Nucleic acid sequence and chromosome assignment of a wheat storage protein gene.

A cloned gliadin gene was isolated from a wheat genomic library, and 2.4 kb of its primary sequence determined. The gene, alpha-1Y, was found by Southern analysis to be located on chromosome 6A, and its derived amino acid sequence identifies it as a member of the A-gliadin subgroup of alpha-gliadins located on the short arm of that chromosome. alpha-1Y is apparently functional, and contains consensus TATA and CAAT boxes, and polyadenylation signals. This gliadin gene has no introns, and its noncoding flanking regions contain several short repeats and inverted sequences. The gene is contained in a 6.2 kb EcoRI genomic fragment whose apparent copy number varies in different wheat cultivars.     

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase

Summary We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.Offprint requests to: R.E. Pearlman     

Molecular structure and sequence homology of a gene related to alpha 1-antitrypsin in the human genome

A 7.7-kb EcoRI genomic DNA fragment highly homologous to the human alpha 1-antitrypsin (AAT) gene has been cloned. This antitrypsin-related sequence is physically linked to the authentic AAT gene and both are present in a single cosmid clone. Nucleotide sequencing of the AAT-related genomic fragment demonstrated extensive homology with the authentic AAT gene in the introns as well as in the exons. The conservation of all RNA splice sites and lack of internal termination codons in the exonic regions suggest that it may not be a classical pseudogene. If expressed, it could result in a protein of 420 amino acid residues exhibiting a 70% overall homology with human alpha 1-antitrypsin. The signal peptide sequence is well conserved in the related gene, but the active site for protease inhibition of Met-Ser in alpha 1-antitrypsin has been changed to Trp-Ser. These data suggest that the putative protein encoded by the AAT-related gene is a secretory serine protease inhibitor with an altered substrate specificity. Interestingly, even the intronic regions in the related gene exhibit a 65% overall nucleotide sequence homology with those of the authentic AAT gene. These results suggest that the AAT-related gene is derived from a recent duplication of the authentic AAT gene and represents a new member of the serine protease inhibitor superfamily.     

The sequence of the Saccharomyces cerevisiae gene PHO2 codes for a regulatory protein with unusual aminoacid composition.

A new centromere vector for the construction of a Saccharomyces cerevisiae gene library, allowing direct selection for DNA insert, will be described. From that library the gene for the regulatory protein PHO2 involved in PHO5 induction has been cloned by complementation of a pho2 mutation. The complementing activity was shown to be located on a 3.6 kb HindIII fragment. This fragment was used to evict the genomic copy and with appropriate genetic crosses we proved, that the cloned gene is PHO2. The DNA sequence of PHO2 was determined. Analysis of the sequence data uncovered striking homology regions with PHO4, another protein necessary for the induction of PHO5. The relevance of the observed homology will be discussed.     

Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger

The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined –16 to –25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616

Abstract A genomic gene encoding a polygalacturonase from Aspergillus oryzae , used in soy sauce production, was cloned and sequenced. The structural gene comprises 1227 bp coding for 363 amino acids with a putative prepropeptide of 28 amino acids and the open reading frame is disrupted by two short introns of 57 bp and 81 bp. The deduced amino acid sequence of the mature protein showed 63, 63, 63 and 64% homology with those of Aspergillus niger polygalacturonase I, Aspergillus niger polygalacturonase II, Aspergillus tubingensis polygalacturonase II and Cochliobolus carbonum polygalacturonase, respectively. There is, however, little homology among fungal, plant and bacterial polygalacturonases.     

Characterization and sequence of a novel nitrate reductase from barley

Summary Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.Scientific Paper No. 9101-14. College of Agriculture and Home Economics Research Center, Washington State University, Research Project Nos. 0233 and 0745     

Identification of new GH 10 and GH 11 xylanase genes from Aspergillus versicolor MKU3 by genome-walking PCR

Xylanases randomly clear the backbone of xylans, which are hemicelluloses representing a considerable source of fixed carbon in nature. Consequently, these enzymes have important industrial applications. To characterize the genes responsible for producing these enzymes, we cloned xylanase genes belonging to the GH11 and GH10 families from Aspergillus versicolor MKU3 using a 2-step polymerase chain reaction (PCR) protocol involving degenerate PCR and genome-walking PCR (GWPCR). We amplified a family 10 xylanase consensus fragment using degenerate PCR primers exhibiting specificity for conserved motifs within fungal family 10 xylanase genes. We identified a single family 10 xylanase gene (xynv10) and determined its entire gene sequence during the second step of GWPCR, which was used to amplify genomic DNA fragments upstream and downstream of xynv10. The xynv10 sequence contains a 1,378-bp open reading frame separated by 8 introns with an average size of 49 bp. We also amplified a partial GH11 xylanase gene sequence (xynv11) using degenerate PCR and genome-walking methods. Amplification of the C-terminal region of xynv11 using a degenerate primer designed from sequences revealed strong homology with the partial GH11 xylanase gene of A. versicolor MKU3. The structural region in xynv11 was approximately 680 bp and has one intron that is approximately 64 bp in length. Further expression and characterization of these genes will give better understanding of the role of these genes in xylan degradation by A. versicolor.     

Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.     

Glucoamylase P gene of Hormoconis resinae: Molecular cloning, sequencing and introduction into Trichoderma reesei

The glucoamylase P gene of the fungus Hormoconis resinae has been cloned and sequenced from a genomic library. The gene consists of a 2153-bp protein coding region including three introns. The usual number of introns in cloned fungal glucoamylase genes has been four and in some cases five. Two of the glucoamylase P gene introns contain a sequence resembling the consensus sequence found near the 3' splice site in the introns of the fungus Trichoderma reesei cellobiohydrolase 1 (cbh1) gene. The H. resinae glucoamylase P gene, under its own promoter, was introduced into T. reesei, but no expression could be detected.