Reconstitution of the R compound allele in maize

The R^r:standard allele in maize, which conditions anthocyanin pigmentation in plant and seed tissues in the presence of appropriate complementary factors, is associated with a tandem duplication. The proximal member of the duplication carries P, the plant pigmenting determiner and the distal member member carries S, the seed pigmenting determiner. Derivatives from R^r that have lost S function are designated r^r. They represent either losses of the distal member of the duplication (P derivatives) or mutations of S to s (P s). Derivatives that have lost P function are designated R^g, and represent either losses of the proximal member of the duplication (S derivatives) or mutations of P to p (p S).—All four possible types of r^r/R^g heterozygotes were tested for their capacity to yield R^r reconstitution by crossing over. No R^r derivatives were obtained from P/S heterozygotes, a result consistent with the view that P and S occupy corresponding positions in homologous chromosome segments. R^r reconstitution was detected in both tandem duplication heterozygotes P s/S and P/p S, and was found to be about ten times more frequent in the latter. The ratio of R^r reconstitution in the two heterozygotes is a function of position of the anthocyanin marker within the duplicated segment. The data from these heterozygotes allow one to measure the distance between P and S, that is to say, the genetic length of the duplicated segment. This distance was found to be 0.16 map units. The highest frequency of R^r reconstitution was obtained from P s/p S heterozygotes, since direct pairing (see PDF) as well as the p//s type of displaced pairing have the potential to produce R^r derivatives. One of the R^g derivatives used in this study, R^g₆, was found to back-mutate in some sublines to R^r. The basis for this instability remains unknown.     

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.     

Meiosis in Male DROSOPHILA MELANOGASTER. II. Nonrandom Segregation of Compound-Second Chromosomes

The segregation of compound-second chromosomes in males from two different stocks has been examined. Segregation is random in males from the C(2L)RM4, dp; C(2R)RM4, px stock. Gametes containing only one of the two compound chromosomes comprise 50% of the gametes, and gametes containing either both elements or neither element make up the other 50% of the gametes.——In males from the C(2L)RM, b; C(2R)RM, cn stock, gametes containing either C(2L)RM, b or C(2R)RM, cn make up the majority of the gametes. Gametes containing both chromosomes or neither chromosome account for only 2-3% of the gametes. The nonrandom segregation is due to the C(2R)RM, cn chromosome.——Viability is reduced in flies carrying the C(2R)RM, cn chromosome. This includes larval lethality, delayed development and premature adult lethality. Cytologically, this chromosome contains a large duplication of 2L material, which includes material proximal to region 38 or 39. It is suggested that the viability and segregational properties associated with this chromosome are due to the duplicated 2L material.     

Tandem duplications in Drosophila melanogaster

In the tandem duplication Dp(1;1)Gr approximately one quarter of the euchromatic part of the X-chromosome is duplicated. Dp(1;1)Gr itself has no phenotypic effect, but it can be made visible by combining different alleles within the tandem duplication and the homologous X-chromosome. In heterozygous females crossing-over between the two X-chromosomes is strongly reduced while at the same time crossing-over in the distal regions of the autosomes is increased.     

Molecular evolution of glycinin and β-conglycinin gene families in soybean (Glycine max L. Merr.)

There are two main classes of multi-subunit seed storage proteins, glycinin (11S) and β-conglycinin (7S), which account for approximately 70% of the total protein in a typical soybean seed. The subunits of these two protein classes are encoded by a number of genes. The genomic organization of these genes follows a complex evolutionary history. This research was designed to describe the origin and maintenance of genes in each of these gene families by analyzing the synteny, phylogenies, selection pressure and duplications of the genes in each gene family. The ancestral glycinin gene initially experienced a tandem duplication event; then, the genome underwent two subsequent rounds of whole-genome duplication, thereby resulting in duplication of the glycinin genes, and finally a tandem duplication likely gave rise to the Gy1 and Gy2 genes. The β-conglycinin genes primarily originated through the more recent whole-genome duplication and several tandem duplications. Purifying selection has had a key role in the maintenance of genes in both gene families. In addition, positive selection in the glycinin genes and a large deletion in a β-conglycinin exon contribute to the diversity of the duplicate genes. In summary, our results suggest that the duplicated genes in both gene families prefer to retain similar function throughout evolution and therefore may contribute to phenotypic robustness.     

Novel insertion mutation in Caenorhabditis elegans.

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.     

Tandem duplications resulting from recombination between ribosomal RNA genes in Escherichia coli.

A set of Escherichia coli K12 mutants, which carry a tandem duplication of the glyT purD region, have been analyzed. Three types of duplications have occurred repeatedly, and we show that they were generated by recombination between the ribosomal RNA gene, rrnE, which lies to one side of the glyT purD region and one of threerrn genes which occur as direct repetitions on the other side of this region. Characterization of these duplication mutants has involved the isolation of the duplicated material in the form of a DNA circle. Class I duplications, which extend from rrnE to rrnE, are 39,500 base-pairs long, class II duplications, which extend from rrnA to rrnE, are 164,000 base-pairs long, and class III duplications, which extend from rrnC to rrnE, are 258,000 base-pairs long.     

