Characterization of a Chlamydomonas reinhardtii gene encoding a protein of the DNA photolyase/blue light photoreceptor family

The organization and nucleotide sequence of a gene from Chlamydomonas reinhardtii encoding a member of the DNA photolyase/blue light photoreceptor protein family is reported. A region of over 7 kb encompassing the gene was sequenced. Northern analysis detected a single 4.2 kb mRNA. The gene consists of eight exons and seven introns, and encodes a predicted protein of 867 amino acids. The first 500 amino acids exhibit significant homology with previously sequenced DNA photolyases, showing the closest relationship to mustard (Sinapis alba) photolyase (43% identity). An even higher identity, 49%, is obtained when the Chlamydomonas gene product is compared to the putative blue-light photoreceptor (HY4) from Arabidopsis thaliana. Both the Chlamydomonas and the Arabidopsis proteins differ from the well characterized DNA photolyases in that they contain a carboxyl terminal extension of 367 and 181 amino acids, respectively. However, there is very little homology between the carboxyl terminal domains of the two proteins. A previously isolated Chlamydomonas mutant, phrl, which is deficient in DNA photolyase activity, especially in the nucleus, was shown by RFLP analysis not to be linked to the gene we have isolated. We propose this gene encodes a candidate Chlamydomonas blue light photoreceptor.     

Isolation, characterization and expression of a cDNA encoding an elongation factor-1α from Porphyra yezoensis (Bangiales, Rhodophyta)

We report the isolation, characterization and expression of a cDNA encoding a polypeptide elongation factor‐1α (EF‐1α) from the marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and the sequence was analyzed. The clone contained an open reading frame for a protein of 449 amino acids which exhibits sequence similarity to the known EF‐1α. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra pur‐purea EF‐1αtef‐c (97%) than to the P. purpurea EF‐1αtef‐s (61%). The mRNA was detected both in the leafy gametophyte and the filamentous sporophyte.     

Molecular and morphologically distinct Scytosiphon species (Scytosiphonales,Phaeophyceae) display similar antioxidant capacities1

The taxonomic status of an alga with complanate thalli, occurring in central Chile and belonging to the genus Scytosiphon, was elucidated. Morphological and molecular features demonstrated that, in addition to the known and widespread constricted S. lomentaria (Lyngb.) Link that occurs along the Chilean coast, there is a Scytosiphon with complanate thalli that occurs only in central Chile—S. gracilis Kogame. Morphological analyses of this previously unreported complanate Scytosiphon showed thalli without constrictions, coherent plurilocular sporangia without ascocysts, and phaeophycean hairs arising from cortical cells. Furthermore, sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the complanate Scytosiphon were 99.8% and 100% identical to those detected in S. gracilis from Korea. ITS‐based comparative analyses showed that complanate Scytosiphon grouped in a different clade than S. lomentaria and S. tenellus Kogame from various parts of the world, including Chilean species. Moreover, molecular analyses suggest the occurrence of two distinct ITS types of S. lomentaria in northern Chile, corresponding to the Korean and Greek types. On the other hand, biochemical analyses of copper‐induced antioxidant responses in S. gracilis and S. lomentaria showed an identical increase in antioxidant enzyme activities. These results suggest that copper tolerance might be a constitutive trait in these species of Scytosiphon.     

Strain‐specific consumption and transformation of alga‐derived dissolved organic matter by members of the Limnohabitans‐C and Polynucleobacter‐B clusters of Betaproteobacteria

We investigated changes in quality and quantity of extracellular and biomass‐derived organic matter (OM) from three axenic algae (genera Rhodomonas, Chlamydomonas, Coelastrum) during growth of Limnohabitans parvus, Limnohabitans planktonicus and Polynucleobacter acidiphobus representing important clusters of freshwater planktonic Betaproteobacteria. Total extracellular and biomass‐derived OM concentrations from each alga were approximately 20 mg l^?1 and 1 mg l^?1 respectively, from which up to 9% could be identified as free carbohydrates, polyamines, or free and combined amino acids. Carbohydrates represented 54%–61% of identified compounds of the extracellular OM from each alga. In biomass‐derived OM of Rhodomonas and Chlamydomonas 71%–77% were amino acids and polyamines, while in that of Coelastrum 85% were carbohydrates. All bacteria grew on alga‐derived OM of Coelastrum, whereas only Limnohabitans strains grew on OM from Rhodomonas and Chlamydomonas. Bacteria consumed 24%–76% and 38%–82% of all identified extracellular and biomass‐derived OM compounds respectively, and their consumption was proportional to the concentration of each OM compound in the different treatments. The bacterial biomass yield was higher than the total identifiable OM consumption indicating that bacteria also utilized other unidentified alga‐derived OM compounds. Bacteria, however, also produced specific OM compounds suggesting enzymatic polymer degradation or de novo exudation.     

