Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.     

Screening of Environmental DNA Libraries for the Presence of Genes Conferring Lipolytic Activity on Escherichia coli

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.     

同源肿瘤克隆细胞蛋白表达和侵袭能力的异质性研究

摘要 目的：比较同源肿瘤细胞来源的不同单克隆表型差异。方法：采用极限稀释法,在悬浮培养条件下获取HCT116结肠癌细胞系的单个细胞,对每孔含单个的细胞进行扩增培养,获得子代单克隆,并以同样方法继续挑取单克隆,连续获得子三代克隆。根据单克隆形态特点,选取第三代的三株代表性的单克隆,采用Western blot和免疫荧光法比较其SOX2、EpCAM和Vimentin蛋白表达差异。采用放疗观察三株单克隆的Vimentin蛋白的动态变化,研究其放疗干预的时间异质性,Transwell体外侵袭实验比较克隆侵袭力的差异。结果：三株由单细胞扩增培养的同源第三代子克隆依然存在明显生物学差异。形态有明显区别的球形与不规则的克隆形态。不规则形态克隆更表现为SOX2低表达及Vimentin的高表达。并且在单个细胞水平上,同个单克隆群体内也存在个体细胞间蛋白的表达差异（Vimentin; EpCAM）。通过观察放疗前后Vimentin蛋白在不同时间点上的荧光强度,发现肿瘤单克隆细胞存在时间异质性。Transwell体外侵袭实验也显示三个同源克隆间存在明显的差异性。结论：同源的、连续单细胞扩增获得的第三代单克隆依然存在明显生物学差异,提示肿瘤内部异质性是其固有特征,并且在治疗干预下,也会引起肿瘤时间异质性的产生。     

3个中山杉杂种无性系及其亲本的光合特性比较

对‘中山杉405’(Taxodium‘Zhongshanshan 405’)、‘中山杉406’(T.‘Zhongshanshan 406’)和‘中山杉407’(T.‘Zhongshanshan 407’)及其母本墨西哥落羽杉(T.mucronatum Tenore)和父本落羽杉〔T.distichum(L.)Rich.〕在7月份、9月份和10月份的光合参数进行了测定,并比较了3个无性系净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)和水分利用效率(WUE)的杂种优势。结果显示:不同月份3个无性系及其亲本的Pn值有明显差异,导致光响应曲线明显不同,但3个无性系的光响应曲线基本一致;3个无性系的Pn值在不同月份都高于落羽杉,且‘中山杉405’的Pn值高于另2个无性系。3个无性系及其亲本的光补偿点总体上表现为7月份最高、10月份最低,其中7月份墨西哥落羽杉的光补偿点显著高于3个无性系及落羽杉(P<0.05);3个无性系和墨西哥落羽杉的光饱和点则为9月份最高、7月份最低,而落羽杉的光饱和点为9月份最高、10月份最低。7月份3个无性系与其亲本的Pn日变化曲线均呈不规则型,低谷出现在中午12:00左右;9月份和10月份3个无性系和墨西哥落羽杉的Pn日变化曲线均呈单峰型,峰值出现在上午10:00;落羽杉的Pn值从上午8:00开始呈持续降低的趋势,且全天都明显低于其他树种。不同月份3个无性系的Pn、Gs、Tr和WUE的杂种优势有明显差异,总体上看,‘中山杉405’的Pn、Gs和Tr在3个月份中均保持较高的杂种优势率,而‘中山杉406’9月份的杂种优势率最高,‘中山杉407’则在7月份杂种优势率最高;3个无性系的WUE仅在10月份有明显的杂种优势,且‘中山杉405’和‘中山杉406’的杂种优势率远高于‘中山杉407’;‘中山杉405’各项指标的杂种优势率总体上高于另2个无性系。研究结果表明:3个中山杉无性系的光合能力接近其母本墨西哥落羽杉、高于父本落羽杉,其中‘中山杉405’杂种优势明显。     

