Optional elements in the chloroplast DNAs of chlamydomonas eugametos and C. moewusii: unidirectional gene conversion and co-conversion of adjacent markers in high-viability crossses.

We have surveyed the tandemly repeated genes encoding U2 snRNA in a diverse panel of humans. We found only two polymorphisms within the U2 repeat unit: a SacI polymorphism (alleles SacI+ or SacI-) and a CT microsatellite polymorphism (alleles CT+ or CT-). Surprisingly, individual U2 tandem arrays are entirely SacI+ or SacI-, and entirely CT+ or CT-, although the SacI and CT alleles can occur in any combination. We also found that polymorphisms in the left and right junction regions flanking the tandem array fall into only two haplotypes (JL+ and JL-, JR+ and JR-). Most surprisingly, JL+ is always associated with JR+, and JL- with JR-. Thus individual U2 arrays do not exchange flanking markers, despite independent assortment and subsequent homogenization of the SacI and CT alleles within the U2 repeat units. We propose that the primary driving force for concerted evolution of the tandem U2 genes is intrachromosomal homogenization; interchromosomal genetic exchanges are much rarer, and reciprocal nonsister chromatid exchange apparently does not occur. Thus concerted evolution of the U2 tandem array occurs in situ along a chromosome lineage, and linkage disequilibrium between sequences flanking the U2 array may persist for long periods of time.

Homologous recombination can result in the transfer of genetic information from one DNA molecule to another (gene conversion). These events are often accompanied by a reciprocal exchange between the interacting molecules (termed "crossing over"). This association suggests that the two types of events could be mechanistically related. We have analyzed the repair, by homologous recombination, of a broken chromosome in yeast. We show that gene conversion can be uncoupled from crossing over when the length of homology of the interacting substrates is below a certain threshold. In addition, a minimal length of homology on each broken chromosomal arm is needed for crossing over. We also show that the coupling between gene conversion and crossing over is affected by the mismatch repair system; mutations in the MSH2 or MSH6 genes cause an increase in the crossing over observed for short alleles. Our results provide a mechanism to explain how chromosomal recombinational repair can take place without altering the stability of the genome.

Asexual reproduction is believed to be detrimental, mainly because of the accumulation of deleterious mutations over time, a hypothesis known as Muller's ratchet. In seed plants, most asexually reproducing genetic systems are polyploid, with apomictic species (plants forming seeds without fertilization) as well as plastids and mitochondria providing prominent examples. Whether or not polyploidy helps asexual genetic systems to escape Muller's ratchet is unknown. Gene conversion, particularly when slightly biased, represents a potential mechanism that could allow asexual genetic systems to reduce their mutation load in a genome copy number-dependent manner. However, direct experimental evidence for the operation of gene conversion between genome molecules to correct mutations is largely lacking. Here we describe an experimental system based on transgenic tobacco chloroplasts that allows us to analyze gene conversion events in higher plant plastid genomes. We provide evidence for gene conversion acting as a highly efficient mechanism by which the polyploid plastid genetic system can correct deleterious mutations and make one good genome out of two bad ones. Our finding that gene conversion can be biased may provide a molecular link between asexual reproduction, high genome copy numbers and low mutation rates.

With the goal of studying directly the inheritance and recombination of physically mapped markers on the chloroplast genome, we have recently identified and localized physical differences between the chloroplast DNAs (cpDNAs) of the interfertile algae Chlamydomonas eugametos and C. moewusii. Here we report the inheritance patterns of 24 polymorphic loci mapping throughout the chloroplast genome in hybrids recovered from reciprocal crosses between the two algae. Most polymorphic loci were found to be inherited mainly from the mt+ parent, with no apparent preference for one or the other parental alternatives in reciprocal crosses. Virtually all hybrids, however, inherited exclusively the long alleles of three loci; i.e. an intron in the large subunit ribosomal RNA gene of C. eugametos, a 21 kbp sequence addition in the inverted repeat of the C. moewusii cpDNA and a 5.8 kbp sequence addition in one of the single-copy regions of C. moewusii cpDNA. As these alleles are derived from opposite parental strains, their unidirectional inheritance in hybrids results necessarily from interspecific recombination of cpDNA molecules. We propose that gene conversion events led to the spreading of the long alleles of the three loci.

