Positive correlation between the occurrence of chromosome breakage and the induction of point mutations associated with male recombination 31.1 MRF system of hybrid dysgenesis in Drosophila melanogaster

One of the weak singed (snw) mutations, induced by the 31.1 MRF in the X-chromosome of a laboratory strain, is highly unstable, often changing to either a strong expression (snst) or reverting to wild type (sn+). The present study shows that the X-chromosome carrying the (snw) mutation and the X-chromosome carrying one of the snst alleles derived from the snw mutation generate different frequencies of deletions associated with the w locus. Moreover, they produce different frequencies of mutations associated with the w locus in males after the reintroduction of the 31.1 MRF second chromosome. The occurrence of the deletions and the induction of the mutations are positively correlated and increase when flies are raised at a higher temperature. These data indicate that the induction of the w mutations follows the generation of chromosome breaks in the w locus. The break-points of the recovered deletions occurred in specific sites in the 3C subdivision. Furthermore both snw and snst X-chromosomes induce different frequencies of non-disjunction in females depending on the culture temperature and the genetic background. The present data also show that the 23.5 MRF second chromosome which exhibits specific differences in its activities from the 31.1 MRF is unable to induce w mutations. This fact supports our previous indications that the 31.1 MRF and the 23.5 MRF are not identical.     

On the toleration of duplications and deletions by the Vicia faba genome

Summary From eight pairs of crosses between differently reconstructed diploid karyotypes of Vicia faba, the progeny after selfing of plants heterozygous for both parental chromosome reconstructions were inspected for occurrence and transmission of duplications and deletions of defined chromosome segments, comprising together about one third of the metaphase genome length. The duplications and deletions studied involved either one or more chromosome segments of the respective karyotype (0.8%–9.1% of the metaphase length). They arose during meiosis in double heterozygotes by crossing over between partially homologous chromosomes or by mis-segregation from multivalents. While most duplications, provided they were not accompanied by deletions and in dependence on the segment involved, were viable and transmissible, even in homozygous state, deletions had lethal effects on gametes of both sexes.     

Chromosome rearrangement patterns of an SD chromosome (SDKona-2) in Drosophila melanogaster caused by hybrid dysgenesis.

The types and frequencies of spontaneous chromosome rearrangements caused by hybrid dysgenesis were studied in a second chromosome autosome of Drosophila melanogaster. This second chromosome, being an SD chromosome, had two important advantages over other autosomes for this study: (i) it had the two inversions characteristic of a standard SD-72 chromosome type, which distinguished it from its homolog in polytene chromosome spreads, and (ii) because of the meiotic drive associated with the segregation distorter system, it was preferentially transmitted to the next generation. The chromosome mutation frequency of this chromosome (given the name SDKona-2) was 8.3 and 11.7% in the F2 and F3 generations, respectively. The types of new chromosome rearrangements observed in the first four generations included paracentric inversions, pericentric inversions, duplications, deletions, reciprocal translocations (involving the third chromosome), and transpositions. Small paracentric inversions were the most common type of new rearrangement. Later, over 35 generations, some of these new rearrangements changed, either by becoming more complex or by being replaced with yet another new chromosome rearrangement. Duplications were unstable and were replaced by paracentric inversions whose breakpoints were on either side of the duplication. Transpositions arose both from a single multibreak event and from a series of two-break events.     

Ribosomal proteins. XXX. Specific protein binding sites on 23S RNA of Escherichia coli

Summary Strains of A. nidulans with a chromosome segment in duplicate (one in normal position, one translocated to another chromosome) are unstable at mitosis. During vegetative growth they produce variants which result from deletions in either of the duplicate segments.Caffeine increased the frequency of deletions from the duplicate segments of an unbalanced haploid a) without changing the proportions of the different deletion types and b) under conditions in which there were few, if any, induced breaks in the same segments of a balanced diploid. One possible explanation is that caffeine stimulates the mechanism which, in unbalanced strains, produces replication errors leading to deletions; an alternative is that it exposes the intrinsic instability of duplication strains by preventing the repair of spontaneous replication errors.     

