Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Gracilaria 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-1-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. Received: 6 April / 2 June 1998     

Cloning and characterization of the nuclear gene and cDNAs for triosephosphate isomerase of the marine red alga Gracilaria verrucosa

cDNAs and an intronless single-copy nuclear gene (TPI1) encoding triosephosphate isomerase have been cloned and sequenced from the marine red alga Gracilaria verrucosa. The predicted amino-acid sequence of TPI1 is readily alignable with those of other known TPIs; 26 of 27 active-site residues and 19 of 26 intersubunit-contact residues are identical between TPIs of G. verrucosa and/or animals and green plants. A partial cDNA sequence of a second TPI gene (TPI2), presumably encoding plastid-localized TPI, was recovered by PCR and demonstrated by phylogenetic analysis to be red algal; no TPI2 cDNA or genomic clones could be recovered. Genomic Southern analysis demonstrated that at least two TPI-like genes are present in the nuclear DNA of G. verrucosa.Issued as NRCC no. 38070 GSDB accession L38662     

cDNA cloning and characterization of the nuclear gene encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase from the marine red alga Gracilaria verrucosa

Using a PCR-generated homologous probe, we have recovered a cDNA (GapA cDNA) encoding the complete 338 amino-acid chloroplast GAPDH of the marine red aga Gracilaria verrucosa, together with its 78 amino-acid transit peptide. This cDNA was readily aligned with chloroplast-localized GAPDH genes (GapA) and (GapB) of green plants. The proline residue which contributes to the specificity of NAD⁺ binding to cytosolic GAPDHs is absent from the deduced polypeptide chain of G. verrucosa GapA as is also the case in the chloroplast GAPDHs of plants. The transit peptide shows a high proportion of random coil, an amino-terminal Met-Ala dipeptide, a high content of hydroxylamino acids, and a net positive charge. The polyadenylation signal appears to the AGTAAA. Genomic Southernhybridization data indicate that only one chloroplast-GAPDH gene may occur in G. verrucosa. Bootstrapped parsimony trees indicate that the G. verrucosa Gap A gene is a sister group to plant chloroplast-GAPDH genes, and are most readily interpreted as showing that red algal and plant chloroplast-localized GAPDHs arose in a single endosymbiotic event.     

The nuclear gene and cDNAs encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase from the marine red alga Gracilaria verrucosa: cloning,characterization and phylogenetic analysis

We have cloned and sequenced the single-copy nuclear gene (GapC) encoding the complete 335-amino acid cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) from the red alga Gracilaria verrucosa. The proline residue which contributes to the specificity of NAD⁺ binding in other GAPC-like proteins is present. Putative regulatory regions, including GC-rich regions, a GATA element, and 11-base T- and T/G-clusters, but excluding TATA- and CCAAT-boxes, were identified upstream. Two types of GapC cDNAs differing in polyadenylation site were characterized. An 80-bp phase-two spliceosomal intron was identified in a novel position interrupting the highly conserved cofactor-coding region I. The G. verrucosa GAPC was easily aligned with other known GAPC-type sequences. Inferred phylogenetic trees place red algae among the eukaryote crown taxa, although with modest bootstrap support and without stable resolution among related GAPC lineages.GSDB accession L38661 Issued as NRCC no. 38071 Present address Human Genetics Center, University of Texas, P.O. Box 20334, Houston, TX 77225, USA     

Circular plasmid DNAs from the red alga Gracilaria chilensis

Summary Total cellular DNA extracted from eight red algal species (from the genera Gracilaria, Gracilariopsis, Porphyra and Gymnogongrus) was centrifuged on Hoechst dye/CsCl gradients. In five species, plasmid-like DNAs banded with the A+T rich organellar DNAs in the CsCl gradients. Based on their electrophoretic migration in different agarose gels, the plasmid-like DNAs are circular. This is the first report of putative plasmid DNAs in the red algae outside the genus Gracilaria. Two similar Gracilaria chilensis plasmid-like DNAs of 3.8 and 3.4 kb (GC2 and GC3) were cloned in pUC19. The cloned GC2 DNA did not hybridize to either the organellar or nuclear genomes of G. chilensis, suggesting that GC2 is a true plasmid. GC2 did hybridize to the plasmid of one other red algal species, Gracilaria sordida.     

Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi.

A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi. A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs. A 2178-bp protein coding region of the T. cruzi PLC gene, composed from cDNA and genomic clones, encodes a putative PLC with a calculated molecular mass of 82,032 Da and an isoelectric point of 5.93. The deduced amino acid sequence of T. cruzi PLC exhibited 23-42% overall identities with the PLCs from other organisms. Among them, PLC from Ictalurus punctatus revealed the highest identity to T. cruzi PLC. The percentage identities of the entire proteins and the catalytic X/Y domains suggested that T. cruzi PLC is more evolutionarily related to the PLCs of higher eukaryotes than to those of lower unicellular eukaryotes. The tetrad analysis of the segregants of the Saccharomyces cerevisiae PLC1/plc1::HIS3 diploid strain transformed with the T. cruzi PLC-expressing plasmid showed that expression of T. cruzi PLC suppressed the growth defect caused by the plc1 disruption in yeasts. Temperature-sensitive phenotype of the S. cerevisiae plc1-mutant haploid strain was also suppressed by the expression of T. cruzi PLC. The phosphatidyl inositol-4,5-biphosphate (PtdIns(4,5)P2) hydrolyzing activity of T. cruzi PLC was demonstrated in the lysate from the plc1-temperature sensitive yeast mutant strain transformed with the T. cruzi PLC-expressing plasmid. The yeast-expressed T. cruzi PLC showed an absolute Ca2+ dependence which was similar to mammalian PLC isoforms: the half-maximal activity at 0.5-1 x 10(-5) M Ca2+ and the maximal activity at 1-2 x 10(-4) M Ca2+.     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Cloning and characterization of the gene encoding Trypanosoma cruzi DNA topoisomerase II.

