The effects of coumarin on the frequency of deletions in a duplication strain of Aspergillus nidulans.

Summary Strains of A. nidulans with a chromosome segment in duplicate show instability resulting from deletions in either of the duplicate segments. In Dp (I, II) strains, with the terminal segment of IR attached terminally to IIR, spontaneous deletions occur most frequently, though not exclusively, from the translocated segment. Coumarin, at concentrations which did not affect viability or growth rate, enhanced the instability of Dp (I, II) strains by selectively increasing only the deletion class of highest spontaneous frequency. This selective action is interpreted tentatively as due to inhibition of the repair of a particular class of DNA lesion occurring spontaneously in the attachment region of Dp (I, II) strains.     

Euploid derivatives of duplications from a translocation in neurospora

Nontandem terminal chromosome duplications derived from N. crassa translocation T(I-->VI)NM103 give rise mitotically to some daughter nuclei which have become euploid by loss of one or the other of the two duplicated segments. Loss of the segment in normal sequence occurs as often as loss of the translocated segment. This is in contrast to all of several other Neurospora duplications that have been studied, where loss of the segment in normal sequence is absent or rare.--T(NM103) has the distal two thirds of linkage group IR exchanged with the right tip of VI. Crosses to normal sequence produce a class of morphologically distinct progeny with IR chromosome duplications. For a few days after germination, test crosses of these progeny are barren (make perithecia but few or no spores, as observed commonly with Neurospora duplications). Growing duplication cultures become fertile by accumulating nuclei which have been reduced to either normal sequence (by loss of the segment in translocation sequence) or translocation sequence (by loss of the segment in normal sequence). Both types usually appear within the first week of growth. Naturally formed mixtures or heterokaryons of NM103 duplication nuclei and their reduced euploid products have been studied by plating and by progeny testing. Determination of nuclear type is based on culture morphology, expression of genetic markers, and crossing behavior. Within the limits of testing, loss is found to begin precisely at the interchange points. The unique finding of frequent breakdown of normal-sequence linkage group I chromosomes is not dependent on the strain from which the chromosome was derived. Many different strains were tested, and for each one evidence was found that nuclei reduced to translocation sequence had been produced from duplication nuclei by loss of the segment in normal sequence.     

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.     

DNA amplification and rearrangements in archival Salmonella enterica serovar Typhimurium LT2 cultures

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.     

The genetic instability and mutagenic interaction of chromosomal duplications present together in haploid strains of Aspergillus nidulans

Previous work was shown that strains of Aspergillus nidulans with a chromosome segment in duplicate (one in normal position, one translocated to another chromosone) are unstable. Deletions occur from either duplicate segment. The present work has shown that when a chromosome I duplication and a chromosome III duplication are together in a haploid, deletions from the intact III duplication generally precede deletions from particular sections of the I duplication. Furthermore, the III duplication can enhance to some (but not major) extent the frequency of deletions from the I duplication. After the III duplication becomes reduced in size as a result of the loss of chromosomal material from the translocated duplicate III segment, such a reduced III duplication can greatly enhance the frequency of deletions from the I duplication. In other words a III duplication of reduced size can promote far more deletions from the I duplication than the intact III duplication. The major increase in the deletional instability of the duplication as promoted by the reduced III duplication is confined to the translocated duplicate I segment. The reduced III duplication can induce deletions from a section of the translocated duplicate I segment in accord with a temporal programme, and it appears that a particular region of the I duplication is far more under the mutagenic influence of the reduced III duplication than another region. Moreover, there is indication that there is a differential effet of two generally different genetic backgrounds on the susceptibility of duplication-regions to deletion.     

