Ouriel Faktor

Population structure of the pestiferous moth Helicoverpa armigera in the Eastern Mediterranean using RAPD analysis

The genetic structure of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), was studied in the eastern Mediterranean. Moths were sampled in six locations (five in Israel, and one in Turkey) and their genetic relationship was analysed using RAPD-PCR. Three 10-oligonucleotide primers revealed 84 presumptive polymorphic loci that were used to estimate population structure. Results reveal low level of genetic distances among Israeli and Turkish populations. The estimated values of FST and θ for the eastern Mediterranean populations were very low across all populations, indicating a high level of gene flow. Four distinct RAPD-product profile types were defined, and found in all Israeli and Turkish populations. Although no isolation by geographical distance was detected, topographical barriers may play a role in such isolation.

The use of bi-cistronic transfer vectors for the baculovirus expression system.

In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.

Enhancer element, repetitive sequences and gene organization in an 8-kbp region containing the polyhedrin gene of the Spodoptera littoralis nucleopolyhedrovirus

SummaryThe Spodoptera littoralis multicapsid nucleopolyhedrovirus (Spli MNPV) shows only a distant genetic relationship to other NPVs. In this report we describe the gene organization of an 8-kbp region of the SpliMNPV which contains the polyhedrin gene. The polyhedrin transcription initiation sites were mapped and the sequence and gene organization of an 8-kbp region of SpliMNPV were determined. The sequences downstream of the polyhedrin gene showed colinearity with the gene organization of other NPVs. An anti-clockwise 1035-bp open reading frame (ORF), capable of encoding a prolinerich polypeptide, was found at the 3′ end of the polyhedrin gene, followed by an 837-bp ORF encoding a putative protein kinase (PK), with an orientation similar to that of the polyhedrin gene. Sequences upstream of the polyhedrin gene were found to be unique to SpliMNPV and contained two regions consisting of highly repetitive sequences. One region, 980 bp in length and termed sequence repeat region 1 (SR1), contained a variety of short direct repeats, SR1 was found to act as a transcriptional enhancer in a transient expression assay. Additional regions containing different repetitive sequences were identified within the proline-rich ORF1035 and in sequences located downstream of the pk gene.

IDENTIFICATION AND NUCLEOTIDE SEQUENCE OF AN ECDYSTEROID UDP-GLUCOSYLTRANSFERASE GENE OF SPODOPTERA LITTORALIS MULTICAPSID NUCLEAR POLYHEDROSIS VIRUS

TheSpodoptera littoralis multicapsid nuclear polyhedrosis virus (SlMNPV) is a member of the Baculoviridae that shows a distant genetic relationship to the prototypeAutographa californica MNPV (AcMNPV). Using anAcMNPV gene-specific probe, we identified and mapped an ecdysteroid UDP-glucosyltransferase (egt) gene in the genome ofSlMNPV. Sequence determination of a part from the hybridizing DNA fragment revealed an open reading frame of 1548 nucleotides that exhibits 38% and 44% identity to theegt amino acid sequences ofAcMNPV andLymantria dispar MNPV (LdMNPV), respectively. Sequences flanking theSlMNPVegt gene, including the promoter region, were found to be unique to the virus. The presence of this nonstructural gene inSlMNPV and several other baculoviruses points to the importance ofegt for the viral infection process.

Differential utilization of regulatory cis-elements for stress-induced and tissue-specific activity of a French bean chalcone synthase promoter

Abstract Expression of the French bean chalcone synthase ( CHS ) gene is induced in response to external stimuli and to the plants tissue-specific and developmental program. We have previously demonstrated the essential role of a cooperative regulatory element (CAR), containing a G-box and H-box, in tissue-specific expression. In this paper, we focus on understanding the requirement for cis -elements in the response of the CHS15 promoter to the abiotic elicitor HgCl 2 , mechanical wounding and infection with tobacco mosaic virus (TMV). Leaves of tobacco plants transformed with a chimeric CHS15 promoter-β-glucuronidase gene construct carrying a mutation at the G-box did not exhibit impaired promoter response to wounding, but did show a 19% reduction in the response to HgCl 2 and TMV. A mutation at the H-box within CAR resulted in a 30% increase in promoter response to wounding and reductions of 36 and 54% in the response to HgCl 2 and TMV, respectively. These results suggest differential utilization of regulatory cis -elements for stress-induced and tissue-specific expression of the bean CHS15 promoter.

TMV-induced expression of tobacco β-glucanase promoter activity is mediated by a single, inverted, GCC motif

Abstract Tobacco basic β -1,3-glucanase has been implicated in plant development and defense responses against pathogens. We examined the functional cis -elements involved in the response of basic β -1,3-glucanase promoter (gglb50) to infection with tobacco mosaic virus (TMV). In plants transformed with chimeric gglb50- β -glucuronidase ( GUS ) reporter gene, significant GUS-activity levels were measured in the leaves and in roots. Activity in the leaves was further induced (10-fold) by TMV infection. Maximal virus-induced activity was directed by a promoter region between −1233 to +19 (gglb-1233). A 5′ deletion to −1038, which removed two TAAGAGCCGCC motifs (GCC-boxes), reduced virus-induced activity. However, when the gglb-1233 promoter was mutated by base substitutions within a third, inverted, GCC-box located between positions −106 and −95, virus-induced promoter activity was abolished. Duplication of a small region containing this inverted GCC-box in the context of the gglb-1233 promoter resulted in increased virus-induced activity. Gel retardation assay demonstrated nuclear-factor binding to the inverted GCC-box. These studies strongly suggest the inverted GCC-box as a regulatory element essential for TMV-induced activity directed by the gglb50 promoter.

