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.     

Nucleotide sequences at the boundaries between gene and insertion regions in the rDNA of Drosophila melanogaster

Ribosomal RNA genes interrupted by type 1 insertions of 1 kb and 0.5 kb have been sequenced through the insertion region and compared with an uninterrupted gene. The 0.5 kb insertion is flanked by a duplication of a 14 bp segment that is present once in the uninterrupted gene; the 1 kb insertion is flanked by a duplication of 11 of these 14 bp. Short insertions are identical in their entire length to downstream regions of long insertions. No internal repeats occur in the insertion. The presence of target site duplications suggests that type 1 insertions arose by the introduction of transposable elements into rDNA. Short sequence homologies between the upstream ends of the insertions and the 28S' boundaries of the rRNA coding region suggest that short type 1 insertions may have arisen by recombination from longer insertions.We have sequenced both boundaries of two molecules containing type 2 insertions and the upstream boundary of a third; the points of interruption at the upstream boundary (28S' site) differ from each other in steps of 2 bp. Between the boundary in the 0.5 kb type 1 insertion and the type 2 boundaries there are distances of 74, 76, and 78 bp. At the downstream boundary (28S' site) the two sequenced type 2 insertions are identical. The rRNA coding region of one molecule extends across the insertion without deletion or duplication, but a 2 bp deletion in the RNA coding region is present in the second molecule. Stretches of 13 or 22 adenine residues occur at the downstream (28S') end of the two type 2 insertions.     

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.     

Modification of chromosome instability in Aspergillus nidulans

Strains of Aspergillus nidulans with a chromosome segment in duplicate show instability at mitosis; their colonies produce faster-growing sectors which arise from nuclei with spontaneous deletions in either duplicate segment. In an attempt to probe the deletion process, the effects of mutations causing sensitivity to UV treatment, and those of manganous ions, have been studied in strains carrying either Dp(I,II) or Dp(III,VIII). For comparison, the effects of Mn2+ on balanced and unbalanced diploids have also been examined. The uvsE allele, which decreases intragenic mitotic crossing over in diploids, increased deletion frequency in strains with either duplication. The uvsB allele, which increases intragenic mitotic crossing over in diploids, increased deletion frequency only in Dp(I,II) strains; in addition, by causing early mitotic crossing over between the homologous segments, it produced some novel deletion products. Mn2+ substantially decreased the deletion frequency in Dp(I,II) strains and decreased mitotic crossing over in diploids; it had no effect on Dp(III,VIII) strains. The results suggest that in haploid duplication strains there are two classes of spontaneous DNA lesions, recombinogenic and non-recombinogenic, both of which, failing repair, lead to deletion.     

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.     

Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster.

In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus.     

The synaptonemal complexes of Caenorhabditis elegans: Pachytene karyotype analysis of the Dp 1 mutant and Disjunction Regulator Regions

The duplication mutant Dp1 presents morphological changes of the pachytene nuclei which include enlarged nuclei (58% greater than the wild type (WT)) and increased total length of the synaptonemal complex (SC) complement (32% greater than WT). The size of the nucleolus is similar to that of the wild type. The SC of the XX bivalent is 2.5 m long which is similar to the length in other strains. Genetic analysis (Herman et al. 1976) has shown that a duplicated segment of the X chromosome has been transposed to linkage group V in the Dp1 mutant. The present analysis, however, did not reveal the expected duplication loop in bivalent V of the heterozygous Dp1 strain. There is an average of three Disjunction Regulator Regions (DRR) per nucleus and approximately 1% X-chromosome nondisjunction which is consistent with the notion that the presence of DRRs somehow facilitates regular disjunction of the X chromosome. Statistical analysis, based on 9 different strains and 81 nuclei, indicates that there is a good correlation between the frequency of occurrence of X-chromosome nondisjunction and the number of DRRs.     

The Tribolium castaneum ortholog of Sex combs reduced controls dorsal ridge development

Several constitutional chromosomal rearrangements occur on human chromosome 17. Patients who carry constitutional deletions of 17q21.3-q24 exhibit distinct phenotypic features. Within the deletion interval, there is a genomic segment that is bounded by the myeloperoxidase and homeobox B1 genes. This genomic segment is syntenically conserved on mouse chromosome 11 and is bounded by the mouse homologs of the same genes (Mpo and HoxB1). To attain functional information about this syntenic segment in mice, we have generated a 6.9-Mb deletion [Df(11)18], the reciprocal duplication [Dp(11)18] between Mpo and Chad (the chondroadherin gene), and a 1.8-Mb deletion between Chad and HoxB1. Phenotypic analyses of the mutant mouse lines showed that the Dp(11)18/Dp(11)18 genotype was responsible for embryonic or adolescent lethality, whereas the Df(11)18/+ genotype was responsible for heart defects. The cardiovascular phenotype of the Df(11)18/+ fetuses was similar to those of patients who carried the deletions of 17q21.3-q24. Since heart defects were not detectable in Df(11)18/Dp(11)18 mice, the haplo-insufficiency of one or more genes located between Mpo and Chad may be responsible for the abnormal cardiovascular phenotype. Therefore, we have identified a new dosage-sensitive genomic region that may be critical for normal heart development in both mice and humans.     

