Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker

The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae. Received: 16 March 2000 / Accepted: 18 July 2000     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome

Summary The nuclear gene coding for the mitochondrial subunit 9 of the F₀F₁-ATP synthase complex was isolated from a genomic library of Podospora anserina. Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid. The P. anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger. In contrast to the situation in Saccharomyces cerevisiae, N. crassa and A. nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P. anserina could be detected. Thus, in P. anserina this gene appears to be exclusively encoded by the nuclear genome.     

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase

Summary The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5kkb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51 600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.     

Organization and transcription of the mitochondrial ATP synthase genes in the yeast Yarrowia lipolytica

Mitochondrial gene organization was studied in a dimorphic yeast, Yarrowia lipolytica. The gene order in a sequenced 6.6-kilobase region closely resembles that of the human mitochondrial genome in that ATP synthase subunit 8 and 6 genes are followed by genes for cytochrome c oxidase subunit 3 (which contains an intron), NADH-ubiquinone oxidoreductase subunit 4, and ATP synthase subunit 9. This region also contains tRNA genes decoding AUA, UGA, CUN and CCN codons, suggesting a unique mitochondrial translation. All the above genes are transcribed from the same DNA strand into multigenic RNAs, starting from a nonanucleotide sequence, 5′-ATA-TAAATA-3′, similar to other yeast mitochondrial promoters.     

Nucleotide sequence of the gene for pre-apocytochrome f in the spinach plastid chromosome

Summary We have characterized a cloned fragment of the spinach plastid chromosome encoding the gene for apocytochrome f. Northern blot analysis and hybrid selection translation discloses that the gene is expressed. From the nucleotide sequence, we deduce that the protein contains 285 amino acids and an amino-terminal signal sequence of 35 amino acid residues. The calculated molecular mass of pre-apocytochrome f is 35.3 kd. The clustering of hydrophobic residues indicates that the processed protein (31.3 kd) possesses only a single anchoring transmembrane domain close to the C terminus, and that 75% of the polypeptide chain including the heme-binding site protrudes into the thylakoid lumen. This topology resembles that reported for beef heart mitochondrial cytochrome c₁.     

Sequence of the Coat Protein Gene of Bermuda Grass Etched-line Virus,and of the adjacent ‘Marafibox’ Motif

The complete nucleotide sequence of the coat protein (CP) gene of Bermuda grass etched-line virus (BELV), including 376 nucleotides (nt) of the region to its 5 side, was determined and compared with sequences of the other viruses associated with the genus Marafivirus, substantiating the assignment of BELV to this group. The CP gene coding sequence was 585 nt in length. Inferred amino acid sequences showed homologies among marafiviral CP gene products ranging from 41% to 59%. A non-coding sequence motif characteristic of the marafiviruses lies in the region adjacent to the CP gene to the 5 side. In contrast to various homology levels in the coding regions of the CP genes, the interspecific sequence homology in this 18 nt motif was almost perfect.     

Nucleotide sequence of the Aspergillus niger trpC gene: structural relationship with analogous genes of other organisms

Summary The nucleotide sequence of the Aspergillus niger tryptophan C (trpC) gene was determined. Northern hybridization and S1-mapping experiments showed the presence of a 2.6 kb trpC poly(A)⁺ RNA with two very short (5 and 6 nucleotides) noncoding 5-regions. Comparison of the predicted amino acid sequence with that of trp gene proteins of pro- and eukaryotic organisms revealed three functional domains (G, C, F) in the A. niger TrpC protein which catalyse the glutarnine amidotransferase reaction (GAT), the indole-3-glycerol phosphate synthase reaction (IGPS) and the N-(5-phosphoribosyl) anthranilate isomerase reaction (PRAI), respectively. These domains are highly conserved and bordered by short areas showing less homology. Within the F domain of the trpC gene in A. niger, A. nidulans and Neurospora crassa, a region encoding 30 amino acids was found which is absent in the analogous genes of Saccharomyces cerevisiae and prokaryotic organisms. This region has features of a mutated in-phase intron.     

The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.)

The mitochondrial atp6 gene in male fertile (N) and CMS (S) pepper has previously been compared and was found to be present in two copies (Kim et al. in J Kor Soc Hort Sci 42:121–127 2001). In the current study, these atp6 copies were amplified by an inverse PCR technique, and the coding region as well as the 5′ and 3′ flanking regions were sequenced. The atp6 copies in CMS pepper were detected as one intact gene and one pseudogene, truncated at the 3′ coding region. When the atp6 genes in pepper were compared to other plant species, pepper, potato, and petunia all possessed a sequence of 12 identical amino acids at the 3′ extended region, which was considered a hallmark of the Solanaceae family. Northern blot analysis showed differences in mRNA band patterns between CMS and restorer lines, indicating that atp6 gene is one of the candidates for CMS in pepper. GenBank accession number: DQ126682 (atp6-1 genomic sequence common to fertile and CMS pepper), DQ126681 (Fertile atp6-2 genomic sequence), DQ126680 (CMS pseudo-atp6-2 genomic sequence)     