黄瓜芽黄突变体抑制消减杂交文库的构建及初步分析

利用抑制消减杂交技术(suppression subtractive hybridization,SSH)分离了黄瓜芽黄突变体及其野生型之间差异表达的cDNA片段.以突变体和野生型分别作检测子和驱赶子,建立正向和反向两个消减杂交cDNA文库;经阳性克隆鉴定,在正向文库中获得特异表达的阳性克隆有133个,在反向文库中得到的阳性克隆有73个.测序后将所得到的159条非重复且非黄瓜的ESTs(登录号:GH270133～GH270291)进行序列同源性比对分析,发现这些ESTs分别与叶绿素合成、光合系统、信号转导、转录因子、氨基酸代谢、糖类代谢、脂类代谢等相关酶及蛋白基因高度同源.     

Production of avian oncoviral subgroups after multiple infection.

The number of different oncoviral env genes that can be expressed by a single chicken embryo fibroblast was investigated. Fibroblasts were infected with one to three subgroups of Rous-associated virus, which is a nontransforming avian oncovirus, then superinfected with a transforming virus, Rous sarcoma virus, of a different subgroup. The subgroups of viruses released by the resulting clones were analyzed. When two viral subgroups were used for preinfection, all the resulting clones produced transforming virus particles having the subgroup of the superinfecting virus, and most clones produced transforming virus particles of all the infecting viral subgroups. However, when cells were preinfected with three viral subgroups, many of the resulting clones did not produce transforming virus particles having the subgroup of the superinfecting virus, and only 1 of 23 clones produced transforming particles of all the infecting viral subgroups. DNA annealing experiments showed that cells infected with three or four viral subgroups had an additional 8 to 20 copies of proviral DNA per cell. Finally, most clones resulting from cells simultaneously infected with three or four viral subgroups were able to produce virus of all infecting subgroups. It appears that the number of exogenous oncoviral env genes that can be expressed by a single cell is limited, and in the range of 4 to 8-20 per cell.     

Variation in herbivore density among host plants and its consequences for community structure

Summary The densities of four species of gall-forming sawflies were found to vary significantly among willow host plant clones. Two of the speices varied among host plants at four sites in each of three years. The other two species varied in density among host plants at most of the sites in two of the three years. Total sawfly density also varied significantly among clones. Individual species densities on willow clones were significantly positively correlated between years when all sites were combined and frequently when sites were considered separately. Most pairwise species combinations were independent in density between years, but some negative correlations existed between the stem galler and the leaf galler. Gall-former densities also were largely independent among clones within years with all sites combined and with sites considered separately. The significant correlations were nearly all positive. At all four sites the combination of significant variation in sawfly densities among willow clones in the field and independence of species densities among clones resulted in significantly different communities (relative abundance of species) among willow clones in three years. Although sawfly abundances differed substantially among the four sites, this remained true. It is argued that the pattern of community structure among clones is the result of variation in host plant quality of clones. We propose an hypothesis to account for patterns of herbivore species associations based on intrapopulation host plant variation.     

A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

Nucleotide sequences of long terminal repeats of the human endogenous retrovirus (LTR HERV-K) on the short arm of chromosome 7: identification,analysis and evaluation of transcriptional activity

Six clones containing long terminal repeat (LTR) sequences of human endogenous retrovirus of the HERV-K family were found in the YAC library (1200 kb) of the short arm of human chromosome 7. The sequence sizes of the three clones corresponded to the full-length LTR (969 bp). The LTR localization was determined using FISH and verified by comparison with the GenBank database. All three DNA fragments containing solitary LTRs were transcribed in normal germline cells (testicular parenchyma tissue). The differences in the expression of these clones in the germline tumor cells (seminoma) were observed.     

