Molecular characterization can quantify and partition variation among genebank holdings: a case study with phenotypically similar accessions of Brassica oleracea var. capitata L. (cabbage) `Golden Acre'

To better characterize and conserve crop genetic resources, the assessment of genetic identity, relatedness, and structure among entries and collections becomes a priority. In the present study, a random amplified polymorphic DNA (RAPD) assay was applied as a quick, cost-effective, and preliminary screen to quantify and partition the molecular variation among accessions. Fourteen phenotypically uniform accessions of Brassica oleracea var. capitata L. (cabbage) similarly designated as `Golden Acre' were tested with nine decamer oligonucleotide primers. These amplifications generated 110 fragments, of which 80 were polymorphic ranging in size from 370 to 1720 bp. The 80 polymorphic fragments were sufficient to distinguish between all 14 accessions. Data based on the partitioning of variation among accessions indicated that `Golden Acre' entries could be reduced to as few as four groups, with the potential loss of variation being only 4.6% of the absolute current genetic variation in those holdings as estimated from RAPD analysis. This proposed grouping would concurrently save approximately 70% [$750–1000 (US) per accession] for each cycle of regeneration (approximately 20–25 years at most) which alternatively could then be used for other priorities in B. oleracea conservation and use. This case represents but one example where targeted use of a molecular-marker assay linked with rigorous statistical analysis will be useful for plant genebank management, particularly for questions at the intraspecific level. Molecular markers will provide genebank curators with additional sources of information to better plan and organize collection holdings and use finite financial support in a more effective manner. Received: 10 June 1996 / Accepted: 23 August 1996     

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.     

筛选转基因动物的新方法——PCR及其产物测序法

用特异性引物对肌球蛋白轻链2启动子(myosin light chain-2,MLC₂)-糜酶融合基因的转基因新生鼠鼠尾DNA进行PCR筛选, 低熔点琼脂糖凝胶电泳回收阳性样品PCR所扩出的DNA条带,纯化后用同一对引物中的一个进行单引物PCR测序,与所转外源基因序列比较,进一步确定整合有外源基因的阳性鼠.PCR及PCR 产物测序法检测转基因动物具有操作方便,灵敏度高及特异性强等优点.     

Evaluation of Antiherpetic Compounds Using a Gastric Cancer Cell Line: Pronounced Activity of BVDU against Herpes Simplex Virus Replication

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV-induced cytopathogenicity in gastric adenocarcinoma MKN-28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV-1 and HSV-2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC₅₀) values of the antiherpes agents in MKN-28 cells were compared with those in human embryo lung MRC-5 cells, all compounds, except for BVDU, showed higher EC₅₀ values in MKN-28 cells. For BVDU the EC₅₀ values in MKN-28 cells were 0.8 (HSV-1) and 0.036 (HSV-2) times the EC₅₀ values in MRC-5 cells. Thus BVDU was 27.5 times more active against HSV-2 in MKN-28 cells than in MRC-5 cells. The MKN-28 gastric cancer cells may be useful for the rapid screening of anti-HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.     

Estimating the sensitivity of cervical cytology: errors of interpretation and test limitations

