Cloning of opal suppressor tRNA genes of a filamentous fungus reveals two tRNASerUGA genes with unexpected structural differences.

The informational suppressors su4-1 and su8-1 of Podospora anserina were isolated by transformation of Schizosaccharomyces pombe UGA mutants. The DNA sequence revealed that they were opal (UGA) suppressor tRNAs. Wild-type alleles were also isolated by hybridization. The DNA sequence showed that they both encode species of tRNASerUGA. The gene SU8 has an 18-bp intervening sequence and its primary sequence is very different from that of SU4.     

球毛壳菌甘油醛-3-磷酸脱氢酶基因克隆及特性分析

用粗糙脉孢菌(Neurospora crassa,XP_327967)和菜豆炭疽病菌(Colletotrichum lindemuthianu,P35143)的甘油醛_3_磷酸脱氢酶基因(Glyceraldehyde 3_phosphatedehydrogenase,GAPDH)氨基酸序列对球毛壳菌(Chaetomium globosum)菌丝ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌GAPDH全长cDNA序列。该序列长1240bp,开放阅读框1014bp,编码337个氨基酸组成的多肽,蛋白分子量为36.1kD。用PCR方法克隆了该基因的DNA序列,序列长为1556bp,由2个内含子和3个外显子组成。BlastP同源性分析表明该基因与鹅掌柄孢壳(Podosporaanserine)同源性最高为95%;与米曲霉(Aspergillusoryzae)同源性最低为87%。GAPDH酵母转化子生物功能分析表明转化子对Na2CO3和高温有高的耐受性,证明GAPDH为抗胁迫基因。该基因的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY522719,AY593253,AAS01412)。     

Cloning, Sequencing, and Transgenic Expression ofPodospora curvicollaandSordaria macrosporaeEF1A Genes: Relationship between Cytosolic Translation and Longevity in Filamentous Fungi

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi,Podospora curvicollaandSordaria macrospora.These fungi are close relatives ofPodospora anserinaand also show senescence syndromes. Comparison of the sequences of the deduced proteins with that ofP. anserinareveals that the three proteins differ in several positions. Replacement of theP. anserinagene by either of the two exogenous genes does not entail any modification inP. anserinaphysiology; the longevity of the fungus is not affected. No alteration ofin vivotranslational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity.     

Evolution of multicopper oxidase genes in coprophilous and non-coprophilous members of the order sordariales

Multicopper oxidases (MCO) catalyze the biological oxidation of various aromatic substrates and have been identified in plants, insects, bacteria, and wood rotting fungi. In nature, they are involved in biodegradation of biopolymers such as lignin and humic compounds, but have also been tested for various industrial applications. In fungi, MCOs have been shown to play important roles during their life cycles, such as in fruiting body formation, pigment formation and pathogenicity. Coprophilous fungi, which grow on the dung of herbivores, appear to encode an unexpectedly high number of enzymes capable of at least partly degrading lignin. This study compared the MCO-coding capacity of the coprophilous filamentous ascomycetes Podospora anserina and Sordaria macrospora with closely related non-coprophilous members of the order Sordariales. An increase of MCO genes in coprophilic members of the Sordariales most probably occurred by gene duplication and horizontal gene transfer events.     

Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. A potential role for an 11 base-pair consensus sequence in the excision process

Three novel mitochondrial excision-amplification plasmids of Podospora anserina were identified and the excision-junction sites on the mitochondrial genome determined. All three plasmids were at least partially derived from a common region of the mitochondrial genome termed EcoRI-7 (E7). The entire 5651 base-pair sequence of E7 is presented. Included within this sequence are the E7-specific excision-junction sites of these novel plasmids, the localizations of nine tRNA genes, and the localization of a class I intron of the large rRNA mitochondrial gene. The E7 region contains the 3' portion of this large rRNA gene. Formation of these three novel plasmids as well as other previously described mitochondrial plasmids was found to be associated with the presence of an 11 base-pair consensus sequence, GGCGCAAGCTC, or its complementary sequence. A possible role for this consensus sequence and its complement in plasmid formation and the senescence process of Podospora is discussed. A possible role for the tRNA genes in plasmid formation is considered.     

球孢白僵菌线粒体序列分析及系统进化树构建

线粒体是真核细胞的重要的细胞器,在细胞能量代谢和细胞衰老方面扮演了重要角色,同时也是研究物种进化关系的重要资源。本文应用球孢白僵菌的线粒体序列分析了白僵菌与粪壳菌纲其他11属的13种真菌间的进化关系,为阐明球孢白僵菌的进化地位提供了新的论据。线粒体编码的rnl,rns,25个tRNAs和14个蛋白质基因均由同一条链编码,大部分的tRNA分布为三个tRNA簇,这与粪壳菌纲的其他物种相似。与NCBI上公布的序列比对发现球孢白僵菌线粒体序列与蝇蚧霉线粒体序列最相似,相似率达73%,用14个蛋白编码基因通过邻接法(Neighbor-joint)和最大简约法(Maximum parsimony)构建的系统进化树也得到相同结论,其支持率均为100%。     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Insertion of an LrDNA gene fragment and of filler DNA at a mitochondrial exon-intron junction in Podospora.

