Hybridization in Actinomycetes]

Ways of transferring genetic information in Actinomycetes and their use for the transmission of genetic material in intervarietal crosses are discussed. The producing of hybrids is shown to be an efficient method to obtain Actinomycetes strains with new properties. Properties of recombinants obtained in intervarietal crosses Streptomyces coelicolor A3(2)XS. lividans 66 are shown to possess new antibiotic properties differing from those of parental strains: they suppress a number of Actinomycetes and bacterial strains which are not affected by parental strains. It is found that at least two groups of genes located on chromosomes of S. coelicolor A3(2) and S. lividans 66 participate in the synthesis of the antibiotic. The obtaining of recombinants between S. coelicolor A3(2) and S. griseus 15 makes possible to select variants which are capable to produce the antibiotic grisine and are resistant to actinophages, specifically attacking S. griseus. Properties of recombinants between S. coelicolor A3(2) and S. griseus 15 make possible to localize on parental chromosomes regions containing genes which control the synthesis of the antibiotic, the formation of a receptor for the adsorption of actinophages, and genes controlling the restriction and t he modification of actinophage development in S. coelicolor and S. griseus. A sex plasmid (SGP1) is found in S. griseus 15.     

Construction and properties of hybrids obtained in interspecific crosses between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15.

Recombinants between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15 were obtained using methods of hybrid construction. Recombinant Rcg1, obtained from a cross between S. griseus and a S. coelicolor UF (SCPI-) strain, phenotypically resembled S. coelicolor UF strains and in crosses with a S. coelicolor NF donor strin produced recombinatn progeny at a frequency of 100%. Recominant Rcg3, like SCP1-carrying S. coelicolor strains, inhibited SCP1-strains of S. coelicolor and in crosses with a UF recipient strain of S. coelicolor generated recombinants at high frequency. In crosses between S. griseus and Rcgi the frequency of recombinant formation was increased about 100-fold relative to crosses between S. griseus and S. coelicolor. Effective transfer of S. grieseus and Rcg3 chromosomal markers into Rcg1 and S. coelicolor, respectively, indicated that S. griseus had donor properties. Studies of the ability of recombinants to support phage growth indicated that parental chromosomal fragments containing genes involved in control of phage-receptor formation and intracellular growth were present in the hybrids. Grisin-producing recombinants, capable of restricting phages attacking S. coelicolor and S. griseus, were obtained.     

Molecular evolution of the Escherichia coli chromosome. V. Recombination patterns among strains of diverse origin.

Incorporation patterns of donor DNA into recipient chromosomes following transduction or conjugation have been studied in the progeny of a variety of Escherichia coli crosses in which donor and recipient nucleotide sequences differ by 1-3%. Series of contiguous or variously spaced PCR fragments have been amplified from each recombinant chromosome and digested with a commercial restriction endonuclease previously shown to distinguish the respective parents in a given fragment. We conclude that entering donor DNA fragments are frequently abridged (cut and shortened) before incorporation, the cutting being due to restriction systems, and the shortening presumably due to exonuclease activity. Analysis of several backcrosses confirms, and extends to conjugation, the importance of restriction in E. coli recombination in nature. The transmission patterns in conjugation are similar to those of transduction, but (as expected) on a much larger scale. Asymmetric results of reciprocal crosses imply that mismatch frequency is not a major factor. Marked differences among the results of simple crosses according to parental strain combinations are consistent with observations that E. coli strains in nature vary dramatically in their restriction-modification systems.     

Genetic recombination in auxotrophic strains ofPhanerochaete chrysosporium

Four auxotrophic strains of the ligninolytio basidiomycetePhanerochaete chrysosporium were obtained by UV mutagenesis. The heterokaryotic mycelium formed by complementation of different auxotrophic isolates was able to fruit and produce basidiospores. From the hasidiospore progeny of the heterokaryons prototrophic strains and strains with a recombined set of parental nutritional requirements were isolated. Genetic recombination hence takes place in fruit bodies produced by the heterokaryotic mycelium.     

Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids

Chromosome association and chiasma formation were studied in pollen mother cells at metaphase I of four allotriplod BC1 plants (2n=3x=24) obtained from the backcross of the hybrid Alstroemeria aurea x A. inodora with its parent A. inodora. We distinguished the chromosomes of both parental species by genomic in situ hybridization (GISH), whereas the individual chromosomes were identified on the basis of their multicolour FISH banding patterns obtained after a second hybridization with two species-specific satellite repeats as probes. All the four BC1 plants possessed two genomes of A. inodora and one of A. aurea. Variable numbers of recombinant chromosomes, resulting from meiotic recombination in the interspecific hybrid, were present in these plants. The homologous A. inodora chromosomes generally formed bivalents, leaving the homoeologous A. aurea chromosomes unassociated. High frequencies of trivalents were observed for the chromosome sets that contained recombinant chromosomes, even when the recombinant segments were small. Chromosome associations in the trivalents were restricted to homologous segments. The implications of the absence of homoeologous chromosome pairing on gamete constitution and prospects for introgression in Alstroemeria are discussed.     

Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.     

Mechanism of conjugation and recombination in bacteria. XX, Recombination DNA synthesis during mating in Escherichia coli K-12.

In crosses of dnaA recipients with HfrH at restrictive temperature a functional recombinant structure and viable recombinants were formed at a nearly normal rate, whereas recombination in crosses with HfrC was markedly inhibited. Recombinational DNA synthesis, as determined by 3H-thymine incorporation, was found in dnaA merozygotes from HfrH crosses. The recombinational synthesis was completed shortly after the end of mating. The replication of recombinant chromosomes in dnaA Lac+ recombinants started 120 minutes after interruption of mating, and at the restrictive temperature was sensitive to acridine orange, thus pointing to the presence of integrated F factor in the recombinant chromosome.     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

Excretion of alpha-keto acids by strains of Streptomyces venezuelae

Cultures of Streptomyces venezuelae released acidic metabolites during nitrogen-limited growth on glucose. The main products were pyruvic acid and alpha-ketoglutaric acid. Variation in the extent of acid production was observed; spores of the parental strain 13s gave approximately 10% of low-producing colonies when plated on acid-base indicator medium. Examination of one low producer, strain PC 51-5, showed that differences in acid production became apparent only in low-glucose media containing manganese. In both strains PC 51-5 and 13s, uptake of alpha-keto-[5-14C]glutaric acid occurred by diffusion and no marked differences in permeability to alpha-ketoglutarate were detected. However, differences were observed in the activity of alpha-ketoglutarate dehydrogenase. In cultures of strain PC 51-5, the specific activity of the enzyme increased throughout growth, whereas in the parental strain activity decreased and could not be detected in older mycelium. Loss of enzyme activity was accompanied by excretion of alpha-ketoglutaric acid and failure to assimilate the product after glucose exhaustion. The results suggest that accumulation of pyruvic and alpha-ketoglutaric acids in S. venezuelae cultures grown in glucose-containing media may be due to regulatory suppression of the dehydrogenases by this carbon source.     

Integration of plasmid RP1 with the chromosome of Escherichia coli K-12 recA. 2 classes of Hfr strains

Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied. Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains. Properties of these Hfrs have been examined. From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1. The other class of Hfr strains is formed due to a stable integration of RP1. In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E. coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids). On the basis of the latter results, a new simple method for R' plasmids selection has been proposed. Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated. Probable mechanisms of the stable Hfr strains formation are discussed.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.     

Transposition of mobile dispersed genes (MDG) after substitution of various chromosome pairs in inbred Drosophila melanogaster strains

Transpositions of MDG-1, MDG-3 and copia were detected as a result of crosses of the inbred maladaptive LA stock with laboratory stocks, in order to construct the genomes carrying different combinations of the LA or non-La chromosomal pairs. Changes of the mobile gene distributions were revealed in chromosomes of hybrid genotypes, as compared to parental chromosomal pairs. A trivial source of variability of chromosomal molecular structure ensured by crossing over was excluded by inversions which serve as suppressors of crossing over in corresponding crosses. Multiple transpositions of mobile genes in definite chromosomal sites were detected in genotypes carrying chromosomal pair 2 originated from the LA stock. No such transpositions were observed, when the pair 2 was substituted by the chromosome 2 originated from the Swedish-b line or in control crosses, where the LA stock was not involved. Both LA chromosomes 2 and 3 were shown to be the targets of transpositions. Comparison of hot spot transposition sites of MDG-1, as a result of crosses, with the earlier described rare events of spontaneous transpositions in the LA stock, coupled with its fitness increase, revealed that the hot spot sites were shared in both series of experiments. The data obtained show that transpositions of mobile genetic elements may change the genetic and molecular structure of the chromosome involved in crosses, in spite of suppression of crossing over by inversions usually suggested as a tool for keeping chromosomal genetic structure intact.     

