Origin and timing of the horizontal transfer of a PgiC gene from Poa to Festuca ovina

A segregating second locus, PgiC2, for the enzyme phosphoglucose isomerase (PGIC) is found in the grass sheep's fescue, Festuca ovina. We have earlier reported that a phylogenetic analysis indicates that PgiC2 has been horizontally transferred from the reproductively separated grass genus Poa. Here we extend our analysis to include intron and exon information on 27 PgiC sequences from 18 species representing five genera, and confirm our earlier finding. The origin of PgiC2 can be traced to a group of closely interrelated, polyploid and partially asexual Poa species. The sequence most similar to PgiC2 is found in Poa palustris with a divergence, based on synonymous substitutions, of only 0.67%. This value suggests that the transfer took place less than 600,000 years ago (late Pleistocene), at a time when most extant Poa and Festuca species already existed.     

A recently silenced, duplicate PgiC locus in Clarkia

Previous electrophoretic analysis showed that 17 diploid species of the wildflower Clarkia (Onagraceae) have two cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9), whereas 15 other diploid species have a single PGIC. Molecular studies revealed that the two isozymes in the former species are encoded by duplicate genes, PgiC1 and PgiC2, whereas the single isozyme in the latter is always encoded by PgiC1. Phylogenetic analysis of the nucleotide sequences implied that PgiC2 was silenced four times independently in the genus. Here we describe a psi PgiC2 from C. mildrediae, a species in which only PgiC1 is expressed. The discovery of the psi PgiC2 is significant because it confirms a formal prediction of the phylogenetic analysis. The psi PgiC2 includes 5,039 nucleotides corresponding to 18 of the 23 exons of PgiC, as well as the intervening introns and 3' nontranslated region. The absence of an increase of nucleotide substitutions in its "exons" suggests that the gene was silenced recently. The present study appears to be the first to establish that a specific duplicate gene locus regularly expressed in a group of related plant species has been silenced in one of them. The multiple independent silencings of PgiC2 suggest that it remained functional but inessential in ancestral lineages. We discuss the possibility that PgiC2 may have been preserved in these lineages by selection against mutants causing defective PGIC1- PGIC2 heterodimers.      

A DNA marker for the duplicated cytosolic PGI genes in sheep's fescue (Festuca ovina L.)

Active duplicate PgiC genes in sheep's fescue, Festuca ovina, are associated with a PCR marker of specific length (about 370 bp, of which 231 are in an intron). Using this marker, the frequency of plants with duplicate genes is estimated to be about 10% in a population from southern Sweden. The close molecular similarity between the electrophoretically different duplicated genes is in accordance with the conclusion reached earlier that they are indeed alleles at the same locus.     

Structure of the natural transgene PgiC2 in the common grass Festuca ovina

Background

A horizontal gene transfer has brought an active nuclear gene, PgiC2, from a polyploid Poa species (P. palustris or a close relative) into the common grass sheep''s fescue (Festuca ovina). The donor and the receptor species are strictly reproductively separated, and PgiC2 occurs in a polymorphic state within F. ovina. The active gene copy is normally closely linked to a very similar pseudogene.

Methodology/Principal Findings

By genome walking we have obtained the up- and downstream sequences of PgiC2 and of corresponding genes in the donor and recipient species. Comparisons of these sequences show that the complete upstream region necessary for the gene''s expression is included in the transferred segment. About 1 kb upstream of PgiC2 a fragment with transposition associated properties has been found (TAF). It is present in P. palustris and its polyploid relatives, though not at the homologous position, and is absent from many other grasses, including non-transgenic F. ovina plants. It is possible that it is a part of a transposing element involved in getting the gene into a transferring agent and/or into the recipient chromosome.

Conclusions/Significance

The close similarity of the up- and downstream regions with the corresponding regions in P. palustris excludes all suggestions that PgiC2 is not a HGT but the result of a duplication within the F. ovina lineage. The small size of the genetic material transferred, the complex nature of the PgiC2 locus, and the associated fragment with transposition associated properties suggest that the horizontal transfer occurred via a vector and not via illegitimate pollination.     

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.     

Contrasting modes of evolution acting on the complex N locus for rust resistance in flax

Three rust resistance specificities, N, N1 and N2, map to the complex N locus of flax. We used a degenerate PCR approach, with primers directed to the nucleotide binding site (NBS) domain characteristic of many plant resistance genes, to isolate resistance gene analogs (RGAs) from flax. One RGA clone detected RFLPs co-segregating with alleles of the N locus. With this probe we isolated four related genes that occur within a 30kbp region and encode proteins with NBS and leucine-rich repeat (LRR) domains and N-terminal Toll/Interleukin-1 Receptor homology (TIR) domains. One of these four genes was identified as the N resistance gene by sequence analysis of three mutant alleles and by transgenic expression. We isolated homologous genes from two flax lines containing the N1 or N2 specificities and from flax lines carrying no N locus resistance specificities. Analysis of shared polymorphisms among this set of 18 N locus sequences revealed three groups of genes with independent lineages. Sequence exchanges have only occurred between genes within each group, but not between groups. Two of the groups contain only one sequence from each haplotype and probably represent orthologous genes. However, the third group contains two genes from each haplotype. We suggest that the re-assortment of variation by recombination/gene conversion at this locus is limited by the degree of sequence identity between genes.     

