Reordering of nine exons is necessary to form a functional actin gene in Oxytricha nova.
During the development of a macronucleus from a micronucleus after cell mating in hypotrichs all the genes (approximately 20,000) are excised from micronuclear chromosomes as individual small DNA molecules. Telomeres are added to the ends of each gene-sized molecule and each is amplified, mostly by approximately 1000-fold, to yield a transcriptionally active macronucleus. As a part of the study of the excision of genes from chromosomes, we have cloned six fragments of chromosomal DNA from Oxytricha nova, each containing a full copy of an actin gene, for comparison with the structure of the actin-encoding DNA molecule in the macronucleus. All six micronuclear actin clones had the same overall organization as judged by restriction mapping. Two micronuclear actin clones were sequenced. These differ from one another at a few nucleotide positions but both prescribe precisely the same actin polypeptide. Both micronuclear actin genes contain nine exons separated by eight intron-like sequences. The macronuclear gene contains these nine exons without intron-like segments. Assigning the order 1 through 9 to the nine micronuclear exons, the order in the macronucleus is 8-7-1-2-4-3-5-9-6. In the micronuclear actin gene, all nine exons possess terminal repeat sequences. These repeat sequences provide precise directions for reordering and joining of the nine exons to yield the exon order in the macronuclear gene. Polymerase chain reaction analysis of micronuclear DNA of the related species, Oxytricha trifallax, shows that the actin gene has an unorthodox arrangement in this species also.
Greslin
A F
AF
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309.
Prescott
D M
DM
Oka
Y
Y
Loukin
S H
SH
Chappell
J C
JC
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016798:Ciliophora / Q000235:genetics*; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase; D005091:Exons; D005784:Gene Amplification; D005796:Genes; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012091:Repetitive Sequences, Nucleic Acid
D000199:Actins; D004259:DNA-Directed DNA Polymerase