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Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 217 records · Page 12Linked to original sources
PubMed1991

Variable methylation of the 5'-flanking DNA of the human pro-opiomelanocortin gene.

The pro-opiomelanocortin gene is widely expressed in human tissues, although both transcriptional initiation sites and regulation appear to be tissue specific. In order to determine how promoter and enhancer choice is effected, we have studied the methylation pattern of the gene in a number of normal tissues, tumours and cell lines. Variability of this pattern was observed in the 5'-flanking DNA, particularly at the HpaII site located at -304 bp upstream from the pituitary CAP site. This site was generally methylated in tissues likely to express the predominant extrapituitary (800 nucleotide) message, while in tissues known to express the normal pituitary (1150 nucleotide) message and longer species, a tendency towards undermethylation was observed. Although the sites at which variable methylation occurs did not correspond to established binding sites for regulatory proteins, many of these regions remain to be determined and thus it is possible that methylation may be influential in the tissue-specific regulation of this gene.

DNA↗
PubMed1991

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Uterine calcium-binding protein activity of nonlaying hens and hens laying hard-shelled or shell-less eggs.

Uterine calcium-binding protein (CaBP) activity was compared at oviposition and 14 h postoviposition in nonlaying (NL) hens and hens laying a high incidence of shell-less (SL) or hard-shelled (HS) eggs fed a commercial laying diet. Epithelial cells were removed from uteri of NL, SL, and HS hens and homogenized in Tris buffer, pH 7.4. Proteins in homogenate were fractionated by the addition of ammonium sulfate and centrifugation, separated by Sephadex chromatography, and measured by ultraviolet spectrophotometry at A280. Calcium-binding protein activity was determined using a 45Ca-chelex binding assay. Two peaks of CaBP activity were detected and designated CaBP-a (28,000 Da) and CaBP-b (16,000 Da). Uterine CaBP-b may differ from CaBP-a only in Ca content. The activities of CaBP (a and b) were higher (P less than or equal to .05) in HS and SL hens than in NL hens at 0 h. There was no significant difference (0 h) between CaBP activity (a versus b) of either HS or SL hens. There was also no difference (0 h) of CaBP-a or CaBP-b activity when SL and HS hens were compared. Uterine CaBP-a activity was lower (P less than or equal to .05) in SL hens than HS hens at 14 h. Under normal dietary conditions, the level of CaBP activity at 14 h may be associated with SL egg production.

Animals↗
PubMed1990

Characterisation of the alleles encoding ovine beta-lactoglobulins A and B.

beta-Lactoglobulin (BLG) is the major whey protein in the milk of ruminants and is produced in the mammary gland during pregnancy and lactation. Here, we compare the nucleotide sequences of two BLG-encoding clones isolated from a sheep genomic library. The two clones are very similar differing by only 1 bp in their coding regions, giving rise to a Tyr/His difference in the gene product, and suggesting that the two clones correspond to A and B allelic variants of BLG. The isoelectric points (pI) of BLGs A and B were estimated as 5.7 and 6.0, respectively. Transgenic mice carrying a particular clone secrete BLG of the expected pI into their milk. Restriction fragment length polymorphism analysis of a small sheep population demonstrated the existence of at least four BLG haplotypes.

Alleles↗
PubMed1990

Factors affecting expression of the recF gene of Escherichia coli K-12.

This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95% of the dnaA gene and lambda bet, N (double amber) and 40% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.

Bacterial Proteins↗
PubMed1990

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro.

We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.

Adenosine Triphosphatases↗
PubMed1990

The synthesis of ACTH and related peptides by tumours.

Despite all we have learned, the reason why certain tumours and particularly non-pituitary tumours synthesize ACTH remains an enigma. There is no clear theory which links the neoplastic process with the expression of peptide hormones but it is interesting to speculate that the amplification of certain oncogenes may be linked to de-repression of hormone genes. Once the gene has been switched on, there should be some mechanism for preventing continuous expression and in the pituitary the POMC gene is normally inhibited by glucocorticoids. Therefore it is crucial to investigate the role of glucocorticoids in non-pituitary tumours and this requires an understanding of the molecular mechanisms involved in glucocorticoid inhibition of the normal POMC gene in the pituitary. The evidence presented in this chapter describing the glucocorticoid receptor binding site in the promoter region of the POMC gene in rat pituitary gives an exciting insight into the regulatory mechanisms and their potential for aberrant control. Taken with the presence of pituitary-specific regions regulating the POMC gene promoter there appear to be multiple approaches to dissecting out the differences in non-pituitary tumours. Thus in a relatively short period of time there has been a marked increase in our understanding of the molecular mechanisms underlying POMC gene expression. At the level of the peptides, progress has been slower. We are now aware that secretion of ACTH implies that a number of other peptides will be found in the circulation, even though there is limited evidence for a specific role for any of the co-secreted peptides. However, it is hard to understand the conflicting reports that N-POC is synthesized by non-small cell and small cell carcinoma of the lung when ACTH, which we assume to be co-secreted, is thought to be synthesized only by small cell carcinoma. The most likely explanation for this is the difficult nature of the radio-immunoassays for these hormones and the problems associated with studying large groups of clearly defined patients. Development of very simple methods for measuring the ACTH precursors has demonstrated that they are released into the circulation in normal subjects and that the levels are markedly elevated in non-pituitary tumours, suggesting that they are the major circulating forms in the ectopic ACTH syndrome. This implies that these tumours cannot process the precursor molecules suggesting that the processing enzymes are lacking.(ABSTRACT TRUNCATED AT 400 WORDS)