Duplication processes in Saccharomyces cerevisiae haploid strains

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.     

Association of Isozymes with a Reciprocal Translocation in Cultivated Rye (SECALE CEREALE L.)

In rye (Secale cereale L. cv. "Ailés") the progeny of a cross between a structural heterozygote for a reciprocal translocation (involving the 1R chromosome) and a homozygote for the standard chromosome arrangement were analyzed for the electrophoretic patterns of eight different leaf isozymes and also for their meiotic configuration at metaphase I.——The Got-3 and Mdh-2b loci are linked to each other and also to the reciprocal translocation. The Mdh-2b locus is located in the interstitial segment of the 3Rq chromosome arm, with an estimated distance of 8 cM to the breakpoint. Therefore, the reciprocal translocation involves the 1R and 3R chromosomes.——Also, the Mdh-1 and 6-Pgd-2 loci are linked (16 ± 3 cM) and have been located on the 2Rq arm. Finally, the Per-3 and Per-4 loci are located on the 2Rp chromosome arm at an estimated distance of 26 ± 4 cM.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.     

Degree of Functional Divergence in Duplicates Is Associated with Distinct Roles in Plant Evolution

Gene duplication is a major mechanism to create new genes. After gene duplication, some duplicated genes undergo functionalization, whereas others largely maintain redundant functions. Duplicated genes comprise various degrees of functional diversification in plants. However, the evolutionary fate of high and low diversified duplicates is unclear at genomic scale. To infer high and low diversified duplicates in Arabidopsis thaliana genome, we generated a prediction method for predicting whether a pair of duplicate genes was subjected to high or low diversification based on the phenotypes of knock-out mutants. Among 4,017 pairs of recently duplicated A. thaliana genes, 1,052 and 600 are high and low diversified duplicate pairs, respectively. The predictions were validated based on the phenotypes of generated knock-down transgenic plants. We determined that the high diversified duplicates resulting from tandem duplications tend to have lineage-specific functions, whereas the low diversified duplicates produced by whole-genome duplications are related to essential signaling pathways. To assess the evolutionary impact of high and low diversified duplicates in closely related species, we compared the retention rates and selection pressures on the orthologs of A. thaliana duplicates in two closely related species. Interestingly, high diversified duplicates resulting from tandem duplications tend to be retained in multiple lineages under positive selection. Low diversified duplicates by whole-genome duplications tend to be retained in multiple lineages under purifying selection. Taken together, the functional diversities determined by different duplication mechanisms had distinct effects on plant evolution.     

Induction and Transmission of a Merodiploid Condition near the Terminal Area of the Chromosome of BACILLUS SUBTILIS

A small fraction (about 0.5%) of the transformants for a particular marker of B. subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids. They possess a redundancy of the metB–ilvA chromosome segment. When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4⁺ transformants. For several of the duplicated loci both copies often are of recipient strain origin. Markers originally belonging to different copies of the diploidized region can be contransferred in PBS1-mediated transduction. The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication. An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered. Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA.     

A rare case of plastid protein‐coding gene duplication in the chloroplast genome of Euglena archaeoplastidiata (Euglenophyta)

Gene duplication is an important evolutionary process that allows duplicate functions to diverge, or, in some cases, allows for new functional gains. However, in contrast to the nuclear genome, gene duplications within the chloroplast are extremely rare. Here, we present the chloroplast genome of the photosynthetic protist Euglena archaeoplastidiata. Upon annotation, it was found that the chloroplast genome contained a novel tandem direct duplication that encoded a portion of RuBisCO large subunit (rbcL) followed by a complete copy of ribosomal protein L32 (rpl32), as well as the associated intergenic sequences. Analyses of the duplicated rpl32 were inconclusive regarding selective pressures, although it was found that substitutions in the duplicated region, all non‐synonymous, likely had a neutral functional effect. The duplicated region did not exhibit patterns consistent with previously described mechanisms for tandem direct duplications, and demonstrated an unknown mechanism of duplication. In addition, a comparison of this chloroplast genome to other previously characterized chloroplast genomes from the same family revealed characteristics that indicated E. archaeoplastidiata was probably more closely related to taxa in the genera Monomorphina, Cryptoglena, and Euglenaria than it was to other Euglena taxa. Taken together, the chloroplast genome of E. archaeoplastidiata demonstrated multiple characteristics unique to the euglenoid world, and has justified the longstanding curiosity regarding this enigmatic taxon.     

