Quantitative analysis of expression of six BmGST genes in silkworm, Bombyx mori.

Glutathione S-transferases (GSTs) are a multifunctional super gene family, some of which play an important role in insecticide resistance. In this research, we used a real-time quantitative RT-PCR method, and a novel strategy, to measure the transcriptional level per gene copy using an exogenous RNA reference and DNA reference. The transcription levels of six BmGST genes in different tissues of fifth instar Bombyx mori larvae and their responses to insecticide and fluoride were investigated. The results show different levels and patterns of expression of the different BmGSTs in the various tissues observed. The BmGSTs can be induced by insecticide and fluoride, but their responses to each are different. The results of this research are helpful in studying the tissue-specific expression of BmGSTs in Bombyx mori, and in developing new pesticide resistant silkworm varieties.

A proteomic-based approach for the characterization of some major structural proteins involved in host-parasite relationships from the silkworm parasite Nosema bombycis (Microsporidia).

Nosema bombycis is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about host-parasite interactions at the molecular level for this spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. Major microsporidian structural proteins from the spore wall (SW) and the polar tube (PT) are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by SDS-PAGE and 2-DE to establish complementary proteomic profiles. Three proteins were shown to be located at the parasite SW. Moreover, 17 polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots were shown to correspond to polar tube proteins (PTPs) by immunofluorescent assay and transmission electron microscopy immunocytochemistry on cryosections. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by MASCOT and SEQUEST searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a nongenomic context.

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori.

To study the minimal length required for the secretion of recombinant proteins and silk proteins in posterior silk gland, the signal peptide (SP) of the fibroin heavy chain (FibH) of silkworm Bombyx mori was systematically shortened from the C-terminal. Its effect on the secretion of protein was observed using enhanced green fluorescent protein (EGFP) as a reporter. Secretion of EGFP fusion proteins was examined under fluorescence microscope. FibH SPs with lengths of 20, 18, 16 and 12 a.a. can direct the secretion of the reporter, yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP was shortened to 12 a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP. When 16 a.a. of the FibH SP were used, the secretion of fusion protein was normal and the cleavage site was between the Gly-Ser linker and Met, the starting amino acid of EGFP. These findings are applicable for the expression of foreign proteins in silkworm silk gland. The cleavage site of the SP is discussed and compared with the predictive results of the SignalP 3.0 online prediction program.

In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector.

In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

Loss of posterior silk gland transcription specificity of fibroin light chain promoter due to absence of 41 bp sequence containing possible inhibitor binding sites.

The gene encoding fibroin light chain protein (FibL) is specifically expressed in the posterior silk gland of silkworm and repressed in other tissues. The binding sites of several transcription factors involved in the silk gland transcription specificity of fibl promoter have been recognized, including SGFB, PSGF and BMFA. Here we report the leak expression of the enhanced green fluorescent protein (EGFP) reporter gene in tissues other than the posterior silk gland in vivo when under the control of a shortened fibl promoter with deletion of the 5' terminal 41 bp sequence, which is located at -650 nt to -610 nt upstream of the fibl transcription starting site. Assay of silk gland specificity of the promoters was performed by observation of green fluorescence in tissues of silkworm larvae following inter-haemocoelic injection of recombinant Autographa californica multiple nuclear polyhedrosis virus carrying the EGFP reporter gene controlled by different lengths of fibl promoters. Our results indicated that availability of the binding sites of several known factors, including SGFB, PSGF and BMFA, is not sufficient for intact silk gland transcription specificity of fibl promoter, and there are possible inhibitor binding sites in the 41 bp sequence (-650 nt to -610 nt) upstream of the transcription starting site which may be required to repress the activity of fibl promoter in other tissues.

Introduction of foreign genes into silkworm eggs by electroporation and its application in transgenic vector test.

Electroporation as a methodology to introduce foreign genes into silkworm eggs was systematically analyzed. The foreign gene in both the newly hatched and 3rd instar larva DNA can be detected by PCR. The amount of foreign gene in 3rd instar larva DNA was about 1/1000 of that in newly hatched larva DNA. The ratio of foreign gene entering into silkworm eggs was voltage dependent and showed significant difference between the tested silkworm strains. When the piggyBac transposon system was applied, the effect of nuclear localization signal (NLS) peptide and the in vitro transcribed transposase mRNA on the transposition rate has been measured. Results showed that the in vitro transcribed transposase mRNA facilitated transposition to take place earlier and NLS could result in higher transposition probability and earlier transposition as well. When linearized vectors containing varied length of flanking homologous sequences around a reporter gene were introduced into silkworm eggs by electroporation, the one with 2.6 kb total arm length gave higher G1 positive ratio than that with total arm length of 1.5 kb and 800 bp.

