DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon
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O R I GI N A L P A P E R
Y. Zhou á E. B. Cambareri á J. A. Kinsey
DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon
Received: 26 June 2000 / Accepted: 2 February 2001 / Published online: 13 April 2001 Ó Springer-Verlag 2001
Abstract Tad is a LINE-like retrotransposon of the ®lamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5¢ noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we ®nd that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background. Keywords Neurospora crassa á Tad á Cytosine methylation á Inhibition of transposition
Introduction Tad is a LINE-like element similar to non-LTR retrotransposons found in a wide variety of organisms (Cambareri et al. 1994; Malik et al. 1999). It was originally identi®ed by analysis of insertion mutations in the am (glutamate dehydrogenase) gene of hybrid strains derived
Communicated by E. CerdaÂ-Olmedo Y. Zhou1 á E. B. Cambareri2 á J. A. Kinsey (&) Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA E-mail: [email protected] Tel.: +1-913-5887088 Fax: +1-913-5887295 Present addresses: Department of Biology, University of Missouri, Kansas City, KS 66160, USA 1
2 Neugenesis Corporation, 517J Industrial Road, San Carlos, CA 94070, USA
from a cross between the Adiopodoume strain of Neurospora crassa and a laboratory strain (Kinsey and Helber 1989). One of the am mutations recovered, am::Tad3-2, resulted from the integration of Tad3-2 into the 5¢ noncoding sequence immediately upstream of the am TATA box. The am::Tad3-2 mutant strain shows an unstable phenotype, switching from Am± (unable to form colonies on glycine-supplemented medium) to Am+ (able to form colonies on glycine-supplemented medium) and back to Am± with high frequency (Kinsey and Helber 1989). Subsequent analysis demonstrated that this switching of phenotype was an epigenetic phenomenon that depended upon the cytosine methylation state of DNA near the 5¢ end of the Tad3-2 element (Cambareri et al. 1996). This ®nding was somewhat surprising since only a small fraction of the Neurospora genome shows cytosine methylation (estimated to be only about 1.5% of the cytosine residues; Russell et al. 1987). Margolin et al. (1998) speculated that cytosine methylation in Neurospora occurs primarily in about 500 heavily methylated, genesized, regions. Only ®ve of these regions have been characterized . Two of these appear to represent sequences that have been subjected to the Repeat Induced Point mutation (RIP) process (Margolin e
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