CLONING, EXPRESSION AND CHARACTERIZATION OF Pro3 GENE OF YEAST Saccharomyces cerevisiae

The proline biosynthetic pathway and Pro genes in Saccharomyces cerevisiae have just begun to be studied recently. In our laboratory, Pro2 gene of S. cerevisiae had been cloned in yeast. As described in this paper, yeast Pro3 gene was also cloned, which can complement yeast Pro3 mutants, and be expressed efficiently in E. coli. The high activities of this gene product, L-pyrroline-5-carboxylate (P5C) reductase, can be detected in both organisms. The activity of the Pro3 gene product in multiple copy plasmids is not higher than that of single copy genes in chromosomes in both yeast and E. coll. The preliminary characterization of the gene is also reported.     

EXPRESSION OF SYNTHESIZED HIRUDIN GENE IN YEAST

Hirudin is a sort of polypeptides secreted from the salivary gland of medicinal leech. It is of potential importance in medicine. We designed and synthesized the hirudin gene based on the amino acid sequence of hirudin HV2, and expressed it using the yeast alpha factor expression system. The yeast strain stably carrying the hirudin expression plasmid was deduced by mutagenesis. After its cultivation in rich nutritious medium for 36—48 h, the hirudin expression product secreted into the culture fluid was 10—20 ATU/ml. The HPLCpure hirudin product could be obtained through a simpler purification procedure, its N-terminal amino acid sequence was identical with the natural product, and it showed potent anticoagulant and antithrombin activity. About 3000 ATU of pure hirudin witha specific activity of 6600 ATU/mg could be obtained from 500 ml of culture fluid.     

MOLECULAR CLONING AND EXPRESSION OF Bacillus thuringiensis subsp. galleriae INSECTICIDAL CRYSTAL PROTEIN GENES IN Escherichia coli

The location of the toxin gene of B. thuringiensis subsp. galleriae (H5ab) on the Mr-130Mdplasmid is determined by molecular cloning. Double digestion fragments (BamHⅠ and SalⅠ)and PstⅠ restriction fragments as well, from the 130 Md plasmid of B. thuringiensis subsp.galleriae, are ligated with the cloning vector pAT 153 respectively and transformed into E.coli strain HB 101. Out of 208 transformants, three colonies (FG2, FG9, FG19) give posi-tive hybridization reaction using the HD-1 delta-endotoxin gene as a probe. They are presum-ed to contain the delta-endotoxin gene of B. thuringiensis subsp. galleriae. Western bolt assaysindicate that Mr-130 kDal and 68 kDal, crystal proteins produced by clone FG 2 react withanticrystal protein antibody. The protein extracts of clone FG2 are lethal to Ostrinia furna-calis (Guenee). This is the first report with regard to the cloning and expression of the B. thuringiensissubsp. galleriae (H5ab) delta-endotoxin gene.     

TRANSFER OF THE ATRAZINE-RESISTANT GENE OF BLACK NIGHTSHADE TO SOYBEAN CHLOROPLAST GENOME AND ITS EXPRESSION IN TRANSGENIC PLANTS

The atrazine-resistant psbA gene of black nightshade was transferred to the chloroplast genome of atrazine-susceptible soybean by means of ovary microinjection during the stage of zygote. The identification was carried out by using the methods of spraying the leaves directly with atrazine solution, examining the change of leaf fluorescence kinetics under a brighter light induction, molecular hybridization, etc. The experimental results show that the transgenic soybean plants do have been obtained for the first time.     

EXPRESSION OF A PARTIAL SYNTHETIC HUMAN TNF cDNA IN E. coli

A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exou codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fer1entator was used to produce rhTNF. About 20g (wet weight) of bacterial pellet per liter medium and 106—10~7 units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer, rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.     

INDUCTION OF UMUC+ GENE EXPRESSION IN Escherichia coli IRRADIATED BY NEAR ULTRAVIOLET LIGHT

Abstract— The induction of umu + gene expression caused by irradiation with near ultraviolet light (BLB; black light blue) was studied in Escherichia coli K-12 strains with special reference to the effects of SOS repair deficiencies. The umuC + gene expression was measured as the enzymic activity of (J-galactosidase which is regulated by the promoter of the umuC + operon carried in a plasmid DNA carrying a promoter of umuC* operon, a umuD + gene and a umuC +- lacZ + gene fusion. A high induction of the umuC + gene expression was observed in the uvrA cells in the case of BLB or UV irradiation as compared with the parental wild-type cells. Caffeine inhibited the induction of the umuC* gene expression due to BLB or UV irradiation in both strains. There was very little induction in lexA and recA mutants. In contrast with UV irradiation, there was no killing of cells by BLB irradiation in any strain (wild, uvrA, lexA and recA). Possible implications of the present experimental results were discussed.     

