烟草花叶病毒复制酶介导抗性的研究进展

在抗病毒植物基因工程中,利用病毒的复制酶基因是一种很有前途的方法。本对烟草花叶病毒（TMV）的基因组结构及其编码的蛋白的功能作了简介,同时较详细地阐述了由TMV复制本科的通读部分、全长复制酶以及突变或缺失的复制酶介导的对病毒抗性的研究进展。     

病原激发子对番茄阴离子过氧化物酶表达与反应性氧迸发的诱导

采用Nortnern印迹法和鲁米诺化学发光法分析抗性的、近等位基因的敏感番茄细胞株阴离子过氧化物酶基因的转录和细胞反应性氧变化,发现病原真菌的激发子和外源H2O2均能促进抗性番茄细胞中阴离子过氧化物酶转录。激发子还能刺激抗性细胞中反应性氧水平暂时急剧升高。敏感番茄细胞对激发子或H2O2没有响应。Ca2 和磷脂酶C的抑制剂硫酸新霉素分别促进和抑制激发子诱导下抗性细胞反应性氧增加。     

武昌鱼肝线粒体DNA限制性内切酶酶解图谱与12SrRNA基因的初步定位

武昌鱼肝线粒体(mt)DNA经六种限制性内切酶BamHI,BgⅢ,BgⅡ,EcoRI,HindⅡHpall单酶完全酶解分別得到2,2,3,3,3和7个片段。用琼脂糖凝胶电泳测得各个酶解片段的长度和分子量,经计算该mtDNA长约16.6kb,分子量10.2×10~6道尔顿(dalton)。用七对限制酶双酶全酶解,构建出五种限制性内切酶图谱。以酵母线粒体15SrRNA基因为探针对武昌鱼肝mtDNA中的12SrRNA基因进行初步定位。     

病原激发子对番茄阴离子过氧化物酶表达与反应性氧迸发的诱导

采用Ｎｏｒｔｈｅｒｎ印迹法和鲁米诺化学发光示分析抗性的,近等位基因的敏感番茄细胞株阴离子过氧化物酶基因的转录和细胞反应性氧变化,发现病原真菌的激发子和外源Ｈ２Ｏ２均能促进抗性番茄细胞中阴离子过氧化物酶转录。激发子还能刺激抗性细胞中反应性氧水平暂时急剧升高。敏感番茄细胞对激发子或Ｈ２Ｏ２没有响应。Ｃａ＾２＋和磷脂酶Ｃ的抑制剂硫酸新霉素分别促进和抑制激发子诱导下抗性细胞反应性氧增加。     

番茄感染TMV诱导的β-1,3-葡聚糖酶的纯化和性质

番茄系统感染TMV诱导对胞外β—1,3—葡聚糖酶活性升高。番茄叶胞外提取液经冰冻干燥浓缩、-20℃丙酮沉淀、CM-Sephadex C-25离子交换层析和PBE 94聚焦层析纯化,获得PAGE和SDS-PAGE均一的β—1,3—葡聚糖酶。测得该酶的分子量为22kD;以昆布多糖为底物,该酶的最适pH5.4,最适温度30～40℃;K_m和V_(max)值分别为5.64mg/ml和 0.328nmol/s。在感染TMV的番茄叶中,β—1,3—葡聚糖酶活力大部分位于胞外,它是番茄叶胞外提取液中主要的病原相关蛋白。     

从番茄过氧化物酶同工酶表型差异探讨杂种优势机理

用聚丙烯酰胺凝胶电泳对番茄进行过氧化物酶同工酶分析表明:(1)不同栽培品种在同一生育期或同一品种在不同生育期、不同部位酶谱表型有明显差异,但把各品种不同生育期的酶谱叠加,所得到的总酶谱是相同的;(2)品种间杂交其F_1的过氧化物酶同工酶谱不存在“杂种酶带”或“互补酶带”;(3)品种间过氧化物酶同工酶的结构基因相同,酶谱表型上的差异可能是基因表达顺序上的差异,这可能是番茄杂种优势产生的生理基础之一。选择早期(芽期)酶谱表型差异适中的品种作杂交亲本,有可能获得生产上可用的高优组合。     

对TMV不同抗性番茄品种叶绿体DNA限制性差异片段的克隆

对ＴＭＶ不同抗性番茄品种ｃｔ－ＤＮＡ经限制性内酶切后,将所获得的差异片段Ｂａｍ６克隆到质粒ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ中,经筛选、菌落原位杂交、斑点杂交及电泳鉴定,证明重组体为抗性品种Ｂａｍ６差异片段克隆,对探讨番茄ｃｔ－ＤＮＡ与抗ＴＭＶ的关系有重要意义。     

