Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

Biochemical and genetic analysis of Streptococcus mutans alpha-galactosidase.

The aga gene coding for alpha-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage lambda. The gene was subcloned into plasmid vectors and shown to specify a novel protein of Mr 80,000. Characterization of alpha-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the alpha-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted Mr of 82,022 and revealed regions of homology to alpha-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all alpha-galactosidase activity and abolished the ability to ferment melibiose; alpha-glucosidase activity was also lost, due to an indirect effect on the dexB gene.     

Distribution of genes and recombination in wheat and other eukaryotes

The genome sizes of eukaryotes may differ as much as 10,400-fold. A part of these differences may be attributed to polyploidy, and increase in gene number and size. Most of the genome size disparity is due to non-transcribed repeated DNA including retrotransposons and pseudogenes. Only a small fraction of the larger genomes such as those of many crop plants, contain genes. Genes are distributed unevenly along the chromosomes, often organized in clusters of varying sizes and gene-densities (gene-rich regions). The regions corresponding to gene-clusters in smaller genome plants such as rice may be divided into many mini gene-clusters in the related larger genomes. The range of gene-density within the mini2019; gene-clusters is about the same among plants with varying genome sizes. Recombination per chromosome is similar among eukaryotes, and thus is considerably independent of DNA content and chromosome size. Relatively little recombination occurs outside the gene-rich regions. Recombination varies dramatically among various gene regions, and is highly uneven within gene regions as well. Consequently, a significant number of genes may be inaccessible to recombination-based manipulations such as map-based cloning.     

Distribution of genes and recombination in wheat and other eukaryotes

The genome sizes of eukaryotes may differ as much as 10,400-fold. A part of these differences may be attributed to polyploidy, and increase in gene number and size. Most of the genome size disparity is due to non-transcribed repeated DNA including retrotransposons and pseudogenes. Only a small fraction of the larger genomes such as those of many crop plants, contain genes. Genes are distributed unevenly along the chromosomes, often organized in clusters of varying sizes and gene-densities (gene-rich regions). The regions corresponding to gene-clusters in smaller genome plants such as rice may be divided into many ‘mini’ gene-clusters in the related larger genomes. The range of gene-density within the ‘mini2019; gene-clusters is about the same among plants with varying genome sizes. Recombination per chromosome is similar among eukaryotes, and thus is considerably independent of DNA content and chromosome size. Relatively little recombination occurs outside the gene-rich regions. Recombination varies dramatically among various gene regions, and is highly uneven within gene regions as well. Consequently, a significant number of genes may be inaccessible to recombination-based manipulations such as map-based cloning.     

Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli

Derivatives of plasmid pBR322 DNA containing tet mutations were constructed by inserting XhoI linkers at various sites in the tetracycline resistance gene. Monomer plasmids containing either the tet-10 allele located at nucleotide position 23 or the tet-14 allele located at nucleotide position 1267 were used to construct a circular dimer containing one copy of each allele and a circular trimer containing one copy of the tet-10 allele and two copies of the tet-14 allele. Genetic recombination of these plasmid DNAs to produce a functional tetracycline resistance gene could be detected as the production of tetracycline-resistant progeny during the growth of transformants or using a restriction mapping assay which detected the rearrangement of the mutant alleles. The structure of individual tetracycline-resistant recombination products was determined by restriction mapping. This analysis suggested that as many as 70% of the plasmid recombination events in Escherichia coli AB1157 could have involved gene conversion events. The formation of these recombination products was most easily predicted by a model involving figure 8 recombination intermediates and the formation of symmetric regions of heteroduplex. Recombination in JC10287 delta(srlR-recA)304 occurred at 5% of the wild-type frequency and appeared to occur by a similar mechanism. Recombination in JC9604 recA56 recB21 recC22 sbcA23 occurred at 20 times the wild-type frequency and appeared to involve multiple independent recombination events.     

Employing site-specific recombination for conditional genetic analysis in Sinorhizobium meliloti

The ability to remove a genetic function from an organism with good temporal resolution is crucial for characterizing essential genes or genes that act in complex developmental programs. The rhizobium-legume symbiosis involves an elaborate two-organism interaction requiring multiple levels of signal exchange. As an important step toward probing rhizobium genetic functions with temporal resolution, we present the development of a conditional gene deletion system in Sinorhizobium meliloti that employs Cre/loxP site-specific recombination. This system enables chemically inducible and irreversible gene deletion or gene upregulation. Recombinase-mediated excision events can be positively or negatively selected or monitored by a colorimetric assay. The system may be adaptable to various bacterial species, in which recombinase activity may be placed under the control of diverse user-defined promoters. This system also shows promise for uses in promoter trapping and biosensing applications.     

Links between replication, recombination and genome instability in eukaryotes

Double-strand breaks in DNA can be repaired by homologous recombination including break-induced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.     

