Laboratoire de Biologie Cellulaire 4, Université Paris-Sud
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon
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Abstract |
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Introduction |
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The ubiquitination system consists of a multienzyme complex in which the conjugating enzyme (E2 or Ubc) catalyses the covalent transfer of Ub to the lysine residues of the substrate proteins. This ubiquitination step follows the conjugation of Ub to a conserved cysteine residue of the E2 proteins, which is critical for the enzyme conjugation activity. Numerous genes encoding E2 proteins have been identified in plants, animals, and fungi, and they constitute a large family with a broad range of biological functions. For instance, 13 E2s have been characterized in Saccharomyces cerevisiae which act on different sets of substrate proteins (Jentsch 1992a, 1992b
). At the amino acid level, structural conservation of the proteins is only observed in the central domain and especially in the vicinity of the cysteine active site.
Recently, a novel family of genes that encode E2-like proteins lacking the critical cysteine residue have been identified (Sancho et al. 1998
; Xiao et al. 1998
) and designated UEV for Ub-conjugating enzyme variant. The first report (Rothofsky and Lin 1997
) concerned the isolation of a human gene called croc-1 whose product has the capacity to activate the transcription of c-fos. It appears that the biochemical activity of UEV is required for the unique Ub Lys63 chain assembly mediated by UBC13 (Hofmann and Pickart 1999
), and unlike the conventional Lys48 polyubiquitination (Chau et al. 1989
), the UBC13-UEV activity does not target protein degradation (Deng et al. 2000
). In addition, differential expression of human UEV genes in proliferating cells versus differentiating cells (Ma et al. 1998
; Sancho et al. 1998
), in carcinogenic cells (Ma et al. 1998
; Xiao et al. 1998
), and their roles in TRAF6-mediated NF
B kinase activation (Deng et al. 2000
) suggest that UEVs may act as regulators in cellular differentiation and proliferation. UEV homologues in other multicellular organisms (plants, animals, fungi) have been reported; however, the cellular processes in which they participate may be different, as in yeast S. cerevisiae MMS2 has been clearly implicated in the error-free branch of the RAD6 DNA postreplication repair pathway (Broomfield, Chow, and Xiao 1998
; Xiao et al. 1999, 2000
; Ulrich and Jentsch 2000
).
From the observed conservation of this large E2 gene family in multicellular organisms, it is clear that their common unicellular ancestor possessed a similar molecular system. In protists, Ub genes have been isolated in several distant groups (e.g., diplomonads, ciliates, trichomonads) but the Ub-conjugating enzyme family is poorly documented because only four E2 sequences are available: a partial genomic E2 sequence of the parabasalid Trichomonas vaginalis (Keeling et al. 1996
) and a complete E2 macronuclear sequence of, respectively, the ciliate Paramecium tetraurelia (Okano et al. 1996
), the slime mold Dictyostelium discoideum (Clark et al. 1997
), and the trypanosomatid Leishmania major (Ivens et al. 1998
). These four sequences are highly divergent except in the central domain of the molecule. Recently, while searching for dormant mRNA in cysts of the ciliate Sterkiella histriomuscorum, we have reported the isolation of a partial cDNA whose deduced protein is most similar to the C-terminus of a putative UEV from the plant Picea mariana (Baroin-Tourancheau et al. 1999
). The human CROC-1 was also among the top 10 matches in the Blast search. The identification of a UEV gene in a ciliate shows that the inactive variants are not specific to animals, plants, and fungi but originated in unicellular ancestors. It is particularly interesting to identify UEV members in protists with regard to the regulatory role(s) this subfamily has evolved in metazoans, fungi, and plants and especially with regard to the origin and evolution of regulatory genes known to be involved in differentiation and proliferation.
In this study, we characterized the complete macronuclear gene ShUEV and analyzed its expression during the life cycle. A phylogenetic analysis, including a total of 45 E2 sequences, confirms the clustering of ShUEV with the other UEVs. The deep genetic divergence between the different classes of proteins (inactive E2 vs. active E2) suggests an early split in their functional assignment. To investigate further the structural and functional conservation of ShUEV, we have shown that the ciliate protein can substitute for the yeast UEV (MMS2) for its DNA repair function. It thus emphasizes both structural and functional conservation of the UEV family during evolution.
