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Y. P. Loh, M. G. Castro, F.-J. Zeng and U. Patel-Vaidya

ABSTRACT

Pro-vasopressin mRNA, neurophysin and arginine vasopressin (AVP) were assayed in the mouse anterior pituitary gland, in mouse anterior pituitary cells in culture and in the AtT-20 corticotrophic tumour cell line. Northern blot analysis revealed the presence of an ∼700 base pair pro-vasopressin mRNA in anterior pituitary and AtT-20 cells. Neurophysin, identified by immunoblots, and AVP, identified by high-performance liquid chromatography and cross-reactivity with AVP antiserum, were detected in anterior pituitary cells and AtT-20 cells. Immunocytochemical staining with anti-neurophysin showed that ∼40–45% of the dissociated anterior pituitary cells in culture and >95% of the AtT-20 cells were stained. Anterior pituitary cells in culture and AtT-20 cells had a basal level of release of AVP in the 0·01–0·1 nm range. These results indicate that anterior pituitary cells and AtT-20 cells have the ability to synthesize and process pro-vasopressin to AVP and neurophysin, endogenously.

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C. F. Voliva and K. Paigen

ABSTRACT

A hybridization probe that is homologous to the B2 short interspersed repetitive element detects an mRNA in mouse kidney and liver that is regulated by androgen. Administration of testosterone induces this mRNA in kidney and represses it in liver. The mRNA was cloned by first using the B2 probe to select 48 cDNA clones from an androgen-induced kidney library. These clones were then tested for their androgen response by hybridizing them with probes made by reverse transcription of basal and testosterone-treated kidney poly(A)+ RNA. Any homology to the B2 sequence was masked by prehybridizing the filters to an excess of non-radioactive RNA synthesized from a B2 sequence cloned into a riboprobe vector. A unique sequence was subcloned from the largest androgen-responsive cDNA clone. A radioactive riboprobe generated from the unique sequence subclone detected an androgen-responsive mRNA in Northern blots with the same electrophoretic mobility as the predominant androgen-responsive mRNA detected with the B2 homologous riboprobe. The riboprobe also detected a unique sequence in Southern blots of genomic DNA. This subclone was then used as the probe to isolate a full-length cDNA clone from a second androgen-induced kidney library.

When sequenced, this full-length cDNA of an androgen-responsive, B2-containing mRNA showed strong homology to the rat and human cytochrome P450J and the rabbit cytochrome P450 3a genes (CYP2E1). It showed only weak homology to the mouse testosterone 15 α-hydroxylase gene (CYP2A3) which is also regulated reciprocally by androgen in kidney and liver. The sequence of mouse P450J is identical to the B2 homologous mRNA previously named B2+mRNAx which is abundant in mouse liver.

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A M Simon, G Veyssière and Cl Jean

ABSTRACT

The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids), an androgen-dependent protein specifically expressed in the mouse seminal vesicle, was isolated and sequenced. A mouse genomic library constructed in the λEMBL12 vector was screened using a full length cDNA probe. One genomic clone was selected, 7·4 kb of which were shown to contain the whole MSVSP99 gene. The complete sequence of the MSVSP99 gene (1·7 kb), plus 0·8 and 0·3 kb of the 5′ and 3′ flanking regions respectively, has been determined. The gene is composed of four exons interrupted by three introns. The size range for the four exons is 47–217 bp, while that of introns is 87–615 bp. The transcription start site was identified as an adenine residue located 21 nucleotides upstream from the ATG start codon. Putative TATA and CAAT boxes were identified, along with a number of regions that shared homologies with known regulatory sequences. These included androgen-responsive elements located in the promoter as well as in the gene sequence. Sequence comparisons with other androgen-responsive genes showed strong homologies between the MSVSP99 gene and the seminal vesicle secretory protein (SVS) family genes (rat SVS II, IV, V and VI). Moreover, some regions were found to be conserved between the MSVSP99 gene and the human semenogelin I and II genes.

