scholarly journals Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens

1974 ◽  
Vol 137 (3) ◽  
pp. 513-524 ◽  
Author(s):  
W. Ian P. Mainwaring ◽  
Peter A. Wilce ◽  
Allan E. Smith
Keyword(s):  
Prostate Gland ◽  
Sodium Dodecyl ◽  
Ventral Prostate ◽  
Experimental Conditions ◽  
Free System ◽  
Cell Free System ◽  
Messenger Ribonucleic Acid ◽  
Selective Incorporation

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6–15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5′-fluoro-orotic acid into this 6–15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6–15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6–15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.

scholarly journals Specific changes in the messenger ribonucleic acid content of the rat ventral prostate gland after androgenic stimulation. Evidence from the synthesis of aldolase messenger ribonucleic acid

1974 ◽  
Vol 144 (2) ◽  
pp. 413-426 ◽  
Author(s):  
W I P Mainwaring ◽  
F R Mangan ◽  
R A Irving ◽  
D A Jones
Keyword(s):  
Ribonucleic Acid ◽  
De Novo ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Mrna Synthesis ◽  
Hormonal Stimulation ◽  
Messenger Ribonucleic Acid ◽  
Stimulation Of

1. Aldolase was selected as a suitable marker for following the androgenic regulation of mRNA synthesis in the prostate gland. 2. Antibodies raised in rabbits against crystalline prostate aldolase were used to monitor the synthesis of this androgen-induced enzyme after hormonal stimulation of castrated animals, by using procedures in vivo and in vitro for the translation of prostate poly(A)-rich mRNA. 3. After androgenic stimulation in vivo the poly(A)-rich mRNA was isolated from the prostate gland and other tissues of castrated rats, and added to a protein-synthesizing system in vitro derived from Krebs II ascites-tumour cells. By using this approach it was found that androgens regulate the synthesis of aldolase mRNA in a highly tissue-specific manner. Stimulation of aldolase mRNA synthesis reached a maximum after 8h of androgenic treatment and then declined. 4. The androgenic control of aldolase mRNA synthesis was also investigated in vivo. After treatment of castrated animals with various steroids in vivo [35S]methionine was injected directly into the prostate gland, and labelled aldolase was selectively precipitated from isolated polyribosomes with anti-aldolase serum. The regulation of aldolase mRNA synthesis in the prostate gland was stringently steroid-specific and could only be evoked by androgens. After a single injection of testosterone, aldolase synthesis reached a maximum after 16h of hormonal stimulation and then declined. 5. Although androgens exert significant control over transcriptional processes in the prostate gland, and appear to regulate the synthesis of aldolase mRNA de novo, the possibility exists for additional means of control at the translational level of aldolase synthesis. The results are discussed in the context of the overall mechanism of action of androgens.

scholarly journals A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

1971 ◽  
Vol 125 (1) ◽  
pp. 285-295 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
Brenda M. Peterken
Keyword(s):  
Prostate Gland ◽  
Ventral Prostate ◽  
Free System ◽  
Tissue Specific ◽  
Receptor Complexes ◽  
Rat Ventral Prostate ◽  
Reconstituted System ◽  
Androgenic Steroids

1. A system has been developed for the specific transfer of [3H]dihydrotestosterone–receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [3H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [3H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.

THE BINDING OF [1,2-3H]TESTOSTERONE WITHIN NUCLEI OF THE RAT PROSTATE

1969 ◽  
Vol 44 (3) ◽  
pp. 323-333 ◽  
Author(s):  
W. I. P. MAINWARING
Keyword(s):  
Molecular Weight ◽  
Equilibrium Dialysis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Rat Tissues ◽  
Receptor Complex ◽  
Rat Prostate ◽  
Binding Component