[Production and application of rabbit anti-imitative spider dragline silk protein polyclonal antibody].

A synthetic spider dragline gene s600 was cloned into fusion protein expression vector pGEX-KG and expressed in Escherichia coli. Protein S600 was purified and rabbit antiserum was prepared. Amino acid composition analysis confirmed the right expression of S600. Western blot analysis revealed that anti-S600 antiserum could react with natural spider silk, so the synthetic dragline protein, designed by the authors, shares similar immunological characteristics with the natural spider silk. An ELISA system was also established for the quantitative detection of synthetic dragline protein expression in silk gland (or in the cocoon) of transgenic silkworm.

[Activating effect of hepatitis B virus preS/S protein on proliferating cell nuclear antigen gene promoter].

BACKGROUND: PreS/S gene was derived from hepatitis B virus (HBV) integration fragment of human hepatocellular carcinoma genome, containing the promoter of preS/S and the C terminal truncated preS/S open reading frame. PreS/S protein may have important roles in processing hepatoma in some HBV-infected patients. The aim of the study was to study the activity of HBV preS/S protein on proliferating cell nuclear antigen (PCNA) promoter and to localize compartment of the preS/S protein in the liver cell line L02. METHODS: The authors studied the effect of the 3 -truncated preS/S on human PCNA promoter by co-transfecting the expression plasmids of luciferase reporter gene, used the immunohistochemical method to localize the preS/S protein. RESULTS: The expression product of the plasmid, pKSH7C-HpaI which contained the 3 -truncated preS/S and the flanking cellular sequences, stimulated the expression of PCNA promoter dose dependently,and its effect was 0.5 folds higher than control. Immunohistochemistry showed that the preS/S protein located in the cytosolic region of the liver cell line L02. CONCLUSIONS: The HBV preS/S protein could stimulate the PCNA promoter of the liver cell, its effect was not direct, which suggests that the effect of preS/S protein on PCNA promoter was probably through the cell signal transduction pathway.

An E2F site in the 5'-promoter region contributes to serum-dependent up-regulation of the human proliferating cell nuclear antigen gene.

The synthesis of proliferating cell nuclear antigen (PCNA) is strictly regulated during the cell cycle. To investigate the contribution of the promoter region to the up-regulation of human PCNA expression at the onset of S phase, we have examined 17 putative elements with reporter assays in quiescent L-O2 cells and following serum stimulation. The E2F-like sequence 5'-TTCCCCGCAA-3' located at -84 to -75 is required for the serum-induced transactivation. In electrophoretic mobility shift assays, nuclear extracts from asynchronous L-O2 cells exhibit two binding activities toward the -75 E2F oligonucleotide, and the minor band, whose formation could be interfered with by E2F-1 antibody, represents an S phase-specific complex. This is the first demonstration of the E2F site in the human PCNA 5' promoter as a serum-responsive element.

[Involvement of PHO80 and PHO85 genes in Saccharomyces cerevisiae ion tolerance].

PHO85 is a versatile gene in Saccharomyces cerevisiae, which is involved in metabolism of inorganic phosphate and usage of carbon source, accumulation of glycogen, regulation of protein stability and cell cycle control. The viability of wild type budding yeast strain YPH499 and its derivative pho85Delta mutant, pho80 mutant, and pap1(pcl-7)Delta mutant in different cations were investigated and their tolerance to the cations(LC(50)) was measured. The results showed that the deletion of PHO85 or PHO80 gene both increased sensibility of Sacchromyces cerevisiae to ions K(+), Mg(2+), Zn(2+), Ca(2+) and Mn(2+), while the deletion of pap1(pcl-7) gene did not lead to such phenotype. The difference between the patterns of relative growth curve of the mutants and wild type strain in the above ions also implied that PHO80 was the unique PCLs in complex with PHO85 CDK, that were contributed to K(+) and Mg(2+) ion homeostasis control and there were some other PCLs besides PHO80 that were involved in Zn(2+), Ca(2+) and Mn(2+) tolerance regulation as cyclin of PHO85 CDK. Furthermore, the amount of the total cellular calcium of pho85Delta mutant, pho80Delta mutant and YPH499 indicated that the ability of calcium accumulation of pho85 mutant and pho80Delta mutant was impaired.

Synthesis of Ser-His-Gly.DNA conjugates.

There are strong interactions between the bases of oligonucleotides. Based on Watson-Crick principle, they can form stable secondary and tertiary structure such as hairpin, duplex, triplex, G-quartet, pseudoknot, which can serve as the scaffold of molecules. Peptides contain active groups such as amino, carboxyl, imidazole, hydroxyl. A protected Ser-His-Gly-Threoninol phosphoramidite was synthesized in this work and was incorporated into a triplex-forming oligonucleotide. The results indicate that the oligonucleotide containing Ser-His-Gly-Threoninol in the middle could still form triplex with the target duplex and the dissociation constant was 0.5 micromol/L.

Small Subunit Ribosomal RNA Genes of Microsporidia.

Microsporidia are ubiquitous intracellular parasitic protozoa infecting all types of animals. Their ribosomes and rRNAs are of prokaryotic size.In order to better understand their phylogenetic relationship and identify the uncertain species, the sequences of the small subunit ribosomal RNA (ssurRNA, 16 S rRNA) genes of nine microsporidia infectious to the silkworm, Bombyx mori, were determined. The results of phylogenetic trees and Southern blotting suggest all the nine strains of microsporidia are various species of the genus Nosema.

Hammerhead Ribozymes Suppress HBV(adr) in HepG2 Cells.

Three hammerhead ribozymes (RS3, RC2 and RC1) targeting to the HBV genome have been designed. Plasmids were constructed by inserting the genes of naked and tRNA-embedded ribozymes into RNA trimming vector pRG523 and then were transferred to eukaryotic expression vector. By the similar cloning method the shotgun-type plasmids carrying homogeneous RS3 or RtS3 unitconnected in tandem were obtained. After co-transfecting the above plasmids and HBV genome containing plasmid into human hepatoma cell line HepG2 respectively and selection by G418, the HBV-inhibiting activity of different kinds of ribozyme in G418-resistant cells was achieved by measuring the decrease of HBV-RNA, progeny DNA and the antigens expressed. The results showed that all the ribozymes were active with more than 70% inhibition activity against the HBV and that tRNA-embedded ribozymes had higher activity than naked ribozymes. It is worth particular interest that shotgun-type ribozymes with the connected unit in tandem with 8 and 12 units constructed in the plasmid revealed the highest activity, reaching >90% inhibition.

Altering Fibroin Heavy Chain Gene of Silkworm Bombyx mori by Homologous Recombination.

A gene unit, which encoded fibroin-like peptide, was synthesized and constructed. The unit was multimerized to about 2 400 bp using BamHI and BglII at each end of the unit, then was fused with gfp reporter gene. The fusion gene, flanked by the 5'and 3'sequence of the fibroin heavy chain gene of silkworm Bombyx mori, was transferred into the eggs of silkworm by electroporation. After the silkworms developed and spinned silk, 73 out of about 5 400 cocoons were brighter than normal ones under UV light. The protein extracted from the brighter cocoon could react with the GFP polyclonal antibodies. Genomic DNA from these silkworms and their progenies were analyzed. The integration of gfp gene into genomic DNA of silkworm and the occurrence of expected homologous recombination event had been proved by Southern hybridization. It was shown that gfp-fibroin like fusion gene had integrated into the genomic DNA of silkworm by homologous recombination and the phenotype of "brighter cocoon" could be used to select transgenic silkworms.

Active cis-elements of PCNA Promoter in HeLa, HepG2, L-02 and MCF-7 Cell Lines.

Proliferating cell nuclear antigen (PCNA) is an auxiliary factor of DNA polymerase delta and epsilon and functions in DNA replication and repair. Using PCR methods, 17 sites within the human PCNA promoter from 60 to 538 were subjected to a 8 bp substitution mutagenesis. Wild type promoter and each mutated promoters were inserted into luciferase expression vector pGL2-Basic. These nonmutated and mutated PCNA promoters were assayed by transient transfection of the plasmids into HeLa, HepG2, L-02 and MCF-7 cell line, respectively. It was found that several sites were common cis acting elements and several sites were cell-specific cis acting elements. Data were further presented using in vitro transcription assay with HeLa and HepG2 nuclear extract.

Transgenic Silkworm of Anti-NPV Ribozyme.

