Biomedical subjects

D K Bideshi

Publications and source records attributed to D K Bideshi.

9 recordsLinked to original sources

PubMed2003

Patterns of Hermes transposition in Drosophila melanogaster.

Transposable elements are being developed as tools for genomics and for the manipulation of insect genotypes for the purposes of biological control. An understanding of their transposition behavior will facilitate the use of these elements. The behavior of an autonomous Hermes transposable element from Musca domestica in the soma and germ-line of Drosophila melanogaster was investigated using the method of transposon display. In the germ-line, Hermes transposed at a rate of approximately 0.03 jumps per element per generation. Within the soma Hermes exhibited markedly non-random patterns of integration. Certain regions of the genome were distinctly preferred over others as integration targets, while other regions were underrepresented among the integration sites used. One particular site accounted for 4.4% of the transpositions recovered in this experiment, all of which were located within a 2.5-kb region of the actin5C promoter. This region was also present within the Hermes element itself, suggesting that this clustering is an example of transposable element "homing". Clusters of integration sites were also observed near the original donor sites; these represent examples of local hopping. The information content (sequence specificity) of the 8-bp target site was low, and the consensus target site resembles that determined from plasmid-based integration assays.

Animals↗

PubMed2003

Recombinant bacteria for mosquito control.

Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria.

Animals↗

PubMed2000

Molecular characterization and phylogenetic analysis of the Harrisina brillians granulovirus granulin gene.

Well over 100 isolates of granulosis viruses (GVs), genus Granulovirus (family Baculoviridae), have been reported, all from lepidopterous insects. Three types of GVs are recognized, those of Type 1, which attack the fat body, Type 2, which attack most tissues, and Type 3, which attack only the midgut epithelium. To determine whether a correlation exists between tissue tropism and lepidopteran family phylogeny, the granulin gene of the Harrisina brillians (HbGV), a virus that attacks the midgut epithelium of H. brillians (family Zygaenidae) was cloned, sequenced, characterized, and compared with granulin genes of GVs that attack species of Tortricidae, Pieridae, and Noctuidae. The HbGV granulin gene encoded a peptide of 248 amino acids with a predicted Mr of 29.6 kDa, and shared a significant level of homology with other granulin (81-95% identical and 90-98% similar) and polyhedrin (49-58% identical and 62-72% similar) proteins. Phylogenetic analyses based on granulin and polyhedrin genes as well as on their 5'-untranslated sequences (5'-UTSs) indicated that HbGV was more closely related to GVs isolated from the tortricids, Cryptophlebia leucotreta (ClGV), Cydia pomonella (CpGV) and Choristoneura fumiferana (CfGV) than to other GVs and NPVs. This analysis provides preliminary evidence for a correlation between GV tissue tropism and the phylogeny of lepidopteran families, suggesting that GVs attacking species of Tortricidae and Zygaenidae are ancestral to those attacking species of the family Noctudiae.

5' Untranslated Regions↗

PubMed2000

The Trichoplusia ni granulovirus helicase is unable to support replication of Autographa californica multicapsid nucleopolyhedrovirus in cells and larvae of T. ni.

Baculovirus DNA helicases are essential for replication and are determinants of host range. Helicases of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Trichoplusia ni granulovirus (TnGV) differ markedly, although both viruses replicate efficiently in the cabbage looper, T. ni. To determine whether the TnGV helicase (P137) could support replication of AcMNPV in T. ni cells or larvae, the native AcMNPV helicase gene (p143) was disrupted and substituted with p137. P137 did not support replication when synthesized by the P143-deficient AcMNPV. Moreover, P137 did not inhibit AcMNPV replication when co-synthesized in the presence of the AcMNPV P143. These results suggest that although TnGV and AcMNPV replicate efficiently in T. ni, specific protein-protein or protein-DNA interactions between baculoviral helicases and viral-specific factors which form the replicase complex are required for virus replication. A novel and rapid method for disrupting AcMNPV genes in E. coli using the commercial Bac-to-Bac AcMNPV baculovirus expression vector is described.

Animals↗

PubMed2000

DNA-independent ATPase activity of the Trichoplusia ni granulovirus DNA helicase.

DNA helicases of baculoviruses are essential for virus replication and have been implicated as molecular determinants of host range. Although these proteins contain seven motifs (I, Ia, II-VI) characteristic of DNA helicases, the two most important characteristics of helicases - duplex-DNA unwinding and ATPase activity - have not been demonstrated. In the present study, a recombinant putative DNA helicase (rP137) of Trichoplusia ni granulovirus (TnGV) was purified from insect cells infected with a recombinant Autographa californica multicapsid nucleopolyhedrovirus that overproduced rP137. The rP137 protein exhibited an intrinsic DNA-independent ATPase activity that required Mg(2+) as a co-factor, an activity that was reduced in the presence of TnGV and phage lambda DNAs. These results provide further evidence that baculovirus helicase genes encode proteins with biochemical properties similar to those of classical DNA helicases.