The expression of enterotoxin A-B+ gene of V. cholerae in E. coli

The cloning in E. coli of a cholerae toxin gene that is A-B+ has been successfully constructed by using DNA recombinant techniques. E. coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.     

ALTERATION IN CHROMATIN CONFORMATION OF PROLIFERATING AND DIFFERENTIATING ENZYME GENES IN NORMAL MOUSE LIVER AND ASCITES HEPATOMA CELLS AND ITS RELATION TO THE GENE EXPRESSION OF ENZYMES

Nuclei from the normal mouse liver were partially digested with micrococcal nuclease, followed by DNA extraction, agarose gel clectrophoresis and dot blot hybridization with ~(32)P-labeled cDNA probes of CPS_1 and ACT complex, It was clearly shown that the CPS_1 genes were distributed on the monomer, dimer. and trimer of nucleosomes, while the genes coding for ACT complex were distributed on the condensed oligonucleosomes. An opposite manner of distribution of CPS_1 and ACT complex genes was, however, noted in the case of ascites hepatoma cells, in which the specific activity of ACT was 13 times higher than that in the normal liver, while that of CPS_1 was remarkably reduced. Similar patterns of change in mRNA level of CPS_1 and ACT complex were observed in the normal mouse liver and ascites hepatoma cells, indicating a close relationship between chromatin structure and gene expression of these enzymes.     

B SUBUNIT OF CHOLERA TOXIN PRODUCED IN Escherichia coli

An engineered E. coli strain containing high expression level of CT-B subunits has beenobtained by the application of recombinant DNA techniques. The B subunit can be secretedinto the medium and reaches 20- 40 μg/ml when this strain is incubated in a 50 1 fermenta-tion tank. The CT-B subunit purified with affinity chromatography in E. coli has the samecharacters as the natural CT- B subunit in molecular weight, N terminal amino acid analysisand antigenicity. The CT-B subunit has good immunogenicity and can be used as a preparation for protect-ing against diarrhea caused by V. cholera and enterotoxigenic E. coli. It can also be usedas a vector for hepatins.     

EXPRESSION OF A ZEIN GENE (Z4) INTRODUCED INTO DICOTYLEDONOUS Solanum nigrum

A maize genomic clone containing a zein gene (Z4) is inserted into the T-DNA of the Ti plasmid pTiT37. Agrobacterium tumefaciens strain harboring this modified Ti plasmid is used to infect stem sections of young plants or explants of dicotyledonous Solanum nigrum. Axenic transformed calli active in nopaline synthesis are obtained and transgenic plants are differentiated from them DNA Southern hybridization and RNA dot-hybridization analyses show that the zein gene is really transferred and integrated into the nuclear genome of transformed Solanum nigrum and that the zein gene can be transcribed into mRNA in the transformed calli and shoots. But the presence of the zein protein cannot be detected in either the transformed calli or the transgenic shoots. The results of thte experiments demonstrate that the promoter of a gene from monocotyledonous plants can function normally in transgenic dicots. The possibility of developmentally-regulated expression of the zein gene in transformed dicots is discussed in     

Expression of Shiga Toxin B Subunit at Cell Surface in E. coli K-12

The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.     

TRANSFER OF FOREIGN GENES BY ELECTROPORATION AND THEIR EXPRESSION IN MAMMALIAN CELLS

Expression of foreign genes transferred into mammalian cells by electroporation has been studied. The pX1TK gene, pSV2Neo gene and pUCEJ oncogene have been introduced into MLTK-cells and NIH/3T3 cells, respectively. Stable transformation transient expression of TK gene by MLTK-cells as well as stable and malignant transformation of NIH/3T3 cells have been obtained. Transient expression frequency is about 80% and stable transformation frequency is about 10~(-4). Integration of foreign genes into the cellular genome was verified with molecular hybridization. Tumor development was observed after inoculation of transformed celts into nude mice.     

