Characterisation of complementary DNAs from the expressed sequence tag analysis of life cycle stages of Laminaria digitata (Phaeophyceae)

Laminariales (Phaeophyceae, Heterokonta) are characterised by a heteromorphic digenetic life cycle with a filamentous, microscopic gametophyte and a highly evolved, macroscopic sporophyte. With the ultimate goal of comparing gene expression in each life cycle stage, complementary DNA libraries were constructed from sporophytes and gametophytes of Laminaria digitata. A set of ca. 500 expressed sequence tags (EST) was generated from each life history phase, by single-run partial sequencing of randomly picked cDNA clones. Comparison of the EST deduced amino acid sequences with database protein sequences assigned a putative identity for 39% of the 412 gametophyte clones and 48% of the 493 sporophyte clones sequenced thus far. These data represent more than 152 different proteins now probably identified in L. digitata. Several of those newly identified proteins are of interest to our understanding of the molecular physiology of kelps, for example their carbon-concentrating mechanisms, cell wall biosynthesis and halogen metabolism. EST analysis also confirmed that genes with long 3-UTRs are widespread in Laminariales and the study of 5-UTRs allowed the identification of a Kozak consensus sequence, c(A/C)A(A/C)CAUGGc(G/T). Several potential developmentally regulated differences in gene expression are discussed.     

A tandem of α-tubulin genes preferentially expressed in radicular tissues from Zea mays

The identification of a cDNA (MR19) corresponding to a maize -tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two -tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published -tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3 probes it appears that both genes are preferentially expressed in the radicular system of the plant. The -tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members.     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

A novel method for efficient expression cloning of fungal enzyme genes

We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays. A cDNA library from the fungus Humicola insolens was constructed in a S. cerevisiae/Escherichia coli shuttle vector in E. coli. Sub-pools of the library were subsequently screened for enzyme activity in S. cerevisiae. More than 130 clones were identified as positive in either an endo--glucanase or an endo-xylanase assay. Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo--glucanases and three types of endo-xylanases. A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A. oryzae. The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.     

Nucleotide and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover (Trifolium repens L.)

The nucleotide sequence and derived amino acid sequence of two different -glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic -glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other -glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic -glucosidase activity. Extensive homology was found between the white clover -glucosidase sequences and a group of prokaryote and mammalian -glycosidases. This group of sequences has no homology with a separate set of -glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.     

cDNA cloning and characterization of a 3′/5′-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum

Enzymatic O-methylation of plant secondary metabolites is an important mechanism for the inactivation of reactive hydroxyl groups and for the modification of their solubility. A cDNA clone (pFOMT3) encoding the gene for the 3/5-O-methylation of partially methylated flavonols was isolated from Chrysosplenium americanum (Saxifragaceae). We used a PCR fragment obtained with degenerate oligonucleotides designed from conserved regions of various O-methyltransferases (OMTs). The pFOMT3 cDNA sequence shows about 67–85% similarity to other plant OMT sequences. The recombinant protein expresses strict specificity for positions 3/5 (meta) of partially methylated flavonols, but does not accept quercetin or caffeic acid for further methylation. Southern blot analysis of the genomic DNA probed with an OMT sequence suggests the presence of a number of related genes in this species, consistent with the multiple enzymatic methylations involved in the biosynthesis of polymethylated flavonols in this plant.     

Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-α-amylase inhibitor family of the common bean and its relatives

The common bean, Phaseolus vulgaris, contains a family of defense proteins that comprises phytohemagglutinin (PHA), arcelin, and -amylase inhibitor (AI). Here we report eight new derived amino acid sequences of genes in this family obtained with either the polymerase chain reaction using genomic DNA, or by screening cDNA libraries made with RNA from developing beans. These new sequences are: two AI sequences and arcelin-4 obtained from a wild accession of P. vulgaris that is resistant to the Mexican bean weevil (Zabrotes subfasciatus) and the bean weevil (Acanthoscelides obtectus); an AI sequence from the related species P. acutifolius (tepary bean); a PHA and an arcelin-like sequence from P. acutifolius; an AI-like sequence from P. maculatus; and a PHA sequence from an arcelin-5 type P. vulgaris. A dendrogram of 16 sequences shows that they fall into the three identified groups: phytohemagglutinins, arcelins and AIs. A comparison of these derived amino acid sequences indicates that one of the four amino acid residues that is conserved in all legume lectins and is required for carbohydrate binding is absent from all the arcelins; two of the four conserved residues needed for carbohydrate binding are missing from all the AIs. Proteolytic processing at an Asn-Ser site is required for the activation of AI, and this site is present in all AI-like sequences; this processing site is also found at the same position in certain arcelins, which are not proteolytically processed. The presence of this site is therefore not sufficient for processing to occur.     

