The effects of tandem duplications on crossing over inDrosophila melanogaster

Each of three tandem duplications,Bar, Beadex ^r49k andDp(I)z-w, when homozygous increases crossing over in their environs in excess of the genetic length of the duplication.Detailed crossing over studies withDp(I)z-w showed that in the duplication homozygotes interference is reduced and when combined with heterologous autosomal inversions, double crossovers occurring in less than 10 map units are readily recovered.These results are interpreted in terms of the concept of effective pairing and suggest that tandem duplications increase crossing over by increasing effective pairing.     

Frequency of x-ray induced mitotic recombination in genotypes with different X-Y-combination-chromosomes in Drosophila melanogaster

Summary In first and second instar larvae of Drosophila melanogaster mitotic recombination has been induced by X-rays. Resulting mosaic spots were analysed in the eyes of adult females. The five examined genotypes varied in the combination of different X-chromosomes and in the presence or absence of the duplication Dp(1;3)N ^264-58. X-chromosomes used have been elongated by proximally or distally linked arms of the Y-chromosome. The portion of Y-heterochromatin in the genome is negatively correlated with the frequency of twin mosaic spots (Fig. 1). The frequency of these spots is lower in flies bearing the duplication than in those without the duplication. With respect to the X-chromosome combinations, there are no marked differences in the frequencies of single mosaic spots (Fig. 2); with respect to the duplication they are absent. In the genotype Y ^S X/X.Y ^S prophases of neuroblast mitoses (Fig. 4) show normal pairing of homologous chromosome sections. Reasons for the different spot frequencies are discussed.

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Vorgelegt von E. Hadorn     

Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP)

Hereditary neuropathy with liability to pressure palsies (HNPP) is in most cases associated with an interstitial deletion of the same 1.5-Mb region at 17p11.2 that is duplicated in Charcot-Marie-Tooth type 1A (CMT1A) patients. Unequal crossing-over following misalignment at flanking repeat sequences (CMT1A-REP), either leads to tandem duplication in CMT1A patients or deletion in HNPP patients. With the use of polymorphic DNA markers located within the CMT1A/HNPP duplication/deletion region we detected the HNPP deletion in 16 unrelated HNPP patients, 11 of Belgian and 5 of French origin. In all cases, the 1.5-Mb size of the HNPP deletion was confirmed by EcoRI dosage analysis using a CMT1A-REP probe. In the 16 HNPP patients, the same 370/320-kb EagI deletion-junction fragments were detected with pulsed field gel electrophoresis (PFGE), while in CMT1A patients, a 150-kb EagI duplication-junction fragment was seen. Thus, PFGE analysis of EagI-digested DNA with a CMT1A-REP probe allows direct detection of the HNPP deletion or the CMT1A duplication for DNA diagnostic purposes.     

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.     

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.     

Different DNA replication behavior of a tandem duplication in Drosophila melanogaster

Salivary gland chromosome DNA replication of a heterozygous tandem duplication Dp(1 1)Gr/+ and a wild type strain in Drosophila melanogaster has been studied by ³H-thymidine autoradiography. Three parameters-labeling frequency, labeling intensity and labeling pattern—have been used to characterize the replication behavior of late labeling spots in the distal part (1A–9A) of the X chromosomes for both genotypes. — Differences in the labeling frequency between homologous subdivisions in both genotypes have been found. Changes of the DNA replication behavior are also indicated by the comparison of labeling patterns in both Drosophila strains. Furthermore, in comparable replication phases the labeling intensities of the Dp(1 1)Gr/+ subdivisions are different from those of the homologous subdivisions in +/+ chromosomes, even where the different DNA amount of both genotypes is taken into consideration.     

Partial hyperploidy of the X-chromosome inDrosophila hydei leading to duplication of male gonadal and genital structures

Summary Introduction of two doses of the X-chromosomalw ^mCo duplication next to a normal X-chromosome in males ofD. hydei leads to duplication of testis tissue and structures derived from the male genital disc. The effect of this partial hyperploidy of the X-chromosome seems restricted to the male. We tentatively conclude that this part of the X-chromosome contains some factor(s) which may specifically affect the reproductive system and analia of males.     

An X chromosome locus in Drosophila melanogaster that enhances survival of the triplo-lethal genotype,Dp-(Tpl)

Only a single locus (Tpl) is known in the Drosophila melanogaster genome that leads to early lethality when present as a heterozygous duplication (three doses) or deficiency (one dose). We report the recovery of third instar larvae (and of occasional adults) carrying a duplication for the triplo-lethal locus, Dp(Tpl). Karyotype analysis of the larvae showed that the individuals surviving were almost entirely 3X;2A metafemales. We examined the question of whether the entire X or a single X locus was a major factor permitting survival. X-Y translocations were used to produce females hyperploid for different portions of the X and carrying Dp(Tpl). Analysis of metaphase chromosomes by quinacrine fluorescence pattern indicates that the X chromosome region between 6D and 7DE must be present in an extra copy to enhance the survival of Tpl duplication-bearing females. Another type of experiment suggests that it is the region between 7C and 7DE which is essential.     