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Materials and Methods |
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Cloning of the Macronuclear Gene
All molecular standard techniques, including RNA extraction, screening of libraries, sequence analysis, Southern and Northern hybridization were described by Sambrook, Fritsch, and Maniatis (1989)
, Baroin-Tourancheau et al. (1999)
, and Villalobo et al. (2001)
.
Genomic Library
Genomic DNA was extracted from vegetative cells isolated after the medium was depleted of food organisms (prestarved cultures) using standard procedures (Sambrook, Fritsch, and Maniatis 1989
). Ten micrograms of DNA was run in a 1% (w/v) agarose gel in TAE (40 mM Tris-acetate, 2 mM EDTA) and DNA fragments in the 0.5- to 1.5-kb size fraction were subsequently electroeluted, phenol-chloroform extracted, and precipitated in ethanol. The pellet was resuspended in 45 µl of 10 mM Tris, 0.1 mM EDTA, pH 8, and treated with 2.5 U Bal31 (USB) at 31°C for 10 min in a 50-µl final volume of 25 mM Tris-HCl, pH 7.5, 50 mM CaCl2, 3 mM NaCl, 50 mM MgCl2. The reaction was stopped by the addition of 50 mM EDTA and precipitated after two extractions with phenol-chloroform. After reparation of the extremities with Klenow enzyme (USB), the molecules were ligated overnight into SmaI-treated pUC18 plasmid (Pharmacia) at 16°C and used to transform competent Escherichia coli JM109. Screening of the library was performed using as a probe the croc-1 cDNA clone previously isolated from a cDNA library of cysts of St. histriomuscorum.
Inverse PCR
Total genomic DNA of St. histriomuscorum was circularized following the conditions previously reported (Villalobo et al. 2001
). A pair of internal outward-facing primers was designed based on the sequences of the genomic clones. The primers, 223 bp apart, were, respectivelyForward: 5'-TCAAGAATGAAATAGTTGCTCAC-3', Reverse: 5'-CATTCCAGTCAGTGAATGAG-3'. An aliquot (about 100 ng) of the circularized DNA was then used as the template in a 50-µl PCR reaction with 5 U of Taq polymerase (Q-BioTaq from Appligene-Quantum) and 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 1 min. The inverse-PCR products were run on a 1% (w/v) agarose gel in TBE (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 8.3) and the fragments of the expected size were cut out of the gel and purified using the QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions. DNA was further cloned in SmaI-treated pUC18 plasmid at 16°C.
Primer Extension
Ten micrograms of total RNA from cysts were reverse transcribed with primers specific to the St. histriomuscorum croc-1 sequence. Reactions were carried out using (-32P)dATP and an unlabelled primer. The sequences of the primers and their location relative to the macronuclear molecule (nucleotide numbers in parentheses) are, respectively5'-TGCAAGTCCATAGGAAACAC-3'(225206) and 5'-CATTCCAGTCAGTGAATGAG-3'(295276). After a 30-min reaction at 50°C with Superscript II reverse transcriptase (GIBCO-BRL), the reaction was inactivated during 15 min at 70°C. RNA was digested for 20 min at 37°C with 5 U RNase H (Amersham). Aliquots of the labeled primer extension products were electrophoresed on a 6% acrylamide sequencing gel. Sequencing reactions were carried out with the (295276) primer mentioned above using the Sequenase Version 2 kit (Amersham-Pharmacia) and were migrated next to the primer extension products.
RT-PCR
Total RNA (3 µg) was reverse transcribed using 3.5 µM random hexamers pdN6 (Amersham) as primers and 200 U M-MLV reverse transcriptase (Eurobio) in a 20-µl reaction containing 1 U of RNasin (Promega). RNA was incubated for 5 min at 65°C prior to the reverse-transcription step. After 50 min at 37°C, the enzyme was inactivated at 95°C for 5 min. The PCR step was subsequently carried out using 1 µl of the RT reaction, a pair of specific primers, and 1.5 U of Taq polymerase in a 25-µl reaction. The sequences of the forward and the reverse primers and their location relative to the macronuclear molecule (nucleotide number in parentheses) are, respectively5'-TTAGATATAACATGGTTGAAT-3'(7696) and 5'-TCAATACATTTCACCATCTGC-3'(579559).
