The feedback response of Escherichia coli rRNA synthesis is not identical to the mechanism of growth rate-dependent control.

Growth rate-independent rrn P1 promoter mutants were tested for their ability to respond to changes in rrn gene dosage. Most were found to be normal for the feedback response. In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the rrn gene dosage was altered. These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood.

Multiple mechanisms are used for growth rate and stringent control of leuV transcriptional initiation in Escherichia coli.

Expression of the Escherichia coli leuV operon, which contains three tRNA(1)(Leu) genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3', 5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp(1). The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp(1). Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

Eukaryotic expression of enzymatically active human immunodeficiency virus type 1 reverse transcriptase.

Reverse transcriptase of human immunodeficiency virus type I is a vitalenzyme in the HIV-1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase-specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA-immunized mice inhibited reverse transcription in vitro.

The DNA polymerase-encoding gene from a thermoacidophilic archaeon Sulfolobus acidocaldarius.

We have cloned, sequenced and characterized the gene encoding a DNA polymerase from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sac). The putative transcription promoter and terminator elements, as well as a potential ribosome-binding site (rbs), have been identified in the flanking regions. One large open reading frame (ORF) found in the sequenced portion of the Sac genome encodes a protein of 875 amino acids (aa). All conserved motifs characteristic of family B of DNA polymerases have been found in the deduced primary structure of this enzyme. The Sac DNA polymerase also contains sequence motifs that form a proofreading exonuclease domain.

Functional analysis of the lymphotoxin-beta promoter. Sequence requirements for PMA activation.

Membrane lymphotoxin (LT) complex is a trimer composed of two subunits , LT-alpha and LT-beta of which the latter is a 33-kDa transmembrane protein. The LT-beta gene is expressed in lymphoid cells and organs, but little is known about its inducible regulation. Previously, the surface expression of LT-beta in Jurkat cells has been shown to increase in response to PMA. In this report, we used this model to study the transcriptional control of the human and murine LT-beta genes. PMA strongly induced the expression of LT-beta mRNA, and the level of induction was not changed markedly by cycloheximide (CHX) treatment. The LT-beta promoter region contains conserved Egr-1, nuclear factor (NF)-kappaB, and Ets binding sites, and PMA-inducible factors bound to these sites were detected by the gel-retardation technique (electrophoretic mobility shift assay (EMSA)). To identify sequences involved in transcriptional control, sets of human and mouse promoter-chloramphenicol acetyltransferase (CAT) constructs were generated and assayed by transient transfections. The PMA response was lost after deletion of the distal Ets binding site at -110. Mutations at either the Ets or NF-kappaB sites that prevented factor binding dramatically reduced PMA-inducible promoter activity, suggesting cooperative interaction between corresponding transcription factors in PMA activation. Mutation at the Egr-1 site also resulted in substantial loss of promoter activity, and the residual activity may be attributed to binding of constitutively expressed Sp-1 to the same site. We propose that the interaction between the members of NF-kappaB and Ets families of transcription factors and their cognate sites in the promoter is the major determinant of inducible expression of the LT-beta gene in Jurkat cells.

[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.

Cloning and expression analysis of the murine lymphotoxin beta gene.

Tumor necrosis factor alpha (TNF-alpha) and soluble lymphotoxin (LT) (also called LT-alpha or TNF-beta) are cytokines with similar biological activities that are encoded by related and closely linked genes. TNF-alpha, a mediator of the inflammatory response, exists in soluble and transmembrane forms. LT-alpha can be secreted or retained at the cell surface by binding to a 33-kDa transmembrane subunit, LT-beta. The recently cloned human LT-beta gene encodes another TNF family member and is linked to the TNF/LT locus within the major histocompatibility complex locus. The cell surface LT is a heterotrimer consisting of LT-alpha and LT-beta, whose physiological function is not yet clearly defined. We now report the sequence analysis of the genomic region and cDNA of murine LT-beta gene, which is closely associated with the TNF-alpha and LT-alpha genes within the murine major histocompatibility complex locus. Unlike the TNF-alpha, LT-alpha, and human LT-beta genes, which contain four exons, the murine LT-beta contains three exons and encodes a 244-amino acid polypeptide with a 66-amino acid insert that is absent from the human homologue. In situ hybridization demonstrates constitutive expression of LT-beta in lymphoid and hematopoietic tissues. LT-beta transcription is maximal in the thymic medulla and in splenic white pulp. LT-beta mRNA is also detected in the skin and in specific regions of the brain. The LT-beta promoter region contains putative Ets-binding sites, suggesting that the expression of LT-beta may be regulated in part by Ets transcription factors whose pattern of lymphoid expression overlaps that of LT-beta.

[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.

[Site-specific mutagenesis of residue Lys-172 of phage T7 RNA polymerase: characterization of transcription properties of mutant proteins]. / Sait-spetsificheskii mutagenez ostatka Lys-172 RNK-polimerazy faga T7: kharakterizatsiia transkriptsionnykh svoistv mutantnykh belkov.

Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides. The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower. Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter. DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts. L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes. The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed.