Occurrence in wild strains of Neurospora crassa of genes controlling genetic recombination.

Each of the main laboratory wild stocks of N. crassa carries one of two alleles at the rec-1 and rec-2 loci and one of three at the rec-3 locus. The constitutions of the stocks are given in Fig. 1. Some of those conserved are evidently not the originals. The third rec-3 gene (rec-3L), found in Lindegren A, controls recombination at the am-1 locus to a level between that of rec-3+ and rec-3, the relative levels being 1 : 8 : 25. At the his-2 locus rec-3L is indistinguishable from rec-3+ in its level of control. This proves that there are minor differences between the control (con) genes, near to am-1 and his-2, which recognize products of rec-3 genes. Further, this is the first clear evidence, though indirect, that the binding sites for products of rec genes are situated in the chromosome regions where recombination is modulated.     

The nature of genetic recombination

The biological evolutionary axiom proposed earlier by the author states that in the absence of genetic recombination the evolution of organic forms would be impossible. In the present paper the literature data are considered, illustrating the role of genetic recombination in evolution. It is urged that a tendency towards an increasing complexity of biological organization results from periodical recombinational combining of the diverged genes as well as the whole genomes of different origin. The alternative mechanism implying the production of duplications from the identical gene copies or whole genomes is considered to be unlikely. According to the biological evolutionary axiom, the origin of life is connected with the appearance of a mode of reparation of crystalline type aggregates--the precursors of DNA by means of exchanges among their constituents. A hypothesis is proposed that in the process of recombination a certain distribution of the 6-amino bases (adenine, cytosine) along the DNA molecule is settled, with respect to the 6-carbonyl bases (guanine, thymine). It is proposed that the relative distribution of the bases mentioned influences electrostatic stability of the DNA molecule as a crystalline associate.     

Frequency of recombination exchanges in Escherichia coli K-12: genetic determinants controlling this frequency

RecF, recQ, ruv, recJ and recN genes of so called RecF pathway of recombination appear to be not silent on the RecBCD pathway also. These genes are responsible for the frequency of recombination exchanges per unit length of DNA. The list: recF::Kmr greater than recQ::Tn3 greater than ruv54 greater than recJ::Tn9 demonstrated the efficiency of inhibition of recombination exchanges by these mutations. The recN262 mutation gives a feeble contrary effect. It slightly increases the frequency of recombination exchanges.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Utility of the FLP-FRT recombination system for genetic manipulation of rice

To develop an FLP-FRT recombination system- (derived from 2 mu plasmid of Saccharomyces cerevisiae) based marker gene removal application for rice, we introduced the gene for FLP recombinase, under the control of the maize ubiquitin-1 promoter, into the rice genome. FLP activity was monitored in callus and regenerated plants by an assay based on the deletion of the FRT-flanked DNA fragment, leading to the activation of the beta-glucuronidase gene. FLP activity was detected both in the callus and leaves of some of the transgenic lines. Based on our comparison of the recombination efficiency of the FLP-FRT system expressed in the transgenic lines with that of the widely used Cre-lox system (derived from bacteriophage P1), we suggest that the FLP-FRT system is a useful tool for the genetic manipulation of rice.     

The genetic control of dark recombination in yeast

The post-UV effects of dark holding and photoreactivating light on survival and intragenic recombination in UV-sensitive strains of yeast have been studied. The results indicate that two liquid holding dark repair pathways exist in this organism: the excision pathway which does not lead to intragenic recombination (“silent” repair) and a dark recombination pathway which does lead to intragenic recombination. A third pathway also exists which is plating-dependent and leads to intragenic recombination via a postreplication pathway.     

Protein-DNA interactions in genetic recombination

The formation of DNA-protein complexes that are capable of interaction with other DNA molecules is necessary for efficient genetic recombination. How do such complexes form, and how are homologous DNA sequences brought into alignment? Physical and biochemical studies of recombination enzymes from bacteria indicate that these proteins provide the structural framework within which the genetic exchanges occur.     