鲤鱼、鲫鱼肌细胞线粒体DNA的限制性内切酶酶切图谱比较

鲤鱼肌细胞线粒体DNA经限制性内切酶Bam HI和Eco RI酶切后,皆被切成3个片段;鲫鱼肌细胞线粒体DNA经上述两种限制性内切酶酶切后,皆被切成2个片段。通过琼脂糖凝胶电泳对这些片段进行测定,并分别画出它们的酶切图谱。鲤鱼肌细胞线粒体DNA的分子量约为10.50×10~6道尔顿,有16.99千碱基对;鲫鱼肌细胞线粒体DNA的分子量约为9.40×10~6道尔顿,有15.21千碱基对。     

三种昆虫核型多角体病毒DNA的限制性内切酶酶解分析

在茶尺蠖、茶毛虫、斜纹夜蛾三种昆虫的四株核型多角体病毒中提取DNA,应用限制性内切酶图谱分析法研究,结果表明三种昆虫核型多角体病毒的内切酶图谱各不相同。而二株茶尺蠖核型多角体病霉的内切酶图谱是一致的。     

玉米黑粉菌mtDNA的研究 Ⅱ.限制性内切酶酶切图谱

本文报道玉米黑粉菌mtDNA的限制性内切酶酶切图谱。分别将mtDNA的Bam HI各片段制成探针,与mtDNA分别用8种酶酶切后的Southern膜进行杂交,用片段重叠法得出各套片段的排列次序,再将克隆化的Bam HI片段进行第二酶切,按分子量拼排出各酶酶切位点在mtDNA上分布的图谱。此外,片段重叠分析时,还发现玉米黑粉菌mtDNA为环状结构;杂交分析时还发现mtDNA内没有明显的重复序列。DNA总长60.7kb。     

Heterogeneity of the Canidae Bsp-repeats family: discovery of the EcoRI subfamily]

A 1600 bp EcoRI fragment was cloned from genome of raccoon dog. The structure obtained is homologous to the Canidae Bsp-repeats family. Comparative blot hybridization of the EcoRI fragment and BamHI repeat from fox genome with restricted hydrolysates of the total of raccoon dog and fox DNAs revealed differences both in structure and genomic organization between these two Bsp-repeats versions. Evidently, the EcoRI fragment contains a sequence lacking from the BamHI fragment of the fox Bsp-repeats. Quantitative differences in contents of two Bsp versions in various canid genomes were revealed as well. The EcoRI version is most abundant in raccoon dog genome, while the BamHI fox version is most representative in polar fox genome. With other species studied, quantitative differences in version contents are not so dramatic, and the EcoRI fragment is always present in lower copy numbers. The discovery of the EcoRI subfamily of the Bsp-repeats is in accordance with the "library hypothesis" advanced by Salser in 1976. Connection of the Bsp-repeats' evolution with centric fusions and breaks characteristic of karyotype evolution of canids is being discussed. Comparative study of cloned EcoRI and BamHI fragments of Bsp-repeats in cytogenetical and molecular aspects may be useful, when investigating the role of tandem repeats in large chromosome rearrangements.     

Physical maps of bovine papillomavirus type 1 and type 2 genomes.

Physical maps of bovine papillomavirus type 1 and type 2 (BPV-1 and BPV-2) DNA were constructed from analysis of the electrophoretic mobilities of restriction endonuclease cleavage fragments from dual digests. BPV-1 DNA was sensitive to Hind III, HindIII, EcoRI, HpaI, AND BamHI, with all but HindII yielding single scissions. BPV-2 DNA was resistant to EcoRI, and HindIII had one cleavage site whereas HpaI, BamHI, and HindII yielded multiple fragments. Of four BPV-1 isolates examined, DNA from one isolate was resistant to HindIII, and another DNA isolate was resistant to BamHI. The three BPV-2 isolates examined were uniformly sensitive to the restriction endonucleases employed.     

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

对TMV不同抗性番茄品种叶绿体DNA的RAPD分析

本实验对ＴＭＶ抗性和敏感的两个番茄品种ｃｔ－ＤＮＡ进行ＲＡＰＤ分析,结果发现在２０个引物中有４个引物的扩增产物存在明显差异,共显示出１４条差异带。这可能是由于ｃｔ－ＤＮＡ碱基顺序变异或小片段ＤＮＡ插入或缺失造成的,深入研究将对探讨番茄抗ＴＭＶ的分子机理,进而用遗传工程方法培育抗病品种具有重要意义。     

Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endoncleases EcoRI, BamHI, and HindIII.

