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M Needham

Publications and source records attributed to M Needham.

32 records · Page 2Linked to original sources
PubMed1988

Characterization of response elements for androgens, glucocorticoids and progestins in mouse mammary tumour virus.

We have characterized steroid response elements in mouse mammary tumour virus (MMTV) by transient transfection. Four partial inverted repeats of the sequence TGTTCT function as response elements for androgen, as well as for glucocorticoid and progestins, although the relative hormone inductions mediated by each oligonucleotide were different. Mutational analysis of the left half of the palindrome showed that a perfect dyad symmetry is not required for optimum activity as a steroid response element. To investigate potential interactions between steroid receptors and transcription factors we have analysed the minimum sequence requirements for a hormone response. Interestingly, a single 15 bp steroid response element and a TATA box are sufficient for steroid inductions. When the distance between the two elements was increased by up to two turns of the helix the hormone induction initially increased and then gradually declined with no obvious periodicity.

Animals↗
PubMed1988

Analysis of regulatory sequences in androgen-responsive genes.

We have analysed the effect of androgens on the activity of promoters from MMTV, and the rat prostate C3(1) and mouse secretory protease inhibitor genes. MMTV promoter activity was stimulated by testosterone as well as progesterone and dexamethasone but not by oestradiol. Deletion analysis indicated that the three steroids acted through DNA sequences between nucleotides -201 and -69 upstream of the MMTV cap site. In contrast, the promoters for the C3(1) gene and the protease inhibitor gene were unaffected by testosterone in a number of cell types, including prostate cells, despite the fact that the MMTV promoter was stimulated in such cells.

Androgens↗
PubMed1987

A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas.

A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas.

Amino Acid Sequence↗
PubMed1987

Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein.

Amino Acid Sequence↗
PubMed1987

Identification of androgen response elements in mouse mammary tumour virus and the rat prostate C3 gene.

The effect of steroid hormones on mouse mammary tumour virus (MMTV) promoter activity has been analysed in the breast cancer cell lines ZR-75-1 and T-47D 11. Both androgens and progestins were stimulatory in both cell lines, whereas glucocorticoids were stimulatory in only ZR-75 cells. A glucocorticoid response was restored to T-47D cells by the transient expression of a glucocorticoid receptor expression vector, suggesting that endogenous receptors were limiting. The effect of androgen was mediated by a region of MMTV previously shown to be essential for glucocorticoid and progestin responses between nucleotides -201 and -69 upstream of the cap site. Similarly, the effect of androgens on the rat prostate C3(1) gene promoter was analysed in the rat prostate cell line T5. Although these cells had functional androgen receptors which could stimulate MMTV-promoter activity, they were incapable of interacting with androgen response elements to stimulate transcription from the C3(1) promoter.

Androgens↗
PubMed1987

Androgen-regulated expression of secretory protein synthesis in mouse ventral prostate.

Two proteins of molecular weights 25 and 12 kDa (p25 and p12 respectively), whose expression is regulated by testosterone, were identified in mouse ventral prostate. An antiserum raised to mouse ventral prostate secretion was used to demonstrate that p25 corresponds to the major secretory glycoprotein in mouse prostatic fluid. This antiserum does not cross-react with the major secretory proteins of the rat ventral prostate. Western blot analysis of mouse ventral prostate proteins using the prostatic secretion antiserum demonstrates that p12 and p25 are detectable at 3 weeks of age, but the maximum level of both proteins is not attained until 5 weeks of age. In addition, synthesis of p25 was also observed in prostate tissue derived from differentiated embryonic urogenital sinus tissue growing as implants under the renal capsule of syngeneic male hosts.

Animals↗
PubMed1987

Structural organization and expression of the mouse estrogen receptor.

