Drosophila ribosomal RNA genes function as an X-Y pairing site during male meiosis

In Drosophila melanogaster males, the sex chromosomes pair during meiosis in the centric X heterochromatin and at the base of the short arm of the Y (YS), in the vicinity of the nucleolus organizers. X chromosomes deficient for the pairing region segregate randomly from the Y. In this report we show that a single ribosomal RNA (rRNA) gene stimulates X-Y pairing and disjunction when inserted onto a heterochromatically deficient X chromosome by P element-mediated transformation. We also show that insert-containing X chromosomes pair at the site of insertion, that autosomal rDNA inserts do not affect X-Y pairing or disjunction, and that the strength of an X pairing site is proportional to the dose of ectopic rRNA genes. These results demonstrate that rRNA genes can promote X-Y pairing and disjunction and imply that the nucleolus organizers function as X-Y pairing sites in wild-type Drosophila males.     

Male sterility and meiotic drive associated with sex chromosome rearrangements in Drosophila. Role of X-Y pairing.

In Drosophila melanogaster, deletions of the pericentromeric X heterochromatin cause X-Y nondisjunction, reduced male fertility and distorted sperm recovery ratios (meiotic drive) in combination with a normal Y chromosome and interact with Y-autosome translocations (T(Y;A)) to cause complete male sterility. The pericentromeric heterochromatin has been shown to contain the male-specific X-Y meiotic pairing sites, which consist mostly of a 240-bp repeated sequence in the intergenic spacers (IGS) of the rDNA repeats. The experiments in this paper address the relationship between X-Y pairing failure and the meiotic drive and sterility effects of Xh deletions. X-linked insertions either of complete rDNA repeats or of rDNA fragments that contain the IGS were found to suppress X-Y nondisjunction and meiotic drive in Xh-/Y males, and to restore fertility to Xh-/T(Y;A) males for eight of nine tested Y-autosome translocations. rDNA fragments devoid of IGS repeats proved incapable of suppressing either meiotic drive or chromosomal sterility. These results indicate that the various spermatogenic disruptions associated with X heterochromatic deletions are all consequences of X-Y pairing failure. We interpret these findings in terms of a novel model in which misalignment of chromosomes triggers a checkpoint that acts by disabling the spermatids that derive from affected spermatocytes.     

On the Roles of Heterochromatin and Euchromatin in Meiosis in Drosophila: Mapping Chromosomal Pairing Sites and Testing Candidate Mutations for Effects on X–Y Nondisjunction and Meiotic Drive in Male Meiosis

Mapping of pairing sites involved in meiotic homolog disjunction in Drosophilahas led to conflicting hypotheses about the nature of such sites and the role of heterochromatin in meiotic pairing. In the female-specific distributive system, pairing regions appear to be exclusively heterochromatic and map to broad regions encompassing many different sequences. In male meiosis, autosomal pairing sites appear to be distributed broadly within euchromatin but to be absent from heterochromatin, whereas the X-pairing site maps in the centric heterochromatin. The X site has been shown to coincide with the intergenic spacer (IGS) repeats within the rDNA arrays shared between the X and Y. It has not been clear whether the heterochromatic location of this pairing site has any significance. A novel assay for genic modifiers of X–Y chromosome pairing was developed based on the intermediate nondisjunction levels observed in males whose X chromosome lacks the native pairing site but contains two transgenic insertions of single rDNA genes. This assay was used to test several mutations in Su(var)(Suppressor of position effect variegation), PcG(Polycomb-Group) recombination defective, and repair-defective genes. No strong effects on disjunction were seen. However, the tests did uncover several mutations that suppress or enhance the meiotic drive (distorted X-Y recovery ratio) that accompanies X–Y pairing failure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

X and Y chromosomal ribosomal DNA of Drosophila: comparison of spacers and insertions.

