Studies on Petunia hybrida Transformed with Flower-meristem-identity Gene AP1

Three deletion mutants of tobacco mosaic virus (TMV) 54-kD putative replicase gene (54K) were obtained by PCR, and cloned into plant expression vector p208, then transformed into Nicotiana tabacum L. cv. SR1 by Agrobacterium tumefaciens (Smith et Townsend) Conn Ti plasmid-mediated transformation. All the transgenic plants with the N-terminal deletion mutant, the C-terminal deletion mutant and the only 261 nucleotides region from the central part of the 54K ORF showed significant resistance against TMV.     

Specific cessation of minus-strand RNA accumulation at an early stage of tobacco mosaic virus infection.

The time course of accumulation of viral plus-strand RNAs (genomic RNA and subgenomic mRNA for the coat protein) and minus-strand RNA in tobacco protoplasts synchronously infected with tobacco mosaic virus (TMV) RNA was examined. In protoplasts infected with the wild-type TMV L RNA, the plus and minus strands accumulated differently not only in quantity but also in the outline of kinetics. The time courses of accumulation of the genomic RNA and coat protein mRNA were similar: they became detectable at 2 or 4 h postinoculation (p.i.), and their accumulation increased until 14 to 18 h p.i. The accumulation rate reached the maximum at about 4 h p.i. and then gradually decreased. In contrast, accumulation of the minus-strand RNA ceased at 6 to 8 h p.i., at which time the plus-strand accumulation was already about 100 times greater and still continued vigorously. This specific halt of minus-strand accumulation was not caused exclusively by encapsidation of the genomic RNA, because a similar halt was observed upon infection with a deletion mutant that lacks the 30K and coat protein genes. Upon infection with a mutant that could not produce the 130K protein (one of the two proteins that are thought to be involved in viral RNA replication), the accumulation levels of both plus and minus strands were lower than that of the parental wild-type virus. Given these observations, possible mechanisms of TMV replication are discussed.     

Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication

We have investigated the function of the 30 kd protein of tobacco mosaic virus (TMV) by a reverse genetics approach. First, a point mutation of TMV Ls1 (a temperature-sensitive mutant defective in cell-to-cell movement), that causes an amino acid substitution in the 30 kd protein, was introduced into the parent strain, TMV L. The generated mutant showed the same phenotype as TMV Ls1, and therefore the one-base substitution in the 30 kd protein gene adequately explains the defectiveness of TMV Ls1. Next, four kinds of frame-shift mutants were constructed, whose mutations are located at three different positions of the 30 kd protein gene. All the frame-shift mutants were replication-competent in protoplasts but none showed infectivity on tobacco plants. From these observations the 30 kd protein was confirmed to be involved in cell-to-cell movement. To clarify that the 30 kd protein is not necessary for replication, two kinds of deletion mutants were constructed; one lacking most of the 30 kd protein gene and the other lacking both the 30 kd and coat protein genes. Both mutants replicated in protoplasts and the former still produced the subgenomic mRNA for the coat protein. These results clearly showed that the 30 kd protein, as well as the coat protein, is dispensable for replication and that no cis-acting element for replication is located in their coding sequences. It is also suggested that the signal for coat protein mRNA synthesis may be located within about 100 nucleotides upstream of the initiation codon of the coat protein gene.     

In-frame deletion in the EGF receptor alters kinase inhibition by gefitinib

The existence of an in-frame deletion mutant correlates with the sensitivity of lung cancers to EGFR (epidermal growth factor receptor)-targeted tyrosine kinase inhibitors. We reported previously that the in-frame 15-bp deletional mutation (delE746-A750 type deletion) was constitutively active in cells. Kinetic parameters are important for characterizing an enzyme; however, it remains unclear whether the kinetic parameters of deletion mutant EGFR are similar to those of wild-type EGFR. We analysed autophosphorylation in response to ATP and inhibition of gefitinib for deletion mutant EGFR and wild-type EGFR. Kinetic studies, examining autophosphorylation, were carried out using EGFR fractions extracted from 293-pDelta15 and 293-pEGFR cells transfected with deletion mutant EGFR and wild-type EGFR respectively. We demonstrated the difference in activities between unstimulated wild-type (K(m) for ATP=4.0+/-0.3 microM) and mutant EGFR (K(m) for ATP=2.5+/-0.2 microM). There was no difference in K(m) values between EGF-stimulated wild-type EGFR (K(m) for ATP=1.9+/-0.1 microM) and deletion mutant EGFR (K(m) for ATP=2.2+/-0.2 microM). These results suggest that mutant EGFR is active without ligand stimulation. The K(i) value for gefitinib of the deletion mutant EGFR was much lower than that of wild-type EGFR. These results suggest that the deletion mutant EGFR has a higher affinity for gefitinib than wild-type EGFR.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants

We have used Southern hybridization analysis to characterize the extent of fim homology in recognized type 1 fimbriae mutants of Escherichia coli K12, including strains HB101, P678-54, and VL584. We have found extensive homology in strain HB101, and confirm that P678-54 lacks the majority of fim DNA. Strain VL584 contains a deletion of the entire fim region. We have used a new allelic exchange procedure to generate novel fim deletion derivatives of strains MG1655, MM294, and YMC9. To increase the utility of the new deletion strains we also isolated recA derivatives of each mutant. These strains facilitate the isolation, characterization, and manipulation of cloned fimbriae genes from diverse sources.     

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.     

Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis model

Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism.     