The gene encoding Trypanosoma cruzi type II topoisomerase (TcTOP2) was isolated from a genomic library with a heterologous probe corresponding to part of the Trypanosoma brucei type II topoisomerase (TBrTOP2) gene. Nucleotide sequencing of TcTOP2 showed that the gene consists of an open reading frame of 3696 nucleotides (1232 amino acids), predicting a polypeptide product of 138,413 Da. Comparison of the amino acid sequence with that of type II topoisomerases from T. brucei (TBrTOP2) and Crithidia fasciculata (CfaTOP2), shows a high degree of conservation with estimated identities of 78% and 69%, respectively. TcTOP2 is a single copy gene in the genome of T. cruzi Dm28c and is expressed as a 4.5-kb mRNA. PCR mapping showed two distinct mini-exon addition sites at positions 225 and 203 upstream from the initiator AUG.     

Cloning and characterization of a novel gene encoding 16 kDa protein (Ac16) from Angiostrongylus cantonensis

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis or meningoencephalitis. A novel gene (AC16) was isolated from a cDNA library of A. cantonensis fourth-stage larvae. The putative 16-kDa protein has 149 amino acids and is homologous to an immunodominant hypodermal antigen (IHA16) from Ancylostoma caninum (identities?=?57%). In this paper, we cloned the gene and purified the recombinant Ac16 (rAC16) protein. Real-time quantitative PCR revealed that Ac16 was expressed significantly higher in the fourth-stage larvae and adult worms derived from rats than that in the fourth-stage larvae derived from mice. Moreover, sera from rat (permissive host) infected with A. cantonensis detected Ac16 by Western blot, while sera from infected mouse (non-permissive host) could not. The results implied that Ac16 was related to the parasitic adaptation of A. cantonensis in different hosts and non-permissive host mouse had no circulating antibody to the antigen Ac16 from A. cantonensis and thus might contribute to understanding the mechanism of parasite immune evasion. Furthermore, we evaluated the ability of Ac16 antibody diagnosing A. cantonensis infection by an indirect enzyme-linked immunosorbent assay. The results showed that the Ac16 antibody had a 79.17% sensitivity to rAC16 and 83.33% to crude adult worm antigens (CA) (P?>?0.05), while the specificity to rAC16 and to CA were 95.89% and 86.30% respectively (P?相似文献   

Cloning and characterization of an Ehrlichia canis gene encoding a protein localized to the morula membrane

A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E. canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent. The mmpA gene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis of E. canis and E. chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E. canis, not in E. chaffeenesis. Sera from dogs which were either naturally or experimentally infected with E. canis recognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E. canis. Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.     

Cloning, characterisation, and heterologous expression of the Candida utilis malic enzyme gene

The Candida utilis malic enzyme gene, CME1, was isolated from a cDNA library and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1,926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of approximately 70.2 kDa. The inferred amino acid sequence suggested a cytosolic localisation for the malic enzyme, as well as 37 and 68% homologies with the malic enzymes of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Expression of the CME1 gene was subject to carbon catabolite repression and substrate induction, similar to the regulatory mechanisms observed for the C. utilis dicarboxylic acid permease. The CME1 gene was successfully expressed in S. cerevisiae under control of the S. cerevisiae PGK1 promoter and terminator. When coexpressed with the S. pombe malate permease gene (mae1), it resulted in a recombinant S. cerevisiae strain able to completely degrade 90% of the extracellular L-malate within 24 h. Nucleotide sequence data reported are available in the DDBJ/EMBL/Genbank databases under the accession number DQ173437.     

Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica

Summary Endothiapepsin is an aspartic protease secreted by Cryphonectria parasitica. It has a milk-clotting activity and is used in the cheese industry. The eapA gene encoding endothiapepsin has been cloned and sequenced. An open reading frame of 419 codons, which encodes a precursor differing from mature endothiapepsin by the presence of an 89 aa residue prepro-sequence, was found. The eapA gene is interrupted by three introns. C. parasitica mutant strains deficient in the production of endothiapepsin (eapA^-) were constructed using a gene-replacement strategy. Two nonsense mutations were introduced at the beginning of the coding sequence by PCR-induced mutagenesis. The mutated DNA fragment was introduced in C. parasitica by co-transformation with a benomyl-resistant (ben^R) selection plasmid. Transformants which have the eapA^- phenotype were obtained. Protein analysis confirmed that they secreted no detectable amount of endothiapepsin. No ectopic integration of the mutated eapA gene occurred in the eapA^- transformants. Moreover, after one conidiation step, eapA^- transformants yielded benomyl-sensitive (ben^S) segregants which were analyzed by Southern blotting experiments. The results revealed no difference with the wildtype strain, suggesting that the eapA^-, ben^S segregants differed from the non-transformed strain only by the presence of the two nonsense mutations in the eapA locus.