The plasmid pGL2Rz including ribozyme gene was linearized and introduced into early eggs of silkworm (G(0)) by gene gun. The luciferase activity in blood of the G(1) generation was detected, then the resistant silkworm was selected by NPV infection from G(2) generation. The transgenic silkworm resistant against NPV 10 times more than control ones was got at the G(4) generation. PCR and Southern blotting proved that the ribozyme gene was integrated into the genome of silkworm multicopily. The expression of ribozyme was also detected by RT-PCR in pupa. The results showed that the transgenic silkworm strain of anti-NPV ribozyme has been got.

Fluorescent Transgenic Silkworm.

The replacement of fibroin heavy chain gene in silkworm by site directed homologous recombination was studied The DNA fragment consisting of an IE promoter driving green fluorescent protein (GFP) gene as reporter flanked by pieces of the 5' and 3' sequences of the fibroin heavy chain gene of silkworm at two sides was transferred into silkworm eggs by electroporation Green fluorescent flecks were seen on three silkworms fifth instar among five thousand silkworms under UV light PCR analysis proved that the GFP gene was integrated into the genome of silkworm Southern hybridization of genomic DNA of one transgenic silkworm showed that the fibroin heavy chain gene was successfully knocked out and replaced by the reporter gene The transgenic silkworms could grow up to the fifth instar as normal but they could not spin silk while control ones do.

Transformation of Neomycin Resistance Gene (neo(R)) into Silkworm (Bombyx mori.L.).

Neomycin resistance gene (neo(R)) flanked by 5' and 3' regions of fibroin H-chain gene of silkworm (Bombyx mori.L.) was transferred into eggs of silkworm by gene gun in the early period of fertilization. The larvae were fed with an artificial diet containing neomycin in early 24 hours post transfettion, and some of them survived. The neo(R) encoding sequence in G(2) generation derived from the survivals was detected by Southern blotting. The results indicated that neo(R) could be used as a selective marker for studies on transgenic silkworm.

A New Strategy for Optimizing the Translation Initiation Rate of Foreign Gene Expression in E. coli.

The translation initiation rate is greatly affected by the secondary structure of the translation initiation region (TIR) of mRNA. A novel system was established for improving the translation initiation rate of a foreign gene in E. coli. As a model, the 5' 114 bp coding sequence (38 amino acids from the start codon) of human proliferating cell nuclear antigen (PCNA) gene was fused with the lacZ' gene at its 5' end in vector pTZ19R. A Shine/Dalgarno (SD) sequence GAGGT was inserted to the -8 position of AUG by site-directed mutation. Then the flanking sequences of SD, which were the 6 nucleotides upstream the SD and the 7 nucleotides between SD and AUG, were randomly changed by PCR using a synthetic primer with partially random sequences. This random mutation led to potential variations in the secondary structure of the TIR of mRNA through base pairing with the 5' coding sequence. The 5' PCNA-LacZ' mRNA could be efficiently and specifically transcribed by inducible T7 RNA polymerase in E. coli strain JM 109 (DE3). There were 269 clones of 5' PCNA-LacZ' fusion plasmid selected first by the blue color on X-gal plate and then by hybridization with a 5' PCNA probe. Eight clones with different blue colors among the 269 clones were chosen for beta-galactosidase activity assay. The results showed that the difference between their enzyme activities was more than 20 fold, but there seemed no apparent difference at the transcription level as assayed by RNA dot hybridization, which suggested that the difference in the expression of the fusion protein was due to the different rate of translation initiation. Thus, by this strategy, an effective translation initiation region for the high expression of human PCNA gene in E. coli was obtained.

Regulatory Effect of HBV Integrated Fragment on PCNA Promoter.

H7C is a HBV integrated fragment isolated from a human hepatocellular carcinoma, containing the promoter of preS2 and the C-terminal truncated preS/S open reading frame. We have studied the effect of the 3'-truncated preS/S on human proliferating cell nuclear antigen (PCNA) promoter by co-transfection of the expression plasmids. Result showed that the product, pKSH7C-Hpa I, which contained the intact H7C and the flanking cellular sequences, stimulated the expression from PCNA promoter dose-dependently, and its effect was 1-2 folds higher than that on SV40 promoter. However, two subclones, pKSH7C-XHX and pKSH7C-XbH, which would not express preS/S, showed no stimulatory effect. Furthermore, when if the -45 bp ATF-like site was mutated, the activation effect became diminished. This showed that the ATF-like site might be important in mediating the transactivating process. This is the first report of the effect of a HBV integrated fragment on the promoter of a replicating protein factor.