The Molecular through Ecological Genetics of Abnormal Abdomen. III. Tissue-Specific Differential Replication of Ribosomal Genes Modulates the Abnormal Abdomen Phenotype in DROSOPHILA MERCATORUM

The abnormal abdomen (aa) syndrome in Drosophila mercatorum is controlled by two major X-linked genetic elements. We have previously shown that the major X-linked element of aa is associated with the presence of large inserts in the 28S gene of the ribosomal RNA (rDNA) genes. We show that, in polytene tissue of wild-type D. mercatorum, the uninterrupted rDNA repeats are overreplicated relative to interrupted repeats. Uninterrupted rDNA repeats are also overreplicated in polytene tissue of hybrid larval offspring from wild-type and aa parents. This overreplication of uninterrupted repeats is not observed in diploid tissues of wild-type hybrids (of wild-type and aa parents) and homozygous aa larvae or in polytene tissue of aa larvae. Furthermore, molecular analysis of an aa line that has reverted to the wild type indicates that the reversion phenomenon is associated with the ability to overreplicate uninterrupted rDNA repeats in polytene tissues. The patterns of differential replication of rDNA genes in wild-type hybrids and aa larvae of D. mercatorum offer a possible mechanism for the tissue-specific control of the aa phenotype and suggest that the molecular basis for the second X-linked genetic element of aa is involved in the control of differential replication in polytene tissues.     

The Molecular through Ecological Genetics of Abnormal Abdomen in DROSOPHILA MERCATORUM. I. Basic Genetics

The abnormal abdomen syndrome (aa) in Drosophila mercatorum is characterized by the persistence of juvenilized cuticle on the adult abdomen. The aa phenotype is shown to depend on at least two X-linked genetic elements that are about one map unit apart near the centromeric end of the X chromosome. These two genetic elements are necessary for aa expression; one behaves as a dominant element and the other as a recessive. Overlaying these genetic studies upon molecular work reported elsewhere, it is argued that the dominant element is the presence of a 5 kb insertion in a majority of the X-linked repeats coding for the 28S ribosomal RNA. The recessive element appears to be a locus controlling differential replication of noninserted over inserted 28S genes during polytenization. The aa syndrome requires both the presence of the inserted repeats and the failure to preferentially amplify noninserted repeats. Given the necessary X-linked elements for aa, a variety of modifiers are revealed. First, aa expression in males is Y-linked, apparently corresponding to a deletion of the 18S/28S rDNA gene cluster normally found on the Y. Moreover, all major autosomes can modify the penetrance of aa.     

The Molecular through Ecological Genetics of Abnormal Abdomen. IV. Components of Genetic Variation in a Natural Population of Drosophila Mercatorum

Natural populations of Drosophila mercatorum are polymorphic for a phenotypic syndrome known as abnormal abdomen (aa). This syndrome is characterized by a slow-down in egg-to-adult developmental time, retention of juvenile abdominal cuticle in the adult, increased early female fecundity, and decreased adult longevity. Previous studies revealed that the expression of this syndrome in females is controlled by two closely linked X chromosomal elements: the occurrence of an R1 insert in a third or more of the X-linked 28S ribosomal genes (rDNA), and the failure of replicative selection favoring uninserted 28S genes in larval polytene tissues. The expression of this syndrome in males in a laboratory stock was associated with the deletion of the rDNA normally found on the Y chromosome. In this paper we quantify the levels of genetic variation for these three components in a natural population of Drosophila mercatorum found near Kamuela, Hawaii. Extensive variation is found in the natural population for both of the X-linked components. Moreover, there is a significant association between variation in the proportion of R1 inserted 28S genes with allelic variation at the underreplication (ur) locus such that both of the necessary components for aa expression in females tend to cosegregate in the natural population. Accordingly, these two closely linked X chromosomal elements are behaving as a supergene in the natural population. Because of this association, we do not believe the R1 insert to be actively transposing to an appreciable extent. The Y chromosomes extracted from nature are also polymorphic, with 16% of the Ys lacking the Y-specific rDNA marker. The absence of this marker is significantly associated with the expression of aa in males. Hence, all three of the major genetic determinants of the abnormal abdomen syndrome are polymorphic in this natural population.     

X chromosome nucleolus organizer mutants which alter major type I repeat multiplicity in Drosophila melanogaster

The nucleolus organizer (NO) of the D. melanogaster X chromosome is composed of ribosomal repeat units which contain two types (I and II) of non-rDNA insertions (In+) and repeats with no insertions (In-). Evidence from other laboratories indicate random interspersion of all types of repeat units within the X NO. An EcoRI and BamHI examination of rDNA from two bobbed mutants, bb2rI and mal12 demonstrates segregation of the major type I repeat units. The 46 rDNA repeats of the bb2rI NO contain no detectable major type I repeats whereas the majority of the 68 rDNA mal12 repeats are major type I and tandemly linked. This observation suggests that gross deletions of rDNA can result in nucleolus organizer regions with predominantly one type of repeat unit. Additivity tests demonstrate that the 46 ribosomal repeats of the bb2rI chromosome revert the phenotype of other bobbed NOs, but the 68 mal12 ribosomal repeats show no or slight additivity. This is in agreement with the observation that In+ repeats do not significantly contribute to functional rRNA. A Southern blot analysis using BamHI which cuts only in type I insertions demonstrates that the majority of major type I In+ repeating units exist in tandem linkage group(s) within the X NO.     

The Molecular through Ecological Genetics of Abnormal Abdomen in Drosophila Mercatorum. V. Female Phenotypic Expression on Natural Genetic Backgrounds and in Natural Environments

The abnormal abdomen (aa) syndrome in Drosophila mercatorum depends on the presence of R1 inserts in a third or more of the X-linked 28S rDNA genes and the absence of selective underreplication of inserted repeats in polytene tissues that is controlled by an X-linked locus (ur) half a map unit from the rDNA complex. This syndrome affects both life history and morphology in the laboratory. Because abnormal morphologies are rarely encountered in nature, the purpose of this study is to see if the female life history traits are still affected under more natural genetic backgrounds and environmental conditions. Two outbred stocks were extracted from the natural population living near Kamuela, Hawaii: KaaX that has only X chromosomes with ur(aa) alleles, and K+X that has only ur(+) alleles. These two stocks have nonoverlapping distributions of insert proportions, indicating strong disequilibrium between the ur locus and the rDNA complex. The KaaX stock had almost no morphological penetrance of ur(aa), indicating that genetic background is important. KaaX expressed longer female egg-to-adult developmental times, increased early adult female fecundity, and decreased female adult longevity compared with K+X. By bagging natural rots of the cactus Opuntia megacantha near Kamuela, Hawaii, it was shown that egg-to-adult developmental time is slowed down by 0.92 days in females bearing ur(aa) alleles in nature, with no detectable slowdown in ur(aa) males. The bagged rot data also indicate that females bearing ur(aa) alleles have a strong fecundity advantage in nature under some ecological conditions but not others.     

The four ribosomal DNA units of the malaria parasite Plasmodium berghei. Identification, restriction map, and copy number analysis

The four ribosomal RNA genes (rDNA units) of the rodent malaria parasite, Plasmodium berghei, were identified and mapped by restriction enzyme analysis and Southern blot hybridization of genomic DNA. Although the four genes share common characteristics, they appear to be internally different from each other in expanse and sequence. One HindIII site near the 3' end of the coding region for the large rRNA is the only restriction site which we have detected that is conserved in all of the genes. The distance between the conserved HindIII site and the coding region for the small rRNA is at least 1-2 kilobases longer in two of the rDNA units than in a third unit. None of the four rDNA units were linked by restriction mapping where about 150 kilobases of the genome were accounted for. The copy number of two of the rDNA units was determined to be approximately 1 per haploid genome by quantitative analysis of cloned (plasmid) DNA versus genomic DNA digests on Southern blots. The other two genes also appear to be present in 1 copy. Restriction analysis confirms both the low copy number and that these genes are not in an easily recognizable tandem array. The low number of rDNA units requires that new copies of the genome produced during intraerythrocytic growth be active in RNA synthesis soon after their replication.     

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units,inverted sequences,pseudogenes and chromosome loci

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.     

Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana.

We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied.     