The role of genes of the system of mismatched base correction in genetic recombination of Escherichia coli

A genetic system was elaborated to study intramolecular recombination of bacteriophages lambda and phi 80. Practically, the ideal complementation of nucleotide sequences in recombining DNA molecules is required to obtain recombinants resulting from RecA-dependent recombination in Escherichia coli cells. a hypothesis is proposed to which the correction of mismatched bases hinders recombinant formation during recombination of fully homologous DNAs. The increased yields of hybrid molecules during interaction of the same DNA in the cells with deficient genes for correction support the hypothesis as well as independent demonstration of mutation in a gene for correction according to the effect of the increased yield of recombinants. A series of Escherichia coli cells mutants with increased formation of recombinant clones has been obtained.     

X-ray-induced recombination during oogenesis in the silkworm (Bombyx mori L.)

Summary The physical induction of recombinational events has been studied in the female silkworm (Bombyx mori), in which crossing-over does not normally occur. Female silkworms heterozygous in the trans type of two egg-color genes,pe (V-0.0) andre (V-31.7), received a single dose of X-rays (1000 R) at various developmental stages. Then they were crossed to marked males homozygous for both genes. The results indicated that X-rays increase the occurrence of recombinational events in silkworm females from first instar larvae to late stage pupae. The spontaneous frequency of exchange type recombinants in the control series was 2.5 x 10^–5, while after irradiation the frequency of these recombinants was up to 38.8 x^10–5. The sensitive stage to X-ray-induced recombinational events was late stage larvae from fourth to fifth instar. Exchange (cross-over) type recombinants were about three times more frequent than interchange types among the 122 recombinants recovered. The biological significance of the present finding is discussed.     

A system for analysis of yeast mutants deficient in the genetic recombination process]

A system of molecular-genetic methods intended for analysis of yeast Rec-mutants was developed. A number of plasmids containing noncomplementing mutations in the ADE2 gene and different selective markers were constructed. The system was based upon the phenomenon of interplasmid intragenic recombination during transformation and mitotic division. Transformation of yeast cells with the plasmid containing two long right repeats allowed to estimate the efficiency of intraplasmid crossing-over. The system also allows to study plasmid-chromosomal interaction. To check up the system proposed, a well known rad50 and rad52 mutants were used. The rad52-1 mutation was found sharply decreased the levels both intra- and interplasmid recombination events. On the other hand, rad50-1 mutation has not influences or stimulates the processes. Thus, the system proposed efficiently distinguishes the mutants deficient in different stages of recombinational process.     

The influence of recombination on human genetic diversity

In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.     

RecA-DNA helical filaments in genetic recombination

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.     

The effect of recombination on the neutral evolution of genetic robustness

Conventional population genetics considers the evolution of a limited number of genotypes corresponding to phenotypes with different fitness. As model phenotypes, in particular RNA secondary structure, have become computationally tractable, however, it has become apparent that the context dependent effect of mutations and the many-to-one nature inherent in these genotype-phenotype maps can have fundamental evolutionary consequences. It has previously been demonstrated that populations of genotypes evolving on the neutral networks corresponding to all genotypes with the same secondary structure only through neutral mutations can evolve mutational robustness [E. van Nimwegen, J.P. Crutchfield, M. Huynen, Neutral evolution of mutational robustness, Proc. Natl. Acad. Sci. USA 96(17), 9716-9720 (1999)], by concentrating the population on regions of high neutrality. Introducing recombination we demonstrate, through numerically calculating the stationary distribution of an infinite population on ensembles of random neutral networks that mutational robustness is significantly enhanced and further that the magnitude of this enhancement is sensitive to details of the neutral network topology. Through the simulation of finite populations of genotypes evolving on random neutral networks and a scaled down microRNA neutral network, we show that even in finite populations recombination will still act to focus the population on regions of locally high neutrality.