Regulation of rRNA gene number in Drosophila melanogaster: new aspects resulting from the use of free duplications

Summary We have isolated a bobbed (bb) mutant on the free duplication Dp(1; f)122bb ⁺ and we have measured the rDNA content of the bb ⁺ and the bb loci in genetic combinations in which none of the phenomena involved in the change of the rDNA redundancy occurs. We have also measured the rDNA content of the two bb loci carried by the free duplications in two different genetic combinations: (1) and females in which there are two attached X chromosomes completely deleted for the nucleolus organizer (NO) regions and there-fore the only rDNA is contributed by the free duplication; (2) X/Dp122bb ⁺ and X/Dp122bb males, in which there are two bb loci, one on the X chromosome and the other on the X free duplication.The bb ⁺ and the bb duplications produced an overall increase of the rDNA content in the two genetic conditions tested.These results are not in favour of both a cis and trans effect of the regulator locus (cr ⁺ locus) hypothesised as being involved in the disproportionate replication of rRNA genes.     

Different behaviour of Xbb+ and Xbb chromosomes in D. melanogaster with only one nucleolus organizer.

Summary We have examined the rDNA content of male and female adult flies having only one nucleolus organizer (NO), using X chromosomes carrying wild or partially deleted bobbed loci (Xbb ⁺/O, Xbb ⁺/X_NO-and Xbb/O, Xbb/X_NO ^-).The results show that in Xbb ⁺/O and Xbb ⁺/X_NO ^-flies, where only somatic gene compensation is supposed to occur, the rDNA increase, although less pronounced than previously reported, is directly proportional to the number of rRNA genes initially present in the nucleolus organizer. In Xbb/O and in Xbb/X_NO ^-flies the rDNA increase is relatively much higher than that observed in flies carrying bb ⁺ instead of bb. It is suggested that this may be due to rDNA premagnification and somatic gene compensation occurring simultaneously in the former flies.On leave of absence from International Institute of Genetics and Biophysics, Naples, Italy     

A Study of Rdna Magnification Phenomenon in a Repair-Recombination Deficient Mutant of DROSOPHILA MELANOGASTER

The rDNA magnification process consists of a rapid and inheritable rDNA increase occurring in bobbed males: in a few generations the bb loci acquire the wild-type rDNA value and reach a bb⁺ phenotype.—We have analyzed the rDNA magnification process in the repair-recombination-deficient mutant mei9^a, both at the phenotypical and rDNA content levels. In mei9^a bb double mutants the recovery of bb⁺ phenotype is strongly disturbed and the rDNA redundancy value fails to reach the wild-type level. The strong effect of this meiotic mutation on rDNA magnification suggests a close relationship between this phenomenon and the repair-recombination processes.     

The compensatory response locus deletion increases the number of nucleoli in Drosophila melanogaster polytene cells

The formation of ribosomal DNA (rDNA) not associated with the nucleolar organizer (NO) regions was studied in polytene cells of Drosophila melanogaster mutants, mal ¹² and bb ^2rl , heterozygous for deficiencies in the NO. In the mutant X chromosome mal ¹² a smaller part of the NO is deleted than in bb ^2rl but it comprises the compensatory response (cr) locus, controlling the compensatory synthesis of rDNA. We found that in the polytene cells of all tissues of mutant X/Xmal ¹² investigated (larval salivary gland, fat body and midgut; adult fly midgut) the number of nucleoli was increased compared with that in the corresponding cells of X/Xbb ^2rl and X/X (wild type). Using in situ hybridization we showed that in the salivary gland cells of the X/Xmal ¹² larvae chromosomal sites containing type I insertion sequences and scattered throughout the genome were more frequent than in the wild type and in X/Xbb ^2rl mutant cells.     

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

Summary D. melanogaster males of bb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb ⁺/O females, yielding bb/O male progeny, and to X_NO-w sn bb ⁺ fameles, yielding bb/X_NO- females. The male and female offspring were scored for the bb ⁺ phenotype.Results show that there is a higher percentage of bb ⁺ flies in the bb/O male progeny than in bb/X_NO- females progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters.These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.     

Check of Gene Number during the Process of rDNA Magnification

THE multiple sequences of rDNA (DNA complementary to ribosomal RNA) of the Drosophila genome are localized at the bobbed locus, located in the X chromosome, position 66 and in the short arm of the Y chromosome^1,2. Wild bobbed (bb⁺) is that locus which, without a partner, gives rise to a normal phenotype. That locus which in similar conditions is incapable of giving rise to a normal phenotype is called a bobbed mutation (bb) and contains fewer genes for rRNA. The number of genes for rRNA in different individuals can vary considerably. One mechanism for rDNA variation is unequal crossing over³. Another mechanism, described by Tartof⁴, becomes apparent when individual flies, carrying only one bobbed locus, are constructed and only if such a locus is on the X chromosome; that is, if one constructs Xbb⁺/O males (and also Xbb/O males) or Xbb⁺/X_NO- females. Such individuals show a higher rDNA content than expected from the analysis of the same locus in Xbb⁺/Xbb⁺ females or in Xbb⁺/Ybb⁺ males. The increase of rDNA in this case is not inheritable⁴.     

Comparison between rDNA magnification and bb lethal mutation frequencies in Drosophila melanogaster

Summary Unequal mitotic sister strand crossing over has been evoked to explain the occurrence of phenotypically bb ^- males in the progeny of phenotypically bobbed males during magnification. If this is the case, complementary bb ¹ loci should be obtained together with the bb ¹. To test this hypothesis we compared the frequency of bb lethal mutations in the sperms of bb males with the percentage of phenotypically bb ⁺ males obtained during magnification of these bb males. We then compared these values with those occurring in phenotypically bb ⁺ control males. We found that, while the number of bb ⁺ males obtained during magnification, though variable, is high, the bb lethal mutation occurs at a very low frequency in all the genetic conditions, whatever the phenotype of the parental male.     

The conditions required for the induction of petite yeast mutants by fluorinated pyrimidines

Summary Newly magnified bobbed loci, combined with bb⁺ or bb^o loci, are in certain cases unstable. This may lead either to a reversion to the original bobbed mutation, or to lethal bobbed mutations. We name this instability modification. Modification occurs very early during the first divisions following fecondation of eggs, in embryos heterozygous for a magnified bobbed locus and a bb⁺ or bb^o locus. The phenomenon of modification is consistent with the model proposed by Ritossa (1972) to account for the phenomenom of magnification.     

BEHAVIORAL THERMOREGULATION OF THE NEW ZEALAND SEA LION (PHOCARCTOS HOOKERI)

Behavioral thermoregulation of New Zealand sea lions (Phocarctos hookeri) was studied at a male haul‐out ground at Papanui Beach, Otago Peninsula, New Zealand. The proportion of time spent by sea lions in each of five postures (prone, curled, oblique, ventral‐up, dorsal‐up) and also with the number of flippers exposed or tucked (hind and fore) at different black‐bulb temperature (T_bb°C) ranges was recorded. Use of prone and curled postures (0–1 flippers exposed) declined as T_bb increased, suggesting that these are adopted to conserve heat; oblique and dorsal‐up postures (3–4 flippers exposed) use increased with T_bb indicating a role in heat dissipation. The transition between heat conserving and heat dissipating postures occurred at about 14°–20°C (T_bb). Both foreflipper and hind flipper exposure increased with T_bb and the trends were similar, but overall hind flipper exposure was 89% of foreflipper exposure. The results show that surface area of flippers exposed to air is largely controlled by postural adjustment. The increase in flipper exposure with T_bb provides evidence of behavioral thermoregulation and that flippers are major sites for heat loss in the New Zealand sea lion, as observed for other otariid species. Nonpostural thermoregulatory behaviors such as flipper waving and sand flipping increased with T_bb, and may provide additional means of dissipating heat. Total body surface areas of six sea lions ranged from 1.72 to 3.39 m² (curvilinear length range from 1.60 to 2.35 m), and total flipper surface area averaged 22.7% of total body surface area. As otariids do not employ their hind limbs for aquatic propulsion, their role in behavioral thermoregulation may provide an explanation for the relatively large size of the hind flippers of the New Zealand sea lion.     

Insertion of a genetic marker into the ribosomal DNA of yeast

Plasmid pBR322 carrying the yeast LEU2+ gene transforms leu⁻ yeast into LEU+ at a low frequency by integration at homologous chromosomal DNA. When one-half of the yeast rDNA repeat unit (BglII-A) is inserted into the plasmid, the frequency of yeast transformation increases 100- to 200-fold, in proportion to the increased amount of homologous repetitive rDNA available for integration. When the other half of the repeat unit (BglII-B) is inserted into the plasmid, the transformation frequency increases by a factor of 10⁴, and the transformants are very unstable. It is likely that this fragment of rDNA contains a yeast origin of replication. This plasmid is a useful vector for cloning fragments of yeast DNA in yeast. We have used the LEU2+ gene, inserted into the rDNA locus, as a genetic marker for mapping the rDNA, in a procedure analogous to the use of antibiotic resistance transposons in the mapping of bacterial genes. Yeast ribosomal DNA is on chromosome XII between asp5 and ura4 as determined by mitotic linkage. Genetic analysis of markers inserted at the rDNA locus should be a useful tool for studying the conservation of sequence homology and the conservation of copy number of repeated genes.     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.     

Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

In Paracoccusdenitrificans the aa₃-type cytochrome c oxidase and the bb₃-type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb₃-type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen-sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR-binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa₃, bb₃. and cbb₃ make up the complete set of terminal oxidases in P. denitrificans. Proton-translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H⁺/e coupling efficiencies of the cbb₃-type     

Malathion resistance in Tribolium strains and their hybrids: Inheritance patterns and possible enzymatic mechanisms (Coleoptera,Tenebrionidae)

(1) The genetics of malathion resistance in two strains of the flour beetle, Tribolium castaneum, was investigated. In CTC-12, resistance is polygenic, while in Kano, it is due to a dominant allele at a single autosomal locus. Reciprocal hybrids with the susceptible control strains bb and pp showed an overdominant response in particular when Kano was the male parent in the original cross. (2) Three possible genetic mechanisms to explain these results are discussed. The model which best explains the genetic results, particularly the difference between the reciprocal crosses, assumes a modifier resistance allele on the Y chromosome. (3) The levels of activity of total esterases, carboxyesterases, mixed-function oxidases, epoxide hydrase, and glutathione transferase in the parent strains and their hybrids were measured quantitatively. Although total esterase activity may not be relevant for the breakdown of malathion, it was inhibited by the pesticide. The activity of the microsomal enzymes was high in CTC-12, low in bb, and intermediate in the hybrids, while carboxyesterases were very active in Kano as well as in the hybrids with bb and low in the latter. These patterns agree with the genetics of resistance in the two strains. A higher level of GSH transferase in the Kano×bb hybrids than in Kano seems to indicate a possible biochemical mechanism for their overdominant resistance.     

Nucleolus organizers in the wild silkworm Bombyx mandarina and the domesticated silkworm B. mori

Two types (R ^a1 and R ^a2) of nucleolus organizers were identified in the genome of Bombyx mandarina (Japan) which occurs in Japan. Genetical analysis of a hybrid with B. mori suggested that the loci of both nucleolus organizers are allelic and correspond to the R ⁰ locus of B. mori. These nucleolus organizers segregated and were inherited by the progeny in a Mendelian fashion. The majority of the R ^a1 rDNA units were 10.6 kb in length and had an additional EcoRI site in the transcribed spacer region when compared with the same size unit of R ⁰. On the other hand, the KpnI site present in the non-transcribed spacer region of the R ⁰ rDNA was not detected in the R ^a1 unit. The 15.1 kb unit observed in the R ^a2 locus was the same as the unit with the type II intron of R ⁰. The four major components of R ^a2 rDNA, with lengths of 10.6, 15.1, 15.7 and 20.8 kb, were also found in the R ⁰ locus, and thus the R ^a2 and the R ⁰ loci were considered to be closely related. Usually the functional unit of rDNA in the nucleolus organizers of homologous or non-homologous chromosomes cannot be easily distinguished. However, in the case of B. mandarina (Japan), distinct functional 10.6 kb units were observed in the allelic R ^a1 and R ^a2 loci. Therefore the existence of the two distinct units suggest the possibility of introducing the chromosomes of the interspecies in the genus Bombyx.     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

Tli1, a resistance locus for carcinogen-induced T-lymphoma

Within 180 days after injection with N-methyl-N-nitrosourea (MNU), 83.5% of AKR/J mice and 37.5% of BALB/cJ mice developed T-lymphoma. The high tumor incidence was a dominant trait, as 93% of MNU-injected F₁ mice developed T-lymphoma. A genome screen of 285 MNU-injected F₂ mice identified a locus, designated T-lymphoma Induced 1 or Tli1, in a ∼10-cM interval on central Chr 1 between D1Mit87 and D1Mit423 with significant linkage to the incidence of MNU-induced T-lymphoma (P= 0.0004). Injection of BALB/cJ.AKR/J–Tli1 congenic mice with MNU confirmed the presence of Tli1 on central Chr 1. Mice homozygous for the BALB/cJ allele (Tli1 ^bb) were over-represented in the tumor-free F₂ mice, while the inheritance of parental alleles of Tli1 in tumor-bearing mice was close to expected. This suggests that the Tli1 ^b allele is recessive and suppresses MNU-induced T-lymphoma development in BALB/cJ mice and in Tli1 ^bb F₂ mice. Furthermore, the kinetics of lymphoma development in BALB/cJ and the Tli1 congenic mice suggests that Tli1 ^b acts to suppress lymphomas developing late after injection with MNU. Two known genes that map in the identified genomic interval on central Chr 1 are candidates for Tli1:IL10, encoding the lymphokine IL10, and Cmkar4, encoding the chemokine receptor CXCR4. Received: 2 November 1998 / Accepted 12 January 1999     

Disproportionately-replicated, nonfunctional rDNA in compound chromosomes of Drosophila melanogaster

Summary The multiplicity of the genes for ribosomal RNA varies in several ways. The series of bb mutants are an example of mutant phenotypes associated with deficiencies of the rDNA. In certain genotypes the amount of rDNA present can disproportionately replicate and in some cases become incorporated into the chromosome and thus inherited. In other cases there is no stabilization of the increased multiplicity of rRNA genes. In still other cases the multiplicity of rDNA does not change. We describe cases of increased, but non-functional, multiplicity of rDNA in compound chromosomes routinely used in analyses of these phenomena and discuss the associated problems.Supported by the National Research Council of Canada.     

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.     

X-Y Exchange and the Coevolution of the X and Y Rdna Arrays in Drosophila melanogaster

The nucleolus organizers on the X and Y chromosomes of Drosophila melanogaster are the sites of 200-250 tandemly repeated genes for ribosomal RNA. As there is no meiotic crossing over in male Drosophila, the X and Y chromosomal rDNA arrays should be evolutionarily independent, and therefore divergent. The rRNAs produced by X and Y are, however, very similar, if not identical. Molecular, genetic and cytological analyses of a series of X chromosome rDNA deletions (bb alleles) showed that they arose by unequal exchange through the nucleolus organizers of the X and Y chromosomes. Three separate exchange events generated compound X·Y ^L chromosomes carrying mainly Y-specific rDNA. This led to the hypothesis that X-Y exchange is responsible for the coevolution of X and Y chromosomal rDNA. We have tested and confirmed several of the predictions of this hypothesis: First, X· Y^L chromosomes must be found in wild populations. We have found such a chromosome. Second, the X·Y^L chromosome must lose the Y^L arm, and/or be at a selective disadvantage to normal X⁺ chromosomes, to retain the normal morphology of the X chromosome. Six of seventeen sublines founded from homozygous X·Y^Lbb stocks have become fixed for chromosomes with spontaneous loss of part or all of the appended Y^L. Third, rDNA variants on the X chromosome are expected to be clustered within the X⁺ nucleolus organizer, recently donated (" Y") forms being proximal, and X-specific forms distal. We present evidence for clustering of rRNA genes containing Type 1 insertions. Consequently, X-Y exchange is probably responsible for the coevolution of X and Y rDNA arrays.     

Inheritance of the group I rDNA intron in Tetrahymena pigmentosa

We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron⁺ and intron^? strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron⁺ and intron^? alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron⁺ and intron^? rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing. During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is that T. pigmentosa has two rdn loci in contrast to the single locus found in T. thermophila. Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis. © 1992 Wiley-Liss, Inc.