Heterokaryon Incompatibility Genes in NEUROSPORA CRASSA Detected Using Duplication-Producing Chromosome Rearrangements

Evidence is presented for five or six previously undetected heterokaryon incompatibility (het) loci, bringing to about ten the number of such genes known in Neurospora crassa. The genes were detected using chromosome duplications (partial diploids), on the basis of properties previously known for het genes in duplications. Duplications homozygous for het genes are usually normal in growth and morphology, whereas those heterozygous are strikingly different. The heterozygotes are inhibited in their initial growth, produce brown pigment on appropriate medium, and later "escape" from their inhibition, as a result of somatic events, to produce wild-type growth. - Five normal-sequence strains were crossed to 14 duplication-producing chromosome rearrangements, and the duplication progeny were examined for properties characteristic of duplications heterozygous for known het genes. Each cross produced duplications for a specific region of the genome, depending on the rearrangement. Normal-sequence strains were wild types from nature, chosen from diverse geographic locations to serve as sources of genetic variation. - The duplication method was very effective. Most of the longer duplications uncovered het genes. The genes are: het-5 (on linkage group IR, in the region covered by duplications produced using rearrangement T (IR LEADS TO VIR)NM103), het-6 (on IIL, covered by T(IIL LEADS TO VI)P2869 and T(IIL LEADS TO IIIR)AR18 duplications), het-7 (tentatively assigned to IIIR, T(IIIR LEADS TO VIL)D305), het-8 (VIL, T(VIL LEADS TO IR)T39M777), het-9 (VIR LEADS TO IVR)AR209), and het-10 (VIIR, T(VIIR LEADS TO IL)5936.     

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.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.     

Birth and death of duplicated genes in completely sequenced eukaryotes

Gene and genome duplications are commonly regarded as being of major evolutionary significance. But how often does gene duplication occur? And, once duplicated, what are the fates of duplicated genes? How do they contribute to evolution? In a recent article, Lynch and Conery analyze divergence between duplicate genes from six eukaryotic genomes. They estimate the rate of gene duplication, the rate of gene loss after duplication and the strength of selection experienced by duplicate genes. They conclude that although the rate of gene duplications is high, so is the rate of gene loss, and they argue that gene duplications could be a major factor in speciation.     

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.     

An insertional translocation in neurospora that generates duplications heterozygous for mating type

In strain T(I-->II)39311 a long interstitial segment is transposed from IL to IIR, where it is inserted in reversed order with respect to the centromere. In crosses of T x T essentially all asci have eight viable, black spores, and all progeny are phenotypically normal. When T(I-->II)39311 is crossed by Normal sequence (N), the expected duplication class is viable while the corresponding deficiency is lethal; 44% of the asci have 8 Black (viable) spores and 0 White (inviable) spores, 41% have 4 Black: 4 White, and 10% have 6 Black: 2 White. These are the ascus types expected from normal centromere disjunction without crossing over (8B:0W and 4B:4W equally probable), and with crossing over between centromere and break point (6B:2W). On germination, 8B:0W asci give rise to only parental types-4 T and 4 N; 4B:4W asci usually give four duplication (Dup) progeny; and 6B:2W asci usually give 2 T, 2 N, 2 Dup. Thus one third of all viable, black ascospores contain duplications.-Recessive markers in the donor chromosome which contributes the translocated segment can be mapped by duplication coverage. Ratios of 2 Dominant: 1 Recessive vs. 1 Dominant: 2 Recessive distinguish location in or outside the transposed segment. Eleven loci including mating type have been shown to lie within the segment, and markers at four loci have been transferred into the segment by meiotic recombination. The frequency of marker transfer indicates that the inserted segment usually pairs with its homologue. Ascus types that would result from single exchanges within the insertion are infrequent, as expected if asci containing dicentric bridges usually do not survive.-Duplication ascospores germinate to produce distinctive inhibited colonies. Later these "escape" to grow like wild type, and genes that were initially heterozygous in the duplication segregate when escape occurs. As with duplications from pericentric inversion In(IL-->IR)H4250 (Newmeyer and Taylor 1967), the initial inhibition is attributed to mating-type heterozygosity, and escape to a somatic event that makes mating type homoor hemizygous.-Twenty additional duplication-generating Neurospora rearrangements are listed and described briefly in an Appendix.     