In Drosophila melanogaster, the genes coding for 18S and 28S ribosomal RNA (rDNA) are clustered at one locus each on the X and the Y chromosomes. We have compared the structure of rDNA at the two loci. The 18S and 28S rRNAs coded by the X and Y chromosomes are very similar and probably identical (Maden and Tartof, 1974). In D. melanogaster, many rDNA repeating units are interrupted in the 28S RNA sequence by a DNA region called the insertion. There are at least two sequence types of insertions. Type 1 insertions include the most abundant 5 kilobase (kb) class and homologous small (0.5 and 1 kb) insertions. Most insertions between 1.5 and 4 kb have no homology to the 5 kb class and are identified as type 2 insertions. In X rDNA, about 49% of all rDNA repeats have type 1 insertions, and another 16% have type 2 insertions. On the Y chromosome, only 16% of all rDNA repeats are interrupted, and most if not all insertions are of type 2.rDNA fragments derived from the X and Y chromosomes have been cloned in E. coli. The homology between the nontranscribed spacers in X and Y rDNA was studied with cloned fragments. Stable heteroduplexes were found which showed that these regions on the two chromosomes are very similar.The evolution of rDNA in D. melanogaster might involve genetic exchange between the X and Y chromosomal clusters with restrictions on the movement of type 1 insertions to the Y chromosome.     

Genetic evidence that nonhomologous disjunction and meiotic drive are properties of wild-type Drosophila melanogaster male meiosis

We have followed sex and second chromosome disjunction, and the effects of these chromosomes on sperm function, in four genotypes: wild-type males, males deficient for the Y-linked crystal locus, males with an X chromosome heterochromatic deficiency that deletes all X-Y pairing sites, and males with both deficiencies. Both mutant situations provoke chromosome misbehavior, but the disjunctional defects are quite different. Deficiency of the X heterochromatin, consonant with the lack of pairing sites, mostly disrupts X-Y disjunction with a decidedly second-level effect on major autosome behavior. Deleting crystal, consonant with the cytological picture of postpairing chromatin-condensation problems, disrupts sex and autosome disjunction equally. Even when the mutant-induced nondisjunction has very different mechanics, however, and even more importantly, even in the wild type, there is strong, and similar, meiotic drive. The presence of meiotic drive when disjunction is disrupted by distinctly different mechanisms supports the notion that drive is a normal cellular response to meiotic problems rather than a direct effect of particular mutants. Most surprisingly, in both wild-type and crystal-deficient males the Y chromosome moves to the opposite pole from a pair of nondisjoined second chromosomes nearly 100% of the time. This nonhomologous interaction is, however, absent when the X heterochromatin is deleted. The nonhomologous disjunction of the sex and second chromosomes may be the genetic consequence of the chromosomal compartmentalization seen by deconvolution microscopy, and the absence of Y-2 disjunction when the X heterochromatin is deleted suggests that XY pairing itself, or a previously unrecognized heterochromatic function, is prerequisite to this macrostructural organization of the chromosomes.     

The role of rDNA genes in X chromosome association in the aphid Acyrthosiphon pisum.

Silver staining of mitotic metaphases of the aphid A. pisum reveals the presence of argentophilic bridges connecting the two X chromosomes. The presence of nucleolar material connecting sex chromosomes seems to be quite a common phenomenon in organisms belonging to very different phyla, and suggests a role of nucleolar proteins in chromosome association and disjunction. In somatic cells of A. pisum, bridges connecting X chromosomes are detectable not only after silver staining but also after CMA3 staining. This finding suggests that GC rich DNA is involved in this type of association. Molecular analysis of rDNA intergenic spacers shows several 247 bp repeats containing short sequences having a high level of homology with the chi sequence of Escherichia coli and with the consensus core region of human hypervariable minisatellites. Moreover, each 247 bp repeat presents a perfect copy of a promoter sequence for polymerase I. These aphid repeats show structural homologies with a 240 bp repeat, which is considered to be responsible for sex chromosome pairing in Drosophila, not only in view of their common presence within rDNA spacers but also for their length and structure. The presence of chi sequences in the IGS of A. pisum, by promoting unequal crossing-over between rDNA genes, could thus give rise to the nucleolar organizing region (NOR) heteromorphism described in different aphid species. Although X pairing at NORs is fundamental in aphid male determination, the presence of heteromorphism of rDNA genes does not inhibit male determination in the A. pisum clone utilized for our experiments.     

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Fragments of rDNA3 from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.      

