<Emphasis Type="Italic">Oscheius tipulae</Emphasis> as an Example of eEF1A Gene Diversity in Nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.     

Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism. 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes. In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species. In each species additional, divergent 4CL genes were found. Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform. Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily. According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species.     

Duplicated Proteasome Subunit Genes in Drosophila Melanogaster Encoding Testes-Specific Isoforms

Using the previously cloned proteasome α-type subunit gene Pros28.1, we screened a Drosophila melanogaster genomic library using reduced stringency conditions to identify closely related genes. Two new genes, Pros28.1A (map position 92F) and Pros28.1B (map position 60D7), showing high sequence similarity to Pros28.1, were identified and characterized. Pros28.1A encodes a protein with 74% amino acid identity to PROS28.1, while the Pros28.1B gene product is 58% identical. The Pros28.1B gene has two introns, located in exactly analogous positions as the two introns in Pros28.1, while the Pros28.1A gene lacks introns. Northern blot analysis reveals that the two new genes are expressed only in males, during the pupal and adult stages. Tissue-specific patterns of expression were examined using transgenic flies carrying lacz-fusion reporter genes. This analysis revealed that both genes are expressed in germiline cells during spermatogenesis, although their expression patterns differed. Pros28.1A expression is first detected at the primary spermatocyte stage and persists into the spermatid elongation phase of spermiogenesis, while Pros28.1B expression is prominent only during spermatid elongation. These genes represent the most striking example of cell-type-specific proteasome gene expression reported to date in any system and support the notion that there is structural and functional heterogeneity among proteasomes in metazoans.     

Characterization of the CYP4A11 gene,a second CYP4A gene in humans

Comparison between the cDNA sequence of CYP4A11 and that deduced from a published genomic clone suggested the presence of an additional CYP4A gene in humans, CYP4A22. PCR amplification of genomic DNA yielded overlapping clones covering 13kb of genomic DNA and extending from 1003bp upstream from CYP4A11 translation initiation to 135bp upstream of the mRNA polyadenylation signal. Sequence and Southern blot analysis showed the presence in humans of two highly homologous CYP4A genes, CYP4A11 and CYP4A22. These two genes share 96% sequence identity and have similar intron/exon sizes and distribution. Short nucleotide insertions (< or =10bp) in introns 1, 3, 9, and 11, and deletions (< or =18bp) in introns 4, 6, and 11 differentiate the two genes. RT-PCR amplification of human kidney RNA followed by restriction fragment analysis showed that CYP4A11 is the predominant isoform expressed in kidney.     

Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis

In green tissues of plants under illumination, photosynthesis is the primary source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is utilized in reductive reactions such as carbon fixation and nitrogen assimilation. In non-photosynthetic tissues or under non-photosynthetic conditions, the oxidative pentose phosphate pathway contributes to basic metabolism as one of the major sources of NADPH. The first and committed reaction is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). We characterized the six members of the G6PDH gene family in Arabidopsis. Transit peptide analysis predicted two cytosolic and four plastidic isoforms. Five of the six genes encode active G6PDHs. The recombinant isoforms showed differences in substrate requirements and sensitivities to feedback inhibition. Plastidic isoforms were redox sensitive. One cytosolic isoform was insensitive to redox changes, while the other was inactivated by oxidation. The respective genes had distinct expression patterns that did not correlate with the activity of the proteins, implying a regulatory mechanism beyond the control of mRNA abundance. Two cytosolic and one plastidic isoform were detected in vivo using zymograms, and the respective genes were identified using T-DNA insertion lines. The activity of a plastidic isoform was detected in all tissues including photosynthetic tissues despite its sensitivity to reduction observed in vitro. Genomic data, gene expression, and in vivo enzyme activity data were integrated with in vitro biochemical data to propose in vivo roles for individual G6PDH isoforms in Arabidopsis.     

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.     

Two Arabidopsis homologs of the animal trithorax genes: a new structural domain is a signature feature of the trithorax gene family.

Two Arabidopsis genes have been characterized as first examples of plant genes homologous to the animal trithorax genes. The Arabidopsis genes are highly similar but display different tissue and development expression patterns. One of them was ubiquitously expressed, with highest levels registered in young seedlings. The other gene was less active in all tested tissues, was not expressed in mature leaves but was highly expressed in roots. A new structural motif common to all TRX-related proteins has been identified. This new architectural element was found only in genes of multicellular species and is present in all genes belonging to the trithorax family. Along with the SET domain and the PHD fingers, this new element is a signature feature for the trithorax gene family.