The broad objective is to understand how organims can manipulate their DNA. The model system is hypotrichous ciliated protozoa, which manipulate their DNA extensively during development of the macronucleus from a micronucleus after cell mating. During macornuclear development all genes are excised from the chromosomes and exist in the mature macronucleus as 20,000 different and separate DNA molecules ranging in size from 400 pairs (bp) to 12,000 bp. An example of cutting and joining of DNA is the chromosomal actin gene, which contains nine exons in the order 1- 2-4-5-6-7-8-9 separated by eight intronlike sequences. The intronlike sequences are eliminated and the nine exons are rearranged and joined in the order 8-7-1-2-4-3-5-9-6. Reordering and removal of intronlike sequences is necessary to yield a functional actin gene. The specific aims are: (1) to identify other genes in which removal of intronlike sequences is accompanied by reordering of exons; (2) to track the temporal order of steps in removal of intronlike sequences and joining of exons in the micronuclear acting gene, followed by telomere addition, during maconuclear development using primarily the polymerase chain reaction (PCR) technique.
