Stichotrich molecular biology

contributed by Glenn Herrick, University of Utah, Salt Lake City, UT, USA

History. Spirotrichs have been studied since the 1800s, the old literature on life history insightfully re-synthesized by Bell (1988). The present era of molecular genetics of Spirotrichs (review: Klobutcher and Prescott 1986) was opened by Dieter Ammermann's doctoral research, when he demonstrated polytene chromosomes in the developing macronucleus (MAC) of Stylonychia (Ammermann 1964) and laid out the time-course of cytogenetic events of this spectacular process (Ammermann 1965). His work attracted David Prescott, and he and his lab launched into the molecular biology of MAC development, first demonstrating (Prescott et al. 1971) the dramatic, now often-called "gene-sized" MAC chromosomes (see Hoffman et al. 1995, but also Seegmiller et al. 1997), each with two telomeres (Wesley 1975).

Stichotrichs, phylogeny and taxonomy (see Snoeyenbos-West et al., 2002; Figure 1).
Hypotrichs. In the formative years of stichotrich molecular biology, it focused on the taxon Hypotrichs (sensu lato), including the current clades Stichotrichs (Stylonychia, Oxytricha, Urostyla, Sterkiella, etc.) and Euplotids (now Hypotrichs, sensu stricto). That taxon, "hypotrichs," made molecular sense, given that Euplotids share with the Stichotrichs many features of macronuclear development, including polytenization of the chromosomes of the developing MAC and the subsequent formation of vesicles around polytene bands (Kloetzel 1970), the generation of "gene-sized" MAC chromosomes, and their final amplification by replication bands that also replicate the vegetative MAC. However, modern taxonomy and phylogeny (Figs. 1 & 2) have split off the Euplotids into the early-diverged Hypotrichs, distinct from the Stichotrichs; indeed, molecular details have emerged that support the split: Hypotrichs have a different genetic code from the Stichotrichs and numerous genes using programmed frameshifts (review: Klobutcher and Farabough 2003), and carrying Internal Eliminated Sequences (see below) mostly of the "TA" variety. A note of caution: Katz and Riley (2001) argue that the process of generation of "gene-sized" MAC chromosomes has evolved independently in a variety of ciliate lineages; hence, this character is not a valid basis for phylogenetic inference. The shared characters between the Euplotids and the Stichotrichs do however represent a valid clade, the Spirotrichs, which includes both the Hypotrichs and Stichotrichs.
While this web-page focuses on Stichotrichs, to the exclusion of Hypopotrichs and, notably, Euplotes, it should be noted that much of what is understood about Spirotrich MACs and MAC development is the result of the efforts of a talented set of Euplotes workers, nearly all of whom recently have, regrettably, abandoned Euplotes for other endeavors. Farther afield, Spirotrich molecular genetics has grown side-by-side with that of the Oligohymenophorans, Tetrahymena and Paramecium, which sport similar (analogous or homologous?) mechanisms of MAC development, making for a lively and exciting research community (see Gall 1986).
Stichotrichs. Significant confusion reigns regarding Stichotrich taxonomy. On one hand, two of the more prominently-studied stichotrichs, Oxytricha nova and Oxytricha trifallax have rightfully earned the term "nomen nudum" (Foissner and Berger 1999), having been isolated and named without any formal taxonomic description. On the other hand, Foissner and Berger (1999) changed the names of these two ciliates, placing them in the genus Sterkiella (S. nova and S. histriomuscorum, respectively); the effect has been to fragment the literature and destroy the continuity of citations of various published facts about the two.
This line was studied by Hammersmith and the late Gary Grimes (e.g., 1981) for its ability to fold in its spectacular cortical structures (Fig. 3) into viable cysts and recreate them when excysting. This O. fallax line was adopted by Spear’s lab, and then Herrick’s lab, because cryogenic storage of these cysts provided a way to circumvent vegetative senescence that had plagued stichotrich research. Hammersmith in 1985-86 made extensive collections in Indiana of candidate O. fallax lines, most of which would be identified from cortical features as O. fallax, but which proved to be several non-fallax species, by mating and molecular tests (Hammersmith and the Herrick lab, unpubl.), reminiscent of the old problem of Paramecium snygens (Sonneborn 1975). In analogy to the naming of those syngens, one of the O. fallax look-alike species was named Oxytricha trifallax (see Williams et al. 1993; Seegmiller et al. 1996). Thanks to their facile encystment, Hammersmith's initial collection of 32 isolates, isolates of 142 related stichotrichs, and numerous O. trifallax offspring lines are being permanently stored in liquid nitrogen in Salt Lake City. Hopefully, the ATTC will take over the maintenance of this collection.
Reference O. trifallax samples of interfertile lines were distributed widely to other stichotrich workers, so that we all might work on the same species, and so that any new isolate could be tested for fertility and molecular similarity to them. Thus, Adl and Berger (1997, 2000), as well as the Herrick lab, have described these lines. Other labs have isolated stichotrichs and named them O. trifallax, but have not proven them to be of this species by breeding and molecular tests. However, these authors have been conscientious to identify the isolates used in each study, allowing results on the same organism to be pooled: the Prescott group has characterized "unscrambling" (see below) in the WR strain; and the Baroin-Tourancheau group have studied excystment in strain BA of S. histriomuscorum (Villalobo et al. 2001, 2002).
Clearly, it is imperative that data from different labs about the same putative species should be pool-able. Given the ease of PCR and sequencing, as well as of performing mating tests for fertility, we hope that future work will be focused on a manageable set of stichotrichs, instructed by these tools.

