Decrease In RNA Synthesis Research Articles

The interaction of cellular protein ANP32A with influenza A virus polymerase component PB2 promotes vRNA synthesis

The subunits PA, PB1, and PB2 of influenza A virus RNA polymerase are essential for efficient viral RNA synthesis and virus replication because of their role in recruiting multiple nuclear proteins. ANP32A is an acidic leucine-rich nuclear phosphoprotein 32 (ANP32) family member and a crucial cellular protein that determines the species specificity of the influenza virus RNA polymerase activity. However, how ANP32A modulates polymerase activity remains largely unknown. In this study, we showed that viral RNA synthesis was increased in A549 cells overexpressing ANP32A and decreased after treatment with ANP32A RNAi. This decrease in RNA synthesis was reversed by rescued ANP32A expression. The results of docking modeling, co-immunoprecipitation, and yeast two-hybrid assays showed that PB2 was the only subunit of the three that interacted with ANP32A. The C-terminal portion of ANP32A and the middle domains (residues 307-534) of PB2 were required for PB2-ANP32A interaction. Glu189 and Glu196 in ANP32A and Gly450 and Gln447 in PB2 were essential for interaction between ANP32A and PB2. These residues were located in conserved regions of the ANP32A or PB2 protein sequences. These data suggest that ANP32A is recruited to the polymerase through direct interaction with PB2 via critical amino acid residue interactions and promotes viral RNA synthesis. Our findings might provide new insights into the molecular mechanisms underlying influenza virus RNA synthesis and replication in infected human cells.

A changing paradigm of transcriptional memory propagation through mitosis.

The highly reproducible inheritance of chromosomes during mitosis in mammalian cells involves nuclear envelope breakdown, increased chromatin compaction, loss of long-range intrachromosomal interactions, loss of enhancer-promoter proximity, displacement of many transcription regulators from the chromatin and a marked decrease in RNA synthesis. Despite these dramatic changes in the mother cell, daughter cells are able to faithfully re-establish the parental chromatin and gene expression features characteristic of the cell type. Pioneering studies of mitotic chromatin signatures showed that despite global repression of transcription, the Hsp70 gene promoter retains an open chromatin conformation, which was proposed to allow the reactivation of the Hsp70 gene upon completion of mitosis - a phenomenon termed mitotic bookmarking. It was later shown that various cell-type-specific transcription factors, such as GATA-binding factor 1 (GATA1) in erythroblasts and forkhead box protein A1 (FOXA1) in hepatocytes, remain bound at a subset of their interphase binding sites in mitosis. Such bookmarking transcription factors remain on chromosomes in mitosis and have been shown to enable a subset of genes to be reactivated in a timely fashion upon mitotic exit. In addition, sensitive new methods to measure transcription revealed that mitotic cells retain residual transcription at a large number of genes. Furthermore, genes recover their interphase level oftranscription in distinct waves. Thus, gene expression is precisely regulated as cells pass through mitosis to ensure faithful propagation of cell identity and function through cellular generations.

Solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral RNA.

Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation.

