猪传染性胸膜肺炎放线杆菌毒素apxII基因无药物抗性标记突变株HBC-/GFP+的构建及其生物学特性研究

通过使用枯草芽胞杆菌一个蔗糖敏感sacB基因发展了一种依靠蔗糖的负向筛选系统 ,这种方法允许没有标记突变的基因进入胸膜肺炎放线杆菌染色体。首先 ,构建了猪传染性胸膜肺炎放线杆菌毒素apxII基因GFP插入失活型的重组质粒pOSAKCG ,其中一个表达盒含有氨苄青霉素基因和以外膜蛋白omlA作为启动子表达sacB基因。重组质粒pOSAKCG通过电穿孔转化 ,它的突变apxIICA基因与野生型亲本菌株胸膜肺炎放线杆菌HB03染色体上野生型apxIICA基因发生同源交换 ,两步法筛选获得了apxII基因突变株HBC^-GFP⁺,PCR和Southernblot对突变株进行初步鉴定 ,进一步对突变株的一些生物学特性 ,包括它的溶血活性、免疫原性、生长特性及其对小鼠的安全性进行了研究。结果表明 ,无药物抗性标记突变株的构建是成功的。该突变株的构建为进一步研究突变株作为载体和疫苗奠定了坚实的基础。     

金属硫蛋白产生菌的诱变育种*

采用亚硝基胍和紫外线诱变,自金属硫蛋白（ＭＴ）产生菌酿酒酵母（ｄｅｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）ＢＤ１０１－２５单倍体中获得遗传稳定的高Ｃｕ^２＋、Ｃｄ^２＋抗性突变株ＢＤ１０１－６９和ＢＤ１０１－３０。并对其重金属解毒、桔抗ｕ．Ｖ．和６０Ｃｏ辐射效应、清除羟基自由基能力等生物学功能进行了研究。与出发菌株相比,上述生物学活性与酵母细胞对Ｃｉ^２＋抗性、ＭＴ表达量表现出正相关性。两个突变株类ＭＴ表达量与生物学活性皆有所提     

D-氨基酸氧化酶在不同毕赤酵母宿主菌中的表达比较

D-氨基酸氧化酶（DAAO）在转化头孢菌素C生产7-ACA和转化DL-氨基酸制备α-酮酸和L-氨基酸上起着重要的作用。采用DNA操作技术,将来源于三角酵母的DAAO基因连接至表达载体pPIC35K上,再将表达质粒pPIC35KDAAO分别整合P. pastoris的宿主细胞KM71和GS115,经筛选获得阳性重组菌PDK13（Mut^S）和PD27（Mut⁺）。重点对两种突变菌的表达条件进行了比较。结果显示：PDK13（Mut^S）株比PD27（Mut⁺）株消耗甲醇慢、诱导时间长,但对通气量要求低、表达水平高,摇瓶活力分别达到2700和2500 IU/L,14L发酵罐内活力分别达到10140和8463 IU/L。初步探索了DAAO对DL-苯丙氨酸的拆分,结果显示基因工程菌表达的DAAO具有良好的转化DL-苯丙氨酸制备苯丙酮酸和L-苯丙氨酸的能力。     

脱卤脱亚硫酸菌脱氯呼吸链的遗传分析*

以携有结合转座子Ｔｎ９１６的Ｅｎｔｅｒｏｃｏｃｃｕｓ　ｆａｅｃａｌｉｓ　ＪＨ２－２为供体,脱卤脱亚硫酸菌ＨＳＳ１（Ｄｅｓｕｌｆｉｔｏｂａｃｔｅｒｉｕｍ　ｄｅｈａｌｏｇｅｎａｎｓ,Ｓｍ抗性突变株）为受体,在厌氧条件下,通过滤膜杂交、结合转移,将Ｔｈ９１６转移并插入到受体菌的染色体上,其转移频率为：１．１×１０^－７～３×１０^－８。　在丙酮酸／乳酸－３－氯－４－羟基苯氧乙酸、Ｔｃ、Ｓｍ培养基上,筛选脱氯呼吸的缺陷型突变株,并用反向ＰＣＲ（Ｉ     

