Mapping unprocessed epitopes using deletion mutagenesis of gene fusions.

To locate the antigenic determinant recognized by a monoclonal antibody directed against a baculovirus capsid protein, a series of overlapping deletions of a fusion protein were immunologically screened with the monoclonal antibody. The immunoreactive fusion protein was derived from a restriction fragment which contained a large portion of a baculovirus capsid protein open reading frame fused in-frame with a truncated trpE gene in a bacterial (pATH3) expression system. To map the epitope, nested sets of 5' and 3' deletion mutants were generated. Mutants were characterized by the DNA insert size or by the size of the expressed fusion protein. Selected N- and C-termini truncated fusion proteins were Western blotted and incubated with the monoclonal antibody to identify mutants which retained the epitope. Plasmid DNA from mutants which flank the 5' and 3' junction of the antigenic determinant were sequenced to determine the epitope junction. By screening forty 3' deletions and sixty-four 5' deletions, the antigenic determinant was localized to a region of seven amino acids.     

Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.     

Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase

The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino-acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene.     

Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene.

The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake.     

The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae.

The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae. The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog. The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein. This protein is essential; deletion of the coding sequence was lethal. The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function. Mutations in the rox3 gene showed an interesting pattern of intragenic complementation. A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation. These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7. Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression. The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.     

Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay

In studies with a trpE promoter-strength measuring system we observed that constructs containing the Escherichia coli trp promoter and its adjacent transcribed region yielded lower levels of trpE protein than were expected. To analyze this observation we introduced mutational changes in the nucleotide sequence preceding the trpE Shine-Dalgarno region and examined their effects on trpE mRNA synthesis, translation and decay. We found that certain deletion, insertion and substitution mutations in the pre-Shine-Dalgarno region caused a two- to fivefold increase in trpE enzyme activity. These increases were accompanied by increases in steady-state levels of trpE mRNA. Pulse-chase analyses of trpE mRNA degradation revealed that the observed steady-state trpE mRNA levels correlated with changes in trpE mRNA stability. These findings are interpreted in terms of alternative models in which the primary effect of mutational changes that elevate trpE expression is to increase trpE mRNA translation, versus increasing trpE mRNA stability.     

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.     

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.     

鸡传染性法氏囊病病毒非结构蛋白基因的克隆、表达及多克隆抗体制备

利用RT-PCR技术从传染性法氏囊病病毒(IBDV)TL2004株感染鸡胚尿囊液中扩增到VP5基因,进而构建了T7启动子控制下的N端GST-Tag融合表达质粒pGEX-VP5。序列测定表明VP5基因全长438bp,编码一个由145个氨基酸组成的VP5蛋白。将pGEX-VP5转化大肠杆菌BL21,在IPTG的诱导下高效表达了GST-VP5融合蛋白(44kD)。通过包涵体纯化的方法,获得的较高纯度的融合蛋白,免疫新西兰兔,Western blot和ELISA分析表明,制备的融合蛋白抗血清效价在1∶12800以上,并具有良好的免疫反应特异性,为进一步研究VP5在IBDV复制与致病中的作用,以及研制IBDVVP5基因缺失疫苗打下了良好的基础。     

Localization of Two Functions of the Phosphoribosyl Anthranilate Transferase of Escherichia coli to Distinct Regions of the Polypeptide Chain

The trpD gene specifies a polypeptide which has both glutamine amidotransferase and phosphoribosyl anthranilate (PRA) transferase activities. Deletions fusing segments of trpD to the gene preceding it in the operon, trpE, were selected in strains carrying various trpD point mutations. The selection procedure required both that a deletion enter trpE and that it restore the PRA transferase activity which the parent trpD point mutant lacked. Deletion mutants were found which had PRA transferase activity although the first third of trpD was deleted. The existence of the mutants proves that a terminal segment of trpD is sufficient to specify a polypeptide having PRA transferase activity. The location of the deletion end points on the genetic map of trpD defines the extent of the trpD segment required for PRA transferase activity. This segment did not overlap the initial region of trpD required to specify the glutamine amidotransferase function of the trpD polypeptide. These results support the hypothesis (M. Grieshaber and R. Bauerle, 1972; H. Zalkin and L. H. Hwang, 1971) that the bifunctional trpD polypeptide might have evolved by fusion of a gene specifying a glutamine amidotransferase with a gene directing PRA transferase synthesis.     

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.     

猪水泡病病毒VP1基因的克隆和表达

以猪水泡病病毒RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了849bp的VP1基因,通过T-A克隆技术,将VP1基因片段克隆至pMD18-T克隆载体质粒中,构建SVDVVP1基因克隆重组质粒,进行核苷酸序列分析。然后亚克隆插入pBAD/ThioTOPO表达载体,经测序鉴定,筛选获得VP1基因正向插入、有正确读码框的阳性克隆,成功构建了猪水泡病病毒VP1基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP1蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002%的L-阿拉伯醛糖进行诱导,5h后表达可达到高峰,表达蛋白为融合蛋白,质量约47.13kDa,表达产量约占菌体总蛋白的16%。Westernblotting检测表明,诱导的蛋白能与猪水泡病阳性血清发生特异性反应。融合蛋白中含有猪水泡病病毒VP1蛋白抗原,为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础。     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

非洲猪瘟病毒VP73基因克隆及在大肠杆菌中的高效表达

参照Genebank中非洲猪瘟VP73基因序列,人工合成的VP73全基因克隆至pMD 18-T克隆载体质粒中,采用PCR扩增得到1188 bp的VP73基因;将VP73基因片段亚克隆插入pBAD/Thio表达载体,经测序鉴定,筛选获得VP73基因正向插入、有正确读码框的阳性克隆,成功构建了非洲猪瘟病毒VP73基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP73蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002 %的L-Arabinose进行诱导,4 h后表达量最高,表达蛋白为融合蛋白,分子量约60 kDa,表达产量约占菌体总蛋白的30％。Western blotting和ELISA检测表明,表达的融合蛋白能与非洲猪瘟阳性血清发生特异性反应,说明表达获得的产物为非洲猪瘟病毒VP73融合蛋白,且具有良好的反应原性,这为应用该表达蛋白抗原制备ASFV免疫血清学诊断试剂和疫苗研究奠定了基础。