Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.
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PMID:RNA polymerase activity in bovine spermatozoa. 2 Apr 46

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids.
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PMID:Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro. 34 18

Spermatogenesis is a complex developmental process which sequentially generates several different germ cell types. These cell types from rainbow trout (Salmo gairdnerii) testis were separated by sedimentation in serum albumin gradients and characterized on the basis of their physical properties, chronological appearance, and protein synthesis. The rate of RNA synthesis, the types of RNA made, and the RNA polymerase activities present were determined for each cell type. The rate of RNA synthesis decreased from a high level in spermatogonia and spermatocytes to a low level in early spermatids and was absent in late spermatids and mature spermatozoa. Newly synthesized RNA in spermatogonia and spermatocytes consisted of a variety of molecular weight species, including 18 S and 28 S ribosomal RNAs. The synthesis of high molecular weight RNAs, especially ribosomal RNAs, decreased drastically in early spermatids, leading to the synthesis of only small molecular weight RNAs. RNA polymerase I and II were present in all cell types but the activities of both showed large decreases between spermatocytes and middle spermatids. Both RNA polymerase activities were almost absent from spermatozoa. The activities of RNA polymerase I and II from unfractionated testis cells at different stages of hormone-induced spermatogenesis were quantitated by fractionation of the solubilized extract on DEAE-cellulose. Both polymerases showed major decreases in activity which began near the chronological mid-point of development. For polymerase I the decrease in activity was over 400 fold, for polymerase II over 200 fold. The number of RNA polymerase II molecules per testis cell, quantitated by the binding of [3H]amanitin to cell extracts, also decreased markedly during spermatogenesis. The reduction in polymerase II activity was accompanied by a parallel 200-fold decrease in[3H]amanitin binding. The reduction in polymerase activity appears, therefore, to be due to an actual reduction in the cellular content of RNA polymerase II molecules. These results suggest that transcription in maturing testes is regulated, at least in part, by the concentrations of the RNA polymerases.
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PMID:RNA synthesis and RNA polymerase activities in germ cells of developing rainbow trout testis. 51 81

In nuclei and nucleoli of the slime mold Physarum polycephalum, ornithine decarboxylase (OrnDCase) (Mr 70,000) is phosphorylated by a protein kinase reaction that is dependent on spermidine and spermine. Putrescine antagonizes the phosphorylation. Phosphorylation of OrnDCase inhibits its capacity to catalyze decarboxylation of ornithine. The protein kinase that catalyzes this phosphorylation has many properties similar to those of nuclear protein kinase II, or type G, which has been studied by other groups. The interaction of this protein kinase with OrnDCase resembles the behavior of the OrnDCase antizyme described by other investigators. Phosphorylated OrnDCase binds to purified, palindromic rDNA isolated from nucleoli. It also stimulates transcription of the ribosomal genes by RNA polymerase I in a chromatin form of rDNA. It does not stimulate transcription in a purified, homologous transcription system comprised of RNA polymerase I, rDNA, and phospho-OrnDCase. Thus, phospho-OrnDCase may have a function in promoting rRNA gene transcription but the detailed mechanism is yet unclear. The polyamine-dependent protein kinase and its natural substrate of 70,000 daltons have been demonstrated in other eukaryotic cells, including bovine spermatozoa and rat liver nuclei, and in Ehrlich ascites tumor cells, where the protein kinase is induced by interferon. This phosphorylation system appears to be widely distributed and conserved among eukaryotic species.
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PMID:Posttranslational control of ornithine decarboxylase by polyamine-dependent protein kinase. 714 Oct 3

In order to investigate the role of germ cells in the sexual transmission of immunodeficiency virus (HIV), spermatozoa from healthy HIV-seronegative men were incubated in vitro with HIV1. After washing, they were cocultured with peripheral blood leukocytes from seronegative blood donors. Reverse transcriptase assays and p24 antigen tests were performed in culture supernatants. Electron microscopy examination of these HIV-incubated spermatozoa was carried out, as well as the search for CD4 molecules on their surface. Although virus bound to and seemed to enter spermatozoa despite the absence of detectable CD4 epitopes on their surface, no replication of HIV was apparent. However, HIV particles on the surface of spermatozoa were capable of infecting CD4 T lymphocytes. Present results would seem to preclude artificial insemination between an HIV-seropositive man and an HIV-seronegative woman.
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PMID:Spermatozoa as potential carriers of HIV. 814 Feb 92

