Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

抗阿特拉津转基因大豆植株后代的遗传分析

本试验用阿特拉津溶液涂抹、荧光诱导动力学检测、分子杂交等方法对抗阿特拉津转基因大豆植株的后代进行了鉴定,在第二代及第三代中检测到了抗性基因的存在,表明从龙葵中得到的此抗阿特拉津 psbA 基因不仅能导人大豆叶绿体基因组中获得表达,而且可以遗传到后代。     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

Post-castration rebound of an androgen regulated prostatic gene

Summary After castration, the rat dorsolateral prostate M-40 mRNA initially decreased then rebounded to precastrated levels. The cellular site of M-40 expression and its renewed expression after castration was defined by in situ hybridization histochemistry. In situ hybridization with either a ³²P-labeled or biotin-labeled M-40 cDNA probe demonstrated that M-40 mRNA levels were higher in the lateral than dorsal prostate. A second androgen regulated gene, RWB, also was highly expressed in the lateral prostate. The biotinylated cDNA probes provided microscopic resolution of the expressing cells, revealing two distinct morphologies of lateral epithelium which expressed both the M-40 and RWB mRNA. These morphologies appeared in ducts which contained either epithelial cell sheets that were highly convoluted or thinner epithelial cells with a minimal degree of convolution. The RWB mRNA decreased in both cell populations in response to androgen withdrawal. The decline and reappearance of M-40 mRNA also appeared in both epithelial cell types. These data demonstrated that after castration the M-40 mRNA initially decreased as expected for an androgen sensitive gene and then progressed to a fully inducible state. The mechanism of this progression remains to be elucidated.     

Molecular biology of wound-inducible proteinase inhibitors in plants

Abstract. The techniques of molecular biology are being employed to investigate at the gene level the systemically mediated, wound-induced accumulation of two defensive proteinase inhibitor proteins in plant leaves. These techniques have added a new dimension to biochemical and physiological studies already underway to understand the mechanism of induction by wounding. The acquisition of cDNAs from the RNAs coding for the two inhibitors facilitated studies of mRNA synthesis in leaves in response to wounding, and provided probes to obtain wound-inducible proteinase inhibitor genes from tomato ( Lycopersicon esculentum ) and potato (Solarium tuberosum) genomes. Successful transformations of tobacco plants with fused genes, containing the 5' and 3' regions of the inhibitor genes with the open reading frame of the chloramphenicol acelyltransferase ( cat ) gene, have provided a wound-inducible chloramphenicol acetyltransferase (CATase) activity with which to seek cis- and transacting elements that regulate wound-inducibility to help to understand the interaction of cytoplasmic and nuclear components of the intracellular communication systems that activate the proteinase inhibitor genes in response to wounding by insect pests.