In vivo recombination between two strains of the genus Nucleopolyhedrovirus in its natural host, Spodoptera exigua.

The DNA restriction map for the enzymes BamHI, BglII, PstI, and XbaI of SeMNPV-US (Se-US), the best-studied Nucleopolyhedrovirus strain isolated from Spodoptera exigua in California, was used as a reference to construct that of SeMNPV-SP2 (Se-SP2), a closely related Spanish strain of the same virus. After coinfection of S. exigua with both the Se-US and Se-SP2 strains per os, a recombinant virus (SeMNPV-SUR1 [Se-SUR1]) was detected after one passage which quickly replaced the parental viruses. A physical map of Se-SUR1 DNA was constructed for BamHI, BglII, PstI, and XbaI and compared to that of the parental viruses, Se-US and Se-SP2. Se-SUR1 is the result of at least four crossover events between Se-US and Se-SP2 and not of selection of a minor variant in one of the parental viruses. Bioassays of the recombinant and parental strains against L2 beet armyworm larvae demonstrated that their 50% lethal dose values were not significantly different. The differences in median lethal time values are too small to explain the replacement of the parental viruses by the recombinant virus upon successive passage in vivo, although it cannot be ruled out as an explanation for the selective advantage of the recombinant strain, Se-SUR1. The consequences of the release of nonindigenous or recombinant baculovirus strains in agro-ecosystems are discussed.     

Genetic exchanges in the macrocysts of Dictyostelium discoideum.

Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2.5% of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10% of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal matA (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds--Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum--and are reminiscent of the results reported for germinated zygospores of Phycomyces blakesleeanus.     

Genetic analysis of the psychostimulant effects of nicotine in chromosome substitution strains and F2 crosses derived from A/J and C57BL/6J progenitors

Previous research utilizing the AcB/BcA recombinant congenic strains (RCS) of mice mapped provisional quantitative trait loci (QTLs) for the psychostimulant effects of nicotine to multiple regions on chromosomes 7, 11, 12, 14, 16, and 17. The current study was designed to confirm these QTLs in an A/J (A) × C57Bl/6J (B6) F2 cross and a panel of B6.A chromosome substitution strains (CSS). The panel of B6.A CSS consists of 21 strains, each carrying a different A/J chromosome on a B6 background. The A × B6 F2, CSS, A, and B6 mice were tested for sensitivity to the effects of nicotine on locomotor activity using a computerized open-field apparatus. In A × B6 F2 mice two QTLs were identified which confirm those previously observed in the AcB/BcA RCS. Significant differences in the expression of nicotine-induced activity were associated with loci on chromosome 11 (D11Mit62) and chromosome 16 (D16Mit131) in the A × B6 F2. At the chromosome 11 QTL, an A allele was associated with lower nicotine-induced activity scores relative to the B6. In contrast, the A allele was associated with greater relative nicotine activity values for the chromosome 16 QTL. A survey of the CSS panel confirmed the presence of QTLs for nicotine activation on chromosomes 2, 14, 16, and 17 previously identified in the AcB/BcA RCS. In the informative CSS strains, A alleles were consistently associated with greater nicotine-induced activity scores compared to the B6. The results of the present study are the first to validate QTLs for sensitivity to the effects of nicotine across multiple strains of mice. QTLs on chromosomes 2, 11, 14, 16, and 17 were confirmed in CSS and/or F2 mice. Significantly, the identification of a QTL on chromosome 16 has now been replicated in three crosses derived from the A and B6 progenitors.     

The methods of protoplast formation and transformation of Streptomyces strains

Factors influencing formation, regeneration and transformation of protoplasts in streptomyces are described. Conditions for formation and regeneration of protoplasts in 4 industrial strains producing the macrolide antibiotic tylosin were studied. It was demonstrated possible to apply the method for transformation of the S. lividans type culture to 3 industrial strains of S. griseus producing grisin, an antibiotic used as a feed additive. Potential increasing of the efficiency of protoplast transformation and transfection in various actinomycetous strains including industrial ones is discussed. The stimulating effect of lyposomes on transformation of protoplasts in S. lividans 66 with DNA of plasmids pVG101 and pIJ350 as well as transfection with DNA of phages SH10 and KS404 was shown. The tylosin resistance genes in S. fradiae strain B45 were cloned which enabled isolating the cluster of the genes participating in tylosin biosynthesis.     

