Stomatogenesis and Formation of Cirri in Fragments of Oxytricha fallax Stein

Of fragments involving a partial removal of the original adoral zone of membranelles (AZM), the monomacro-nucleate ones become reorganized monostomes resulting from a simple fusion of the remnant AZM to the oral primordium induced, and the binucleate ones become dividers by initiation of the oral primordium posteriorly from the posterior terminal of the remnant AZM. The cirral primordium in any fragment arises alongside its corresponding oral area. Weisz's idea of the dominance and inhibition of the original oral system extending over the oral primordium site is applicable in stomatogenesis of the present species. This application is found also in cirral formation.
In fragments from early stage dividers, a formed oral primordium is easily absorbed by influence of the intact original AZM. This event also occurs after complete removal of the AZM. Such results led to the hypothesis that the oral primordium in the normal divider may be formed under some stoma-togenic activation of the AZM followed by escape from inhibition also arising from the same source. Irrevocable furrow formation and irreversibility of the oral primordium in stomatogenesis occur in later stages of division. Nevertheless division in these stages is blocked when certain operations are performed, forming monsters possessing the AZM of the opisthe translocated to the side opposite to that of the proter. In other monsters obtained from a fusion of the AZM of the proter to that of the opisthe, division occurs belatedly, prior to which secondary oral and cirral primordia are produced.     

Differentiation During Encystment and Excystment in Oxytricha fallax*

During encystment of Oxytricha fallax, a wall composed of 4 distinct layers, each derived from a different kind of endoplasmic vesicle, is formed between the 2 unit membranes that cover the vegetative cell. Numerous autophagic vacuoles arise in the endoplasm and later (during excystment undergo internal changes comparable to those characteristic of food vacuoles. Mitochondria aggregate into a band. The 2 macronuclei fuse and their nucleoli become homogeneous. Except for the 2 cell membranes, all visible cortical structures, including cilia, kinetosomes, and microtubules, disappear. Despite the absence of visible ciliature in the mature cyst, the various primordia of the normal vegetative ciliature arise during excystment in the same positional relations to one another as is characteristic of developments during cell division.     

Isolation and mapping of the rRNA genes in the macronucleus of Oxytricha fallax

The DNA in the macronucleus of the protozoan Oxytricha, unlike like that of typical eukaryotes, exists as short, gene-sized molecules. Within the macronucleus the rRNA genes are contained in molecules 7,380 nucleotide pairs in length. This rDNA has been substanially purified by selective denaturation of non-ribosomal DNA followed by Sl nuclease digestion. Results from restriction nuclease digestion and rRNA:DNA hybridization show that the rDNA is a linear, non-palindromic molecule which contains one gene each for the 19s and 25s rRNAs. A total of less than 600 base pairs of DNA lies between the 19s and 25s genes or at the 3 end of the 25s gene. The non-coding portion of the ribosomal DNA is almost entirely limited to an approximately 1,400 base pair region at the 5 end of the molecule.     

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.     

Multiple Sequence Versions of the Oxytricha fallax 81-MAC Alternate Processing Family1

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclcar homologs have been classified according to DNA sequence into three highly homologous (95.9–97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclcar chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclcar DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be “macronuclear-homologous” but “micronucleus-limited” and not “macronucleusdestined.” Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidai senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidai life to mairitain 1) a constant absolute amount of 81-MAC sequences in the macronuclcus and 2) a constant sioichiometry within the family, both according to version and chromosome size.     

Three different macronuclear DNAs in Oxytricha fallax share a common sequence block.

The three members of a cross-hybridizing family of macronuclear DNAs (4,890, 2,780, and 1,640 base pairs) from the protozoan Oxytricha fallax have in common a conserved sequence block 1,300 to 1,550 base pairs long. Adjacent to the common block in the two larger DNAs are sequences which are unique to them, whereas the smallest DNA contains few if any additional sequences. The family reappears when the macronucleus is replaced after conjugation and can be detected in another O. fallax subspecies. In a random collection of cloned macronuclear DNAs, 6 of 15 hybridize to macronuclear DNA families. This high frequency suggests that families sharing common sequence blocks have an important role in macronuclear function.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

Micronuclear DNA sequences of Oxytricha fallax homologous to the macronuclear inverted terminal repeat.

