Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

A protocol for the efficient screening of putatively transformed plants forbar,the selectable marker gene,using the polymerase chain reaction

Amplification of thebar gene usingTaq DNA polymerase in PCR is often not successful, possibly due tobar's high GC content. We describe a PCR protocol in which reliable amplification at a sensitivity of one gene copy per genome (in this study, barley) present in the reaction was achieved using a novel pair of primers and Expand^tm High Fidelity DNA polymerase mix (Boehringer Mannheim). This method should allow for rapid screening of plants putatively transformed withbar.     

Development of a serum-free medium using computer-assisted factorial design and analysis

Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA Analysis of variance - BSA bovine serum albumin - CHO Chinese hamster ovary - FBS fetal bovine serum - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - PBS phosphate buffered saline - SFM serum-free medium     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.     

Genetic analysis of agronomic traits in a cross between sugarcane (Saccharum officinarum L.) and its presumed progenitor (S. robustum Brandes & Jesw. ex Grassl)

Saccharum robustum Brandes & Jesw. ex Grassl has been suggested as the immediate progenitor species of cultivated sugarcane (S. officinarum L.) [4]. Chromosome pairing and assortment in these two species were previously studied by genetic analysis of single-dose DNA markers in parents in and 44 F₁ progeny of a cross between euploid, meiotically regular 2n=80S. officinarum LA Purple andS. robustum Mol 5829 [2]. This same population was subsequently clonally propagated and evaluated in replicated trials for quantitative traits important to sugarcane breeders. Numbers of stalks, tasseled stalks, and stalks with smut, and the average diameter of two stalks were determined one day prior to harvest. At harvest, plant material from each plot was weighed and evaluated for pol (sucrose content) and fiber percentages. Clones were significantly different (P<0.01) for all traits analyzed. Associations of 83 single-dose arbitrarily primed PCR genetic markers with quantitative trait loci (QTL) of recorded traits was determined by single-factor ANOVA, and multiple regression. QTL analysis revealed markers significantly (P<0.05) associated with the expression of each trait analyzed. Markers associated with QTL after multiple regression were tested for digenic linear × linear epistatic interactions. The various multilocus models explained between 23% and 58% of the total phenotypic variation and 32% and 76% of the genotypic variation for the various traits. Digenic interactions were uncommon. Implications for marker-assisted selection in sugarcane and sugarcane domestication are discussed.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

Detection and Analysis of an Estrous－associated Oviductal Glycoprotein DNA Fragment from Primates by PCR

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应用PCR技术检测细小病毒H－1DNA在人肝癌与裸鼠正常组织中复制的差异

应用ＰＣＲ技术检测细小病毒Ｈ－１ＤＮＡ在人肝癌与裸鼠正常组织中复制的差异黄青山,马承武,郭兰萍,陈献华,罗祖玉（上海复旦大学生理与生物物理学系,上海２００４３３）关键词：自主性细小病毒Ｈ－１及ＭＶＭ,聚合酶链式反应（ＰＣＲ）,人肝癌模型,抑瘤作用,肿...