MOLECULAR CHARACTERIZATION OF RICE Wx GENE

The complete nucleotide (nt) sequence of the rice waxy(Wx) gene, which is responsible for the synthesis of amylose in endosperm and pollen, has been determined by a combination of restriction mapping and nt sequence analysis of two overlapping genomic DNA clones. The entire gene is about 5.5 kb in length. The alignment of the nt sequence of the Wx gene from rice with those of maize (Klsgen, R. B. et al.) and barley (Rohde, W. et al.) revealed the presence of thirteen introns and fourteen exons. The full-length of Wx protein in cluding transit peptide is 609 amino acid (aa) residues. The calculated molecular weight of rice Wx preprotein is about 72 kD. There is no significant difference between the similarity scores of the aa sequence deduced from the rice Wx gene compared with those of maize and barley. However, the nt sequences of the 5'-end upstream, 3'-end downstream and introns of the rice Wx gene, as well as the aa sequence of the transit peptide region of the Wx preprotein have low similarity scor     

STRUCTURE IN THE PRECORE REGION OF HEPATITIS B CORE GENE AFFECTING ITS EXPRESSION IN E.coil

Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI,containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence.They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation. Transformants in E.coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert's chemical degradation method and by M13 chain termination method. Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter,SD sequence and identical N-terminus.The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants.The findings w     

CLONING AND ANALYSIS OF HISTONE 3 GENE AND RUBISCO SMALL SUBUNIT GENE OF RICE

We have isolated and determined the DNA sequence of several genes from the nucleus of rice (Oryza sativa, IR26). We screened a genomic library of rice IR26 and isolated a 14.8 kb segment containing an H3 gene and an H3-like pseudogene. Sequence analysis showed that the coding sequence of the rice H3 gene is 405 bp in length, and the 5' and 3' noncoding regions contain several regulatory sequences common to eukaryotic or histone genes. The codon usage of the rice H3 gene is highly unusual in that the third codon position is 98% G and C. Southern blotting analysis suggested that the copy number of the H3 gene is around 50 per diploid rice genuine. From the same rice geaomic library, we have identified rbcS, which codes for the small subunit of ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco). The rbcS sequence is interrupted by an intron at the same location in both rice and wheat. The first 18 amino acids of the transit peptide in rice and wheat rbcS are identical     

Cloning and Sequencing of Trichosanthin Gene and Its Expression in Escherichia coli and Tobacco Plant

A Trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature Trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a highlevel expression plasmid (pJLA502) of E. coli under the control of a P_RP_L promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the Trichosanthin gene has been     

THE NUCLEOTIDE SEQUENCE OF SURFACE ANTIGEN GENE OF HEPATITIS B VIRUS SUBTYPE adr

The nucleotide sequence of the XhoI-BamHI fragment (1279bp), which contains the sur-face antigen gene (S gene) of HBVadr, was determined by Maxam and Gilbert's method.By comparing the differences both of the nuclectide sequence in the S gene and its codedamino acid sequence between adr and those reported for adw, ayw and adyw, some new var-iation sites were discovered. The differences were mainly distributed in the two hydrophilieregions. However, at those sites which might show biological function, there were no varia-tions among different subtypes, they are relatively conservative in heredity and evolution.Comparing the variation of the nucleotide sequence in the S gene region with that in thenon-S gene region, it is shown that the frequency of variation in the non-S gene regiondoubled that in the S gene region. Tim S gene region is more conservative.     

Cloning, Sequencing and Expression in E. coli of Interferon-ω1 Gene

Human interferon ω1 (huIFN-ω1) gene was isolated and cloned from chromosome DNA derived from a Chinese fetal liver via polymerase chain reaction (PCR). By determining its nucleotide sequence we proved that the 88th codon should be GGA, coding for Gly. After engineering the original IFN-ω1 gene clone to a form that may be expressed as a nonfused protein, we also took the IFN-ω1 gene under the control of the PRPL promoter with an expression vector pBV220 in E. coli. The antivirus activity of the recombinant IFN-ω1 is about 6.5×10~7 units/L CULTURE (OD_(600)=0.75). Since IFN-ω1 not only has antivirus activity but also shows considerably high homology with animal trophoblast proteins which have been proved antiluteolysins as a maternal recognition signal for pregnancy, we believe that study on it will be practically and theoretically significant.     

THE COMPLETE NUCLEOTIDE SEQUENCE OF THE CLONED DNA OF HEPATITIS B VIRUS SUBTYPE adr IN pADR-1

The complete nucleotide sequence of the cloned hepatitis B virus DNA subtype adr in pADR-1 wasdetermined by Maxam and Gilbert's method. It is 3215 base pairs in size, which is 27 bp longer thanthe sequence of the adr pHBr330, as reported by Ono et al. The nucleotide difference between pADR-1and adr pHBr330 is about 2% while those between pADR-1 and adw as well as ayw are 9.3% and 9.7%respectively. In this paper, the heterogeneity and homogeneity of the S gene, the C gene and the othercoding regions in pADR-1 and in the other subtypes are compared and discussed.     