Expression of ShUEV in S. cerevisiae
The expression vector pYES2 (YEp, URA3, Invitrogen) was used to clone ShUEV under the control of the galactose-inducible GAL1 promoter. The dTs of the four internal stop codons within the ShUEV coding sequence (respectively, positioned at 190, 268, 400, 472 nt in the macronuclear sequence) were replaced by dC prior to cloning. These substitutions were obtained through four rounds of amplification-circularization with four distinct pairs of corrected outward-facing primers (186169, 187205; 396379, 397414; 465447, 466483; 229202, 266283) performed on a 503-bp macronuclear amplification product. The last round allows elimination of the 36-bp internal intron from the product. A final amplification with the pair (84157, 579559) was done to give the amplification product free of the 5' end intron. The 34-meric forward oligonucleotide corresponds to nucleotide 84 through 157 of the macronuclear sequence minus the 40-nt intron. It was finally cloned into pYES2 at the EcoRI site and the construct was confirmed by sequencing.
Saccharomyces cerevisiae strain FY86 (MAThis3-
200 ura352 leu2-
1 GAL+) and its mms2::LEU2 null mutant FY86m2L were created and cultured as previously described (Xiao et al. 1998
). Yeast cells were transformed with pYES-ShUEV by a lithium acetate method (Ito et al. 1983
). For the gradient plate assay, 30 ml of molten YPD or YPGal agar were mixed with the appropriate concentration of methyl methanesulfonate (MMS) to form the bottom layer; the gradient was created by pouring the media into tilted square Petri dishes. After brief solidification, the Petri dish was returned flat and 30 ml of the same molten agar without MMS was poured to form the top layer. A 0.1-ml sample was taken from an overnight culture, mixed with 0.9 ml of molten 1% agar and immediately imprinted onto freshly made gradient plates via a microscope slide. Gradients plates were incubated at 30°C for the time indicated before taking photographs.
Construction of the Phylogenetic Trees
Management, formatting of the sequences for the tree building programs, and construction of distance trees were carried out using the MUST package (Philippe 1993
). The alignment of the sequences was manually performed with the ED program of the package. The following sequences (with their respective accession numbers) were included in the analyses: P. tetraurelia, D5099; L. major, CAB75567; D. discoideum, U67838; T. vaginalis, U38786; Picea mariana, AF051209, AAC32141; Arabidopsis thaliana, AAD21451, L19354, L19355, U33757, U33758; Pisolithus tinctorius, L38756; Lycopersicon esculentum, CAA58111; S. cerevisiae, P53152, CAA90451(YD6652), K02962, NP_010462, NP_010344, S28951, AAB64489, NP_010219, AAB67357, NP_013409, NP_010377, NP_010339, P29340; Pichia pastoris, U12511; Caenorabditis elegans, A48145, T16646; Homo sapiens, AAC05381, U39360, U39361, XP_004699, NP_003339, NM_014501; Drosophila melanogaster, AA246265, CAA44453, BAA34575, AAA28309, P35128; Mus musculus, NP-033484, AAG22084, AAG22085; Gallus gallus, L77699; African swine fever virus, NP_042834. The terminal parts of the sequences are highly variable and can only be aligned between closely related genes. Only unambiguously aligned regions were retained for phylogenetic analyses. A distance matrix was calculated considering all amino acid differences without weighting the transition probabilities. This matrix served as a basis for phylogenetic reconstructions using the neighbor-joining procedure (Saitou and Nei 1987
). Evaluation of the statistical validity of the nodes was performed by applying the bootstrap procedure (1,000 replicates) on the neighbor-joining method. The alignment was also used in a parsimony analysis. Parsimony analysis was done using PAUP 3.0 program for the Macintosh (Swofford, Illinois History Survey, Champaign, Ill.). In parsimony trees, bootstrap proportions were recorded from 100 resamplings.