伪狂犬病病毒TK-/gG-缺失株的构建及其生物学特性研究

将伪狂犬病病毒TK^-/gG^-/LacZ⁺突变株的基因组DNA与含有缺失的gG基因的转移质粒pUSKBB共转染猪肾传代细胞PK-15,待完全病变后收获病毒进行空斑试验,用PCR筛选gG缺失的重组病毒。空斑纯化3次后,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的TK^-/gG^-缺失株。遗传稳定性试验表明该重组病毒能在PK-15细胞上稳定遗传,动物试验表明该缺失株对Balb/c小鼠极为安全且能保护Balb/c小鼠抵抗致死量PRV强毒的攻击。该突变株的获得为我国伪狂犬病的控制和根除奠定了基础。     

高含量赖氨酸酵母的选育和培养条件的研究

本文报道一株能积累赖氨酸的酿酒酵母S1为原始菌株,经10^-3mol/L的s-(β-氨基乙基)L-半胱氨酸(SAEC)处理,选得SAEC抗性突变株。再经二次紫外诱变,选得7株SAECr突变株,其中3株具有较高积累赖氨酸能力,并对其培养条件进行了研究。突变株MSU1的游离赖氨酸含量为菌体干重的7.83%,而突变株MSU5平均赖氨酸含量可达8.91%。     

经基因工程改良的抗白叶枯病水稻粳型恢复系“C418-Xa21”及其杂交稻

通过农杆菌介导的转化系统,将业已克隆的水稻抗白叶枯病基因Xa21导入重要的粳型杂交稻恢复系“C418”。PCR和抗性分析表明单拷贝整合的Xa21在T₁代的分离比为3∶1。在T₂代通过PCR和抗性分析选择了Xa21纯合的转基因株系“C41-Xa21”。将选择的转基因纯合系“C418-Xa21”与常用的雄性不育系“屉锦A”杂交,产生了带有转基因Xa21的杂交稻“屉优41-Xa21”(简称转基因杂交稻)。分子分析表明转基因Xa21在杂交稻“屉优418-Xa21”中能稳定遗传;抗性分析表明转基因恢复系“C418-Xa21”和转基因杂交稻“屉优418-Xa21”对白叶枯病具有高度的广谱抗性,并保持了受体对照的优良农艺性状。另外我们还发现转基因杂交稻“屉优418-Xa21”对白叶枯病的抗性水平高于转基因恢复系“C418-Xa21”,这可能是遗传背景的差异所致。抗白叶枯病转基因粳型恢复系和杂交稻的育成将有益于杂交稻在我国北方稻区的推广。     

产金属硫蛋白酵母菌原生质体电融合研究

采用原生质体电融合技术,由不能水解淀粉、细胞生物量低,只抗Cu²⁺、金属硫蛋白(MT)中cys含量高的酿酒酵母(Saccharomyces cerevisiae,a)单倍体BD101-25和具淀粉水解能力、细胞生物量高、Cu²⁺、Cd²⁺抗性高、金属硫蛋白中cys含量低的异常毕赤酵母(Pichiaamomala,a)单倍体BD102-13获得4个融合株。融合组合有两亲株属间融合和单一亲株种内融合两类。融合株细胞体积、DNA含量均近似于两亲株细胞之和,并具有水解淀粉能力,Cu²⁺、Cd²⁺抗性高,细胞生物量高,MT中cys含量高等特点。属间融合株Cu²⁺、Cd²⁺抗性的遗传性状稳定。     

同源重组法构建多功能农药降解基因工程菌研究

构建遗传稳定的多功能农药降解基因工程菌可以为农药污染的生物修复提供良好的菌种资源,然而,构建遗传稳定且不带入外源抗性的基因工程菌是一个难点。通过以受体菌的16S rDNA为同源重组指导序列、sacB基因为双交换正筛选标记构建同源重组载体,二亲结合的方法将甲基对硫磷水解酶基因（mpd）整合到呋喃丹降解菌Sphingomonas sp.CDS1染色体的16S rDNA位点,分别成功构建了含1个和2个mpd基因插入到rDNA位点且不带入外源抗性的基因工程菌株CDSmpd和CDS-2mpd。同源重组单交换的效率为3.7×10^－7～6.8×10^－7。通过PCR和Southern杂交的方法验证了同源重组事件。基因工程菌遗传稳定,能同时降解甲基对硫磷和呋喃丹。甲基对硫磷水解酶（MPH）的比活在各生长时期均高于原始出发菌株,比活最高达6.22 mu/μg。     