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N. Divecha, H. Mansouri, D. Peat, G. Cope, L. Partridge and C. J. McDonald

ABSTRACT

Members of the proline-rich protein (PRP) family of mouse parotid glands were analysed before and after stimulation with the β-agonist isoprenaline by using a monoclonal antibody raised against the induced PRP A30 (GP-27). Antibody NAL1 reacted strongly with isoprenaline-induced B-type PRP precursors and their salivary counterparts, but not against the A-type PRPs A10 (Gp-66) and A20 (GP-45) or human salivary proteins, and it is likely that NAL1 recognizes a proline-rich repeat variant unique to this group of rodent PRPs. PRP-related antigens were observed in the parotid glands (N10 and N20) and saliva of normal mice. The antigens were located immunocytochemically in secretory granules of parotid acinar cells of both normal and isoprenaline-stimulated mice. The total amount of PRP antigens increased 16-fold from 2·5 to 40% of parotid protein after 10 days of isoprenaline treatment, as estimated by enzyme-linked immunosorbent assay. Immunoblotting showed that new PRP species appeared during the period of increase. After treatment with isoprenaline, B-type PRPs appeared first, followed by A30 and another member of the family. These results show that the mouse PRP family is larger than previously thought and can be divided immunologically into sub-groups. That a subset of PRPs are produced in the normal mouse indicates that there is differential β-adrenergic regulation within the family, and also has implications for the role of PRPs in the normal maintenance of healthy dentition and other processes.

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J. W. Barlow, L. E. Raggatt, C. C. Drinkwater, I. G. Lyons and R. I. Richards

ABSTRACT

We have used a DNA-cellulose competition assay to investigate the binding of thyroid hormone receptors to fragments of the mouse glandular kallikrein genes and the human and rat GH genes. Nuclear extracts from human lymphoblastoid IM-9 cells were incubated with [125I]tri-iodothyronine ([125I]T3) and DNA-cellulose. The ability of cloned gene fragments to compete for radiolabelled receptors bound to DNA-cellulose was compared with that of DNA from pBR322. As previously observed, a 900 bp fragment from the human GH gene showed preferential binding to the thyroid hormone receptor. High-affinity binding was observed with a synthetic fragment of the rat GH gene encompassing positions − 163 to − 192 but not with a similar fragment from positions −224 to −192. Preferential binding was also observed with fragments of the mouse glandular kallikrein gene, mGK-6. Binding to the entire gene and fragments containing 2300 and 776 bp of the promoter region was identical. Detectable but reduced binding was seen with a shorter fragment. These results suggest that the T3 receptor binds to multiple sites within the first 776 bp of the mGK-6 gene promoter. Potential thyroid hormone response elements can be identified within this region of the gene. In contrast, the kallikrein gene mGK-3, which shows a different response to thyroid hormone from that of mGK-6, showed no significant binding in the comparable promoter region.

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V Compère, D Lanfray, H Castel, F Morin, J Leprince, B Dureuil, H Vaudry, G Pelletier and M C Tonon

−80 °C until use. In situ hybridization was performed as described previously ( Compère et al . 2003 ). The vector used for the production of the cRNA probe was constructed by insertion of a 266 bp fragment of mouse DBI cDNA (GenBank no. NM_007830

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PR Manna, DW Eubank, E Lalli, P Sassone-Corsi and DM Stocco

Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.

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M. Welsh, D. L. Eizirik and E. Strandell

ABSTRACT

To elucidate the role of thermal stress on the function of pancreatic β cells, isolated mouse pancreatic islets were incubated for 30 min at 42°C. This resulted in decreased glucose-stimulated insulin secretion, inhibited total protein and pro-insulin synthesis and the induction of heat-shock proteins with molecular weights of 64 and 88 kDa. Six days later, the islets exposed to heat shock showed a lower DNA content, indicating islet cell death. However, the insulin secretory response and rates of oxygen consumption in the presence of glucose were normal. It is suggested that the induction of heat-shock proteins does not permanently impair β-cell function, but rather protects these cells from lasting damage.

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P J O'Shaughnessy and K Dudley

ABSTRACT

The structure of RNA encoding the mouse testis FSH receptor was studied using reverse transcription and the polymerase chain reaction. Four major bands were observed by ethidium bromide staining and by hybridization to an FSH-receptor cDNA probe. The largest of these bands was the expected size (779 bp) while the other bands were spaced approximately 70 bp apart. Using alternative primers, each of the products was shown to contain exons 1, 9 and 10. Exons 2-8 in the FSH receptor gene are between 68 and 77 bp in size, suggesting that these multiple products arise by alternate splicing of the region encoding the extracellular domain of the receptor. A similar pattern of splicing was observed in cDNA from the testes of hypogonadal mice, showing that this alternative splicing pattern is not gonadotrophin-dependent.

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Knut R Steffensen, Soek Ying Neo, Thomas M Stulnig, Vinsensius B Vega, Safia S Rahman, Gertrud U Schuster, Jan-Åke Gustafsson and Edison T Liu

) in the presence of E. coli DNA polymerase I, DNA ligase and RNase H. The purification of cDNA and in vitro transcription was carried out as before. Mouse universal reference RNA (Stratagene), which comprised of total RNA from 11 different mouse