The broad (br ) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these three isoforms correlated well with those of yolk protein precursor (YPP) genes. These BR isoforms were activated by 20E in fat bodies cultured in vitro and behaved as early genes, with a self-repressive autoregulatory loop that can be blocked by the protein inhibitor, cyclohexamide. Multiple binding sites for all four BR isoforms were present in the 5′-regulatory region of the major YPP gene, vitellogenin (Vg). Effects of BR isoforms on the expression of Vg have been demonstrated by cell transfection analysis. In particular, BR isoforms by themselves had no effects on the Vg promoter. However, Z1 and Z4 each repressed Aedes aegypti ecdysone receptor (EcR)/Ultraspiracle (USP)-mediated 20E activation of the Vg promoter, while Z2 enhanced activation of the Vg promoter by AaEcR/AaUSP in the presence of 20E. Z3 had no obvious effect in the same experiment. These results suggested that BR isoforms are essential for proper activation and termination of the Vg gene in response to 20E. Overall, our study implicated br in the regulation of mosquito vitellogenesis.
L Chen, J Zhu, G Sun and A S Raikhel
J. S. Fleming, P. J. Greenwood and C.-L. C. Chen
Clusterin or sulphated glycoprotein-2 is a major component of the rete testis fluid, synthesized by the rete testis epithelial cells and Sertoli cells. Differences in the two-dimensional polyacrylamide gel electrophoresis pattern of clusterin-like proteins have been reported in rete testis fluid from Booroola rams carrying the fecundity gene FecB, when compared with that from non-carrier rams. In order to determine whether the FecB gene influences the expression of the clusterin gene, we used a rat clusterin cRNA probe to investigate mRNA species in the tissues of homozygous (BB) or non-carrier (+ +) Booroola sheep. Northern blots of polyadenylated RNA showed hybridization to the cRNA probe in the testis, ovarian follicles, corpora lutea and stroma, pituitary and liver. A major mRNA transcript was observed at 2·3 kb and a minor transcript in some tissues at 0·8 kb. Densitometry of the autoradiographs revealed no FecB-specific differences in the densities of the hybridization signals from + + and BB testis or ovarian follicle, corpora lutea or stromal RNA. We conclude that the gene for ovine clusterin is expressed widely in the tissues of sheep and that its expression is not affected by the presence of the FecB gene.
Mahuya Bose, Dilip Debnath, Yue Chen and Himangshu S Bose
Nicotine, a pharmacologically active constituent of tobacco smoke, decreases sex steroid production and impairs reproductive function. The rate-limiting step in steroid hormone biosynthesis is the transport of substrate cholesterol from the outer to inner mitochondrial membrane by the steroidogenic acute regulatory protein (StAR). StAR is a 37 kDa cytoplasmic phosphoprotein processed as a 32 kDa intermediate to a mature 30 kDa inactive mitochondrial protein. StAR’s cholesterol transport capacity is proportional to its residency time at the outer mitochondrial membrane (OMM). Nonsteroidogenic COS-1 cells transfected with StAR/F2, steroidogenic MA-10 cells induced with cAMP or transfected with StAR or the isolated steroidogenic mitochondria preincubated with nicotine reduced StAR expression, import and activity. Mitochondria isolated from steroidogenic tissues or cells, pretreated with nicotine, also reduced the association of StAR with the OMM, but had no effect on the import of signal sequence substituted SCC/N-62StAR. The fluorescence emission maximum of StAR was unchanged with nicotine, but StAR’s free energy of unfolding and the surface area (m) increased in the presence of nicotine. Nicotine also blocked StAR from proteolysis with trypsin, suggesting that nicotine partially stabilised protein conformation by insertion into the molten globule conformation of StAR.
M. Tanaka, T. M. Telecky, S. Fukada, S. Adachi, S. Chen and Y. Nagahama
The enzyme aromatase P-450 (P450arom) catalyses the conversion of androgen to oestrogen. A cDNA insert encoding P450arom was isolated from a rainbow trout (Oncorhynchus mykiss) ovary cDNA library. The insert was sequenced and found to contain an open-reading frame predicted to encode a protein of 522 amino acid residues. The deduced polypeptide is 52% homologous with human, mouse and rat P450arom and 53% homologous with that of chicken. The insert was confirmed to encode P450arom by introducing it into COS-1 monkey kidney tumour cells (COS-1 cells) and detecting the conversion of testosterone to oestradiol-17β by radioimmunoassay. The N-terminal region of the deduced polypeptide was 19 amino acids longer than that of the other four species, and was found by hydropathy plotting to be very hydrophobic.
Northern blot analysis revealed 2.6kb RNA transcripts which were present in the trout ovary during vitellogenesis and hybridized to the cDNA insert. In preparations from subsequent stages of ovarian development, no RNA transcripts hybridized to the probe. Since the RNA transcripts are present only during the stage of oestradiol-β production by the ovarian follicles, oestradiol-17β production may be regulated, in part, by the amount of P450arom mRNA present.
