Genetic instability in Drosophila melanogaster

Summary An unstable long tandem duplication which includes the white locus twice, marked with w ^sp in the left and w ^17G in the right locus, when kept in males has been found to produce red-eyed sons which have lost the long duplication and with it the w ^sp and w ^17G mutants. Such exceptions were produced also when w ^17G had been exchanged for w ^a.Stocks originating from these exceptions are unstable, producing: 1) zeste males, also unstable, 2) w ^- deletions, stable, 3) transpositions of the white locus to sites in other chromosomes.The instability is interpreted as the effect of an IS element, within or adjacent to the white locus, which is supposed to retain a duplication of the proximal zeste interacting part of this locus. According to the orientation of the IS element the duplicated part can be active or inactive, giving a zeste or red eye phenotype.The frequency of exceptional offspring after X-ray treatment of the red and zeste unstable stocks have been compared to stable stocks with corresponding genotypes.     

An analysis of white-mottled mutants inDrosophila hydei,with observations on X-Y exchanges in the male

Chromosomes and phenotypes of four different sex-linkedwhite-mottled mutants of the position-effect variogation type were studied. Three mutants (w ^m1,w ^m2,w ^m3) are X-chromosomal rearrangements which shift the w⁺ locus into a position close to heterochromatin, but which have different ouchromatic and heterochromatic breaks. The fourth, a spontaneous derivative ofw ^m1, is an insertional duplication of part of the X chromosome, including thew ⁺ andN ⁺loci. The duplicated segment is inserted into the distal part of the long arm of the heterochromatic Y chromosome. It is designated,w ^m CoY, orXw ^m Co when transferred to the X chromosome.Three chromosomal types (w ^m1,w ^m CoY) and (Xw ^m Co) having the same cuchromatic break near thew ⁺ locus, cause large-spotted eyes whereas two others (w ^m2,w ^m3) produce a popper-and-salt type of mottling. From the position of the various eu- and heterochromatic breaks, it appears that the distance of thew ⁺ locus to the point of reunion with heterochromatin, rather than the amount or type of adjoining heterochromatin, dietates the phenotypic action of the displacedw ⁺ locus, in the sense of a spreading effect on two proposed functional subunits within thew ⁺ locus.The pigmentation background against which the mottling effect is produced, i.e., a givenw-allele with its characteristic colour, or other eye colour mutations, does not seem to affect the type of mottling. Drosopterins and ommochromes react in the same way to modifing factors like temperature and supernumerary Y chromosomes. Two mutants (w ^m2 andw ^m CoY) while reacting in the same manner to Y chromosomes showed an opposite temperature response.By exchange between the heterochromatin of the Y and X chromosome inw/w ^m CoY males thew ^m Co duplication was transferred between the sex chromosomes with a certain regularity. It is not yet known wether the exchanges are mitotic or meiotic in origin but their heterochromatic nature has been demonstrated cytologically.     

Mutations affectingβ-alanine metabolism influence inducibility of the 93D puff by heat shock inDrosophila melanogaster

Effect of mutations at the ebony or black locus on induction of heat shock puffs in polytene nuclei of salivary glands ofDrosophila melanogaster larvae were examined by [³H]uridine autoradiography. The levels of-alanine in the body are known to be increased by mutation at the ebony locus but decreased by mutation at the black locus. The presence of mutant allele/s at either locus in the homo- or heterozygous condition prevented induction of the 93D puff by heat shock. Elimination of the mutant allele at the ebony or black locus by recombination or by reversion of a P element insertion mutant allele of ebony restored the heat shock inducibility of the 93D puff. In vivo or in vitro administration of excess-alanine to salivary glands of wild-type larvae also resulted in the 93D site being refractory to heat shock induction. In agreement with earlier results, noninduction of the 93D puff during heat shock due to the-alanine effect was accompanied by unequal puffing of the 87A and 87C loci. The selective inducibility of the 93D puff by benzamide was not affected by ebony or black mutations or by excess-alanine in wild-type larvae     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Electron microscopic analysis of the yeast mitochondrial DNA segment conferring chloramphenicol resistance

Summary Mitochondrial DNAs from six ^- mutants carrying the genetic locus Ribl and deleted for the rest of the genome were analyzed. Distribution of circular molecules from one mutant followed exactly the frequency rule, 1/n, for multimers with discreet classes n, 2n, 3n, etc. Another, genetically unstable mutant displayed a continuous spectrum of circular molecules of various lengths. Four other mutants contained multiple series of circular molecules. Partial denaturation maps show that the mutants analyzed show a common segment ca. 1.0 m long and differ by characteristic deletions of extremites of this segment. Short terminal deletions of the right i.e. pointing towards the Rib3 locus, terminus of this segment are correlated with modifications of the recombination properties related to the locus.     