The macronucleus of the protozoan Oxytricha fallax is generated from a micronucleus following conjugation. While the micronucleus contains high molecular weight DNA, the macronucleus contains only short linear DNA molecules which all end in the same 20 bp inverted terminal repeat (Ma-ITR). The Ma-ITR was radioactively labeled and purified for use as a probe in hybridizations to micronuclear and macronuclear DNA. Sequences homologous to the Ma-ITR were detected in micronuclear DNA. The copy number of the repeat in the micronuclear genome is approximately that required to encode the macronuclear DNA termini. The micronuclear copies are found embedded in repeated long sequence blocks.     

The Structure and Development of the Dorsal Bristle Complex of Oxytricha fallax and Stylonychia pustulata*

SYNOPSIS. Oxytricha fallax and Stylonychia pustulata possess 6 rows of dorsal bristle units. Each dorsal bristle unit consists of a pair of kinetosomes; the anterior kinetosome has a cilium and the posterior kinetosome a ciliary stub. The kinetosome pair, located at the bottom of a cortical pit surrounding the cilium and ciliary stub, is surrounded by an asymmetrical fibrillar mass. Future rows 1-4 are formed from 2 sets of primordia originating within mature dorsal rows 1-3. Rows 5 and 6 originate from the anterior regions of both right marginal cirral primordia. Old dorsal bristle units utilized in formation of primordia are presumably maintained in the new rows of the proter and opisthe; those outside the primordia are resorbed. The morphogenetic pattern of the Oxytrichidae is similar to those of the Urostylidae and Holostichidae, but quite different from that of the Euplotidae.     

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.     

Telomere formation on macronuclear chromosomes of Oxytricha trifallax and O. fallax : alternatively processed regions have multiple telomere addition sites

Background  

Ciliates employ massive chromatid breakage and de novo telomere formation during generation of the somatic macronucleus. Positions flanking the 81-MAC locus are reproducibly cut. But those flanking the Common Region are proposed to often escape cutting, generating three nested macronuclear chromosomes, two retaining "arms" still appended to the Common Region. Arm-distal positions must differ (in cis ) from the Common Region flanks.     

Two two-gene macronuclear chromosomes of the hypotrichous ciliates Oxytricha fallax and O. trifallax generated by alternative processing of the 81 locus

We describe the first known macronuclear chromosomes that carry more than one gene in hypotrichous ciliated protozoa. These 4.9- and 2.8-kbp chromosomes each consist almost exclusively of two protein-coding genes, which are conserved and transcribed. The two chromosomes share a common region that consists of a gene that is a member of the family of mitochondrial solute carrier genes (CR-MSC; [Williams and Herrick (1991): Nucleic Acids Res 19:4717–4724]. Each chromosome also carries another gene appended to its common region: The 4.9-kbp chromosome also carries a gene that encodes a protein that is rich in glutamine and charged amino acids and bears regions of heptad repeats characteristic of coiled-coils. Its function is unknown. The second gene of the 2.8 kbp chromosome is a mitochondrial solute carrier gene (LA-MSC); thus, the 2.8-kbp chromosome consists of two mitochondrial solute carrier paralogs. Phylogenetic analysis indicates that the two genes were duplicated before ciliates diverged from the main eukaryotic lineage and were subsequently juxtaposed. The CR- and LA-MSC genes are each interrupted by three introns. The introns are not in homologous positions, suggesting that they may have originated from multiple group II intron transpositions. These chromosomes and their genes are encoded in the Oxytricha germline by the 81 locus. This locus is alternatively processed to generate a nested set of three macronuclear chromosomes, the 4.9- and 2.8-kbp chromosomes and a third (1.6 kbp) which consists almost exclusively of the shared common gene, CR-MSC. Such alternative processing is common in macronuclear development of O. fallax [Cartinhour and Herrick (1984): Mol Cell Biol 4:931–938]. Possible functions for alternative processing are considered; e.g., it may serve to physically link genes to allow co-regulation or co-replication by a common cis-acting sequence. Dev. Genet. 20:348–357, 1997. © 1997 Wiley-Liss, Inc.