Javier Avalos, Bina Mehta, Irina Obraztsova, Nicolas Prados, Javier Ruiz-Albert, Klaus Holzmann, Luis M. Corrochano and Enrique Cerda-Olmedo. Departamento de Genetica, Facultad de Biologia, Universidad de Sevilla, 41012 Sevilla, Spain.
Protocols for transformation of Phycomyces protoplasts have been improved: the production and regeneration of protoplasts has been optimized and new vectors have been developed with a bacterial geneticin resistance gene under the control of regulatory Phycomyces sequences. A plasmid has been recovered from a -carotene superproducing strain obtained after microinjecting DNA from a plasmid genomic library into Phycomyces sporangiophores. The plasmid, containing a 3.1 Kb insert of Phycomyces DNA, confers upon microinjection the superproducing phenotype. No similarity was found by comparing the 3.1 Kb sequence with gene databases. The gene hmgA, encoding the key enzyme in terpenoid biosynthesis HMG-COA reductase, has been cloned from a lambda genomic library. A 3 Kb segment containing the entire gene is now being sequenced.
Southern analysis indicates the presence of one copy of the hmgA gene in Phycomyces.
Highly repetitive DNA sequences of Phycomyces have been characterized. About 5% of the total genome is a repetition of any of two consensus sequences of 31 bp, named PrAl and PrA2. Higher order repetitions of groups of PrA units have been identified, surrounded by more complex repetitive sequences.
We have isolated two sequences of Phycomyces with similarities to ORFs encoded in two different transposable elements: Tcl from Caenorhabditis elegans and Txl from Xenopus laevis. Preliminary southern analysis suggest the presence of 10-20 copies of the Tcl-like transposable element in the Phycomyces genome.
Flipper, a Bacterial-like Transposable Element in Botrytis cinerea
Caroline Levis, Dominique Fortini and Yves Brygoo. INRA, Pathologie Vegetale, Route de St-Cyr, 78026 Versailles Cedex France
Botrytis cinerea (Botrylinia fuckeliana) is a phytopathogenic fungus having a broad host spectrum whinc includes grapevine, tomato, flower bolbs and otnamentals. There is no apparent specialization in the fungus for this wide host range. From a wild strain isolated from sweet pepper, a mutant deficient int he nitrate reductase gene nia) was selected through its spontanewus resistance to chlorate. A Southern Blot hybridization with the nia gene of Bortytis cinerea showed that a size polymorphism appeared between the mutant strain and the wild strain. By PCR we have shown that the mutation correspond to an insertion of 1.7 Kb in the coding region. The sequence of this insertion is characterized by two ITS and one ORF which has strong similarity with bacterial-like transposable elements, Pol2 of Magnaorthe grisea and Fot1 of Fusarium oxysporum. This new transposable element hot yet reported in discomycetes is named Flipper. Analysis by Southern blot hybridization have shown that the number of Flipper copies varied from zero to about 20 copies depending on the strains.
Evolutionary Origin of the Impala Element Within the Fusarium oxysporum Species
A. Hua-Van, M.J. Daboussi, P. Capy, C. Gerlinger, T. Langin. Institut de Genetique et Microbiologie, Universite de Paris-Sud, Bat. 400, 91405 Orsay CEDEX, France
The impala element was first identified as an insertion within the niaD gene. Impala is a DNA transposon (1,280 hp in length, 27 hp ITRS, TA insertion specificity) belonging to the Tcl-mariner superfamily (Langin et al. MGG, 1995). This inserted copy is transposase defective due to the presence of several stop codons but it can be mobilized by a source of transposase. In order to identify an autonomous element, the 8 impala copies present in the genome of the F24 strain have been cloned and sequenced. Two copies carrying an ORF able to encode an active transposase have been identified. In addition, this analysis revealed the existence of a high level of nucleotide polymorphism leading to the identification of four subfamilies. Within a subfamily, the polymorphism is low (0.20 to 1.4%) whereas between subfamilies, it ranges between 9 to 30%. To gain insights on the evolutionary origin of the impala family, an analysis of the distribution of the different subfamilies has been carried out within the F. oxysporum species using Southern blots and PCR amplification. Impala elements belonging to the different subfamilies have been detected in most of the formae speciales analyzed. These results indicate that the impala element is an ancient component of the F. oxysporum genome with a presence of the different subfamilies before host specialization.
