Characterization of lignin peroxidase-encoding genes from lignin-degrading basidiomycetes

Two closely linked lignin peroxidase (LPO)-encoding genes (lpo) from Phanerochaete chrysosporium were isolated. Nucleotide sequence studies indicated that the two genes are separated by 1.3 kb of flanking DNA and transcribed in opposite directions. Cloned P. chrysosporium lpo gene probes have been shown to hybridize to multiple sequences present in the DNAs of the white-rot fungi, Bjerkandera adusta, Coriolus versicolor and Fomes lignosus, but no hybridization was detected with DNA from Pleurotus ostreatus. Thus, lpo gene families appear to be common in a number of lignin-degrading basidiomycetes, some of which have not yet been shown to produce LPO proteins.     

Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species

Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes tersicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.     

Cloning and characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor

Six putative lignin peroxidase (LIP) genes were isolated from a lambda EMBL3 phage library of the white-rot fungus, Trametes versicolor, using the Phanerochaete chrysosporium LIP cDNA CLG5 as the probe. Sequence analysis of one of the genes, VLG1, showed that its coding region is interrupted by six small introns (49-64 bp) and that it encodes a mature LIP protein (341 aa; Mr: 36,714) that is preceded by a 25 aa signal sequence. This protein has a relatively high degree of aa homology to the N-termini of the LIP proteins purified from T. versicolor and has an aa homology of 55-60% to the LIP proteins of P. chrysosporium, which is comparable to that found between P. chrysosporium and Phlebia radiata LIP proteins.     

A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei

The nucleotide sequence of a cDNA coding for a lignin peroxidase (Lgp) of the white-rot fungus, Phlebia radiata, has been determined. By amino acid (aa) sequencing, it has been shown that the protein product of this gene is the LIII Lgp of Pb. radiata. The isolated gene and the putative aa sequence are about 60% homologous to published Lgp sequences from the fungus, Phanerochaete chrysosporium. The aa thought to be involved in the catalysis of LIII are revealed by comparison with the yeast cytochrome c peroxidase. The P. radiata Lgp-encoding gene (lgp3) was expressed in the fungus, Trichoderma reesei, under the cellobiohydrolase-encoding cbh1 gene promoter. Lgp3 mRNA was produced by the T. reesei transformants. No Lgp protein, however, could be detected.     

Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus

We utilized a cloned gene (TRP5) encoding tryptophan synthetase (TSase) from Saccharomyces cerevisiae to identify and clone the corresponding gene (TRP1) from the basidiomycete Coprinus cinereus. The primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for TSase. A transformation assay was used to demonstrate that 321 nt, which do not include CAAT or TATAAA elements and precede the translation initiation codon, are sufficient for expression in a variety of chromosomal locations. The coding region (2584 nt) is interrupted at nine positions, and putative splicing signals (5'-GTRNGT...YAG-3') are present in each case. The predicted translation product contains 702 amino acids (aa) and is very similar to other TSases, except in the region of aa 257-296 that connects the alpha and beta functional domains. Both the number and the identity of the aa differ in this region between C. cinereus. S. cerevisiae, and Neurospora crassa. Comparison of exon boundaries in the C. cinereus sequence to the three-dimensional structure of Salmonella typhimurium TSase indicates that there is no simple correlation between exons and major functional domains in this protein.     

An in silico analysis of cytochrome c from Phanerochaete chrysosporium: its amino acid sequence and characterization of gene structural elements

An in silico approach was used to investigate cytochrome c and the cytochrome c gene of Phanerochaete chrysosporium. The cytochrome c gene contains four introns. Omission of the introns reveals a DNA sequence coding for a complete predicted amino acid sequence for P. chrysosporium cytochrome c consistent with those of other cytochromes c. Fungal cytochromes c often have a short N-terminal peptide preceding a Gly that is the N-terminal amino acid in many cytochromes c. Thus a microexon codes for an N-terminal pentapeptide (MetProTyrAlaPro) in P. chrysosporium that is identical to the N-terminal pentapeptide of Schizosaccharomyces pombe, a well studied yeast, the genome of which bears more similarity to higher eukaryotes than to other fungi. The fourth intron, when omitted, reveals the presence of another microexon resulting in a sequence for the C-terminal portion of the protein and the stop codon. Interestingly, two interpretations for the sequence of this intron leads to predictions that the C-terminal sequence ends with either AlaValAsn or AlaTyr. Selected aspects of the molecular architecture of cytochrome c and regulatory control elements of the P. chrysosporium cytochrome c gene were analyzed and compared to those present in other fungi and to those present in genes for lignin peroxidases and cytochromes P-450, two important families of hemeproteins produced by this fungus.     

