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.

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.

The Role of DNA Polymerase delta in HeLa Cell DNA Replication Studied by Antisense Technology.

We have investigated the biological role of DNA polymerase delta in HeLa cell DNA replication using antisense technology with the Lipofectin Delivery and the GPT selection method. Both of the oligonucleotides designed to inhibit the expression of DNA polymerase delta and alpha can specifically reduce the DNA replication level in HeLa cells. This is the first report directly proving that DNA polymerase delta plays an important role in mammalian cell DNA replication.

Purification and Characterization of DNA Helicase BstH2 from Bacillus Stearothermophilus.

In the purification of DNA helicase BstH1 we have partially purified the second DNA helicase BstH2 from Bacillus Stearothermophilus through Polymin P precipitation, ammonia sulfate precipitation and chromatographic steps with DEAE-cellulose, phosphocellulose, Blue-Sepharose, FPLC Superose 12, Mono Q and second Mono Q. The ATPase activity of BstH2 depends on Mg(2+) and is differentially stimulated by different types of nucleic acids. BstH2 has a maximal ATPase activity at 55 degrees. The ATPase activity is greatly inhibited by E. coli SSB or higher ionic strength. The DNA helicase activity of BstH2 depends on ATP and Mg(2+). BstH2 can unwind partial duplex DNA as well as blunt-ended duplex DNA. E. coli SSB stimulates the unwinding reaction catalyzed by BstH2.

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.

Purification and Characterization of DNA Helicase BstH1 from Bacillus stearothermophilus.

We have partially purified a DNA helicase BstH1 from Bacillus stearothermophilus through Polymin P precipitation, ammonia sulfate precipitation and column chromatographic steps with Pheny1-Sepharose, DEAE-cellulose, phosphocellulose, FPLC Mono Q and Superose 12. Bsth1 possesses a DNA-Dependent ATPase activity in the presence of Mg(2+). The ATPase activity of BstH1 is differentially stimulated by the presence of different types of nucleic acids. BstH1 has an optimal ATPase activity at 55 degrees. The DNA helicase activity of BstH1 requires a 3'-terminal single-stranded DNA binding site to initiate the unwinding reaction in the 3' right curved arrow 5'direction. BstH1 can unwind blunt-ended duplex DNA in a concentration-dependent manner.

Computer Analysis of the Cleavage Reaction of Hammerhead Ribozyme.

We have analyzed various in vitro cleavage reactions of hammerhead ribozyme by a computer program. Three energy changes: deltaE(s'), deltaE(r'), and deltaE, were calculated. deltaE(s') was the hypothetical energy requirement for the substrate to be activated from the most stable state to the relaxed state. deltaE(r') was the same energy requirement for the ribozyme. deltaE was the calculated free energy decrease by the formation of Rz-S complex from the relaxed ribozyme and the substrate. We found that high deltaE(s') or deltaE(r') reduced the cleavage efficiency of the reaction, and that the deltaE was related to both cleavage efficiency and the optimal temperature of the reaction. deltaE(s'), deltaE(r') can be calculated in advance according to the sequences of the ribozyme and the substrate. Our model, therefore, can be use in the designing of ribozymes.

Expression of Anti-PCNA mRNA Ribozyme by T7 Vaccinia Virus System in HeLa Cells.

We have designed and synthesized a hammer-head Rz targeted to human proliferating cell nuclear antigen (PCNA) mRNA at site No.524. Results showed that Rz524 can cleave the transcribed substrate RNA site-specifically in vitro. A self trimming expression plasmid of this Rz was constructed and co-introduced into HeLa cells with T7 vaccinia virus. Dot-blotting hybridization showed that the Rz was expressed in HeLa cells.

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.

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.

[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.

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.

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.

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.

[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.

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 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.