We are studying the mechanisms by which gene expression is controlled in the C. elegans germline. Many genes have been shown to express in the germline, with a variety of expression patterns and regulated properties. This observed diversity leads to the question of whether any single mechanism would apply to all germline express on. From this perspective it has been of great interest to follow attempts over the last few years (in this lab and others) to obtain efficient expression of transgenes in the germline These experiments had been surprisingly and uniformly negative: while many transgenes functioned well in somatic tissue, expression in the germline was rarely (or never) seen at detectable levels. In particular, expression of reporter constructs in the germline had never been observed, despite the fact that transgene DNA was demonstrably present. These results suggest that some aspect(s) of transgene structure or context can lead to silencing in the germline. Recently we have found a gene which can function in the germline both in res:Je assays and in driving expression of reporter constructs. Our initial studies with the gene,
let-858, had suggested that it might be no different from other genes that had been examined previously: standard transgene arrays (made with a lacZ or gfp-tagged
let-858 mixed with the
rol-6 plasmid pRF4) give transgenic lines which express in soma but express only at marginal levels in germline. The germline silencing of these "simple" arrays is evident at least as early as the Z2 and Z3 gene cell precursors in L1 larvae, and continues throughout the development of the germline. This block to germline function is also reflected in mutant rescue assays: the embryonic lethality of
let-858 mutan is rescued by such "simple" arrays, but the rescued homozygotes grow to be sterile adults with no proliferation of the germline. To introduce the transgene in a context which is less repetitive and perhaps rare similar to a true chromosomal context, we have performed injections with an excess of complex DNA (genomic DNA fragments from C. elegans). The resulting transgenes show strong expression in germline as well as some, and can rescue the
let-858 mutation to fertility. Our working model is that the
rol-6/let-858::gfp ("simple") injections result in the formation of highly repetitive extrachromosomal arrays that are read as heterochromatic by the germline and silenced. The dilution of these plasmids by random genomic fragments results in more "complex" arrays that are viewed by the germline as euchromatic, so that the silencing is alleviated. We had hoped that the "desilencing" of germline expression of complex arrays might allow expression of virtually any germline gene in transgenic animals; so far, however, this has not been successful with constructs other than
let-858. We are thus drawn to the conclusion that the
let-858 segment used for these experiments has some special signals which can promote desilencing and/or gene activation in the germline. We have therefore been asking which sequences within or around the
let-858 gene are critical for germline expression. From these studies it is clear that multiple elements play a role in this expression, including signals both upstream of and internal to the gene. To complement these studies and further analyze at a genetic level the factors involved in germline silencing, screens have been undertaken to isolate mutations that allow efficient expression of "simple" arrays in the germline (J. Goodliffe & A.F. pers. comm.)