Detection of gene duplications and block duplications in eukaryotic genomes

Several eukaryotic genomes have been completely sequenced and this provides an opportunity to investigate the extent and characteristics (e.g., single gene duplication, block duplication, etc.) of gene duplication in a genome. Detecting duplicate genes in a genome, however, is not a simple problem because of several complications such as domain shuffling, the existence of isoforms derived from alternative splicing, and annotational errors in the databases. We describe a method for overcoming these difficulties and the extents of gene duplication in the genomes of Drosophila melanogaster, Caenorhabditis elegans, and yeast inferred from this method. We also describe a method for detecting block duplications in a genome. Application of this method showed that block duplication is a common phenomenon in both yeast and nematode. The patterns of block duplication in the two species are, however, markedly different. Yeast shows much more extensive block duplication than nematode, with some chromosomes having more than 40% of the duplications derived from block duplications. Moreover, in yeast the majority of block duplications occurred between chromosomes, while in nematode most block duplications occurred within chromosomes.     

Rapid evolution in a pair of recent duplicate segments of rice

Gene duplication has been considered the most important way of generating genetic novelties. The subsequent evolution right after gene duplication is critical for new function to occur. Here we analyzed the evolutionary pattern for a recently duplicated segment between rice chromosomes 11 and 12. This duplication event was estimated to occur about 6 million years ago, during the divergence of the B- and C-genome rice species. The duplicate segment in chromosome 12 has significantly higher frequency of sequence rearrangement rate than non-duplicated regions. The rearrangement rate is approximately 6.5 breakages/Mb per million years, about six times higher than the fastest rate ever reported in eukaryotes. The genes within both segments experienced accelerated nucleotide substitution rates revealed by synonymous (Ks) and non-synonymous divergence (Ka) between Oryza sativa indica and O. sativa japonica. Analysis using EST data also implicates rapid divergence in expression between these segmental duplicate genes. These overall rapid changes from different perspective for the first time provide evidence that relaxation of selection also occurs in large-scale duplications.     

Genomic changes arising in long-term stab cultures of Escherichia coli

Genomic scans of clones isolated from long-term stab cultures of Escherichia coli K-12 showed the loss of two large segments of the genome, with each lost segment being approximately 20 kb long. A detailed analysis of one of the deletions, located between 5.4 and 5.9 min, revealed that similar deletions had arisen in several other stab cultures. All deletions of this type exhibited a right terminus ending precisely at an IS5A element and a left terminus that varied over an approximately 5-kb range but was bordered in all but two cases by sequences belonging to the preferred consensus target sequence for IS5, YTAR. The ubiquity of such deletions in independent stab cultures and the increase in their frequency over time argue that they have a selective advantage. It is speculated that the loss of the crl locus is responsible for the selective advantage of the deletions.     

Pervasive and persistent redundancy among duplicated genes in yeast

The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication (~100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.     

Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum

Background

Duplications of stretches of the genome are an important source of individual genetic variation, but their unrecognized presence in laboratory organisms would be a confounding variable for genetic analysis.

Results

We report here that duplications of 15 kb or more are common in the genome of the social amoeba Dictyostelium discoideum. Most stocks of the axenic 'workhorse' strains Ax2 and Ax3/4 obtained from different laboratories can be expected to carry different duplications. The auxotrophic strains DH1 and JH10 also bear previously unreported duplications. Strain Ax3/4 is known to carry a large duplication on chromosome 2 and this structure shows evidence of continuing instability; we find a further variable duplication on chromosome 5. These duplications are lacking in Ax2, which has instead a small duplication on chromosome 1. Stocks of the type isolate NC4 are similarly variable, though we have identified some approximating the assumed ancestral genotype. More recent wild-type isolates are almost without large duplications, but we can identify small deletions or regions of high divergence, possibly reflecting responses to local selective pressures. Duplications are scattered through most of the genome, and can be stable enough to reconstruct genealogies spanning decades of the history of the NC4 lineage. The expression level of many duplicated genes is increased with dosage, but for others it appears that some form of dosage compensation occurs.

