An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.
Developmental gene regulation in trypanosomatids proceeds exclusively by post-transcriptional mechanisms. Stability and abundance of heat shock protein (HSP)70 and HSP83 transcripts in Leishmania increase at mammalian-like temperatures, and their translation is enhanced. Here we report that the 3-untranslated region (UTR) of HSP83 (886 nucleotides) confers the temperature-dependent pattern of regulation on a chloramphenicol acetyltransferase (CAT) reporter transcript. We also show that the majority of the 3-UTR sequences are required for increasing mRNA stability during heat shock. Processing of the HSP70 and HSP83 primary transcripts to poly(A)؉ mRNA was more efficient during heat shock; therefore, even when stability at 33°C was reduced by deletions in the 3-UTR, transcripts still accumulated to comparable and even higher levels. Translation of heat shock transcripts in Leishmania increases dramatically upon temperature elevation. Unlike in other eukaryotes in which the 5-UTR confers preferential translation on heat shock transcripts, we show that translational control of HSP83 in Leishmania originates from its 3-UTR. The 5-UTR alone cannot induce translation during heat shock, but it has a minor contribution when combined with the HSP83 3-UTR. We identified an element located between positions 201 and 472 of the 3-UTR which is essential for increasing translation of the CAT-HSP83 reporter RNA at 33-37°C. This region confers preferential translation during heat shock even in transcripts that were less stable. Thus, investigating the traditionally conserved heat shock response reveals that Leishmania parasites use unique pathways for translational control.Unique and unusual features characterize the genomes of trypanosomatids that comprise an ancient group of eukaryotes. Genes transcribed by RNA polymerase II are found in polycistronic transcription units, and gene clusters repeated in tandem frequently encode abundant proteins. There is no evidence for transcriptional activation of developmentally regulated genes, and mRNA abundance is determined exclusively by post-transcriptional mechanisms. These include differential RNA processing (1) and control of mRNA decay (2-4). Polycistronic transcripts mature by trans-splicing that adds a short capped leader (39-mer) to the 5Ј-end of mRNAs and by 3Ј processing that includes cleavage and polyadenylation. Transsplicing and polyadenylation are coupled mechanistically in trypanosomatids and share regulatory signals that consist of polypyrimidine tracks and potential AG splice sites (5, 6). The 3Ј-untranslated sequences lack the consensus eukaryote AAUAAA signal for cleavage and polyadenylation, and no other consensus element of that nature has been identified.Leishmania parasites exist in the alimentary canal of female sand flies as flagellated promastigotes. They develop into virulent metacyclics that are uniquely adapted for transmission by the fly into a mammalian host where they differentiate into amastigotes, an obligate intracellular life form that resides within ...
Leishmania parasites are ancient eukaryotes, characterized by unusual molecular mechanisms. We have used the gene encoding for Hsp83 as a model system for studying regulatory mechanisms that control developmental gene regulation. We previously showed that protein coding genes are regulated exclusively by post-transcriptional mechanisms, while no transcriptional activation could be observed even for the conserved Hsp83 gene. We now show that processing and maturation of the Hsp83 polycistronic primary transcripts is more efficient at elevated temperatures. The mature transcripts are more stable during heat shock, with regulation conferred by 3' UTRs. Poly(A) tails of Hsp83 are approximately 30 nucleotides long, as common for other low eukaryotes. The mechanism that signals differential degradation is still unclear, since it was not possible to detect differences in deadenylation of Hsp83 transcripts at varying temperatures. Heat shock transcripts are preferentially translated at 33-37 degrees C, but unlike Drosophila, translational regulation is controlled by a region within the 3' UTR. Using this traditionally conserved system emphasizes that regulatory mechanisms in Leishmania differ from those prevailing in other eukaryotes.
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