60Co γ射线对不同细胞形态出芽短梗霉的辐射诱变效应

使用⁶⁰Co γ射线辐照诱变的方法处理出芽短梗霉Aureobasidium pullulans AP92菌株的原生质体、茵丝体片段、分生孢子悬液,经初筛、复筛与对突变株的遗传稳定性研究,发现采用原生质体进行诱变,所获突变株的正突变率、单株产多糖的提高幅度、正突变株的产多糖遗传稳定性均明显高于菌丝体与分生孢子。比较出发菌株AP92与经原生质体诱变获得的正突变株A8l的性能,有如下明显改善：产多糖能力从16-35g/L提高到29.69g／L'糖转化率从 32.7％升到61.4％,残糖从13.26g/L降到3.06g/L,而发酵周期则可缩短约24小时。结果证明,对原生质体进行⁶⁰Co γ射线诱变,是优化出芽短梗霉菌种的有效途径。     

乳酸抗性酵母的筛选及其生长特性的研究

以酿酒酵母 (saccharomycesceevisiae)单倍体YNN -2 7(αtep ura )为亲株 ,在含有 4 %乳酸的梯度平板上直接进行紫外线诱变处理 ,筛选到突变株YNN -2 7-2 4。通过对该突变株乳酸抗性产生原因分析、在含有不同浓度的乳酸和潮霉素B(hygromycinB)的YPDL和YPDLH培养基中的重复特性的研究发现 ,该突变株对乳酸和潮霉素B产生的抗性 ,不是因对环境条件的适应而产生 ,而是由基因突变所引起。与突变株YNN2 7-2 4相比 ,乳酸对亲株生长的影响在于延长了其生长的延迟期 ,而其生长速率没有发生改变。用Mini-photo 51 8测定供试菌株在生长过程中的吸光度 ( 660nm)以研究酵母菌的生长特性 ,是一种行之有效的方法 ,具有较高的灵敏度和较好的再现性。     

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation

Summary The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-t C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-t mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.     

大肠杆菌抗氟乙酸变株的选育及应用

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E.coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon…     

PCR—SSCP与测序技术相结合检测小麦耐盐突变体

根据位于小麦第四同源群上与耐盐有关的gf-2.8基因的编码区序列设计1对引物,分别以两个耐盐突变体及其亲本的总DNA为模板进行PCR扩增,在5个供试材料中均扩增出1条约685bp的目的条带,SSCP电泳显示突变体974915与其他供试材料之间存在差异。测序表明冀麦24和其耐盐突变体8901-17的扩增产物序列与gf-2.8基因的发表序列相同,这表明突变体8901-17的突变位点不在该基因上,而另一耐盐突变体974915的序列中则至少存在2个单碱基突变,有一处突变导致了氨基酸的变化,该突变位点位于gf-2.8基因的保守区域内。     

In vitro mutagenesis of HLA-B27. Amino acid substitutions at position 67 disrupt anti-B27 monoclonal antibody binding in direct relation to the size of the substituted side chain.

The class I MHC molecule HLA-B27 bears an unpaired Cys residue at position 67, which is predicted to face the Ag binding pocket, based on the x-ray crystallographic model of HLA-A2. To investigate the potential of this residue in the antigenic structure of HLA-B27, a panel of 11 mutant HLA-B27 genes has been created, each bearing a separate amino acid substitution at position 67. The genes were transfected into mouse L cells and the resulting cells analyzed by cytofluorography with a panel of antibodies reactive with the wild-type B27 molecule. Although previous studies had indicated that all mAb that bound the B27 molecule on human lymphocytes bound comparably to L cells transfected with the wild-type B27 gene in the absence of h beta 2-m (human beta 2-microglobulin), the first of the mutant B27 genes was found to express several mAb epitopes in the presence but not in the absence of a h beta 2-m gene. Therefore, subsequent analysis of the B27 mutant panel was conducted in L cells coexpressing the h beta 2-m gene. Under these circumstances, all of the mutants bound the monomorphic anti-class I HLA mAb W6/32 and B.9.12.1, as well as the broadly polymorphic mAb B.1.23.2. Binding to the mutant transfectants of three anti-B27 mAb that cross-react with HLA-B7, ME1, GS145.2, and GSP5.3, was directly proportional to the size of the substituted amino acid side chain. The binding of another anti-B27 mAb, B27M2, that recognizes a B27 determinant that includes the region of amino acids 77-81, was not affected by the Cys67- greater than Tyr67 substitution. Rabbit antibodies to a synthetic peptide composed of B27 amino acids 61-84 bound to both the wild-type B27 and to the Tyr67 mutant. This binding, but not the binding of ME1 or B27M2, was inhibited by the synthetic peptide. These data are interpreted as suggesting that the large amino acid substitutions at position 67 induce a limited conformational change that disrupts the epitopes of the three anti-B27, B7 mAb, that are themselves at least partially conformational. The potential implications of these findings for the role of HLA-B27 in disease pathogenesis are discussed.     

The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection

The phenotypic properties of ICP27 temperature-sensitive and deletion mutants and the results of transient expression assays have demonstrated that ICP27 has a modulatory effect on viral gene expression induced by ICPs 0 and 4. In order to identify the regions of the ICP27 molecule that are responsible for its enhancing and repressing activities, 10 nonsense and 3 in-frame deletion mutations were introduced into the coding sequence of the cloned ICP27 gene. These mutant genes were tested in transient expression assays for their ability to complement an ICP27 null mutant and to enhance and repress expression from a spectrum of herpes simplex virus type 1 promoters in reporter CAT genes when expression was induced by ICP0 or ICP4. The results of assays with cloned mutant genes demonstrate that the ICP27 polypeptide contains two regions, located between amino acid residues 327 and 407 and residues 465 and 511, that contribute to its repressing activity. The amino acid region located between the two repressing regions (residues 407 to 465) is able to interfere with ICP27 repressing activity. None of the mutant genes exhibited efficient enhancing activity for any of the herpes simplex type 1 promoters tested, demonstrating that amino acids comprising the carboxy-terminal half of the ICP27 molecule, including the terminal phenylalanine residue, are required for wild-type enhancement as well as for efficient complementation of an ICP27 null mutant. Phenotypic characterization of an in-frame deletion mutant, vd3, and a previously isolated null mutant, 5dl 1.2 (A. M. McCarthy, L. and P. A. Schaffer, J. Virol. 63:18-27, 1989), demonstrated that ICP27 is required to induce the expression of all classes of viral genes very early in infection and confirmed the requirement for ICP27 later in infection (i) to repress early gene expression, (ii) to induce wild-type levels of delayed-early or gamma 1 gene expression, and (iii) to induce true late or gamma 2 gene expression. The vd3 mutant, which specifies an ICP27 peptide lacking the repressing region between residues 327 and 407, is able to (i) repress early gene expression, consistent with the repressing ability of the d3 mutation in transient expression assays, (ii) induce the synthesis of significant but reduced levels of delayed-early (gamma 1) proteins and no gamma 2 proteins (thus vd3 exhibits a late protein phenotype intermediate between that of the wild-type virus and 5dl 1.2), and (iii) confer altered electrophoretic mobility on ICP4, demonstrating a role for ICP27 in the posttranslational modification of this essential regulatory protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpes simplex virus ICP27 activation of stress kinases JNK and p38

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0-/4-) and d103 (4-/22-/47-) mutants activated p38 and JNK, while the d106 (4-/22-/27-/47-) and d107 (4-/27-) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.     

Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27(-/-) mice and CTX

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.     

Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes (pf-17, pf- 24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes. Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology. Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci (pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components. In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants. In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides.