Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

The replication of bacteriophage f1: Gene V protein regulates the synthesis of gene II protein

Two filamentous phage gene products are required for the replication of phage DNA. One of these, the gene II protein, is a site-specific endonuclease required for all phage-specific DNA synthesis. The other, the gene V protein, is a single-stranded DNA-binding protein required only for single-strand synthesis. Purified gene V protein, when added to an in vitro protein synthesizing system programmed by f1 DNA, specifically inhibits the synthesis of gene II protein. Inhibition seems to be translational, since synthesis of gene II protein from an RNA template is also inhibited by gene V protein. Gene V protein control of gene II expression can account for the regulation of the level of expression of the filamentous phage genome.     

HCV E蛋白基因的变异及变异的意义

目的分析不同来源HCV的不同亚型（不同型别之间的不同亚型）及同一亚型之间E蛋白基因的变异及变异的意义。方法使用GenBank公开发表的不同来源HCV的不同亚型及同一亚型之间全基因序列为材料,用CLustalw2软件分析序列之间的同源性。结果不同亚型之间E蛋白基因变异达到了70．5％（E1蛋白基因）和59．8％（E2蛋白基因）;不同亚型的E2蛋白基因有两个高度变异区,相同的亚型之间只有一个高变区;伺一亚型之间E蛋白基因的变异率为11．8％-16．8％（E1蛋白基因）和18．4～29．9％（E2蛋白基因）。结论根据相同亚型之间E蛋白基因的变异较小的特点,可以利用不同亚型病毒的抗原表位发展多价混合多肽疫苗或多基因混合疫苗,可能会对多种不同亚型病毒的感染起到免疫保护作用。     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.     

In vitro binding of the bacteriophage f1 gene V protein to the gene II RNA-operator and its DNA analog.

We have investigated the binding of the f1 single-stranded DNA-binding protein (gene V protein) to DNA oligonucleotides and RNA synthesized in vitro. The first 16 nucleotides of the f1 gene II mRNA leader sequence were previously identified as the gene II RNA-operator; the target to which the gene V protein binds to repress gene II translation. Using a gel retardation assay, we find that the preferential binding of gene V protein to an RNA carrying the gene II RNA-operator sequence is affected by mutations which abolish gene II translational repression in vivo. In vitro, gene V protein also binds preferentially to a DNA oligonucleotide whose sequence is the DNA analog of the wild-type gene II RNA-operator. Therefore, the gene V protein recognizes the gene II mRNA operator sequence when present in either an RNA or DNA context.     

Translational control of bacteriophage f1 gene II and gene X proteins by gene V protein

The gene II region of bacteriophage f1 DNA codes for two proteins, the 46 kd gene II protein and the 13 kd gene X protein, which results from an in-phase start at codon 300 of gene II. Using antigens II protein IgG, we show that the intracellular concentration of both proteins is controlled by the phage gene V protein. In wild-type f1-infected cells, the amount of gene II protein reaches a plateau of about 1500 molecules per cell at 20 min after infection, as measured by blot immunoassay. Similarly, the amount of gene X protein reaches a peak of about 500 molecules per cell around 10 min after infection. In contrast, when the gene V protein is inactive, both gene II and gene X proteins continue to accumulate at a high rate for at least 40 min after infection. This difference is caused by decreased synthesis of gene II and gene X proteins in the presence of gene V protein, which represses the translation of these two proteins.     

Essential amino acid residues in the single-stranded DNA-binding protein of bacteriophage T7. Identification of the dimer interface

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase.     

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.     

Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.

Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene. Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562. Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative. Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA. The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA. In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively. Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25. Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA.     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.     

棘孢木霉丝裂原活化蛋白激酶基因task1的克隆及序列分析

为深入研究丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)的功能,从棘孢木霉(Tricho-derma asperellum)中克隆了丝裂原活化蛋白激酶(MAPK)基因task1,并对其序列进行分析。该基因编码355个氨基酸,全长1 757 bp,理论分子质量41.1 kD,理论等电点为6.64,与深绿木霉(T.atroviride)MAPK基因tmk1、里氏木霉(T.reesei)MAPK基因tmkA和绿色木霉(T.virens)MAPK基因tmkA在氨基酸和核苷酸水平上同源性都很高,蛋白结构预测为丝氨酸/苏氨酸蛋白激酶。     

甜菜坏死黄脉病毒75kDa通读蛋白基因构建与表达

利用DNA重组技术,将甜菜坏死黄脉病毒(BNYVV)内蒙分离物的CP基因和54kDa通读区片段拼接。构建了BNYVV 75kDa通读蛋白基因。序列分析表明,构建的75kDa通读蛋白基因与野生型相比．只有4个核苷酸发生了改变(包括将CP基因的终止密码子TAG改造为ATG),相应地2个氨基酸也发生了改变。将75kDa通读蛋白基因及其54kDa片段分别克隆到pJw2上,构建了这两十基因的原核表达载体。SDS—PAGE和western blotting检测结果表明,75kDa通读蛋白基因在E coli BL21(DE3)中经温度(42℃)诱导后除可特异地表达75kDa蛋白外。还产生两种小蛋白。75kDa通读蛋白基因的54kDa片段只表达出37kDa的蛋白。     

The role of bacteriophage T7 gene 2 protein in DNA replication.

The in vivo function of the gene 2 protein of bacteriophage T7 has been examined. The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers. This modulation is not however a direct interacton between the two proteins. In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production. This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein.     

Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR

Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein.

Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers.     

基孔肯雅病毒非结构蛋白基因合成中多位点缺失突变的校正方法

目的:建立一种PCR方法,以快速校正基孔肯雅病毒非结构蛋白基因合成过程中发生的多位点缺失突变。方法:用PCR方法合成基孔肯雅病毒非结构蛋白基因;对测序的克隆进行序列比对,分析不同克隆上缺失突变发生的位置,以保守区域互相重叠的寡核苷酸为上下游引物、以该区域测序正确的克隆为模板进行PCR扩增,得到所需片段,再将这些片段用PCR方法进一步组装成完整的基因序列并进行测序。结果:测序结果表明,经过2次PCR扩增,校正了基孔肯雅病毒非结构蛋白基因合成过程中发生的5个位点缺失突变。结论:得到序列正确的基孔肯雅病毒非结构蛋白基因。在进行基因合成过程中如发生多位点缺失突变,可利用该方法同时对以上突变进行校正,无须再合成引物,降低了实验操作难度,并提高了实验效率。