Variation in the life cycle and morphology of the tobacco host-race of Myzus persicae (Hemiptera: Aphididae) in relation to its geographical distribution

Morphological variation and life cycle category were examined in 121 clones of Myzus persicae (Sulzer). The clones were collected from tobacco from three localities in Greece (Xanthi, Nea Efessos and Naphplion), one in Germany (Rheinstetten), one in France (Bergerac) and one in Spain (Madrid). Before morphometrics, all aphids were laboratory-reared on potato. The morphological variation was investigated using both canonical variates analysis and a novel non-parametric classification tree method. The life cycle category was examined by rearing the clones for three generations under short day conditions. In Nea Efessos a relative high proportion of clones was found to overwinter as eggs on the primary host. In the other regions all collected clones were non-holocyclic. Intermediate genotypes were found in all regions at percentages ranging from 4.0 to 24.0%. Androcyclic clones were found only in Xanthi, Greece (4.0%) and Rheinstetten, Germany (16.7%). The canonical variates analysis and the tree classification method revealed important intrapopulation polymorphisms in clones from Bergerac, Nea Efessos and Madrid. Both methods separated the populations originating from Greece from those collected elsewhere in western Europe. The observed morphological variation was probably due to genetic differences, since all clones were reared in a common environment. The results are discussed in relation to factors responsible for genetic divergence in M. persicae populations.     

Abundance patterns of lily pollen cDNAs: characterization of three pollen-preferential cDNA clones

Twenty-five clones were randomly selected from a mature pollen cDNA library of Easter lily (Lilium longiflorum Thunb.) in order to study the abundance of pollen-expressed mRNAs and the functional roles of the proteins encoded by these mRNAs. Plaque hybridization experiments were conducted to estimate indirectly the expression level of the mRNAs. Based on the hybridization frequency in the mature pollen library, the cDNA clones were divided into three abundance groups. Eight clones belonged to a high abundance class in which each cDNA clone was present in the mature lily pollen library at a frequency between 0.3 and 3%. Six of these clones were not found in cDNA libraries made from carpel, leaf, or root, suggesting that they are preferentially expressed in pollen. Fourteen clones belonged to a medium abundance class and were present in the mature pollen library at a frequency between 0.01 and 0.08%. The remaining three clones, which were present at a frequency below 0.01%, were grouped as a low abundance class. Almost all of the cDNA clones which belong to either the medium or low abundance class were also detected in the leaf library. Northern blot hybridization with three of the highly abundant cDNA clones confirmed their preferential expression in anther. In situ hybridization experiment with one of the clones showed the pollen-specific expression of the clone in mature anther. DNA sequence analysis revealed that the clone LMP131 encodes a peptide which is highly homologous to the tomato pollen-preferential gene, LAT59, which encodes a putative pectate lyase. The clone LMP134 encodes a peptide that shows an extensive similarity to a variety of thioredoxins. The third clone LMP132 encodes a 182-residue protein that has no significant homology to known sequences.     

Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.     

Nucleotide Sequences of Long Terminal Repeats of the Human Endogenous Retroviruses (LTR HERV-K) on the Short Arm of Chromosome 7: Identification,Map Locations,and Transcriptional Activity

Six clones containing long terminal repeat (LTR) sequences of human endogenous retrovirus of the HERV-K family were found in the YAC library (1200 kb) of the short arm of human chromosome 7. The sequence sizes of the three clones corresponded to the full-size LTR (969 bp). The LTR localization was determined using FISH and verified by comparison with the GenBank database. All three DNA fragments containing solitary LTRs were transcribed in normal germline cells (testicular parenchyma tissue). The differences in the expression of these clones in the germline tumor cells (seminoma) were observed.     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

Rapid and reliable screening of a tomato YAC library exclusively based on PCR

An improved procedure is presented to select clones from a tomato yeast artificial chromosome (YAC) library. The procedure is based exlcusively on the polymerase chain reaction (PCR). We combined DNA from approximately 36,000 YAC clones in pools containing 96-single YAC clones from one master plate and further in super pools representing 10 master plates. This pooling strategy allows the selection of single YAC clones homologous to a target sequence after three rounds of PCR using super pools, single pools, and single YAC clones as a template. Single YAC clones were spheroplasted prior to the third PCR round in order to omit the conventional radioactive colony hybridization step. To date, we applied this PCR-based selection strategy to isolate clones homologousto ten different sequence-tagged sites (STS) that are linked to genes targeted for map-based cloning. The selection of YAC clones can be readily accomplished within three days. The PCR-based screening strategy is easy to set up and contributes to a further acceleration of the construction of YAC contigs.     