Abstract 586: Targeting Pim kinases by SGI-1776 in mantle cell lymphoma

Abstract Proviral integration site for Moloney murine leukemia virus (PIM) kinases are oncoproteins that promote tumor progression. Pim-1, -2 and -3 are highly conserved Ser/Thr kinases that are important for normal B-cell development and are over-expressed in B-cell malignancies such as mantle cell lymphoma (MCL). Pim kinases phosphorylate a number of substrates; including: cMyc and Histone H3, which drive transcription, eukaryotic elongation factor 4E-BP-1 that regulates translation and Bad that activates cell survival. Furthermore, cell cycle protein activation by Pim kinase is involved with proliferation. An imidazo-[1, 2-b]-pyridazine compound, SGI-1776 (SuperGen) has high inhibitory specificity to Pim kinases at nM levels. Based on this knowledge we hypothesized that Pim kinase are druggable targets in MCL and inhibition of Pim kinase-mediated phosphorylation of downstream substrates will disrupt transcriptional and translational processes and trigger apoptosis. To test this hypothesis, Pim kinase expression profile and actions of SGI were studied in MCL cell lines, Jeko and Mino. All three Pim kinase were expressed in both cell lines. SGI was used in concentration (0.1 to 10 µM) and time dependent (up to 24 hrs) fashion. Treatment with SGI resulted in decreased Pim-1 and -3 protein levels in both cell lines; however Pim-2 protein was declined only in Jeko. Pim kinase pathways were investigated to identify mechanism of action of SGI. In both Jeko and Mino, total and phospho cMyc (Ser62) protein expression decreased at 3 and 10 µM, respectively. Interestingly, Histone H3 (Ser10) phosphorylation decreased only in Jeko. Consistent with cMyc data, there was 50% decrease in RNA synthesis (uridine incorporation) at 5 µM SGI in Jeko and 10 µM in Mino. When evaluated in Mino cells, 4E-BP-1 was not affected up to 3 µM of SGI. SGI inhibition on transcription/translation in principle would affect short-lived proteins such as Mcl-1 that are survival factors. Significant decreases of Mcl-1 mRNA (80% and 50%) and protein (50% and 40%) levels were observed in Jeko and Mino at 3 and 10 µM, respectively. In contrast, other anti-apoptotic proteins Bcl-2, Bcl-XL and XIAP were not affected by SGI treatment. In concordance with the survival role of Mcl-1, apoptosis (Annexin V+) was induced at 3, 5 and 10 µM SGI (15%, 30% and 80%) in both cell lines. Similarly, PARP cleavage was detected. Total and phospho Bad (Ser112) protein was decreased at 3 µM in Jeko, suggesting a role of this Pim substrate in apoptosis. Consistent with the observations in cell lines, there was induction of cell death in primary MCL cells from patients (n = 5). Preliminary data suggest inhibition of RNA synthesis in these malignant lymphocytes. Taken together, Pim kinases are potential therapeutic targets in MCL and SGI-1776 is effective to inhibit global RNA synthesis, as well as inducing apoptosis. MCl-1 appears important factor in Pim kinase pathway and warrant more detailed investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 586. doi:10.1158/1538-7445.AM2011-586

Differential Responses of Bacillus subtilis rRNA Promoters to Nutritional Stress

The in vivo expression levels of four rRNA promoter pairs (rrnp(1)p(2)) of Bacillus subtilis were determined by employing single-copy lacZ fusions integrated at the amyE locus. The rrnO, rrnJ, rrnD, and rrnB promoters displayed unique growth rate regulation and stringent responses. Both lacZ activity and mRNA levels were highest for rrnO under all growth conditions tested, while rrnJ, rrnB, and rrnD showed decreasing levels of activity. During amino acid starvation induced by serine hydroxamate (SHX), only the strong rrnO and rrnJ promoters demonstrated stringent responses. Under the growth conditions used, the rrn promoters showed responses similar to the responses to carbon source limitation induced by α-methyl glucoside (α-MG). The ratio of P2 to P1 transcripts, determined by primer extension analysis, was high for the strong rrnO and rrnJ promoters, while only P2 transcripts were detected for the weak rrnD and rrnB promoters. Cloned P1 or P2 promoter fragments of rrnO or rrnJ were differentially regulated. In wild-type (relA(+)) and suppressor [relA(S)] strains under the conditions tested, only P2 responded to carbon source limitation by a decrease in RNA synthesis, correlating with an increase in (p)ppGpp levels and a decrease in the GTP concentration. The weak P1 promoter elements remain relaxed in the three genetic backgrounds [relA(+), relA, relA(S)] in the presence of α-MG. During amino acid starvation, P2 was stringently regulated in relA(+) and relA(S) cells, while only rrnJp(1) was also regulated, but to a lesser extent. Both the relA(+) and relA(S) strains showed (p)ppGpp accumulation after α-MG treatment but not after SHX treatment. These data reveal the complex nature of B. subtilis rrn promoter regulation in response to stress, and they suggest that the P2 promoters may play a more prominent role in the stringent response.

Abstract 1649: Pim kinase inhibitor, SGI-1776, induces apoptosis in acute myeloid leukemia primary cells

