A Micronuclear Locus Containing Three Protein-Coding Genes Remains Linked During Macronuclear Development in the Spirotrichous Ciliate Holosticha

Abstract

We have discovered a three-gene macronuclear chromosome in a spirotrichous ciliate of the genus Holosticha. From 5' to 3', this chromosome contains genes encoding a member of the small G-protein family, an NAD kinase domain-containing protein, and the large subunit of DNA polymerase alpha. These three genes are separated by 16 and 38 nucleotides, respectively, and are oriented in the same direction in both the macronuclear and the micronuclear genomes. Probes made to these genes all hybridize to a single, strong band of size 7.0 kbp on a Southern Blot of Holosticha sp. macronuclear DNA, corresponding to the size of the three-gene macronuclear chromosome. Mapping the 5' and 3' ends of each of these genes using RACE showed that the transcripts of these genes exist as discrete mRNAs that are capped and polyadenylated. No nucleotides appeared to be added at the 5' ends of these transcripts, indicating that these transcripts are not generated by alternative or trans-splicing, but rather that each gene is transcribed from its own distinct promoter. Analysis of these linked genes may help define the evolutionary pressures leading to the extensive chromosome fragmentation seen in spirotrichous ciliates.