Restless, an Active Transposon from the Filamentous Fungus Tolypocladium inflatum
Frank Kempken and Ulrich Kuck. Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat, Universitatsstra e 150, D-44780 Bochum, Germany (fax +49-234-7094-184)
Transposons in eukaryotes can be divided in two major groups (McDonald 1993; Flavell et al. 1994). Class I transposable elements are able to transpose via an RNA intermediate, thereby employing a reverse transcriptase. Class II elements transpose via DNA transposition intermediates, demonstrating their activity by direct excision and subsequent integration into target sequences. Transposons of both classes have previously been identified in filamentous fungi (reviewed in Daboussi and Langin 1994). We previously described the isolation and characterization of a new type of fungal class II transposons from Tolypocladium inflatum (synonym: Beauveria nivea) which so far has not been found in any other fungus. It carries short inverted repeats and eight basepair target site duplications, and encodes a large open reading frame which is interrupted by a single intron sequence showing a rare intron consensus sequence. The predicted amino acid sequence deduced from this frame shows significant homology to transposases of the hAT transposon family. Its transcription and splicing characteristics were analyzed in detail. Based on cDNA sequencing, alternate RNA splicingmay lead to two different proteins, both encoded by the transposon. The function of these proteins is currently under investigation. In addition we will present data concerning the activity of the transposable element.
References:
Daboussi M-J, Langin T (1994) Transposable elements in the fungal pathogen Fusarium oxysporum. Genetica 93:49-59
Flavel AJ, Pearce SR, Kumar A (1994) Plant transposable elements and the genome. Curr Opin Genet Dev 4:838-844
McDonald JF (1993) Evolution and consequences of transposable elements. Curr Opin Genet Develop 3:855-864
Evolution of the Fot1 Element in the Fusarium Genus: Horizontal Versus Vertical Transmission?
Thierry Langin and Marie-Josie Daboussi. Institut de Genetique et Microbiologie, Universite Paris-Sud. 91405 Orsay. Cedex. France.
Fot1 is a DNA transposon identified in filamentous fungi Fusarium oxysporum and recognized recently in other species. To understand the evolutionary origin of Fot1, we have undertaken an extensive survey of the F. oxysporum species. The presence of Fot1 was detected in strains belonging to 15 formae speciales with a copy number ranging from I to more than 100. PCR amplications using the ITRs demonstrated that Fot1 exists in this species essentially as full-length copies. Partial sequencing of Fot1 copies in different strains showed that the nucleotidic polymorphism is very low (<1%). All these results suggest that Fot1 was present in this species before the host specialization and has been vertically transmitted. The existence of a few number of strains devoid of Fot1 copies can be interpretated as the result of stochastic loss. The presence of Fot1 in different Fusarium species was analysed by Southern blot analysis at high stringency conditions. Although this element appeared to be absent in most of the Fusarium species examined, Fot1 -related elements were detected in species genetically distant from F. oxysporum. This discontinuous distribution can be explained by two non-exclusive hypotheses: (1)- Fot1 is an ancient element present in the common ancestor of Fusarium species and the discontinuity is the result of a complete loss in some species; (2)- the presence of Fot1-related elements in distant species would correspond to a recent acquisition resulting from horizontal transfer. To gain insights on the mechanisms of spreading of Fot1 within the Fusarium genus, the nucleotide divergence of Fot1 elements from different species and compared to those observed for non-transposable chromosomal sequences.