Abstract Small molecule SGI-1776 was found to be potent and selective inhibitor of Pim family serine-threonine kinases [IC50: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM)] without effects on other kinases except Flt-3 and haspin. In preclinical studies in chronic lymphocytic leukemia, SGI-1776 treatment resulted in apoptosis induction that was associated with a decrease in RNA synthesis, Mcl-1 protein level, and c-Myc phosphorylation (Chen, LS et al, Blood 2009, 114: 4150). Increased levels of Pim-2 are reported in acute myeloid leukemia (AML) as well as Flt-3 activating mutation, providing a rationale to test SGI-1776 in AML. We evaluated SGI-1776 using FLT3-ITD AML cell lines (MOLM-13, MV4-11), xenograft models, and in primary cells from AML patients, and our results indicate that SGI-1776 is effective in AML. In MOLM-13 and MV4-11 cell lines, the IC50 for viability was < 10 nM after treatment with SGI-1776 for 72 h, which was associated apoptosis induction and reduction in phosphoBad (Ser112) and Bad protein. In xenograft models of both cell lines, treatment with 135mg/kg SGI-1776 resulted in a consistent decline in tumor volume after 14 days. To further these investigations in primary cells, we obtained cells from AML patients including FLT-3 positive and mutated FLT3-ITD patients, and evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of AML cells (n = 4) with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 5%,12% and 19% respectively, measured using Annexin-staining compared with untreated cells. Apoptosis induction was verified via PARP cleavage immunoblot, and apoptosis was observed in cells from FLT3 negative or mutated FLT3-ITD patients. In the samples evaluated, there was disparity in cytogenetic factors, Flt-3 status and prior chemotherapeutic treatment history. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. There was no observed change in phosphorylation of Pim targets histone H3 (Ser10) or p21 (Thr145), however protein levels of both total and phospho-Bad(Ser112) decreased following SGI-1776 treatment. In contrast, there was no change in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and levels of pro-apoptotic proteins, Bak and Bax, were also unaffected. However, there was a decrease in phospho-c-Myc(Ser 62), which is a Pim target and associated with stabilization of c-Myc protein. Consequently, we evaluated the potential inhibition of c-Myc driven transcription by SGI-1776 by measuring total RNA synthesis. Following treatment with 1, 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to 75%, 42% and 25% of control respectively, measured using a radioactive uridine incorporation assay (n = 4). Taken together, SGI-1776 was demonstrated to be an effective agent against AML in cell lines, xenograft models, and primary cells, and may be an effective new agent in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1649.

Mechanism of Action of SNS-032, a Novel Cyclin Dependent Kinase Inhibitor, in Chronic Lymphocytic Leukemia: Comparison with Flavopiridol.

Cyclin dependent kinases (Cdks) not only drive cell cycle progression, but also control transcription. For example, Cdk7 and Cdk9 phosphorylate specific sites on the C-terminal domain of RNA polymerase II (pol II) to promote transcription initiation and elongation. Our previous experience with flavopiridol, a non-selective pan-Cdk inhibitor, in primary chronic lymphocytic leukemia (CLL) cells demonstrated inhibition of RNA pol II-driven mRNA synthesis, reduction of the expression of short lived anti-apoptotic proteins, and induction of apoptosis in CLL cells in vitro (Blood 106:2513–19, 2005). Here we studied a novel Cdk inhibitor SNS-032, a 2-aminothiazole derivative with potent and selective inhibitory activity against Cdk2, Cdk7 and Cdk9. We hypothesized that similar to flavopiridol, SNS-032 would induce apoptosis in CLL cells through transcriptional inhibition of anti-apoptotic proteins. To test this hypothesis, we first studied toxicity of SNS-032 in primary CLL samples. At the clinically relevant concentration of 0.3 μM, SNS-032 induced 61 ± 13% apoptosis after 24 hr (mean ± S.D., n=19). This occurred in a time- and concentration-dependent manner that plateaued after 10–12 hr. Additional studies demonstrated this was not due to inactivation of SNS-032. A similar amount of apoptosis was induced in samples with a deletion of p53 gene, indicating a p53-independent cell death. This is important since clinical resistance to fludarabine in CLL is associated with loss of p53 function. Peripheral blood mononuclear cells from healthy donors were less sensitive to SNS-032, suggesting SNS-032 is selective against CLL cells. In direct comparisons SNS-032 was more potent than flavopiridol; 0.1 μM SNS-032 induced a similar amount of apoptosis as did 3 μM flavopiridol. Secondly, we investigated the inhibition of transcription by SNS-032. SNS-032 strongly inhibited the phosphorylation of pol II at both the Ser2 and Ser5 sites of its C-terminal domain, which are the substrates of Cdk9 and Cdk7, respectively. Inhibition of pol II function was associated with a decrease in RNA synthesis, measured by the incorporation of [3H]uridine. After 6 hr incubation, 0.1 μM SNS-032 inhibited 80% of RNA synthesis. Again, SNS-032 was about 30-fold more potent than flavopiridol at inhibiting transcription. As the most sensitive targets of transcription inhibitors are mRNAs and proteins with short intrinsic half-lives, we measured the mRNA levels of short-lived anti-apoptotic proteins by real-time RT-PCR. SNS-032 significantly reduced the mRNA levels of Mcl-1, XIAP and Bcl-2. The protein levels of Mcl-1 and XIAP were also reduced accordingly, whereas Bcl-2 protein remained stable, consistent with a longer half-life of the protein. As CLL cells exemplify a biological context of a tumor that is dependent upon the continued expression of anti-apoptotic proteins for survival, we conclude that inhibition of their expression led to the SNS-032-induced cell death. Thus, while the inhibition of transcription induced by both SNS-032 and flavopiridol is associated with apoptosis in CLL cells, SNS-032 is about 30-fold more potent. This may be due to differences in potency against Cdks and/or differential protein binding. These in vitro results provide rationale for a phase 1 trial of SNS-032 administered to patients with advanced B-lymphoid malignancies that is currently ongoing in multiple centers.

