9723970 Goodenough Like most sexual eukaryotes, the green alga Chlamydomonas reinhardtii transmits its organelle DNA to its sexual progeny in a uniparental fashion, with all 4 products of meiosis inheriting the chloroplast DNA contributed by the mating-type plus (mt+) parent and all 4 inheriting the mitochondrial DNA contributed by the mating-type minus (mt-) parent. These non-Mendelian patterns, first noticed by Sager over 40 years ago, have been shown to be the result of the selective degradation of minus chloroplast genomes and plus mitochondrial genomes in the early zygote, but nothing is yet known about how the selection or the destruction process occurs at a molecular level in this or any other system. Most studies have focused on the uniparental inheritance of chloroplast DNA. The dominant Protector/Destroyer model for the operation of this system proposes that the mt+ locus encodes Protector(s) which binds to and/or modifies chloroplast DNA in the plus gamete. A Destroyer function then degrades all non-protected, minus-derived genomes in the zygote. Key to this model is the observation that when mt- gametes also carry an mt+ locus, they differentiate as minus gametes, but, when they are mated, their chloroplast DNA is not degraded in the zygote, the result being uniparental (BP) inheritance of chloroplast markers. This observation indicates that the mt+ locus encodes gene(s) involved in protecting chloroplast DNA from degradation, and documents that these genes can be expressed even when the cells undergo minus gametic differentiation.. We have recently cloned the mt+ (and the mt-) locus and have, we believe, identified the one gene in the locus that is necessary for plus gametes to mate. There remain blocks of DNA with no identified function. The working premise of this proposal is that these sequences contain genes involved in chloroplast DNA protection. The goal of the proposed research is to identify and characterize these genes and hence better understand how protection i s achieved. We propose a two-stage screen. 1) Plus-specific genes in the cloned mt+ sequences are first identified by northern analysis. 2) The genes are then transformed into mt+ recipients, and the transformants are tested for their capacity to engage in BP inheritance. The first state of this screen has been carried out successfully in pilot experiments. A mt+- linked gene with plus-specific expression has been identified in northern blots and its cDNA has been sequenced. The gene product has features several suggesting that it is a transcriptional regulator. Animal and fungal lineage's have devised uncharacterized mechanisms to monitor their mitochondrial genomes; plant lineage's keep track of both mitochondrial and chloroplast chromosomes. During the first few hours of zygote development formed by the fusion of gametes of plus and minus mating type cells of Chlamydomonas, the chloroplast genomes contributed by the mt- parent are destroyed and the mitochondria genomes contributed by the mt+ are destroyed. The results of this work will allow greater insight into this remarkable process of genetic discrimination.
