Studies on the production of daunomycinonederived glycosides and related metabolites inStreptomyces coeruleorubidus andStreptomyces peucetius

Strains ofStreptomyces coeruleorubiduě ISP 5145,JA 10092 and 39–146, differing mutually in antibiotic activity, were found to produce identical spectrum of metabolites (at least nine antibiotically active glycosides, 13-dihydrodaunomycinone, ε-rhodomycinone and a larger number of unidentified compounds); only trace quantities of daunomycin and daunomycinone could be detected. A fraction of glycosides with a higher R_f (0.4–0.7), isolated from strain 39–146, could be transformed to daunomycin by mild hydrolysis and to daunomycinone by total hydrolysis.Streptomyces peucetius IMI 101 335 differed fromStreptomyces coeruleorubidus in an increased production of ε-rhodomycinone and a lower content of glycosides; the zone of daunomycin was most pronounced among the glycoside spots.Streptomyces coeruleorubidus 39-146 exhibited the highest activity in a medium containing 3.5% soluble starch, 3.0% soybean meal, 0.3% NaCl and 0.3% CaCo₃; glucose was a more useful carbon source for the remaining strains The activity ofStreptomyces coeruleorubidus was inhibited by 1-propanol, Na-propionate,5,5-diethylbarbiturate and bacitracin. Ferrous sulphate stimulated the production of glycosides only in strain JA 10092, decreasing simultaneously the production of aglycones.     

Site-specific recombination for genetic engineering in plants

Site-specific recombination has been developed into a genetic engineering tool for higher eukaryotes. The manipulation of newly introduced DNA is now possible in the course of genetic transformation procedures, thus making the process more predictable and reliable. Also, a wide variety of chromosomal rearrangements using site-specific recombination have been documented both in metazoan and plant species. Applying such methods to plants opens new avenues for large-scale chromosome engineering in the future.     

Evidence for protein phosphorylation inStreptomyces lincolnensis

Protein phosphorylation was investigated inStreptomyces lincolnensis underin vivo conditions. In cells grown in the presence of³²P-orthophosphate, proteins ofM=12, 22, 45, 68 and 90 kDa were labeled with³²P (detected by gel electrophoresis and autoradiography). These proteins were shown to contain O-phosphoserine and a small proportion of O-phosphotyrosine. Taken together the results indicate thatStreptomyces lincolnensis harbors several protein kinases including a protein-tyrosine kinase activity.     

GARD: a genetic algorithm for recombination detection

MOTIVATION: Phylogenetic and evolutionary inference can be severely misled if recombination is not accounted for, hence screening for it should be an essential component of nearly every comparative study. The evolution of recombinant sequences can not be properly explained by a single phylogenetic tree, but several phylogenies may be used to correctly model the evolution of non-recombinant fragments. RESULTS: We developed a likelihood-based model selection procedure that uses a genetic algorithm to search multiple sequence alignments for evidence of recombination breakpoints and identify putative recombinant sequences. GARD is an extensible and intuitive method that can be run efficiently in parallel. Extensive simulation studies show that the method nearly always outperforms other available tools, both in terms of power and accuracy and that the use of GARD to screen sequences for recombination ensures good statistical properties for methods aimed at detecting positive selection. AVAILABILITY: Freely available http://www.datamonkey.org/GARD/     

Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination

Bacteriophage P1 encodes its own site-specific recombination system consisting of a site at which recombination takes place called loxP and a recombinase called Cre. A number of lambda and plasmid substrates containing two loxP sites have been constructed. Using these substrates we have shown both in vivo and in vitro that a fully functional loxP site is composed of no more than 60 bp. In vitro, when an extract containing Cre is used, recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs efficiently. The most surprising result from the in vitro studies is that 50% of the products of recombination between loxP sites on a supercoiled DNA substrate are present as free supercoiled circles. The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule.     

Evidence for phosphoprotein phosphatase inStreptomyces granaticolor

The existence of phosphrotein phosphatase (PPP) in aerial mycelium ofS. granaticolor was demonstrated. Using inhibitors of serine and/or threonine PPP and specifically labeled substrate it was found that the PPP is of the serine and/or threonine type.     

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.     

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.     

Mitotic recombination counteracts the benefits of genetic segregation

The ubiquity of sexual reproduction despite its cost has lead to an extensive body of research on the evolution and maintenance of sexual reproduction. Previous work has suggested that sexual reproduction can substantially speed up the rate of adaptation in diploid populations, because sexual populations are able to produce the fittest homozygous genotype by segregation and mating of heterozygous individuals. In contrast, asexual populations must wait for two rare mutational events, one producing a heterozygous carrier and the second converting a heterozygous to a homozygous carrier, before a beneficial mutation can become fixed. By avoiding this additional waiting time, it was shown that the benefits of segregation could overcome a twofold cost of sex. This previous result ignores mitotic recombination (MR), however. Here, we show that MR significantly hastens the spread of beneficial mutations in asexual populations. Indeed, given empirical data on MR, we find that adaptation in asexual populations proceeds as fast as that in sexual populations, especially when beneficial alleles are partially recessive. We conclude that asexual populations can gain most of the benefit of segregation through MR while avoiding the costs associated with sexual reproduction.     

The genetic system controlling recombination in the silkworm

The nature of recombination modifiers was investigated in Bombyx mori lines selected for high (H) and low (L) recombination rates between the p^S and Y loci in chromosome 2. Since the mean recombination rates for the H x L and L x H F₁ crosses were approximately intermediate between those of high and low lines, the cytoplasmic maternal effect and difference in the activity of recombination modifiers between marked and unmarked second chromosomes were not detected. The H x (L x H), H x (H x L), L x (L x H) and L x (H x L) backcrosses indicated the presence of additive and dominance effects of marked and unmarked second chromosomes and the remaining chromosomes.——Recombination rates between the p^S and Y loci in chromosome 2 and half-nonrecombination rates between the pe and re loci in chromosome 5 of high and low lines indicated that these recombination modifiers caused changes in the recombination frequency between p^S and Y in chromosome 2, but not between pe and re in chromosome 5.——There were no differences in viability between individuals having the second chromosomes of the recombinant types [p^S +, p^Y (H); p^S +, + Y (L)] and those of the nonrecombinant types [p^S Y, p + (H); p^S Y, + + (L)] in both high and low lines. Mean recombination rates measured in cis [p^S Y/p + (H); p^S Y/+ + (L)] and trans [p^S +/p Y (H); p^S +/+ Y (L)] males were the same in the high but not in the low line. No segregation of a single recombination modifier was indicated by the distribution of recombination rates measured in trans males [p^S +/p Y (H); p^S +/+ Y (L)] of high and low lines. Accordingly, the recombination modifiers distributed on chromosome 2 in the heterozygous condition were not gross chromosomal aberrations, but polygenic factors in the low line.     

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.     

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.