Adenosine Triphosphatases↗

PubMed2000

Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield.

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.

Animals↗

PubMed1999

Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence.

Previously we demonstrated that the yield of Cry3A (70 kDa) can be increased as much as 10-fold when cry3A including its upstream STAB-SD mRNA stabilizing sequence is expressed in Bacillus thuringiensis under the control of cyt1A promoters. To determine whether the cyt1A promoters/STAB-SD combination (cyt1AP/STAB) has broader applicability, we used it to synthesize two other Cry endotoxins in the 70-kDa mass range, Cry2A and Cry11A. Combination of cyt1AP/STAB with orfs 2 and 3 of the cry2A operon yielded about 4. 4-fold the amount of Cry2A obtained with the wild-type cry2A operon. The yield of Cry11A obtained with a construct that contained the cyt1AP/STAB, cry11A and the 20-kDa protein gene was 1.3-fold the amount obtained with a construct similar to the wild-type operon. These results demonstrate that the cyt1AP/STAB combination can enhance synthesis of different Cry proteins significantly, but that the level of enhancement varies with the specific protein synthesized.

Animals↗

PubMed1999

A baculovirus anti-apoptosis gene homolog of the Trichoplusia ni granulovirus.

An inhibitor of apoptosis (iap) gene homolog (Tn-iap) of the Trichoplusia ni granulovirus (TnGV) was cloned, sequenced and mapped on the genome of TnGV. Tn-iap encoded a protein (Tn-IAP) of 301 amino acids with a predicted molecular mass of 35 kDa. The Tn-IAP contained the two sequence motifs, BIRs and RING finger, characteristic of IAP proteins, and shared identities of 21-27% and similarities of 28-53% with IAP proteins of Cydia pomonella GV (Cp-IAP), Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (MNPV) (Op-IAP1, 3), Autographa californica MNPV (Ac-IAP1), Bombyx mori NPV (Bm-IAP1), Lymantria dispar MNPV (Ld-IAP3) and Buzura suppressaria single nucleocapsid NPV (Bs-IAP1). However, Tn-IAP shared no significant homology with baculovirus IAP2 proteins. Using an antisense Tn-iap probe, two major transcripts of approximately 800 nt and 1600 nt were detected by Northern blot analysis of RNA extracted from the fat body of T. ni larvae infected with the TnGV. Unlike Cp-IAP and Op-IAP3, however, Tn-IAP did not rescue virion occlusion in SF21 cells infected with a p35-deficient AcMNPV mutant. Tn-IAP's synthesis in vivo but failure to rescue p35-deficient AcMNPV in SF21 cells suggests it is a functional IAP that is only effective in certain cell types.

Amino Acid Sequence↗

PubMed1998

Molecular characterization and expression of the Trichoplusia ni granulovirus helicase gene.

A putative DNA helicase gene from the granulovirus of Trichoplusia ni (TnGV) was cloned, sequenced, and compared with the corresponding gene of several multinucleocapsid nucleopolyhedroviruses (MNPVs) including those from Autographa californica (AcMNPV), Orgyia pseudotsugata (OpMNPV), Bombyx mori (BmNPV), and Spodoptera exigua (SeMNPV). The TnGV helicase gene (p137) encoded a helicase of 1158 amino acids with a predicted mass of 137 kDa. Comparison of p137 with AcMNPV p143 revealed 44.5% identity at the nucleotide level, and, respectively, 28.6% identity and 53.0% similarity at the amino acid level. Similar levels of identity and similarity were obtained when TnGV p137 was compared with the corresponding helicase genes of BmNPV, OpMNPV and SeMNPV. Using an antisense probe made from an internal 1.6 kb region of p137, a major transcript of approximately 3600 nt was detected by Northern blot analysis in fat body tissue from TnGV-infected larvae of T. ni. As both TnGV and AcMNPV replicate efficiently in larvae of T. ni, these results demonstrate that baculovirus putative DNA helicases which have diverged markedly can function efficiently in the same host. Three genes flanking TnGV p137, designated ORF68, ORF219 and ORF157, corresponded in order and orientation with AcMNPV ORFs 93, 94 and 96. However, the amino acid similarity between corresponding genes ranged from only 50.4 to 62.5%, providing further evidence that related baculovirus proteins which have diverged markedly can function efficiently in the same host.

Amino Acid Sequence↗