The objective of this study was to estimate: (i) the sensitivity of cytologists in recognizing abnormal smears; (ii) the sensitivity of cervical cytology as a method of detecting abnormal smears among those obtained in the presence of cervical intraepithelial neoplasia (CIN). Study subjects were 61 women with a histologically confirmed CIN identified through colpohistological and cytologic screening. For objective (i) new smears were taken from study subjects just before treatment, mixed with routine preparations, interpreted by unaware cytologists and then blindly reviewed by a group of three expert supervisors, who reached a consensus diagnosis. Cytologists classified as positive for squamous intraepithelial lesion (SIL) 30 of the 34 smears judged as positive by supervisors (100% of smears classified as high-grade and 67% of smears classified as low-grade SIL by the supervisors). Our approach, based on creating a set of smears with a high a priori probability of being positive, proved to be an efficient way of estimating errors of interpretation. For objective (ii), smears taken at the moment of diagnosis, just before biopsy, were also reviewed by the same supervisors. These CIN cases were identified among asymptomatic women independently of cytological findings and results are therefore not subject to verification bias. Among the 33 histological CINII/III, four (12%) smears had no atypical cells (three negatives and one unsatisfactory) at review. The same proportion was 26% (four negatives and one unsatisfactory) among the 19 histological CINI. No significant differences in smear content were found between the seven ‘false negatives’ and a sample of ‘true positives’ and ‘true negatives’ for a number of formal adequacy criteria (including presence of endocervical cells). Strong differences were found between positive smears taken just before biopsy and those taken just before treatment (in 11 women the first smear only was positive, while the opposite was never observed), suggesting an effect of punch biopsy in removing lesions.     

Costs and benefits of cervical screening. II. Is it worthwhile reducing the screening interval from 5 to 3 years?

A reduction in screening interval from 5 years to 3 years would greatly increase the cost of the programme, but would save few extra lives. The cost per life saved would be around £250 000 at 1995 prices, or around £8000 per life per year saved. There would in addition be human costs for the women screened. The opportunity cost of reducing the interval may be too great, since it is likely that the Health Service would achieve greater health benefits by investing the funds in other health care activities.     

基于系谱和SSR标记的高山杜鹃杂交种亲缘关系分析

该研究对高山杜鹃(Rhododendron L.)的总DNA提取方法进行了改良,然后综合利用高山杜鹃EST数据库和该实验室的马缨杜鹃高通量测序数据,引用已发表文献的SSR引物,从154对SSR引物中筛选出了26对多态性高、重复性好、条带清晰的SSR引物。再从中随机选择10对SSR引物进行荧光标记,对69份不同高山杜鹃的种质进行遗传多样性分析。结果表明,平均有效等位基因数6.959 2个;位点多态性信息含量(PIC)、观测杂合度(HO)、期望杂合度(HE)和Neis基因多样性(H)分别为0.795 2、0.543 5、0.826 5和0.820 2;杂交种间的非加权配对算术平均(UPGMA)聚类结果与其谱系分析结果基本一致,可实现对一些未知来源的育成品种资源进行祖先亲本类型的推测。     

纤维素降解真菌DF14101的筛选与鉴定

纤维素是植物细胞壁的主要成分,也是人类宝贵的天然可再生资源之一。由于纤维素不易降解,严重限制了生物质废弃物中纤维素的有效利用。从海口、儋州、屯昌市郊的森林、农田以及香蕉园采集土壤和腐烂秸秆样品中,筛选获得1株产纤维素酶能力较强的真菌DF14101。在以香蕉秸秆粉为碳源的培养基中,28℃、180r/min培养5 d时,该菌株发酵液的内切葡聚糖酶酶活(CMCase)为43.98 U/m L,滤纸酶活(FPA)为14.05 U/m L。结合形态学特征和ITS序列系统发育分析结果,将该菌株鉴定为草酸青霉(Penicillium oxalicum)。     

L-天冬氨酸-α-脱羧酶高产菌株初筛方法的建立

通过试管法、平板法、Berthelot法和纸层析法对L-天冬氨酸-α-脱羧酶(PanD)高产菌株的初筛方法进行研究。分别检测转化体系中底物L-天冬氨酸减少引起的pH变化及产物CO2和β-丙氨酸的增加量,并用高效液相色谱法比较这四种方法的准确性。结果表明:CO2能溶于转化液,难以用倒置管收集完全;PanD系胞内酶,难以用平板点种法改变平板的pH;β-丙氨酸和L-天冬氨酸在Berthelot检测中吸收值均偏低,不适用于PanD高产菌的筛选;纸层析法可以直接检测β-丙氨酸和L-天冬氨酸,成本低,操作简单,具一定的精确度,可以较大规模地筛选PanD高产菌株。