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81.
Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly 总被引:6,自引:0,他引:6
Michael Syvanen Zonghan Zhou Jonathan Wharton Claire Goldsbury Alan Clark 《Journal of molecular evolution》1996,43(3):236-240
One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier
work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST
loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with
genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both
the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3
genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral
genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed
sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest
that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the
divergence of sequence between the amplified copies.
Received: 22 November 1995 / Accepted: 23 February 1996 相似文献
82.
Replication of a plasmid lacking the normal site for initiation of one strand. 总被引:2,自引:1,他引:1 下载免费PDF全文
The origin of replication of the plasmid R1162 contains an initiation site for the synthesis of each DNA strand. When one of these sites (oriL) is deleted, synthesis on the corresponding strand is no longer initiated efficiently in vitro by the R1162-encoded replication proteins, and the plasmid is no longer stably maintained in the cell. However, in vivo the two strands of the plasmid duplex molecule are active at a similar level as templates for DNA synthesis, and newly synthesized copies of each strand are incorporated into daughter molecules at a similar rate. No secondary, strong initiation sites on the delta oriL strand were detected in the region of the origin. The delta oriL plasmid induces the SOS response, and this is important for plasmid maintenance even in a recombination-proficient strain. Our results indicate that an SOS-induced host system can maintain an R1162 derivative lacking one of its initiation sites. 相似文献
83.
Summary DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.Abbreviations ABA
Abscisic Acid
- 5-azaC
5-azacytidine
- GUS
-glucuronidase
- Dc8
carrot promoter 相似文献
84.
85.
Barbara Kroczynska Rengang Zhou Clifford Wood Jan A. Miernyk 《Plant molecular biology》1996,31(3):619-629
The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)+ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP
maltose binding protein
- PCR
polymerase chain reaction
- Stress70c
the cytosolic member of the 70 kDA family of stress-related proteins 相似文献
86.
应用MUCAP试剂快速检测沙门氏菌 总被引:5,自引:0,他引:5
报告了用4-甲基伞形酮辛酯(4-Methylumbelliferyl-caprylate, MUCAP)快速检测沙门氏菌的特异性、敏感性和实用性。经HE,DHL,SS和麦康凯琼脂平板分离的65株沙门氏菌标准菌株和48株从食品中分离的沙门氏菌,用MUCAP测试均呈阳性反应;394株非沙门氏菌中呈阳性反应的假单胞菌、气单胞菌、邻单胞菌可通过氧化酶试验与沙门氏菌区分开;与粘质沙雷氏菌的交叉反应改用加1%蔗糖的分离平板也可排除。此方法的敏感性和特异性均达到97%以上,而且操作简便、快速,数分钟内即可完成。 相似文献
87.
马桑菌株的生物学特性 总被引:3,自引:0,他引:3
对从湖北、四川、云南等地采集的尼泊尔马桑(Coriaria nepalensis Wall.)根瘤中分离的20株内生菌纯培养物所进行的研究表明,其形态均具有弗兰克氏菌属的特征。在粗细不一的分枝菌丝体上有泡囊和孢囊,有的还有串珠状结构的菌丝体。不同菌株在液体培养基上生长速度很不一致;菌丝体多呈絮状或颗粒状沉淀,个别还呈薄膜状沉淀;绝大多数菌株的菌丝体具有不同色调的色素;在S培养基中大多数都能产生可溶性色素。上述这些培养特征常因培养基成分和培养方式的不同而有差异。大多数菌株的胞壁组分属Ⅱ型,少数菌株为Ⅲ型;全细胞糖型变化很大,20个菌株可划分为6个糖型,而且绝大多数糖型与已知弗兰克氏菌有所不同。此外,有65%的菌株还具有明显的抗菌活性。 相似文献
88.
The effects of external pH on calcium channel currents in bullfrog sympathetic neurons. 总被引:4,自引:0,他引:4 下载免费PDF全文
We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance. 相似文献
89.
In the search for candidate genes for the tuberous sclerosis (TSC1) disease locus on chromosome 9q34, we have isolated an overlapping series of 22 plasmid and phage cDNA clones covering nearly 7 kb and with an open reading frame of 5070 bp encoding a protein of 1690 amino acids. The putative protein product is a member of the kinesin superfamily and is homologous to the mouse KIF1A and theCaenorhabditas elegansunc-104 genes. Both KIF1A and unc-104 function in the anterograde axonal transport of synaptic vesicles. The human homolog is therefore termed H-ATSV (axonal transporter of synaptic vesicles, HGMW-approved nomenclature ATSV) Screening of DNA from 107 tuberous sclerosis patients and 80 unaffected individuals with H-ATSV cDNA probes by pulsed-field gel electrophoresis/Southern blotting following digestion by rare-cutting methylation-sensitive restriction enzymes showed variant banding patterns in three patients with tuberous sclerosis. However, further analysis indicated that these variant fragments represent a rare polymorphism probably associated with methylation of clustered restriction sites. There is no evidence to support H-ATSV as a candidate gene for TSC1. 相似文献
90.
Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures
Zhou W Bibila T Glazomitsky K Montalyo J Chan C Distefano D Munshi S Robinson D Buckland B Aunins J 《Cytotechnology》1996,22(1-3):239-250
Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×109 cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions. 相似文献