Molecular Characterization of Duplicate Cytosolic Phosphoglucose Isomerase Genes in Clarkia and Comparison to the Single Gene in Arabidopsis

The nucleotide sequence of PgiC1-a which encodes a cytosolic isozyme of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5.     

Gene dosage effects on the synthesis of maltase in yeast.

Inbred strains of Saccharomyces cerevisiae carrying MAL1, MAL2, or MAL6 in a common background were used to construct (i) homo- or heterozygous diploids carrying one or two active alleles of a single MAL locus (MAL1, MAL2, or MAL6) and (ii) triploids carrying one, two, or three active alleles of MAL2. The diploid and triploid strains were used to investigate gene dosage effects of the differential rate of maltase synthesis (delta enzyme activity/delta growth) and the kinetics of induction (for MAL2). All three MAL loci exhibited a gene dosage effect on the differential rate of maltase synthesis; MAL2 also exhibited a gene dosage effect on the kinetics of induction. The dosage effects of the MAL1 and MAL6 loci were additive, but the effects of the MAL2 locus were not; the magnitude of the MAL2 gene dosage effect decreased with increasing dosage. These results are compatible with the current genetic evidence that the MAL genes are regulatory loci if the product(s) of the MAL1 and MAL6 locus is produced in limiting amounts but the product(s) of the MAL2 locus is produced in excess, except at very low genes dosages.     

Characterization of duplicated two cytosolic phosphoglucose isomerase (PgiC) loci in Arabidopsis halleri ssp. gemmifera

Arabidopsis halleri ssp. gemmifera has two cytosolic phosphoglucose isomerase (PgiC) loci. A 48-bp deletion was observed in the junction of exon 17 and intron 17 for a locus (PgiC2). PCR-RFLP analysis using cDNA template did not detect the PgiC2 locus. Another locus (PgiC1) has common structure with A. thaliana and expressed normally. A phylogenetic tree of PgiC sequences revealed that duplication of the two loci in A. gemmifera occurred after species splitting of A. thaliana and A. gemmifera. More than 12 kb region encompassing PgiC was sequenced for both loci. In both PgiC1 and PgiC2, sequence homologous to A. thaliana PgiC 5' upstream region was not detected. A gene located on chromosome 4 of A. thaliana was detected in the 5' upstream of PgiC2. This result suggested that the microsyntheny around the PgiC region between A. thaliana and A. gemmifera is not established.     

DNA polymorphism, haplotype structure and balancing selection in the Leavenworthia PgiC locus.

A study of DNA polymorphism and divergence was conducted for the cytosolic phosphoglucose isomerase (PGI:E.C.5.3.1.9) gene of five species of the mustard genus Leavenworthia: Leavenworthia stylosa, L. alabamica, L. crassa, L. uniflora, and L. torulosa. Sequences of an internal 2.3-kb PgiC gene region spanning exons 6-16 were obtained from 14 L. stylosa plants from two natural populations and from one to several plants for each of the other species. The level of nucleotide polymorphism in L. stylosa PgiC gene was quite high (pi = 0.051, theta = 0.052). Although recombination is estimated to be high in this locus, extensive haplotype structure was observed for the entire 2.3-kb region. The L. stylosa sequences fall into at least two groups, distinguished by the presence of several indels and nucleotide substitutions, and one of the three charge change nucleotide replacements within the region sequenced correlates with the haplotypes. The differences between the haplotypes are older than between the species, and the haplotypes are still segregating in at least two of five species studied. There is no evidence of recent or ancient population subdivision that could maintain distinct haplotypes. The age of the haplotypes and the results of Kelly's Z(nS) and Wall's B and Q tests with recombination suggest that the haplotypes are maintained due to balancing selection at or near this locus.     

Multiple Versions of the a Mating Type Locus of Coprinus Cinereus Are Generated by Three Paralogous Pairs of Multiallelic Homeobox Genes

The A mating type locus of Coprinus cinereus determines mating compatibility by regulating essential steps in sexual development. Each A locus contains several genes separated into two functionally independent complexes termed Aα and Aβ, and the multiple alleles of these genes generate an estimated 160 A mating specificities. The genes encode two classes of homeodomain-containing proteins designated HD1 and HD2. In this report we describe two newly cloned loci, A2 and A5, and compare them with A42, A43 and A6 that we have described previously. An Aβ-null locus, retaining just a single active HD1 gene from the α-complex, was generated by mutation. Using this as a transformation host, gene combinations that promote A-regulated development were identified. We demonstrate that each A locus contains members of three paralogous pairs of HD1 and HD2 genes. Different allelic versions of gene pairs are compatible but paralogous genes are incompatible. The genes present in four uncloned A loci were deduced using Southern analyses and transformations with available cloned genes. The combined analysis of nine A factors identifies sufficient A gene alleles to generate at least 72 A mating specificities.     