Cleavage of bovine adenovirus type 3 (BAV3) DNA by restriction endonucleases EcoRI, BamHI, and HindIII yielded 7 (A to G), 5 (A to E), and 12 (A to L) fragments, respectively. The order of these fragments has been determined to be GDACBFE for EcoRI fragments, AEBDC for BamHI fragments, and JEBKACDHFGIL for HindIII fragments, and cleavage sites of these enzymes have been mapped on the genome of BAV3. BAV3 preparation contains incomplete virus whose genome has a deletion of about 13% of complete virus genome. Restriction endonuclease digestion of the incomplete virus DNA revealed that EcoRI E and F, BamHI C and HindIII G, I, and L fragments were deleted. Therefore, the deleted region of incomplete virus DNA is located near the right-hand end of the BAV3 DNA molecule, a result consistent with our previous electron-microscopic observations on heteroduplex molecules formed between complete and incomplete BAV3 DNA.     

Structure-based sequence alignment of type-II restriction endonucleases

The type-II restriction endonucleases generally do not share appreciable amino acid sequence homology. The crystal structures of restriction endonucleases EcoRI and BamHI have shown these enzymes to possess striking 3D-structural resemblance, i.e., they have a similar overall fold and similar active sites, though they possess <23% sequence identity. Structural superimposition of EcoRI, BamHI, EcoRV, and PvuII based on active site residues led to sequence alignments which showed nine possible sequence motifs. EcoRV and PvuII show a more similar pattern than EcoRI and BamHI suggesting that they belong to a different subgroup. The motifs are characterized by charged and/or hydrophobic residues. From other studies on the structure of these endonucleases, three of the motifs could be implicated in DNA binding, three in forming the active site and one in dimer formation. However, the motifs were not identifiable by regular sequence alignment methods. It is found that motif IX in BamHI is formed by reverse sequence order and the motif IX in PvuII is formed from the symmetry related monomer of the dimer. The inter-motif distance is also quite different in these cases. Of the nine motifs, motif III has been earlier identified as containing the PD motif involving one of the active site residues. These motifs were used in a modified profile analysis procedure to identify similar regions in eight other endonuclease sequences for which structures are not known.     

Unusually infrequent cleavage with several endonucleases and physical map construction of Bacillus subtilis bacteriophage phi 1 DNA.

HaeIII, BalI, StuI, BamHI, SlaI, and EcoRII did not cut the genome of Bacillus subtilis phage phi 1 at all, whereas ThaI, BglII, EcoRI, SalI, and Bsu1247I cut the genome once or twice. The physical map of the phi 1 genome was constructed with the latter restriction endonucleases.     

Supercoiling and the mechanism of restriction endonucleases

We have used topoisomerase I in the presence of netropsin and ethidium bromide to generate DNA molecules of varying superhelical density. Digestion by endonuclease EcoRI is sensitive to supercoiling, being maximal for the relaxed form. Endonucleases AvaI and BamHI, by contrast, are relatively unaffected. The results are interpreted in terms of the base composition of the DNA in the vicinity of these sites. dA + dT-rich regions are more susceptible to deformation than are dG + dC-rich ones. Analysis of the rates of disappearance of linear molecules confirms a two-step mechanism for EcoRI cleavage but suggests that BamHI and AvaI cleave both strands simultaneously.     

Distribution and evolution of two satellite DNAs in the genus Beta

Summary EcoRI monomers of a highly repetitive DNA family of Beta vulgaris have been cloned. Sequence analysis revealed that the repeat length varies between 157–160 bp. The percentage of AT-residues is 62% on average. The basic repeat does not show significant homology to the BamHI sequence family of B. vulgaris that was analyzed by us earlier. Both the EcoRI and BamHI sequences are investigated and compared to each other with respect to their genomic organization in the genus Beta. Both repeats were found to be tandemly arranged in the genome of B. vulgaris in a satellite-like manner. The EcoRI satellite DNA is present in three sections (Beta, Corollinae and Nanae) of the genus, whereas the BamHI satellite DNA exists only in the section Beta. The distribution of the EcoRI and BamHI satellite families in the genus is discussed with respect to their evolution.     

Mapping the Mutation Site of an Autographa californica Nuclear Polyhedrosis Virus Polyhedron Morphology Mutant

A polyhedron morphology mutant of Autographa californica nuclear polyhedrosis virus, designated M5, was compared with wild-type virus by genotypic analysis with EcoRI, BamHI, HindIII, SstI, and SmaI restriction endonucleases. M5 DNA revealed several alterations relative to the wild-type pattern: (i) EcoRI fragment I was 400 base pairs larger; (ii) BamHI fragment F was missing; (iii) HindIII fragment F was 400 base pairs larger; (iv) an extra restriction fragment was obtained with both HindIII and SmaI; and (v) SstI fragment G was 400 base pairs larger. M5 virions contained two size classes of circular DNA, one of 100% of the wild type and one of about 58% of the wild-type molecule. A revertant of M5, designated M5R, was isolated on the basis of polyhedron morphology. The genome of M5R contained the insertion of DNA in EcoRI fragment I and in HindIII fragment F, but was similar to the wild type in its other restriction fragment patterns. M5-infected cell cultures synthesized a polyhedrin polypeptide smaller in size than the wild type or M5R.