Structure and evolution of Cyclops: a novel giant retrotransposon of the Ty3/Gypsy family highly amplified in pea and other legume species.

We characterized a novel giant Gypsy-like retrotransposon, Cyclops, present in about 5000 copies in the genome of Pisum sativum. The individual element Cyclops-2 measures 12 314 bp including long terminal repeats (LTRs) of 1504 bp and 1594 bp, respectively, showing 4.1% sequence divergence between one another. Cyclops-2 carries a polypurine tract (PPT) and an unusual primer binding site (PBS) complementary to tRNA-Glu. The element is bounded by 5 bp target site duplications and harbors three successive internal regions with homology to retroviral genes gag (424 codons) and pol (1382 codons) and an additional open reading frame (423 codons) of unknown function indicating the element's potential capacity for gene transduction. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAse H and integrase in the same typical order (5'-PR-RT-RH-IN-3') known for retroviruses and Gypsy-like retrotransposons. The reading frame of the pol region is disrupted by several mutations suggesting that Cyclops-2 does not encode functional enzymes. A phylogenetic analysis of the reverse transcriptase domain confirms our differential genetic assessment that Cyclops from pea is a novel element with no specific relationship to the previously described Gypsy-like elements from plants. Genomic Southern hybridizations show that Cyclops is abundant not only in pea but also in common bean, mung bean, broad bean, soybean and the pea nut suggesting that Cyclops may be an useful genetic tool for analyzing the genomes of agronomically important legumes.

Non-phosphorylating GAPDH of higher plants is a member of the aldehyde dehydrogenase superfamily with no sequence homology to phosphorylating GAPDH.

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NADP-specific, EC 1.2.1.9) operates in the cytosol of autotrophic eukaryotes where it generates NADPH for biosynthetic processes from photosynthetic glyceraldehyde 3-phosphate exported from the chloroplast by the phosphate translocator. Here we report the first cloning and characterization of cDNAs encoding complete polypeptide chains of nonphosphorylating GAPDH from pea and maize by using oligonucleotide probes derived from amino acid sequences determined for the purified enzyme. Unexpectedly, nonphosphorylating GAPDH cannot be aligned with the well-known sequences of phosphorylating GAPDH, but shares about 30% amino acid identity with various specialized and non-specialized aldehyde dehydrogenases (ALDHs) of eubacteria and eukaryotes. A phylogenetic analysis of this ALDH superfamily reveals a complex evolutionary pattern with numerous major branches carrying genes from eubacteria, eukaryotes, or both, encoding enzymes that are specific or non-specific for particular aldehyde substrates. This topology suggests a concomitant emergence of multiple substrate specificities from non-specialized ALDH during an early evolutionary phase of intense metabolic diversification. Although unrelated at the sequence level, non-phosphorylating aldehyde dehydrogenases and phosphorylating GAPDH resemble one another with respect to catalytic hydride transfer and covalent thiol ester formation. Whether or not this reflects an ancestral relationship can only be decided when crystallographic data for ALDH enzymes have become available.

Five identical intron positions in ancient duplicated genes of eubacterial origin.

In 1985 Cornish-Bowden wrote "although there is now much to suggest that introns are an ancient relic of primordial genes, convincing proof must await the discovery of clearly corresponding intron arrangements in genes that arose by duplication before the separation of prokaryotes and eukaryotes". Genes for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases of eukaryotes are descendants of an ancient gene family that existed in the common ancestor of extant eubacteria. During eukaryotic evolution, both genes were transferred to the nucleus from the antecedents of present-day chloroplasts and mitochondria, respectively. Here we report the discovery of five spliceosomal introns at positions that are precisely conserved between nuclear genes for this chloroplast/cytosol enzyme pair. These data provide strong evidence in favour of the 'introns early' hypothesis, which proposes that introns were present in the earliest cells, consistent with the idea that introns facilitated the assembly of primordial genes by accelerating the rate of exon shuffling.