Identification of Autonomous Copies of the Fusarium oxysporum Fot1 Transposable Element
Q. Mighelli, M.J. Daboussi, C. Gerlinger, T. Langin, R. Lauge. Institut de Genetique et Microbiologie, Universite Paris-Sud, Bat. 400, 91405 Orsay CEDEX, France
Insertional mutagenesis is a powerful method for gene isolation that does not require prior knowledge on gene product. One of the tags efficiently used is represented by transposons. The discovery of two families of' DNA transposons (Fot1 and Impala) in the genome of the fungal plant pathogen F. oxysporum, offers the opportunity to develop a transposon gene tagging system, a strategy not yet available in fungi. These elements transpose at high rate by excision and reinsertion, which are features pertinent when designing gene tagging experiments. To gain insights on the autonomy of the Fot1 copies we cloned them as insertions within the nitrate reductase gene (niad) by developing a phenotypic assay based on the restoration of the nia function af'ter excision of the Fot1 elements. Plasmids carrying the niad gene disrupted by two different copies of' Fot1 were introduced by co-transformation with the pAN7-1 plasmid into stable nia- mutants deriving from two strains free of active Fot1 copies. NiaD+ colonies have been recovered, thus indicating that the gene function was restored after excision of the Fot1 copy. Molecular analysis of these niad+ revertants indicates that in more than 50% of them the excised copy has reinserted in a new genomic position, indicating that both Fot1 copies encode all functions necessary for transposition. These results constitute the first identification of autonomous copies of transposons in filamentous fungi.
Molecular Epidemiology of Aspergillus fumigatus with AfuT1, a Retrotransposon-like Element
Neuveglise Cecile, Sarfati Jacqueline, Debeaupuis Jean-Paul, Latge Jean-Paul, Paris Sophie.
Laboratoire des Aspergillus, Institut Pasteur, 25, rue du Docteur Roux, 75015 Paris, France.
Epidemiological studies of the opportunistic fungus Aspergillus fumigatus has required the development of molecular methods for fingerprinting strains isolated from patients and clinical environment. Repeated DNA sequences specific of A. fumigatus have been cloned and used as probes for Southern blot hybridization. This method allowed to show that immunocompromised patients with invasive aspergillosis are mostly contaminated by a single strain, whereas several strains are isolated from cystic fibrosis patients. However, when the latter patients develop an aspergillosis, the infection is due to a single strain. Moreover, the nosocomial origin of invasive aspergillosis was suggested in some cases.
The repetitive sequence has been characterized. It is a retrotransposon of 6914 bp, bounded by Long Terminal Repeats (LTR) of 282 bp, with sequence and feature characteristic of retroviruses and retrotransposons. The organization of the domains homologous to the reverse transcriptase, RNase H and endonuclease domains of retroviruses revealed that Afut1 is a member of the gypsy group. However, Afutl is a defective element because of an accumulation of transitions from C:G to T:A generating multiple stop codons in the putative coding domains.
Tan and Vader - Transposable Elements Found from A. niger var. awamori
Eini Nyyssonen, Maria Amutan and Nigel Dunn-Coleman. Genencor International, Inc., 180 Kimball Way, South San Francisco, CA 94080.
A.niger var. awamori has transposable elements which we refer to as Vader (Amutan et al, 1996) and Tan (Nyyssonen et al, 1996). Vader was isolated using a transposon entrapment strategy, in which unstable nidD (nitrate reductase) mutants were screened for transposons. Four of the isolated nidD mutants were shown to contain a small insertion element. This 441 bp, ATrich insertion element, Vader, is flanked by 44 bp inverted repeats and is present in approximately 15 copies in the genomes of the two A. niger var. awamori strains examined (Amutan et al., 1996). Vader contained no ORF and hence it was deduced that the mobility of Vader was dependent upon a transposase activity present elsewhere in the genome. A synthetic oligomer corresponding to the inverted repeat was used to clone a 2.3 kb element, Tan, which is present as a single copy in the A. niger var. awamori genome. Tan has a single ORF (1668 bp) encoding a putative transposase, which is bounded by IRs and Vader contiguous with it (i.e. IR-ORF-IR-IR-Vader-IR). Protein alignment of this 555 aa Tan encoded protein with other proteins in the EMBL and Genbank databases revealed 31.2% and 27.9% identities throughout the entire length of the protein to the Fusarium oxysporum Fot1 and Magnaporthe grisea Pot2 ORFS, respectively. This result suggests that Tan was the element which had provided the activity for the Vader element to transpose. We hypothesize that at some stage the independent Vader element, although inactive by itself, has arisen from Tan resulting in current strains with only one copy of Tan providing transposase activity and numerous mobile copies of Vader dispersed in the genome.
