Pré-visualização17 páginas
and a posterior zone in which the DNA is replicated (Lin & Prescott, 1985; Olins & Olins; Raikov, 1982, 1996). A similar substructure has been observed in the oligotrich Strombidium (Salvano, 1975) and in other oligotrichs and chore- otrichs (Laval-Peuto, 1994; Laval-Peuto et al., 1994). Our recent assignments to the spirotrichs also demonstrate replication bands : certainly in Licnophora (Da Silva Neto, 1994a; Villeneuve- Brachon, 1940) and probably in Plagiotoma (Dworakowska, 1966). Thus, replication bands are characteristic of what one might call the \u201chigher\u201d subclasses of spirotrichs , but to our knowledge, they have not been observed in the macronu- clei of Phacodinium (Subclass Phacodiniidia ) and Protocruzia (Subclass Protocruziidia ). Spirotrich macronuclear division very likely involves par- ticipation of intramacronuclear microtubules , as observed for the stichotrichs Gastrostyla and Stylonychia (Walker & Goode, 1976). However, we do not yet have any details on the ultrastruc- ture of the unusual \u201cchromosomal\u201d structures during \u201cmitosis-like\u201d macronuclear division of Protocruzia (Ammermann, 1968; Ruthmann & Hauser, 1974). Micronuclear division involves participation of intramicronuclear microtubules (Walker, 1976b). The chromosome-like structures of Protocruzia , which may be the basal taxon of the spirotrich clade (Bernhard et al., 2001; Shin et al., 2000), are remi- niscent of the polytene chromosomes that appear during the development of the \u201chigher\u201d spirot- rich macronuclear anlage following conjugation (Alonso, 1978; Ammermann, 1971; Ammermann et al., 1974; Jareño, 1976; Kuhlmann & Heckmann, 1991; Prescott, 1994). Following amplification of the DNA in the polytene chromosomes , DNA is eliminated and then followed by a further ampli- fication to complete development of the mature macronucleus (Ammermann et al., 1974; Jahn & 7.6 Nuclei, Sexuality and Life Cycle 169 170 7. Subphylum 2. INTRAMACRONUCLEATA: Class 1. SPIROTRICHEA Klobutcher, 2002; Prescott, Murti, & Bostock, 1973). The mature macronucleus is composed of gene-sized pieces, 0.5-25 kb in size, a fact that has been confirmed in several stichotrich gen- era, Halteria , and the hypotrich Euplotes (Lawn, Heumann, Herrick, & Prescott, 1978; Prescott et al., 1973; Riley & Katz, 2001; Steinbrück, 1990). Typically, these macronuclear \u201cchromosomes\u201d contain a single gene, although two-gene chromo- somes have been described (McFarland, Chang, Kuo, & Landweber, 2006). Different gene-sized pieces may be differentially amplified and their copy number may be controlled through the veg- etative cell cycles of the hypotrichs and stichotrichs (Baird & Klobutcher, 1991; Steinbrück, 1983). Creation of these gene-sized pieces by processing the macronuclear chromosomes generates liter- ally thousands of chromosome ends or telomeres to which the telomeric sequence CCCCAAAA is added (Klobutcher, Swanton, Donini, & Prescott, 1981). Telomerases are the enzymes responsible for addition of these sequences (Blackburn, 1992; Greider & Blackburn, 1987), and telomerase tran- scripts are tightly regulated during macronuclear development (Price, Adams, & Vermeesch, 1994; Shippen-Lentz & Blackburn, 1989). The telomeric sequences of hypotrichs and stichotrichs have dif- ferent numbers of GT/CA repeats and show some differences in primary sequence (Prescott, 1994; Steinbrück, 1990). Since much of the amplified micronuclear DNA in the polytene chromosomes is eliminated during anlage development, this raised the question of what molecular signals were used to recognize the non-genic eliminated sequences. It is now clear that transposon-like elements dis- tributed throughout the genome are the first sets of sequences to be eliminated in hypotrichs and sti- chotrichs (Baird, Fino, Tausta, & Klobutcher, 1989; Herrick et al., 1985; Jahn, Kirkau, & Shyman, 1989; Prescott, 1994). These may have originated from the invasion of the hypotrich / stichotrich micronuclear genome by transposons that origi- nally populated the micronuclear genome but that now are excised by host-directed mechanisms (Klobutcher & Herrick, 1997). The chromosome fragmentation process appears to use unique sites in Euplotes but multiple, closely spaced sites in Oxytricha (Baird & Klobutcher, 1989). Not only are sequences eliminated between the ends of micronuclear genes , but sequences are also elimi- nated within micronuclear genes leading to what are called internally eliminated sequences or IESs and macronuclear destined sequences or MDSs (Klobutcher, Jahn, & Prescott, 1984; Prescott, 1994, 1998). The macronuclear destined sequences are then ligated to construct the functional genes. The story becomes even more bizarre: the macro- nuclear destined sequences in some genes are actually scrambled so that their linear order in the micronuclear genome would not yield a functional gene if ligation occurred simply by joining the cut ends (Greslin, Prescott, Oka, Loukin, & Chappell, 1989). Genes have now been discovered with up to 48 scrambled, macronuclear destined sequences , stimulating intriguing explanations as to how the spirotrichs have solved the complex computa- tional problem of assembling a functional gene (Landweber & Kari, 1999; Landweber, Kuo, & Curtis, 2000; Prescott, 1999). Internally eliminated sequences were apparently added successively into genes, first without scrambling . Scrambling occurred later likely by recombination pathways that gave rise to divergent arrangements in the descendant lineages (Hogan, Hewitt, Orr, Prescott, & Müller, 2001; Wong & Landweber, 2006). A final unusual aspect to the stichotrich genome is the change in the universal genetic code with devi- ations from the universal stop codons - UAA, UGA, and UAG. Helftenbein (1985) demonstrated that tubulin genes of Stylonychia use the universal UAA stop codon to code for the amino acid glutamine. There are UAA and UAG internal codons in a puta- tive Oxytricha gene (Herrick, Hunter, Williams, & Kotter, 1987), a feature that presumably evolved independently in Tetrahymena and Paramecium (see Chapter 15 ). In contrast, the hypotrich Euplotes continues to use UAA as the stop codon (Harper & Jahn, 1989; Miceli, La Terza, & Melli, 1989) and codes cysteine using UGA (Meyer et al., 1991). We do not know how often codon deviations have occurred during the evolution of the spirotrichs. Do the oligotrichs and choreotrichs have a codon useage similar to hypotrichs or stichotrichs ? The most plausible explanation for these deviations in the spirotrichs , and other ciliates, is the evolution of translational release factors with a higher spe- cificity for one or other of the universal stop codons (Caron, 1990). There is now evidence, in both hypotrichs and stichotrichs , of coevolution between these genetic code changes and the recognition site of eukaryotic release factor 1 , which is the protein that recognizes stop codons and terminates transla- tion (Inagaki & Doolittle, 2001; Lozupone, Knight, & Landweber, 2001). Micronuclear genome organization has been the subject of several studies. The micronuclear genome shows considerably more sequence com- plexity than the macronuclear genome that is derived from it. In both hypotrichs and stichot- richs , \u201cmacronuclear\u201d genes are typically clustered together along the micronuclear chromosomes, and long stretches of eliminated unique sequences are uninterrupted by repetitive sequences (Jahn, Nilles, & Krikau, 1988a; Jahn, Prescott, & Waggener, 1988b; Klobutcher, 1987). Conjugation in spirotrichs is characterized as tem- porary. In rare instances, total conjugation , that is the fusion of both conjugants, has been recorded in tin- tinnids (Gold, 1971) and