We consider two examples of reversible inactivation of transgenes by cytosine methylation. In Ascobolus immersus, the transformation of a methionine auxotroph (met-2-) by a plasmid carrying met-2+ allele resulted in its integration (Goyon and Faugeron, 1989). When the transformant carrying an extra copy of a gene was crossed to a wild type, both the normal and the ectopic copies were inactivated. Since both copies of each allele in the meiotic tetrad were inactivated, the inactivation must occur before the premeiotic chromosome division. The frequency of inactivation of the transforming DNA was doubled if the met-2- genes are repeated in tandem than if the duplicated genes are at the ectopic site. No point mutations are associated with methylation. Gene inactivation is spontaneously reversible; where the progeny have a met-2- phenotype (Faugeron et al., 1989), the reversion rate is increased by growing the fungus in the presence of 5-azacytidine, an analog of cytidine that prevents cytosine methylation, suggesting that methylation plays a major role in this inactivation and accompanying gene silencing. This phenomenon is called MIP for methylation induced premeiotically.
In another study, N. crassa was transformed with the hygromycin phosphotransferase (hph) gene (Pandit and Russo, 1992). When the primary (heterokaryotic) transformant having more than one copy of the transgene was grown in the absence of hygromycin, the expression of the hygromycin (hph) transgene was silenced, as determined by the very low percentage of colonies formed from conidial plating on hygromycin supplemented
Parents I N i n
Translocation Normal sequence
Progeny Genotype Phenotype
Figure 9.6 Diagram of Neurospora crosses that led to discovery of MSUD. Only two linkage groups are shown. The translocation is indicated by inverse parentheses that is indicated by looping out. Crosses between normal sequence strains and translocation strains yield three kinds of progeny: normal sequence (like one parent), translocation sequence (like the other parent) and a novel class that is duplicated for the translocated segment. The duplication progeny arise when one component of the translocation segregates with a normal sequence chromosome; deletion progeny resulting from segregation of the complimentary component are inviable. Duplication strains produce perithecia that lack ascospores (barren phenotype). (From Kasbekar, D.P (2002). J. Biosci, pp. 633-635.)
(+Hyg) media. Silencing is reversed if the transformant is grown in the presence of hygromycin (Figure 9.7) or 5-azacytidine, an inhibitor of methylation. This observation suggests that methylation plays a role in the reversible silencing of the hph gene.
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