Reduction of prostatic binding protein-messenger ribonucleic acid sequences in rat prostate by castration

Messenger RNA coding for the three subunits of prostatic binding protein was isolated from polysomal RNA of rat ventral prostate by oligo (dT)-cellulose affinity chromatography and purified by repeated sedimentations through sucrose gradients under denaturing conditions. The purified mRNA migrated as a 9S peak in sucrose gradient centrifugation and hybridized with its cDNA within 2 log Rot units. In a cell-free reticulocyte lysate system, the mRNA directed the synthesis of three polypeptides of 12000, 9000, and 8000 daltons. These translation products were identified as the subunits of prostatic binding protein by immunoreaction with antibodies to this protein. Quantitation of prostatic binding protein-mRNA sequences in normal and castrated rats by hybridization with the cDNA probe showed that 3-day castration reduced the prostatic binding protein-mRNA sequences to less than 2% of the normal level. Similar hybridization was performed by using the cDNA to determine the level of prostatic binding protein coding sequences in polysomal poly(A) RNA following castration. The results showed a first-order rate constant of 3.92 X 10-2 h-1 for reduction of prostatic binding protein-mRNA sequences in polysomes. The period of castration required to reduce the level of these sequences to 50% of the normal level was calculated to be 17.6 h.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Eimeria tenella unsporulated oocyst-specific cDNA clones.

A cDNA library was constructed with poly(A)+ RNA from unsporulated oocysts of Eimeria tenella in pUC18. After screening, 4 cDNA clones that hybridized to RNA of unsporulated and sporulating oocysts but not to RNA of either sporulated oocysts or second generation merozoites were isolated and characterized. Each of the cDNA clones is unique. The loci for 2 of the clones are on E. tenella chromosome 7, the site of the third is located on chromosome 6 and the last clone hybridizes, for the most part, to chromosome 5 but also to other E. tenella chromosomes. The cognate RNAs for each of the cDNA clones show differential patterns of hybridization during oocyst sporulation with the levels of RNA being low at the start of sporulation (0 hr), increasing to peak levels between 6.5 and 23 hr after the onset of sporulation and, in each case, decreasing to low hybridization levels at 48 hr after initiation of sporulation. These results establish that specific mRNA levels are differentially regulated during sporulation.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Rat tropoelastin is synthesized from a 3.5-kilobase mRNA

A lambda gt11 cDNA library was constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats and screened with a human tropoelastin cDNA clone. DNA sequence analysis of several overlapping rat clones confirmed the presence of DNA sequences coding for murine tropoelastin and DNA sequences coding for the 3'-untranslated region of the rat tropoelastin mRNA. Northern blot analysis of total RNA from aortic tissue of neonatal rats using oligonucleotide probes derived from these rat tropoelastin cDNAs demonstrated the presence of a 3.5-kilobase tropoelastin mRNA. The size of this rat tropoelastin mRNA agrees with previous reports for the size of the mRNA coding for tropoelastin in tissue from several vertebrate species but contrasts with several reports suggesting the presence of a higher molecular weight mRNA species responsible for the synthesis of tropoelastin in rodent tissue.     

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.     

The repetitive structure of the profilaggrin gene as demonstrated using epidermal profilaggrin cDNA

Filaggrin is the histidine-rich basic protein that aggregates keratin filaments in fully differentiated cells of the epidermis. Filaggrin is synthesized in the granular cell layer as a high molecular weight precursor protein (profilaggrin) that consists of multiple repeated copies of filaggrin. cDNA clones for rat and mouse epidermal profilaggrin have been constructed from sucrose gradient-enriched RNA in order to study the repetitive structure of profilaggrin. These clones hybridize to high molecular weight epidermal mRNA (23 kilobase pairs, rat and 19 kilobase pairs, mouse) and exhibit limited cross-hybridization between species. Several rat clones direct the synthesis of a portion of rat profilaggrin in bacteria. One of these, rat profilaggrin cDNA clone R4D6, is 2400 base pairs in length. The R4D6 cDNA is shown to contain repetitive sequence by restriction mapping and southern hybridization analysis of restriction digests of this plasmid, using subfragments of the plasmid as hybridization probes. Southern hybridization analysis of rat genomic DNA, digested to completion with several restriction enzymes, reveals a simple hybridization pattern of fragments equal in size to those of the cDNA. Partial digestion of rat genomic DNA results in a ladder of bands based on a 1200-base pair repeat, equal to the size of the repeating unit of the cDNA clone, and consistent with the expected repeating size of profilaggrin. Together, these results show that the profilaggrin mRNA and gene have repetitive structure and that the gene apparently lacks introns in the coding region.     

Molecular cloning of complementary deoxyribonucleic acid for an androgen-regulated epididymal protein: sequence homology with metalloproteins

Acidic epididymal glycoprotein (AEG) is a 31,000 molecular weight secretory protein of the rat epididymis. Screening of a rat epididymal cDNA library with affinity-purified AEG antiserum yielded cDNA for AEG. Identity of the clones was verified by comparison of amino acid sequence of the purified protein with the sequence derived from the nucleotide sequence of the cDNA isolates. Two classes of AEG cDNA, approximately 1500 base pairs (bp) and 950 bp in length, differed by 538 bp in the 3'-untranslated region and by four single nucleotide mismatches, one of which was in the coding region. Northern blot hybridization of epididymal RNA revealed two species of AEG mRNA, corresponding in length to each type of cDNA. Analysis of RNA from individual animals provided evidence that the two mRNA species are the products of allelic genes. In vivo studies demonstrated that the level of total AEG mRNA is regulated by androgen. Amino acid sequence homology of AEG with metal-binding domains of several proteins suggests that AEG is a metalloprotein.     

Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.     

Regulation of gene expression in rat prostate by androgen and beta-adrenergic receptor pathways

Denervation of rat ventral prostate has been accomplished by excising prostatic tissue fragments and implanting them under the renal capsules of intact syngeneic rats. This resulted in a substantial reduction of expression of a major organ-specific secretory protein, prostatic binding protein (PBP). The depressed level of PBP and its subunits and mRNAs could be restored, however, to as much as 80% of control levels by the administration of a pharmacological dose of exogenous androgen, testosterone propionate (TP), and/or a beta-adrenergic agonist, isoproterenol (ISO). Furthermore, compared to ascorbate-treated controls, TP and ISO increased the synthesis of total cellular protein and PBP by the prostatic renal implants. TP and/or ISO also remodelled the luminal epithelial structure and elevated secretory functions. ISO alone had no effect, however, in castrated animals, indicating that androgen plays a dominant role in the restoration of tissue PBP content. Concomitant to increased PBP content and remodelling of prostatic histomorphology, androgen was also found to raise the depressed levels of beta 2-adrenergic and androgen receptors in the prostatic isografts maintained in intact hosts. In contrast, although an established rat prostatic epithelial cell line (NbE-1) contains high affinity androgen receptor, androgen failed to restore beta-adrenergic receptor as well as PBP content in this cultured cell line. These results, taken together, suggest that a tight coupling between androgen receptor and beta 2-adrenergic receptor pathways may be a prerequisite for PBP expression and functional differentiation in the rat ventral prostate gland.     

Xenopus laevis serum albumins are encoded in two closely related genes.

cDNA clones containing sequences complementary to Xenopus laevis albumin mRNA have been identified in a collection of cDNA clones made from poly(A)+ RNA prepared from male Xenopus laevis liver. Although all the albumin cDNA clones crosshybridise, restriction enzyme and heteroduplex analysis show that there are 2 closely related albumin mRNA sequences. The 2 albumin mRNAs are only mismatched by 8% but could be isolated by positive selection using stringent hybridization conditions. Oocytes injected with the 2 purified mRNAs, secreted either the 68,000 or 74,000 dalton albumin into the culture medium showing that the 2 albumins of X. laevis serum are encoded in the 2 closely related mRNAs. Measurements of the abundance of albumin mRNA show that the 2 albumin mRNAs together account for about 9% of total poly(A)+ RNA in male Xenopus laevis liver but the mRNA coding for the 74,000 dalton mRNA is about twice as abundant as that coding for the 68,000 dalton mRNA.     

Induction by cAMP of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. Identification and characterization of a cDNA clone for the enzyme

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.     

Effects of estradiol on prostate epithelial cells in the castrated rat.

There is evidence that estrogens can modulate the activity of prostate epithelial cells. To determine whether estradiol can have a direct influence on rat prostate, this study examined the effects of estradiol-17beta (E(2)) administered alone or in combination with dihydrotestosterone (DHT) to castrated rats for 3 weeks on prostate binding protein (PBP) C1 mRNA expression and androgen receptor (AR) localization. PBP C1 mRNA levels were measured by semi-quantitative in situ hybridization using a (35)S-labeled cDNA probe. In intact animals, strong hybridization signal could be observed in prostate sections after 12 hr of exposure to Kodak X-Omat films. In castrated rats, no PBP C1 mRNA could be detected even with longer exposure times, an effect that was prevented by administration of DHT. E(2) administered alone induced a detectable hybridization signal, and the concomitant administration of E(2) and DHT induced an increase in PBP C1 mRNA that significantly exceeded that obtained in animals that received only DHT. In prostate epithelial cells of intact animals, AR immunostaining was restricted to the nucleus. In castrated animals the alveoli were decreased in size and the epithelial cells were atrophied. AR staining was weak and was detected in both cytoplasm and nucleus. DHT administration completely obviated the effect of castration on epithelial cell histology and on AR immunostaining distribution and intensity. Interestingly, E(2) administration alone induced moderate hypertrophy of epithelial cells compared to the histological appearance of cells in untreated castrated rats. Moreover, in E(2)-treated animals the nuclear staining was much stronger than that detected in untreated castrated rats, whereas the cytoplasmic staining was not modified by the treatment. In animals that received both DHT and E(2), the staining was similar to that seen in DHT-treated rats. These results suggest that E(2) can influence the activity of rat prostate epithelial cells by mechanisms that remain to be fully clarified.     

Identification of two cDNA clones coding for androgen-dependent polypeptides in rat ventral prostate

cDNA clones were obtained by transformation of $\text{E. coli}$ x1776 with pBR322 containing insert of ds cDNA synthesized from total rat prostate poly(A) RNA. Two prostate-specific cDNA clones were isolated by colony hybridization and identified by message selection/translation as encoding polypeptides of M_r: 13,500 and 9,300. Hybridization of poly(A) RNA from normal and castrated rat prostates to the cloned cDNAs indicated that the levels of mRNAs coding for M_r: 13,500 and 9,300 polypeptides are regulated by testosterone.     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.     

Cytoplasmic RNA sequences complementary to cloned chick delta-crystallin cDNA show size heterogeneity.

Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.