Stichotrich molecular biology
contributed by Glenn Herrick, University of Utah, Salt Lake City, UT, USA
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
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;
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,
for other endeavors. Farther afield, Spirotrich molecular
genetics has grown side-by-side with that of the Oligohymenophorans, Tetrahymena
, which sport similar (analogous or homologous?) mechanisms
of MAC development, making for a lively and exciting research community (see
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
(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
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 Spears lab, and then Herricks 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
species was named Oxytricha trifallax
(see Williams et al
Seegmiller et al. 1996). Thanks to their facile encystment, Hammersmith's initial
collection of 32 isolates, isolates of 142 related stichotrichs, and numerous
offspring lines are being permanently stored in liquid nitrogen
in Salt Lake City. Hopefully, the ATTC will take over the maintenance of this
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
, 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 conjugants 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
Initially the chromosomes of the anlage are endo-reduplicated, evidenced by
visible polyteny in a variety of ciliates; the ploidy can reach ~64 (Ammermann
. 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
-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
The entry of Laura Landwebers 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,
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 Landwebers 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 doesnt 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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Herrick lab "poster" <http://www.biology.utah.edu/posters2.php?id=23&area=home
Landweber lab <http://www.princeton.edu/~lfl/
- PPT, 3.6 Mb.