A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.     

On the eigenvalue distribution of genetic and phenotypic dispersion matrices: Evidence for a nonrandom organization of quantitative character variation

A quantitative genetic model of random pleiotropy is introduced as reference model for detecting the kind and degree of organization in quantitative genetic variation. In this model the genetic dispersion matrix takes the form of G = BB ^T, where B is a general, real, Gaussian random matrix. The eigenvalue density of the corresponding ensemble of random matrices (_G) is considered. The first two moments are derived for variance-covariance matrices G as well as for correlation matrices R, and an approximate expression of the density function is given. The eigenvalue distribution of all empirical correlation matrices deviates from that of a random pleiotropy model by a very large leading eigenvalue associated with a size factor. However the frequency-distribution of the remaining eigenvalues shows only minor deviations in mammalian skeletal data. A prevalence of intermediate eigenvalues in insect data may be caused by the inclusion of many functionally unrelated characters. Hence two kinds of deviations from random organization have been found: a mammal like and an insect like organization. It is concluded that functionally related characters are on the average more tightly correlated than by chance (= mammal like organization), while functionally unrelated characters appear to be less correlated than by random pleiotropy (insect like organization).     

Thermo-inducible expression of cloned early genes of bacteriophage Mu.

An EcoRI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c. The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28 degrees C at high copy number, but at 42 degrees C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B, the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42 degrees C, therefore the leftmost EcoRI site on the Mu DNA is located between genes kil and gam or sot, or within the gam or sot gene.     

PRD-Class Homeobox Genes in Bovine Early Embryos: Function,Evolution, and Overlapping Roles

Eutherian Totipotent Cell Homeobox (ETCHbox) genes are mammalian-specific PRD-class homeobox genes with conserved expression in the preimplantation embryo but fast-evolving and highly divergent sequences. Here, we exploit an ectopic expression approach to examine the role of bovine ETCHbox genes and show that ARGFX and LEUTX homeodomain proteins upregulate genes normally expressed in the blastocyst; the identities of the regulated genes suggest that, in vivo, the ETCHbox genes play a role in coordinating the physical formation of the blastocyst structure. Both genes also downregulate genes expressed earlier during development and genes associated with an undifferentiated cell state, possibly via the JAK/STAT pathway. We find evidence that bovine ARGFX and LEUTX have overlapping functions, in contrast to their antagonistic roles in humans. Finally, we characterize a mutant bovine ARGFX allele which eliminates the homeodomain and show that homozygous mutants are viable. These data support the hypothesis of functional overlap between ETCHbox genes within a species, roles for ETCHbox genes in blastocyst formation and the change of their functions over evolutionary time.     

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.     

青弋江流域大型底栖动物群落结构及水质评价

研究河流大型底栖动物群落结构特征及水环境质量对水生态的保护与修复具有重要意义。以青弋江流域为研究对象,于2020年9月进行了大型底栖动物及水体理化因子的调查,采用相对重要性指数、生物多样性指数及Jaccard相似性系数分析了大型底栖动物群落结构特征,运用丰度/生物量比较曲线法及冗余分析法探究了底栖动物群落受到的干扰程度及其主要影响环境因子,最后利用水生态环境质量综合指数对河流水质进行评价。研究结果表明：(1)共采集鉴定大型底栖动物61种,隶属于3门6纲17目,平均丰度为265.9个/m~2,平均生物量41.6 g/m~2。相对重要性指数分析显示,日本沼虾(Macrobrachium nipponensis)、中国圆田螺(Cipangopaludina chinensis)、羽摇蚊(Chironomus plumosus)及扁蜉(Ecdyrus)为群落中的主要优势种。(2)由群落相似性分析知,Jaccard相似性系数较低,调查点位之间存在较强的空间异质性。(3)冗余分析表明,电导率、氨氮浓度及流速是影响底栖动物群落结构的主要环境因子。(4)生物指数分析结果显示该流域水质为轻度污染或中度污...     

异质水分环境下野牛草相连分株间光合同化物的生理整合及其调控

异质环境下,克隆植物通过生理整合机制使资源在分株间实现共享,提高了其对异质性环境的适应能力,具有重要的生态进化意义,研究生理整合机制及其调控机理可为进一步发掘克隆植物应用潜力提供理论依据。以野牛草3个相连分株为材料,对其中一个分株用30%聚乙二醇6000(PEG-6000)模拟水分胁迫,通过Hoagland营养液培养试验,研究了异质水分环境下光合同化物在野牛草相连分株间的生理整合及分株叶片与根系内源激素ABA与IAA含量的变化规律。结果表明,14C-光合同化物在克隆片断内存在双向运输,但以向顶运输为主,异质水分环境下,受胁迫分株光合同化物的输出率明显降低,而与其相邻分株合成的光合同化物向受胁迫分株方向运输率明显增加;异质水分环境下,各分株ABA含量均明显增加,但以受胁迫的分株叶片及根系ABA的含量增加幅度最大,各分株IAA含量较对照均显著下降(P0.05),且以受胁迫分株IAA含量下降幅度最大;各分株叶片与根系ABA/IAA均显著提高(P0.05),相邻分株ABA/IAA增加幅度低于受胁迫分株。异质水分环境影响野牛草克隆分株间光合同化物的生理整合,且ABA与IAA在分株间光合同化物运输与分配过程中具有重要的调节作用。     

珠江口南沙河涌浮游植物群落结构时空变化及其与环境因子的关系

随着城市生态健康理念的提出,城市河涌生态健康也受到了前所未有的关注。为更好的了解河涌的水环境和浮游植物现状,于2015年3月至2016年2月对珠江口南沙河涌8个站位水环境和浮游植物群落结构进行调查。结果显示:共发现浮游植物164种(属),隶属7门73属,其中以绿藻种类最多,达33属79种,占48.17%;硅藻次之,17属41种,占25%。优势种为梅尼小环藻(Cyclotella meneghiniana)、假鱼腥藻属(Pseudanabaena sp.)和小球藻(Chlorella vulgaris)。浮游植物细胞密度在0.19×10~6—101.34×10~6个/L内变动,呈现单峰型,在4月发生拟菱形弓形藻(Schroederia nitzschioides)水华,14涌密度高达87.38×10~6个/L,随后因强降雨细胞密度骤降。浮游植物群落的季节演替基本符合PEG(Plankton Ecology Group)模型,从冬季的硅藻,到春夏季的绿藻,再到秋季的蓝藻。One-way ANOVA分析显示,各月份浮游植物细胞密度差异显著(P0.01)。Pearson相关性分析表明绿藻细胞丰度变化主导着浮游植物总丰度的变化(r=0.454,P0.01)。运用Margalef物种丰富度指数、Shannon物种多样性指数、Pielou均匀度指数对水体进行评价表明,调查水体呈中度污染。相关加权营养状态指数表明,河涌全年处于富营养化状态。浮游植物聚类分析表明,时间异质性较高,总体相似性较低;空间上相似性较高,人为活动可能是导致空间差异的关键因子。冗余分析显示,叶绿素a、溶解氧、盐度、水温、总氮和p H与浮游植物群落结构关系最为密切。p H对硅藻门浮游植物影响较大,碱性条件适宜直链藻生长,春季水华形成的驱动因子是盐度、温度和总氮。