Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

Cloning and Sequencing of the xynA Gene Encoding Xylanase A of Aspergillus kawachii

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.     

Cloning and sequence analysis of the flagellin gene ofCampylobacter jejuni TGH9011

Flagella are essential for motility and have been implicated to be one of the pathogenic determinants. The flagellum ofCampylobacter jejuni is a polymeric structure of a 62-kd protein. Using a high-affinity flagellin antibody to screen a lambda gt 11 phage genomic expression library ofC. jejuni strain TGH9011 (Serotype LIO36), a recombinant phage clone lambda gt 11RK that expresses theC. jejuni flagellin protein was isolated. The recombinant lambda gt 11 RK produced a 56-kd protein upon induction with isopropylthiogalactoside, which reacted specifically with anti-flagellin antibody. The flagellin gene was sequenced, and comparative analysis of the nucleotide and amino acid sequence identified a region of the flagellin that shows hypervariability among differentCampylobacter species and strains.     

Characterization of a pollen-expressed gene encoding a putative pectin esterase ofPetunia inflata

From a pollen tube cDNA library ofPetunia inflata, we isolated cDNA clones encoding a protein, PPE1, which exhibits sequence similarity with plant, bacterial, and fungal pectin esterases. Genomic clones containing thePPE1 gene were isolated using cDNA for PPE1 as a probe, and comparison of the cDNA and genomic sequences revealed the presence of a single intron in thePPE1 gene. During pollen development,PPE1 mRNA was first detected in anthers containing uninucleate microspores; it reached the highest level in mature pollen and persisted at a high level inin vitro germinated pollen tubes. The observed expression pattern of thePPE1 gene suggests that its product may play a role in pollen germination and/or tube growth.     

Isolation and sequence analysis of a chalcone synthase cDNA ofMatthiola incana R. Br. (Brassicaceae)

A cDNA clone (pcM12) of the chalcone synthase (CHS) ofMatthiola incana R. Br. (Brassicacease) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5 and 3 untranslated regions. The deduced amino acid sequence shows that theMatthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.     

P-related sequences inDrosophila bifasciata: A molecular clue to the understanding of P-element evolution in the genusDrosophila

Summary Two P-elements (bif1 and bif2) were isolated from a genomic library ofDrosophila bifasciata. Both elements are internally deleted and have lost the coding capacity for a functional transposase. One of the elements (bif2) contains an insert consisting of a repetitive sequence. The terminal inverted repeats and the segments necessary for passive mobility are well conserved. Element bif2 has retained rudiments of the coding sequence of exon 0 and exon 3, but the reading frame is destroyed by insertions and deletions. The comparison of theD. bifasciata P-elements with P-elements ofDrosophila melanogaster andDrosophila nebulosa reveals that the two latter sequences are more similar to each other than either of them is to theD. bifasciata elements. This finding contradicts the phylogenetic relationship of the species and can be taken as an indirect but unequivocal evidence for recent horizontal gene transfer from a relative ofD. nebulosa to the gene pool ofD. melanogaster. The P-elements ofD. bifasciata are phylogenetically ancient and have evolved independently for about 50 million years. A higher substitution rate at the third codon position as well as a predominance of conservative replacements at the amino acid level indicates that the P-elements ofD. bifasciata have been under selective constraint over a long period and that immobilization has occurred only recently.     

Cloning and functional annotation of rare mRNA species from drought-stressed hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

To better understand gene expression at very low levels, we have designed a method to eliminate cDNA clones representing abundant mRNAs. A cDNA library for drought-stressed hot pepper (Capsicum annuum) (Choi et al., 2002) underwent double-negative screening, once with probes made from a drought-stressed plant, the second time, with probes from a non-stressed plant. The cDNA clones that showed very weak or negative signals were isolated for further analysis, which resulted in 1399 cDNA clones from about 20,000 screened clones. When nucleotide sequences were determined, we obtained 1142 tentative unique genes, with a redundancy rate of 20.41%. An homology database search for the deduced amino acid sequences revealed that about 79% of the cDNA clones could not be matched for functioning with previously characterized sequences. However, when these uncategorized clones were subjected to classification based on functional domains, most could be cited. Notably, clones with possible functions in RNA transport, protein synthesis, and regulation of protein activity showed a dramatic increase in appearance while those coding for transposable elements, viral proteins, and plasmid proteins occupied a much smaller portion compared with those in theArabidopsis thaliana genome. In addition, those coding for proteins targeted to the endoplasmic reticulum were dramatically more abundant in our clones compared with theArabidopsis database.     

Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit

A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.     

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacteriumSulfolobus solfataricus

ASulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (G1–G6) and one of them (G4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of theS. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.This work was supported by grants from the CNR, Target Project on Biotechnology and Bioinstrumentation, and MURST (Rome).     

Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine,serine and threonine

An intact cDNA fromArabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein ofM _r 27140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to theEscherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates theA. thaliana andE. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Primary structure of human pepsinogen gene

A recombinant clone, which covers the pepsinogen gene in a single insert, has been isolated by screening a library of human genomic DNA, using a swine pepsinogen cDNA as a probe. Sequence analysis of coding DNA segments of the clone revealed that the pepsinogen gene occupies approximately 9.4-kilobase pairs of the genomic DNA and is separated into nine exons by eight introns of various lengths. The predicted amino acid sequence of human pepsinogen consists of 373 residues and is 82% homologous with that of swine pepsinogen. In addition, the predicted sequence contained a single sequence of 15 amino acid residues at the NH2 terminus, showing that the protein is synthesized as prepepsinogen. The structure of the gene, in which two homologous sequences including the two active site aspartyl residues of pepsin are present in different coding segments, is in support of the view that the pepsinogen gene evolved by duplication of a shorter ancestral gene.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.