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually approximately 15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.

Relationship between mRNA levels and protein accumulation in a chloroplast promoter-mutant of Chlamydomonas reinhardtii

The photosynthetic chloroplast mutant G64 of Chlamydomonas reinhardtii was shown to contain a single point mutation within the 5' region of the psbD gene encoding the D2 protein of the photosystem II reaction center. The mutation affects the sequence element TATAATAT which has previously been hypothesized to function as the psbD promoter. Run-on analysis confirmed that transcription of psbD in the mutant was reduced to approximately 10% of the wild-type level. However, psbD mRNA accumulated to approximately 35%, despite the prominent decrease in RNA synthesis. This suggests that RNA-stabilization effects can compensate to some extent for a reduction in transcriptional activity. Interestingly, a direct correlation between transcript levels and the accumulation of the psbD gene product, the D2-protein, was observed in G64. The data suggest that posttranscriptionally acting regulatory factors determine the rate-limiting steps of chloroplast psbD gene expression.

Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

Влияние метилирования ДНК на изменение структуры хроматина при тиреоидной патологии

Impaired regulation of thyroglobin (TG) gene expression is observed in various forms of thyroid pathology. The impairment in question can be explained by the transcription events that depend not only on the primary sequence of nucleotides in the DNA structure, but also on structural and functional alterations in the chromatin of thyroid cells. DNA methylation is the mechanism regulating TGgene transcription. It has a significant effect on the structure of DNA itself and on DNAase-sensitive sites, as well as on the interaction of DNA with various proteins including RNA polymerase. In the context the yield of hydrolyzed DNA from the nuclei, DNAase I-hypersensitivity of methylated chromatin in the nuclei of thyroid cells was 10% while that of поп-methylated chromatin n isolated nuclei was 50%. In nodal euthyroid goiter, DNAase I-hypersensitivity of methylated chromatin was 7%> and that of nonmethylated one was 10%. In congenital goiter, the hypersensivity of поп-methylated chromatin in the isolated nuclei was 0. An investigation of the RNA-polymerase activity of thyroid cellular nuclei under the influence of methylation indicated a substantial decrease in RNA synthesis irrespective of the type of an abnormality.

Targeted Disruption of the GAS41 Gene Encoding a Putative Transcription Factor Indicates That GAS41 Is Essential for Cell Viability

The glioma-amplified sequence (GAS) 41 protein has been proposed to be a transcription factor. To investigate its functional role in vivo, we attempted to knock out the GAS41 gene by targeted disruption in the chicken pre-lymphoid cell line DT40. Heterozygous GAS41+/- cell lines generated by the first round of homologous recombination express approximately half the normal level of GAS41 mRNA. However, a homozygous GAS41-/- cell line with both GAS41 alleles disrupted was not obtained following the second round of transfection, indicating that the GAS41 gene is essential for cell viability. Indeed, homozygous GAS41-/- cell lines with two disrupted GAS41 alleles can be generated following substitution of the endogenous gene by stable integration of GAS41 cDNA controlled by a tetracycline-regulated CMV promoter. Inactivation of this promoter by tetracycline withdrawal results in rapid depletion of GAS41, causing a significant decrease in RNA synthesis and subsequently cell death. Thus, our results indicate that GAS41 is required for RNA transcription.

Inhibition of Various Steps in the Replication Cycle of Vesicular Stomatitis Virus Contributes to Its Photoinactivation by AIPcS4 or Pc4 and Red Light

Abstract— Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene‐labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8‐oxo‐7,8‐dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Inhibition of RNA Synthesis by Bradykinin Involves Both the B1and B2Receptor Subtypes

The efficacy of angiotensin converting enzyme inhibitors in the treatment of heart disease is due in part to the accumulation of bradykinin (BK). Since BK can exert its effect by influencing cell proliferation, we chose to study the effect of BK on the growth of A10 vascular smooth muscle cells. Ligand binding studies to determine which BK receptor subtypes are present on A10 cells showed that both B1and B2receptors were present in approximately equal numbers. Examination of RNA synthesis demonstrated that BK inhibits uridine incorporation in a dose-dependent manner. This decrease in RNA synthesis was blocked by both B1and B2receptor antagonists, as well as by addition of indomethacin, a cyclooxygenase inhibitor. The latter result suggested that prostaglandins mediate the biological actions of BK. Consequently, we examined the direct effect of two prostaglandins, PGE2and PGI2(prostacyclin), on A10 cells. PGE2caused a decrease in RNA synthesis, thus mimicking the effect of BK, while PGI2did not. Therefore, the inhibition of RNA synthesis in A10 vascular smooth muscle cells by BK requires both B1and B2receptor subtypes and this action of BK is apparently mediated byde novosynthesis of prostaglandins.

