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The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

The gap3 genes of the Synechococcus and Anabaena cyanobacteria fulfill so far unknown function. A homolog of this gene has recently been found in the nuclear genomes of diplonemids, which are heterotrophic flagellates closely related to kinetoplastids and euglenoids. To understand the function of the gap3 gene in the cyanobacteria, we performed Northern blotting experiments with the gap3 probes under different growth conditions. Under the standard photosynthetic growth conditions (high illumination and 1% CO2 in the gas phase), the expression of the gap3 gene was very low, but significantly increased during cell adaptation to the low CO2 concentration (0.03%). The gap3 operon was expressed as a polycistronic transcript of about 7 kb in size, which included ORF2 (1259 bp) immediately downstream of gap3. ORF2 probably encodes a putative transporter of HCO3. The nucleotide sequence of ORF2 has been submitted to GenBank under accession number AF 428100.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are essential to glycolysis, the major route of carbohydrate breakdown in eukaryotes. In animals and other heterotrophic eukaryotes, both enzymes are localized in the cytosol; in photosynthetic eukaryotes, GAPDH and TPI exist as isoenzymes that function in the glycolytic pathway of the cytosol and in the Calvin cycle of chloroplasts. Here, we show that diatoms--photosynthetic protists that acquired their plastids through secondary symbiotic engulfment of a eukaryotic rhodophyte--possess an additional isoenzyme each of both GAPDH and TPI. Surprisingly, these new forms are expressed as an TPI-GAPDH fusion protein which is imported into mitochondria prior to its assembly into a tetrameric bifunctional enzyme complex. Homologs of this translational fusion are shown to be conserved and expressed also in nonphotosynthetic, heterokont-flagellated oomycetes. Phylogenetic analyses show that mitochondrial GAPDH and its N-terminal TPI fusion branch deeply within their respective eukaryotic protein phylogenies, suggesting that diatom mitochondria may have retained an ancestral state of glycolytic compartmentation that existed at the onset of mitochondrial symbiosis. These findings strongly support the view that nuclear genes for enzymes of glycolysis in eukaryotes were acquired from mitochondrial genomes and provide new insights into the evolutionary history (host-symbiont relationships) of diatoms and other heterokont-flagellated protists.

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.

Cryptomonads are complex microalgae which share characteristics of chromophytes (chlorophyll c, extra pair of membranes surrounding the plastids) and rhodophytes (phycobiliproteins). Unlike chromophytes, however, they contain a small nucleus-like organelle, the nucleomorph, in the periplastidial space between the inner and outer plastid membrane pairs. These cellular characteristics led to the suggestion that cryptomonads may have originated via a eukaryote-eukaryote endosymbiosis between a phagotrophic host cell and a unicellular red alga, a hypothesis supported by rRNA phylogenies. Here we characterized cDNAs of the nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from the two cryptomonads Pyrenomonas salina and Guillardia theta. Our results suggest that in cryptomonads the classic Calvin cycle GAPDH enzyme of cyanobacterial origin, GapAB, is absent and functionally replaced by a photosynthetic GapC enzyme of proteobacterial descent, GapC1. The derived GapC1 precursor contains a typical signal/transit peptide of complex structure and sequence signatures diagnostic for dual cosubstrate specificity with NADP and NAD. In addition to this novel GapC1 gene a cytosol-specific GapC2 gene of glycolytic function has been found in both cryptomonads showing conspicuous sequence similarities to animal GAPDH. The present findings support the hypothesis that the host cell component of cryptomonads may be derived from a phototrophic rather than a organotrophic cell which lost its primary plastid after receiving a secondary one. Hence, cellular compartments of endosymbiotic origin may have been lost or replaced several times in eukaryote cell evolution, while the corresponding endosymbiotic genes (e.g., GapC1) were retained, thereby increasing the chimeric potential of the nuclear genome.

The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) gene family of maize is differentially expressed in response to anaerobic stress. While GapCl and GapC2 are downregulated, GapC3 and GapC4 are anaerobically induced. We have sequenced and analyzed a 3073 bp promoter fragment of GapC4. The promoter confers anaerobic induction of a reporter gene construct in a transient gene expression system in maize. Deletion analysis of the GapC4 promoter revealed a 270 bp long DNA region required for anaerobic induction. This region contains sequence motifs resembling the cis-acting sequences of the anaerobically induced maize Adh1 and Adh2 genes. Furthermore, the 3073 bp GapC4 promoter fragment displays homology to long terminal repeats of maize retrotransposons and to the 3' region of the maize anthocyanin regulatory locus C1.

