbandry↗
PubMed1989

Expression of pro-opiomelanocortin gene and quantification of adrenocorticotropic hormone-like immunoreactivity in human normal peripheral mononuclear cells and lymphoid and myeloid malignancies.

Using Northern blotting with a human genomic DNA probe for the pro-opiomelanocortin (POMC) gene, we have shown specific mRNA in normal human peripheral mononuclear cells (PBMC); the presence of specific mRNA was also observed in a T lymphocyte cell line derived from a patient with lymphoma. We then demonstrated that PBMC translate the message into protein. Thus, using a radioimmunoassay with an antibody for ACTH, a median of 29 pg of ACTH-like immunoreactivity (ACTH-LIR) was found in 10(7) PBMC. ACTH-LIR was also detected in seven different cell lines derived from patients with lymphoid and myeloid malignancies, two of them JM and U937 showing the highest values 135 and 108 pg/10(7) cells, respectively. The chromatographic characterization of this ACTH-LIR showed, at least, three molecular forms of immunoreactive ACTH with molecular weights of the order of 31,000 POMC, 22,000 ACTH, and 4,500 ACTH, in addition to high-molecular-weight material (greater than 43,000). We conclude that PBMC produce ACTH-LIR which may act as a paracrine immunomodulator in a similar way to lymphokines and/or may signal the adrenal gland to secrete glucocorticoids.

Adrenocorticotropic Hormone↗
PubMed1989

Pro-opiomelanocortin mRNA size heterogeneity in ACTH-dependent Cushing's syndrome.

As an approach to understanding the abnormalities of pro-opiomelanocortin (POMC) gene regulation in human ACTH-secreting tumours, we have analysed the POMC mRNA content of nine such tumours using the Northern blot technique. Most of the tumours and normal human pituitary contained easily detectable quantities of POMC mRNA. The length of this message in most tumours was similar to, or slightly larger than, that in the normal pituitary (1150-1200 bases). Ribonuclease H studies suggested that the origin of any size heterogeneity was a longer poly(A) tail in the tumour RNA. Some tumours, however, expressed a short POMC mRNA (800 bases) which may lack the first two exons of the POMC gene as has been described. A third POMC mRNA size variant (1500 bases) was also seen in low levels in two cases, and as the principal mRNA species in one case. Primer extension and S1 nuclease protection studies suggested that most transcripts in the tumours analysed originated from the conventional promoter, and thus the use of an alternative promoter is not an adequate explanation for the expression of this gene in ectopic ACTH-secreting tumours.

ACTH Syndrome, Ectopic↗
PubMed1989

Pro-opiomelanocortin gene expression and peptide secretion in human small-cell lung cancer cell lines.

Expression of the RNA coding for the ACTH-beta-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.

Adrenocorticotropic Hormone↗
PubMed1988

Characterization of the gene encoding ovine beta-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins.

Beta-lactoglobulin is the major whey protein in the milk of ruminants and is expressed in the mammary gland during pregnancy and lactation. Here we describe the isolation and characterization of genomic clones encoding ovine beta-lactoglobulin. Two very similar but non-identical, types of beta-lactoglobulin clone were obtained. DNA sequence analysis of one of these showed that the gene is 4900 bases long and contains seven exons. It codes for a protein of 180 amino acid residues, containing an 18-residue signal peptide, within exons I to VI; exon VII is non-coding. We show that the genes encoding serum retinol binding protein, major urinary protein, alpha-1-acid glycoprotein and apolipoprotein D have a similar organization of exons and introns to beta-lactoglobulin. In particular, a comparison between beta-lactoglobulin and retinol binding protein shows that both genes encode equivalent elements of three-dimensional protein structure within analogous exons. These proteins are all members of a large, diverse family of secretory proteins, many of which function in binding small hydrophobic molecules.

Amino Acid Sequence↗
PubMed1988

Effect of DNA sequence changes on UV mutability of a purine anticodon triplet of glyU cloned on M13 phage.

Mutant forms of the glyU (glycyl tRNA) gene cloned in M13mp8 were subjected to uninduced targeted UV mutagenesis; i.e. phage particles were irradiated and used to infect unirradiated umuC+ or irradiated umuC mutant cells. The irradiated phage carried GAG at the anticodon triplet and transitions to GAA were scored. The uninduced targeted mutation rate was reduced by altering the sequence of the gene in the vicinity of the target purine (Pu) residue. In particular a triplet of pyrimidines (PyPyPy) 5' to the target G was changed to PyPuPy in order to prevent formation of cyclcobutane and 6-4 pyrimidine dimers close to the target. On this basis we suggest a mechanism for one type of uninduced regionally targeted UV mutagenesis.

Anticodon↗
PubMed1988

Ectopic hormone production.

Current understanding of the phenomenon of ectopic hormone production is largely based on a histopathological and immunocytochemical analysis of peptide hormone secreting tumours arising in non-endocrine tissues. Recent advances in the study of gene regulation show that the tissue-specific expression of genes is a highly sophisticated process and is unlikely to be disturbed by a spontaneous event such as point mutation in DNA. Study of several genes for frequently found ectopic hormones, i.e. prop-opiomelanocortin, vasopressin/neurophysin II, gastrin-releasing peptide, parathyroid hormone-related peptide, calcitonin gene-related peptide and beta-chorionic gonadotropin, suggests they are transcribed as they would be in their natural cell of origin. It is argued therefore that these data are compatible with the concept that the tumour cell of origin was capable of expressing these peptides, if only in a minor or transient manner. In one example, the ectopic ACTH syndrome, it is also necessary to explain the non-suppression of this gene's expression by elevated levels of glucocorticoids. Recent work suggests that this may result from physically present, but biologically inactive glucocorticoid receptors, a phenomenon that has occasionally been noted in hormonally inactive tumour tissue and cell lines.