Life cycle, nuclear dimorphism. Stichotrichs, like all ciliates, are eukaryotic microbes that employ two different nuclei in each cell, the germline nucleus or micronucleus (MIC), and the somatic macronucleus (MAC), a specialized gene expression organelle. Following pairing, each conjugant’s MIC undergoes meiosis (see Figure 4 for a mystifying meiotic figure in O. trifallax), and generates haploid gametic nuclei from post-meiotic mitosis; cross-fertilization generates a new zygotic diploid nucleus in each conjugant, which then divides mitotically, generating identical anlagen of the new MIC and MAC of each exconjugant. Once MAC development is complete, the cell proliferates by binary fission into a clone of cells (Figure 4): the MIC replicates mitotically, bearing conventional large linear eukaryotic chromosomes, with conventional telomeres (Dawson and Herrick 1984); in another part of the cell cycle the macronucleus replicates by means of replication bands that pass through it, and the contents are randomly mixed and distributed to the two daughter nuclei amitotically; thus there is no conventional copy-control mechanism, although most feel that the copy numbers of MAC chromosomes are regulated. The processes of vegetative proliferation are controlled solely by the small fraction (as little as 5%) of germline sequences that survive MAC development, and the MIC genome is not transcribed in vegetative cells (review: Herrick 1994).
MAC development (Review: Jahn and Klobutcher 2002). Over the course of ~3 days the MAC develops from its anlage. Cytogenetic stages were described by Ammermann (1965).
Initially the chromosomes of the anlage are endo-reduplicated, evidenced by visible polyteny in a variety of ciliates; the ploidy can reach ~64 (Ammermann et al. 1974; Klobutcher and Prescott 1986). Thus, each sequence that survives subsequent elimination (MAC-destined) is represented by numerous polytene chromatids; in principle, each could be processed differently. In the late polytene-stage anlage, many transposons and short sequences are eliminated by precise excision (Internally Eliminated Sequences, or IESs); short IESs are believed to have evolved from long transposon insertions that have shrunk, retaining only minimal terminal cis-acting sequences necessary for excision of the IES (Klobutcher and Herrick 1997). Excision is immediately followed by massive chromatid fragmentation and de novo telomere formation ("chromosome healing," Melek and Shippen 1996). "Spacer DNAs" are cut away from MAC-destined telomere addition sites. Once cut from MAC-destined sequences, IESs and spacer DNAs are degraded, apparently within vesicles that form around polytene bands (). The surviving sequences reside on many small MAC chromosomes, which are then highly amplified to the level of the mature MAC, by action of multiple passes of replication bands across the maturing nucleus, possibly adjusting copy numbers of chromosomes.
IES excision has been studied in two contexts, as conventional deletion intervals, and as part of the process of unscrambling "scrambled genes." Unscrambling rearranges MAC-destined segments that do not lie in the same order, nor orientation, in MIC DNA as they do in the mature MAC chromosome (review: Prescott 2000); Prescott and Rozenberg (2003) present an innovative model for unscrambling, in which the unscrambled DNA of the parental MAC provides a template or gig to align the MAC-destined segments, prior to recombination.
Following chromatid cleavage, single telomere addition sites are used at some loci, but are scattered across regions of other loci; such heterogeneity of telomere addition site use is often attributed to exonucleolytic erosion of the raw chromatid end prior to telomerase action; implicit is that breakage and telomerase action are not coupled (Williams et al. 2002).
The mature Oxytricha MAC genome is typical of those of other Spirotrichs (Figure 5). It consists of ~1000 copies of each of ~20,000 different tiny, acentric linear chromosomes. They range in length from ~0.5 kbp to >15 kbp (average ~2.4 kbp). Those studied are comprised almost exclusively of transcription units. Many carry only one gene (Hoffman et al. 1995), but some carry two or three genes each; for example two chromosomes generated from the 81-MAC locus by alternative processing of chromatids bear two genes each (Figure 6; Seegmiller et al. 1997).

New directions. The entry of Laura Landweber’s lab into Stichotrichs marks the beginning of a new era, pulling together the excitement of unscrambling and technical innovations from the Lipps lab.
Molecular genetics tools for Stichotrichs. Lipps' group has pioneered in Stichotrichs a microinjection/transformation procedure that has allowed them to test the function of DNA motifs during Stylonychia MAC development (Wen et al. 1995; Jönsson et al. 2001). RNAi has been harnessed for Euplotes (Möllenbeck et al. 2003), and adapted to Stylonychia as well (H. Lipps, pers. comm.).
Genomics. Toward sequencing a stichotrich MAC genome, a pilot project was performed on O. trifallax, in collaboration between the Doak and Herrick lab, and R. Weiss and D. Dunn of the Utah Genome Center. Sequences were analyzed with the assistance of A. Cavalcanti in Landweber’s lab. This data set aided the submission of two white papers to NHGRI: both have now been given high priority. The first project is to generate a BAC library of MIC DNA (white paper: <http://www.genome.gov/page.cfm?pageID=10001852>); this is in progress in the lab of Pieter de Jong. The second and main project is to sequence the complete MAC genome and at least 50 mbp of the MIC (white paper: <http://genome.gov/page.cfm?pageID=10002154>). In the MIC sequence, we hope to sequence as much MAC destined sequence as possible. Sequencing will be performed by the Whitehead sequencing center. The approach to the MAC sequencing is presently being decided; the MAC genome is non-conventionally structured and doesn’t fit well into current procedures designed for genomes of large mitotic chromosomes (primarily shotgun cloning and sequencing). Sequencing is expected to start in early 2004.



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Web sites:
Herrick lab "poster" <http://www.biology.utah.edu/posters2.php?id=23&area=home>
Landweber lab <http://www.princeton.edu/~lfl/>


Resources:
Stichotrich molecular biology - PPT, 3.6 Mb.