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject     

Characterization of a new lignin peroxidase gene (GLG6) from Phanerochaete chrysosporium

Sequence analysis of a new lignin peroxidase (LIP) gene, GLG6, from P. chrysosporium showed that it encodes a mature LIP protein with a predicted Mr of 36,454. A 28 aa signal peptide precedes the mature protein. The coding region of GLG6 is interrupted by nine introns ranging in size from 50-57 bp. GLG6 encoded-LIP has 72%, 88% and 82% homology, respectively, to the LIP isozymes H2, H3, and H10. Comparison of the N-terminal sequence of GLG6-encoded LIP to that of the LIP proteins H2, H8, H10 also showed that the former is relatively less related to the H2 protein than it is to the H8 and H10 proteins. Expression of GLG6, similar to the other LIP genes, occurs only during secondary metabolism.     

Nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 12

The nucleotide (nt) sequence of a cloned DNA segment containing the early 2b region of the class A adenovirus Ad12 has been determined. When compared to the corresponding region of Ad2 or Ad7, there is a high degree of nt and predicted amino acid (aa) sequence homology within the r-strand regions that encode the preterminal protein and the viral DNA polymerase. A gene coding region comparable to the Mr 13,600 gene product found in Ad2 can be identified; this hypothetical gene product shares 30% aa homology with its Ad2 counterpart and has a very similar hydropathy profile.     

Isolation and nucleotide sequence of the ribosomal protein S16-encoding gene from Aspergillus nidulans.

A genomic clone has been isolated from Aspergillus nidulans which is homologous to the ribosomal (r) protein S16-encoding gene of Saccharomyces cerevisiae (S16A) and the r-protein S19-encoding gene of rat (S19). The amino acid (aa) sequences, deduced from nucleotide (nt) sequence analysis, show that in both cases more than 63% of the aa are conserved. The proposed A. nidulans r-protein S16 gene (rps16) differs from that of S. cerevisiae in that it occurs as a single copy in the haploid genome (rather than two copies as in yeast) and contains two putative introns (rather than one). The mRNA leader is long compared to many Aspergillus genes, commencing 293 nt upstream from the coding region, and contains an open reading frame of 13 codons.     

Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod

The complete nucleotide sequence and exon/intron structure of the rat embryonic skeletal muscle myosin heavy chain (MHC) gene has been determined. This gene comprises 24 X 10(3) bases of DNA and is split into 41 exons. The exons encode a 6035 nucleotide (nt) long mRNA consisting of 90 nt of 5' untranslated, 5820 nt of protein coding and 125 nt of 3' untranslated sequence. The rat embryonic MHC polypeptide is encoded by exons 3 to 41 and contains 1939 amino acid residues with a calculated Mr of 223,900. Its amino acid sequence displays the structural features typical for all sarcomeric MHCs, i.e. an amino-terminal "globular" head region and a carboxy-terminal alpha-helical rod portion that shows the characteristics of a coiled coil with a superimposed 28-residue repeat pattern interrupted at only four positions by "skip" residues. The complex structure of the rat embryonic MHC gene and the conservation of intron locations in this and other MHC genes are indicative of a highly split ancestral sarcomeric MHC gene. Introns in the rat embryonic gene interrupt the coding sequence at the boundaries separating the proteolytic subfragments of the head, but not at the head/rod junction or between the 28-residue repeats present within the rod. Therefore, there is little evidence for exon shuffling and intron-dependent evolution by gene duplication as a mechanism for the generation of the ancestral MHC gene. Rather, intron insertion into a previously non-split ancestral MHC rod gene consisting of multiple tandemly arranged 28-residue-encoding repeats, or convergent evolution of an originally non-repetitive ancestral MHC rod gene must account for the observed structure of the rod-encoding portion of present-day MHC genes.     

Structure of a duck H3 variant histone gene: a H3 subtype with four cysteine residues

A duck recombinant DNA phage library was screened for H3 histone genes, and the sequence of a variant H3 gene, which appears not to be part of a histone gene cluster, has been determined. As derived from the nucleotide sequence, this gene codes for a 135-amino acid (aa) protein (as any other H3) and shows 10 aa substitutions compared with most published H3 structures. Six of these aa changes are based on one nucleotide (nt) substitutions in arginine codons. This results in three new histidines and, in addition to the highly conserved cysteine at position 110, three more cysteines are found in this H3 histone subtype.     

Nucleotide sequence of the glnA-glnL intercistronic region of Escherichia coli

The nucleotide (nt) sequence of a 682-bp fragment containing the 3' end of the glnA gene, the region between the glnA and glnL genes, and the 5' end of the glnL gene from Escherichia coli was determined. This segment contains the region coding for the last 107 amino acids (aa) of glutamine synthetase, including the adenylylation site of this enzyme. The analysis of this sequence revealed two REP sequences, a Rho-independent terminator, the putative glnL promoter and the possible binding site for the glnG product, NRI.