In the hypotrichous ciliate Oxytricha nova the cloned precursor gene from the micronuclear genome that encodes actin I is composed of highly disordered blocks of deoxynucleotide sequences. We present and illustrate in detail a recombination model that explains how the actin I gene may be unscrambled during macronuclear development after cell mating. The model was described in a previous publication (Greslin et al.: Proc Natl Acad Sci USA 86:6264-6268, 1989). Here we show the data, described in the earlier publication, that support the model. The data show that scrambling is not an artifact of cloning. They rule against the presence of an unscrambled copy of the actin I gene in the micronucleus, which means that unscrambling must be a part of macronuclear development. Finally, the data prove that the actin I gene in O. trifallax is scrambled in a pattern that resembles the pattern in O. nova.

All of the genes in the macronucleus of Oxytricha sp. occur on physically separate "gene-sized" DNA molecules. We have inserted the DNA molecule that codes for rRNA into a bacterial plasmid in order to study its structure and function. Using restriction nuclease mapping and hybridization of 125I-rRNAs to gel separated DNA fragments we have determined that the intact rDNA is 8,140+/-50 base pairs (bp) in length. Reading from one end, the molecule consists of approximately 1,540 bp of non-coding DNA, approximately 2,100+/-50 bp that code for 19S rRNA, approximately 3,700+/-50 bp that code for 25 rTNA, and approximately 620+/-50 bp of non-coding DNDA. The 5.8S rRNA coding sequence (approximately 150 bp) occurs at one end of the 25S RNA coding region but which end is not known yet. All three rRNAs are encoded in the same strand of the DNA molecule, and transcription is in the order: 19S leads to 25S.

During the development of a macronucleus from a micronucleus after cell mating in hypotrichs all the genes (=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 ampliflied, mostly by %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 actinencoding 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.The genetic apparatus of hypotrichous ciliates consists of two morphologically and functionally different nuclei, the micronucleus and the macronucleus. The micronucleus contains high molecular weight DNA organized into chromosomes that divide mitotically and undergo meiosis during the sexual phase of the organism's life cycle (1). In contrast, the macronucleus contains only low molecular weight DNA molecules, ranging in size from =500 base pairs (bp) to 15 kilobase pairs (kbp) with a number average size of 2200 bp (2). There are -20,000 different molecules, and each appears to contain only one transcription unit or gene-encoding region.Each of the 20,000 molecules is present, on average, in =1000 copies (3). Transcription is very active in the macronucleus but is not detectable in the micronucleus. The micronucleus is not essential for vegetative growth in at least some species of hypotrichs, including Oxytricha nova.The micronucleus functions as a germ-line nucleus. When two cells mate, the micronucleus divides meiotically, and haploid micronuclei are exchanged between the paired cells. An exchanged nucleus fuses with a stationary haploid micronucleus to form a new diploid zygotic micronucleus. Shortly thereafter the old macronuclei and the remaining haploid micronuclei are destroyed. After conjugation, the new diploid micronucleus divides mitotically without cell division. One of the two new micronuclei develops to a new macronucleus.Our long-term objective ...

We have cloned and sequenced a 1.6-kb macronuclear molecule encoding actin from the hypotrichous ciliate Oxytricha nova. High-stringency Southern hybridization to native and digested macronuclear DNA shows that there is only one 1.6-kb actin-encoding molecule in O. nova. The 227-nucleotide 5' leader sequence contains AT-rich stretches punctuated by short GC regions. The AT-rich regions contain TATA-like sequences. However, other known eukaryote transcription regulatory sequences were not found. The 249-nucleotide 3' trailer sequence is also AT-rich and does not contain any obvious known eukaryotic mRNA processing signals. Sequence comparison with a closely related species, O. fallax, shows an 87% sequence similarity in the coding regions and an almost total lack of similarity in the noncoding regions of the molecules. However, a few small sequence similarities and motifs appear in the noncoding regions of the actin-encoding molecules of these two species. The actin-encoding molecule of O. nova could encode a polypeptide 374 amino acids long, the same size as some vertebrate cytoplasmic actins. Contrary to a previous report, we show that the actin-encoding molecule of O. fallax also codes for a polypeptide 374 amino acids long.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.