© 1992 Oxford University Press

610 Nucleic Acids Research, Vol. 20, No. 3

Nucleotide sequence of the 5.8S ribosomal RNA Lentinula edodes

gene

of

H.S.Kwan, K.M.Pang, S.W.Chiu and S.C.Cheng1 Department of Biology, The Chinese University -of Hong Kong, Shatin, N.T. and 1Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic, Kowloon, Hong Kong Submitted December 20, 1991 The gene encoding the 5 .8S rRNA is a part of the rDNA cluster and flanked by two internal transcribed spacer (ITS) regions. During our characterization of four Lentinula edodes (Shiitake mushroom) strains based on ITS sequence differences, we amplified part of the rDNA cluster using the primers ITS2, ITS3, ITS4 and ITS5 by polymerase chain reaction (1). In addition to the determination of the DNA sequence of the ITS regions, we also determined the DNA sequence of the 3' end of the 18S rDNA, the 5' end of the 25S rDNA, and the entire 5.8S rDNA. The arrangement of the rRNA genes and ITS regions of this mushroom follows that of other eukaryotes. We present here the DNA sequence of the 5.8S rDNA and its flanking sequences (Figure 1). An analysis of the possible stem-loop structures led to a putative secondary structure of the 5.8S rDNA (Figure 2). This secondary structure is very similar to that originally predicted for Xenopus laevis (2). We found that the 5.8S rDNA of Lentinula edodes is very closely related to those of Saccharomyces cerevisiae (3) (92 % homology), Schizosaccharomyces pombe (4) (87 % homology), and Neurospora crassa (5) (89% homology). The base differences between the 5.8S rDNA sequence of L. edodes and each of those of the other three fungi occur mainly in the last stem-loop that is closest to the 3' end of the 5.8S rRNA (Figure 1).

EMBL accession no. X63130 LEN NEU YSP YSC LEN NEU YSP YSC

1 ATCTTGTTAT TGGGACTTTA TTGACCCTTT AAACTTAATA CAACTTTCAG A A A A A 51 CAACGGATCT CTTGGCTCT

GCATCGATGA AGAACGCAGC GAAATGCGAT

T T

LEN 101 AAGTAATGTG AATTGA NEU C YSP G YSC C

TTCAGTGAAT CATCGAATCT TTGAACGCAC C C

LEN 151 CTTGCGCCCT CTGGTATTCC GGAGGCATGC CTGTTTGAGT GTCATTAAAT NEU A T T G *C A AA YSP TO * A* T CA C C YSC A * T A G C LEN 201 TCTCAACTTT ATAAGTTTTT ACTTATTAAA GCTTGGATGT TGGAGGCTTG

Figure 1. Nucleotide sequence of the 5.8S rRNA gene and its flanking sequences. Base differences among Lentinula edodes and three other fungi are indicated. LEN, Lentinula edodes; NEU, Neurospora crassa; YSP, Schizosaccharomyces pombe; YSC, Saccharomyces cerevisiae. Underlined sequence, 5.8S rRNA gene; *, missing base; Bold-faced type, additional base.

UGCCUGUUUGAGUGUCAUUAAA

A

ACKNOWLEDGEMENT This work was supported by a research grant from The Croucher Foundation.

REFERENCES 1. White,T.J., Bruns,T., Lee,S. and Taylor,J. (1990) In Innis,M.A., Gelfand,D.H., Sninsky,J.J. and White,T.J. (eds), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. pp. 315 -322. 2. Vaughn,J.C., Sperbeck,S.J., Ramsey,W.J. and Lawrence,C.B. (1984) Nucleic Acids Res. 12, 7479-7502. 3. Yeh,L-C.C. and Lee,J.C. (1991) J. Mol. Biol. 217, 649-659. 4. Schaack,J., Mao,J. and Soell,D. (1982) Nucleic Acids Res. 10, 2851 -2864. 5. Selker,E. and Yanofsky,C. (1979) Nucleic Acids Res. 6, 2561-2566.

Gc

uu C CUCGCA AU U-A C-G G-C A-U U-A G-C

AA

AG

AAC-G

CU

AAA

A

UG

G-C C-G A-U

C CU

A

AUAA

G G U U

G

GIM9 G

C

C-G

G

A-U

C

G A

A A

Figure 2. Predicted secondary structure of the 5.8S rRNA.

3'