Expression of a Bacillus thuringiensisdelta-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum, Mills).

AIMS: To introduce a cry gene into microorganisms that naturally colonize the phylloplane of tomato plants to improve the persistence of the Cry proteins for controlling a South American tomato moth (Tuta absoluta, Meyrick, 1917). METHODS AND RESULTS: A cry1Ab gene isolated from a native Bacillus thuringiensis strain (LM-466), showing a relevant activity against T. absoluta larvae, was cloned into the shuttle vector pHT315 (Arantes and Lereclus 1991). The construct was introduced by electroporation into native Bacillus subtilis and Bacillus licheniformis strains, both natural inhabitants of the tomato phylloplane. Western analysis and toxicity assays against the target larvae proved that the successful expression of the gene was accomplished in host bacteria. Recombinant toxin displayed a similar LC50 value in comparison to native donor strain LM-466. Both transformed Bacillus survived for at least 45 days on the tomato leaf surface. CONCLUSIONS: Plant-associated microorganisms that naturally colonize the phylloplane could be useful as recombinant microbial delivery systems of toxin genes of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Modified microorganisms capable of surviving on leaf surfaces for several weeks with insecticidal activity should allow for a reduction in pesticide application.

PCR-RFLP identification of endemic Chilean species of Rhagoletis (Diptera: Tephritidae) attacking Solanaceae.

Four species of the genus Rhagoletis are native to Chile: R. nova (Schiner), R. conversa, (Brèthes), R. penela Foote and R. tomatis Foote. Currently, identification of these species is based on morphological criteria, but their strong similarity makes precise recognition difficult. To clarify species separation for quarantine purposes, a reliable method based on a PCR-RFLP procedure is reported. A DNA region containing mitochondrial NADH dehydrogenase genes was selected as a target sequence for the analysis. The amplification products (c. 1 kb) were digested with either SspI or DdeI, yielding specific patterns that differentiated each of the endemic species. Complete nucleotide sequences were determined, confirming empirical restriction maps. This report updates information on the geographical distribution of Rhagoletis species in Chile.

Two related, low-temperature-induced genes from Brassica napus are homologous to the human tumour bbc1 (breast basic conserved) gene.

In order to identify genes involved in cold acclimation, we have constructed a cDNA library from Brassica napus (cv. Samouraï) cold-acclimated etiolated seedlings. By differential screening, a cDNA clone named pBnC24 (Brassica napus Cold), corresponding to a new cold-inducible plant gene, was isolated. Northern blot hybridizations using total RNA from acclimated and unacclimated seedlings confirmed that BnC24 represents a cold-regulated gene. In contrast with a number of cold-inducible plant genes, BnC24 does not seem to be responsive to abscisic acid (ABA). In addition, further screening of the 'cold-acclimated' cDNA library using pBnC24 cDNA as a probe, allowed the isolation of a second type of homologous cDNA. Sequence analysis showed that the two BnC24 genes encode basic 24 kDa proteins, which are highly hydrophilic and rich in alanine, lysine and arginine. The nucleotide and deduced amino acid sequences of these clones do not show any homology with other previously described cold-induced plants genes. However they have strong homology with a recently discovered human tumour gene, bbcl (breast basic conserved), which seems to be highly conserved in eukaryotes.

Changes in Protein Synthesis in Rapeseed (Brassica napus) Seedlings during a Low Temperature Treatment.

Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18 degrees C for 48 hours, one half of the seedlings was transferred to 0 degrees C for another 48 hour period, the other half being kept at 18 degrees C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [(35)S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0 degrees C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments.

A coupled DNA-directed in vitro system to study gene expression based on di- and tripeptide formation.

In this report, a simplified coupled DNA-directed in vitro system has been described that is based on the formation of the first di- or tripeptide of the gene product. This system is gene specific and quantitative, and the assay (especially the extraction procedure) is very rapid. The fact that both transcription and translation initiation occur in this system makes it ideally suited for studies on the regulation of prokaryotic gene expression. The ideal templates are plasmids, DNA fragments or purified mRNAs that direct the synthesis of a limited number of products with different second amino acids. An essential requirement is that the initial sequence of the protein products be known, although this system could be used to determine the second amino acid in cases where there is some doubt from the DNA sequence as to where a particular protein initiates. A difficulty arises when a plasmid contains more than one gene whose protein products have the same initial dipeptide. One solution to the problem is to measure tripeptide formation if the third amino acid is different. A second procedure, if the code word for the second amino acid differs between the genes, is to use purified isoacceptor tRNA species to distinguish the products. Another important application of tripeptide synthesis is that it can be used as a measure of the amount of active mRNA present in a mixture of mRNAs. The use of a ribosomal high-salt wash instead of the purified initiation and elongation factors greatly simplifies this system and should make it suitable for routine analysis in most laboratories.

Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

A simplified translation system coupled to DNA transcription that involves assaying the synthesis of the first dipeptide of a gene product has been described recently [Robakis, N., Meza-Basso, L., Brot, N. & Weissbach, H. (1981) Proc. Natl. Acad. Sci. USA 78, 4261--4264]. Using this dipeptide system, we have investigated the expression of genes carried on plasmids coding for beta-lactamase, ribosomal protein L12, and the chloroplast large subunit (LS) of ribulosebisphosphate carboxylase (RbuBPCase). Although all three nascent gene products begin with the sequence fMet-Ser, the formation of fMet-Ser can be used to distinguish between the synthesis of beta-lactamase and either L12 or the LS of RbuBPCase by using different serine isoacceptor tRNA species. In beta-lactamase, the serine codon is AGU, which utilizes the serine isoacceptor species tRNASer3; in L12 and the LS of RbuBPCase, the serine codewords are UCU and UCA, respectively, both of which are recognized by the serine isoacceptor species tRNASer1. By using either pure tRNASer1 or pure tRNASer3, the expression of each gene can be quantitated. In this system, guanosine-5'-diphosphate-3'-diphosphate inhibits the expression of the beta-lactamase and L12 genes but stimulates the synthesis of the LS. In addition, the ratio of fMet-Ser/fMet-Ala (L12/L10) synthesized was about 1 as compared with the ratio of 4 that has been obtained previously in vivo or in vitro protein-synthesizing systems in which the entire gene product was measured.

Translational control of ribosomal protein L10 synthesis occurs prior to formation of first peptide bond.

A simplified DNA-directed in vitro system has been developed to study the regulation of the synthesis of ribosomal protein L10 by measuring the formation of the first dipeptide, fMet-Ala. The results show that the inhibition of L10 synthesis by L10 (autoregulation) occurs at or prior to the formation of the first peptide bond.