Levels Of RNase Research Articles

Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E.

Polyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively. Here, we show that the half-lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp-lacZ and rne-lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5' untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase.

The global regulator RNase III modulates translation repression by the transcription elongation factor N.

Efficient expression of most bacteriophage lambda early genes depends upon the formation of an antiterminating transcription complex to overcome transcription terminators in the early operons, p(L) and p(R). Formation of this complex requires the phage-encoded protein N, the first gene product expressed from the p(L) operon. The N leader RNA contains, in this order: the NUTL site, an RNase III-sensitive hairpin and the N ribosome-binding site. N bound to NUTL RNA is part of both the antitermination complex and an autoregulatory complex that represses the translation of the N gene. In this study, we show that cleavage of the N leader by RNase III does not inhibit antitermination but prevents N-mediated translation repression of N gene expression. In fact, by preventing N autoregulation, RNase III activates N gene translation at least 200-fold. N-mediated translation repression is extremely sensitive to growth rate, reflecting the growth rate regulation of RNase III expression itself. Given N protein's critical role in lambda development, the level of RNase III activity therefore serves as an important sensor of physiological conditions for the bacteriophage.

RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters.

The rne gene of Escherichia coli encodes RNase E, an essential endoribonuclease that is involved in both mRNA decay and rRNA processing. Here we present evidence that the gene is transcribed from three promoters: p1, p2 and p3. The p2 and p3 promoters map 34 and 145 nt upstream from the previously characterized rne promoter, p1, generating unusually long 5' UTRs of 395 and 506 nt respectively. Based on promoter-lacZ transcriptional fusions, p1 is a more efficient promoter than either p2 or p3. Low copy number or single copy number vectors carrying rne transcribed from either p1, p2 or p3 alone complement the rne 1018::bla deletion mutation at 30 degrees C, 37 degrees C and 44 degrees C. However, normal autoregulation requires the presence of all three promoters. A comparison among intracellular levels of RNase E, the half-lives of the rpsO, rpsT and rne mRNAs, and growth rates, indicates that the cell contains a considerable excess of RNase E protein. In addition, when the rne transcript is stabilized at low RNase E levels, it is not efficiently translated.

Gene Expression Analysis in Human Monocytes, Monocyte-Derived Dendritic Cells, and α-Galactosylceramide-Pulsed Monocyte- Derived Dendritic Cells

In vitro proliferation and functional activation of Vα24NKT cells following stimulation with α-galactosylceramide (α-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with α-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with α-GalCer (α-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in α-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, α-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with α-GalCer, suggesting a novel immunomodulating effect of α-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNFα for two extra days.

The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227–427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT).We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244–322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.

Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A

Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMRNaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by alkaline lysis. The RNaseA protein is stable throughout incubation at high pH (∼12–12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following alkaline lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18Δ lacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements.

The RNase III Family: A Conserved Structure and Expanding Functions in Eukaryotic dsRNA Metabolism

The last few years have witnessed the appreciation of dsRNA as a regulator of gene expression, a potential antiviral agent, and a tumor suppressor. However, in spite of these clear effects on the cell function, the mechanism that controls dsRNA maturation and stability remains unknown. Recently, the discovery of eukaryotic orthologues of the bacterial dsRNA specific ribonuclease III (RNase III) suggested a central role for these enzymes in the regulation of dsRNA and eukaryotic RNA metabolism in general. This article reviews the structure-function features of the eukaryotic RNase III family and their roles in dsRNA metabolism with an emphasis on the yeast RNase III. Yeast RNase III is involved in the maturation of the majority of snRNAs, snoRNAs, and rRNA. In addition, perturbation of the expression level of yeast RNase III alters meiosis and causes sterility. These basic functions of the yeast RNase III appear to be widely conserved which makes it a good model to understand the importance of eukaryotic dsRNA metabolism.

Caspase-dependent apoptosis by 2',5'-oligoadenylate activation of RNase L is enhanced by IFN-beta.