Genetic basis of the major malate dehydrogenase isozymes in maize

The mitochondrial MDH isozymes in the scutellum of the mature maize (Zea mays L.) kernel are encoded by three independently inherited nuclear genes. Mdh1 is located on chromosome 8, close to the breakpoint (8L.35) of a waxy-marked reciprocal translocation between chromosomes 8 and 9. Mdh2 is located in the distal region of the long arm of chromosome 6. Mdh3 is on the long arm of chromosome 3, approximately 2.6 map units from sh2. A modifier of the mitochondrial MDH isozymes (Mmm) maps approximately 27.5 units proximal to Adh1 in the central portion of the long arm of chromosome 1. Independently assorting duplicate genes code for the soluble MDH isozymes. Mdh4 is located in the same region of chromosome 1 as Mmm, approximately 29 map units proximal to Adh1. Mdh5 maps approximately 20 units distal to a2 in the short arm of chromosome 5.——Intergenic and interallelic heterodimer formation occurs among gene products that occupy the same subcellular compartment. MDH isozymes were purified and analyzed by native-SDS two-dimensional polyacrylamide gel electrophoresis. The proposed mitochondrial MDH intergenic heterodimer bands were found to be composed of two subunits, which differ in their migrations on SDS gels; whereas, genetically defined homodimers contained only one type of subunit.——This evidence is discussed in terms of two genetic models proposed for the maize mitochondrial MDH isozymes.     

The Tandem Repeats Enabling Reversible Switching between the Two Phases of β-Lactamase Substrate Spectrum

Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (de novo TRs) that occurred in the coding region of a β-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic. Under selective pressure with one of the original substrates (amoxicillin), a high level of reversion occurred in the mutant β-lactamase genes completing a cycle back to the original substrate spectrum. The de novo TRs coupled with reversion makes a genetic toggling mechanism enabling reversible switching between the two phases of the substrate spectrum of β-lactamases. This toggle exemplifies the effective adaptation of de novo TRs for enhanced bacterial survival. We found pairs of direct repeats that mediated the DNA duplication (TR formation). In addition, we found different duos of sequences that mediated the DNA duplication. These novel elements—that we named SCSs (same-strand complementary sequences)—were also found associated with β-lactamase TR mutations from clinical isolates. Both direct repeats and SCSs had a high correlation with TRs in diverse bacterial genomes throughout the major phylogenetic lineages, suggesting that they comprise a fundamental mechanism shaping the bacterial evolution.     

Healing of Broken Linear Dicentric Chromosomes in Yeast

In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura⁺ Mal⁺ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura⁺ Mal ⁺, others Ura⁺ Mal^- and others Ura ^- Mal⁺. The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.—Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic break-age of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome III partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres.     

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.     

Cis-acting mutation and duplication: History of molecular evolution in a P450 haplotype responsible for insecticide resistance in Culex quinquefasciatus

A cytochrome P450 gene, Cyp9m10, is more than 200-fold overexpressed in a pyrethroid resistant strain of Culex quinquefasciatus, JPal-per. The haplotype of this strain contains two copies of Cyp9m10 resulted from recent tandem duplication. In this study, we discovered and isolated a Cyp9m10 haplotype closely related to this duplicated Cyp9m10 haplotype from JHB, a strain used for the recent genome project for this mosquito species. The isolated haplotype (JHB-NIID-B haplotype) shared the same insertion of a transposable element upstream of the coding region with JPal-per strain but not duplicated. The JHB-NIID-B haplotype was considered to have diverged from the JPal-per lineage just before the duplication event. Cyp9m10 was moderately overexpressed in larvae with the JHB-NIID-B haplotype. The overexpressions in JHB-NIID-B and JPal-per haplotypes were developmentally regulated in similar pattern indicating both haplotypes share a common cis-acting mutation responsible for the overexpressions. The isolated moderately overexpressed haplotype conferred resistance, however, its efficacy was relatively small. We hypothesized that the first cis-acting mutation modified the consequence of the subsequent duplication in JPal-per lineage to confer stronger phenotypic effect than that if it occurred before the first cis-acting mutation.     

A major locus qS12, located in a duplicated segment of chromosome 12, causes spikelet sterility in an indica-japonica rice hybrid

Chromosome segment duplications are integral in genome evolution by providing a source for the origin of new genes. In the rice genome, besides an ancient polyploidy event known in the rice common ancestor, it had been identified that there was a special segmental duplication involving chromosomes 11 and 12, but the biological role of this duplication remains unknown. In this study, by using a set of chromosome segment substitution lines (CSSLs) and near isogenic lines (NILs) derived from the indica cultivar 9311 and japonica cultivar Nipponbare, a major QTL (qS12) resulting in hybrid male sterility was mapped within ~400 kb region adjacent to the special duplicated segment on the short arm of chromosome 12. Compared to the japonica cultivar Nipponbare, the two sides of the qS12 candidate region were inverted in the indica cultivar 9311. Among 47 of the 111 rice genotypes evaluated by molecular markers, the inverted sides were detected, and found completely homologous to indica cultivar 9311. These results suggested that the two inverted sides protect the sequence in the qS12 regions from recombination. On the short-arm of chromosome 12, two QTLs S-e and S25, in addition to qS12, were previously detected as a distinct segregation distortion and pollen semi-sterility loci. We propose these three hybrid sterility loci are the same locus, and the duplicated segment on chromosome 12 may play a prominent role in diversification, i.e., sub-speciation of cultivated rice.