Repeat-induced point mutation (RIP) in crosses with wild-isolated strains of Neurospora crassa: evidence for dominant reduction of RIP

Seventy-one wild-isolated strains of Neurospora crassa were examined for their ability to support repeat-induced point mutation (RIP) in the erg-3 locus. RIP was exceptionally inefficient but detectable in crosses with the strain FGSC 430 from Adiopodoume, Ivory Coast. We could find no consistent differences in ascospore yields when wild isolates identified as "low-RIP" or "high-RIP" strains were crossed with strains bearing the segmental duplication Dp(IIIR > [I; II])AR17. This suggested that RIP may not be responsible for the barren phenotype of crosses involving segmental duplication strains.     

The molecular basis for a cytosolic malic enzyme null mutation. Malic enzyme mRNA from MOD-1 null mice contains an internal in-frame duplication that extends the coding sequence by 522 nucleotides

Many tissues from wild type mice express cytosolic malic enzyme activity and contain two mRNAs (2.0 and 3.1 kilobases (kb)) that encode a single 64-kDa malic enzyme subunit polypeptide. MOD-1 null mutant mice lack cytosolic malic enzyme activity but express 2.5- and 3.6-kb mRNAs that hybridize with wild type malic enzyme cDNAs and are induced in liver by a starvation/carbohydrate refeeding regimen. To investigate the basis of the MOD-1 null mutation, a lambda gt11 cDNA library was constructed using mRNA from the livers of induced MOD-1 null mice as a template. A recombinant phage with a 2-kb insert was isolated by screening with wild type malic enzyme cDNA probes. The subcloned insert exhibited an atypical (non-wild type) restriction pattern and was subjected to sequence analysis. MOD-1 null malic enzyme cDNA contains an internal tandemly duplicated sequence that corresponds to nucleotides 1027-1548 in the coding region of wild type murine malic enzyme cDNA (Bagchi, S., Wise, L. S., Brown, M. L., Bregman, D., Sul, H. S., and Rubin, C. S. (1987) J. Biol. Chem. 262, 1558-1565). An open reading frame is retained throughout the duplicated sequence. The discovery of a 522-nucleotide in-frame duplication accounts for the increased size of MOD-1 null malic enzyme mRNAs and suggests that a variant malic enzyme polypeptide that is 19 kDa larger than the wild type subunit might be found in mutant mice. Western immunoblot analysis disclosed that MOD-1 null liver cytosol contains an 82-kDa protein that is recognized by anti-malic enzyme antibodies. Under stringent conditions, an anti-sense 32P-oligonucleotide that spans the abnormal junction between the reiterated sequences hybridized with the 2.5 and 3.6-kb MOD-1 null malic enzyme mRNAs but failed to form stable complexes with wild type malic enzyme mRNAs. Thus, both MOD-1 null malic enzyme mRNAs contain the duplication deduced from cDNA sequence analyses. The MOD-1 null mutation might originate from an unequal crossover between homologous regions of two different introns in the malic enzyme gene, thereby causing the duplication of one or more exons.     

Analysis of Subtelomeric Heterochromatin in the Drosophila Minichromosome Dp1187 by Single P Element Insertional Mutagenesis

We investigated whether single P element insertional mutagenesis could be used to analyze heterochromatin within the Drosophila minichromosome Dp1187. Forty-five insertions of the P[lacZ,rosy+] element onto Dp1187 (recovered among 7,825 transpositions) were highly clustered. None was recovered in centromeric heterochromatin, but 39 occurred about 40 kb from the distal telomere within a 4.7-kb hotspot containing tandem copies of a novel 1.8-kb repetitive DNA sequence. The DNA within and distal to this region lacked essential genes and displayed several other properties characteristic of heterochromatin. The rosy+ genes within the inserted transposons were inhibited by position-effect variegation, and the subtelomeric region was underrepresented in polytene salivary gland cells. These experiments demonstrated that P elements preferentially transpose into a small subset of heterochromatic sites, providing a versatile method for studying the structure and function of these chromosome regions. This approach revealed that a Drosophila chromosome contains a large region of subtelomeric heterochromatin with specific structural and genetic properties.     