ACTH Syndrome, Ectopic↗
PubMed1990

High-level expression of biologically active human alpha 1-antitrypsin in the milk of transgenic mice.

Reduced circulating levels of alpha 1-antitrypsin (alpha 1 AT) are associated with certain alpha 1 AT genotypes and increased susceptibility to emphysema. Unfortunately, the amounts of alpha 1 AT that would be required for replacement therapy are beyond the capacity of plasma fractionation and mammalian cell culture systems. Thus, we have examined the potential of transgenic animals as an alternative means of producing human alpha 1 AT. A hybrid gene constructed by using sequences from the ovine milk protein gene beta-lactoglobulin fused to an alpha 1 AT "minigene" was used to generate transgenic mice. Of 13 independent transgenic mice and mouse lines, 5 expressed the hybrid gene in the mammary gland, 5 in the salivary glands, and 2 in both these tissues. Human alpha 1 AT was secreted into the milk of each of the 7 mice and mouse lines that expressed the hybrid gene in the mammary gland. Four of these mammary-expressing transgenic mice and mouse lines produced concentrations of at least 0.5 mg of alpha 1 AT per ml in their milk; one line (AATB 35) produced 7 mg of this protein per ml. alpha 1 AT from transgenic mouse milk was similar in size to human plasma-derived alpha 1 AT and showed a similar capacity to inhibit trypsin. Expression at equivalent levels in transgenic sheep or cattle would yield sufficient alpha 1 AT for therapeutic purposes.

Animals↗
PubMed1990

Physical analysis of spontaneous and mutagen-induced mutants of Escherichia coli K-12 expressing DNA exonuclease VIII activity.

We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VIII (ExoVIII). One mutation, sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and the trg gene. Western blot analysis shows that the protein produced is larger than wild type ExoVIII. The nucleotide sequence shows that a translational gene fusion has occurred. The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call sfcA. Analysis of the protein sequence encoded by sfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme. We discuss the possibility that sfcA is identical to maeA which encodes NAD-linked malic enzyme from Escherichia coli. Restriction nuclease analysis of a second deletion, sbcA81, by Southern blot technique indicates that about 105 kb of DNA have been deleted and a transcriptional gene fusion has occurred between recE and the regulatory region of an E. coli chromosomal gene. We also examined eight other sbc mutations that result in ExoVIII production. Five have no effect on restriction nucleotide fragment sizes detected by complementarity to lambda rev as probe. These are presumed point mutations. Three seem to produce additional restriction nucleotide fragments complementary to lambda rev. The possible nature of these sbc mutations is discussed.

Amino Acid Sequence↗
PubMed1990

Defective glucocorticoid regulation of proopiomelanocortin gene expression and peptide secretion in a small cell lung cancer cell line.

A human small cell lung cancer cell line (COR L103) that actively expresses the proopiomelanocortin (POMC) gene has been used as a model of extrapituitary ACTH-secreting tumors to investigate the phenomenon of resistence of ACTH production to glucocorticoids. After both short term (24 h) and long term (10 days) exposure to hydrocortisone at concentrations of 500 and 1000 nM, the accumulation of intracellular POMC mRNA, ACTH, and ACTH precursor peptides in the culture medium was not suppressed. These finding contrast with those in the pituitary corticotroph cell line AtT20, in which POMC mRNA, ACTH, and ACTH precursors were suppressed under the same conditions. Two other genes that are regulated by glucocorticoids in other cell types, the tyrosine amino transferase gene and the glucocorticoid receptor gene, were expressed in COR L103 cells. However, neither gene appeared to be regulated by hydrocortisone in this small cell lung cancer cell line. Further studies demonstrated that glucocorticoid receptor binding could be detected in the nucleus and cytoplasm, with a Kd of 5 X 10(-9) M. It is concluded that nonsuppression of POMC by glucocorticoids is probably part of a more global defect of glucocorticoid signaling in these cells, but that this defect lies distal to steroid binding in the nucleus.

Adrenocorticotropic Hormone↗
PubMed1990

In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA.