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri : phylogenetic origins and recent insertion of introns

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene. Received: 27 November 1998 / Accepted: 21 April 1999     

The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassiceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.     

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

Multiple P450alk (cytochrome P450 alkane hydroxylase) genes from the halotolerant yeast Debaryomyces hansenii

The halotolerant alkane-assimilating yeast Debaryomyces hansenii was examined for P450 alkane hydroxylase genes known to be required for alkane assimilation in Candida. Four distinct P450alk gene segments and an allelic segment were isolated using PCR based on degenerate primers derived from the CYP52 family of alkane-inducible P450 genes. A screen of a genomic library (15–20 kb inserts) constructed for this study, using a probe based on the PCR-isolated segments, yielded seven clones. This has led to the isolation and sequence of two full-length genes DH-ALK1 and DH-ALK2. These genes, each with an ORF of 1557 bp (519 aa), contained no apparent introns and showed 64% nucleotide sequence homology (61% based on the deduced amino acid sequences). The deduced proteins had predicted molecular weights of 59,254 Da (DH-ALK1) and 59,614 Da (DH-ALK2) and have been designated CYP52A12 and CYP52A13 by the P450 Nomenclature Committee. Phylogenetic analysis based on Neighbor Joining Tree showed that DH-ALK1 and DH-ALK2 constitute new genes located on two distinct branches and are most related to the gene CYP52A3 (60% deduced aa homology) and are least related to the gene CYP52C2 (41% deduced aa homology), both of C. maltosa. The isolated genes will provide tools to better understand the diversity of the P450alk family in eukaryotic microorganisms adapted to varied environmental conditions.     

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.     

Regular Spliceosomal Introns Are Invasive in Chlamydomonas reinhardtii: 15 Introns in the Recently Relocated Mitochondrial cox2 and cox3 Genes

In the unicellular green alga, Chlamydomonas reinhardtii, cytochrome oxidase subunit 2 (cox2) and 3 (cox3) genes are missing from the mitochondrial genome. We isolated and sequenced a BAC clone that carries the whole cox3 gene and its corresponding cDNA. Almost the entire cox2 gene and its cDNA were also determined. Comparison of the genomic and the corresponding cDNA sequences revealed that the cox3 gene contains as many as nine spliceosomal introns and that cox2 bears six introns. Putative mitochondria targeting signals were predicted at each N terminal of the cox genes. These spliceosomal introns were typical GT–AG-type introns, which are very common not only in Chlamydomonas nuclear genes but also in diverse eukaryotic taxa. We found no particular distinguishing features in the cox introns. Comparative analysis of these genes with the various mitochondrial genes showed that 8 of the 15 introns were interrupting the conserved mature protein coding segments, while the other 7 introns were located in the N-terminal target peptide regions. Phylogenetic analysis of the evolutionary position of C. reinhardtii in Chlorophyta was carried out and the existence of the cox2 and cox3 genes in the mitochondrial genome was superimposed in the tree. This analysis clearly shows that these cox genes were relocated during the evolution of Chlorophyceae. It is apparent that long before the estimated period of relocation of these mitochondrial genes, the cytosol had lost the splicing ability for group II introns. Therefore, at least eight introns located in the mature protein coding region cannot be the direct descendant of group II introns. Here, we conclude that the presence of these introns is due to the invasion of spliceosomal introns, which occurred during the evolution of Chlorophyceae. This finding provides concrete evidence supporting the ``intron-late' model, which rests largely on the mobility of spliceosomal introns. Received: 22 August 2000 / Accepted: 28 February 2001     

Rapid isolation and sequence analysis of the beta-tubulin gene from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

Beta-tubulin, one of the cytoskeletal proteins, has been highly conserved throughout the evolution of eukaryotes. Degenerate PCR and inverse PCR (iPCR) were used to isolate the full-length beta-tubulin gene and its 5′ and 3′-flanking regions (2799bp) from the marine red alga Porphyra yezoensis. This gene, designated as TubB1, is devoid of introns. The canonical cis-acting elements such as TATA box, CAAT box and polyA signal AAUAAA are not found in flanking sequences, but another putative polyA signal CAYTG is found downstream of the stop codon. Comparison of the deduced 458 amino acid sequences shows higher similarity to the Protoflorideophycidae Cyanidioschyzon merolae (82%) than to the red alga Chondrus crispus (79%). Codon bias indicates strong expression of TubB1. Phylogenetic analysis suggests that the beta-tubulin of P. yezoensis and C. merola go together with fungi and not with green plants. These nucleotide sequence data have been deposited in the DDB/EMBL/Genbank databases under the accession number AY221630.     