Spontaneous IR Duplications Generated at Mitosis in ASPERGILLUS NIDULANS: Further Evidence of a Preferential Site of Transposed Attachment

A radiation-induced translocation, T(IIR----IIIL), has been shown to be nonreciprocal and to have most of IIR, including its terminus, attached uninverted to the terminus of IIIL.--Progeny with the IIR segment in duplicate, obtained from crosses of T(IIR----IIIL) to strains with a standard genome, were unstable at mitosis; like earlier duplication strains, they suffered deletions from either duplicate segment. Frequent mitotic crossing over occurred between the duplicate IIR segments so that, following deletions, more than two classes of stable, balanced products arose from each heterozygous duplication strain.-- Spontaneous, mitotically arising duplications of the IR segment, bearing the rate-limiting adE20 allele, can be selected on adenine-free medium on which they emerge as vigorous sectors from the stunted adE20 colony. It was shown previously that most such duplications, when selected from a strain with standard genome, had the terminal IR segment attached to the end of IIR. Selection has now been made from an adE20 strain carrying T(IIR----IIIL), and seven of the 13 independent IR duplications were linked to the III-IIR translocation complex. In three strains analyzed further, the duplicate IR segments, which included the IR terminus, were attached uninverted to the terminus of IIR; the segments of IR were of approximately equal genetic length.--This supports earlier suggestions that there is a preferential site for the initiation of IR duplications and a preferential site, the IIR terminus, for their attachment.     

Simultaneous detection of structural and numerical chromosome abnormalities in sperm of healthy men by multicolor fluorescence in situ hybridization

Both structural and numerical chromosome aberrations in sperm represent important categories of paternally transmitted genetic damage. Therefore, a new multiprobe fluorescence in situ hybridization (FISH) method, using DNA probes for three targets (centromere and telomere of chromosome 1, centromere of chromosome 8), was developed to detect human sperm carrying three types of chromosomal defects: (1) terminal duplications or deletions in chromosome 1p, (2) aneuploidy involving chromosomes 1 or 8, and (3) diploidy. Baseline frequencies were determined for three healthy donors who had been previously evaluated for sperm cytogenetics by the human-sperm/hamster-oocyte cytogenetic technique (hamster technique). Among ∼120 000 sperm analyzed by the new FISH method, the average baseline frequencies of sperm carrying telomeric duplications and deletions of 1p were 3.2 ± 1.9 and 2.9 ± 3.6 per 10⁴, respectively. Diploid sperm was found in an average frequency of 6.6 ± 4.0 per 10⁴. Average frequencies of disomic sperm for chromosomes 1 or 8 were 1.7 ± 2.2 and 1.9 ± 2.3 per 10⁴, respectively. Inter-individual differences were observed for deletions of 1p but not for the other sperm phenotypes. A good correlation was obtained between the frequencies of sperm with structural chromosome aberrations detected with the new assay and the frequency of sperm carrying premeiotic or meiotic cytogenetic damage detected with the hamster technique. The observed levels of numerical aberrations with the new FISH assay were within range of the baseline frequencies reported by the hamster technique. The newly developed FISH assay has promising applications in genetic, clinical, physiological and toxicological studies. Received: 26 February 1996 / Revised: 6 May 1996     

Variability of the immunoglobulin heavy chain constant region locus: a population study

The Immunoglobulin Heavy chain Constant region (IGHC) locus is a multigene family composed of highly homologous segments often involved in unequal crossings over that lead to deleted and duplicated haplotypes. The frequencies of these haplotypes in 558 individuals from Lombardy, Veneto, Puglia and Sardinia were determined by Pulsed Field Gel Electrophoresis (PFGE), followed by Southern blotting with four IGHC probes, and compared with those observed in 110 subjects from Piedmont. Twenty deletions and 60 duplications were characterized, all in heterozygous individuals except for 2 homozygous deletions. The differences in frequency between the five populations were not significant. The deletions/duplications involved one or more genes: GP-A2, A1-E and G4 duplications, and A1-E and GP-A2 deletions were the most common. Four new duplications are described: three, involving the genes from GP to A2, from G2 to G4, and G4, are counterparts of known deletions. The fourth duplication spans from GP to G2. A G1 deleted heterozygous individual never previously described in Italy is reported. All the rearranged haplotypes seem to be the result of unequal crossing over. The difference between the number of duplications and deletions was significant in Sardinia, Lombardy, Puglia and in the total of 668 subjects (P < 0.001). This may be due to selection or genetic drift.     

Telomeres and chromosome instability

Genomic instability has been proposed to play an important role in cancer by accelerating the accumulation of genetic changes responsible for cancer cell evolution. One mechanism for chromosome instability is through the loss of telomeres, which are DNA-protein complexes that protect the ends of chromosomes and prevent chromosome fusion. Telomere loss can occur as a result of exogenous DNA damage, or spontaneously in cancer cells that commonly have a high rate of telomere loss. Mouse embryonic stem cells and human tumor cell lines that contain a selectable marker gene located immediately adjacent to a telomere have been used to investigate the consequences of telomere loss. In both cell types, telomere loss is followed by either the addition of a new telomere on to the end of the broken chromosome, or sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles that result in DNA amplification and large terminal deletions. The regions amplified by B/F/B cycles can then be transferred to other chromosomes, either through the formation of double-minute chromosomes that reintegrate at other sites, or through end-to-end fusions between chromosomes. B/F/B cycles eventually end when a chromosome acquires a new telomere by one of several mechanisms, the most common of which is translocation, which can involve either nonreciprocal transfer or duplication of all or part of an arm of another chromosome. Telomere acquisition involving nonreciprocal translocations results in the loss of a telomere on the donor chromosome, which subsequently becomes unstable. In contrast, translocations involving duplications do not destabilize the donor chromosome, although they result in allelic imbalances. Thus, the loss of a single telomere can generate a wide variety of chromosome alterations commonly associated with human cancer, not only on the chromosome that originally lost its telomere, but other chromosomes as well. Factors promoting spontaneous telomere loss and the resulting B/F/B cycles are therefore likely to be important in generating the karyotypic changes associated with human cancer.     

Parental and chromosomal origin of unbalanced de novo structural chromosome abnormalities in man

We report the parental origin, and where possible the chromosomal origin of 115 de novo unbalanced structural chromosome abnormalities detectable by light microscopy. These consisted of 39 terminal deletions, 35 interstitial deletions, 8 rings, 12 duplications and 21 unbalanced translocations. In all categories the majority of abnormalities were of paternal origin, although the proportions varied from a high of 84% in the interstitial deletions and rings to a low of 58% in the duplications. Among the interstitial deletions and duplications, there were approximately equal numbers of intra- and interchromosomal abnormalities, while the majority of unbalanced translocations were isodisomic for the duplicated chromosome. The examination of the parental ages in the four main classes of abnormality showed terminal deletions of maternal origin to be associated with a significantly reduced maternal age. Thus, there is a clear propensity for structural chromosome abnormalities to occur in male germ cells, although the chromosomal origin seems similar irrespective of the parental origin.     

Segregation and transmission of chromosomes from a reciprocal translocation in Gallus domesticus cockerels

Segregation behavior of a reciprocal translocation involving the long arm of chromosome No. 1 and a microchromosome was studied in secondary spermatocytes and embryos produced by heterozygous cockerels. The types and frequencies of the various balanced and unbalanced chromosome complements were determined. Complementary products of segregation did not occur in the expected ratios of 1:1 in secondary spermatocytes. The excess of spermatocytes with deficiency of the long arm and duplication of the short arm might be the result of lagging of the long arm at meiosis I, the centromere of the long arm being derived from a microchromosome. In the samples of secondary spermatocytes and embryos 52.5% and 49.6%, respectively, contained balanced chromosome complements. A significantly higher proportion of duplications and deletions of the long arm was seen in embryos than in secondary spermatocytes. Conversely, a lower proportion of duplications and deletions of the short arm was seen in embryos than in secondary spermatocytes. Apparently, spermatogenic cells bearing different unbalanced genomic contents are not equally viable or fertile.     