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain. To do this analysis we chose to examine the relative Ig H chain V region gene usage of pre-B cell lines derived from (C57BL/6 x BALB/c)F1 fetal liver cells by Southern blot analysis. We found a high frequency of Vh-gene rearrangements (77% of the alleles had VDJ rearrangements) and these rearrangements occurred to Vh-genes throughout the Vh locus and were not confined to the D-proximal Vh-genes as has been previously observed with lines from other mouse strains. The Vh-gene usage pattern is similar on both alleles indicating that at least one of the determinants of which Vh-gene is used is trans-acting and acts similarly on each allele. Furthermore, one allele, Ighb (donated by the C57BL/6 parent), rearranged Vh-genes more frequently than the other allele, Igha (donated by the BALB/c parent) suggesting that one of the determinants of Vh-gene rearrangement may be acting in an allele-specific manner.     

DNA sequence polymorphism at the human tumor necrosis factor (TNF) locus. Numerous TNF/lymphotoxin alleles tagged by two closely linked microsatellites in the upstream region of the lymphotoxin (TNF-beta) gene.

TNF-alpha and lymphotoxin (LT, TNF-beta) genes are tandemly arranged and map within the MHC centromeric to HLA-B and telomeric to the class III genes. Both cytokines encoded by these genes are potent immunomodulators. On the other hand, some MHC-linked autoimmune diseases are characterized by abnormal levels of their expression or inducibility. A search for the putative disease-associated TNF/LT alleles depends on the informative genetic markers at the TNF locus. Previously, a low degree of genetic polymorphism at the human TNF locus has been reported, mostly bi-allelic RFLP. To localize and define additional polymorphic markers, we probed the collection of genomic clones with synthetic tandemly repeated dinucleotides, corresponding to the sequences known as microsatellites. We mapped and characterized three (TC/GA) and one (AC/GT) repeats within cloned 40-kb DNA comprising the human TNF locus. Using a polymerase chain reaction-based technique, we analyzed three of these four microsatellites and observed their length of polymorphism. Using DNA samples from blood donors, two families, and three human cell lines, we detected 13 distinct alleles of the AC/GT microsatellite neighboring human TNF genes. The variability was further increased by simultaneous analysis of the second linked microsatellite. This linked TC/GA repeat showed at least five alleles, whereas the least polymorphic TC/GA repeat located in the first intron of LT (TNF-beta) gene had two alleles. TNF alleles defined by microsatellites were stably inherited and segregated in the Mendelian way. Therefore, we describe thus far the most informative level of DNA sequence polymorphism in this part of human MHC. We propose a nomenclature for microsatellite tagged LT/TNF alleles based on their size and variability, which could also be extended to include RFLP and other not yet identified polymorphic markers. Microsatellite tagged polymorphism described here can be used in systematic linkage studies of HLA-associated diseases.     

A new fertility restorer locus linked closely to the Rfo locus for cytoplasmic male sterility in radish

In this study, we have investigated a new fertility restorer (Rf) locus for cytoplasmic male sterility (CMS) in radish. We have obtained a CMS-Rf system consisting of sterile line '9802A1', maintainer line '9802B1' and restorer line '9802H'. F(1) plants from cross between sterile line '9802A1' and restorer line '9802H' were all male fertile, self pollination of F(1) plants produced an F(2) segregating population consisting of 600 individuals. The segregating population was found to fit a segregation ratio 3:1 for male fertile and sterile types, indicating that male fertility is restored by a single dominant gene (termed Rfo2) in the CMS-Rf system. Based on the DNA sequence of Rfo/Rfk1 (AJ535623), just one full length gene in the sterile line '9802A1', in the restorer line '9802H' and in the male fertile line '2006H', was cloned, respectively. The three sequences correspond to the same gene with two alleles: Rfob in '9802H' and rfob in '9802A1' and '2006H'. These two alleles differ from Rfo/Rfk1 and rfk1 (AJ535624) alleles by two synonymous base substitutions, respectively. Based on the differences between the Rfob and rfob genes, one PCR-based marker was developed, and designated Marker 1, which is identical to the corresponding region of Rfob by sequence analysis. In the F(2) segregating population described above, the Marker 1 was present in 5 sterile plants and in 453 fertile plants, absent in 4 fertile plants and in 138 sterile plants, and was found to fit a segregation ratio 3:1 indicating that Rfob was single copy in '9802H'. Linkage analysis showed that the Rfo2 locus for our CMS-Rf system was distant from the Rfo locus by about 1.6 cM. The sterile line '9802A1' was pollinated by the male fertile line '2006H' and the resulting F(1) plants were all male fertile. These results indicated that the male fertility of radish CMS can be restored by a new Rf locus, which linked tightly to the Rfo locus.