FAB-MAPPING of recombinant-DNA protein products

A new method is described for screening and establishing the primary structure of proteins. The procedure is both rapid and sensitive and allows study of the C-terminus of the protein with equal facility to the N-terminus. This new strategy which is illustrated here for the polypeptide hormone Insulin, has obvious applications in recombinant DNA biotechnology research, in post-translational modification and site-directed mutagenesis studies, or any other aspect of modern protein chemistry requiring accurate definition of primary structure.     

Rapid screening for plasmid DNA

Summary A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.     

Bioassay strain development for the analysis of bacitracin in bacitracin/chlortetracycline combinations

A variant strain was developed fromMicrococcus luteus ATCC 10240 for the purpose of bioassay analysis of bacitracin in the presence of chlortetracycline (CTC). Strain EN5 resulted from four sequential mutation steps, using quantitative resistance to CTC and retained bacitracin sensitivity as a selective criterion. Strain EN5 was tested for bioassay response, stability, and identity. The strain measured bacitracin activity with no interference from 40 g ml^–1 added CTC.     

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression. Combinations of IL-15/IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7– effector memory (CD45RO+ CCR7– CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO– CCR7– CD3+), and naive (CD45RO– CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10⁷ PBMCs.     

Isolation of a new drug-resistance plasmid from a strain of Vibrio parahaemolyticus

A new R plasmid, pSA55, with a molecular weight of 112 megadaltons (Md), was isolated from a strain of Vibrio parahaemolyticus with multiple drug resistance. The pSA55 plasmid conferred on its host resistance to chloramphenicol, tetracycline, streptomycin, kanamycin, ampicillin, trimethoprim and 2,4-diamino-6,7-diisopropyl pteridine, and belongs to incompatibility group C. The plasmid was transferable to Escherichia coli, V. parahaemolyticus, V. alginolyticus and NAG bivrio at a frequency of 10(-3) approximately -7, and was stably inherited by the transconjugants of these species. The conjugal transfer of pSA55 plasmid was significantly affected by the growth culture phase. The resistance pattern and resistance levels of transconjugants were the same as those of the donor strain. We did not observe fluctuations in minimal inhibitory concentrations with transfer, unlike the case of V. cholerae. The relationship between the pSA55 plasmid and the Kanagawa phenomenon was not clarified in the present study.     

Kinetic evaluation of claimed synergistic paraben combinations using a factorial design

D. GILLILAND, A. LI WAN PO AND E. SCOTT. 1992. The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

A small plasmid for recombination-based screening.

We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].     

New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris

We describe the isolation and characterization of three new biosynthetic genes-ARG4, ADE1, and URA3-from the methylotrophic yeast Pichia pastoris. The predicted products of the genes share significant sequence similarity to their Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5'-phosphate decarboxylase, respectively. Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shuttle vectors designed to express foreign genes in P. pastoris. In addition, we have constructed a series of host strains containing all possible combinations of ade1, arg4, his4, and ura3 auxotrophies to be used with these new vectors.     

Kinetic evaluation of claimed synergistic paraben combinations using a factorial design.

The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

中国菊属的一些新组合

菊属植物具有重要的园艺和药用价值,但在属名的使用上一直混乱。这一情况一直到最近才得以部分解决。该文报道了中国菊属的三个新组合,并给出了中国产菊属全部种类的名称和地理分布。     

Nisin高产菌株的高通量筛选

【目的】乳酸链球菌素(nisin)是一种天然生物活性抗菌肽,对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,而用作食品的防腐剂。本研究通过建立高通量筛选方法,实现高效快速省力的高产菌株筛选,为工业上筛选高产菌株提供研究方案。【方法】通过对Lactococcus lactis ATCC11454菌株进行紫外诱变,获得2511株突变株。利用Biomek FXP自动工作站建立96微孔板的高通量筛选方法,突变株经高通量挑选、菌种培养及菌液稀释后,加入到生长至对数中期的藤黄微球菌中,采用改进后的比浊法快速检测nisin生物活性。用此方法对突变株进行初筛、复筛后可得到nisin高产菌株,并通过摇瓶发酵评估高通量筛选方法。【结果】确定比浊法检测的条件为:nisin活性稀释在10–25 IU/m L范围内,与藤黄微球菌反应2 h后检测藤黄微球菌的菌体量(OD600)。2511株突变株经过2轮高通量筛选,最终获得约50株产量提升的菌株,对其中8株进行摇瓶精确测量,显示产量均有提高,并且其中一株产量提升了30%,成功建立了高通量筛选nisin高产菌株的方法。【结论】利用比浊检测法,在其基础上成功建立高通量筛选高产nisin菌的方法,经过初筛复筛,整个周期由1人耗时5 d即可完成2511株突变株的筛选工作。相较于传统的选育方法,高通量筛选具有快速、稳定、高效的特点,提高了筛选效率,缩短了选育周期,是工业上筛选高产nisin菌的有效手段。     

An improved method for screening Frankia viability in strain CcI3

An improved method for determining the viability of the nitrogen-fixing actinomycete Frankia is presented. This method uses tetrazolium red as a vital stain, which proved more effective than a previously used method of acridine orange staining.     

Application of factorial and Doehlert designs for optimization of protopectinase production by a Geotrichum klebahnii strain

Two successive factorial designs followed by two response surface methodology were applied to optimize protopectinase production by Geotrichum klebahnii. Factorial designs were used to study the effect of 11 variables (mineral pool and pH) on enzyme production. Only pH and Fe²⁺ had significant effect on the protopectinase production in the conditions of the assay, the interaction between them not being significant. According to this result pH and Fe²⁺ were multivariate according to a Doelhert design. The central points were pH 3.5 and Fe²⁺ 1.8 μmol L⁻¹. The results show a negative effect of pH and a positive (quadratic) effect of Fe²⁺ on enzyme production. The second Doelhert design was centered at pH 3.3. A relative maximum was obtained at pH 2.8 and Fe²⁺ 0.540 μmol L⁻¹, where the enzyme activity obtained was 236 U/mL. This value is two times higher than the value reported elsewhere.     

A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.