Isolation and characterization of a laccase gene fromPodospora anserina

The genome of the filamentous ascomycetePodospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene ofNeurospora crassa. One of these regions contains a gene (lac2) encoding a protein that displays 62% identity with theN. crassa laccase. In shaken cultures,lac2 mRNA is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. Addition of aromatic xenobiotics (guaiacol, hydroquinone, benzoquinone) to the medium during the growth phase results in a rapid, drastic and temporary increase in the abundance oflac2 mRNA. The promoter region oflac2 contains two sequences which display complete homology with the eukaryotic Xenobiotic Responsive Element and two sequences homologous to the eukaryotic Antioxidant Responsive Element. The identity and function of the laccase encoded bylac2 are discussed.     

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa

Summary Theqa-2 gene ofNeurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. TheQUTE gene ofAspergillus nidulans corresponds to theqa-2 gene ofN. crassa. The plasmid pEH1 containing theQUTE gene fromA. nidulans was used to transform aqa-2 ^– strain ofN. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to theQUTE gene probe. Northern blot analyses indicated thatQUTE mRNA was produced in the transformants. The functional integrity of theQUTE gene inN. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-typeN. crassa. The evidence that theQUTE structural gene ofA. nidulans is inducible when introduced into theN. crassa genome implies that theN. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5 to theQUTE gene.     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.     

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.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximatly tenfold in the presence of oxygen and three- to fourfold under heatshock conditions.     

DNA reassociation kinetics and hybridization in different angiosperms

Reassociation kinetics ofDaucus carota andPetroselinum crispum (Apiaceae), andDatura innoxia (Solanaceae) are presented. Hybridization of³H-labelled DNA of two carrot cultivars indicate strong qualitative homologies of DNA sequences; nevertheless, certain quantitative differences in some Cotregions seem to exist. However, homologous sequences ofDaucus DNA with DNA ofDatura, and, suprisingly, even with DNA ofPetroselinum are very restricted: between 8% in the repeated regions and ca. 7–9% in the unique regions.     

A complete sequence of the rice sucrose synthase-1 (RSs1) gene

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5 flanking sequence and 0.9 kb of 3 flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139–142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5 flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.     

Palindromic repeated sequences (PRSs) in the mitochondrial genome of rice: evidence for their insertion after divergence of the genus Oryza from the other Gramineae

We have identified a family of small repeated sequences (from 60 to 66 bp in length) in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare). There are at least ten copies of these sequences and they are distributed throughout the mitochondrial genome. Each is potentially capable of forming a stem-and-loop structure and we have designated them PRSs (palindromic repeated sequences). Their features are reminiscent of the small dispersed repeats in the mitochondrial DNA (mtDNA) of some lower eukaryotes, such as Saccharomyces cerevisiae, Neurospora crassa and Chlamydomonas reinhardtii. Some of the PRSs of rice mtDNA are located in the intron of the gene for ribosomal protein S3 (rps3) and in the flanking sequence of the gene for chloroplast-like tRNA^Asn (trnN). An analysis of PCR-amplified fragments of these regions from the DNA of some Gramineae suggests that the PRSs were inserted into these regions of the Oryza mtDNA after the divergence of Oryza from the other Gramineae.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom Lentinula edodes

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.     

Structure of the dnaA region of Pseudomonas putida: conservation among three bacteria, Bacillus subtilis, Escherichia coli and P. putida

Summary We have cloned from Pseudomonas putida a gene homologous to Escherichia coli dnaA, and determined the sequence of the gene and its neighboring region. The dnaA gene and at least three other genes, dnaN, recF and gyrB, were found to be highly homologous to the genes in the dnaA regions of the E. coli and Bacillus subtilis chromosomes. A non-translatable region of some 600 bp immediately upstream of the dnaA gene is also conserved in the three bacteria and contains 3, 12, and 14 DnaA-boxes (TTATCCACA and closely related sequences) in E. coli, P. putida and B. subtilis, respectively. The present results confirm our hypothesis that the dnaA region is the replication origin region of the ancestral bacterium and that the essential feature of the dnaA and DnaA-box combination is conserved in most eubacteria and plays a central role in initiation of chromosomal replication.     

毛木耳漆酶基因的克隆、序列分析及其鉴定

本文利用PCR和RACE技术首次从毛木耳AP4菌株中获得编码漆酶基因的cDNA及其基因组全长序列,基因组大小为2514 bp.通过比较该漆酶基因的cDNA和基因组DNA的全长序列,发现该基因包含14个外显子和13个内含子.cDNA序列的全长为1972 bp,其包含一个完整的ORE长度为1860 bp,编码619氨基酸,推测的分子量大小为68 kD,等电点pI为5.15.在氨基酸序列的氨基末端存在一个信号肽序列,同时该基因还包括含铜氧化酶的三个功能结构域KOG1263、SufI和pfam00394.氨基酸序列与GenBank中登录的真菌漆酶蛋白序列比对表明:该氨基酸序列与其它真菌漆酶蛋白序列有较高的同源性,氨基酸序列相同性最高达41%,相似性为58%,并且含有真菌漆酶的四个保守的Cu-bind结构域.将获得的漆酶基因lacl与毕赤酵母表达载体pPIC9K连接,构建重组质粒pYH3660,将其转化到毕赤酵母中,经甲醇诱导该基因在第10天产酶高达123 IU/L,并通过Native SDS-PAGE电泳获得预期大小的漆酶蛋白条带.结构分析和功能验证均表明:本研究获得的基因lacl为漆酶基因.     

The hydrogenase gene cluster ofRhizobium leguminosarum bv.viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity

Plasmid pAL618 contains the genetic determinants for H₂ uptake (hup) fromRhizobium leguminosarum bv.viciae, including a cluster of 17 genes namedhupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA located downstream ofhypE has now been sequenced, thus completing the sequence of the 20 441-bp insert DNA in plasmid pAL618. An open reading frame (designatedhypX) encoding a protein with a calculated M_r of 62 300 that exhibits extensive sequence similarity with HoxX fromAlcaligenes eutrophus (52% identity) andBradyrhizobium japonicum (57% identity) was identified 10 bp downstream ofhypE. Nodule bacteroids produced byhypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 µM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity. These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein. The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by ahypX gene provided in trans. From expression analysis ofhypX-lacZ fusion genes, it appears thathypX gene is transcribed from the FnrN-dependenthyp promoter, thus placinghypX in thehyp operon (hypBFCDEX). Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis. Similarities were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N₁₀-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C₁), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase-family, was located in the C-terminal half. These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C₁ donor N₁₀-formyl-tetrahydrofolate and transfer of the C₁ to an unknown substrate, and catalysis of a reaction involving polarization of the C=O bond of an X-CO-SCoA substrate. These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.     

TheNeurospora crassa erg3 gene encodes a protein with sequence homology to both yeast sterol C-14 reductase and chicken lamin B receptor

Theerg3 gene ofNeurospora crassa was sequenced (EMBL accession no. X77955) and found to encode a protein of 490 amino acid residues with significant homology to the yeast sterol biosynthetic enzyme C-14 reductase (39% identity) and also to the C-tenninal region in the sequence reported for the chicken lamin B receptor (41% identity). The possibility that a single protein may possess both lamin B receptor and sterol C-14 reductase functions might account for non-sterol-biosynthetic effects of mutations in sterol biosynthesis genes and of inhibitors of sterol biosynthetic enzymes.     

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.     

DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: Analysis with mutated binding sites

NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UAS_NTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5 or 3 sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.