Biochemical genetic analysis of formycin B action in Leishmania donovani

Formycin B is cytotoxic toward Leishmania and is a potential chemotherapeutic agent for leishmaniasis. In order to determine the mechanism of action of formycin B, we have isolated and characterized clonal populations of formycin B-resistant Leishmania donovani. These formycin B-resistant clones are also cross-resistant to formycin A and allopurinol riboside-mediated growth inhibition. Incubation of the formycin B-resistant cells with [3H]formycin B indicates that, unlike wild type cells, the resistant populations cannot accumulate phosphorylated metabolites of exogenous [3H]formycin B. This is due to a defective transport system for formycin B in the resistant cells. However, wild type and mutant cells incorporate [3H]formycin A equally efficiently into [3H]formycin A-containing nucleotides and into RNA. These data suggest that formycin B cytotoxicity in Leishmania is not mediated by its incorporation as the adenosine analog into RNA. A plausible alternative hypothesis is proposed for the mechanism of action of the pyrazolo (4,3-d)pyrimidine C-nucleosides based upon depletion of an essential intracellular metabolite.     

Cre/lox位点特异性重组系统在高等真核生物中的研究进展

来自于P1噬菌体的Cre/lox系统通过位点特异性重组可以迅速而有效地实现各种生理环境下的基因定点插入、删除、替换和倒位等操作。Cre/lox系统作为目前基因打靶技术的核心工具,已被广泛应用于拟南芥、水稻、小鼠、果蝇、斑马鱼等高等真核模式生物。文章较为全面地介绍了Cre/lox系统的基本概况及其在高等真核生物中的应用,讨论了Cre/lox系统在研究中存在的主要问题和今后的发展方向,为利用该系统在不同高等生物中进行基因操作提供有用的参考。     

Biochemical studies of homologous and nonhomologous recombination in human cells.

Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro. Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein. Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold. Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons. We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa. Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells. A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.     

Biochemical analysis of att-defective mutants of the phage lambda site-specific recombination system

Three mutations previously mapped to the common core region of the bacteriophage lambda att site have been sequenced. All were found to be due to the deletion of a T residue from a string of six T residues within the 15 base-pair core, the region of homology between the recombining sites. As judged by DNAase I protection experiments, binding of the Int protein is the same in the mutant and wild-type core sites. However, a difference in the Int binding to mutant cores is observed when the small neocarzinostatin molecule is used as a nuclease probe. The differences between mutant and wild type lead to the suggestion that Int is interacting with sequences at the core-arm junctions. Accordingly, the mutants are proposed to be defective in the spacing of Int monomers bound at two recognition sequences spanning the core-arm junctions. The anomalous electrophoretic mobility of wild-type att fragments and, more specifically, the effect of the single base core deletion on electrophoretic mobility are discussed in the text and in the Appendix. The mutant att²501, defective in both att and int functions, was sequenced and found to be a 335 base-pair deletion removing the coding region for 25 amino acids from the carboxy-terminal end of Int, as well as the entire att site. The postulated origin of the 501 mutation is also consistent with the model of two juxtaposed Int recognition sites.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12. IV. Analysis of recombinants formed by a recombination deficient (recB21 recC22) strain

Summary Residual genetic recombination is carried out by recB ^- recC ^- mutants of E. coli. Recombinants (for one gene) formed by a recB ^- recC ^- parent were shown to be as recombination deficient as their parent, when recombination of a second gene is measured. Therefore the resididual recombination cannot be attributed to a genetically recombination proficient fraction of the parent recB ^- recC ^- culture. I conclude that each recB ^- recC ^- parent cell is capable of carrying out genetic recombination. This conclusion is consistent with the existence of an alternate (and minor) recombination mechanism in E. coli K12, independent of the recB ⁺ recC ⁺ mediated steps.The previous paper in this series was Capaldo-Kimball and Barbour, Involvement of Recombination Genes in Growth and Viability, J. Bact. April (1971).     

Biochemical and genetic analysis of carbohydrate accumulation in Allium cepa L

Onion and shallot (Allium cepa L.) exhibit wide variation in bulb fructan content, and the Frc locus on chromosome 8 conditions much of this variation. To understand the biochemical basis of Frc, we conducted biochemical and genetic analyses of Allium fistulosum (FF)-shallot (A. cepa Aggregatum group) alien monosomic addition lines (AALs; FF+1A-FF+8A) and onion mapping populations. Sucrose and fructan levels in leaves of FF+2A were significantly lower than in FF throughout the year, and the springtime activity of acid invertase was also lower. FF+8A showed significantly higher winter sucrose accumulation and sucrose phosphate synthase (SPS) activity. Inbred high fructan (Frc_) lines from the 'W202Ax Texas Grano 438' onion population exhibited significantly higher sucrose levels prior to bulbing than low fructan (frcfrc) lines. Sucrose synthase (SuSy) activity in these lines was correlated with leaf hexose content but not with Frc phenotype. Markers for additional candidate genes for sucrose metabolism were obtained by cloning a major SPS expressed in onion leaf and exhaustively mining onion expressed sequence tag resources. SPS and SuSy loci were assigned to chromosome 8 and 6, respectively, using AALs and linkage mapping. Further loci were assigned, using AALs, to chromosomes 1 (sucrose phosphate phosphatase), 2 (SuSy and three invertases) and 8 (neutral invertase). The concordance between chromosome 8 localization of SPS and elevated leaf sucrose levels conditioned by high fructan alleles at the Frc locus in bulb onion or alien monosomic additions of chromosome 8 in A. fistulosum suggest that the Frc locus may condition variation in SPS activity.