[Technical improvements in genetic manipulation of Pichia pastoris and their application in hirudin expression].

Pichia pastoris has become an increasingly popular host for heterologous protein production. However, there is neither a high-efficient transformation method nor a fast colony-PCR assay for the yeast yet. In this paper, we report a transformation procedure by electroporation, which reaches the value of up to 2800 transformants/microgram DNA. By using a cold and heat treatment and a modified PCR buffer, we established a simple and reliable colony-PCR protocol to detect recombinant P. pastoris clones, which is comparable to the conventional assay for E. coli colonies. With these two novel techniques, we have successfully achieved the expression of hirudin, an antithrombin agent, in Pichia pastoris. The secreted hirudin maintains a biological activity of 82 antithrombian units per milliliter supernatant from the media.

[Cucumber mosaic virus cDNA vector expressed foreign genes in tobacco cells].

Full-length cDNA of SD strain CMV (SD-CMV) RNA 3 was cloned and sequenced. An Nsi I site was created at the sequence around the start codon of coat protein (CP) gene and a replacement cassette was constructed. The CP gene was replaced by green fluorescent protein (GFP) gene, beta-glucuronidase (GUS) gene or mouse dihydrofolate reductase (DHFR) gene, respectively. The cDNAs of Fny-CMV RNAs 1 and 2 and the chimeric SD-CMV RNA 3 were cloned between the 35S promoter and terminator separately. Tobacco protoplasts transfected with the CMV cDNA vectors expressed the three reporters, implying that CMV could be used as an expression vector.

Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity.

Cholera toxin B subunit (CTB) mature protein was stably expressed in transgenic tobacco plants under the control of the CaMV 35S promoter and TMV Omega fragment. Fusion of the PR1b signal peptide coding sequence to the CTB mature protein gene increased the expression level by 24-fold. The tobacco-synthesized CTB (tCTB) was purified to homogeneity by a single step of immunoaffinity chromatography. The purified tCTB is predominantly in the form of pentamers with molecular weight identical to the native pentameric CTB, indicating that the PR1b-CTB fusion protein has been properly processed in tobacco cells. Furthermore, by immunodiffusion and immunoelectrophoresis, we have shown that the antigenicity of the purified tCTB is indistinguishable from that of the native CTB protein.

[A fusion protein of rotavirus VP6 and cholera toxin B subunit: expression in Escherichia coli and analysis of biological activities].

Rotavirus infection is a major cause of dehydrating diarrhea in infants worldwide. The non-toxic cholera toxin B subunit(CTB), known as an immunomodulatory carrier, might help to stimulate mucosal immune response when coupled to rotavirus antigens in oral immunization. Here we report for the first time the construction of a translational fusion of CTB gene 5' to the VP6 gene of a human rotavirus A(field strain T114), and expression of the CTB-VP6 fusion protein in E. coli BL21(DE3). The expressed fusion protein has a molecular weight of 56 kD, as expected, and accounts for about 15% of the total E. coli protein. Western blottings with the hyperimmune serum against rotavirus strain WA and the antibody against cholera toxin indicated that the fusion protein retains the antigenicity identical to the native CTB and VP6. The GM1-ELISA analysis proves that the renatured CTB-VP6 has strong affinity for GM1 ganglioside.

Structure analysis of the rice yellow stunt rhabdovirus glycoprotein gene and its mRNA.

The nucleotide sequence of the glycoprotein (G) gene of rice yellow stunt rhabdovirus (RYSV) was determined. The G gene is 2158 nucleotides long and contains an open reading frame of 2007 nucleotides encoding a protein with a calculated molecular mass of 75,358 Da. Furthermore, the 5'- and 3'-terminal sequences of the G protein mRNA were defined by the RACE method. Non-viral nucleotides appear to be present at the 5' end of G mRNA. The G protein contains an N-terminal signal peptide of 32 amino acids, C-terminal transmembrane and cytoplasmic domains, ten potential glycosylation sites and four stretches of a-d hydrophobic heptad-repeats.

Structure of the nucleocapsid protein gene of rice yellow stunt rhabdovirus.

The complete nucleotide sequence of the N protein gene of rice yellow stunt rhabdovirus (RYSV) was determined by sequencing of cDNA clones derived from the viral genomic RNA. The 3' end of the N gene (messenger sense) was defined by sequence analysis of cDNA clones generated from the N protein mRNA by 3'RACE. The 5' end sequence of the gene was putatively assigned as 5'-AACAC-3'; this sequence is found in the presumed 3' leader/N gene junction region. The mRNA encoding the RYSV N protein is 1714 nt comprising a 15-nt untranslated 5' leader sequence followed by an open reading frame (ORF) of 1563 nt and a 136-nt untranslated 3' region. The calculated molecular mass of the N protein encoded by the ORF is 58,400 Da, which is larger in size than N proteins of other rhabdoviruses. Amino acid composition analysis shows that the RYSV N protein is rather basic with a predicted isoelectric point of 10.04; indeed, a large highly basic region could be found at the carboxy terminal portion of the protein. Amino acid sequence comparison between N proteins of RYSV and sonchus yellow net virus, both of which belong to the same genus Nucleorhabdovirus, revealed an overall 30% identity, with three relatively conserved blocks of 14-20 amino acid residues. Moreover, the hydropathy profiles of the two proteins are generally similar. The structural similarities between the N protein of RYSV and that of lettuce necrotic yellows virus, the type member of the genus Cytorhabdovirus, and those of animal rhabdoviruses, are less significant. Nucleotide sequence determination of 5' and 3' regions flanking the RYSV N gene identified a 14-nt common sequence that is very similar to the consensus gene junction sequences of other plant and animal rhabdoviruses.

