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R Gentz

Publications and source records attributed to R Gentz.

82 records · Page 5Linked to original sources
PubMed1986

Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process.

After binding to a promoter Escherichia coli RNA polymerase is in contact with a region of about 70 bp. Around 20 bp of this sequence are transcribed. Information encoded within this transcribed region is involved in late steps of the functional program of a promoter. By changing such 'down-stream' sequences promoter strength in vivo can be varied more than 10-fold. By contrast, information for early steps of the promoter program such as recognition by the enzyme and formation of a stable complex resides in a central core region of about 35 bp. Our data show that the strength of a promoter can be limited at different levels of the overall process. Consequently promoters of identical strength can exhibit different structures due to an alternate optimization of their program.

Base Sequence↗
PubMed1986

Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals.

A 240-bp DNA fragment encoding a peptide, designated ENV(80), homologous to a conserved part of the gp41 transmembrane glycoprotein of human immunodeficiency virus (HIV) was chemically synthesized and inserted into different plasmid expression vectors. Escherichia coli transformants containing these plasmid constructs produced upon induction high amounts of either an ENV(80) peptide of relative molecular mass (Mr) of 10,000 or the same ENV(80) peptide N-terminally fused to E. coli chloramphenicol acetyltransferase (CAT) or to mouse dihydrofolate reductase (DHFR) having Mr of 36,000 and 31,000 respectively. All polypeptides containing the ENV(80) sequences were strongly reactive with antibodies present in sera from AIDS virus-infected individuals, but not with control sera. The strategy of gene assembly allowed the expression of ENV(80) subfragments fused to DHFR. The serodiagnosis of 15 positive sera by Western blot analysis using these bacterially synthesized ENV(80) subfragments revealed the presence of several immunoreactive epitopes on the 80-amino acid polypeptide which were recognized differently by the various patients.

Acquired Immunodeficiency Syndrome↗
PubMed1986

lac Repressor blocks transcribing RNA polymerase and terminates transcription.

Operator sequences are essential elements in many negatively controlled operons. By binding repressors, they prevent the formation of active complexes between RNA polymerase and promoters. Here we show that the Escherichia coli lac operator-repressor complex also efficiently interrupts ongoing transcription. This observation suggests a mechanism of action for operators located distal to promoter sequences.

DNA, Bacterial↗
PubMed1985

Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis.

Using purified sigma 55 RNA polymerase from Bacillus subtilis in an in vitro transcription system, we have shown that both promoters and terminators of Gram negative origin are recognized by this enzyme. Furthermore, when B. subtilis is transformed with a shuttle vector containing certain of these promoters, synthesis of the Staphylococcus aureus CAT protein is achieved, and levels up to 25% of the total cellular protein can be obtained. These findings indicate a closer evolutionary relationship of the expression machinery of these two bacterial species than has been assumed so far. On the basis of these results, the construction of new expression vectors for B. subtilis is likely to be facilitated, since a variety of well-characterized signal elements from Escherichia coli are available.

Acetyltransferases↗
PubMed1985

Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5.

Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.

Base Sequence↗
PubMed1981

Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal.

Downstream placement of a strong transcriptional termination signal has made possible the cloning of bacteriophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively. By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo.

Base Sequence↗
PubMedDate not supplied

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects.

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin, Larp33 was expressed in Escherichia coli after cloning of a 2.2 kb Sau3 digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L. (L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-gamma, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis.

Animals↗