Search for antisense copies of beta-globin mRNA in anemic mouse spleen.

BACKGROUND: Previous studies by Volloch and coworkers have reported that during the expression of high levels of beta-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS) globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. RESULTS: We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. CONCLUSIONS: Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

Efficient site-specific nonribozyme opening of hepatitis delta virus genomic RNA in infected livers.

Examination of the 1,679-nucleotide (nt) unit-length hepatitis delta virus (HDV) RNAs in the livers of two HDV-infected woodchucks showed that 96% of the antigenomic RNA but only 50% of the genomic RNA was circular. We subsequently found that at least half of the linear unit-length genomic RNA was open at a unique location. Using a modified form of RNA ligation-mediated amplification of cDNA ends, we showed that the 5' end was located at nt 1212. Like the previously described ribozyme cleavage site at nt 686, the new site produced a 5'-OH. Nevertheless, we showed that this novel site was not produced by activity of the HDV genomic ribozyme. We speculate that the 5' end at nt 1212 reflects a preferred site of posttranscriptional endonucleolytic cleavage of genomic RNA.

Immunogenic properties of reverse transcriptase of HIV type 1 assessed by DNA and protein immunization of rabbits.

Genetic immunization may be one way to prime individuals for a subsequent broad anti-HIV-1 immune response. Reverse transcriptase of HIV-1 (RT) presents a selective target for attempts to arrest replication of HIV-1. Rabbits immunized with a plasmid carrying the gene for reverse transcriptase HIV-1 (RT DNA) developed potent antibody and cellular responses to the gene product. The immunogenic properties of RT DNA and recombinant reverse transcriptase were compared in rabbits. The specific immune responses were similar to those reported previously for HIV-1 infected humans. The array of B and T cell epitopes recognized in RT DNA-immunized rabbits was broader than in rabbits immunized with the recombinant RT. We localized seven novel B and T cell epitopes and concordance between B cell and helper T cell epitopes was observed. B cell epitopes of RT induced proliferation of peripheral blood mononuclear cells and were active as helper T cell epitopes. T cell-proliferative responses to the epitopes of RT preceded or paralleled the production of antibodies of the same specificity. Subdomains of reverse transcriptase involved in the enzymatic activity of RT were highly immunogenic. Anti-RT IgG partially inhibited reverse transcription in vitro.

Origin of hepatitis delta virus mRNA.

Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5' end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5'-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5' ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5' end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3'-end addition to the template. These new findings support the interpretation that the 5' end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

Characterization of the 5' ends for polyadenylated RNAs synthesized during the replication of hepatitis delta virus.

The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5' rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5' ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5' ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5' ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5' ends. The observed end located at nt 1630, and most of the novel 5' ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5' ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5' ends could be controlled by nucleotide sequences at and around nt 1630.

Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase.

Synthesis of deoxynucleotide-containing RNA-like single-stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter-containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8-10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1-+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non-coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG-containing) was achieved with high yields.

Interaction of tRNA-derivatives and oligonucleotide primers with AZT-resistant mutants of HIV-1 reverse transcriptase.

While the molecular basis of HIV-1 AZT resistance has been widely studied, a biochemical explanation of this process is not well known. No significant changes in the binding affinity of reverse transcriptase (RT) mutants for AZT-triphosphate has been found. Here we analyzed the interaction of wild type and AZT-resistant mutant forms of HIV-1 RT with different primers. Site-directed mutagenesis was used to introduce point mutations on the retroviral enzyme. Primers were either synthetic oligonucleotides or tRNA(Lys3) derivatives containing d(pT)n or r(pU)n at the 3' end. In all cases, determination of kinetic parameters was done in the presence or absence of compounds known to modify protein conformation, such as dimethyl sulfoxide (DMSO), urea, and Triton X-100. Although we found similar K(m) values for all RTs, there was generally an increase in the affinity when enzymes were tested in the presence of DMSO, urea, and Triton X-100. Then, we analyzed the nucleation and elongation steps of the polymerization process. The efficiency of formation of the first base pair was determined by measuring K(m1), the affinity between RT and the 3' terminal nucleotide of the primer. An important difference was found: in the presence of DMSO, urea, and Triton X-100, the K(m1) values for mutated enzymes were higher than those of wild type RTs. Thus, the presence of compounds able to change protein conformation led to a marked destabilization of the interaction of mutated RTs with the 3' terminal nucleotide of the primer. From these results, it can be hypothesized that resistance to AZT is not due to the direct influence of mutations on RT, but rather to conformational changes of the mutated RT in complex with the template-primer altering the ability of the enzyme to select or reject an incoming dNTP.