References:
Amutan, M., Nyyssonen E., Stubbs, J., Diaz-Torres, M. and DunnColeman, N. 1996. Identification and cloning of a mobile transposon from Aspergillus niger var. awamori. Curr. Genet., in press.
Nyyssonen, E., Amutan, M., Enfield L., Stubbs, J. and Dunn-Coleman, N. 1996. Tan, a novel transposable element in A. niger var. awamori. Mol. Gen. Genet., in press.
Mobile Genetic Elements Involved in the Control of Senescence of Podospora anserina
Josef Hermanns* and Heinz D. Osiewacz. Johann Wolfgang Goethe-Universitat, Botanisches Institut, Marie-Curie-Str. 9, 60439 Frankfurt, Germany. *Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
In the ascomycete Podospora anserina life-span is controlled by nuclear and mitochondrial (mt) genetic traits. In particular, a mitochondrial plasmid of circular structure (p1DNA or senDNA) was demonstrated to be crucially involved in the control of senescence (1,2). The plasmid is derived from the first intron (mobile intron) of the mitochondrial cytochrome oxidase subunit I gene (3). Intron mobility was demonstrated by molecular approaches (4) and appears to be involved in the destabilization of the juvenile mtDNA leading to gross DNA rearrangements and to impaired cellular functions.
The analyses of an extrachromosomal long-lived mutant of wild-type strain A led to the identification of an extrachromosomal DNA species (pAL2-1) with the typical characteristics of linear plasmids (5,6). This element was shown to interfere vhth the age-related liberation and/or amplification of p1DNA. In addition, pAL2-1 is able to integrate into the mtDNA leading to integrated plasmid copies with long terminal repeated DNA sequences at the integration site. Finally, the ability of pAL2-1 to modulate the life-span of P. anserina was demonstrated by plasmid transfer experiments. These experiments revealed that the loss-of-plasmid results in a decreased life-span, whereas gain-of-plasmid strains displayed a long-lived phenotype. Interestingly, all strains to which the plasmid was transferred were found to contain pAL2-1 in both, the autonomous and the integrated form.
(1) Stahl U, Lemke PA, Tudzynski P, Kiick U, Esser K (1978) Mol Gen Genet 162:341-343
(2) Cummings DJ, Belcour L, Grandchamp C (1979) Mol Gen Genet 171:239-250
(3) Osiewacz HD, Esser K (1984) Curr Genet 8:299-305
(4) Sellem CH, Lecellier G, Belcour L (1993) Nature 366:176-179
(5) Osiewacz HD, Hermanns J, Marcou D, Triffi M, Esser K (1989) Mutat Res 219: 9-15
(6) Kempken F, Hermanns J, Osiewacz HD (1992) J Mol Evol 35:502-513
The experimental work was supported by a grant of the Deutsche Forschungsgemeinschaft (Bonn, Germany) to HDO.