The p10 gene of Spodoptera littoralis nucleopolyhedrovirus: nucleotide sequence, transcriptional analysis and unique gene organization in the p10 locus.

The p10 gene of the Spodoptera littoralis (Spli) multicapsid nucleopolyhedrovirus (MNPV) was identified. With a coding sequence of 315 nucleotides (nt), corresponding to a protein of 104 amino acids, the SpliMNPV p10 gene is the longest p10 gene known. This gene codes for a putative protein with an Mr of 11130 and was found to be most closely related to the Spodoptera exigua (Se) MNPV p10 (49.4% amino acid identity) and most distant from the Autographa californica (Ac) MNPV p10 (20.0% amino acid identity). Characterization of the proteins secondary structure and a comparison with other p10 protein species suggested that this p10 has an extended alpha-helical domain with high probability of forming a large coiled-coil structure. The p10 mRNA was about 1500 nt long, as determined by Northern blot analysis. Primer extension assay mapped three transcription start sites to a conserved baculovirus late promoter motif, TAAG. In the SpliMNPV genome, the p10 gene is not flanked by genes similar to p26 and p74, as found in SeMNPV, AcMNPV, Choristoneura fumiferana MNPV and Orgyia pseudotsugata MNPV. Instead, an open reading frame (ORF) of 945 bp is located downstream from the p10 gene and is followed by another ORF in opposite orientation, encoding the p74 protein. Upstream of the p10 sequences, an ORF of 552 bp was identified that potentially encodes a 184 amino acid protein of Mr 20925, which showed 52.2% identity with the encoded product of the SeMNPV xb187 gene.

GENOMIC LOCALIZATION AND NUCLEOTIDE SEQUENCE OF A LEF-8 GENE OF THE SPODOPTERA LITTORALIS NUCLEOPOLYHEDROVIRUS

A gene similar to lef-8 of the Autographa californica (Ac) nucleopolyhedrovirus (MNPV) was identified in the Spodoptera littoralis (Spli) MNPV. The SpliMNPV lef-8-like gene was localized on the genomic map between 26.9 and 29 map units and is flanked by a chitinase gene and p47 gene. This gene arrangement differs from that of similar genes in the AcMNPV genome, where the lef-8 gene is located about 62 kbp from the chitinase gene and about 7 kbp from the p47 gene. Sequence analyses of the lef-8 gene revealed an ORF of 2730 nucleotides, predicted to encode a protein with Mr 104876. The putative protein is 60.9% identical to the AcMNPV LEF-8 and contains an identical sequence of a conserved motif of DNA-dependent RNA polymerases. Sequences downstream of the lef-8 gene contain two sequence repeats which resemble a repeated motif of the SpliMNPV enhancer element and other repetitive sequences from the viral genome.

Transcriptional analysis and promoter activity of the Spodoptera littoralis multicapsid nucleopolyhedrovirus ecdysteroid UDP-glucosyltransferase gene

The ecdysteroid UDP-glucosyltransferase gene (egt) of Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) is a homologue of the Autographa californica MNPV (AcMNPV) egt gene, which has been found to block insect moulting. Infection of larvae with an egt-deleted AcMNPV resulted in enhanced mortality as compared to infection with the wild-type virus. Consequently, deletion of an egt gene has been proposed as a tempting approach for enhancing the insecticidal properties of baculoviruses. In a previous report we described the mapping and sequencing of the SpliMNPV egt gene. Here we use time-course Northern blot and biochemical analyses to show the production of egt transcripts and protein. The SpliMNPV egt transcription start sites were mapped to 22 and 25 nucleotides downstream of the TATA box by primer extension. Transient expression assays of chimeric egt promoter-chloramphenicol acetyltransferase (cat) reporter gene constructs revealed low promoter activity that was transactivated by AcMNPV immediate-early viral protein IE-1.

A polymerase chain reaction for the detection of nucleopolyhedroviruses in infected insects: the fate of the Spodoptera littoralis virus in Locusta migratoria

The Spodoptera littoralis nucleopolyhedrovirus (SlNPV) is a potential pest control agent of Spodoptera spp. As part of our studies to establish the use of this virus, a polymerase chain reaction (PCR)-based method was developed for the detection of viral DNA in infected insects. PCR amplification of the polyhedrin sequences enabled the detection of low levels of viral DNA directly from viral occlusion bodies or from total larval DNA. The use of different sets of synthetic DNA primers allowed us to differentiate between SlNPV and the Autographa californica NPV (AcNPV) and to identify a new AcNPV variant isolated from a cotton pest, Pectinophora gossypiella NPV. The PCR method was also used to test for the possible infection of Locusta migratoria larvae by SlNPV, reported by Bensimon et al., 1987. The progress of SlNPV infection in L. migratoria larvae was monitored by PCR for 2 weeks. The reaction revealed decreasing amounts of viral DNA in infected larvae. During this time, no signs of disease were observed in the infected locusts.