The beta-tubulin gene family of pea: primary structures, genomic organization and intron-dependent evolution of genes.

One gene and two cDNAs encoding three different beta-tubulins (TUB1, TUB2, TUB3) of pea have been cloned and sequenced. The derived amino acid sequences show between 92% and 96% identity relative to one another and to most other beta-tubulins of higher plants and green algae. Two notable extremes are the high similarity of 98% between pea TUB3 and maize beta-tubulin 2 and the relatively low similarity (90%) of the hypocotyl-specific beta-tubulin 1 of soybean to the pea sequences. These similarities do not reflect the molecular phylogeny but rather differences in evolutionary rate of beta-tubulins which are differentially regulated during plant development. Genomic Southern blots reveal a beta-tubulin gene family in pea with at least four separate members including two TUB1 genes, one TUB2 gene and one TUB3 gene. This contradicts an earlier report by Raha et al. (Plant Mol Biol 9: 565-571, 1987) suggesting a tandem repeat organization of tubulin genes in pea. The pea TUB1 gene has two introns in identical positions compared to the beta-tubulin genes from Arabidopsis and soybean. In an attempt to reconstruct the universal ancestor of all present-day tubulin genes the intron positions in 38 different alpha- and beta-tubulin genes from plants, animals, fungi and protozoa were compared. This comparison shows that the primordial gene probably had many introns (more than 20) separating 'protoexons' of 15 to 20 codons in agreement with the 'exon theory of genes'. It also supports the view that, during the course of evolution, introns have shifted and were deleted preferentially in the 3' part of the genes. Similar observations have been made previously for other genes. They can be interpreted in terms of a homologous recombination of genes with their modified (incorrectly spliced) and reverse-transcribed pre-mRNAs.

Differential intron loss and endosymbiotic transfer of chloroplast glyceraldehyde-3-phosphate dehydrogenase genes to the nucleus.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GAPA and GAPB, which are encoded in the nucleus by two related genes of eubacterial origin. In the present work the genes encoding chloroplast GAPA and GAPB from pea have been cloned and sequenced. The gene for GAPB is split by eight introns. Two introns interrupt the region encoding the transit peptide and six are found within the region encoding the mature subunit, four of which are in identical or similar positions relative to genes for cytosolic GAPDH of eukaryotic organisms. As opposed to this, the gene encoding pea GAPA has only two introns in the region encoding the mature subunit. These findings strongly support the "intron early" hypothesis and suggest that the low number of introns in the gene for chloroplast GAPA is due to differential loss of introns during the streamlining period of the chloroplast genome following the GAPB/GAPA separation. We deduce from this that eubacteria and chloroplasts contained GT-AG introns until relatively recently and that the duplication event leading to the genes encoding GAPB and GAPA and their respective transit peptides occurred in the chloroplast progenitor prior to the successive transfer and functional reintegration of these genes into the nuclear environment. These conclusions imply that GAPA/GAPB transit peptides are of eubacterial origin.

Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GapA and GapB. cDNA clones containing the entire coding sequences of the cytosolic precursors for GapA from pea and for GapB from pea and spinach have been identified, sequenced and the derived amino acid sequences have been compared to the corresponding sequences from tobacco, maize and mustard. These comparisons show that GapB differs from GapA in about 20% of its amino acid residues and by the presence of a flexible and negatively charged C-terminal extension, possibly responsible for the observed association of the enzyme with chloroplast envelopes in vitro. This C-terminal extension (29 or 30 residues) may be susceptible to proteolytic cleavage thereby leading to a conversion of chloroplast GAPDH isoenzyme I into isoenzyme II. Evolutionary rate comparisons at the amino acid sequence level show that chloroplast GapA and GapB evolve roughly two-fold slower than their cytosolic counterpart GapC. GapA and GapB transit peptides evolve about 10 times faster than the corresponding mature subunits. They are relatively long (68 and 83 residues for pea GapA and spinach GapB respectively) and share a similar amino acid framework with other chloroplast transit peptides.

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba).

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].