Previous studies from our laboratory have shown that messenger RNAs (mRNAs) coding for a cAMP-specific phosphodiesterase (PDE4A) are present in mature rat and mouse germ cells. However, no information is available about the properties of the expressed proteins. To determine their structure and regulation, the PDE4A isoforms expressed in the rat testis were identified and compared to the variants expressed in the brain. Western blot analysis using an antiserum specific for PDE4A demonstrated the presence in testis extracts of two distinct proteins with apparent masses of 98.8 and 86 kDa. The electrophoretic mobilities of these proteins differ from those of proteins detected in the brain extracts (113 and 76 kDa). Reverse transcriptase-PCR of the different splicing mRNA variants expressed in testis confirmed the presence of at least one novel PDE4A mRNA that is distinct from the PDE4A splicing variants identified in the brain and other tissues. Expression of the complementary DNA encoding this variant in a heterologous system resulted in an increase in PDE activity and the appearance of an immunoreactive protein with a mass of 98.8 kDa. No 86-kDa protein could be generated with this transfection. Upon fractionation of testis extracts by HPLC diethylaminoethyl-chromatography, a peak of cAMP-PDE activity coeluted with the two immunoreactive species. During testicular development, the 98.8-kDa protein is present in trace amounts at 10 days, and its level increases with the age of the animals, reaching a plateau at 40 days. The 86-kDa protein appears at 20 days of age and reaches its maximum at 40 days. Studies on the cellular site of expression demonstrated that the two polypeptides are most abundant in round spermatids and are expressed in trace amounts in pachytene spermatocytes, whereas they could not be detected in Sertoli or interstitial cells. The 98.8-kDa, but not the 86-kDa, protein was also expressed in epididymal spermatozoa. These data demonstrate the expression of novel cAMP-specific PDEs coded by the PDE4A gene. The expression of these isoforms is maximal in round spermatids and is maintained in mature spermatozoa. The genesis of the lower mol wt species remains to be determined.
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PMID:Developmental regulation of unique adenosine 3',5'-monophosphate-specific phosphodiesterase variants during rat spermatogenesis. 864 Dec

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.
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PMID:Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm. 891 43

Human membrane cofactor protein (MCP) is a widely distributed cell-associated complement-regulatory protein, and recent findings suggest that MCP may be involved in sperm-egg interaction. We have isolated four cDNA clones and one reverse transcriptase-PCR product homologous to human MCP from guinea pig testis. These clones defined five isoform classes generated from a single copy gene by alternative splicing. Reverse transcriptase-PCR revealed that two classes for the clones termed GMP1 and GM2 were predominant. GMP1 consisted of four short consensus repeats (SCRs), regions corresponding to the human serine/threonine/proline-rich C (STP(C)) domain and a human region of unknown significance, a hydrophobic region presumed to be a transmembrane domain, and a cytoplasmic region. Identity with human MCP in the SCR region was 56% at the amino acid level and 71% at the nucleotide level. GM2 had the same structure as GMP1, except that it lacked the fourth SCR, which is presumed to be essential for C3b binding of human MCP. Northern blotting analysis of various tissues revealed a significant level of MCP transcripts in testis. Guinea pig MCP is likely to have only one STP domain that is homologous to human STP(C) and is similar in this respect to human spermatozoa MCP. Gene analysis revealed a single base deletion and a lack of consensus sequences for splicing in the guinea pig regions corresponding to human STP(A) and STP(B), respectively. These results suggest that guinea pig MCP plays a more restricted role in reproduction than does human MCP.
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PMID:Molecular cloning of guinea pig membrane cofactor protein: preferential expression in testis. 894

Run-on transcription experiments with sonicated chromatin from spermatozoa of healthy men revealed the presence of in vitro transcription. The number of actively transcribing RNA polymerase B molecules in sperm chromatin in vitro, isolated from healthy individuals, and the effect of prostaglandin El, were investigated. The results indicate that the number of RNA polymerase B molecules actively engaged in transcription increased one and a half times when 5 units of prostaglandin E1 was added to the reaction mixtures. These results correlate well with the authors' previous findings that the incorporation of labelled uridine triphosphate into pre-mRNA was higher when prostaglandin E1 was added to the reaction mixtures in run-on transcription experiments, and when sperm chromatin from healthy individuals was used as a template.
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PMID:Quantification of actively transcribing RNA polymerase B molecules in human sperm chromatin and the effect of prostaglandin E1. 924 19

We have investigated the influence of Prostaglandin E1 on the in vitro transcription of chromatin, isolated from spermatozoa of patients suffering from different pathologies, leading to infertility, namely, azoospermia, teratospermia and chronic prostatitis. Our studies indicate that prostaglandin E1 has a stimulatory effect both on in vitro transcription, on the number of RNA polymerase molecules and the polyribonucleotide elongation rates as compared to sperm chromatin from healthy patients. The results on the incorporation of alpha-32P-ATP in to RNA in the presence and absence of Prostaglandin E1 correlate well with the data on the number of actively transcribing RNA polymerase molecules and the rate of RNA elongation, which might be due to low levels of prostaglandin E1 in human semen.
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PMID:The effect of prostaglandin E1 on in vitro transcription of sperm chromatin, isolated from patients with azoospermia, teratospermia and chronic prostatitis. 967 33


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