Cloning, sequencing and expression of a swine IgG H chain gene inE. coli

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass Ig γ3, and is the same as reported by Sun et al., but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector pET-3b (NSEB)( - ). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 % .     

Variation and Characterization Analysis of Partial Fragment of Fibroin Gene From Silkworm, Antheraea pernyi

A 1.4Kb DNA fragment containing 3‘ flanking sequence of fibroin gene of silkworm, Antheraea perny/, was obtained from the silk gland‘s mRNA of 5th larva. Analysis of this sequence with another A. pemyi fibroin protein (accession No. 1383241) revealed that it consists of a completely open reading frame (ORF), which includes 14 polyalanine-containing units (motifs) and 100bp 3‘-UTR. The sequence of the predicted amino acid reveals the highest level of overall iden-tity (90%) with 1383241. It was found that it loses a repeat region at the upstream of TAA codon and some mutations. A putative polyadenylation signal AATAAA tail was found in position 1300, which follows the termination codon.     

Cloning and sequencing of cDNA fragment of gene of wheat (Triticum aestivum. L) induced by dehydration

cDNA fragment of the gene (dehydration induced,di1) of wheat (Triticum aestivum. L) induced by 30% PEG-6000 (−1.13 MPa) treatment was isolated with mRNA differential display technique. Northern blot analysis showed that the expression ofdi1 gene improved at 10 h reached the highest at 48 h under 30% PEG-6000 treatment. cDNA fragment ofdi1 gene has been cloned and sequenced (211 bp). DNA sequence analysis shows that there is no homologue in GenBank todi1 cDNA.     

Construction of the glucose isomerase deficient strain ofStreptomyces M1033 by homologous recombination

After the establishment of the transformation conditions ofStreptomyces diastaticus No.7 Strain M1033, the integration plasmid pXW for homologous recombination, which contains a 600 bp fragment of incompleteGI (G138P. G247D) gene, has been constructed in order to realize the stable overexpression of theGI (G138P. G247D) which is valuable for large-scale industrial production. The Gigene’s disruption has been realized by pXW’s integration into M1033 chromosomes via homologous recombination andGI deficient strain ofStreptomyces M1033 has been obtained. The reliability of introduction of mutation has been proved by analysis of recombinant fragment and affirmance of existence of the mutation, as well as detection of the stability of the deficient strain.     

Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

PCR amplification of Sox genes inMisgurnus anguillicaudatus andParamisgurnus dabryanus (Cypriniforms; Cobitidae)

Using the primers which specially amplify the conservative motif of Human SRY gene, we studied the PCR amplification of Sox genes in genomic DNA of two species of mud loach:Misgurnus anguillicaudatus andParamisgurnus dabryanus. Four bands with the length of 200,550,940 and 1000 bp respectively, were presented in the PCR products ofMisgurnus anguillicaudatus. Three bands with the length of 200,550 and 900 bp were presented in that ofParamisgurnus dabryanus. Southern blotting results indicated that the 200 and 550 bp bands are specially positive. There is no difference between male and female individuals as well as between these two species. Chang zhongjie: Born in Nov. 1965, Ph. D. graduate student     

万安玻璃红鲤鱼PKR的克隆鉴定与系统进化分析

本研究以万安玻璃红鲤鱼为材料,通过同源克隆的方法克隆了万安玻璃红鲤鱼双链RNA激活的蛋白激酶（PKRCcvwPKR）,并首次克隆到CcvwPKR的剪接异构体。CcvwPKR全长为2145 bp,编码714个氨基酸,CcvwPKR剪接异构体全长1431 bp,编码476个氨基酸。CcvwPKR的剪接异构体是CcvwPKR第477个密码子的G突变为T（gaa477taa）,从而使得CcvwPKR的翻译提前终止。氨基酸序列比对和空间结构SMART预测显示,CcvwPKR和CcvwPKR剪接异构体都含有三个双链RNA结合模体（dsRBM）,CcvwPKR剪接异构体缺失PKR的C端的激酶结构域。系统进化分析表明,万安玻璃红鲤鱼PKR在进化上与鲤鱼和鲫鱼密切相关。万安玻璃红鲤鱼和目前已有研究的鲤科鱼类PKR一样,分子结构都含有三个dsRBM,三个dsRBM使得其具有更强的结合dsRNA的作用,推测可能与其较强的抗病能力有关。     

GCC box in<Emphasis Type="Italic">Arabidopsis PDF1.2</Emphasis> promoter is an essential and sufficient cis-acting element in response to MeJA treatment

