Magnification of the Ribosomal Genes in Female DROSOPHILA MELANOGASTER

The genetically induced increase in the number of 18S + 28S ribosomal genes known as magnification has been reported to occur in male Drosophila but has not previously been observed in females. We now report that bobbed magnified (bbm) is recovered in progeny of female Drosophila carrying three different X bobbed (Xbb) chromosomes and the helper XYbb chromosome, which is a derivative of the Ybb- chromosome. Using different combinations of bb or bb+ X and Y chromosomes, we show that magnification in females requires both a deficiency in ribosomal genes and the presence of a Y chromosome: X/X females that are rDNA-deficient but do not carry a Y chromosome do not produce bbm; similarly, X/X/Y females that carry a Y chromosome but are not rDNA-deficient do not produce bbm. Bobbed magnified is only recovered from rDNA-deficient X/XY, X/X/Y or XX/Y females. We have also found that females carrying a ring Xbb chromosome together with the XYbb- chromosome do not produce bbm, indicating that ring X chromosomes are inhibited to magnify in females as in males. We postulate that the requirement for a Y chromosome is due to sequences on the Y chromosome that regulate or encode factor(s) required for magnification, or alternatively, affect pairing of the ribosomal genes.--These studies demonstrate that magnification is not limited to males but also occurs in females. Magnification in females is induced by rDNA-deficient conditions and the presence of a Y chromosome, and probably occurs by a mechanism similar to that in males.     

Autosomal Modifiers of the Bobbed Phenotype Are a Major Component of the Rdna Magnification Paradox in DROSOPHILA MELANOGASTER

rDNA magnification in Drosophila melanogaster is defined experimentally as the ability of bb/Ybb- males to produce exceptional progeny that are wild type with respect to rDNA associated phenotypes. Here, we show that some of these bobbed-plus progeny result not from genetic reversion at the bb locus but rather from variants at two or more autosomal loci that ameliorate the bobbed phenotype of rDNA deficient males in Drosophila. In doing so we resolve several aspects of a long-standing paradox concerning the phenomenon of rDNA magnification. This problem arose from the use of two genetic assays, which were presumed to be identical, but paradoxically, produced conflicting data on both the kinetics of reversion and the stability of magnified bb+ chromosomes. We resolve this problem by demonstrating that in one assay bobbed-plus progeny arise primarily by genetic reversion at the bobbed locus, whereas in the other assay bobbed-plus progeny arise both by reversion and by an epistatic effect of autosomal modifiers on the bobbed phenotype. We further show that such modifiers can facilitate the appearance of phenotypically bobbed-plus progeny even under conditions where genetic reversion is blocked by magnification defective mutants. Finally, we present a speculative model relating the action of these modifiers to the large increases in rDNA content observed in males undergoing magnification.     

One-Step and Stepwise Magnification of a BOBBED LETHAL Chromosome in DROSOPHILA MELANOGASTER

Bobbed lethal (bbl) chromosomes carry too few ribosomal genes for homozygous flies to be viable. Reversion of bbl chromosomes to bb or nearly bb+ occurs under magnifying conditions at a low frequency in a single generation. These reversions occur too rapidly to be accounted for by single unequal sister chromatid exchanges and seem unlikely to be due to multiple sister strand exchanges within a given cell lineage. Analysis of several one-step revertants indicates that they are X-Y recombinant chromosomes which probably arise from X-Y recombination at bb. The addition of ribosomal genes from the Y chromosome to the bbl chromosome explains the more rapid reversion of the bbl chromosome than is permitted by single events of unequal sister chromatid exchange. Analysis of stepwise bbl magnified chromosomes, which were selected over a period of 4-9 magnifying generations, shows ribosomal gene patterns that are closely similar to each other. Similarity in rDNA pattern among stepwise magnified products of the same parental chromosome is consistent with reversion by a mechanism of unequal sister strand exchange.     

The Effect of mei-41 on Rdna Redundancy in DROSOPHILA MELANOGASTER

The recombination and repair defective mutant, mei-41, exhibits three rather striking effects on the genetic properties and chromosomal stability of rDNA in Drosophila. First, mei-41 inhibits rDNA magnification. However, mei-9, another recombination and repair defective mutation has no similar effect. This indicates that magnification requires some, but not all, of the gene products necessary for meiotic exchange. Second, under magnifying conditions, mei-41 induces interchanges between the X rDNA and either arm of the Ybb- chromosome. These interchanges occur at high frequency and are independent of rDNA orientation. Third, in mei-41 bb+/Ybb+ males, bobbed mutants in the X, but not the Y, also arise at high frequency. Evidence suggests that these events involve the rDNA type I insertion. The recombination and repair defective properties of mei-41 together with our results regarding its unusual and specific effects involving rDNA are explained in a simple model that has general implications for chromosome structure.     

Transposition of the bobbed locus in Drosophila melanogaster

Due to the complete absence of ribosomal DNA (genetic symbol bb-), the Xbb- chromosome of Drosophila is lethal both in homozygous conditions and in compound with the Xbb- chromosome. However, in the cross between the C(1)RM/Ybb- females and the Xbb-/BSYbb+ males, characterized by the development of lethal Xbb-/Ybb- zygotes, two fertile males were detected. These males possessed all the markers of the Xbb- chromosome but lacked the Y chromosome BS marker. Genetic analysis of their progeny showed that genes responsible for restoration of viability and fertility of these exceptional males were associated with the X chromosome. The crossover tests showed that in one case these genes were tightly linked to the w locus (the bbAM1 allele), and in the second case they were located 12.6 map units to the right of the Tu locus (the bbAM7 allele). It has also been shown that the bb locus was transposed to the X chromosome within the short arm of Y chromosome. Transposition of the BSYbb+ chromosome-specific rDNA sequences to the X chromosome was confirmed by means of Southern blotting. These data indicate that replacement of the bb locus is realized by transposition rather than recombination.     

Genetic Modulation of RNA Metabolism in Drosophila. IV. Measurement of Rates of RNA Synthesis by Density Labeling

Accumulation of RNA was measured in adult males of two genotypes: car bb/Ybb- and car bb/YbbSuVar-5. The two genotypes have similar amounts of rDNA, which is reduced in comparison to wild type (CLARK, STRAUSBAUGH and KIEFER 1977). Although genotypically bobbed, car bb/YbbSuVar-5 flies have a wild-type phenotype; car bb/Ybb- flies are both phenotypically and genotypically bobbed (CLARK, STRAUSBAUGH and KIEFER 1977). The wild-type phenotype observed in the car bb/YbbSuVar-5 flies is thought to be the result of an increased rate of rRNA synthesis due to the presence of the YbbSuVar-5 chromosome (SHERMOEN and KIFFER 1975; CLARK, STRAUSBAUGH and KIEFER 1977; CLARK and KIEFER 1977). To further define this phenomenon, the absolute accumulation of RNA was measured in the two genotypes, using density labeling methods. The accumulation of RNA is 1.4 to 1.8 times higher in car bb/YbbSuVar-5 flies than in car bb/Ybb- flies, demonstrating that there is genetic regulation of synthesis in this genotype. The use of density-labeled nucleosides has clearly shown that there is no difference in precusor pool sizes or use between the two genotypes studied.     

Origin of X/O progeny from crosses of sex-ratio trait males of Drosophila pseudoobscura

Males of Drosophila pseudoobscura carrying the sex-ratio chromosome (SR) were studied to determine the cause of X/O male progeny that they produce. It was found that among 3671 X/O progeny virtually all resulted from nullo-X sperm. The experiment also revealed a dramatic clustering of the frequency of X/O progeny among SR/Y males. This is interpreted to indicate that premeiotic events in the male germ line are the cause of nullo-X sperm.     

Male-Sex-Ratio Trait in Drosophila Pseudoobscura: Frequency of Autosomal Aneuploid Sperm

Males with the SR X chromosome show the "sex-ratio" (sr) phenotype in which they produce almost entirely daughters. The few sons (about 1%) are invariably sterile X/O males and result entirely from nullo-XY sperm. The "male-sex-ratio" (msr) phenotype is a modified form of sr in which SR/Y males produce a higher frequency of sterile X/O sons. The msr trait is due to the presence of the SR X-chromosome in males which are also homozygous for one or more autosomes from the L116 strain. Here the frequency of nullo-3 and diplo-3 sperm from msr males was measured by crossing to a compound-3 strain and found to be 13.8% and 3.2%, respectively, of the total viable sperm. The sr males produced very low levels of nullo-3 sperm at a frequency not different from control X/Y males and a slightly elevated frequency of diplo-3 sperm over X/Y males. The msr males were found to have only 12% the fecundity of sr males and in matings to cause a high frequency of brown inviable eggs. These results indicate that high rates of autosomal aneuploidy are not restricted to chromosome 3 but also occur for chromosomes 2, 4 and 5. The overall frequency of autosomal aneuploid sperm is estimated to be approximately 50%. Microscopic studies of meiosis in testes from msr males indicates meiotic nondisjunction and meiotic chromosome loss are responsible for the msr phenotype. Last, microscopic studies of sperm cysts from msr males reveal high levels of spermiogenic failure.     

Differential magnification of rDNA gene types in bobbed mutants of Drosophila melanogaster

Summary In Drosophila melanogaster a partial loss of ribosomal genes leads to the bobbed phenotype. Magnification is a heritable increase in rDNA that may occur in males carrying a deleted X chromosome with a strong bobbed phenotype. The restriction patterns of X chromosome total rDNA, insertions and spacers from magnified bobbed strains were compared with those of the original bobbed mutations. It was found that magnification modifies restriction patterns and differentially affects gene types, increasing specific genes lacking insertions (INS^-). Increases in copy number of genes with type I insertions are generally lower than the total number of INS^- genes, while type II insertion genes are not perceptibly increased. The recovery of homogeneous progeny from a single premagnified male indicates that the magnification event might take place and become stable very early in the germ line, arguing against magnification being due to extrachromosomal amplification. Additionally, some gene types increase 3.5-fold while others are eliminated, indicating that they could not result from a single unequal cross-over. These results are in good agreement with the existence of partial clustering of rDNA genes according to type, and suggest that magnification could result from local amplification of genes.     

Inheritance of the snakeskin color pattern in the guppy, Poecilia reticulata

We made single-pair reciprocal crosses between the Green Snakeskin and Yellow Snakeskin domesticated strains of the guppy, Poecilia reticulata. The two snakeskin strains differ by a single autosomal gene, with the Green Snakeskin strain having the wild-type background coloration caused by the dominant gene (B), whereas the Yellow Snakeskin is homozygous for the recessive blond allele (bb). The snakeskin body and tail patterns characterizing males of these two strains are determined by two genes--Ssb and Sst--that are closely linked on the Y chromosome. The greenish-yellow tail color of the Green Snakeskin strain is mediated by an X-linked dominant gene, Grt. The recessive wild-type allele, Grt+, gives the hyaline tail color. In the Yellow Snakeskin strain, the Grt gene is expressed as a golden-yellow color as a result of the presence of the bb homozygous condition. The putative genotypes of the males and females of the Green Snakeskin strain are BB XGrt YSsb,Sst and BB XGrt XGrt, respectively. Males and females of the Yellow Snakeskin strain have the putative genotypes bb XGrt YSsb,Sst and bb XGrtXGrt, respectively. As a result of crossing over between the X and Y chromosomes, a few males and females of these two snakeskin strains may carry one or both snakeskin pattern genes (Ssb and Sst) on the X chromosome.     

Genetic factors affecting normal growth of testes inDrosophila hydei

Summary A marked growth in the length of testes ofDrosophila hydei males occurred during pupal development. This growth continued over the first 8 days of adult life and in the young adults sperm were not produced until the testes increased approximately threefold in length to about 28 mm. The length of testes is correlated with genetic factors on the X and Y chromosomes. In males lacking a Y chromosome (X/O) or the short arm (Y^S) of the Y chromosome (X/Y^L) the testes were about half the length of testes of control males (X/Y) or double Y males (X/Y/Y). Males with deletions of the distal Y^L chromosome arm had testicular lengths equivalent to the controls. Males with short testes (X/O and X/Y^L) showed disruptions to spermatogenesis at meiosis and an absence of normal spermatid elongation. Reduction of active ribosomal RNA genes on the X chromosome in X/O caused an increased expression ofbobbed (bb) and a corresponding reduction in length of testes. Severelybobbed X/O males had very few cysts of spermatogonia and these cysts did not develop into primary spermatocytes.     

A Genetic Locus Having TRANS and CONTIGUOUS CIS Functions That Control the Disproportionate Replication of Ribosomal RNA Genes in DROSOPHILA MELANOGASTER

The results of deficiency mapping experiments reveal the presence of a compensatory response (cr+) locus that is located distal to the cluster of ribosomal RNA (rRNA) genes and is responsible for disproportionately replicating these genes when cr+ locus is present in a single dose, as in X/O males or X/sc4-sc8 females. The cr+ locus is novel in that it exhibits both trans and contiguous cis acting properties in somatic cells. It acts in trans to detect the presence of its partner locus in the opposite homolog, and if that partner locus is absent, it acts in cis to drive the disproportionate replication of those rRNA genes (rDNA) that are contiguous with it. The ability of cr+ to function is independent of the number of ribosomal RNA genes present. Furthermore, it can be shown that the cr+ locus is not required for the magnification or reduction of germ line rDNA. Finally, the implication of cr+ for position-effect variegation and the apparent reversion of the abnormal oocyte (abo) phenotype are discussed.     

Spontaneous Formation of Compound X Chromosomes in Drosophila Melanogaster

Males carrying different X chromosomes were tested for the ability to produce daughters with attached-X chromosomes. This ability is characteristic of males carrying an X chromosome derived from 59b-z, a multiply marked X chromosome, and is especially pronounced in males carrying the unstable 59b-z chromosomes Uc and Uc-lr. Recombination experiments with one of the Uc-lr chromosomes showed that the formation of compound chromosomes depends on two widely separated segments. One of these is proximal to the forked locus and is probably proximal to the carnation locus. This segment may contain the actual site of chromosome attachment. The other essential segment lies between the crossveinless and vermilion loci and may contain multiple factors that influence the attachment process.     

Amplification of rDNA and type I sequences in Drosophila males deficient in rDNA

rDNA magnification is a heritable change in rDNA content that occurs in D. melanogaster males when chromosomes deficient in rDNA are placed together for several generations. We have examined the restriction endonuclease cleavage pattern of the rDNA from an X chromosome undergoing magnification, and find no evidence for the selective amplification of either uninterrupted rDNA units or those containing insertion sequences. In addition, we observe an amplification of rDNA in the first generation of extremely bobbed male progeny to a level exceeding that of wild-type flies, but that reduces to the wild-type level in subsequent generations. The type I rDNA insertion elements also occur as tandem arrays, independently of rDNA. Southern hybridizations indicate that the majority of these sequences are located in the heterochromatin surrounding the nucleolus organizer on the X chromosome, and we find that they, too, amplify transiently in the first generation of magnifying males.     

UV-mutagenesis in Bacillus subtilis. IV. Analysis of revertants to methionine prototrophy]

UV light induces in Bacillus subtilis met5 ade6 two classes of revertants to prototrophy to methionine which can be easily distinguished by their phenotype: double (Met+Ade+) and solitary (Met+) revertants. Crosses of revertants with the wild type, carried out in transformational experiments, showed that original (direct) mutation met5 is presented in chromosome of double revertants. Consequently they are extragenic suppressor revertants. In the chromosome of solitary revertants Met+ an extragenic suppressor was not detected; reversions Met+ seem to be of an intragenic nature. It is possible to use reversions to prototrophy to methionine as a model to study UV-mutagenesis in suppressor and non-suppressor genes.     

DNA Rearrangements Associated with Reversion of Bacteriophage Mu-Induced Mutations

Excision of transposable genetic elements from host DNA is different from the classical prophage lambda type of excision in that it occurs at low frequency and is mostly imprecise; only a minority of excision events restores the wild-type host sequences. In bacteriophage Mu, a highly efficient transposon, imprecise excision is 10-100 times more frequent than precise excision. We have examined a large number of these excision events by starting with mucts X mutants located in the Z gene of the lac operon of Escherichia coli. Mucts X mutants are defective prophages whose excision occurs at a measurable frequency. Imprecise excision was monitored by selecting for melibiose+ (Mel+) phenotype, which requires only a functioning lacY gene. Mel+ revertants exhibit an array of DNA rearrangements and fall in four main classes, the predominant one being comprised of revertants that have no detectable Mu DNA. Most of these revertants can further revert to Lac+. Perhaps 5 base-pair duplications, originally present at prophage-host junctions, are left in these lacZ-Y+ revertants, and they can be further repaired to lacZ+. Another class has, in addition to the loss of Mu DNA, deletions that extend generally, but not always, to only one side of the prophage. The other two classes of revertants, surprisingly, still have Mu DNA in the lacZ gene. One class has deletions in the Z gene, whereas, no deletions can be detected in the other. Many of the revertants in the last class can further revert to lacZ+, indicating that the lacY gene must have been turned on by a rearrangement within Mu DNA. Apparently, all of the detectable precise and most of the imprecise excision events require functioning of the Mu A gene. We suggest that a block in large-scale Mu replication allows the excision process to proceed.     

Ribosomal DNA organization before and after magnification in Drosophila melanogaster

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb(-) chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb(2) mutant and in some magnified bb(+) alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb(2) allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids.     

Analysis of the Autosomal Mutation abo and Its Interaction with the Ribosomal DNA of DROSOPHILA MELANOGASTER: The Role of X-Chromosome Heterochromatin

The autosomal recessive, maternal-effect mutation abnormal oocyte (abo: 2-38) preferentially lowers the viability os XO progeny. The severity of the sex-ratio distortion is reduced by duplications of maternal or zygotic heterochromatin (SANDLER 1970, 1977; PARRY and SANDLER 1974). Utilizing X-chromosome inversions that contain modifications in the quantity and arrangement of the heterochromatic functions, Xhabo and cr+, wer have extended our investigations of abo's influence on XO male recovery and rDNA redundancy (KRIDER, YEDVOBNICK and LEVINE 1979).--XO males bearing In(1)SCS1LSC4R or In(1)Wm4LSC4R are recovered twice as frequently as X chromosomes containing a single Xh region, implying that these inversions possess a duplication of Xhabo. abo mutant females heterozygous for In(1)SCS1LSC4R and wild-type X chromosomes generate XO progeny that do not contain elevated rDNA redundancies. XO males containing In(1)Wm4 exhibit male recoveries and rDNA elevations similar to those of males bearing a wild-type X chromosome, when both derive from a common abo/abo mother. Reciprocal crosses baetween In(1)Wm4 and Canton-S males to attached-X abo females show significant, though reuduced, sex ratios in the absence of an rDNA effect. The observation that abo can elevate the rDNA redundancy of In(1)Wm4, a chromosome that does not compensate, suggests that abo and cr+ functions are not directly related.     

Radiation and transposon-induced genetic damage in Drosophila melanogaster: X-ray dose-response and synergism with DNA-repair deficiency.

The interaction of X-ray-induced and transposon-induced damage was investigated in P-M hybrid dysgenesis in Drosophila melanogaster. The X-ray dose-response of 330-1320 rad was monitored for sterility, fecundity and partial X/Y chromosome loss among F2 progeny derived from the dysgenic cross of M strain females xP strain males (cross A) and its reciprocal (cross B), using a weaker and the standard Harwich P strain subline. The synergistic effect of P element activity and X-rays on sterility was observed only in cross A hybrids and the dose-response was nonlinear in hybrids derived from the strong standard reference Harwich subline, Hw. This finding suggests that the lesions induced by both mutator systems which produce the synergistic effect are two-break events. The effect of increasing dose on the decline of fecundity was synergistic, but linear, in hybrids of either subline. There was no interaction evident and thus no synergism in X/Y nondisjunction and in partial Y chromosome loss measured by the loss of the Bs marker alone or together with the y+ marker. Interaction was detected in the loss of the y+ marker alone from the X and Y chromosomes. The possible three-way interaction of X-rays (660 rad), post-replication repair deficiency and P element mobility was assessed by measuring transmission distortion in dysgenic males derived from the II2 P strain. X-Irradiation of spermatids significantly increased the preferential elimination of the P-element-bearing second chromosome in mei-41, DNA-repair-deficient dysgenic males, but had no effect in their DNA-repair-proficient brothers. These findings indicate that the post-replication repair pathway is required for processing lesions induced by the combined effect of P element mobility and X-rays, and that the unrepaired lesions ultimately lead to chromosome loss.