Nucleotide sequence of a Trichoderma longibrachiatum DNA fragment encoding the 5.8S rRNA gene.

Trichoderma longibrachiatum IMI061758 possesses a powerful cellulose enzyme complex composed by different cellobiohydrolase, endoglucanase and /3-glucosidase activities. Recently we have cloned the T. longibrachiatum egll gene encoding a /S-{l,4)-endoglucanase activity (1). From an industrial point of view, we are interested in the design of strains which have an increased egll gene dosage. To construct these strains, we have developed an integrative transformation system for T. longibrachiatum (2), but the transformants are mitotically unstable. To solve this problem, one of the strategies could be the development of new vectors in which a repetitive sequence of the fungal genome has been included which favours the stability of the transformants. In this report we present the cloning and sequencing of a T. longibrachiatum DNA fragment encoding part of the ribosomal repeat unit. Due to the taxonomical difficulties related to the Trichoderma group (3), this sequence could be also of interest for the classification of this fungal species. Amplification of the 5.8S rRNA gene and the noncoding adjacent regions was accomplished by PCR using the ITS1 and ITS4 primers described in (4). The amplicon was cloned in both orientations into the pCRTM1000 vector (Invitrogen, San Diego CA), and the resulting plasmids were used to construct a restriction map of the insert. Series of nested deletions were obtained by the exonuclease HI-SI nuclease digestion method, and selected deletion mutants or directed subcloned fragments were used as templates for sequencing. The nucleotide sequence (Figure 1) was determined in both strands using the dideoxynucleotide chain termination method. All the restriction sites used for subcloning were sequenced through. The 5.8S rRNA encoding gene (nt 234 to 379) was located by its highly similarity with other filamentous fungal 5.8S rRNA genes. It shows minor changes with respect to the Acremonium chrysogenum, Fusarium sambucinum or Neurospora crassa genes (8, 4 and 5 nt changes respectively). With respect to the noncoding regions, only minor stretches of identity could be detected, particularly in the case of the F.sambucinum sequences. ACKNOWLEDGEMENTS