Molecular characterization of the rbcS multi-gene family of Petunia (Mitchell)

Summary The small subunit (RbcS) of ribulose bisphosphate carboxylase (RuBPCase) is encoded by eight genes in Petunia (Mitchell). These genes can be divided into three subfamilies (51, 117 and 71) based upon hybridization to three petunia rbcS cDNA clones. The nucleotide sequence of six of the eight petunia rbcS genes is presented here and the structure of the genes is discussed with respect to their genomic linkage and their expression levels in petunia leaf tissue. The rbcS genes belonging to the same subfamily encode an identical mature RbcS polypeptide, however the different subfamilies encode distinguishable polypeptides. All the genes, except one, contian two introns within the mature subunit coding region; one gene contains one extra intron within the coding region. There are large regions of nucleotide sequence homology within the introns of genes within a subfamily, but significantly less homology between the introns of genes of different subfamilies. A complex pattern of homology within the multiple genes of the 51 subfamily is observed. There are regions within these genes which share high levels of sequence homology; this homology does not extend throughout the whole gene and the regions of homology do not always occur in adjacent genes. Two 3 rbcS gene fragments which we isolated from the petunia genome show high levels of homology to two of the intact rbcS genes.     

Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron

Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5′ and 3′ sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.     

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.GenBank accession number: M98495     

Characterization of Two Alcohol Dehydrogenase (Adh) Loci from the Olive Fruit Fly, Bactrocera (Dacus) oleae and Implications for Adh Duplication in Dipteran Insects

We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera. Received: 30 May 2000 / Accepted: 17 August 2000     

Characterization of a rice pollen-specific gene and its expression

Pollen development requires a large number of genes expressed in both sporophytic and gametophytic tissues. We have isolated a pollen-specific gene, PS1, from rice. PS1 is a unique gene in the rice genome and encodes a 164 amino acid long protein. RNA blot analysis shows that PS1 mRNAs accumulate specifically in rice anthers. When introduced into rice tissues by microprojectile bombardment, the PS1 promoter drives expression of a marker gene, β-glucuronidase, specifically in rice pollen. The PS1 gene and the deduced amino acid sequence of the PS1 protein share significant levels of homology with another monocot pollen-specific gene—the maize Zm-13 gene and its deduced protein, respectively. PS1 also shows some homology with the dicot tomato anther-specific gene LAT-52. Interestingly, the structure of the PS1 gene is more similar to that of the LAT-52 gene than to Zm-13. The coding regions of both PS1 and LAT-52 are interrupted by a single intron, and the positions of the introns are conserved in these genes. Moreover, there is considerable sequence homology in the introns of the PS1 and LAT-52 genes in regions immediately upstream of the 3' splice sites. The upstream regulatory sequences of the PS1 gene show several regions of homology with other pollen- or anther-specific genes from a number of plant species. The conservation of coding sequences of PS1 from rice, Zm-I3 from maize, and LAT-52 from tomato suggests a functional conservation of their gene products. Similarities in the regulatory regions of PS1 and other anther- or pollen-specific genes among monocotyledonous and dicotyledonous species indicate that at least some regulatory features controlling gene expression in male reproductive tissues are conserved. This is supported by the preservation of pollen-specific expression from the rice PS1 promoter when it is introduced into tobacco plants by Agrobacterium Ti plasmid-mediated transformation.     

Structure of Amylase Genes in Populations of Pacific Cupped Oyster (Crassostrea gigas): Tissue Expression and Allelic Polymorphism

Using the previously determined complementary DNA Sequence of Crassostrea gigas amylase (Y08370), we designed several oligonucleotide primers and used them with polymerase chain reaction (PCR) technology to characterize oyster amylase gene sequences. Two genes encoding 2 different amylases were characterized and sequenced. The 2 genes are similarly organized with 8 exons and 7 introns. Intron insertions are found at the same location in the 2 genes. Sizes and nucleotide sequences are different for the different introns inside each gene and different for the corresponding introns in the 2 genes. Comparing the 2 genes, around 10% of the nucleotides are different along the exons, and comparing the 2 deduced protein sequences, a mean value of 10.4% of amino acids are changed. Genes A and B encode mature proteins of, respectively, 500 and 499 amino acids, which present 94% similarity. A microsatellite (TC₃₇) that constitutes the largest part of intron 4 of gene A has been used as a polymorphic marker. A method consisting of a PCR step followed by EcoRI digestion of the obtained fragments was used to observe polymorphism in these 2 genes. Six and 4 alleles for genes A and B, respectively, have been sequenced, leading to a maximum of 2.9% base change. The 2 genes are ubiquitously expressed in the different digestive tissues with quantitative differences. Gene A is strongly expressed in the digestive gland and at a lower level in stomach, while gene B is preferentially expressed in the labial palps. The microsatellite repeat was used in the analysis of 4 populations of Crassostrea gigas from the French Atlantic coast. A high level of polymorphism observed with 30 different alleles of gene A inside the populations should allow their characterization using the mean value of the microsatellite allelic distribution. These populations showed a low level of differentiation (F _st between 0 and 0.011); however, the population of Bonne Anse appeared to be distinguished from the other populations.