The 2',5'-oligoadenylate (2-5A) system is an interferon (IFN)-regulated RNA decay pathway that provides innate immunity against viral infections. The biologic action of the 2-5A system is mediated by RNase L, an endoribonuclease that becomes enzymatically active after binding to 2-5A. RNase L is also implicated in mediating apoptosis in response to both viral and nonviral inducers. To study the cellular effects of RNase L activation directly, 2-5A was transfected into the human ovarian cancer cell line, Hey1B. Activation of RNase L by 2-5A resulted in specific 18S rRNA cleavage and induction of apoptosis, as measured by TUNEL and annexin V binding assays. In contrast, the dimeric form of 2-5A, ppA2'p5'A, neither activated RNase L nor caused apoptosis. Treatment with IFN-beta prior to 2-5A transfection enhanced cellular RNase L levels (< or = 2.2-fold) and increased the proportion of cells undergoing apoptosis (by < or =40%). However, rRNA cleavages after 2-5A transfections were not enhanced by IFN-beta pretreatments, indicating that basal levels of RNase L were sufficient for this activity. Apoptosis in response to RNase L activation was accompanied by cytochrome c release from mitochondria. Induction of apoptosis by either 2-5A alone or by the combination of 2-5A and IFN-beta was effectively blocked with either the pancaspase inhibitor, Z-VAD-fmk, or with the caspase 3 inhibitor, DEVD-fmk. Therefore, activation of RNase L by 2-5A leads to cytochrome c release into the cytoplasm and then to caspase activation and apoptosis. These results suggest potential uses for 2-5A in augmenting the anticancer activities of IFN.

Correlation between activity of ribonucleases and potato virus Y biosynthesis in tobacco plants

Tobacco plants, leaf discs and protoplasts infected with two strains of the potato virus Y (PVYOand PVYN) were studied during the period of the acute infection stage. The following characteristics of the host plants were monitored: dynamic changes in the ribonuclease (RNase) activities in relation to the virus multiplication; changes in subcellular localization of RNases, phosphomonoesterase (PME) and phosphodiesterase (PDE); and connection of RNase activities with host plants resistance. Activities of RNases in both the crude homogenates and the partially purified enzyme preparations from the diseased discs were markedly increased during the experimental period when compared to the discs from healthy plants. The courses of both curves were coincident and correlated with the multiplication curve of the PVYOand PVYNstrains. Changes in the content and in the subcellular localization of RNase, PME and PDE isozymes were determined in mesophyll protoplasts prepared from the healthy and PVYNinfected plants of Nicotiana tabacum L. cv. Samsun. In the chloroplast and cytosol fractions of the infected protoplasts, activities of RNase as well as PME and PDE were increased. The enhanced content of the RNase isozymes was also found in the infected leaves after electrophoretic separation. A linear correlation (P<0.001) was found between the content of the potato virus YNand the tobacco host resistance to virus multiplication. This was established on the basis of enhanced levels of RNases, using susceptible, tolerant and resistant plants.

Human pancreatic ribonuclease 1

Human pancreatic ribonuclease (RNase 1) is a pancreatic enzyme that is present at high levels in the serum of most patients with pancreatic adenocarcinoma. For this reason, the authors studied its patterns of expression at the single-cell level in pancreatic adenocarcinoma tissues by immunohistochemical analysis and in situ hybridization (ISH). Immunohistochemical analysis with polyclonal antibodies against RNase 1 and by ISH with digoxigenin-labeled RNase 1 probe were used to detect RNase 1 in the neoplastic cells of ductal type pancreatic adenocarcinomas. Fifteen of 18 carcinoma samples were positive for RNase 1, demonstrating that the expression of ribonuclease that the authors observed previously in human pancreatic adenocarcinoma cell lines was not an artifact of cell culture. The authors also found RNase 1 in some of the metaplastic ducts and atrophic islets in 4 of 6 chronic pancreatitis samples, and they observed RNase 1 immunostaining in hyperplastic ducts adjacent to one of the well-differentiated adenocarcinomas. The expression levels of RNase 1 by tumor cells from pancreatic adenocarcinomas are consistent with the high RNase 1 levels found in the serum of most patients with pancreatic adenocarcinoma. This expression of RNase 1, which is an acinar protein, demonstrates that the patterns of gene expression in pancreatic adenocarcinoma are distinct from those of normal pancreatic duct cells. Conversely, RNase 1 expression levels in altered ductal cells from some chronic pancreatitis tissues and hyperplastic ducts from carcinoma tissues suggest that abnormal expression levels may be an early event in pancreatic tumorigenesis.

