We propose to continue our analysis of the organization of genes and other sequences in chromosomes of hypotrichous ciliates and the extensive changes, including especially excision of all genes from chromosomes and destruction of most of the sequences of the genome, that occur when a macronucleus is produced from a micronucleus after cell mating. The work consists of 8 projects. (1) Cloning of DNA intermediates during genome processing to determine the pathway of processing, time of removal of intron-like sequences from genes, time of addition of telomeres to gene-sized molecules, and sequencing of micronuclear DNA, polytene DNA, and DNA of intermediate fragments to search for sequences that may signal processing. (2) Analysis of the arrangement of genes in micronuclear DNA will be continued. (3) Micronuclear and macronuclear versions of genes will be compared to test how general is the occurrence of removal of intron-like sequences from genes during macronuclear development. This includes a search for sequence commonalities among the intron-like sequences and a study of multiple versions of genes. (4) Macronuclear genes will be analyzed for intron presence. If the intron-like sequences removed during macronuclear development represent introns, macronuclear DNA may totally lack introns. (5) Macronuclear genes will be sequenced to identify commonalities that may give insight about macronuclear gene function. (6) Sequencing genes that flank will be compared among different species to test the degree of sequence instability of these """"""""non-essential"""""""" regions. Sequences of macronuclear rDNA molecules of different species will be compared to measure the degree of constraint on sequence divergences on non-transcribed, transcribed non-coding, and coding regions of the gene-sized molecules. (7) We will determine whether regulation of expression of the gene for heat shock 70 protein is transcriptional or translational as part of the question of transcriptional control in hypotrichs. (8) The structure of micronuclear chromosomal telomeres will be determined through cloning and the protein(s) that binds to the telomeres of macronuclear gene-sized molecules characterized.
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