SummaryWe have developed a semi-nested PCR assay which measures gene conversion in mouse MHC class genes. Detectable frequencies occur in DNA from sperm, but not in somatic DNA, such as liver or fibroblast DNA. The frequency of gene conversion events in MHC class II genes varies strongly from one MHC haplotype to another, with the highest detected frequencies as high as 1/40 000 for an individual heterozygous for both donor and acceptor sequences. Deletions or insertions in one gene relative to the other seem to lower the efficiency of gene conversion considerably. However, not all the variation in gene conversion frequency can be readily ascribed to the MHC genes themselves, implying that variation in the non-MHC background genes also might be of importance. Stretches within MHC genes amenable to gene conversion are located in CpG clusters, whereas MHC genes not involved in gene conversion have background CpG levels. This feature extends to some, but not all, of the non-MHC genes that have been reported to undergo gene conversion in mammals. DNA damage, either chemical or radiation-induced, increases the frequency of gene conversion of MHC class 11 genes in cultured cells of the fibroblastoid lineage. The effect of chemical DNA damage seems roughly dose-dependent, whereas irradiation has maximal effect at low doses.

In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54- and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the bar(au) gene that causes a golden-yellow (aurea) leaf color, so that the loss of the bar(au) gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the bar(au) gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/attP-flanked bar(au) gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked bar(au) gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the bar(au) gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency.

Many of the thylakoid proteins involved in photosynthesis, as well as the catalytic subunit of ribulose 1,5‐bisphosphate carboxylaseoxygenase (rubisco), are encoded in the chloroplast, which is inherited maternally. Different chloroplast genomes might result in different photosynthetic rates or differential regulation of photosynthetic enzymes. Nine soybean lines [Glycine max (L.) Merr. and G. gracilis], representing four different chloroplast genomes, were reciprocally crossed to a common high‐photosynthetic parent. Reciprocal F1'S in a field experiment behaved similarly for CO2‐exchange rate (CER), photosynthetic electron transport, rubisco activity, and soluble protein content. Hence, these traits were not influenced by the different chloroplast genomes, implying that the chloroplast genomes used were not different with respect to genes involved in ratelimiting photosynthesis or any sequence changes were not expressed. Partial dominance for low CER seemed evident in the progeny of parents having large differences in CER. This effect seems to be under nuclear influence because it occurred in both F1 progeny of reciprocal crosses. This partial dominance for low CER appeared in both Glycine species and was mainly due to low protein content resulting in low rubisco activity per leaf area. The performance of the two species in photosynthesis and the related traits was similar.

The polymorphic HLA-DR beta-chains are encoded within the human major histocompatibility complex (MHC) by multiple loci resulting from gene duplications. Certain DR haplotypes can be grouped into families based on shared structural factors. We have studied the molecular basis of HLA-DR polymorphism within such a group which includes the haplotypes DR3, DR5 and DRw6. Molecular mapping of the DR beta-chain region allows true allelic comparisons of the two expressed DR beta-chain loci, DR beta I and DR beta III. At the more polymorphic locus, DR beta I, the allelic differences are clustered and may result from gene conversion events over very short distances. The gene encoding the HLA-DR3/Dw3 specificity has been generated by a gene conversion involving the DR beta I and the DR beta III loci of the HLA-DRw6/Dw18 haplotype, as recipient and donor gene, respectively. Based on which allele is found at DR beta III, the less polymorphic locus, two groups of haplotypes can be defined: DRw52a and DRw52b. The generation of HLA-DR polymorphism within the DRw52 supertypic group can thus be accounted for by a succession of gene duplication, divergence and gene conversion.

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561bp in length with 31.3% GC content that contained 108 genes including 77 protein-coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip-flop organization. The flip-flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C.fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C.fulvescens genome, we found three major conflicting signals for C.fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70-gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data-rich approach (i.e., organelle genome data) from broader taxon sampling.

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disease, mainly characterized by exocrine pancreatic insufficiency, hematological dysfunction and skeletal abnormalities. The SDS disease locus was mapped to chromosome 7q11 and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is a member of a highly conserved protein family with putative orthologs in diverse species including archaea and eukaryotes. It is widely expressed in many tissues and its function is still unknown. In the present study we analyzed the genotype of 15 unrelated Italian SDS patients. After sequencing the whole coding region we were able to complete all genotypes of the SDS patients tested. A total of eleven distinct mutations were identified. The most frequent mutations are due to gene conversion events between SBDS and its unprocessed pseudogene, named SBDSP. We described four new gene conversions involving exon 2 and three novel mutations that are not a result of gene conversion events. In two out of the fifteen cases, the family analysis evidenced an apparently unexpected inheritance of SDS alleles between parents and affected children. In the first case we found a new large gene conversion event, that caused the failure of the amplification of the father's allele and in the second what could be explained as a de novo gene conversion. Both cases have important implications for genetic counseling and molecular genetic analysis. In a disorder caused by gene conversions of variable extension these findings emphasize the necessity of testing patient's parents and the significance of the choice of primers.