We have characterized the cDNA and genomic sequences that encode actin from the multicellular red alga Chondrus crispus. Southern-blot analysis indicates that the C. crispus actin gene (ChAc) is present as a single copy. Northern analysis shows that, like the GapA gene, the actin gene is well expressed in gametophytes but weakly in protoplasts. Compared to actin genes of animals, fungi, green plants and oomycetes, that of C. crispus displays a higher evolutionary rate and does not show any of the amino-acid signatures characteristic of the other lineages. As previously described for GapA, ChAc is interrupted by a single intron at the beginning of the coding region. The site of initiation of transcription was characterized by RNAse protection. The promoter region displays a CAAT box but lacks a canonical TATA motif. Other noticeable features, such as a high content of pyrimidines as well as a 14-nt motif found in both the 5'-untranslated region and the intron, were observed.

We characterized a nuclear gene and its corresponding cDNA encoding beta-tubulin (gene TubB1) of the marine red alga Chondrus crispus. The deduced TubB1 protein is the most divergent beta-tubulin so far reported with only 64 to 69% amino acid identity relative to other beta-tubulins from higher and lower eukaryotes. Our analysis reveals that TubB1 has an accelerated evolutionary rate probably due to a release of functional constraints in connexion with a specialization of microtubular structures in rhodophytes. It further indicates that isoform diversity and functional differentiation of tubulins in eukaryotic cells may be controlled by independent selective constraints. TubB1 has a short spliceosomal intron at its 5' end which seems to be a characteristic feature of nuclear protein-coding genes from rhodophytes. The splice junctions of the four known rhodophyte introns comply well with the corresponding consensus sequences of higher plants in agreement with previous suggestions from phylogenetic inference that red algae and green plants may be sister groups. The paucity and asymmetrical location of introns in rhodophyte genes can be explained by differential intron loss due to conversion of genes by homologous recombination with cDNAs corresponding to reverse transcribed mRNAs or partially spliced pre-mRNAs, respectively. The identification of an intron containing TubB1 cDNA in C. crispus confirms that pre-mRNAs can escape both splicing and degradation in the nucleus prior to transport into the cytoplasm. Differential Southern hybridizations under non-stringent conditions with homologous and heterologous probes suggest that C. crispus contains a second degenerate beta-tubulin gene (or pseudogene?) which, however, is only distantly related to TubB1 as it is to the more conserved homologues of other organisms.

Algae are a heterogeneous group of photosynthetic eukaryotes traditionally separated into three major subdivisions: rhodophytes, chlorophytes, and chromophytes. The evolutionary origin of rhodophytes or red algae and their links to other photosynthetic and nonphotosynthetic eukaryotes have been a matter of much controversy and speculation. Here we present the first cDNAs of nuclear protein genes from red algae: Those encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Chondrus crispus. A phylogenetic analysis including GAPDH gene sequences from a number of eukaryotic taxa, cyanobacteria, and purple bacteria suggests that chloroplasts and rhodoplasts together form a monophyletic group of cyanobacterial descent and that rhodophytes separated from chlorophytes at about the same time as animals and fungi. The composite GAPDH tree further demonstrates that chloroplast and cytosolic GAPDH genes are closely related to their homologs in cyanobacteria and purple bacteria, respectively, the presumptive ancestors of chloroplasts and mitochondria, thereby firmly establishing the endosymbiotic origin of these nuclear genes and their fixation in eukaryotic cells before the rhodophyte/chlorophyte separation. The present data are in conflict with phylogenetic inferences based on plastid-encoded rbcL sequences supporting a polyphyletic origin of rhodoplasts and chloroplasts. Comparison of rbcL to GAPDH phylogenies suggests that rbcL trees may be misleading because they are composed of branches representing ancient duplicated (paralogous) genes.

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.

Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi. Here we characterize the genomic sequences of genes GapC and GapA of C. crispus with respect to promotor structures, intron/exon organization, genomic complexity, G + C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively. To our knowledge this is the first report on nuclear protein genes of red algae. The GapC gene is G + C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome. The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5' end. This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes (GapA and GapB) of higher plants. It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of -61.3 kJ. CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated. Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles. Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC.

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.

A Toxoplasma gondii genomic library was constructed in lambda EMBL3. Repeated fragments were detected by hybridization with radiolabeled total DNA from the parasite and one recombinant was chosen due to its strong hybridization signal. By using electrophoretic and hybridization analysis, four cross-hybridizating restriction fragments were selected and sequenced. The determined nucleotide sequence of these fragments (TGR1A, TGR1E, TGR2, and TGR4) has shown a complex system of conserved and degenerated repeats in which TGR1E corresponds to the most conserved element. This last sequence was used to investigate restriction fragment length polymorphisms among several T. gondii strains by Southern blotting.

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.