The expression of Arabidopsis PDF1.2 gene isregulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element responsive to ET, however, the responsive mechanism of JA in such plant defense gene expression is unclear. In this paper, the authors define the essential cis-acting element in PDF1.2 promoter responsive to methyl jasmonate (MeJA) through fragment deletions and site-directed mutageneses combiningAgrobacterium-mediated transient reporter gene expression in tobacco leaves. Firstly, the MeJA inducible expression o fPDF1.2 was confirmed by using the upstream -1.86 kb fragment of PDFI.2 gene. Secondly, the upstream -300— -243 bp fragment of the promoter was evidenced to respond to MeJA. To further characterize this promoter region, three point mutations were introduced into the -300— -243 bp fragment of the promoter. This result showed that the mutation of GCC box abolished MeJA induction, whereas the mutations of the G box-like and the imperfect palindrome sequence did not significantly decrease MeJA inducible effect, indicating that GCC box in PDFI.2 is essential for MeJA induction. The sufficient responsiveness to MeJA of this GCC box was further investigated by 4xGCC fused upstream to the CaMV 35S minimal promoter. This result suggested that the fused promoter was able to activate reporter gene expression in response to MeJA. Thus these results indicate that the GCC box in PDFI.2 is an essential and sufficient element to confer MeJA induction.     

用加端PCR技术改造h－IGF－1cDNA

用ＤＮＡ合成仪合成了分别带有ＰｓｔＩ位点和ＳａｌＩ位点及终止密码子的２个用于扩增ｈＩＧＦ－１ｃＤＮＡ的ＰＣＲ引物．利用合成的引物，７００ｂｐ长的ｈＩＧＦ－１ｃＤＮＡ模板和Ｔａｑ聚合酶进行ＰＣＲ扩增．扩增产物经电泳鉴定后克隆进Ｍ１３ｍｐ１８载体，进行核苷酸序列分析．结果显示：ＰＣＲ产物含已发表的ｈＩＧＦ－１成熟蛋白的编码序列和５＇端的ＰＳｔＩ位点及３＇端的ＳａｉＩ位点及终止密码ＴＡＧ．用加端ＰＣＲ技术成功地扩增和改造了ｈＩＧＦ－１的编码序列．     

Experimental proof for the regulation ofSalmonella typhimurium purB bypurR

Salmonella typhimurium purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway forde novo synthesis of inosine 5′-monophosphate (IMP) and also the final reaction in the two-step sequence from IMP to adenosine monophosphate (AMP). The nucleotide sequence ofpurB was obtained by the genetic map and sequence homologous analysis. The conservedpur operator inpurB was identified to be located 185 bp downstream of the initiation codon and overlaps codons 62 – 67 in the protein-coding region. The binding of PurR to this operator was demonstrated by gel retardation experiment and site-directed mutagenesis, indicating that thepurB is under the control ofpurR. We also answered why previous study had conflicting report concerning the regulation ofpurB bypurR by identifying the junction site ofpurB tolacZ in apurB- MudJ (lacZ,Kan ^r) fusion strain. This result strongly supports that thepurB is the second gene in theycfC-purB operon.     

Cyclic AMP response element binding protein (CREB) participates in the heat-inducible expression of humanhsp90β gene

Human heat shock protein 90b gene ( hsp90b ) is a constitutively expressed heat shock gene existing in most of cell types tested that can be further induced by heat shock. Chloramphenical acetyl transferase (CAT) reporter plasmids driven by different regulatory fragments of hsp90b gene were constructed and transfected into Jurkat cells to explore the role of a cAMP response element (CRE) in the upstream of the gene. Results show that, in comparison with the wild type construct, a severe reduction (~2/3) in the increased folds of promoter activity induced by heat shock at 42℃ for 1 h was observed in a construct with CRE-containing fragment (-173/-91bp) deleted. Electrophoretic mobility shift assays (EMSA) showed that phosphorylated CRE-binding protein (CREB) in the nuclear extract of heat shocked Jurkat cells is specifically bound to the fragment. Additionally, both of the phosphorylation on CREB and the activity of protein kinase A (PKA) were found in Jurkat cells to be enhanced with extending time of heat shock treatment. Our results indicate that in addition to the intronic HSE/HSF pathway, phosphorylated CREB also participates in the heat shock induced expression of human hsp90b gene via its interaction with CRE which may be regulated by PKA-sig- naling pathway.     

Characterization of the flagellar biosynthesis regulatory geneflbD inAzospirillum brasilense

A flagellar gene cluster fragment includingflbD ofAzospirillum brasilense was cloned and sequenced. TheflbD mutant strain was found to be nonmotile—losing both polar and lateral flagella (Fla⁻Laf⁻). Motility and flagella were regained by complementation with plasmid-borne multicopyflbD, but altered with larger swarming circle and fewer lateral flagella on the semisolid plate. This result indicated that FlbD plays an important role in the regulation of both polar and lateral flagellar biosynthesis inA. brasilense.     

