DNA cloning in Streptomyces: a bifunctional replicon comprising pBR322 inserted into a Streptomyces phage

The Gram-positive, mycelial, differentiating streptomycetes are responsible for the production of many important antibiotics. The availability of gene cloning systems in this microbial group would have many industrial applications besides allowing more penetrating study of the genetics of Streptomyces coelicolor A3(2) (which, as the best understood streptomycete genetically, serves as a model for much other Streptomyces genetics). Recent successes (see previous paper) in introducing Streptomyces DNA into S. coelicolor and Streptomyces lividans on plasmid vectors would be nicely complemented by the availability of Streptomyces bacteriophage vectors (discussed in ref. 5): for example, many phages have wide and easily defined host ranges; heat-inducible prophages might be used to give high copy number of cloned DNA; efficient phage promoters might be used to increase gene expression; there may be differential stabilities for particular DNA sequences cloned in plasmids vis-à-vis phages; selective insertion of DNA, utilizing packaging constraints, may be possible with phages; and in situ hybridization of radioactive probes to DNA in plaques is likely to be simple. We describe here the use of the moderately wide host range temperate phage, phi C31, for this purpose.     

Expression in Escherichia coli of hepatitis B virus DNA sequences cloned in plasmid pBR322.

Fragments of hepatitis B virus DNA isolated from Dane particles have been inserted into the Escherichia coli plasmid pBR322 and cloned. Cells carrying the hybrid plasmid synthesise antigenic material that reacts specifically with antisera to hepatitis B viral antigens.     

靶序列三链DNA对质粒pBR322的Ampr基因表达的抑制

利用ＣａＣｌ２法成功地将寡核苷酸导入大肠杆菌ＪＭ１０９细胞。质粒ｐＢＲ３２２转化结果表明,寡革酸片段５’－ＡＧＣＧＧＧＡＡＴＡＡＧＧＧＡＡ－３’在转化前（或后）与靶序列结合均能抑制β－内酰胺酶基因的表达,细菌生长受到抑制。体外聚丙烯酰胺凝胶电泳结果表明,该片段能与靶序列形成三链结构。这些结果表明多聚嘌呤寡核苷酸是通过与靶序列形成三链ＤＮＡ来抑制β－内酰胺酶基因的表达。     

DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages.

The DNA sequences for the origins of replication of the lambdoid bacteriophages phi80, 434, phi21, and lambdaimm21 (identical to phi21) have been determined and compared to the lambda structure. Two presumptive elaborate binding sites for two initiator proteins have been identified in their outer sections, while a replicational primer start site seems to be located in their centres.     

DNA arrangement in isometric phage heads.

DNA is wound tightly into phage heads in such a way that it tends to form layers concentric with the rigid protein shell. In P22 and wild-type lambda, DNA completely fills the internal volume, with a highly uniform local packing of adjacent segments; in lambda deletion mutants containing less than a full genome, the local packing distance increases correspondingly.     

Functional expression of HLA-DP genes transfected into mouse fibroblasts

The HLA class II antigens are a highly polymorphic family of dimeric cell-surface glycoproteins, expressed predominantly on the surface of immunocompetent cells. They are intimately involved with the induction of the T-cell response to extrinsic antigen and are important predisposing factors for a wide spectrum of autoimmune diseases. We describe here the expression of a class II product from the HLA-DP (new WHO nomenclature, formerly SB) subregion after transfer of cloned genes into mouse fibroblasts. The transfected DP antigen is recognized by several HLA class II monoclonal antibodies and, though present in a mouse cell background, is able to function in the presentation of influenza antigen to cloned DP-restricted human T lymphocytes.     

从大肠杆菌中获得高浓度质粒DNA的方法

通过改进菌体的培养以及质粒小提方法,可以简便快捷地得到浓度很高的质粒DNA,经琼脂糖凝胶电泳、紫外分光光度法、酶切、PCR的验证,所提质粒可以应用于分子生物学的研究,特别是可以满足瞬时基因枪转化的要求。     

用M13DNA探针检测小鼠DNA指纹

用ＨｉｎｆＩ酶切，随机引物法标记的Ｍ１３ｍｐ１８ＤＮＡ作探针，对无关小鼠个体及一个小鼠家系三代进行了ＤＮＡ指纹分析，无关个体平均每个个体得到１４条谱带（＞４ｋｂ）。无关个体具有相同指纹图概率为９×１０￣（－８），显示高度个体材异性，对小鼠家系内后代与亲本图谱进行了比较，表明图谱特征按孟德尔方式遗传。对家系内个体间遗传相似度Ｄ进行了计算，亲缘关系近则Ｄ值高，据此对本技术在动物个体识别，亲缘关系分析等方面的应用进行了讨论。     

Focus formation in rat fibroblasts exposed to a tumour promoter after transfer of polyoma plt and myc oncogenes

The gene encoding the large-T protein of polyoma virus (plt), the E1A genes of adenoviruses, the viral myc gene (v-myc) or rearranged forms of the cellular c-myc gene confer on rat embryo fibroblast (REF) cells in primary culture a series of new properties ('immortality', reduced serum requirement and sensitivity to transformation by viral and activated cellular oncogenes) but do not induce the appearance of transformed foci. We now report that focus formation can be induced after transfer of these genes into either REF or established FR3T3 rat cells by subsequent exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Frequencies of transformation are in the same range as those usually observed for transformation with complete polyoma DNA or with a mixture of cloned myc and ras oncogenes. These results further characterize the 'immortalized' state induced by plt and myc as one in which the cells maintain a normal growth control in many respects but can be further acted upon to produce a neoplastic progeny.