Human pancreatic ribonuclease 1

BACKGROUND Human pancreatic ribonuclease (RNase 1) is a pancreatic enzyme that is present at high levels in the serum of most patients with pancreatic adenocarcinoma. For this reason, the authors studied its patterns of expression at the single-cell level in pancreatic adenocarcinoma tissues by immunohistochemical analysis and in situ hybridization (ISH). METHODS Immunohistochemical analysis with polyclonal antibodies against RNase 1 and by ISH with digoxigenin-labeled RNase 1 probe were used to detect RNase 1 in the neoplastic cells of ductal type pancreatic adenocarcinomas. RESULTS Fifteen of 18 carcinoma samples were positive for RNase 1, demonstrating that the expression of ribonuclease that the authors observed previously in human pancreatic adenocarcinoma cell lines was not an artifact of cell culture. The authors also found RNase 1 in some of the metaplastic ducts and atrophic islets in 4 of 6 chronic pancreatitis samples, and they observed RNase 1 immunostaining in hyperplastic ducts adjacent to one of the well-differentiated adenocarcinomas. CONCLUSIONS The expression levels of RNase 1 by tumor cells from pancreatic adenocarcinomas are consistent with the high RNase 1 levels found in the serum of most patients with pancreatic adenocarcinoma. This expression of RNase 1, which is an acinar protein, demonstrates that the patterns of gene expression in pancreatic adenocarcinoma are distinct from those of normal pancreatic duct cells. Conversely, RNase 1 expression levels in altered ductal cells from some chronic pancreatitis tissues and hyperplastic ducts from carcinoma tissues suggest that abnormal expression levels may be an early event in pancreatic tumorigenesis. Cancer 2000;89:1252–8. © 2000 American Cancer Society.

Control of Transfer RNA Maturation by Phosphorylation of the Human La Antigen on Serine 366

Conversion of a nascent precursor tRNA to a mature functional species is a multipartite process that involves the sequential actions of several processing and modifying enzymes. La is the first protein to interact with pre-tRNAs in eukaryotes. An opal suppressor tRNA served as a functional probe to examine the activities of yeast and human (h)La proteins in this process in fission yeast. An RNA recognition motif and Walker motif in the metazoan-specific C-terminal domain (CTD) of hLa maintain pre-tRNA in an unprocessed state by blocking the 5′-processing site, impeding an early step in the pathway. Faithful phosphorylation of hLa on serine 366 reverses this block and promotes tRNA maturation. The results suggest that regulation of tRNA maturation at the level of RNase P cleavage may occur via phosphorylation of serine 366 of hLa.

Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.

2-5A Antisense Directed against Telomerase RNA Produces Apoptosis in Ovarian Cancer Cells

Objective. RNase L is converted to an active form upon binding short 2′,5′-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase—an RNA–protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B.Methods. Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained.Results. Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death.Conclusions. The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.

The barrier-to-autointegration protein is a host factor for HIV type 1 integration.

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein-DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5-20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5-10 microM) human host factor HMG I(Y) was required. Similarly high levels (3-8 microM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein-DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.

Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes.

Ternary complexes of RNA polymerase containing the DNA template and nascent RNA are the intermediates in transcript elongation in all cells. We have footprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase. When complexes are treated with elevated levels of ribonucleases A and T1, the nascent transcript can be cleaved to within 3-4 nucleotides of the 3'-terminus. Ternary complexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an active ternary complex. However, cleavage by ribonuclease I is restricted, and gives a limited digest product of about 16 nt. At lower concentrations of ribonuclease T1, two regions of partial protection are seen. The first region extends through the first 15-16 nucleotides from the 3'-OH terminus; the second region extends from position 30 out to position 45. We interpret these regions of partial protection as defining two RNA product binding sites on the RNA polymerase that bind the product to the enzyme during elongation. Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length.