Stability of large segmental duplications in the yeast genome

The high level of gene redundancy that characterizes eukaryotic genomes results in part from segmental duplications. Spontaneous duplications of large chromosomal segments have been experimentally demonstrated in yeast. However, the dynamics of inheritance of such structures and their eventual fixation in populations remain largely unsolved. We analyzed the stability of a vast panel of large segmental duplications in Saccharomyces cerevisiae (from 41 kb for the smallest to 268 kb for the largest). We monitored the stability of three different types of interchromosomal duplications as well as that of three intrachromosomal direct tandem duplications. In the absence of any selective advantage associated with the presence of the duplication, we show that a duplicated segment internally translocated within a natural chromosome is stably inherited both mitotically and meiotically. By contrast, large duplications carried by a supernumerary chromosome are highly unstable. Duplications translocated into subtelomeric regions are lost at variable rates depending on the location of the insertion sites. Direct tandem duplications are lost by unequal crossing over, both mitotically and meiotically, at a frequency proportional to their sizes. These results show that most of the duplicated structures present an intrinsic level of instability. However, translocation within another chromosome significantly stabilizes a duplicated segment, increasing its chance to get fixed in a population even in the absence of any immediate selective advantage conferred by the duplicated genes.     

Analysis of complex Y chromosome aberrations using a single DNA probe (Y-367)

A specific cloned DNA sequence (Y-367) detects at least four loci in the euchromatic long arm and in the short arm of the human Y chromosome. Deletion mapping assigns one locus to the distal euchromatic long arm, another to a region close to the centromere on either Yq or Yp, and two additional loci to the Y short arm. Y-367 may thus be used for the rapid screening of even complex Y chromosome aberrations. This is exemplified in a 45,X male with Y chromosome material on the long arm of chromosome 10 by the detection of an inversion of a portion of Yp and by the confirmation of duplications and deletions in two individuals with duplications of part of the Y chromosome.     

Physical and genetic characterization reveals a pseudogene, an evolutionary junction, and unstable loci in distal Xq28

A large portion of human Xq28 has been completely characterized but the interval between G6PD and Xqter has remained poorly understood. Because of a lack of stable, high-density clone coverage in this region, we constructed a 1.6-Mb bacterial and P1 artificial chromosome (BAC and PAC, respectively) contig to expedite mapping, structural and evolutionary analysis, and sequencing. The contig helped to reposition previously mismapped genes and to characterize the XAP135 pseudogene near the int22h-2 repeat. BAC clones containing the distal int22h repeats also demonstrated spontaneous rearrangements and sparse coverage, which suggested that they were unstable. Because the int22h repeats are involved in genetic diseases, we examined them in great apes to see if they have always been unstable. Differences in copy number among the apes, due to duplications and deletions, indicated that they have been unstable throughout their evolution. Taking another approach toward understanding the genomic nature of distal Xq28, we examined the homologous mouse region and found an evolutionary junction near the distal int22h loci that separated the human distal Xq28 region into two segments on the mouse X chromosome. Finally, haplotype analysis showed that a segment within Xq28 has resisted excessive interchromosomal exchange through great ape evolution, potentially accounting for the linkage disequilibrium recently reported in this region. Collectively, these data highlight some interesting features of the genomic sequence in Xq28 and will be useful for positional cloning efforts, mouse mutagenesis studies, and further evolutionary analyses.     

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.     

Genomic rearrangements resulting in PLP1 deletion occur by nonhomologous end joining and cause different dysmyelinating phenotypes in males and females

In the majority of patients with Pelizaeus-Merzbacher disease, duplication of the proteolipid protein gene PLP1 is responsible, whereas deletion of PLP1 is infrequent. Genomic mechanisms for these submicroscopic chromosomal rearrangements remain unknown. We identified three families with PLP1 deletions (including one family described elsewhere) that arose by three distinct processes. In one family, PLP1 deletion resulted from a maternal balanced submicroscopic insertional translocation of the entire PLP1 gene to the telomere of chromosome 19. PLP1 on the 19qtel is probably inactive by virtue of a position effect, because a healthy male sibling carries the same der(19) chromosome along with a normal X chromosome. Genomic mapping of the deleted segments revealed that the deletions are smaller than most of the PLP1 duplications and involve only two other genes. We hypothesize that the deletion is infrequent, because only the smaller deletions can avoid causing either infertility or lethality. Analyses of the DNA sequence flanking the deletion breakpoints revealed Alu-Alu recombination in the family with translocation. In the other two families, no homologous sequence flanking the breakpoints was found, but the distal breakpoints were embedded in novel low-copy repeats, suggesting the potential involvement of genome architecture in stimulating these rearrangements. In one family, junction sequences revealed a complex recombination event. Our data suggest that PLP1 deletions are likely caused by nonhomologous end joining.     