半衰期延长获得PAI－1抗性的t—PA突变体的构建、表达及特性分析

用基因重级及定位突变技术成功地构建了ｔ－ＰＡ的Ｋ１区缺失突变体ｔ－ＰＡｄｅｌＫ１、ＰＡＩ－１结合位点缺失突变体ｔ－ＰＡｄｅｌ（２９６－３０２）及两的组合突变全ｔ－ＰＡｄｅｌ（Ｋ１,２９６－３０２）,并在ＣＯＳ－７细胞中实现三的暂时性表达,在ＣＨＯ细胞中实现了ｔ－ＰＡｄｅｌ（Ｋ１,２９６－３０２）的稳定性表达。对表达产物的生物学特性分析表明,ｔ－ＰＡｄｅｌ（２９６－３０２）及ｔ－ＰＡｄｅｌ（Ｋ１     

两个缺陷型TMV粒子在烟草中的互补表达

根据对ＴＭＶ高效复制和基因表达的顺式作用元件的分析,在体外重组包装了２个缺失型ＴＭＶ粒子：ＴＭＶＲＰ和ＴＭＶＣＰ。前者缺失了ＴＭＶ外壳蛋白ＣＰ基因的３′端及后序区域,后者缺失了大部分复制酶基因。把两者分别或共同电击感染烟草原生质体：１．用ＣＰ抗体进行免疫印渍检测,单独感染的原生质体内的ＣＰ在１６小时内无增加,而在共同感染的原生质体内,ＣＰ在感染２小时后就开始明显增加。２．用ＲＴ一两次ＰＣＲ法专一地检测新生负链ＲＮＡ的合成情况,在单独感染的原生质体内没有检测到,但在混合感染的原生质体内在感染１小时后就检测到ＣＰ基因特异的负链ＲＮＡ的形成,并用Ｓｏｕｔｈｅｒｎ杂交得到进一步验证。这些结果表明,复制酶缺失型ＴＭＶＣＰ内的ＣＰ基因不能表达,但可以在ＴＭＶＲＰ存在时,通过其所表达的复制酶互补作用得到复制从而有效表达．     

Escherichia coli dnaJ deletion mutation results in loss of stability of a positive regulator, CRP.

The dnaJ deletion mutant K7052(lambda dnaK) has a temperature-sensitive defect in the synthesis of beta-galactosidase. We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP. In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature. Introduction of a CRP over-producing plasmid into the dnaJ deletion mutant suppressed the defect of beta-galactosidase synthesis. The lower content of CRP in the mutant was found to result from extreme instability of the protein. These results strongly suggested that the heat shock protein dnaJ is involved in the stabilization (or degradation) of CRP.     

Enhanced anti-angiogenic effect of a deletion mutant of plasminogen kringle 5 on neovascularization

Kringle 5 (K5), a proteolytic fragment of plasminogen, has been proved to be an angiogenic inhibitor. Previously, we have evaluated the effect of K5 on the vascular leakage and neovascularization in a rat model of oxygen-induced retinopathy. In this study, we expressed K5 and a deletion mutant of K5 (K5 mutant) in a prokaryocyte expression system and purified them by affinity chromatography. K5 mutant was generated by deleting 11 amino acids from K5 while retaining the three disulfide bonds. The anti-angiogenic activity of intact K5 and K5 mutant were compared in endothelial cells and retinal neovascularization rat model. K5 mutant inhibited the proliferation of primary human retinal capillary endothelial cells (HRCEC) in a concentration-dependent manner, with an apparent EC50 of approximate 35 nmol/L, which is twofold more potent than intact K5. In the even higher concentration range, K5 mutant did not inhibit pericytes from the same origin of HRCEC, which suggested an endothelial cell-specific inhibition. K5 mutant had no effect on normal liver cells and Bel7402 hepatoma cells even at high concentration range either. Intravitreal injection of the K5 and mutant in the oxygen-induced retinopathy rat model both resulted in significantly fewer neovascular tufts and nonperfusion area than controls with PBS injection, as shown by fluorescein angiography. Furthermore, K5 mutant exhibited more strong inhibition effect on neovascularization than intact K5 by quantification of vascular cells. These results suggest that this K5 deletion mutant is a more potent angiogenic inhibitor than intact K5 and may have therapeutic potential in the treatment of those disorders with neovascularization, such as solid tumor, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and hyperplasia of prostate.     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium.     

应用RNAi技术培育抗2种病毒病的转基因烟草

分别提取烟草普通花叶病(TMV)和烟草黄瓜花叶病（CMV）的病毒RNA。经反转录和外壳蛋白阅读框PCR扩增,获得TMV和CMV外壳蛋白基因cDNA, 分别进行两种病毒已知株系cDNA序列比对获得各自的保守序列,设计干涉序列,将干涉片段扩增产物连接到pMD18-T的相邻酶切位点,制备融合序列,并将其正向和反向序列插入pUCCRNAi载体,再转化到pCAMBIA2300-35S-OCS表达载体中。利用农杆菌LBA4404侵染烟草K326,获得3份含有TMV和CMV外壳蛋白基因干涉序列的转化材料,经分子鉴定证实干涉序列已导入烟草,并采用荧光定量PCR技术对其mRNA表达差异进行分析。抗病性调查表明转化烟株对TMV和CMV抗性都显著增强。     

Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium.     

Production of pokeweed antiviral proteins nontoxic to cells by Mutagenesis of PAP-cDNA

Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E.coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells. Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses of 1,000 transformants: among them, three, ten and five mutants produced unprocessed, mature and truncated PAPs, respectively. Fourteen PAP mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesisin vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation. These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity.