Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ～ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.     

The complete sequence of a 10.8 kb segment distal of SUF2 on the right arm of chromosome III from Saccharomyces cerevisiae reveals seven open reading frames including the RVS161, ADP1 and PGK genes.

We have entirely sequenced a 10,835 bp segment of the right arm from chromosome III contained in the J11D and J11D-K3B GF clones. The segment contains seven open reading frames longer then 100 amino acids. Three of them, RVS161 (Urdaci et al., 1990; Crouzet et al., 1991), ADP1 (Purnelle et al., 1991) and PGK1 (Hitzeman et al., 1982) have been described previously. YCR10C encodes a putative membrane protein. YCR8W (encoding a putative protein kinase) and YCR14C extend inside the D10H (Skala et al., 1991) and 62B5-2D clones respectively. Four ARS elements previously reported by Palzkill et al. (1986) are located between RVS161 and YCR10C.     

重组人EC-SOD包涵体的稀释复性及重折叠后蛋白的纯化

目的确定重组人ECSOD包涵体体外稀释复性和复性后蛋白纯化的适宜方法。方法通过对pH、温度、包涵体蛋白浓度、尿素和精氨酸浓度及氧化还原对比率的定性定量分析,研究重组人ECSOD包涵体体外稀释复性的基本条件。采用金属离子亲和柱(IMAC)和肝素亲和柱(Heparinsepharose)对复性蛋白进行纯化,比较纯化效率的差异。结果最佳复性条件为pH8.5,尿素浓度为1.5molL,精氨酸浓度大于0.3molL,GSH∶GSSG=4∶1;复性温度及包涵体蛋白终浓度越高,复性效率越低。IMAC和Heparinsepharose亲和柱均可用来纯化复性蛋白溶液,但Heparinsepharose纯化效率较高。结论确定了重组人ECSOD包涵体体外稀释复性的最佳条件及复性后蛋白纯化的方法。     

重组人白细胞介素-10在大肠杆菌中的表达及生物学活性分析

目的在大肠杆菌中表达重组人白细胞介素-10(Interleukin-10,IL-10),并检测其生物学活性。方法将IL-10基因重组到质粒pET11c中,转化BL21(DE),提取质粒,经酶切鉴定和测序分析;在25℃用低浓度的IPTG诱导表达,对包涵体IL-10稀释复性;经ELISA检测其含量,MC/9细胞增殖法检测其生物学活性。结果工程菌IL-10/pET11c/BL21诱导表达的目的蛋白以可溶性和包涵体两种形式存在,Westernblot鉴定证实为IL-10蛋白,两种形式的IL-10均具有一定的生物学活性。结论已成功地在大肠杆菌中表达了IL-10,为进一步纯化和制备IL-10的基因工程药物打下了基础。     

CpG-ODN对乙肝核酸疫苗及重组疫苗免疫效果的影响

目的研究CpG寡聚脱氧核苷酸(CpGODN)对乙肝核酸疫苗及重组(CHO细胞)疫苗免疫效果的影响。方法核酸疫苗实验组用CpGODN与乙肝核酸疫苗联合免疫BALBc小鼠,阳性对照组注射核酸疫苗,阴性对照组注射质粒pVAX1;重组疫苗分4个实验组,用CpGODN与不同剂量的重组疫苗配伍后分别免疫BALBc小鼠,阳性对照组注射重组疫苗,阴性对照组注射生理盐水。应用ELISA检测小鼠血清中的抗HBs水平。结果CpG与乙肝核酸疫苗组抗体阳转率比阳性对照组提高3倍,抗体水平提高2倍。CpG与重组疫苗组的阳转率和阳转时间均高于或早于CpG与核酸疫苗实验组。加入CpGODN后,即使重组疫苗用量减少3倍,诱导抗体水平和阳转率均无明显改变。结论CpGODN可提高乙肝核酸疫苗和重组疫苗的免疫效果,并使抗体产生的时间提前。     

Mg~(2+)和氨基酸对重组大肠杆菌BL21(DE3)生长及羧肽酶原B表达的影响

目的研究Mg2+和氨基酸对重组菌株生长及表达pCPB的影响。方法通过摇瓶和发酵罐培养,观察不同Mg2+浓度和氨基酸对重组菌的生长和羧肽酶原B表达的影响。结果添加1g/L Mg2+能提高质粒的稳定性;添加氨基酸能使羧肽酶原B表达量由18·7g/L提高到24g/L。结论添加适量的Mg2+和氨基酸能促进重组菌的生长和提高目的蛋白的表达量。     

Molecular interaction networks in the analyses of sequence variation and proteomics data

Protein–protein interaction networks are typically generated in standard cell lines or model organisms as it is prohibitively difficult to record large interaction datasets from specific tissues or disease models at a reasonable pace. Although the interaction data are of high confidence, they thus do not reflect in vivo relationships as such. A wealth of physiologically relevant protein information, obtained under different conditions and from different systems, is available including information on genetic variation, protein levels, and PTMs. However, these data are difficult to assess comprehensively because the relationships between the entities remain elusive from the measurements. Here, we exemplarily highlight recent studies that gained deeper insight from genetic variation, protein, and PTM measurements using interaction information pointing toward the importance and potential of interaction networks for the interpretation of sequencing and proteomics data.     

pEgr-ssEndostatin的构建及其在体外的辐射诱导表达

为探讨pEgr-ssEndostatin重组质粒转染B16细胞后接受不同辐射剂量以及接受同一剂量不同时程endostatin表达的规律。采用RT-PCR方法从鼠肝脏中扩增出Endostatin cDNA,连入信号肽,构建了pEgr-ssEndostatin重组质粒,用脂质体介导的转染法转染小鼠B16细胞,用ELISA方法检测B16细胞上清中endostatin的表达水平。结果表明的分泌型Endostatin序列与报道完全一致,Egr-1启动子和Endostatin cDNA正确插入表达载体pcDNA3．1;pEfr-ssEndostatin具有辐射诱导表达特性。提示小鼠Endostatin基因成功地被克隆和表达,Egr-1启动子具有辐射激活和诱导下游基因表达增强的功能。     

两个重组质粒在快生型大豆根瘤菌中的稳定性的比较

将两个复制子相同的重组质粒导入快生型大豆根瘤菌中，考察了得到的转移接合子在培养和共生条件下的稳定性。发现带有质粒ＲＫ２ ｐａｒ基因的重组质粒ｐＴ２ＴＸ３Ｋ在快生型大豆根瘤菌中无论在培养还是共生条件下都可以稳定存在，而不带有该基因的重组质粒ｐＣＫ３在两种条件下都有一定程度的丢失。     

西南菌种站部分酿酒增香酵母产酯性能初探

酯类是白酒中的主要香气成分,本文主要介绍西南菌种站保存的部分酿酒增香酵母在不同发酵培养条件下的产酯能力。试验对7株产酯酵母进行3种培养基、3种培养时间的多配方处理发酵,采用皂化法测定总酯。实验结果表明,菌株SICC 2．644与SICC2．645产酯水平较高,分别达到230．2mg／100mL和171．2mg／100mL;大部分菌株于麦芽汁发酵培养能获得较为理想的产酯测试效果。