The Molecular through Ecological Genetics of Abnormal Abdomen in Drosophila Mercatorum. VI. the Non-Neutrality of the Y Chromosome Rdna Polymorphism

An association between quantitative variation of rDNA on the Y chromosome and male expression of the juvenilized, adult cuticle of the abnormal abdomen syndrome has been found for Drosophila mercatorum. Many pleiotropic effects of this syndrome have been described previously for females, but little was known about possible pleiotropic effects in males. The effects on males open up new avenues for the action of natural selection operating on the system. In females, the syndrome causes an increase in egg-to-adult development time, precocious sexual maturation, increased fecundity and decreased longevity. In addition to the cuticle phenotype, in males abnormal abdomen causes delayed sexual maturation, increased longevity, and decreased mating success, yet no change in egg-to-adult development time. Thus the syndrome has opposing fitness effects in the two sexes, which may help explain the genetic polymorphism observed in this system. Although investigated intensively, associations between naturally occurring Y-linked polymorphism and fitness phenotypes have not been found in Drosophila melanogaster.     

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.     

26S and 18S rRNA synthesis in bobbed mutants of Drosophila melanogaster

For the most part, bobbed mutations of Drosophila melanogaster consist of deletions of 26S and 18S rDNA located on the X and Y chromosomes. Studies on the synthesis of rRNA of third instar larvae and one day old adult females of three severe bobbed genotypes, indicate that no decrease can be detected, compared ot wild type strains. One of the bobbed mutants studied was a rather unusual type: these flies possess a quantity of rDNA that should confer upon them a near wild type phenotype whereas they actually show an extreme bobbed phenotype. The two other bobbed mutants are of a classical type: their severe bobbed phenotype corresponds to large deletions of rDNA. Two hypotheses can be proposed to explain the extreme bobbed phenotype of the flies, in spite of the fact that rRNA synthesis occurs normally. A regulatory phenomenon may interfere at the stages studied, but in earlier stages a net decrease in rRNA synthesis may have occurred producing an irreversible effect in the tissues affected by bobbed mutations (abdominal cuticle, bristles). The second hypothesis is that the rRNA produced may not be functional, perhaps because it is specific of earlier stages.     

The 10 kb Drosophila virilis 28S rDNA intervening sequence is flanked by a direct repeat of 14 base pairs of coding sequence

Most repeat units of rDNA in Drosophila virilis are interrupted in the 28S rRNA coding region by an intervening sequence about 10 kb in length; uninterrupted repeats have a length of about 11 kb. We have sequenced the coding/intervening sequence junctions and flanking regions in two independent clones of interrupted rDNA, and the corresponding 28S rRNA coding region in a clone of uninterrupted rDNA. The intervening sequence is terminated at both ends by a direct repeat of a fourteen nucleotide sequence that is present once in the corresponding region of an intact gene. This is a phenomenon associated with transposable elements in other eukaryotes and in prokaryotes, and the Drosophila rDNA intervening sequence is discussed in this context. We have compared more than 200 nucleotides of the D. virilis 28S rRNA gene with sequences of homologous regions of rDNA in Tetrahymena pigmentosa (Wild and Sommer, 1980) and Xenopus laevis (Gourse and Gerbi, 1980): There is 93% sequence homology among the diverse species, so that the rDNA region in question (about two-thirds of the way into the 28S rRNA coding sequence) has been very highly conserved in eukaryote evolution. The intervening sequence in T. pigmentosa is at a site 79 nucleotides upstream from the insertion site of the Drosophila intervening sequence.     

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.     

Homogenization of geographical variants at the nontranscribed spacer of rDNA in Drosophila mercatorum

rDNA nontranscribed spacer (NTS) lengths of Drosophila mercatorum have been measured in individuals from several geographic regions. Individuals from the different geographic subpopulations share some length fragments but are in general distinct. The length differences, both within and between individuals, arise from different copy numbers of a 250-bp repeating unit that is localized to one part of the NTS. In addition to the length differences caused by the 250-bp repeat, there is a Y chromosome (male)-specific length variant elsewhere in the NTS that is approximately 70 bp shorter than the NTS fragment from the X chromosome. Sexual dimorphism seems to be present in all Drosophila. Also, D. mercatorum has fewer NTS length variants per individual than does D. melanogaster while possessing comparable levels of restriction- site polymorphism. The mechanisms that may cause this pattern of variation are selection, gene conversion, and unequal recombination.      

Cloning of heat-shock locus 93D from Drosophila melanogaster.

Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.     

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.