Rearrangements of Fusarium Retrotransposon Sequences Induced under Stressing Conditions
Nuria Anaya and M.I.G. Roncero. Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, Avda. San Alberto Magno sln, E-14071 Cordoba, Spain
Retrotransposons are mobile elements which replicate via an RNA intermediate and have been described in different fungal species (Mc Hale et al., 1989, 1992; Dobinson et al., 1993; Julien et al., 1992). Recently a retrotransposon has been identified in F. oxysporum f. sp. lycopersici. (Anaya and Roncero 1995). This element designated skippy is 7,846 bp in length, it contains all conserved features of gypsy class retrotransposons and all the characteristic sequences presumably required for integration and functional transposition. The mobility of this retroelement as for other retroelements from filamentous fungi remained still unproven. Rearrangement of the retroelement skippv (skp) has been induced under stressing growth conditions in the presence of potassium chlorate. Three fungal strains, one showing wild type phenotype str and two of them resistant to chlorate and deficient for nitrate reductase, were studied by Southern analysis of their genomic DNA. Polymorphism in their hybridization banding pattern was detected in comparison with the wild type (wt) grown in the absence of chlorate. Results are consistent, with three different events occurring in the str strain: genomic amplification of skp in tandem copies, integration of new skp elements, and deletion of resident skp elements. The analysis of mutants nidD65 and nidD94 showed that only integration and deletion of copies of the elements had accursed. Amplification of genomic DNA from str using divergent primers belonging to the retroelement originated a new band of 589 bp corresponding to one LTR that was not present in wt strain.
Isolation and Characterization of a Transposable Element from Fusarium oxysporum fsp. lycopersici
Gomez Gomez M, Anaya N, Roncero MIG, Hera C. Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, Cordoba 14071, Spain
Fusarium oxysporum, like many other plant pathogens displays considerable phenotypic variability, a characteristic of organisms harboring transposable genetic elements. Bacterial-like transposable elements have been previously isolated from races of F. oxysporum f. sp. melonis (reviewed by Daboussi and Langin, 1994). We have used the strategy of transposon tagging for isolating mobile elements in F. oxysporum f.sp. lycopersici. A total number of ninety nitrate deficient spontaneous mutants from strain 42-87 (race 2), positively selected for their chlorate resistant phenotype, were characterised by Southern analysis using the cloned nitrate reductase homologous gene as a probe. Among them two mutants (#66, #108) showed to harbor an insert of about 2,5 kilobases as deduced from their hybridization pattern compared with that of the parental strain. Total genomic DNA from mutants #66 and #1 08 was subjected to amplification by PCR using two specific primers from the 5' and 3' ends of the nitrate reductase gene. An unique band, with the expected size of 8kb, was amplified from each mutant and they were cloned in PGEMT vector for subsequent subcloning. The physical maps of the DNA inserts interrupting the nitrate reductase gene showed an identical restriction pattern. Their nucleotide sequences are being determined for identification of ITRs and transposase coding gene, The presence of homologous elements in other formae speciales is been studied in order to identify elementfree strains and to establish the autonomy of this F. oxysporum f. sp. lycopersici transposable element.
Yeast Transposable Elements
F.X.Wilhelm. Institut de Biologie Moleculaire et Cellulaire 15, rue R.Descartes. 67084 Strasbourg. France
Ty elements are a family of LTR retrotranposons found in the yeast Saccharomyces cerevisiae. To date five distinct families designated Tyl-Ty5 have been described. They can be divided into two classes related to the copia or gypsy class of plant and animal elements. The yeast elements are structurally and functionally similar to animal retroviruses. Like retroviruses they contain large internal coding regions flanked by long terminal direct repeats (LTR). They transpose through an RNA intermediate and use the same replication and integration strategy as metazoan retroviruses. The structure and life cycle of the Ty elements will be briefly described.
Transposable elements contribute to the fluidity and shaping of the eukaryotic genome. Chromosomal rearrangements which result in duplication or deletion are induced by recombination between regions of homology. Insertion of a retroelement DNA in or near a cellular gene can affect its regulation or inactivate it. Although Ty element insertion can occasionally be beneficial, the average fitness of host yeast,cells declines with increasing Ty copy number. The balance must be maintained between the level of transposition and the viability of the host cell. The recent studies showing that Ty retrotransposons integrate selectively into certain regions of the genome which can readily tolerate insertions will be discussed.
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