几株球形芽孢杆菌二元毒素基因的检测及对敏感和抗性尖音库蚊的毒性

根据Bacillus sphaericus 2362二元毒素基因核苷酸序列合成的一组寡聚核苷酸为引物,通过PCR扩增出1.1 kb的DNA片段作探针检测了C₃-41、IAB881、IAB872、BS-197和lPl-G菌株中二元毒素基因。Southern杂交表明C₃-41、IAB881、IAB872和BS-197菌株中3.5kb Hind III及LPl-G中4.7kb Hind III的酶切片段分别带有与探针有高度同源性的二元毒素基因。SDS-PAGE和Western印迹杂交表明所有菌株都能产生41.9kD和51.4kD的毒素蛋白。C₃.41、IAB881、BS.197和2362的全发酵液和碱抽提液对敏感尖音库蚊Culex pipienssubsp.Pipiens幼虫毒性高,但对抗性幼虫几乎无毒,LPl-G对敏感和抗性蚊幼虫具有相同的中度毒杀作用;IAB872对敏感幼虫毒性高,对抗性幼虫的毒力同LPl-G相似。这两株菌对抗性蚊幼虫的毒性可能是由Mtx毒素蛋白所导致的。     

Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR

The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.     

Sequence heterogeneity of murine complementary DNA clones related to the C4 and C4-Slp isoforms of the fourth complement component

Two classes of mRNA encoding the murine C4 protein were identified by sequence analysis of clones isolated from a liver complementary DNA library. The divergence found within a 357 base pair sequence available for comparison is limited to five nucleotide replacements located in the region corresponding to the carboxy-terminal end of the C4d peptide fragment. One of the nucleotide substitutions influences the presence of a site for the Hind III restriction endonuclease. That this restriction site indeed discriminates the two non-allelic genes encoding the mouse C4 and C4-Slp isoforms has been demonstrated by Southern blot analysis and nucleotide sequencing at the genomic level. Circumstantial evidence supports the identification of the gene lacking the Hind III site in the region corresponding to the carboxy-terminal end of the C4d fragment as the one encoding the C4-Slp isotype.     

The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase.

The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found.     

Structure and gene organization in the transformed Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and a tRNA Glu 3 gene.

The sequence of a 1.9 kb Bam x Hind III fragment from yeast has been determined. This fragment is part of a yeast 6.7 kb Hind III segment cloned into pBR322 (pY20). The fragment carries a single gene for a glutamate tRNA which has no intron. According to genetic analyses [1] this fragment also contains a yeast chromosomal replicator. We have analyzed the sequence for potential open reading frames and for several structural features which are thought to be involved in the initiation of DNA replication. Hybridization studies have revealed that portions of this sequence are repeated within the yeast genome.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

The heterogeneity of rat-liver mitochondrial DNA

Two types of mitochondrial DNA (mtDNA) can be distinquished in an inbred strain of rats of the Wistar type. The population of DNA molecules of the liver of one single rat is homogeneous. This was shown for a number of 100 animals and confirms the data of other investigators. The two types of mitochondrial DNA, designated A and B, differ in their number of cleavage sites for the restriction endonucleases Eco RI (2sites), Hind II (1 site) and Hha I (1 site). No differences were found for the restriction enzymes Bam HI, Hap II, Hind III and Hpa I. The degree of sequence divergence of the two types of DNA is calculated to be roughly 5% on the basis of these observations. From 20 rats part of the liver was taken and the mtDNA was characterized. Heterologous and homologous crosses between type A and type B rats were made. Analysis of the offspring revealed strictly maternal inheritance of the A and B mtDNA traits. For purposes of base-sequence analysis and RNA.DNA hybridization the strain could easily be "purified" genetically.     

Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome.

The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP3 corresponds to the carbosyl terminal part of VP2, and considering various evidence which indicates that the SV40 Hind E segment is part of the amino acid sequence of VP2-VP3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP1 (Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the vp3 poly peptide, and the amino acid composition is in good agreement with published values (Greenaway and Levine, 1973).     

Restriction fragment length polymorphism of the rat albumin gene in Sprague-Dawley rats and its application in genetic study of analbuminemia.

Restriction fragment length polymorphism of the rat albumin gene was discovered in a stock of Sprague-Dawley rats by Southern blots of rat liver DNAs using cloned albumin cDNA, prAlb-1 (1), as a probe. The polymorphic DNA fragments were observed when rat DNAs were digested with either Hind III or Pst 1 and the difference in length of the DNA fragments in Hind III or Pst 1 digests was estimated as 1.4 kbp. When DNAs were digested with EcoR I, restriction fragment length polymorphism was not observed. Therefore, this polymorphic DNA was concluded to be located in the flanking sequence. Structural analysis of the cloned albumin gene showed that the polymorphism was located in the 3'-flanking sequence. With this polymorphism as a marker of the albumin structural gene, the phenotype of analbuminemia, which is an autosomally recessive trait, was found to be linked to the structural gene of albumin.     

球形芽孢杆菌C3—41二元毒素基因在苏云金芽孢杆菌以色列亚种中的？…

球形芽孢杆菌Ｃ３－４１是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于２３６２菌株,Ｓｏｕｔｈｅｒｎ杂交证明Ｃ３－４１总ＤＮＡ中３．５ＫｂＨｉｎｄＩＩＩ片段上带有４１．９和５１．４ｋＤ二元毒素基因。     

Nucleotide sequence definition of a major human repeated DNA, the Hind III 1.9 kb family

A human Hind III 1.9 kb repeated DNA fragment was isolated and cloned in pBR322. A cloned member that hybridized predominantly to the 1.9 kb Hind III band in a digest of whole human DNA was chosen for sequencing. It is an 1894 bp fragment that shows no significant internal repeats. Few pCG residues are observed in the sequence and there are numerous stop codons. Detailed sequence comparisons confirm this is a novel class of repeats that is not related to previously characterized human satellite DNAs or Alu sequences. At least a portion of the sequence described is conserved in evolution.     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

由基因序列开发番茄枯萎病抗性基因Ⅰ-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

由基因序列开发番茄枯萎病抗性基因I-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。