Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield.
(49/828)
Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC(50)] = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold. (+info)
Nonsense mutations in the human beta-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation.
(50/828)
Generally, nonsense codons 50 bp or more upstream of the 3'-most intron of the human beta-globin gene reduce mRNA abundance. In contrast, dominantly inherited beta-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human beta-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG-->TGA), 39 (C-->T), or 127 (C-->T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C-->T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C-->T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG-->TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3'-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells. (Blood. 2000;96:2895-2901) (+info)
RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro.
(51/828)
Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the sigma subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the beta and beta' subunits and the downstream DNA duplex. (+info)
Superimposed promoter sequences of the adenoviral E2 early RNA polymerase III and RNA polymerase II transcription units.
(52/828)
The human adenovirus type 2 E2 early (E2E) transcriptional control region contains an efficient RNA polymerase III promoter, in addition to the well characterized promoter for RNA polymerase II. To determine whether this promoter includes intragenic sequences, we examined the effects of precise substitutions introduced between positions +2 and +62 on E2E transcription in an RNA polymerase III-specific, in vitro system. Two noncontiguous sequences within this region were necessary for efficient or accurate transcription by this enzyme. The sequence and properties of the functional element proximal to the sites of initiation identified it as an A box. Although a B box sequence could not be unambiguously located, substitutions between positions +42 and +62 that severely impaired transcription also inhibited binding of the human general initiation protein TFIIIC. Thus, this region of the RNA polymerase III E2E promoter contains a B box sequence. We also identified previously unrecognized intragenic sequences of the E2E RNA polymerase II promoter. In conjunction with our previous observations, these data establish that RNA polymerase II and RNA polymerase III promoter sequences are superimposed from approximately positions -30 to +20 of the complex E2E transcriptional control region. The alterations in transcription induced by certain mutations suggest that components of the RNA polymerase II and RNA polymerase III transcriptional machines compete for access to overlapping binding sites in the E2E template. (+info)
Sequence of the genome of Salmonella bacteriophage P22.
(53/828)
The sequence of the nonredundant region of the Salmonella enterica serovar Typhimurium temperate, serotype-converting bacteriophage P22 has been completed. The genome is 41,724 bp with an overall moles percent GC content of 47.1%. Numerous examples of potential integration host factor and C1-binding sites were identified in the sequence. In addition, five potential rho-independent terminators were discovered. Sixty-five genes were identified and annotated. While many of these had been described previously, we have added several new ones, including the genes involved in serotype conversion and late control. Two of the serotype conversion gene products show considerable sequence relatedness to GtrA and -B from Shigella phages SfII, SfV, and SfX. We have cloned the serotype-converting cassette (gtrABC) and demonstrated that it results in Salmonella serovar Typhimurium LT2 cells which express antigen O1. Many of the putative proteins show sequence relatedness to proteins from a great variety of other phages, supporting the hypothesis that this phage has evolved through the recombinational exchange of genetic information with other viruses. (+info)
Inhibition of RNA polymerase III elongation by a T10 peptide nucleic acid.
(54/828)
The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs). We analyzed the effect of a T10 PNA on in vitro transcription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues. At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner. The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription. Thus, an impairment of pol III recycling fully accounts for the observed inhibition. As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA adduct before reaching the terminator region. On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs. Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription. (+info)
Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.
(55/828)
Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage. (+info)
Unbalanced X-chromosome inactivation with a novel FVIII gene mutation resulting in severe hemophilia A in a female.
(56/828)
This report is of a 14-month-old girl affected with severe hemophilia A. Both her parents had normal values for factor VIII activity, and von Willebrand disease type 2N was excluded. Karyotype analysis demonstrated no obvious alteration, and BclI Southern blot did not reveal F8 gene inversions. Direct sequencing of F8 gene exons revealed a frameshift-stop mutation (Q565delC/ter566) in the heterozygous state in the proposita only. F8 gene polymorphism analysis indicated that the mutation must have occurred de novo in the paternal germline. Furthermore, analysis of the pattern of X chromosome methylation at the human androgen receptor gene locus demonstrated a skewed inactivation of the derived maternal X chromosome from the lymphocytes of the proband's DNA. Thus, the severe hemophilia A in the proposita results from a de novo F8 gene frameshift-stop mutation on the paternally derived X chromosome, associated with a nonrandom pattern of inactivation of the maternally derived X chromosome. (Blood. 2000;96:4373-4375) (+info)