Restriction fragment length polymorphism segregation analysis of the Li locus in Trifolium repens L.

The Li locus in white clover controls the presence of cyanogenic -glucosidase (linamarase) activity in leaf tissue, such that plants homozygous for the null allele (li) have no linamarase activity in this tissue. The isolation of a cDNA clone from linamarase mRNA is described. The cDNA clone is used to further characterise alleles of the Li locus. Northern blot analysis shows that plants homozygous for the null allele (li li) produce very reduced levels of mRNA which hybridises to the cDNA. Heterozygous plants (Li li), which have intermediate levels of enzyme activity, produce intermediate levels of mRNA. Southern blot analysis of Hind III digested genomic DNA shows that the white clover genome contains three genes with homology to the linamarase cDNA and that at least two of these genes segregate independently. Analysis of the cosegregation of linamarase activity and the presence of genomic restriction fragments identifies the genomic sequence specifying linamarase structure and indicates either a structural or cis acting control function of the Li locus.     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.     

Characterization of cDNA clones for rye endosperm β-amylase and analysis of β-amylase deficiency in rye mutant lines

Summary In order to characterize a -amylase deficiency in the endosperm of mutant rye lines, homologous cDNA probes were prepared. A rye cDNA library was constructed from a normal line and screened with a barley -amylase probe. Three partial cDNA clones specific for endosperm -amylase in rye were isolated and characterized. The largest of these clones was used to investigate the expression of endosperm -amylase in mutant and normal lines by Northern hybridization. These experiments, as well as in vitro translation experiments, demonstrate the absence of endosperm -amylase mRNA in mutant lines. Sequencing of three different cDNA clones revealed a single nucleotide difference, which suggests that two genes encoding endosperm -amylase genes might exist in rye. From Southern blots we anticipate that these two genes are tightly linked. Results of these experiments and previous data indicating that the mutation was located within the -amylase locus on chromosome 5 are consistent with the hypothesis that the mutation results from a deletion simultaneously affecting the two genes. However, due to extensive polymorphism within normal lines used as control, additional experiments will be required to further substantiate this conclusion. The deduced amino acid sequence reveals the occurrence of three short glycine-rich repeats containing 11 or 12 residues close to the carboxyl terminus of the protein. A comparison of the nucleotide sequence between rye and previously described barley cDNA clones revealed ca. 90% homology at the amino acid level, except in this C-terminal repeated part, where it drops to 45%.     

Cloning and nucleotide sequence of the α-galactosidase cDNA from Cyamopsis tetragonoloba (guar)

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the -galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH₂ terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH₂ terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/ factor and killer factor. A comparison of the derived amino acid sequence of this -galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the -galactosidase from Escherichia coli.     

Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of β-glycosidases

Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3 portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA-and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a -glycosidase enzyme family comprising both -glycosidases and phospho--glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these -glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between -glycosidases and cellulases.     

Characterization of cDNA clones of the family of trypsin/α-amylase inhibitors (CM-proteins) in barley (Hordeum vulgare L.)

Summary Recombinants encoding members of the trypsin/-amylase inhibitors family (also designated CM-proteins) were selected from a cDNA library prepared from developing barley endosperm. Inserts in two of the clones, pUP-13 and pUP-38, were sequenced and found to encode proteins which clearly belong to this family, as judged from the extensive homology of the deduced sequences with that of the barley trypsin inhibitor CMe, the only member of the group for which a complete amino acid sequence has been obtained by direct protein sequencing. These results, together with previously obtained N-terminal sequences of purified CM-proteins, imply that there are at least six different members of this dispersed gene family in barley. The relationship of this protein family to the B-3 hordein and to reserve prolamins from related species is discussed in terms of their genome structure and evolution.     