The ATPase subunit 6 gene of tobacco mitochondria contains an unusual sequence

Summary We have isolated and characterized the F₀-ATPase subunit 6 gene (atp6) from tobacco mitochondria. The tobacco sequence exists as a single copy, is transcribed and contains an open reading frame (ORF) capable of encoding a peptide of 395 amino acids. The first 130 amino acids of the tobacco putative polypeptide show limited homology with the N terminus predicted for the maize ATPase subunit 6. Although poorly conserved at the sequence level, the tobacco and maize amino termini are hydrophilic and have a high percentage of charged amino acids. This portion of the predicted peptide may represent a presequence that is common to the ATPase subunit 6 of plants. Significant homology between tobacco and maize begins with amino acid 131, in a region that is highly conserved among fungal ATPase 6 subunits. In the remainder of the predicted protein, tobacco and maize share approximately 81% homology. A 41 by sequence and a 175 by conserved region found upstream from the tobacco atp6 coding region are homologous with sequence elements found in the 5 flanking regions of other plant mitochondrial genes and may be important for regulation and expression of the atp6 gene.     

<Emphasis Type="Italic">Iranian johnsongrass mosaic virus</Emphasis>: the complete genome sequence,molecular and biological characterization,and comparison of coat protein gene sequences

Iranian johnsongrass mosaic virus (IJMV) is one of the most prevalent viruses causing maize mosaic disease in Iran. An IJMV isolate, Maz-Bah, was obtained from the maize showing mosaic symptoms in Mazandaran, north of Iran. The complete genomic sequence of Maz-Bah is 9544 nucleotides, excluding the poly(A) tail. It contains one single open reading frame of 9165 nucleotides and encodes a large polyprotein of 3054 amino acids, flanked by a 5′-untranslated region (UTR) of 143 nucleotides and a 3′-UTR of 236 nucleotides. The entire genomic sequence of Maz-Bah isolate shares identities of 84.9 and 94.2 % with the IJMV (Shz) isolate, the lone complete genome sequence available in the GenBank at the nucleotide (nt) and deduced amino acid (aa) levels, respectively. The whole genome sequences share identities of 51.5–69.8 and 44.9–74.3 % with those of other Sugarcane mosaic virus (SCMV) subgroup potyviruses at nt and aa levels, respectively. In phylogenetic trees based on the multiple alignments of the entire nt and aa sequences, IJMV isolates formed a separate sublineage of the tree with potyviruses infecting monocotyledons of cereals, indicating that IJMV is a member of SCMV subgroup of potyviruses. IJMV is most closely related to Sorghum mosaic virus and Maize dwarf mosaic virus and less closely related to the Johnsongrass mosaic virus and Cocksfoot streak virus. To further investigate the genetic relationship of IJMV, 9 other isolates from different hosts were cloned and sequenced. The identity of IJMV CP nt and aa sequences of 11 Iranian isolates ranged from 86.4 to 99.8 % and 90.5 to 99.7 %, respectively, indicating a high nt variability in CP gene. Furthermore, in the CP-based phylogenetic tree, IJMV isolates were clustered together with a maize potyvirus described as Zea mosaic virus from Israel (with 86–89 % nt identity), indicating that both isolates probably are the strains of the same virus.     

DNA sequence analysis of an allelic variant of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxIA) from serotype 10

The structural gene encoding Actinobacillus pleuropneumoniae-RTX toxin I (ApxIA), one of the major hemolytic and cytolytic toxins of the organism, was cloned from serotype 10. The nucleotide sequence data showed that the gene from serotype 10 was 98% identical to that from serotype 1 at both DNA and amino acid levels. The sequence difference was found to localize at the 3′ terminal region of the gene. We then analyzed the 3′ terminal region of the apxIA gene from other serotypes, 5a, 5b, 9 and 11, using polymerase chain reaction-amplified DNA fragments. Results of DNA sequence indicated that apxIA gene can be divided into the original form including serotypes 1, 9 and 11, and the allelic variant including serotypes 5a, 5b and 10. These gene products, ApxIA proteins, appear to have different second structures at the carboxyl terminal proximal region.     

Molecular cloning of the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans

Summary The Aspergillus nidulans 3-phosphoglycerate kinase gene (PGK) has been isolated from a phage genomic library, using the equivalent yeast gene as a hybridization probe. The location of the PGK gene within the cloned DNA has been physically mapped. The DNA sequence of a small region of the putative PGK has been determined and found to code for amino acids corresponding to the N-terminal end of the PGK protein. In contrast to the yeast PGK gene the Aspergillus gene contains a 57 base pair intron occurring between the coding sequences for amino acid 22 and 23.A DNA fragment encompassing the PGK gene was shown to hybridize a 1,700 base poly(A) mRNA, sufficient to encode the PGK polypeptide.     

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.