THE INTERACTION OF A GENE (nur) CONTROLLING NEAR-UV SENSITIVITY AND THE polA1 GENE IN STRAINS OF E. COLI K12

Abstract— When stationary cell populations of the Escherichia coli W3110 strain and the polA1 mutant (p3478) derived directly from it were compared for their sensitivity to near-UV (NUV, 300–400 nm) inactivation, the polA1 strain proved to be more sensitive. By appropriate matings and transductions, four essentially isogenic strains have been developed which carry all four possible combinations of genes conferring far-UV (FUV, 200-300 nm) sensitivity ( polA1 vs. polA ⁺) and NUV sensitivity ( nur vs. nur ⁺). Stationary cells of strains carrying either the polA1 or polA ⁺ allele in combination with the nur allele are indistinguishable in their sensitivity to NUV inactivation and are equivalent in their NUV sensitivity to the original polA1 mutant strain (p3478). With the two strains carrying the nur ⁺ allele, stationary cell populations of the polA1 strain are clearly more sensitive to NUV inactivation than is the polA ⁺ strain. The NUV sensitizing effect of the polA1 mutation in a nur ⁺ genetic background is about the same as that of the nur mutation at the 0.37 survival level. This may mean that the polA1 and nur mutations sensitize E. coli stationary cell populations to NUV inactivation by a common mechanism.     

CLONING IN Yersinia enterocolitica OF K99 ANTIGEN GENE FROM Escherichia coli

A recombinant plasmid pFS239 containing the geoe coding for K99 antigen of Escherichia coli and wide-host-range plasmid pKT230 has been cloned in E. coli C600. pFS239 has been transferred to Yersinia enterocolitica strains D29, L15 and L15 (pYV15) through triparental mating. In Y. enterocolitica transconjugants the expression of VW antigens and calcium dependence which represent the propertics associated with the virulence plastmid of Y. enterocolitica remains unchanged.     

STRUCTURE OF THE MITOCHONDRIAL URFA6L GENE AND tRNA~(Lys) GENE FROM CARP

The carp mitochondrial URFA6L gene consists of 165 base pairs. The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene. Their nucleotide sequences exhibit 68% homology. The carp URFA6L gene encodes a protein of 54 amino acids. The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids(proline, tryptophan, leueine, isoleueine and tyrosine). A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function asATPase8. The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in cloverleaf secondary structure. Similar to amphibian and mammalian mitochondrial tRNA~(Lys) genes, the carp mitochondrial tRNA~(Tys) gene also has some unusual structural features as compared with its cytoplasmic counterpart     

铵梯脲混合炸药中改性硝酸脲和硝酸铵含量的测定

论述了铵梯脲混合炸药中改性硝酸脲和硝酸铵组分含量的一种快速准确的化学分析测定方法。该法在研制和生产铵脲类炸药的过程中,对选择最佳配比,控制生产和质量把关等有着重要的指导作用。同时笔者对测定方法,反应原理,影响因素等作了深入的探讨与研究。     

NUCLEOPHILIC ADDITION OF WATER AND ETHANOL TO COORDINATED DI-(2-PYRIDYL)KETONE IN RHENIUM(V) AND TECHNETIUM(V) COMPLEXES. THE CRYSTAL STRUCTURE OF DICHLORO(HYDROXY-DI-(2-PYRIDYL)METHOXIDE)OXORHENIUM(V)

Abstract

Complexes of general formula MOCl₂[(C₅H₄N)₂C(O)(OR)] (M = Re, Tc; R = H, Et) were prepared by the reaction of trans-ReOCl₃(PPh₃)₂ and (n-Bu₄N)[TcOCl₄] with di-(2-pyridyl)ketone (DPK) in ethanol (R = Et) under nitrogen and in benzene, containing trace amounts of water, in air (R = H). The compounds were characterized by elemental analysis, vibrational, optical and proton nuclear magnetic resonance spectroscopy. The evidence suggests that the coordinated DPK ligand has undergone addition of water and ethanol at the carbonylic carbon atom, and that the (C₅H₄N)₂C(o)(OR) moiety acts as a uninegative, terdentate N,O,N-donor ligand. The X-ray crystal structure of the complex ReOCl₂[(C₅H₄N)₂C(O)(OH)] is also reported. Crystal data: C₁₁H₉N₂O₃Cl₂Re, orthorhombic, space group Pcca; a = 14.935(5), b = 11.896(8), c = 14.937(11)Å, and U = 2653.8 (2.7)ų to give Z = 8 for D _calc = 2.37 g cm_?3. The structure was solved by Patterson and Fourier methods and refined by least-squares methods to R = 0.076. The complex has an Re = O bond distance of 1.67(2)Å, and a deprotonated diolate oxygen of the ligand is coordinated in the position trans to the oxo group.