Escape from repeat-induced point mutation of a gene-sized duplication in Neurospora crassa crosses that are heterozygous for a larger chromosome segment duplication

In Neurospora crassa the ability of an ectopic gene-sized duplication to induce repeat-induced point mutation (RIP) in its target gene was suppressed in crosses that were heterozygous for another larger chromosome segment duplication. Specifically, the frequency of RIP in the erg-3 gene due to a 1.3-kb duplication was reduced if the chromosome segment duplications Dp(IIIR > [I;II]) AR17, Dp(VIR > IIIR) OY329, or Dp(IVR > VII) S1229 were present in either the same or the other parental nucleus of the premeiotic dikaryon. We suggest that the larger duplications act as sinks to titrate the RIP machinery away from the smaller duplication. In contrast, RIP efficiency was relatively unaffected in comparably unproductive interspecies crosses with N. intermedia and N. tetrasperma. These findings offer a novel explanation for the observed persistence of the transposable element Tad in only a subset of Neurospora strains.     

Site specific insertion of a type I rDNA element into a unique sequence in the Drosophila melanogaster genome.

We describe a cloned segment of unique DNA from the Oregon R strain of Drosophila melanogaster that contains a short type I insertion of the kind principally found within rDNA. The predominant type I rDNA insertion is 5kb in length, but there are also a co-terminal sub-set of shorter type I elements that share a common right hand junction with the rDNA. The insertion that we now describe is another member of this sub-set. The right hand junction of the type I sequence with the unique DNA is identical to the right hand junction of the type I sequences with rDNA. There is no significant feature within the insertion sequence that could have determined the position of the left junction with the sequence into which it is inserted. Like the corresponding short type I insertions in rDNA, the insertion into the unique DNA is flanked on both sides by a duplicated sequence, which in this case is 10 base pairs long. The cloning of a sequence corresponding to the uninterrupted unique location was facilitated by the observation that the Karsnas strain of D. melanogaster contains only uninterrupted sequences of this kind. The duplicated sequence at the target site for the insertion is only present as a single copy in the uninterrupted DNA. The sequence of the target site for the insertion (ACTGTTCT) in the unique segment shows a striking homology to the target in rDNA (ACTGTCCC).     

Drosophila 5 S RNA processing requires the 1-118 base pair and additional sequence proximal to the processing site.

Using an in vitro processing system, we have identified a required sequence surrounding the Drosophila melanogaster 5 S RNA processing site at nucleotide 120. Mutations in this region vary the processing rate from complete inhibition to a level equal to or greater than wild type. Analysis of mutants at +1 and in the region 118-122 separates the inhibitory effect into two parts. 1) Nucleotide 118 C, the base-paired nucleotide in helix I proximal to the processing site, plays an essential role. Changing it to a purine inhibits processing. The +1-118 base pair must be intact, but this alone is not sufficient for processing, since compensatory changes at +1 do not restore down-processing mutants at 118 to the wild type level. 2) The processing site has to be pyrimidine rich; multiple contiguous purines inhibit processing. On the other hand, multiple pyrimidines can largely negate the inhibitory effect of a mutation at position 118. Thus a base-paired C at 118 followed by a stretch of pyrimidines is the processing signal, which may be recognized by the processing enzyme and/or a required accessory factor.     

Construction and phenotypic analysis of mice carrying a duplication of the major histocompatibility class I (MHC-I) locus

Copy number variation (CNV) has been associated increasingly with altered susceptibility to human disease. Large CNVs are likely to incur disease risk or resilience via predictable changes in gene dosage that are relatively straightforward to model using chromosomal engineering in mice. The classical class I major histocompatibility locus (MHC-I) contains a dense set of genes essential for innate immune system function in vertebrates. MHC-I genes are highly polymorphic and genetic variation in the region is associated with altered susceptibility to a wide variety of common diseases. Here we investigated the role of gene dosage within MHC-I on susceptibility to disease by engineering a mouse line carrying a 1.9-Mb duplication of this region [called Dp(MHC-I)]. Extensive phenotypic analysis of heterozygous (3N) Dp(MHC-I) animals did not reveal altered blood and stem cell parameters, susceptibility to high-fat diet, death by cancer, or contact dermatitis. However, several measures of disease severity in a model of atherosclerosis were improved, suggesting dosage-sensitive modulators of cardiovascular disease. Homozygous Dp(MHC-I)/Dp(MHC-I) mice demonstrated embryonic lethality. These mice serve as a model for studying the consequences of targeted gene dosage alteration in MHC-I with functional and evolutionary implications.