Many peripheral tissues express the proopiomelanocortin (POMC) gene as an 800-base mRNA that lacks the 5' end of the 1200-base pituitary transcript. The missing region encodes the peptide signal sequence, and thus, it is unlikely that any translation product would be secreted. We have found that a RNA transcript equivalent to this short message, generated by transcription in vitro from a T7 polymerase promoter, is translatable in a rabbit reticulocyte lysate, generating peptides of 27.5, 22.5, and 15.5 kD. None of these peptides appears to be processed or protected from proteinase-K digestion by a microsomal membrane fraction. In vivo studies were undertaken by transfecting into GH3 cells one of two expression vectors containing sequences that would produce either a full-length mRNA or a short (800-base) mRNA. The neomycin resistance gene was cotransfected with these plasmids, and 30 permanent cell lines were produced after selection in G418. Cell lines containing the full-length RNA secreted large quantities of ACTH and beta-endorphin immunoreactivity, whereas those expressing the short transcript secreted neither of these peptides. However extractable peptide was present in this latter type of cell line, thereby suggesting that the 800-base mRNA was translated, and that no peptide reached the secretory vesicle. These findings raise important questions about the role of peripheral POMC gene expression.

Adrenocorticotropic Hormone↗
PubMed1990

Gene expression in the mammary gland.

We have demonstrated that the ovine genomic clone SS1 can be used to generate transgenic mice that produce significant quantities of BLG protein in milk. The smallest BLG construct so far examined that retains the ability to direct BLG to mouse milk encompasses approximately 7.3 kb of genomic DNA, of which about 0.8 kb is derived from the promoter region. Gene expression is tissue-specific and regulated in a temporal and developmental fashion that is similar to that reported for sheep. We conclude, therefore, that the cis-acting sequences determining mammary expression of the ovine BLG gene are correctly interpreted in mice, despite the absence of an equivalent gene in this species, and that conclusions drawn from future work on BLG expression in the mammary gland of transgenic mice will be broadly applicable in sheep and other ruminant species. Work is currently in progress to define other sequences within the promoter of BLG that are required for regulated expression in transgenic mice. These and other studies into the DNA-protein interactions within the promoter which are required for efficient tissue-specific, regulated expression should lead to a greater understanding of milk protein gene expression in the mammary gland. Furthermore, in the current absence of a suitable in-vitro system, the mouse will be most useful for evaluating the expression of further constructs designed to express foreign proteins in milk of domestic ruminants.

Animals↗
PubMed1990

Modification of milk composition.

Revolutionary new opportunities for the modification of milk composition have been created by the development of methods for gene transfer and targeted mutation of genes may extend the range of opportunities still further. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue-specific hormonally induced expression in the mammary gland. Fragments of the ovine beta-lactoglobulin gene fused to cDNA for the human therapeutic proteins clotting factor IX and alpha-1 antitrypsin have directed production of these proteins in the milk of transgenic mice and sheep. Factor IX was biologically active and co-migrated with authentic proteins, but was present at too low a concentration for commercial exploitation. Recent observations suggest that fusion genes containing genomic clones direct production of higher concentrations of protein. Mouse whey acidic protein genomic sequences also directed production of low concentrations of human tissue plasminogen activator in the milk of transgenic mice. Targeted expression of this kind may be used for the production of therapeutic and industrial proteins, to increase the concentration or modify the nature of milk proteins, reduce the concentration of lactose, change the composition of fat or direct production of bacteriocidal proteins in milk in order to combat mastitis.

Animals↗
PubMed1990

Anthropometric measurements of white and black southern adolescent girls.

Anthropometric measurements of 691 white and 550 black 12-, 14-, and 16-year-old girls from three income groups, residing in the southern United States, were evaluated, Heights, weights, mid-upper arm circumferences, and arm muscle areas of 14-year-old girls were significantly higher than those of 12-year-old girls and significantly lower than those of girls 16 years of age; triceps skinfolds, arm fat areas, and body mass indexes of 12-year-olds were significantly lower than those of older subjects. Blacks had significantly higher weights, body mass indexes, and arm muscle areas than whites. Black 12-year-old girls were significantly taller than white 12-year-old girls but significantly shorter than older girls of either race; white 16-year-old girls were significantly taller than blacks of that age. Body mass indexes of black 12-year-olds and white 14-year-olds were significantly higher than those of white 12-year-olds, and significantly lower than those of black 14- and 16-year-olds. Medium-income blacks and whites of all income levels had lower (usually significantly) weights, body mass indexes, mid-upper arm circumferences, arm muscle areas, and arm fat areas than low- and high-income blacks did. Anthropometric values of white, but not of black, girls were generally similar to those reported in surveys of primarily white girls.

Adipose Tissue↗
PubMed1989

Intraspecific evolution of a gene family coding for urinary proteins.

The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1-4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5' to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.

Animals↗