Cap 7

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 “higher” 
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 “chromosomal” structures 
during “mitosis-like” 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 “higher” 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 “chromosomes” 
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 , “macronuclear” 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