THE POLYHEDRIN GENE OF Bombyx mori NUCLEAR POLYHEDROSIS VIRUS

Cloned SalI fragments of Bombyx mori nuclear polyhedrosis virus (BmSNPV) DNA were screened with the polyhedrin gene of Autographa californica nuclear polyhedrosis virus as a probe. One positive clone, pBN61, with an insert of 1.65 Kb, was obtained. The Hind-Ⅲ, HpaⅡ and AluⅠ maps of the insert were constructed. Part of its nucleotide sequence has been determined. The 46 amino acid sequence, as determined from the nucleotide sequence, was compared with the reported sequence of BmSNPV polyhedrin. Only ono amino acid difference has been found. It is likely that clone pBN61 contains the whole BmSNPV polyhedrin gene.     

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.     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

Isolation and Characterization of Two Novel Gene Encoded Alkaline Esterases from an Alkaline Soil Metagenomic Library

<正>The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity.In this study,an alkaline soil metagenomic library was constructed to search for new lipolytic enzymes.Two novel gene encoded alkaline esterases(designated as estA and estB) were isolated by functional screening from the library.The estA gene consisted of 834 bp and coded for 277 amino acids with a molecular mass of 29998.Amino acid sequence homology analysis indicates that EstA belongs toα/βhydrolase fold family 4.4(abH4.4),with EstA being the smallest member of that family yet reported.The estB gene consisted of 1185 bp and encoded 394 amino acids with a theoretical molecular mass of 40090.Its conserved domain analysis shows that EstB belongs to the GDSL hydrolase superfamily.Both EstA and EstB exhibit only moderate identity(38%) in amino acid sequence to the known lipolytic enzyme genes in the database.The two genes were respectively expressed in Escherichia coli and the protein products were purified for functional characterization.While the expressed EstA did not exhibit the functional properties that were superior to those of other esterases previously reported,the EstB was stable at temperature up to 45℃and its maximum activity was measured to be 53.6 U/mg at pH=10.Both the enzymes have further enriched the diversity of the lipolytic enzymes database and also appear to be promising biocatalysts for potential biotechnological application.     

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     

MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF A TRANSFORMING GENE FROM HUMAN GASTROCARCINOMA CELL LINE

Mouse and rat fibroblasts were transfected with total DNA from human gastrocarcinoma cell line BGC-823. It was shown by hybridization assay that the genome of one of the rat secondary foci contains transforming genes from the human gastrocarcinoma cell line, which are homologous to the protooncogene c-Ha-ras in the normal cells. The genomic library of the rat secondary foci was constructed, using λ phage EMBL3 as the vector. The transforming gene Ha-ras of the human gastrocarcinoma cell was thus cloned by screening the library with the probes of human Alu repeat sequence and c-Ha-ras. The nucleotide sequences of the first and second exons were analysed by M13-dideoxy method. The result shows that the nucleotide sequence of the transforming gene is the same as that of the normal protooncogene except one nucleotide difference in the first exon.     

CLONING AND SEQUENCE ANALYSIS OF 3''''-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se     

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.     

TOTAL SYNTHESIS OF Trichosanthes TRYPSIN INHIBITOR AND ITS ANALOGUE

Trichosanthes trypsin inhibitor (TTI) is a peptide consisting of 27 amino acid residues with three pairs of disulfide bonds. This paper reports the total synthesis and disulfide bond refolding of this inhibitor and its analogue. After purification, the amino acid sequence and stoichiometrical inhibitory activity against trypsin of the synthetic inhibitor were compatible with those of the natural inhibitor. The analogue of this inhibitor in which residue Met in position 6 was replaced by Ala was also synthesized. The antitrypsin activity of this synthetic analogue was also approximate to that of the natural inhibitor.     

SYNTHESIS OF THE LEU-ENKEPHALIN GENE

The synthesis of Leu-enkephalin gene by an alternative approach was described in thispaper. The sequence of the synthetic gene, 26 base-pairs in length, was derived from the ami-no acid sequence of the hormone peptide Leu-enkephalin. It bears single-stranded cohesive ter-mini for the restriction endonucleases EcoR I and BamH I so that it may be inserted into apBR 322 plasmid. The 4 deoxyoligonucleotide fragments, varying in length from octanucleotideto octadecanucleotide, were synthesized by an improved phosphotriester method developed inour laboratory. All chemically synthetic fragments were pure and had the correct sequences.The assembly of the Leu-enkephalin gene was carried out in a one-step ligation reaction cata-lysed by T_4-DNA ligase with good yield, Finally, the purified products from polyacrylamidegel electrophoresis had the correct joining-points as predicted.     

Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap／GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial (RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.