Impact of RNase L overexpression on viral and cellular growth and death.

The biologic actions of interferons (IFNs) are complex and involve multiple biochemical mechanisms, including the 2-5A system, a regulated RNA decay pathway. The 2-5A system is implicated in the antipicornavirus activity of IFN and in the control of apoptosis. To further investigate involvement of the 2-5A system in the control of viral and cellular growth and death, human RNase L cDNA was stably expressed in murine 3T3 cells from a constitutive cytomegalovirus (CMV) promoter. A clonal cell line, 3T3/pLZ, was isolated that overexpressed RNase L by >100-fold compared with levels of the endogenous murine RNase L. Interestingly, human RNase L levels in 3T3/pLZ cells decreased 3-fold as cells entered a confluent, growth arrest state, suggesting autoregulation. Overexpression of human RNase L greatly enhanced both the cell growth inhibitory activity of IFN and the proapoptotic activity of staurosporine. Furthermore, high levels of RNase L suppressed the replication of diverse viruses: encephalomyocarditis virus, vesicular stomatitis virus, human parainfluenza virus-3, and vaccinia virus. Additional reductions in viral growth were obtained by treating 3T3/pLZ cells with IFN (a + beta) before infections. These results directly demonstrate the anticellular and antiviral potential of the 2-5A system.

Potato virus-Y multiplication in susceptible tobacco cultivar and transgenic breeding line producing coat protein mRNA

Changes in ribonucleases (RNases) and glucose-6-phosphate dehydrogenase (G6P DH) activities, their content and subcellular localisation were studied in relation to virus multiplication in susceptible (cv. Samsun) or resistant (transgenic breeding line NCTG 83) tobacco plants infected with the potato virus YN (necrotic strain of PVY). Activities of RNases and G6P DH from diseased susceptible tobacco plants were markedly increased during the experimental period and significantly correlated with the multiplication curve of the PVYN. In contrast, the activities of RNases and G6P DH were not changed after PVY inoculation of resistant breeding line NCTG 83 producing the CP mRNA of PVY. Changes in the content and in the subcellular localisation of RNases and G6P DH isozymes were also determined in mesophyll protoplasts isolated from healthy as well as PVYN infected plants of both cultivars by differential centrifugation of broken protoplasts on day eight post inoculation (the culmination of multiplication curve of PVY and enhanced activity of both enzymes). The chloroplasts fraction from infected protoplasts showed an enhanced content of RNases (192.4% when compared with that from healthy control ones), and of G6P DH (174.4 %). The cytosol fraction from infected protoplasts contained slightly enhanced levels of G6P DH (117.4 %) and considerably enhanced levels of RNases (141.7 %). No significant differences in the activities, contents and subcellular localisation of RNases and/or G6P DH isozymes were observed in the resistant line NCTG 83. This is in accordance with no detectable contents of PVY.

Qualitative and quantitative analyses of seminal ribonuclease in reproductive tract fluids of bulls.

Bovine seminal ribonuclease (BS RNase) is synthetized in the ampulary gland and seminal vesicles of the bull as shown by indirect immunofluorescence and by using a sensitive sandwich-linked immunosorbent assay (ELISA). The ampullary and seminal vesicle BS RNase concentrations in samples of 15 bulls were 656 micrograms/ml and 1285 micrograms/ml, respectively. Seminal plasma BS RNase levels in 22 breeding bulls were slightly lower than in the seminal vesicles--1132 micrograms/ml. There were no significant differences in the concentration of this enzyme in seminal plasma of 16 bulls ranked as having high and low fertility. Even with its immunosuppressive activity this enzyme does not seem important for the protection of sperm cells in the female reproductive tract.

Regulation of human immunodeficiency virus replication by 2',5'-oligoadenylate-dependent RNase L.

Activation of RNase L by 2',5'-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1 nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.