Complementary DNA clones corresponding to the mouse uterus estrogen receptor mRNA have been isolated and characterized. Nucleotide sequence analysis predicts that full-length cDNA has the potential to code for a polypeptide of 599 amino acids, and comparison with the protein sequences of the rat, human, and chicken estrogen receptors reveals overall homologies of 97%, 88% and 77%, respectively. Genomic clones for the mouse estrogen receptor have been isolated from a cosmid library and used in conjunction with the cDNA clones to study the expression of the receptor in vivo by RNase mapping, primer extension, and Northern blotting. These analyses demonstrate that transcription initiates at multiple sites which span a region of at least 62 base pairs and that the estrogen receptor is encoded by mRNA of approximately 6.5 kilobases in size. There are 10 major starts in total, one of which is situated 31 nucleotides downstream from a TATA box-like motif and coincides with the start of the cDNA clone pMOR8. The ability of the cDNA clone to produce a functional protein was verified by transfection into COS-1 cells which lack endogenous estrogen receptor. The mouse estrogen receptor, in a SV40-based expression vector, was cotransfected with a chimeric marker plasmid consisting of an estrogen response element from the vitellogenin A2 gene linked to the thymidine kinase promoter and the chloramphenicol acetyl transferase gene. In the presence of estradiol chloramphenicol acetyl transferase activity is stimulated by up to 80-fold, while tamoxifen and 4-hydroxytamoxifen act primarily as antiestrogens in this in vitro assay.

Amino Acid Sequence↗
PubMed1983

Prostatic steroid-binding protein. Isolation and characterization of C3 genes.

Prostatic steroid-binding protein, whose expression is stimulated by androgens, consists of two subunits, one containing the polypeptides C1 and C3 and the other containing the polypeptides C2 and C3. We have isolated and sequenced cDNA clones specific for C3 mRNA and used them to isolate and characterize genomic clones for two C3 genes. Both genes are 3.2 kilobases with identical exon/intron arrangements, which is similar to the organization of the C1 and C2 genes, suggesting that they may have arisen by duplications of an ancestral gene. Finally, homologous human genes have not been detected.

Amino Acid Sequence↗
PubMed1982

Prostatic steroid binding protein: organisation of C1 and C2 genes.

Prostatic steroid binding protein, whose expression is stimulated by androgens, consists of two subunits: one containing the polypeptides C1 and C3 and the other containing C2 and C3. We have characterised genomic clones containing the C1 and C2 genes by restriction enzyme analysis and DNA sequencing. Both genes are 3.2 Kb, have similar exon/intron arrangements and share considerable DNA sequence homologies in their coding regions, intervening sequences and 5' upstream DNA sequence which suggests that they have probably arisen from the duplication of an ancestral gene. The 5' termini of C1 and C2 mRNA have been mapped; the sequence TATAAA appears 30 nucleotides upstream but a CAAT-like sequence at -60 - -80 is absent. Finally, homologous human genes have not been detected.

Androgen-Binding Protein↗
PubMedDate not supplied

Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif.

Steroid hormones induce the transcriptional activity of their cognate receptors by recruiting a variety of cofactors. One of these, steroid receptor co-activator-1 (SRC-1) interacts with the ligand binding domains of a number of different receptors by means of LXXLL motifs. We have investigated the relative interaction of four such motifs in SRC-1a using a yeast two-hybrid assay. We demonstrate that ERalpha, ERbeta and ERbeta2 preferentially interact with motif 2 while GR, AR, PPARalpha and PPARgamma preferentially interact with motif 4. We show that the interactions depend not only on the LXXLL motif itself but also on residues flanking the motif.

Amino Acid Motifs↗
PubMedDate not supplied

Expression of a bovine GABAA receptor alpha1-subunit cDNA in murine erythroleukaemia cells.

A plasmid vector has been constructed by insertion of the cDNA encoding the alpha1 subunit of the bovine GABAA receptor into the LCR/MEL expression vector pNV1 downstream of the human globin locus control region between the promoter and the second intron of the beta-globin gene to produce pNVGABAalpha. This plasmid was transfected into murine erythroleukemia (MEL) cells using electroporation to obtain recombinant cells. Parental and recombinant cells were tested by both RNA dot blot and electrophysiological analysis for the presence of bovine GABAA receptor alpha1 subunit mRNA. Parental MEL cells did not express GABA-gated chloride channels but recombinant cells were sensitive to pressure-applied GABA. The GABA responses reversed at the equilibrium potential predicted for chloride ions. These results show that the alpha1 subunit of the bovine GABAA receptor inserts in the plasma membrane of the MEL cells and forms homo-oligomeric chloride channels that are gated by GABA. Our studies suggest, therefore, that the LCR/MEL system can be used for the expression of neurotransmitter receptor genes.

Animals↗
PubMedDate not supplied

Keeping possum.

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Adult↗