Deoxyribonucleotide-containing RNAs: a novel class of templates for HIV-1 reverse transcriptase.

Deoxyribonucleotide-containing RNA-like polynucleotides (dcRNAs) were synthesized by mutant T7 RNA polymerase and their structures confirmed by sequencing. dcRNAs annealed with a 20mer oligodeoxyribonucleotide primer were tested as templates/primers in the reverse transcription reaction catalyzed by HIV-1 reverse transcriptase (RT). All dcRNAs were shown to be efficient templates for both wild-type RT and RT mutants, containing 'AZT-resistant' mutations. Differences in the patterns of the DNA products of RNA- and dcRNA-driven reverse transcription were demonstrated. The kinetic characteristics for dcRNAs utilization were compared with the corresponding parameters for RNA/DNA and DNA/DNA templates/primers. The respective K m values for dcRNAs appear to be intermediate between those for RNA and DNA templates. A correlation equation connecting apparent K m value for template/primer and the number of deoxyribonucleotide substitutions in RNA template is proposed.

[Stability of human immunodeficiency virus to azidothymidine. II. Kinetic characteristics of "AZT-resistant" mutant forms of reverse transcriptase]. / Issledovaniia ustoichivosti virusa immunodefitsita cheloveka k azidotimidinu. II. Kineticheskie kharakteristiki "AZT-ustoichivykh" mutantnykh form obratnoi transkriptazy.

Prolonged treatment of AIDS patients with azidothymidine results in the development of resistance to the drug which correlates with the appearance of point mutations in the reverse transcriptase (RT) coding region within the HIV-1 pol gene. Kinetic studies of interactions of wild type RT and its mutants harbouring the above mutations with substrates and azidothymidine 5'-triphosphate (AZTTP) have been carried out. The complete mutant containing all the above described mutations possess the highest resistance on all the templates tested. Significant increases in resistance for mutants 67,70,215 and 67,215 on all the templates have also been observed. Inhibition of mutant enzymes by AZTTP depends on the template used.

[Kinetic analysis of interaction of human immunodeficiency virus reverse transcriptase with alkaloids]. / Kineticheskii analiz vzaimodeistviia obratnoi transkriptazy virusa immunodefitsita cheloveka s alkaloidami.

The interactions of HIV-I reverse transcriptase with some alkaloids were studied. Among nine compounds tested three--berberine, palmatine and sanguiritrine--inhibited RT. The dependence of the inhibition on the type of template-primer was also demonstrated. The kinetic analysis as well as circular dichroism experiments suggest the complex mechanism of RT inhibition by alkaloids. This mechanism includes both enzyme-alkaloid and alkaloid-template interactions; the latter effect also results in RT inhibition.

[Studies of the resistance of the human immunodeficiency virus to azidothymidine. I. Kinetic studies of the interaction between reverse transcriptase and a series of its mutant forms with substrates and azidothymidine-5'-trisphosphate]. / Issledovaniia ustoichivosti virusa immunodefitsita cheloveka k azidotimidinu. I. Kineticheskie issledovaniia vzaimodeistviia obratnoi transkriptazy i riada ee mutantnykh form s substratami i azidotimidin-5'-trifosfatom.

Prolonged therapy of AIDS patients with azidothymidine results in the development of resistance to the drug. This phenomenon is accompanied by the appearance of point mutations in the pol gene coding for reverse transcriptase (RT). Kinetic studies of interactions of wild type RT and its forms containing the above-mentioned mutations with substrates and azidothymidine 5'-triphosphate have been carried out. Considerable differences in the affinities of RT and the mutants for RNA and DNA heteropolymer templates were established. The mutations did not affect the RT affinity for dNTP; however, its location influenced considerably the inhibition of the reaction with azidothymidine 5'-triphosphate.

Interactions of the HIV-1 reverse transcriptase 'AZT-resistant' mutant with substrates and AZT-TP.

To investigate the biochemical basis of the HIV-1 resistance to AZT we obtained the RT mutant containing four amino acid substitutions by an oligonucleotide-directed mutagenesis technique. Enzymatic properties of the wild type and mutant RTs were compared. 'AZT-resistant' mutations in RT were shown to be associated with the reduced capability of AZT-TP to block the DNA- but not RNA-directed DNA synthesis.