Genomic rearrangement at 10q24 in non-syndromic split-hand/split-foot malformation

Split-hand/split-foot malformation (SHFM) is a congenital limb malformation characterized by a median cleft of hand and/or foot due to the absence of central rays. Five loci for syndromic and non-syndromic SHFM, termed SHFM1-5, have been mapped to date. Recently, a 0.5 Mb tandem genomic duplication was found at chromosome 10q24 in SHFM3 families. To refine the minimum duplicated region and to further characterize the SHFM3 locus, we screened 28 non-syndromic SHFM families for tandem genomic duplication of 10q24 by Southern blot and sequence analysis of the dactylin gene. Of 28 families, only two showed genomic rearrangements. Representative patients from the two families exhibit typical SHFM, with symmetrically affected hands and feet. One patient is a familial case with a 511,661 bp tandem duplication, whereas the second is a sporadic case arising from a de novo, 447,338 bp duplication of maternal origin. The smaller duplication in the second patient contained the LBX1, BTRC, POLL, and DPCD genes and a disrupted extra copy of the dactylin gene, and was nearly identical to the smallest known duplicated region of SHFM3. Our results indicate that genomic rearrangement of SHFM3 is rare among non-syndromic SHFM patients and emphasize the importance of screening for genomic rearrangements even in sporadic cases of SHFM.     

Unequal crossing-over within the B duplication of Drosophila melanogaster: a molecular analysis.

The B mutation is associated with a tandem duplication of 16A1-16A7. It is unstable, mutating to wild type and to a more extreme form at a frequency of one in 1000 to 3000. The reversion to wild type is associated with the loss of one copy of the duplication, whereas the mutation to extreme B is associated with a triplication of the region. The instability of B has been attributed to unequal crossing-over between the two copies of the duplication. Recent molecular data show that there is a transposable element, B104, between the two copies of the duplication and support the hypothesis that this element generated the duplication via a recombination event. These data suggest that unequal crossing-over within the duplication may not be the cause of the instability of B. Instead, the instability may be caused by a recombination event involving the B104 element. This issue was addressed using probes for the DNA on either side of the B104 element at the B breakpoint. All of the data indicate that the B104 element is not involved in the instability of B and support the original unequal crossing-over model.     

Cytogenetics of nucleolus-rranspositions in Drosophila melanogaster

Summary The results from cytological identification of 125 radiation-induced specificlocus mutations revealed that the relative frequency of nucleolus-associated rearrangements could be increased substantially by selection of specific types of position-mutations for cytological analysis.One of the five nucleolus-involved rearrangements investigated was an inversion, In(1)lz ^sB, and four were deletion-insertions: Tp(1)ct ^6a1, Tp(1)lz ⁴⁹¹, Tp(1)lz ¹⁴⁴ and Dp(1;3)in ^61j2.The cytomorphology of the transposed nucleolus and associated bands was easier to resolve in the salivary gland chromosomes of the rearrangements than when the nucleolus was in its normal position in the proximal region of the X-chromosome. However, the extent of the NO and adjacent chromatin involved in the transposition could not be positively delimited because of the tendency for a variegation-type alteration in the morphology of the bands adjacent to the transposed nucleolus.This investigation was supported in part by U.S. Public Health Service Research Grant, GM 15009, and in part by a grant from the Finnish National Research Council for Sciences.     

Distribution of polymorphic gene duplication at theGpdh locus in natural populations ofDrosophila melanogaster

Summary The glycerol-3-phosphate dehydrogenase (GPDH, E. C. 1.1.1.8) gene ofDrosophila melanogaster contains a tandem duplication of a 4.5-kb-long DNA fragment. Survey of theGpdh gene region by the Southern blot analysis revealed the following features of this gene duplication: (1) The duplication was not observed in chromosome lines that carryIn(2L)t, a cosmopolitan chromosomal inversion in this species. The duplication and the inversion are in linkage disequilibrium. (2) The duplication is polymorphic in the Japan and US natural populations examined. Its frequency is 0.26 on an average inIn(2L)t-free chromosomes. (3) Triplication is absent or has not become frequent in the populations surveyed. Possible evolutionary factors of this duplication polymorphism are discussed.     