The model haloarchaeon Haloferax volcanii is polyploid with about 20 copies of its major chromosome. Recently it has been described that highly efficient intermolecular gene conversion operates in H. volcanii to equalize the chromosomal copies. In the current study, 24 genes were selected that encode proteins with orthologs involved in gene conversion or homologous recombination in archaea, bacteria, or eukaryotes. Single gene deletion strains of 22 genes and a control gene were constructed in two parent strains for a gene conversion assay; only radA and radB were shown to be essential. Protoplast fusions were used to generate strains that were heterozygous for the gene HVO_2528, encoding an enzyme for carotinoid biosynthesis. It was revealed that a lack of six of the proteins did not influence the efficiency of gene conversion, while sixteen mutants had severe gene conversion defects. Notably, lack of paralogous proteins of gene families had very different effects, e.g., mutant Δrad25b had no phenotype, while mutants Δrad25a, Δrad25c, and Δrad25d were highly compromised. Generation of a quadruple rad25 and a triple sph deletion strain also indicated that the paralogs have different functions, in contrast to sph2 and sph4, which cannot be deleted simultaneously. There was no correlation between the severity of the phenotypes and the respective transcript levels under non-stressed conditions, indicating that gene expression has to be induced at the onset of gene conversion. Phylogenetic trees of the protein families Rad3/25, MutL/S, and Sph/SMC/Rad50 were generated to unravel the history of the paralogous proteins of H. volcanii. Taken together, unselected intermolecular gene conversion in H. volcanii involves at least 16 different proteins, the molecular roles of which can be studied in detail in future projects.

It is possible to measure gene conversion of MHC genes with the help of a semi-nested PCR assay. Several considerations are of utmost importance when such an assay is set up. Using this assay, we have found that gene conversion occurs in MHC class II genes in mouse sperm, but not in somatic cells tested. Although this gene conversion occurs in germline cells, it is already completed in spermatogonia, and consequently is mitotic event unlinked to meiosis. The frequency of gene conversion events in MHC class II genes varies strongly from one allele to another, with the highest detected frequencies as high as 1/40,000 for an individual heterozygous for both donor and acceptor sequences. Deletions or insertions in one gene relative to the other seem to lower the efficiency of gene conversion considerably. Stretches within MHC genes amenable to gene conversion are located in CpG clusters, whereas MHC genes not involved in gene conversion have background CpG levels. DNA damage, either chemical or radiation induced, increases the frequency of gene conversion of MHC class II genes in cultured cells of the fibroblastoid lineage. The effect of chemical DNA damage seems roughly dose dependent, whereas irradiation has a maximal effect at low doses.

With the intent of further exploring the nature of gene conversion in mammalian cells, we systematically addressed the effects of the molecular nature of mutation on the efficiency of intrachromosomal gene conversion in cultured mouse cells. Comparison of conversion rates revealed that all mutations studied were suitable substrates for gene conversion; however, we observed that the rates at which different mutations converted to wild-type could differ by two orders of magnitude. Differences in conversion rates were correlated with the molecular nature of the mutations. In general, rates of conversion decreased with increasing size of the molecular lesions. In comparisons of conversion rates for single base pair insertions and deletions we detected a genotype-directed path for conversion, by which an insertion was converted to wild-type three to four times more efficiently than was a deletion which maps to the same site. The data are discussed in relation to current theories of gene conversion, and are consistent with the idea that gene conversion in mammalian cells can result from repair of heteroduplex DNA (hDNA) intermediates.

The enigmatic loss of light-independent chlorophyll biosynthesis from an Antarctic green alga in a light-limited environment.

GC-favoring gene conversion enables fixation of deleterious alleles, disturbs tests of natural selection and potentially explains both the evolution of recombination as well as the commonly reported intra-genomic correlation between G+C content and recombination rate. In addition, gene conversion disturbs linkage disequilibrium, potentially affecting the ability to detect causative variants. However, the importance and generality of these effects is unresolved, not simply because direct analyses are technically challenging but also because prior within- and between-species discrepant results can be hard to appraise owing to methodological differences. Here we report results of methodologically uniform whole-genome sequencing of all tetrad products in Saccharomyces, Neurospora, Chlamydomonas and Arabidopsis. The proportion of polymorphic markers converted varies over three orders of magnitude between species (from 2% of markers converted in yeast to only ~0.005% in the two plants) with at least 87.5% of the variance in per tetrad conversion rates being between-species. This is largely owing to differences in recombination rate and median tract length. Despite three of the species showing a positive GC-recombination correlation, there is no significant net AT->GC conversion bias in any, despite relatively high resolution in the two taxa (Saccharomyces and Neurospora) with relatively common gene conversion. The absence of a GC bias means: 1) that there should be no presumption that gene conversion is GC biased, nor 2) that a GC-recombination correlation necessarily implies biased gene conversion, 3) that Ka/Ks tests should be unaffected in these species and 4) it is unlikely that gene conversion explains the evolution of recombination.