Conclusion

The genetic variation described here must underlie some of the phenotypic variation observed between strains from different laboratories. We suggest courses of action to alleviate the problem.     

Chromosome I Duplications in Caenorhabditis Elegans

We have isolated and characterized 76 duplications of chromosome I in the genome of Caenorhabditis elegans. The region studied is the 20 map unit left half of the chromosome. Sixty-two duplications were induced with gamma radiation and 14 arose spontaneously. The latter class was apparently the result of spontaneous breaks within the parental duplication. The majority of duplications behave as if they are free. Three duplications are attached to identifiable sequences from other chromosomes. The duplication breakpoints have been mapped by complementation analysis relative to genes on chromosome I. Nineteen duplication breakpoints and seven deficiency breakpoints divide the left half of the chromosome into 24 regions. We have studied the relationship between duplication size and segregational stability. While size is an important determinant of mitotic stability, it is not the only one. We observed clear exceptions to a size-stability correlation. In addition to size, duplication stability may be influenced by specific sequences or chromosome structure. The majority of the duplications were stable enough to be powerful tools for gene mapping. Therefore the duplications described here will be useful in the genetic characterization of chromosome I and the techniques we have developed can be adapted to other regions of the genome.     

Independent large scale duplications in multiple M. tuberculosis lineages overlapping the same genomic region

Mycobacterium tuberculosis, the causative agent of most human tuberculosis, infects one third of the world's population and kills an estimated 1.7 million people a year. With the world-wide emergence of drug resistance, and the finding of more functional genetic diversity than previously expected, there is a renewed interest in understanding the forces driving genome evolution of this important pathogen. Genetic diversity in M. tuberculosis is dominated by single nucleotide polymorphisms and small scale gene deletion, with little or no evidence for large scale genome rearrangements seen in other bacteria. Recently, a single report described a large scale genome duplication that was suggested to be specific to the Beijing lineage. We report here multiple independent large-scale duplications of the same genomic region of M. tuberculosis detected through whole-genome sequencing. The duplications occur in strains belonging to both M. tuberculosis lineage 2 and 4, and are thus not limited to Beijing strains. The duplications occur in both drug-resistant and drug susceptible strains. The duplicated regions also have substantially different boundaries in different strains, indicating different originating duplication events. We further identify a smaller segmental duplication of a different genomic region of a lab strain of H37Rv. The presence of multiple independent duplications of the same genomic region suggests either instability in this region, a selective advantage conferred by the duplication, or both. The identified duplications suggest that large-scale gene duplication may be more common in M. tuberculosis than previously considered.     

Chromosomal translocation and segmental duplication in Cryptococcus neoformans

Large chromosomal events such as translocations and segmental duplications enable rapid adaptation to new environments. Here we marshal genomic, genetic, meiotic mapping, and physical evidence to demonstrate that a chromosomal translocation and segmental duplication occurred during construction of a congenic strain pair in the fungal human pathogen Cryptococcus neoformans. Two chromosomes underwent telomere-telomere fusion, generating a dicentric chromosome that broke to produce a chromosomal translocation, forming two novel chromosomes sharing a large segmental duplication. The duplication spans 62,872 identical nucleotides and generated a second copy of 22 predicted genes, and we hypothesize that this event may have occurred during meiosis. Gene disruption studies of one embedded gene (SMG1) corroborate that this region is duplicated in an otherwise haploid genome. These findings resolve a genome project assembly anomaly and illustrate an example of rapid genome evolution in a fungal genome rich in repetitive elements.     

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.