Unequal crossing-over in Escherichia coli

Unequal crossing-over between sister chromosomes in the process of DNA replication in Escherichia coli leads to the formation of tandem duplications, thus enhancing the activity of certain genes. In conjugational matings between genetically marked E. coli strains, unequal crossing-over leads to the formation of heterozygous tandem duplications. Studying these duplications as model systems allowed the conclusion that unequal crossing-over between direct DNA repeats of sister chromosomes is the main pathway of the formation of selected recombinants in E. coli strains carrying duplications. This was inferred from the data on the segregation of homozygous diploid recombinants by heterozygous duplications. Unequal crossing-over between sister chromosomes occurs as adaptive exchange providing the survival of the greater part of bacterial cells on a selective medium. The known phenomenon of adaptive mutagenesis may also be a consequence of unequal exchanges at the level of DNA mononucleotide repeats.     

Molecular characterization of four intra-t mouse recombinants

Recombination in the proximal region of mouse chromosome 17 is greatly reduced in heterozygotes carrying the wild-type and thet complex-type chromosomes. The reason for this is the presence of two non-overlapping inversions in thet complex. Rare crossing-over does, however, occur within thet complex of thet/+ heterozygotes. Here we characterize four such exceptional intra-t recombinants,t ^Tu1 throught ^Tu4 . To map the positions of the genetic exchange in these four recombinants, we analyzed them with DNA probes specific for 16 loci distributed over thet complex. The analysis revealed that in three of the four recombinants, an equal crossing-over occurred in the short region between the two inversions, producing chromosomes carrying either the proximal inversion only (t ^Tu1 andt ^Tu4 ) or the distal inversion only (t ^Tu2 ). In the fourth recombinant (t ^Tu3 ), unequal crossing-over occurred within the proximal inversion between lociD17Leh119 andD17Leh66, producing a chromosome in which the region containing lociTcp-1, T, andD17Tu5 has been duplicated. The duplication of theBrachyury locus leads to the suppression of the tail-shortening effect normally produced by the interaction of the dominant (T) and recessive (tct) alleles at this locus so that theT/t ^Tu3 mice have normal tails.     

Sequence signatures of a recent chromosomal rearrangement in <Emphasis Type="Italic">Drosophila mojavensis</Emphasis>

The X-chromosome inversion, Xe, distinguishes Drosophila mojavensis and D. arizonae. Earlier work mapped the breakpoints of this inversion to large intervals and provided hypotheses for the locations of the breakpoints within 3000-bp intergenic regions on the D. mojavensis genome sequence assembly. Here, we sequenced these regions directly in the putatively ancestral D. arizonae X-chromosome. We find that the two inversion breakpoints are near an inverted gene duplication and a common repetitive element, respectively, and these features were likely present in the non-inverted ancestral chromosome on the D. mojavensis lineage. Contrary to an earlier hypothesis, the inverted gene duplication appears to predate the inversion. We find no sequence similarity between the breakpoint regions in the D. mojavensis ancestor, excluding an ectopic-exchange model of chromosome rearrangements. We also found no evidence that staggered single-strand breaks caused the inversion. We suggest these features may have contributed to the chromosomal breakages resulting in this inversion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995     

Non-random loss of uninterrupted ribosomal DNA repeating units upon induction of a bobbed mutation

To investigate the physical organization of ribosomal RNA genes of two bobbed (bb) loci carried by the Dp(1;f)122 free duplication, a wild type and a deleted one derived from it, genomic DNAs from XXNO-/Dp122bb+ and XXNO-/Dp122bb adult females were analyzed by restriction enzyme digestions. We found that in the bb mutant there was a loss of uninterrupted genes, while genes interrupted by type I and type II insertions remained apparently unchanged. This is an indication that at least in this wild type bb+ locus, carried by the 122 free duplication, the different repeating units are not distributed randomly. In fact, after digestion of the rDNA carried by the bb+ duplication with the enzyme BamHI that cuts only in type I insertions, we have obtained long uncleaved fragments of DNA containing uninterrupted genes.     

Lineage-Specific Homogenization of the Polyubiquitin Gene Among Human and Great Apes

Ubiquitin is a highly conserved protein, and is encoded by a multigene family among eukaryote species. The polyubiquitin genes, UbB and UbC, comprise tandem multiple ubiquitin coding units without a spacer region or intron. We determined nucleotide sequences for the UbB and UbC of human, chimpanzee, gorilla, and orangutan. The ubiquitin repeat number of UbB was constant (3) in human and great apes, while that of UbC varied: 6 to 11 for human, 10 to 12 for chimpanzee, 8 for gorilla, and 10 for orangutan. The heterogeneity of the repeat number within closely related hominoid species suggests that a lineage-specific unequal crossover and/or gene duplication occurred. A marked homogenization of UbC occurred in gorilla with a low level of synonymous difference (p_s). The homogenization of UbC also occurred in chimpanzee and less strikingly in human. The first and last ubiquitin coding units of UbC were clustered independently between species, and less affected by homogenization during the hominoid evolution. Therefore, the homogenization of ubiquitin coding units is likely due to an unequal crossing-over inside the ubiquitin units. The lineage-specific homogenization of UbC among closely related species suggests that concerted evolution has a key role in the short-term evolution of UbC.