Meiotic pairing and disjunction of mini-X chromosomes in drosophila is mediated by 240-bp rDNA repeats and the homolog conjunction proteins SNM and MNM

In most eukaryotes, segregation of homologous chromosomes during meiosis is dependent on crossovers that occur while the homologs are intimately paired during early prophase. Crossovers generate homolog connectors known as chiasmata that are stabilized by cohesion between sister-chromatid arms. In Drosophila males, homologs pair and segregate without recombining or forming chiasmata. Stable pairing of homologs is dependent on two proteins, SNM and MNM, that associate with chromosomes throughout meiosis I until their removal at anaphase I. SNM and MNM localize to the rDNA region of the X-Y pair, which contains 240-bp repeats that have previously been shown to function as cis-acting chromosome pairing/segregation sites. Here we show that heterochromatic mini-X chromosomes lacking native rDNA but carrying transgenic 240-bp repeat arrays segregate preferentially from full-length sex chromosomes and from each other. Mini-X pairs do not form autonomous bivalents but do associate at high frequency with the X-Y bivalent to form trivalents and quadrivalents. Both disjunction of mini-X pairs and multivalent formation are dependent on the presence of SNM and MNM. These results imply that 240-bp repeats function to mediate association of sex chromosomes with SNM and MNM.     

A 7.1 kb linear DNA molecule of Theileria parva has scrambled rDNA sequences and open reading frames for mitochondrially encoded proteins.

Theileria parva, an intralymphocytic protozoan parasite of cattle, contains a linear 7.1 kb DNA element with terminal inverted repeat sequences. The molecule is transcribed into low molecular weight RNA, and both DNA strands encode short stretches of unique sequences, usually < 100 nucleotides, which are similar to large (LSU) or small (SSU) ribosomal subunit RNA. Phylogenetically conserved conformational rRNA domains were assembled from the discontinuous rDNA sequences using comparative secondary structure modelling. For example, a minimum of four predicted sequences, two derived from each DNA strand, is required to assemble domain V of LSU rRNA which participates in peptidyl transferase activity. The discontinuities in the identified rRNA domains fall within regions of no known functional significance. Hence, it is likely that the element encodes fragmented rDNA genes and the mature rRNA is unconventional, consisting of several fragments of RNA, primarily held together by intermolecular and intramolecular base pairing. The element also has ORFs for components of the last two mitochondrial electron transport enzyme complexes. The structure of the parasite DNA element, its protein coding capacity and scrambled rDNA gene sequences, are reminiscent of the mitochondrial genome of Chlamydomonas reinhardtii. We propose that the 7.1 kb element is equivalent to the mitochondrial DNA of T. parva, although a number of its features are unusual for this family of extrachromosomal DNA molecules.     

Duplicated rDNA sequences of variable lengths flanking the short type I insertions in the rDNA of Drosophila melanogaster.

We describe cloned segments of rDNA that contain short type I insertions of differing lengths. These insertions represent a coterminal subset of sequences from the right hand side of the major 5kb type I insertion. Three of these shorter insertions are flanked on both sides by a short sequence present as a single copy in uninterrupted rDNA units. The duplicated segment is 7, 14 and 15 nucleotides in the different clones. In this respect, the insertions differ from the 5kb type I insertion, where the corresponding sequence is found only at the right hand junction and where at the left hand side there is a deletion of 9 nucleotides of rDNA (Roiha et al.,1981). One clone is unusual in that it contains two type I insertions, one of which is flanked by a 14 nucleotide repeat. The left hand junction of the second insertion occurs 380 nucleotides downstream in the rDNA unit from the first. It has an identical right hand junction to the other elements and the 380 nucleotide rDNA sequence is repeated on both sides of the insertion. We discuss the variety of sequence rearrangements of the rDNA which flank type I insertions.     

Analysis of hemolytic determinants of plasmid pHly185 by Tn5 mutagenesis.

A nonconjugative hemolysin plasmid, pHly185, was isolated from Escherichia coli 185. Tn5 mutagenesis produced nonhemolytic derivatives that either expressed no hemolytic activity or did not secrete activity. These Tn5 insertions were located in three contiguous EcoRI fragments. Insertions inactivating hemolysin structural gene(s) were identified on all three EcoRI fragments. Mutations affecting the secretory function were clustered in one portion of a single EcoRI fragment.