马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。     

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.     

Geminiviruses: Genome structure and gene function

The plant pathogenic single‐strand DNA‐containing geminiviruses have been the recent focus of intense investigation, owing both to their agronomic importance and to their potential as vectors for the expression of foreign genes in plants. Molecular genetic studies have provided detailed information on the genomic organization of many of these viruses. A greater genetic complexity has been demonstrated among the members of this viral family than had previously been suspected, as well as an apparently rapid rate of evolution of genetic diversity. We now recognize fundamental differences in the genome structure and organization of the whitefly‐ and leafhopper‐transmitted viruses, as well as among those geminiviruses infecting dicotyledonous or monocotyledonous hosts. This knowledge has provided new insights into the evolution of these viruses. The viral genes involved in replication and in systemic movement in the plant have been defined, and viral origins for single‐strand (ss) and double‐strand (ds) DNA replication have been mapped to small nucleotide regions. With the structural features of the viral genomes now well defined, current efforts are focused on elucidating the molecular aspects of viral gene regulation and interactions with host‐cell components that lead to the production of disease. Recent progress in determining the mechanism of replication and systemic movement and the contributions of these to symptom and disease development are discussed in the context of the potential for genetically engineering disease‐resistant plants.     

Estimates of gene flow, genetic substructure and population heterogeneity in bracken (Pteridium aquilinum)

Bracken [ Pteridium aquilinum (L.) Kuhn] is a cosmopolitan species and is a noxious weed in many areas. Because of its abundance, particularly in Britain, bracken affords an ideal system for investigating various aspects of population genetics and evolution. High mobility of dispersal units (spores) suggests that rates of gene flow among distant populations should be high. Gene flow is a major evolutionary force that influences the genetic structure of populations. To examine the effects of gene flow on population heterogeneity and population substructuring in bracken, starch gel electrophoresis of enzymes was used to provide the necessary genetic database. Allele frequency data at 21 loci were obtained for seven British populations, one Majorcan and one from the eastern United States. A model was employed to estimate the amount of gene flow ( Nm ) at several levels. Gene flow among British populations was extremely high ( Nm = 36.51), one of the highest estimates reported for plants. Among eight European populations gene flow was lower (but still considered high) at Nm = 2.47. Trans-Atlantic gene flow was low ( Nm = 0.0926).
F -statistics were used to assess population heterogeneity and substructuring. The data indicate that, compared with other species, there is very little genetic differentiation among British populations of bracken. Indeed, it appears that the whole island is behaving as a single randommating population. This result is consistent with high levels of gene flow. Only one population (on the Isle of Arran) showed statistically significant genetic substructuring. Habitat heterogeneity on the island and age structure are hypothesized as possible causes of this result.
The data reported here support previous studies demonstrating that bracken is genetically polymorphic and is an outcrossing species.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.     

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

Summary The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.     

Genetic assay of misincorporation

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.