Molecular cloning and sequencing analysis of a β-tubulin gene from Lupinus albus

Genomic -Dash library constructed from Lupinus albus nuclear DNA was screened using a fragment of the -tubulin cDNA ( 8–31) clone of Chlamydomonas reinhardtii as probe. One of the positive recombinant phages was isolated, subcloned and analysed by sequencing. We present here nucleotide and derived amino acid sequences of the -tubulin gene, designated as L1 and identified by similarity with other -tubulins. The L1-encoded protein reveals a very high degree of similarity with other plant tubulins and contains consensus sequences for binding guanine base, phosphate and Mg²⁺. Northern analysis of total RNA isolated from roots, leaves, flowers and pools revealed that Lupinus albus -tubulin genes are constitutively expressed in all studied plant tissues.     

Expression of a gene (Ids2) induced by Fe deficiency treatment in the roots of Hordeum vulgare L.

A ZAPII-cDNA library constructed from poly(A)⁺-RNA isolated from Fe-deficient barley roots was used for differential screening of barley roots grown in the presence and absence of Fe. Among seven clones that hybridised specifically to the probe for Fe deficiency, one clone (Ids2) was sequenced. Using a part of the cDNA sequence as a probe, a genomic-DNA library was probed and a corresponding DNA clone was isolated and sequenced. The predicted amino acid sequence resembled 2-oxoglutarate-dependent dioxygenase. Ids2 had a metal regulatory element as well as Cu regulatory elements of CUP1 gene of yeast MT.     

Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules

Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P₂ (AAT-P₂), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P₂ and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P₂ were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P₂-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5 end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P₂.     

The structure of aPhaseolus vulgaris cDNA encoding the iron storage protein ferritin

A cDNA containing the entire coding region for the iron storage protein ferritin has been isolated from the French bean plant,Phaseolus vulgaris L. cv. Tendergreen. Ferritin protein was purified from young leaves and shoot meristem tissue and used to raise antisera in mice. A gt11 cDNA library was constructed from seed-derived poly(A)⁺ RNA, and screened with the mouse anti-ferritin serum. A 1.2 kb immunopositive phage DNA insert was isolated and sequenced. The derived amino acid sequence shows substantial similarity with other ferritin sequences. The 5 untranslated region contains two out-of-frame AUG codons, a region of extreme pyrimidine composition bias and potentially stable secondary structure.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

A reassessment of mammalian αA-crystallin sequences using DNA sequencing: Implications for anthropoid affinities of tarsier

A-crystallin, a major structural protein in the ocular lenses of all vertebrates, has been a valuable tool for molecular phylogenetic studies. This paper presents the complete sequence for human A-crystallin derived from cDNA and genomic clones. The deduced amino acid sequence differs at two phylogenetically informative positions from that previously inferred from peptide composition. This led us to examine the same region of the A-crystallin gene in 12 other mammalian species using direct sequencing of PCR-amplified genomic DNA. New sequences were added to the database, and corrections were made to all anthropoid sequences, defining clear synapomorphies for anthropoids as a clade distinct from prosimians. Within the anthropoids there are further synapomorphies delineating hominoids, Old World monkeys, and New World monkeys. Significantly, sequence revisions and the addition of new sequence for a prosimian, the sifaka, eliminate the previous support for the proposed anthropoid affinities of the tarsier inferred from A-crystallin protein sequences. In addition, DNA sequences provide greater resolution of certain relationships. For example, although they are identical in protein sequence, comparison of DNA sequences clearly separates mouse and the common tree shrew, grouping the tree shrew closer to prosimians. These results show that adding DNA sequences to the existing A-crystallin database can enhance its value in resolving phylogenetic relationships.U05569 Genbank accession number for human CDNA sequence - U24057 chimp - U24058 Colobus - U24059 capuchin - U24060 elephant - 024061 rhesus - U24062 sifaka - U24063 Pteropus - U24064 Tupaia - U24065 Tonatia - U24066 tarsier - U24067 tamarin - U24068 Galago