Study of RecA-Independent Homologous Recombination and a Chromosomal Rearrangement in the Escherichia coli Strain Carrying an Extended Heterozygous Tandem Duplication

A heterozygous tandem duplication in the Escherichia coli deo operon region deoAdeoB::Tn5/ deoCdeoDthr::Tn9 with the total length approximately 150 kb, which was obtained in the conjugational mating in the HfrH strain, was examined. By means of digestion with the NotI enzyme, pulsed-field gel electrophoresis, and the conjugational transfer of the duplication in the F^– strain, the chromosomal rearrangement, which occurred in the duplication region upon its stabilization in the bacterial genome, was studied. In a more stable strain, two new NotI sites were shown to appear in the chromosomal region located close to the duplication, which might have resulted from the transposition of the IS50 sequence from Tn5. The data were also obtained indicating the possibility of secondary transposition of the chromosomal segment between the two new NotI sites (approximately 30 kb) in the region located near the duplication. With the use of rec ⁺ and recA strains, two types of haploid and diploid segregants generated by the duplication were studied: DeoD⁺ (the deoD⁺ allele is not expressed in the original duplication due to the polar effect of the deoB::Tn5 insertion) and DeoC DeoD. The segregation of DeoD⁺ clones was shown to be RecA-dependent, whereas the DeoC DeoD segregants selected on the medium that contained thymine at a low concentration (i.e., under conditions of thymine starvation) appeared at a rather high frequency. However, the relative frequency of haploid clones, which have lost the duplication, strongly decreased in the recA genome among segregants of both types.     

A mutable white-inversion in Drosophila melanogaster

Summary From a zeste mutant stock with a mutable white locus a new mutant (z w ^w ) was isolated. It has a white-eyed phenotype and a short X-chromosome inversion (In(1)w ^w ) which extends from salivary chromosome bands 3B2-C1 to 4B4-C1. In giant chromosomes of heterozygotes the inversion is unusually tightly paired. Probably because of this intimate pairing the recombination frequencies for regions near the inversion are not decreased in comparison to those for structurally normal chromosomes. The inversion chromosome is mutable. The mutations which arise have pigmented eyes and can be subdivided into two groups. One group is characterized by a re-inversion to normal chromosome structure. The mutability of the white locus appears to be independent of the inversion and reinversion. The process of reinversion is discussed.     

Studies on the size of the diploid region inBacillus subtilis merozygotes from strains carrying thetrpE26 mutation

Summary Simultaneous selection of transformants fortrpE26 and a second unlinked marker ofB. subtilis in many cases yields double heterogenotic clones. Several chromosome areas analyzed in this way found to be involved in the diploid condition. Diploids for areas on the left hand side oftrpE26 on the map (and as near as thearo B locus) are in general unstable while stable merodiploids can be obtained for areas on the right hand side of this marker (as far as theilvA locus). Merozygotes for regions other than the aromatic segment are also formed by transformation of already diploid (stable and unstable) clones. Stable diploids give rise to new heterogenotes only for markers on the right hand side oftrpE26. Through reversion of untransformed markers in unstable and stable diploids it was found that these clones are homodiploid for loci situated at a long distance from (or between) the areas which were involved in the transformation. This indicates that the diploid state covers a continuous segment of the chromosome, the length of which can be determined. The segregation pattern of unstable multiple merodiploids suggests that exchange of genetic material must take place between the two homologous regions. The data presented are in agreement with the hypothesis that the merodiploids possess a very long duplication on their chromosome. In the case of the stable clones this duplication is shorter.     

Activin A and transforming growth factor-β stimulate heart formation in axolotls but do not rescue cardiac lethal mutants

In the Mexican axolotl (salamander), Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts lack organized sarcomeric myofibrils and do not contract throughout their lengths. We have previously shown that RNA purified from normal anterior endoderm or from juvenile heart tissue is able to rescue mutant embryonic hearts in an in vitro organ culture system. Under these conditions as many as 55% of formerly quiescent mutant hearts initiate regular contractions within 48 hours. After earlier reports that transforming growth factor-1 and, to a lesser extent, platelet-derived growth factor-BB could substitute for anterior endoderm as a promoter of cardiac mesodermal differentiation in normal axolotl embryos, we decided to examine the effect of growth factors in the cardiac mutant axolotl system. In one type of experiment, stage 35 mutant hearts were incubated in activin A, transforming growth factors-1 or 2, platelet-derived growth factor, or epidermal growth factor, but no rescue of mutant hearts was achieved. Considering the possibility that growth factors would only be effective at earlier stages of development, we tested transforming growth factors-1 and 5, and activin A on normal and mutant precardiac mesoderm explanted in the absence of endoderm at neurula stage 14. We found that, although these growth factors stimulated heart tube formation in both normal and mutant mesodermal explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor- superfamily peptides initiate a cascade of responses in mesoderm that result in both changes in cell shape (the basis for heart morphogenesis) and terminal myocardial cytodifferentiation. The cardiac lethal mutation appears to be deficient only in the latter process.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.F.J. Mangiacapra and M.E. Fransen contributed equally to this work     

Investigations on the organization of genetic loci in Drosophila melanogaster: lethal mutations affecting 6-phosphogluconate dehydrogenase and their suppression.

Summary The molecular nature of lethal and semilethal mutations in the Pgd locus of D. melanogaster coding for 6-phosphogluconate dehydrogenase (6PGD) was studied. All the 11 mutations affect the structural gene of the Pgd locus: 3 semilethal mutations resulted in altered 6PGD molecules with decreased catalytic activities; the rest 8 lethals were null alleles characterized by mutant polypeptides capable of reacting with antisera against highly purified 6PGD.Null or low activity alleles for glucose-6-phosphate dehydrogenase induced by ethyl methanesulfonate were shown to be suppressors for the lethal mutations in the Pgd locus.A monocistronic type of organization of the Pgd locus is suggested taking into account the biochemical mechanism of suppression of the Pgd-lethals and their location in the structural gene coding for 6PGD.     

Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12.

Summary A ColE1 hybrid plasmid, pNU1, carrying the amp operon coding for chromsomal -lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning.By reciprocal recombination between pNU1 and chromosome of two unstable -lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the -lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.     

Rearrangements at the mating type locus in fission yeast

Summary Crosses involving the partially defective mating type mutant B102 (functional in conjugation, defective in meiosis) have confirmed the notion that, in Schizosaccharomyces pombe, certain mating type mutations can arise by transposition. A copy of the mat2 ^P segment (specifying + mating type) is transposed and inserted into the mat1 ^M segment (usually specifying mating type). The mat1 ^M segment affected by the insertion loses its former function entirely. The function is, however, fully regained upon excision of the transposed and inserted mat2 ^P segment.At either position, the mat2 ^P segments can undergo inactivations to different states of residual activity. These events can occur about as frequent as other mutations of the mating type locus (ca. 10^–4 per cell division). In certain diploid strains, such inactivations were significantly correlated with recombination. Spontaneous reversions to full activity were also observed.     

Evolution of incompatibility systems in plants: Complementarity and the mating locus in flowering plants and fungi

Summary The restriction of sexual pairing by a specificity gene is considered to be an ancient development in the plant kingdom. The diversity and general parallelism of incompatibility systems seen amongst the phyla at the present time can be rationalized in terms of the association of various derived forms of the ancestral specificity unit with differing spectra of accessory factors controlling sexual physiology in the different phyla. Sexual morphogenesis has become divided into distinct phases under the control of complementary genes. These phases are initiated by a regulatory system of Co-ordinator genes which control the order in which groups of morphogenetic genes are expressed during development. The entire sexual cycle will be completed only if all the complementary groups are activated in the appropriate sequence. The present article discusses essential features of the evolution of the breeding locus in different phyla. These features are consistent in themselves with the present data and are not dependent on the proposed ancient origin of the specificity gene.The above hypothesis throws light on the (1) evolution of the complex mating loci in flowering plants and fungi; (2) evolution of complementary incompatibility and heteromorphic incompatibility in flowering plants; (3) anomalous cross-compatibility behaviour of mutants in the fungus Schizophyllum commune; (4) nature of homothallism in higher fungi; (5) mode of origin of new functional self-incompatibility alleles; and (6) homogenic and heterogenic incompatibility.     

A preliminary genetic analysis of TE146, a very large transposing element of Drosophila melanogaster

TE146 is a transposing element (TE) consisting of six polytene chromosome bands that has inserted into the no-ocelli (noc 250) locus. This member of Ising's TE family carries two copies of the white and roughest loci. TE146 is lost from noc with a spontaneous frequency of approximately 1 in 22000 chromosomes. All spontaneous losses are accompanied by the reversion of the noc mutation associated with the TE. The TE is associated with fold-back (FB) sequences. The losses of TE146 retain fold-back homology at noc. Of 26 -ray-induced losses of TE146, 16 are gross deletions, removing loci neighboring noc and ten are not. The non-deleted -ray-induced losses are either noc ^– and rst ⁺ or noc ⁺ and rst ^–. The white⁺ genes of TE146 are dosage compensated since w/Y; TE146/+ and w/w; TE146/+ flies are sexually dimorphic for eye color. These w ⁺ genes are also suppressed by zeste since z w; TE146/+ flies have zeste-colored eyes.     

Study of Homologous Recombination and Chromosomal Rearrangements inEscherichia coliStrains Carrying a Heterozygous Tandem Duplication

Heterozygous tandem duplications formed in conjugational matings in Escherichia coliprovides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deooperon of E. colicarrying deoA deoB::Tn5/deoC deoD thr::Tn9alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3and recF::Tn3mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ(or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of adaptive recombination.     

Cannabis 25 1, biotransformation of cannabidiol and cannabidiolic acid by Pinellia ternata tissue segments

Two marihuana constituents, cannabidiol (1) and cannabidiolic acid (4) were each converted mainly to two metabolites using tissue segments of Pinellia ternata tuber. The structures of the metabolites formed from 1 were determined to be 1-O-D-glucopyranoside and 1-O-D-diglucopyranoside by ¹H nmr, ¹³C nmr and fabms. Those from 4 were determined as 4-O-D-glucopyranoside and 10-hydroxyl 4-O-D-glucopyranoside. In time course experiments, 1 was absorbed rapidly by the tissues and glucosylated. Hydroxylation subsequent to the glucosylation occurred at the pentyl group in 4.1 For Part 24, see Taura F, Morimoto S, Shoyama Y, Mechoulam R (1995) J Am Chem Soc 117:9766–9767