S K Nair, T J Thomas, N J Greenfield, A Chen, H He and T Thomas
Estrogen receptors (ERα and ERβ) are ligand-activated nuclear receptors that mediate the action of estrogens. These receptors activate transcription by similar mechanism(s), although the overall amino acid sequence identity is only 47%. In order to compare the structural and conformational features of ERα and ERβ, we monitored their intrinsic tryptophan fluorescence during thermal unfolding. The 50% unfolding temperatures (TM) of ERα and ERβ were 39±1 and 40±2°C, respectively. Estradiol had no significant effect on the TM of ERα or ERβ. In contrast, binding of the estrogen-response element increased the TM of ERα and ERβ by 10 °C. Thermal unfolding of estradiol-bound ERα and ligand-free ERβ showed two-step transitions, with the formation of intermediates that were stable between 36–48 and 34–42°C, respectively. We confirmed the presence of intermediate states during thermal unfolding by circular dichroism spectroscopy. Atomic force microscopy showed that the ERβ intermediate consisted of discrete globular particles, whereas the ERα intermediate showed a speckled appearance, with sparse well-defined particles. Fluorescence-quenching studies showed the presence of two classes of tryptophan in unliganded ERα and ERβ. Binding of estradiol to ERβ exposed its tryptophans, whereas estradiol reduced the accessibility of the tryptophans of ERα. Our results illustrate the differential effects of ligands on the unfolding of ERα and ERβ, and identify partially unfolded intermediates. Differences in the conformational flexibility and stability of ERα and ERβ may represent functional differences of ligand-bound ERs in recruiting coactivator proteins and initiating transcription.
M. Iwashita, K. Hirota, S.-I. Izumi, H.-C. Chen and K.J. Catt
Specific receptors for gonadotrophin-releasing hormone (GnRH) were extracted from the rat pituitary gland with several detergents and characterized by binding studies with the potent GnRH antagonist [Ac-d-pCl-Phe1,2, d-Trp3, d-Lys6, d-Ala10]-GnRH (GnRHant). The particulate GnRH receptors were most effectively solubilized with 5 mm 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS), which extracted 63% of the original membrane binding activity when assayed with 125I-labelled GnRHant. The binding affinities of particulate and CHAPS-solubilized receptors analysed with 125I-labelled GnRHant were 1·5 ± 0·4 × 109 m −1 and 1·2 ± 0·2 × 109 m −1 respectively. Gel filtration of the CHAPS-solubilized receptor revealed a major peak of specific binding activity with M r of about 700 000. A hormone—receptor complex of similar M r was observed when CHAPS-solubilized receptors were labelled with photoreactive radioiodinated [d-Lys6]-des-Gly10-GnRH-N-ethylamide and then analysed by gel chromatography. However, when pituitary particles were photolabelled and solubilized in 2% Triton X-100 before analysis on Sephacryl S-300, the M r of the receptor was approximately 250 000, similar to the value obtained by sucrose density gradient centrifugation of the CHAPS-solubilized receptor. After solubilization in sodium dodecyl sulphate (SDS) the photolabelled receptor was eluted from Sephacryl S-300 as a 60 kDa peak which on SDS-gel electrophoresis contained a 52 kDa component, corresponding to the major binding subunit extracted directly from photolabelled pituitary membranes. The difference in higher molecular weight forms observed under non-denaturing and denaturing conditions could reflect the need for additional membrane components to maintain the active conformation of the GnRH receptor site. Whereas the minimum M r of the solubilized receptor is about 250 000 under non-denaturing conditions, analysis of the photolabelled GnRH-receptor complex by SDS chromatography and electrophoresis indicates that a binding subunit with M r of 50 000–60 000 is present in the GnRH holoreceptor.
Angela Delaney, Vasantha Padmanabhan, Geoffrey Rezvani, Weiping Chen, Patricia Forcinito, Crystal S F Cheung, Jeffrey Baron and Julian C K Lui
Body size varies enormously among mammalian species. In small mammals, body growth is typically suppressed rapidly, within weeks, whereas in large mammals, growth is suppressed slowly, over years, allowing for a greater adult size. We recently reported evidence that body growth suppression in rodents is caused in part by a juvenile genetic program that occurs in multiple tissues simultaneously and involves the downregulation of a large set of growth-promoting genes. We hypothesized that this genetic program is conserved in large mammals but that its time course is evolutionarily modulated such that it plays out more slowly, allowing for more prolonged growth. Consistent with this hypothesis, using expression microarray analysis, we identified a set of genes that are downregulated with age in both juvenile sheep kidney and lung. This overlapping gene set was enriched for genes involved in cell proliferation and growth and showed striking similarity to a set of genes downregulated with age in multiple organs of the juvenile mouse and rat, indicating that the multiorgan juvenile genetic program previously described in rodents has been conserved in the 80 million years since sheep and rodents diverged in evolution. Using microarray and real-time PCR, we found that the pace of this program was most rapid in mice, more gradual in rats, and most gradual in sheep. These findings support the hypothesis that a growth-regulating genetic program is conserved among mammalian species but that its pace is modulated to allow more prolonged growth and therefore greater adult body size in larger mammals.