2000 CurrGenet

Page 1

Ó Springer-Verlag 2000

Curr Genet (2000) 37: 412±419

ORIGINAL PAPER

Sergi Maicas á Ana C. Adam á Julio Polaina

The ribosomal DNA of the Zygomycete Mucor miehei

Received: 21 December 1999 / 1 March 2000

Abstract The ribosomal DNA from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the di erent rRNA species were identi®ed by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70±80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi. Key words Autonomous replication sequence á Filamentous fungus á Ribosomal RNA

Communicated by L. A. Grivell S. Maicas á A. C. Adam á J. Polaina (&) Instituto de AgroquõÂ mica y TecnologõÂ a de Alimentos, Consejo Superior de Investigaciones CientõÂ ®cas. Apartado de Correos 73. E-46100 Burjassot, Valencia, Spain e-mail: jpolaina@iata.csic.es Tel.: +34-963-90-00-22; Fax: +34-963-63-63-01

Introduction Mucor (Rhizomucor) miehei is a signi®cant organism from a biotechnological point of view because of its aspartic protease (MMP), a milk-clotting enzyme used as a substitute for calf chymosin in the cheese industry (Tonouchi et al. 1986; Foltmann 1987). Despite its biotechnological interest, M. miehei remains poorly characterised from a genetical point of view. At least in part, this may be due to its morphological characteristics. The fungus does not form discrete colonies on solid medium. It has a di use, ``cotton-like'' mycelial growth, which makes its manipulation di cult. The only available information about its life cycle is that it is homothallic (Ohnuki et al. 1982). The analysis of the rDNA region of M. miehei is important for several reasons. (1) It should help establishing taxonomical relationships to other species (Buckler et al. 1997). (2) It may have functional implications since replication origins (ARS sequences) are known to be present within rDNA sequences (Amin and Pearlman 1985; Wendland et al. 1999). Additionally, (3) the repeated rDNA sequences can be useful tools for the genetic manipulation of fungi, as they can be used as targets for gene integration by homologous recombination (Lopes et al. 1989; Adam et al. 1995). Integrative transformation by homologous recombination has been described for other Mucor species (Arnau et al. 1991; Wada et al. 1996). Fungi, like other eukaryotes, contain in their genomes multiple copies of a ribosomal DNA unit arranged in tandem. The organisation of the rDNA varies in di erent fungal species. The size of the repeated unit ranges from 7.7 to 12 kb (Rozek and Timberlake 1979; van Heerikhuizen et al. 1985). Within the unit there is a cistron containing the 16S/18S, 5.8S and 25S/28S sequences. These three sequences are transcribed as a high-molecular-weight (35S) precursor (Tague and Gerbi 1984; Rustchenko and Sherman 1994). The remaining rDNA functional sequence, 5S, is transcribed independently and may or may not be genetically linked


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