Genetic and cytological characterisation of fusion chromosomes of Dictyostelium discoideum

Abnormally large chromosomes which appear to result from the fusion of 2 chromosomes of the normal karyotype have been found in diploids of Dictyostelium discoideum formed by parasexual fusion of haploid strains HU483 (n=7) and HU245 (n=7). These fusion chromosomes appear to be the products of the tandem translocation of most, if not all, of one acrocentric chromosome to the telomere of a second acrocentric. Thus the chromosome number of the diploids is reduced from the normal 2n=14 to 2n=13 with the formation of an abnormally large acrocentric fusion chromosome. Experimental haploidisation of such diploids results in two types of products, those with a normal 7 chromosome karyotype and those with an abnormal 6 chromosome karyotype which contains the fusion chromosome. Genetic analysis of haploid segregants indicates that linkage groups II and VII are involved in this fusion. Phenotypes of recombinant diploids obtained following mitotic crossing-over establishes that linkage group II is proximal to linkage group VII. Cytological examination of the karyotypes of haploid strains bearing the fusion chromosome suggest that chromosome 2 may correspond to linkage group II and chromosome 3 to linkage group VII. Haploid strains bearing the fusion chromosome grow and develop normally so little or no genetic information can have been lost in the fusion event. While the nature of this event is unknown it may have involved aberrant recombinational DNA repair since the parental haploid strain HU483 bears the radB13 DNA repair mutation.     

Synergistic gene interactions control the induction of the mitochondrial uncoupling protein (Ucp1) gene in white fat tissue

Among a selected group of mouse strains susceptible to dietary obesity, those with an enhanced capacity for Ucp1 and brown adipocyte induction in white fat preferentially lost body weight following adrenergic stimulation. Based on the generality of this mechanism for reducing obesity, a genetic analysis was initiated to identify genes that control brown adipocyte induction in white fat depots in mice. Quantitative trait locus (QTL) analysis was performed using the variations of retroperitoneal fat Ucp1 mRNA expression in progeny of genetic crosses between the A/J and C57BL/6J parental strains and selected AXB recombinant inbred strains. Three A/J-derived loci on chromosomes 2, 3, and 8 and one C57BL/6J locus on chromosome 19 were linked to Ucp1 induction in retroperitoneal fat. Although A/J-derived alleles seemed to contribute to elevated Ucp1 expression, the C57BL/6J allele on chromosome 19 increased Ucp1 mRNA to levels higher than parental values. Thus, novel patterns of C57BL/6J and A/J recombinant genotypes among the four mapped loci resulted in a transgressive variation of Ucp1 phenotypes. Although the extent of the interchromosomal interactions have not been fully explored, strong synergistic interactions occur between a C57BL/6J allele on chromosome 19 and an A/J allele on chromosome 8. In addition to selective synergistic interactions between loci, variations in recessive and dominant effects also contribute to the final levels of Ucp1 expression.     

Major genes control hormone-induced ovulation rate in mice

The present study examined the magnitude of genetic variation, mode of inheritance and number of loci controlling major genetic differences in hormone-induced ovulation rate in mice. Mice were injected with 5 i.u. PMSG at 28 days of age and 5 i.u. hCG at 30 days, and hormone-induced ovulation rate was determined from counts of oviducal eggs in cumulus the next morning. Six-fold genetic differences in induced ovulation rate were detected amongst strains, ranging from a low mean (+/- s.e.) value of 8.8 (+/- 0.9) in A/J up to 53.5 (+/- 2.2) in C57BL/6J. The number of ova differed significantly amongst strains and amongst F1 crosses (P less than 0.0001): 70% of the variation in hormone-induced ovulation rate was amongst strains. Of 9 F1 crosses examined, 4 showed positive heterosis, 3 showed no heterosis and 2 showed negative heterosis for hormone-induced ovulation rate. Analysis of parental, F1 and F2 generations revealed that the induced ovulation rate of A/J and C57BL/6J mice differed due to the action of about 3 or 4 loci, and A.SW/SnJ and SJL/J mice differed due to the action of about 2 to 3 loci. Analysis of recombinant inbred strains formed from A/J and C57BL/6J confirmed that these strains differed due to the action of a small number of loci. This study demonstrates the existence of a small number of major genes controlling hormone-induced ovulation rate in young mice.