The major goals of this study are to identify meiotic sites in Drosophila and to use them to gain insight into the mechanisms of meiotic pairing. We have utilized cytogenetic methods to show that pairing capacity is widely distributed throughout the euchromatin of chromosome 2, a major autosome, but that a strong pairing site maps to the base of chromosome arm 2L in the same region occupied by the repeated structural genes of the histone. We have also shown that X-Y pairing is mediated by 240bp intergenic spacer repeats from the rDNA loci on the X and Y. X chromosomal insertions of these repeats can restore pairing capacity to X chromosomes deleted for the native pairing region in the heterochromatin. By contrast, fragments of the rDNA transcription unit without promoters or spacers have no pairing ability. Each of the spacer repeats has a functional copy of the pre-rRNA promoter and we have shown by in vitro mutagenesis and functional testing that disabling the """"""""spacer promoters"""""""" in arrays of these repeats also prevents them from stimulating X-Y pairing. We propose that pairing sites in Drosophila consist of transcribed sequences or the promoters thereof. The proposed experiments involve development of a novel mini- chromosome assay that can be used to test any candidate sequence for pairing capacity. It will be used to test transgenic insertions of histone repeats and a variety of other candidate sequences, including arrays of promoter sequences from both Drosophila and yeast. The idea that transcription is required for pairing will be tested by attempting to """"""""activated"""""""" sequences that lack autonomous pairing capacity by transcription from heterologous promoters. The possibility that pairing involves formation of joint molecules will be investigated by assessing sensitivity of pairing to mismatches between potential partners, with special attention to whether mismatches upstream or downstream of the initiation site are especially disruptive. This latter test is aimed at discovering evidence for or against direct involvement of nascent transcripts in the pairing process. We will also search for sites outside the promoter region of the 240bp spacer repeats that are essential for pairing function of the repeats.
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