SUMMARY The specificity of the binding of [1,2-3H]testosterone to nuclei of various rat tissues in vivo has been studied. A significant amount of radioactivity was retained in the nuclei of androgen-dependent tissues only, particularly the ventral prostate gland. The bound radioactivity was only partially recovered as [1,2-3H]testosterone; the remainder was identified as [3H]5α-dihydrotestosterone. Efforts were made to characterize the binding component, or 'receptor', in prostatic nuclei. On digestion of nuclei labelled in vivo with [1,2-3H]testosterone, with enzymes of narrow substrate specificity, only trypsin released tritium, suggesting that the receptor is a protein. On the basis of subfractionation studies of labelled nuclei, the receptor is an acidic protein. The androgen—receptor complex could be effectively extracted from the prostatic nuclei in 1 m-NaCl and from the results of fractionations on a calibrated agarose column, the complex has a molecular weight 100,000–120,000. The specificity of the binding of steroids to such 1 m-NaCl extracts in vitro was investigated by the equilibrium dialysis procedure. Under these conditions, the specificity of the binding of [1,2-3H]testosterone demonstrated in vivo could not be simulated. The receptor is probably part of the chromatin complex but its precise intranuclear localization cannot be determined by biochemical procedures alone.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Vijayalakshmi NAGARAJ ◽  
David NORRIS
Keyword(s):  
Homologous Recombination ◽  
Specific Activity ◽  
Yeast Cells ◽  
Active Components ◽  
Free System ◽  
Cell Free System ◽  
Molecule Formation ◽  
A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

scholarly journals Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

1985 ◽  
Vol 5 (6) ◽  
pp. 1238-1246 ◽  
Author(s):  
J J Li ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Dna Molecules ◽  
Free System ◽  
Cell Free System ◽  
Cell Extracts ◽  
Sv40 Origin

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

scholarly journals Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651 ◽  
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication

1985 ◽  
Vol 5 (6) ◽  
pp. 1238-1246
Author(s):  
J J Li ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Dna Molecules ◽  
Free System ◽  
Cell Free System ◽  
Cell Extracts ◽  
Sv40 Origin

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

scholarly journals Studies on the solubilized ribonucleic acid polymerase from rat ventral prostate gland

1971 ◽  
Vol 123 (4) ◽  
pp. 619-628 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
F. R. Mangan ◽  
B. M. Peterken
Keyword(s):  
Time Course ◽  
Prostate Gland ◽  
Exchange Chromatography ◽  
Ventral Prostate ◽  
Enzyme Preparations ◽  
Rat Ventral Prostate ◽  
Rapid Stimulation ◽  
Androgenic Steroids

1. By using ultrasonic treatment in media of high ionic strength, the RNA polymerase activities associated with prostatic nuclei and nucleoli can be completely solubilized. Such enzyme preparations are entirely dependent on the provision of added DNA for full activity. 2. The solubilized enzymes from the nucleolar and extranucleolar regions can be separated by ion-exchange chromatography. 3. Based on differences in the optimum DNA templates, pH optima and the effects of ammonium sulphate on the activities in vitro, Mn2+- and Mg2+-specific enzymes are associated with both the nucleolar and extranucleolar regions of prostatic nuclei. 4. Androgenic hormones administered in vivo have a particularly pronounced effect on the activity of Mg2+-dependent enzyme associated with the isolated prostatic nucleolus. 5. Time-course experiments in vivo show that androgens induce a rapid stimulation of the solubilized Mg2+-dependent nucleolar enzyme before a pronounced activation of nucleolar chromatin can be measured. 6. The implications of these findings to the mechanism of action of androgenic steroids are discussed.

scholarly journals In vitro synthesis of pp60v-src: myristylation in a cell-free system.

1988 ◽  
Vol 8 (10) ◽  
pp. 4295-4301 ◽  
Author(s):  
I Deichaite ◽  
L P Casson ◽  
H P Ling ◽  
M D Resh
Keyword(s):  
Amino Acids ◽  
Synthetic Peptide ◽  
Covalent Attachment ◽  
Free System ◽  
In Vitro Synthesis ◽  
Cell Free System ◽  
Reticulocyte Lysates ◽  
A Cell

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.

Sign in / Sign up

Export Citation Format

Share Document