Cloning and characterization of genes involved in carbohydrate metabolism in the marine red alga Gracilaria gracilis

The molecular biology of carbohydrate metabolism in red algae is poorly known. Enzymological studies are few, and no gene for the biosynthesis of sugar nucleotides and polysaccharides has so far been characterized. To isolate genes involved in carbohydrate metabolism in Gracilaria gracilis, genomic libraries were screened with homologous probes prepared either by PCR with degenerate primers, or from cDNAs previously isolated for generating expressed sequence tags (ESTs) from G. gracilis. Genes involved in carbohydrate metabolism, photosynthesis, protein synthesis and degradation, amino acid metabolism, and stress response were among those tagged by the ESTs. Three genes were characterized. These encode galactose-1-phosphate uridylyltransferase (GALT, named GgGALT1), a key enzyme for scD-galactose metabolism; UDPglucose pyrophosphorylase (UGPase; GgUGP), a key enzyme for sugar nucleotide synthesis; and starch branching enzyme (SBE; GgSBE1), which helps determine the structure of floridean starch. The three genes are devoid of introns. Each possesses a polyadenylation signal, TAAA, which occurs in all G. gracilis genes so far characterized, as well as a potential TATA box. Southern hybridization experiments indicate that the three genes are single-copy, but that other genes related to GgGALT1 and GgSBE1 exist. GgGALT1 and GgUGP are each located close to another gene, hinting that occurrence of closely-spaced genes, atypical in eukaryotic genomes, may not be uncommon in the G. gracilis genome. The deduced proteins show high sequence similarity with their homologs in other organisms, but intriguing differences, such as nonconservative substitutions at functionally important sites, were observed. The protein encoded by GgSBE1 lacks an N-terminal portion that could contain a possible target peptide, consistent with the cytosolic localization of floridean starch synthesis. The GgUGP and GgSBE1 proteins are as phylogenetically related to plant as they are to their animal and fungal homologs.Doctor of Philosoph

Characterization of the UDP-glucose pyrophosphorylase gene from the marine red alga Gracilaria gracilis

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in carbohydrate metabolism, particularly polysaccharide biosynthesis, in red algae. In this report, we characterize at the genomic and cDNA levels the putative UGPase gene of the agarophytic red alga Gracilaria gracilis. The gene is single-copy, devoid of introns, and produces two kinds of transcripts that differ in size by 332 basepairs. The large and small transcripts appear to utilize distinct polyadenylation signals. The putative protein has 495 amino acids, and is about 50% identical in sequence to its homplogs in plants, animals and fungi. Sequencing of the,genomic clone revealed that another gene, potentially encoding a DNA helicase and containing a 76 bp-intron near its 3' end, occurs 376 bp downstream of the UGPase gene

Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Graeilaria gracilis

The metabolism of D-galactose is a major feature of red-algal physiology. We have cloned and sequenced a gene from the red alga Gracilaria gracilis that encodes a key enzyme of D-galactose metabolism, galactose-l-phosphate uridylyltransferase (GALT). This gene, designated GgGALT1, is apparently devoid of introns. A potential TATA box, four potential CAAT boxes, and a repeated sequence occur in the 5'-flanking region. The predicted 369-aa peptide shares significant sequence similarity with GALTs from other organisms (human, 47%; Saccharomyces cerevisiae, 49%; Solanum tuberosum, 49%). Southern-hybridization analysis reveals two related, but apparently not identical, GALT genes in the nuclear genome of G. gracilis. Sequence analysis indicates that the GgGALT1 enzyme lacks a rubredoxin "knuckle" motif, which in bacterial and fungal GALTs is involved in binding zinc. An open reading frame encoding a potential peptidyl tRNA hydrolase occurs 179 bp downstream from the GgGALT1 gene

Cloning and characterization of a nuclear gene encoding a starch branching enzyme from the marine red alga Gracilaria gracilis

The biosynthesis of starch in red algae occurs in the cytosol, in contrast to green plants where it takes place in the plastid. We have cloned a nuclear gene from the red alga Gracilaria gracilis that encodes a homolog of starch-branching enzymes (SBEs); this gene, which is apparently intron-free, was designated as GgSBE1. A potential TATA box, CAAT boxes, and other potential regula tory elements were observed in its 5' flanking region. The encoded 766-aa peptide shares significant sequence similarity with SBEs from green plants (at least 40%), and with glycogen-branching enzymes (GBEs) from human (46%) and Saccharomyces cerevisiae (45%). Southern-hybridization analysis indicates that the gene is single-copy, although weaker signals suggest that related genes exist in the genome of G. gracilis. Phylogenetic analyses indicate that GgSBE1 groups within the eukaryote branching enzymes (BEs) and not with eubacterial GBEs, suggesting that its gene has not been derived directly from an endosymbiotic cyanobacterium, but instead is ancestrally eukaryotic

Occurrence of closely spaced genes in the nuclear genome of the agarophyte Gracilaria gracilis

Little is known about the structure and organisation of nuclear genomes in red algae. In particular, it is not known whether genes are densely or loosely packed, whether gene order is conserved, whether their genes tend to occur in one or multiple copies and whether their nuclear genes tend to be compact or interrupted by numerous introns. Sequencing of cloned genomic DNA from Gracilaria gracilis has begun to provide provisional answers to some of these questions. Four pairs of closely spaced genes have been found in G. gracilis upon sequencing genomic clones that contain genes for UDPglucose pyrophosphorylase, galactose-1-phosphate uridylyltransferase, the beta subunit of tryptophan synthetase, and methionine sulphoxide reductase (a fifth pair of closely spaced genes, encoding polyubiquitin and aconitase, was reported earlier). An open reading frame with significant similarity to another known gene occurs close (< 1.7 kbp) to each of these genes. In two pairs the intergenic region is less than 400 bp in length, and for these the location of the putative polyadenylation signals indicates that the gene transcripts, encoded on opposite strands, have overlapping (hence complementary) 3' regions. These somewhat unexpected findings begin to establish a basis for genome-level characterisation of red algae

Expressed sequence tags (ESTs) from the marine red alga Gracilaria gracilis

Expressed sequence tags (ESTs) are partial sequences of cDNAs, and can be used to characterize gene expression in organisms or tissues. We have constructed a 200-sequence EST database from vegetative thalli of Gracilaria gracilis, the first ESTs reported from any alga. This database contains recognizable ESTs corresponding to genes of carbohydrate metabolism (seven), amino acid metabolism (three), photosynthesis (five), nucleic acid synthesis, repair and processing (three), protein synthesis (14), protein degradation (six), cellular maintenance and stress response (three), other identifiable protein-coding genes (13) and 146 sequences for which significant matches were not found in existing sequence databases. We have already used this EST database to recover genes of carbohydrate biosynthesis from G. gracilis