Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.

The organization of macronuclear rDNA molecules of four hypotrichous ciliated protozoans.

We have compared the structure of macronuclear DNA molecules that contain rRNA genes of four hypotricous ciliates, Stylonychia pustulata, Euplotes aediculatus, Oxytricha fallax and Oxytricha nova. The macronuclear rDNA, like all macronuclear DNA in hypotrichs, exists as achromosomal molecules of approximately single-gene size. The rDNA molecules have been cloned intact as recombinant plasmids and analyzed by restriction mapping and Southern hybridization. The sites of restriction enzymes BamHI, EcoRI, HindIII, PstI, PvuII and XhoI have similar but not identical patterns in Stylonychia and the two Oxytricha rDNAs. The restriction pattern of Euplotes rDNA is unlike those of the other three, with only one site of seventeen in the same position. Despite this divergence in nucleotide sequence, the overall structure of the rDNA molecules in the four hypotrichs is constant. The size of all the rDNA molecules is the same, 7.49 kb. Also, the positions of the regions coding for 19S and 25S rRNA are alike. The 25S coding region is at the 5' end of the DNA template strand (3' end of the RNA transcript), within 500 base pairs of the terminus of the DNA molecule. The 19S coding region is adjacent to the 25S region with less than 500 base pairs of spacer lying between the two genes. The largest non-coding sequence is at the 3' end of the DNA molecule adjoining the 19S RNA gene. The 3' non-coding regions show greater sequence divergence among the different rDNAs than do the coding regions. The similarity in size and organization of these molecules and the variability in the restriction patterns suggest that the gene structure is under tighter evolutionary constraint than is the primary nucleotide sequence.

Gene-sized DNA molecules of the macronuclei in three species of hypotrichs: size distributions and absence of nicks. DNA of ciliated protozoa. VIII.

Macronuclear DNA s from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from approximately 400 bp to approximately 20,000 bp. By EM, the number average molecular sizes for double-stranded DNA were 2,200 bp for Oxytricha sp., 2,514 bp for Stylonychia pustulata and 18,836 bp for Euplotes aediculatus. Contrary to previous reports we present evidence that the macronuclear DNA s in each of these three organisms lack single-stranded interruptions.

Arrangement of coding and non-coding sequences in the DNA molecules coding for rRNAs in Oxytricha sp. DNA of ciliated protozoa. VII.

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.

All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of approximately 2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ... In Euplotes aediculatus, the sequences is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A-A-C-T-T-T-T-G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region: 5'-C4A4C4A4C4A4C4(X)17T-T-G-A-A ... 3'-G2T4G4T4G4T4G4T4G4T4G4(X)17A-A-C-T-T ... The length of the terminal repeat sequence for O. nova was confirmed in cloned macronuclear DNA molecules.

Sequence of the small subunit ribosomal RNA gene from the hypotrichous ciliate Euplotes aediculatus.

We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.