Cloning of human cytokine signal transduction inhibitor genehumSOCS-2

An EST (gb/AA115239) with high identity to the mouse cytokine signal transduction inhibitor genemmSOCS-2 was selected in GenBank EST database by the homologous screening method. The cDNA with the same sequence of the EST was got in human placenta cDNA library by PCR and a 1011 bp cDNA fragment was selected using above cDNA as probes to perform walking hybridization in placenta cDNA library. The cDNA fragment contains one 594 bp open reading frame (ORF) which encodes 198 amino acid residues. It was proved to be novel after NCBl database screening. Homology comparison showed that this gene has 93% identity tommSOCS-2 at the amino acid level and it has high identities to other related genes in SH₂ domain and SOCS box, so it was namedhumSOCS-2 and the accession number in GenBank is gb/AF020590. The expression analysis showed that the gene is expressed obviously higher in prostate than in other 15 human tissues.     

Recovery of DNA from agarose gel with home-made silica milk

An usefulness of silica milk made with waste ultraviolet light tube for recovery of DNA fragment from agarose gel was represented. The glass milk is a water suspension of 50% fine silica powder prepared by grinding the crushed waste ultraviolet light tube with a porcelain mortar. It was showed that one microliter of the glass milk could bind more than 1 μg of DNA fragment, and DNA fragment in length from 125 bp to 23 kb could be efficiently recovered from agarose gel. The bound DNA could be eluted from the particle of SiO₂ in the glass milk with a yield of about 60%–80%. The eluted DNA could be used in all manipulations in molecular cloning. Biography: Wang Zhuo-hua (1977-), female, M. D, research direction: genetic engineering.     

Nodulation and molecular characterization of pure cultures isolated from root nodules ofCoriaria nepalensis

Four strains with typical morphology of actinomycete genus ofFrankia were isolated from root nodules ofCoriatia nepalensis. They were shown to nodulate the seedlings of host plant and hybridize withFrankia 16S rRNA targeted olionucleotide probes, indicating that they did belong to the genusFrankia. Furthermore, by nifHDK probe hybridizations, the homologous fragments of nifHDK genes were detected among the bacteria, and they were located in various sizes of restriction fragments of total DNA, showing diverse patterns of restriction fragment length polyrnorphisms of nifHDK gene (nifHDK-RFLPs). The PCR-based amplification and cloning of nifH gene throw light on the molecular phylogeny ofCoriaria -infectiveFrankia.     

四环素产生菌质粒DNA的分离检测及遗传学分析

用７％蔗糖的ＹＥＭＥ液体培养基培养四环素产生菌金色链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｕｒｅｏｆａｃｉｅｎｓ）９１－０６，以链霉菌遣传操作法为基础，从该菌体中分离到质粒ＰＳＡ３１４，分子量约为２８．５ｋｂ（１８．８ＭＤ）。该质粒经酶切分析表明，具有２个ＢａｍＨＩ位点和１个ＥｃｏＲＩ位点，用吖啶橙处理获得无质粒珠，其在孢子形成和菌落形态方面出现明显差异。     

Desulfurization metabolite of Rhodococcus erythropolis LSSE8-1 and its related desulfurizational gene fragments

Rhodococcus erythropolis LSSE8-1 is a newly isolated biodesulfurizaion strain from the soil of Chishui gas field, Guizhou Province, China. The analysis of its metabolism product shows that the strain is a kind of biocatalyst able to oxidize dibenzothiophene (DBT) to 2-hydroxydi-phenyl (HBP), and therefore the sulfur in DBT is selectively removed. By using DBTO2 (dibenzothiophene 5,5-dioxide) as substrate, both DBT and HBP are found in the culture,which shows that the reaction from DBT to DBTO2 is reversible in the cell. While using 0.5 mmol/L DBT as control,0.01-0.4 mmol/L DBTO2 shows poisonous effect to the cell,which will explain why there is no DBTO2 accumulation in the process of biodesulfnrization. After treatment by lysozme,the plasmid DNA of the strain is isolated by alkaline method to be used as the template of PCR reaction. Three dsz gene fragments of 1.3, 1.0 and 1.2 kb respectively were amplified.Each fragment is ligate with PGEM-T vector, and cloned into E. coil DH5a. The clone DNA is sequenced and the result shows that dsz related genes are highly conservative. The identities of dszA and dszB with respect to IGTS8 are 100%,and the identity of dszC with that of IGTS8 is 99%.