Inversions of chromosome arms 4AL and 2BS in wheat invert the patterns of chiasma distribution

In many species, including wheat, crossing over is distal, and the proximal regions of chromosome arms contribute little to genetic maps. This was thought to be a consequence of terminal initiation of synapsis favoring distal crossing over. However, in an inverted rye chromosome arm, the pattern of metaphase I chiasmata was also inverted, suggesting that crossover frequencies were specific to chromosome segments. Here, wheat chromosome arms 2BS and 4AL, with essentially entire arms inverted in reverse tandem duplications (rtd), were studied in the MI of meiosis. Inversion–duplication placed the recombining segments in the middle of the arms. While the overall pairing frequencies of the inverted–duplicated arms were considerably reduced relative to normal arms, chiasmata, if present, were always located in the same regions as in structurally normal arms, and relative chiasma frequencies remained the same. The frequencies of fragment or fragment + bridge configurations in AI and AII indicated that of the two tandemly arranged copies of segments in rtds, the more distal inverted segments were more likely to cross over than the segments in their original orientations. These observations show that also in wheat, relative crossover frequencies along chromosome arms are predetermined and independent of the segment location. The segments normally not licensed to cross over do not do so even when placed in seemingly most favorable positions for it.     

Genetic duplications in bacteria and their relevance for genetic toxicology

Tandem genetic duplications of various lengths occur at high frequency and at many chromosomal locations in bacteria. Most duplications are formed and lost by recombinational mechanisms. Since they readily give rise to haploid segregants, duplications are characteristically unstable. Various selection procedures permit measurements of duplication frequencies, and several mutagens have been shown to induce the formation of duplications in haploid and the loss of duplications from merodiploid bacteria. Although the data base is not extensive, it includes agents that interact with DNA by a variety of molecular mechanisms. Grounds on which the induction of genetic duplications in bacteria can be relevant for genetic toxicology are discussed.     

The Instability of Neurospora Duplication Dp(IL-->IR)H4250 , and Its Genetic Control

Previous work (Newmeyer and Taylor 1967) showed that a nontandem duplication, Dp(IL-->IR)H4250, is regularly produced by recombination in crosses heterozygous for the effectively terminal pericentric inversion In(IL-->IR)H4250. The duplications initially have strongly inhibited growth because they are heterozygous for mating type, which behaves like a vegetative-incompatibility (het) locus. Such cultures "escape" from the inhibition as a result of events that eliminate the mating-type heterozygosity. The product of a given escape event may be barren or fertile. (Neurospora duplications are characteristically barren; that is, when crossed, they make many perithecia but few ascospores.)-The present paper reports on a genetic analysis of the instability of Dp(IL-->IR)H4250 . Most of the barren escape products behave as if due either to mitotic crossovers, which make mating type and distal markers homozygous, or to very long deletions which uncover mating type and all distal markers; presumably the latter would retain enough duplicated material to render them barren. It is difficult to distinguish between these two possibilities, but homozygosis seems more probable and has been clearly demonstrated in one case. Only a few barren escapes could be due to short deletions or to changes at the mating-type locus.-The fertile escape products appear to be euploid. Most of these behave as if they arose by precise deletion of one or the other duplicated segment, thus restoring one of the parental sequences. A large majority of the precise deletions restore normal sequence; only a few restore inversion sequence. Preferential restoration of the normal sequence has also been found by other workers for Neurospora duplications from several other rearrangements. A hypothesis is presented to explain these findings; it is posulated that the precise deletions result from mitotic crossing over in homologous material located at chromosome tips and tip-break-points.-There is a smaller group of fertile escapes that are unlike either parental sequence; at least one of these involves a chromosome break outside the duplicated region.-Duplications in which the vegetative incompatibility is suppressed by the unlinked modifier tol are extremely barren; they only rarely lose a duplicated segment so as to become fertile.-The instability of Dp(IL-->IR)H4250 , with and without tol, is markedly altered by factors in the genetic background. The two factors studied in detail have qualitatively different effects.     

Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination—Deficient Escherichia Coli strains

The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied. The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling. Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome. The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells. The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9]. In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions. These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells. In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.