Selective Inactivation of Two Components of the Multiprotein Transcription Factor TFIIIB in Cycloheximide Growth-arrested Yeast Cells

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.

Dephosphorylation of nuclear non-histone proteins in submandibular glands of rats treated with isoproterenol.

Protein phosphatase that removed [32P]phosphate from non-histone proteins, i.e., phenol-soluble acidic proteins, more rapidly and strongly than from histone proteins was present in nuclei of rat submandibular glands, but was not associated with chromatin. Cyclic AMP (10(-4)-10(-2) mM) stimulated the dephosphorylation of non-histone proteins, but not that of histone proteins. After a single injection of isoproterenol (IPR), the dephosphorylation of non-histone proteins in rat submandibular gland nuclei increased within 15 min, reached a maximum in 30 min and returned to normal control levels within 4 h. The stimulation of dephosphorylation of non-histone proteins induced by IPR was not observed after prior treatment of the animals with dichloroisoproterenol. The dephosphorylation of histone proteins was not affected by the injection of IPR. Stimulation of beta-adrenoceptors with IPR in rat submandibular glands resulted in increase in cyclic AMP and decrease in RNA synthesis in the tissues in the first few hours after the injection. This decrease in RNA synthesis was temporary and was preceded by the increase in cyclic AMP level and in the dephosphorylation of non-histone acidic proteins in the tissues. These results suggest that protein phosphatase in nuclei plays an important part in the events controlling RNA synthesis by regulating the state of phosphorylation of non-histone acidic proteins. In addition, the phosphatase may be regulated by a function of the cytoplasmic membranes.

Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting

The Bacillus subtilis sigA gene encodes the primary sigma factor of RNA polymerase and is essential for cell growth. We have mutated conserved region 2.3 of the sigma A protein to substitute each of seven aromatic amino acids with alanine. Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation. Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant sigma factor to support cell growth by gene replacement and complementation. The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only the Y189A variant of the sigma A protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A sigma A proteins efficiently complement a temperature-sensitive allele of sigA. Overexpression of three of the sigma A proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifically impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role in both DNA melting and promoter recognition.

Morphological and functional changes accompanying the acquisition of meiotic competence in ovarian goat oocyte.

In order to determine at which follicular size goat oocytes were capable of resuming and completing meiosis, we evaluated their ability to mature in vitro and measured their maturation promoting factor (MPF) activity by histone H1 kinase assay. The results indicated that goat oocyte meiotic competence developed progressively in follicles ranging from 0.5 to 3 mm; the oocytes acquired the ability to resume meiosis in follicles of 0.5-0.8 mm, to reach metaphase I (MI) in follicles of 1-1.8 mm, and to reach metaphase II (MII) in follicles larger than 3 mm. The presence of MPF activity was first observed in oocytes arrested at early prometaphase I and reached a maximum level in oocytes blocked in metaphase I (MI). In the second part of this study, RNA synthesis and nucleolar changes were analyzed during the growth period. The acquisition of meiotic competence was accompanied by nucleolar compaction and a dramatic decrease in RNA synthesis. Changes in protein patterns were also analyzed, but only slight differences were observed among oocytes from the different classes.

Mechanism of bactericidal action of phenethyl alcohol inEscherichia coli

The mechanism of bactericidal action of phenethyl alcohol (PEA) inE. coli, which was previously demonstrated to be dependent on protein synthesis, has been investigated. Mutants resistant to PEA were selected, but the resistance observed was associated with a change in permeation. PEA effects on DNA, RNA, and protein synthesis were studied with bacteriostatic and bactericidal concentrations Similar results (inhibition of DNA synthesis and decrease in RNA synthesis) were obtained with lethal concentrations of PEA in cells pretreated with chloramphenicol, and with bacteriostatic concentrations of PEA in unpretreated cells. The PEA intracellular accumulation reached a maximum within 4 min and was not inhibited by KCN or by 2,4-dinitrophenol. The presence of phenylacetaldehyde was demonstrated in both stationary and exponential growth phase cells exposed to PEA but not in cells pretreated with chloramphenicol. These results suggested that the bactericidal mechanism of action of PEA involves its conversion into the corresponding aldehyde.