A rearrangement of the mitochondrial genome of a long lived mutant of Podospora anserina is presented. It consists in the insertion of 191 bp of the LrDNA gene (coding for the large ribosomal RNA) at the junction between exon1 and intron alpha of gene co1 (coding for subunit 1 of cytochrome oxidase). This insertion is accompanied by a 53 bp deletion of the junction and the presence of extra A and T nucleotides at both sides of the inserted sequence. We discuss possible mechanisms of production of this rearrangement. The presence of extra nucleotides at the recombination junctions suggests that it may pass through a stage of free DNA ends originating from a DNA break at the junction between exon1 and intron alpha of gene co1. The possibility that such a DNA break plays a major role in the instability of the mitochondrial genome is envisaged.     

Transformation by integration in Podospora anserina. III. Replacement of a chromosome segment by a two-step process

Summary We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.     

The genome sequence of Podospora anserina, a classic model fungus

The completed genome sequence of the coprophilous fungus Podospora anserina increases the sampling of fungal genomes. In line with its habitat of herbivore dung, this ascomycete has an exceptionally rich gene set devoted to the catabolism of complex carbohydrates.     

Intron open reading frames as mobile elements and evolution of a group I intron

Group I introns are proposed to have become mobile following the acquisition of open reading frames (ORFs) that encode highly specific DNA endonucleases. This proposal implies that intron ORFs could behave as autonomously mobile entities. This was supported by abundant circumstantial evidence but no experiment of ORF transfer from an ORF- containing intron to its ORF-less counterpart has been described. In this paper we present such experiments, which demonstrate the efficient mobility of the mitochondrial nad1-i4-orf1 between two Podospora strains. The homing of this mobile ORF was accompanied by a bidirectional co-conversion that did not systematically involve the whole intron sequence. Orf1 acquisition would be the most recent step in the evolution of the nad1-i4 intron, which has resulted in many strains of Podospora having an intron with two ORFs (biorfic) and four splicing pathways. We show that two of the splicing events that operate in this biorfic intron, as evidenced by PCR experiments, are generated by a 5'-alternative splice site, which is most probably a remnant of the monoorfic ancestral form of the intron. We propose a sequential evolution model that is consistent with the four organizations of the corresponding nad1 locus that we found among various species of the Pyrenomycete family; these organizations consist of no intron, an intron alone, a monoorfic intron, and a biorfic intron.      

Size and complexity of the nuclear genome of the ectomycorrhizal fungus Paxillus involutus

The basidiomycete Paxillus involutus is forming ectomycorrhizal symbiosis with a broad range of forest trees. Reassociation kinetics on P. involutus nuclear DNA indicated a haploid genome size of 23 Mb including 11% of repetitive DNA. A similar genome size (20 Mb) was estimated by genomic reconstruction analysis using three single copy genes. To assess the gene density in the P. involutus genome, a cosmid containing a 33-kb fragment of genomic DNA was sequenced and used to identify putative open reading frames (ORFs). Twelve potential ORFs were predicted, eight displayed significant sequence similarities to known proteins found in other organisms and notably, several homologues to the Podospora anserina vegetative incompatibility protein (HetE1) were found. By extrapolation, we estimate the total number of genes in the P. involutus haploid genome to approximately 7700.     

Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences.

The CapR protein is an ATP hydrolysis-dependent protease as well as a DNA-stimulated ATPase and a nucleic acid-binding protein. The sequences of the 5' end of the capR (lon) gene DNA and N-terminal end of the CapR protein were determined. The sequence of DNA that specifies the N-terminal portion of the CapR protein was identified by comparing the amino acid sequence of the CapR protein with the sequence predicted from the DNA. The DNA and protein sequences established that the mature protein is not processed from a precursor form. No sequence corresponding to an SOS box was found in the 5' sequence of DNA. There were sequences that corresponded to a putative -35 and -10 region for RNA polymerase binding. The capR (lon) gene was recently identified as one of 17 heat shock genes in Escherichia coli that are positively regulated by the product of the htpR gene. A comparison of the 5' DNA region of the capR gene with that of several other heat shock genes revealed possible consensus sequences.     

Variable DNA splicing sites of a mitochondrial intron: relationship to the senescence process in Podospora

The unavoidable phenomenon of senescence in Podospora was previously shown to be correlated with the presence of a senescence-specific DNA originating from amplification of some regions of the mitochondrial chromosome. The most frequently amplified region (α) corresponds to the first intron of the gene coding for subunit one of cytochrome oxidase. Eleven long-lived mitochondrial mutants were isolated. Here we report sequencing experiments that show that three of them are deleted for most of intron α and for a few base pairs belonging to the upstream adjacent exon. We also report an analysis of the residual mitochondrial DNA associated with amplification of senescence-specific DNA α which allows us to identify, in senescent cultures, mitochondrial chromosomes lacking sequence α. These results taken together suggest that excision of intron α from the mitochondrial DNA occurs systematically during the aging process in Podospora. They furthermore provide the first example of inaccurate intron excision at the DNA level.