Intraspecific diversity and distribution of the cosmopolitan species Pseudo‐nitzschia pungens (Bacillariophyceae): morphology,genetics, and ecophysiology of the three clades

Three clades of Pseudo‐nitzschia pungens, determined by the internal transcribed space (ITS) region, are distributed throughout the world. We studied 15 P. pungens clones from various geographical locations and confirmed the existence of the three clades within P. pungens, based on ITS sequencing and described the three subgroups (IIIaa, IIIab, and IIIb) of clade III. Clade III (clade IIIaa) populations were reported for the first time in Korean coastal waters and the East China Sea. In morphometric analysis, we found the ultrastructural differences in the number of fibulae, striae, and poroids that separate the three clades. We carried out physiological tests on nine clones belonging to the three clades growing under various culture conditions. In temperature tests, only clade III clones could not grow at lower temperatures (10°C and 15°C), although clade I and II clones grew well. The estimated optimal growth range of clade I clones was wider than that of clades II and III. Clade II clones were considered to be adapted to lower temperatures and clade III to higher temperatures. In salinity tests, clade II and III clones did not grow well at a salinity of 40. Clade I clones were regarded as euryhaline and clade II and III clones were stenohaline. This supports the hypothesis that P. pungens clades have different ecophysiological characteristics based on their habitats. Our data show that physiological and morphological features are correlated with genetic intraspecific differentiation in P. pungens.     

Resistance to potato leafroll luteovirus can be greatly improved by combining two independent types of heritable resistance

Twelve potato clones were exposed to infection by aphids with potato leafroll luteovirus (PLRV) in three field trials in order to assess their resistance to infection. Up to 92% of the plants of some clones became infected, although other clones were relatively resistant to infection and one clone remained virus-free in all three trials. The resistance of the same 12 clones to PLRV multiplication was assessed in glasshouse-grown plant: lants were graft-inoculated and their daughter tubers were used to grow plants with secondary infection. High concentrations of PLRV were found in some clones (c. 1700 ng/g leaf) while in others much less virus accumulated (as little as 60 ng/g leaf). However, clones in which little virus accumulated were not necessarily those which were most resistant to infection in the field, and there was no association between the two types of resistance. Nevertheless, both types of resistance were found in some clones. The clone G8107(1), which remained virus-free in all the field exposure trials, was also the most resistant to PLRV multiplication. The combination of these two types of resistance in cultivars should help to eliminate the spread of PLRV in crops.     

Control of laminin synthesis during differentiation of F9 embryonal carcinoma cells

Molecular clones complementary to the mRNA species for the A, B1 and B2 chains of murine laminin were identified by hybrid-selection and in vitro translation. Northern blot analysis demonstrated that the three clones, p59 (A), p2 (B1) and p16 (B2) hybridized to mRNA species 9.8, 6.0, and 8.0 kb in length, respectively. The three clones were used as probes to monitor the steady-state levels of laminin mRNA species during differentiation of F9 embryonal carcinoma cells induced by treatment with retinoic acid and dibutyryl cyclic AMP. The steady-state levels of the three mRNA species appeared to increase in a coordinate manner. Undetectable levels at the beginning of induction were followed by a dramatic increase in the levels of the three mRNA species between 48 and 72 h. The kinetics parallel the increase in laminin synthesis and the striking morphological changes previously reported.