Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.     

Identification of Brassica oleracea monosomic alien chromosome addition lines with molecular markers reveals extensive gene duplication

Summary Chromosomes of Brassica oleracea (2n=18) were dissected from the resynthesized amphidiploid B. napus Hakuran by repeated backcrosses to B. campestris (2n=20), creating a series of monosomic alien chromosome addition line plants (2n=21). Using morphological, isozyme and restriction fragment length polymorphism markers (RFLPs), 81 putative loci were identified. Of nine possible synteny groups, seven were represented in the 25 monosomic addition plants tested. Sequences homologous to 26% of the 61 DNA clones utilized (80% were cDNA clones) were found on more than one synteny group, indicating a high level of gene duplication. Anomalous synteny associations were detected in four 2n=21 plants. One of these plants showed two markers from one B. oleracea chromosome associated with a second complete B. oleracea synteny group, suggesting translocation or recombination between non-homologous chromosomes in Hakuran or the backcross derivatives. The other three 2n=21 plants each contained two or more B. oleracea synteny groups, suggesting chromosome substitution.     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

Construction and packaging of pseudotype retrovirus containing human N—ras cDNA antisense sequence and its biological effects on human hepatoma cells

N-ras is one of the transforming genes in human hepatic cancer cells.It has been found that N-ras was overexpressed at the mRNA and protein level in hepatoma cells.In order to explore the biological roles of N-ras in human hepatic carcinogenesis and the potential application in control of cancer cell growth,a preudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged.A recombinant retrovirus vector containing antisense or sense sequences of N-ras cDNA was constructed by pZIP-NeoSV(X)1.The pseudotype virus was packaged ang rescued by transfection and infection in PA317 and ψ 2 helper cells.It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PRF/5 hepatoma cells accompanied with inhibition of p21 expression,while the retrovirus containing sense sequence had none.The pseudotype virus had no effect on human diploid fibroblasts.     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

野生大豆基因文库的构建

以氯化铯密度梯度离心法纯化噬菌体λEMBL4,将纯化的EMBL4 DNA用BamH1/SalI双酶切制成载体。用CTAB(十六烷基三甲基溴化铵)法提取野生大豆(种名待定)大分子DNA,Sau3A部分酶解,从琼脂糖凝胶中回收10—22kb“目的”DNA片段,与载体连接,体外包装成重组噬菌体。所得重组子值为8×10(?)pfu(噬菌斑形成单位),达到了构建野生大豆基因文库要求的理论值。以栽培大豆7S贮藏蛋白a′-cDNA作探针,用噬菌斑原位杂交法从文库中筛选出一个阳性克隆。     

Probabilistic reconstruction of ancestral protein sequences

Using a maximum-likelihood formalism, we have developed a method with which to reconstruct the sequences of ancestral proteins. Our approach allows the calculation of not only the most probable ancestral sequence but also of the probability of any amino acid at any given node in the evolutionary tree. Because we consider evolution on the amino acid level, we are better able to include effects of evolutionary pressure and take advantage of structural information about the protein through the use of mutation matrices that depend on secondary structure and surface accessibility. The computational complexity of this method scales linearly with the number of homologous proteins used to reconstruct the ancestral sequence.