Insect resistance of transgenic tobacco plants expressing delta-endotoxin gene of Bacillus thuringiensis.

The initiative B.thuringiensis delta-endotoxin (Bt toxin) gene clones TH12 and TH48 contain two different classes of homologous genes, the 5.3 kb class and 6.6 kb class, respectively. Bt toxin genes of both classes, modified at the 5'-end and truncated at the 3'-end, can still be expressed to produce the insecticidal, truncated toxin proteins in E. coli. The modified Bt toxin genes were inserted into the plant binary expression vector pBin 437 (a derivative plasmid of pBin 19) and were transferred into tobacco by Ti plasmid-mediated gene transfer system. Southern blot and DNA slot blot analysis indicate that the Bt toxin genes have been integrated into tobacco genome at a copy number of 1 to 5. Northern blot analysis of polyA+ RNAs from progeny of the transgenic plants revealed that Bt toxin genes of both 5.3 kb and 6.6 kb classes were expressed in transgenic plants, though the transcripts were degraded to RNAs of lower molecular weights. In insecticidal test, 5 plants from the progeny of 5.3 kb class gene-transformed SR1 tobacco plants and 3 plants from those of 6.6 kb class gene-transformed plants were found to be toxic to the testing larvea of H.assulta. In comparison with the control, mortality of the insects fed on transgenic plants reached 40-50% and the growth of the survived insects was remarkably inhibited. These results indicate that the modified Bt genes of the 5.3 kb and 6.6 kb classes were expressed in transgenic plants and could confer on the transgenic plants a new character of insect resistance.

Characterization of an endogenous quail intestinal lectin.

Soluble extracts of quail intestine scrapings contain a lectin activity specific for chicken and rabbit trypsinized, glutaraldehyde-fixed erythrocytes. The lectin displayed a specificity for the simple sugar haptens lactose and galactose and for mucin. Quail lectin was purified by affinity chromatography on either asialofetuin- or mucin-Sepharose, followed by DEAE-Sepharose chromatography, and demonstrated an apparent molecular weight of 14,500 on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a pI of 6.2 upon isoelectric focusing. Immunohistochemical localization of this lectin in the intestine was carried out using polyclonal antibody raised in rabbits and tested for specificity in Western blots. Immunoperoxidase staining for quail lectin showed the lectin to be prominent in secretions at the mucosal surface and in goblet cells.

Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.

The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5- to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-specific promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.

Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants.

The 35S promoter is a major promoter of the cauliflower mosaic virus that infects crucifers. This promoter is still active when excised from cauliflower mosaic virus and integrated into the nuclear genome of transgenic tobacco. Previous work has shown that the -343 to -46 upstream fragment is responsible for the majority of the 35S promoter strength (Odell, J.T., Nagy, F., and Chua, N.-H. [1985]. Nature 313, 810-812). Here we show by 5', 3', and internal deletions that this upstream fragment can be subdivided into three functional regions, -343 to -208, -208 to -90, and -90 to -46. The first two regions can potentiate transcriptional activity when tested with the appropriate 35S promoter sequence. In contrast, the -90 to -46 region by itself has little activity but it plays an accessory role by increasing transcriptional activity of the two distal regions. Finally, we show that monomers and multimers of a 35S fragment (-209 to -46) can act as enhancers to potentiate transcription from a heterologous promoter.

Bacteria-lectin interactions in phytohemagglutinin-induced bacterial overgrowth of the small intestine.

The mechanism of phytohemagglutinin-induced bacterial overgrowth of the small bowel in the rat was studied. Interaction of the lectin with bacterial isolates selected at random from those that comprised the major population of the overgrowth was determined. In both bacterial agglutination assays and glycocalyx stabilization, no specific association between lectin and bacteria was seen. In three independent binding assays phytohemagglutinin was not found to increase bacterial adherence to washed intestinal mucosa. Phytohemagglutinin would not appear to act, therefore, as a direct ligand to mediate bacterial adherence or to modify the mucosal surface to increase bacterial adherence.

Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA.

Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP-mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism.

Study on the genome of cauliflower mosaic virus (Xinjiang isolate). Restriction mapping of virion and cloned viral DNA.

The genomic DNA of CaMV (Xinjiang isolate) was mapped on the virion DNA with a number of restriction endonucleases by double digestions and partial digestions of one-end 32P-labelled fragments. It contained the unique sites of BglI, SalI and XhoI. BamHI and HhaI each cut it at two sites, EcoRI at five sites (three of which were located), HpaII at seven sites, BglII at eight sites, and HaeIII at ten sites. The sites of all the enzymes used above, as well as HindIII which cut at ten sites were also mapped on the cloned CaMV DNA. Virion DNA and cloned viral DNA gave the same results in the number and location of cleavage sites of the restriction enzymes tested on both DNAs. Like most of the other isolates, CaMV-Xinjiang isolate has three single-stranded discontinuities. From the digestion patterns of SalI, XhoI, EcoRI and HhaI, it is concluded that the restriction map of CaMV-XJ genome is closely related to that of BKMV isolate from East Germany.