[Hydrolysis of 5'-phosphonates and nucleoside phosphates by phosphatases of various origin and human and calf serum]. / Gidroliz 5'-fosfonatov i fosfatov nukleozidov fosfatazami razlichnogo proiskhozhdeniia i syvorotkami krovi cheloveka i telenka.

The hydrolysis of 5'-phosphonates of 2'-deoxythymidine and its 3'-modified analogs, inhibiting the HIV reproduction, by the E. coli alkaline, calf intestine and human placenta phosphatases as well as by the Crotalus atrox venom 5'-nucleotidase were studied. Transformations of 5'-phosphonates of adenosine and its analogs during incubation with human and fetal calf blood sera were investigated. The nucleotide derivatives modified at the phosphate residue were not hydrolyzed by any of the phosphatases studied except for the cobra venom 5'-nucleotidase, the effectiveness of the latter depended on the substitutes at both phosphate and sugar residues. 2'-Deoxyadenosine incubation with blood sera resulted in its transformation to 2'-deoxyinosine and then to hypoxanthine. 2'-Deoxyadenosine 5'-phosphonates were stable during incubation with blood sera under the same conditions.

[Effects of trophoblastic beta 1-glycoprotein (TBG) on the functional activity of different cell lines]. / Vliianie trofoblasticheskogo beta 1-glikoproteina (TBG) na funktsional'nuiu aktivnost' razlichnykh kletochnykh linii.

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.

[An immunochemical study of M-HeLa cells as producers of human placental alkaline phosphatase]. / Immunokhimicheskoe issledovanie kletok linii M-HeLa kak produtsentov platsentarnoi shchelochnoi fosfatazy cheloveka.

While analyzing M-HeLa cells by IFA technique secretory and membrane-bound forms of human placental alkaline phosphatase (HPAP) were detected. Activity of secretory HPAP increased if cell density and incubation time were increased too. After short influence of heat shock (15 min at 42 degrees C) activity of secretory HPAP increased for 45% and intracellular HPAP 3 for 37%. It is proposed that HPAP take part in organization of first response to heat shock and support cellular thermotolerance.

[Placental alkaline phosphatase as a marker of malignant neoplasms]. / Platsentarnaia shchelochnaia fosfataa--marker zlokachestvennykh novoobrazovanii.

Activity of placental alkaline phosphatase (PAP) was studied in blood serum of 53 healthy persons and of 72 oncologic patients, using solid-phase immunoenzymatic analysis with polyclonal antibodies towards PAP. The enzyme was detected both in blood serum of healthy persons and of oncologic patients. The blood serum under study was preheated at 65 degrees in order to inactivate the intestinal phosphatase--the only isoenzyme cross-reacting with antibodies to thermostable PAP. This controlled heating treatment decreased distinctly the possibility of pseudo-positive reactions. The limiting values of the PAP activity were about 0.15 un/L in blood sera of healthy persons. Higher values were considered as an evidence of pathological state. After screening analysis of blood sera from patients with various forms of malignant tumors the PAP activity above 0.15 un/L was observed in 20% of the patients; the enzymatic activity exceeded these values in 54% of patients with ovary carcinoma. The data obtained suggest that the procedure developed as an adequate means for estimation of thermostable PAP isoenzymes as well as that the rate of PAP activity might serve as marker of malignancy in ovary and testis carcinomas independently on level of total activity of alkaline phosphatase.

[Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase]. / Issledovanie éffektivnosti metodov vydeleniia spetsificheskikh krolich'ikh antitel klassa G dlia immunofermentnogo opredeleniia platsentarnoi shchelochnoi fosfatazy cheloveka.

Four different chromatographic methods of IgG isolation from rabbit antisera to placental alkaline phosphatase (HPAP) have been compared. The antibodies were obtained by ion-exchange chromatography and affinity chromatography on protein-A-sepharose, on the sepharose with immobilized antigen. IgG samples were characterized by the content of specific antibodies to HPAP and checked in enzyme immunoassay (EIA). IgG purified on immobilized antigen were found to be the optimal both from the point of view of the specific antibodies content and EIA sensitivity, but satisfactory results could be also obtained with ion-exchange and protein-A-chromatography purified IgG. The last two isolation methods are simpler and provide 3-10 ng/ml sensitivity of HPAP detection, which is lower, as compared with the test employing affinity antibodies (1 ng/ml), but allows the detection of HPAP in serum samples.