Calcitonin↗
PubMed1988

Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination.

A mutation partially suppressing the UV sensitivity caused by recF143 in a uvrA6 background was located at codon 37 of recA where GTG (valine) became ATG (methionine). This mutation, originally named srf-803, was renamed recA803. Little if any suppression of the recF143 defect in UV induction of a lexA regulon promoter was detected. This led to the hypothesis that a defect in recombination repair of UV damage was suppressed by recA803. The mutant RecA protein (RecA803) was purified and compared with wild-type protein (RecA+) as a catalyst of formation of joint molecules. Under suboptimal conditions, RecA803 produces both a higher rate of formation and a higher yield of joint molecules. The suboptimal conditions tested included addition of single-stranded DNA binding protein to single-stranded DNA prior to addition of RecA. We hypothesize that the ability of RecA803 to overcome interference by single-stranded DNA binding protein is the property that allows recA803 to suppress partially the deficiency in repair caused by recF mutations in the uvrA6 background. Implications of this hypothesis for the function of RecF protein in recombination are discussed.

Bacterial Proteins↗
PubMed1988

Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII.

Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure. This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J. W. Joseph and R. Kolodner, J. Biol. Chem. 258:10411-10417, 1983). The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C. Chu, A. Templin, and A. J. Clark, manuscript in preparation). Polyclonal antibodies directed against purified exoVIII were also prepared. Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis. This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII. Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present. The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity. One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide. The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation).

Alleles↗
PubMed1987

Genetic analysis of UV mutagenesis of the Escherichia coli glyU gene.

By genetic analysis we examined UV mutagenesis of the Escherichia coli glyU gene. When carried by M13 phage mp9, glyU is subject to induced UV mutagenesis which is dependent on the umuC+ and recF+ genes. When carried by M13 phage mp8, glyU is not subject to induced UV mutagenesis. This difference is correlated with the nature of the target nucleotides: CTC in the mp9 derivative and GAG in the mp8 derivative. Thus, we conclude that the induced (umuC and recF dependent) mutagenesis is locally targeted on pyrimidine cyclobutane or 6-4 dimers. glyU carried by M13 is equally subject to uninduced UV mutagenesis whether carried by mp8 or mp9. This uninduced mutagenesis is independent of the umuC+, recF+ and recA+ genes and we hypothesize that it is regionally targeted on pyrimidine cyclobutane or 6-4 dimers in the vicinity of the target CTC and GAG nucleotides. The role of recF in UV mutagenesis was tested in two ways. First, mutagenesis of glyU carried by M13 mp9 in a recA730 genetic background was found to be recF dependent. Because recA730 renders induced UV mutagenesis partially constitutive, we conclude that the RecF product plays a direct role in UV mutagenesis rather than, or in addition to, any indirect regulatory role it may play. Second, UV mutagenesis of E. coli chromosomal glyU was found to be recF independent while UV mutagenesis of M13-bourne glyU was recF dependent. We conclude that the mechanism of induced UV mutagenesis of the E. coli chromosome is at least partly different from that of M13 phage and we discuss the biochemical basis for such a difference.

Bacterial Proteins↗
PubMed1987

Plasmid and bacteriophage vectors for excision of intact inserts.

Plasmid (pPolyIII) and bacteriophage lambda (EMBL301) vectors are described in which sites for the rare-cutting enzymes SfiI and NotI (8-bp, recognition sequences) flank the polylinker cloning region. Intact DNA inserts for introduction into cultured cells or into the early embryo are readily excised from the vectors. General-purpose miniplasmid cloning vectors pPolyI and pPolyII are also described, and the utility of the bacteriophage lambda vector is demonstrated in the construction of a bovine genomic library.

Animals↗
PubMed1987

Folacin status in adolescent females.

The folacin status of 103 adolescent females aged 12, 14, and 16 y was evaluated. In the third year, longitudinal data regarding folacin status was obtained from 50 girls aged 14 and 16 y who had participated in the first year. In the first year, 11.7% and 47.6% of the girls had serum and erythrocyte folacin levels less than 3 ng/mL (6.8 nmol/L) and 140 ng/mL (317 nmol/L), respectively. Serum folacin levels and dietary folacin intake decreased with increasing age but not significantly. During the first year, 75% and 66% of the subjects that were folacin deficient based upon serum and erythrocyte folacin values, respectively, were the same subjects considered to be deficient during the third year. Folacin status, as indicated by serum and erythrocyte folacin values, was not related to per capita income. These findings demonstrate that folacin status is less than adequate in females during the adolescent years.

Adolescent↗
PubMed1987

Longitudinal assessment of vitamin B-6 status in southern adolescent girls.

The vitamin B-6 status of 62 black and 50 white adolescent girls living in Virginia and Alabama was assessed in 1981 and again in 1983, using the parameters coenzyme stimulation of erythrocyte alanine aminotransferase activities and dietary intakes of the vitamin. The subjects were 12 or 14 years old in 1981. The height and weight measurements of the subjects were within normal ranges. The mean daily vitamin B-6 intake of the girls from food was 1.25 mg both years, as estimated by two nonsequential 24-hour food recalls. Approximately half of the girls reported consuming less than 0.02 mg vitamin B-6 per gm protein during both years. Almost half of the girls had coenzyme stimulation values indicative of marginal or deficient status. Coenzyme stimulation and dietary values of the race, age, and income groups were similar. Changes in the status grouping of the girls between the 2 years as reflected by the coenzyme stimulation measurement were associated with changes in their vitamin B-6 intakes in 70% of the cases. Vitamin B-6 inadequacy seems to be prevalent among both black and white adolescent girls.

Adolescent↗
PubMed1986

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA.

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.

Cell-Free System↗