Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer

Summary In P. hybrida and B. nigra an enhancement of transformation rates (direct gene transfer) of about six to seven-fold was obtained after irradiation of protoplasts with 12.5 Gy (X-ray). The effect of protoplast irradiation was similar in experiments where protoplasts were irradiated 1h before transformation (X-ray/DNA) or 1h after completion of the transformation procedure (DNA/X-ray). Increased X-ray doses up to 62.5 Gy resulted in further enhancement of percentages of transformed colonies, indicating a correlation between relative transformation frequencies (RTF) and the doses applied. Estimation of degradation rates of plasmid sequences in plant protoplasts yielded a reduction of plasmid concentration to 50% 8–12 h after transformation. In 1-day-old protoplasts, the level of plasmid fragments dropped to 0%–10% compared to 1h after transformation. The results demonstrate that the integration rates of plasmid sequences into the plant genome may in part be governed by DNA repair mechanisms. This could be an explanation for the observed genotypic dependence of transformation rates in different plant species and plant genotypes. Gene copy number reconstructions revealed enhanced integration rates of plasmid sequences in transformed colonies derived from irradiated protoplasts.     

质粒YRP7的基本特性鉴定

质粒YRP7用氯霉素法扩增,碱变性裂解法提取,酸酚法及核糖核酸酶纯化后,得到了高产量(5.6mg/L培养液),高纯度(A260:A280=2.0)的质粒制品,经转化实验及酶切分析确定YRP7具有下列特征:大小为5.41±0.10kb,可赋予宿主细胞AmP~r、Tet~r的表型,对大肠杆菌C600的转化频率为10~(-6)、转化效率为1.5×10~6转化子/mgDNA。限制性内切酶BamH Ⅰ、ECoRⅠ、Hind Ⅲ及PstⅠ在其分子上的切点数分别为1、2、2、2,并确定了各酶切片段的分子大小,对BanHⅠ的单切点,经插入失活法证实其位于Tet~r的基因上。由上述特征可确定,质粒YRP7是一个比较理想的克隆载体。     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG

Abstract The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum . The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated. The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG). The plasmid vector pSO246 was stable in BCG for at least 50 generations.     

Vertical canopy structure of Dutch chalk grasslands in relation to their management

Analysis of leaf canopy overtopping relationships was carried out using a non-destructive point quadrat method followed by a destructive stratified harvest of the above-ground phytomass in Dutch chalk grasslands with different management: summer sheep grazing and annual autumn mowing. The two methods of analysis are compared: e.g. relative leaf overtopping can be recorded by the point quadrat method but it is obscured in vertical vegetation profiles based on stratified phytomass distribution. However the stratified harvest method describes the relationship between canopy phytomass and light microclimate, recorded by measuring Photosynthetically Active Radiation (PAR) at different heights in the vegetation. Plant growth form during peak standing crop is of greater significance than Raunkiaerian life form in determining structure of chalk grassland vegetation. In annually mown grasslands, the tall graminoid growth form, shown by Brachypodium pinnatum, reduces PAR in the lower canopy and suppresses most other growth forms except those which can reach the higher canopy levels, e.g. clumped herbs such as Origanum vulgare. In contrast, grazing can result in a reduction of dominance from the tall graminoids and reduction of the abundance of taller, grazing-sensitive herbs, e.g. clumped and climbing herbs such as Origanum and Lathyrus pratensis, and an increase in grazing-tolerant species, e.g. smaller rosette herb growth forms, e.g. Leontodon hispidus and shorter rhizomatous or stoloniferous graminoids, e.g. Carex flacca and Briza media. Changes in both the overtopping hierarchy at the peak of the growing season and the intensity of overtopping in the course of a growing season are of conclusive importance in determining the relative abundance of species in the vegetation.     

Plasmid transfer to indigenous marine bacterial populations by natural transformation

Abstract Horizontal gene transfer among microbial populations has been assumed to occur in the environment, yet direct observations of this phenomenon are rare or limited to observations where the mechanism(s) could not be explicitly determined. Here we demonstrate the transfer of exogenous plasmid DNA to members of indigenous marine bacterial populations by natural transformation, the first report of this process for any natural microbial community. Ten percent of marine bacterial isolates examined were transformed by plasmid DNA while 14% were transformed by chromosomal DNA. Transformation of mixed marine microbial assemblages was observed in 5 of 14 experiments. In every case, acquisition of the plasmid by members of the indigenous flora was accompanied by modification (probably from genetic rearrangement or methylation) that altered its restriction enzyme digestion pattern. Estimation of transformation rates in estuarine environments based upon the distribution of competency and transformation frequencies in isolates and mixed populations ranged from 5 × 10⁻⁴ to 1.5 transformants/1 day. Extrapolation of these rates to ecosystem scales suggests that natural transformation may be an important mechanism for plasmid transfer among marine bacterial communities.     

Changes in UsnRNA biosynthesis during rat liver regeneration

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4–U6) gradually decreased by 30–50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20–30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30–50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30–50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H. Thus, it may be concluded that the changes in UsnRNAs' level during the proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver.This paper was published in Molecular and Cellular Biochemistry131:67–73, 1994. Kluwer Academic Publishers regret the publication of the only partly corrected version.     

Global analysis of a model of plasmid-bearing,plasmid-free competition in a chemostat

A model of competition between plasmid-bearing and plasmid-free organisms in a chemostat was proposed in a paper of Stephanopoulis and Lapidus. The model was in the form of a system of nonlinear ordinary differential equations. Such models are relevant to commercial production by genetically altered organisms in continuous culture. The analysis there was local (using index arguments). This paper provides a mathematically rigorous analysis of the global asymptotic behavior of the governing equations in the case of uninhibited specific growth rate.Research supported by the National Council of Science, Republic of ChinaResearch supported by National Science Foundation Grant, DMS-9204490Research supported by the Natural Science and Engineering Council of Canada. This author's contribution was made while on research leave visiting the Department of Ecology and Evolutionary Biology at Princeton University. She would especially like to thank Simon Levin for his guidance as well as for providing an exceptional working environment     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.