Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination

Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be transferred from A. tumefaciens to Saccharomyces cerevisiae and integrates into the yeast genome via homologous recombination. We show here that when the T-DNA lacks homology with the S. cerevisiae genome, it integrates at random positions via illegitimate recombination. From 11 lines the integrated T-DNA was cloned back to Escherichia coli along with yeast flanking sequences. The T-DNA borders and yeast DNA flanking the T-DNA were sequenced and characterized. It was found that T-DNA integration had resulted in target DNA deletions and sometimes T-DNA truncations or filler DNA formation. Therefore, the molecular mechanism of illegitimate recombination by which T-DNA integrates in higher and lower eukaryotes seems conserved.

Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1− and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT− cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT− products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1− cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1− cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1− cells are repaired by illegitimate recombination.

The role of double-stranded break repair in the creation of phenotypic diversity at cereal VRN1 loci

Nonhomologous repair of double-stranded breaks, although fundamental to the maintenance of genomic integrity in all eukaryotes, has received little attention as to its evolutionary consequences in the generation and selection of phenotypic diversity. Here we document the role of illegitimate recombination in the creation of novel alleles in VRN1 orthologs selected to confer adaptation to annual cropping systems in barley and wheat.

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of ≈103. Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.

Molecular origin of the mosaic sequence arrangements of higher primate α-globin duplication units

The human adult α-globin locus consists of three pairs of homology blocks (X, Y, and Z) interspersed with three nonhomology blocks (I, II, and III), and three Alu family repeats, Alu1, Alu2, and Alu3. It has been suggested that an ancient primate α-globin-containing unit was ancestral to the X, Y, and Z and the Alu1/Alu2 repeats. However, the evolutionary origin of the three nonhomologous blocks has remained obscure. We have now analyzed the sequence organization of the entire adult α-globin locus of gibbon (Hylobates lar). DNA segments homologous to human block I occur in both duplication units of the gibbon α-globin locus. Detailed interspecies sequence comparisons suggest that nonhomologous blocks I and II, as well as another sequence, IV, were all part of the ancestral α-globin-containing unit prior to its tandem duplication. However, sometime thereafter, block I was deleted from the human α1-globin-containing unit, and block II was also deleted from the α2-globin-containing unit in both human and gibbon. These were probably independent events both mediated by independent illegitimate recombination processes. Interestingly, the end points of these deletions coincide with potential insertion sites of Alu family repeats. These results suggest that the shaping of DNA segments in eukaryotic genomes involved the retroposition of repetitive DNA elements in conjunction with simple DNA recombination processes.

The evolution of plant nuclear genes

We analyze the evolutionary dynamics of three of the best-studied plant nuclear multigene families. The data analyzed derive from the genes that encode the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS), the gene family that encodes the enzyme chalcone synthase (Chs), and the gene family that encodes alcohol dehydrogenases (Adh). In addition, we consider the limited evolutionary data available on plant transposable elements. New Chs and rbcS genes appear to be recruited at about 10 times the rate estimated for Adh genes, and this is correlated with a much smaller average gene family size for Adh genes. In addition, duplication and divergence in function appears to be relatively common for Chs genes in flowering plant evolution. Analyses of synonymous nucleotide substitution rates for Adh genes in monocots reject a linear relationship with clock time. Replacement substitution rates vary with time in a complex fashion, which suggests that adaptive evolution has played an important role in driving divergence following gene duplication events. Molecular population genetic studies of Adh and Chs genes reveal high levels of molecular diversity within species. These studies also reveal that inter- and intralocus recombination are important forces in the generation allelic novelties. Moreover, illegitimate recombination events appear to be an important factor in transposable element loss in plants. When we consider the recruitment and loss of new gene copies, the generation of allelic diversity within plant species, and ectopic exchange among transposable elements, we conclude that recombination is a pervasive force at all levels of plant evolution.

Transposons and genome evolution in plants

Although it is known today that transposons comprise a significant fraction of the genomes of many organisms, they eluded discovery through the first half century of genetic analysis and even once discovered, their ubiquity and abundance were not recognized for some time. This genetic invisibility of transposons focuses attention on the mechanisms that control not only transposition, but illegitimate recombination. The thesis is developed that the mechanisms that control transposition are a reflection of the more general capacity of eukaryotic organisms to detect, mark, and retain duplicated DNA through repressive chromatin structures.

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Carrier transfer in the optical recombination of quantum dots

We report a study of dynamic effects detected in the time-resolved emission from quantum dot ensembles. Experimental procedures were developed to search for common behaviors found in quantum dot systems independently of their composition: three quantum dot samples were experimentally characterized. Systems with contrasting interdot coupling are compared and their sensitivity to the excitation energy is analyzed. Our experimental results are compared and contrasted with other results available in literature. The optical recombination time dependence on system parameters is derived and compared to the experimental findings. We discuss the effects of occupation of the ground state in both valence and conduction bands of semiconductor quantum dots in the dynamics of the system relaxation as well as the nonlinear effects.

A two-phase approach for detecting recombination in nucleotide sequences

Genetic recombination can produce heterogeneous phylogenetic histories within a set of homologous genes. Delineating recombination events is important in the study of molecular evolution, as inference of such events provides a clearer picture of the phylogenetic relationships among different gene sequences or genomes. Nevertheless, detecting recombination events can be a daunting task, as the performance of different recombination-detecting approaches can vary, depending on evolutionary events that take place after recombination. We recently evaluated the effects of post-recombination events on the prediction accuracy of recombination-detecting approaches using simulated nucleotide sequence data. The main conclusion, supported by other studies, is that one should not depend on a single method when searching for recombination events. In this paper, we introduce a two-phase strategy, applying three statistical measures to detect the occurrence of recombination events, and a Bayesian phylogenetic approach in delineating breakpoints of such events in nucleotide sequences. We evaluate the performance of these approaches using simulated data, and demonstrate the applicability of this strategy to empirical data. The two-phase strategy proves to be time-efficient when applied to large datasets, and yields high-confidence results.

A two-phase strategy for detecting recombination in nucleotide sequences

Genetic recombination can produce heterogeneous phylogenetic histories within a set of homologous genes. Delineating recombination events is important in the study of molecular evolution, as inference of such events provides a clearer picture of the phylogenetic relationships among different gene sequences or genomes. Nevertheless, detecting recombination events can be a daunting task, as the performance of different recombination-detecting approaches can vary, depending on evolutionary events that take place after recombination. We previously evaluated the effects of post-recombination events on the prediction accuracy of recombination-detecting approaches using simulated nucleotide sequence data. The main conclusion, supported by other studies, is that one should not depend on a single method when searching for recombination events. In this paper, we introduce a two-phase strategy, applying three statistical measures to detect the occurrence of recombination events, and a Bayesian phylogenetic approach to delineate breakpoints of such events in nucleotide sequences. We evaluate the performance of these approaches using simulated data, and demonstrate the applicability of this strategy to empirical data. The two-phase strategy proves to be time-efficient when applied to large datasets, and yields high-confidence results.

Measurements of neutral atom diffusion and electron-ion recombination by laser enhanced ionisation and planar laser induced fluorescence in an air-acetylene flame

The spatial and temporal evolution of a depleted atomic distribution created by laser enhanced ionisation (LEI) was employed to determine both a diffusion coefficient for sodium (Na) and an electron (e(-)) and sodium ion recombination rate coefficient in an analytical air-C2H2 flame. A depleted distribution of neutral sodium atoms was produced in a flame by ionising approximately 80% of the irradiated sodium atoms in a well defined region using a two step LEI excitation scheme. Following depletion by ionisation, planar laser induced fluorescence (PLIF) images of the depleted region recorded the diffusion and decay of the depleted Na distribution for different depletion-probe delays. From measurements of the diffused width of the distribution, an accurate diffusion coefficient D = (1.19 +/- 0.03) x 10(-3) m(2) s(-1) for Na was determined in teh burnt gases of the flame. Measurements of the integrated fluorescence intensity in the depleted region for different depletion-probe delays were related to an increase in atomic sodium concentration caused by electron-ion recombination. At high concentrations (greater than or equal to 50 mu g ml(-1)), where the electron and ion concentrations in the depleted region were assumed equal, a recombination rate coefficient of 4.2 x 10(-9) cm(3) s(-1) was calculated. (C) 1997 Elsevier Science B.V.

Occurrence of TRGV-BJ hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.

Sexual recombination in Phytophthora cinnamomi in vitro and aggressiveness of single-oospore progeny to Eucalyptus

Fifty single oospore progeny were established from an in vitro mating of A1 and A2 mating type isolates of Phytophthora cinnamomi from South Africa. Forty-nine progeny were identified as F-1 hybrids using seven random amplified polymorphic DNA (RAPD) primers, and one was a selfed isolate of the A1 mating type parent. Among the hybrid progeny, 24 and 25 were A1 and A2 mating type, respectively. Aggressiveness of progeny and parental isolates was assessed on 1-year-old seedlings of Eucalyptus smithii. The mean aggressiveness of hybrid oosporic isolates, expressed as lesion length, was significantly (P = 0.0001) lower than that of the parental isolates. No significant difference in aggressiveness of A1 and A2 mating type F-1 hybrid isolates was observed. This is the first report demonstrating sexual recombination in vitro in P. cinnamomi.