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Results |
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Molecular Cloning and Sequence Analysis of ShUEV
In hypotrichous ciliates such as St. histriomuscorum (formerly known as Oxytricha trifallax), the somatic nucleus (macronucleus) is organized in tiny chromosomes ranging in size from 0.5 to 20 kb. In most cases, each macronuclear molecule encodes a single transcription unit flanked by its 5' and 3' noncoding sequences and is ended by the telomeric 5'-C4A4-3' direct repeats with short 3' overhangs. Probing a Southern blot with high stringency yielded a single band at about 800 bp (data not shown). The screening of a genomic library with this specific probe allowed us to isolate several truncated clones, all lacking 5' and 3' subtelomeric and telomeric regions. In order to clone the missing telomeric region, we took advantage of the gene-sized DNA molecules in using an inverse PCR-based strategy that has been successfully applied in Histriculus cavicola (Oxytrichidae) and in St. histriomuscorum (Perez-Romero et al. 1999
; Villalobo et al. 2001
). DNA molecules covalently circularized by self ligation were amplified using a pair of specific primers oriented toward the telomeres. A product of the expected size (550 bp), cloned in pUC18, contained the 5' and 3' flanking regions up to the 5'-C4A4-3' terminal repeats. The deduced macronuclear molecule was obtained by assembling the sequences of the genomic and the inverse-PCR clones. The final molecule was 787-bp long excluding both 3' terminal single stranded regions of the telomeres.
Examination of the sequence indicates a long open reading frame starting at nucleotide 1 and ending at TGA (position 577) which is the stop codon used in Oxytricha species. In many ciliates, the standard stop codons UAA and UAG are sense codons and encode glutamine. The genetic code of St. histriomuscorum also obeys this deviant rule because in the sequences of ß-tubulin, several highly conserved glutamine residues are encoded by TAA and TAG (data not shown). The nucleotide sequence matches that of the cDNA and shows the presence of a small 36 bp intron in the macronuclear gene (230265 nt). Its A + T content (nearly 90%), with the characteristic starting (GT) and ending (AG) dinucleotides is significantly much higher than the 60% A + T content of the surrounding coding region. Typically, in hypotrichs, introns, 5' leader, and 3' trailer noncoding DNA regions are AT rich (>70%) (Prescott 1994
; Hoffman et al. 1995
). This difference in A + T content renders the flanking domains easily distinguishable from putative ORFs. Downstream of the TGA stop codon of the predicted UEV amino acid sequence, the 3' trailer of the macronuclear gene is indeed nearly 90% A + T. Similarly, the 5' leader region is also recognized with a high A + T content and putative TATA boxes. However, the identification of its precise boundary is unexpectedly difficult because no in-frame ATG initiation codon is found upstream of the partial cDNA sequence. The first in-frame ATG codon is located within the intron at position 244. To rule out any amplification or sequencing artifacts, clones obtained from two different inverse-PCR experiments using different sets of primers have been sequenced and were found to be identical. This likely suggests the presence of a small intron very close to the 5' end of the coding sequence. In searching for such a putative intron, we have found that 11 nucleotides downstream of the ATG (87 nt, in-frame +3), the nucleotide sequence (5'-CAG/GTAAG-3') displays the exact 5' junction sequence of a known intron in O. nova (Hicke et al. 1990
). Forty nucleotides downstream, the corresponding exact (5'-TAG/G-3') 3' junction sequence strongly supports the existence of a 5' end intron. Its A + T content (75%) is lower than the first detected one but is still compatible with the high A + T content of the noncoding sequences in ciliates. Additional support for this second intron comes from the determination of the transcription initiation site.
Mapping Introns and the Transcriptional Initiation Site of ShUEV
Total RNA isolated from cysts was reverse-transcribed separately with two different primers bracketing the 36-bp internal intron (see Materials and Methods). In the cDNA, these two primers are expected to be 35 bp apart because of the removal of the intron. Figure 1A
displays the autoradiograph of an aliquot of the reverse-transcription reactions run in polyacrylamide gel. Firstly, the difference in length between the extension products was found to exactly equal the 35-bp distance between primers on the transcript. The splicing of the 36-bp intron is thus confirmed, which excludes the ATG within it (244 nt) to be the translation initiation site. Secondly, two distinct experiments gave identical 5' ends. Assuming the existence of an additional 5'-end intron, we mapped the putative transcription start at a T residue in the 5'-ATAA-3' sequence 40 nt from the telomere. These features are in good agreement with recent compilations of transcription initiation sites in hypotrichs (Ghosh et al. 1994
; Hoffman et al. 1995
) in which it turns out that although no conserved promoter sequences (strict TATA boxes) are found, the vicinity of the sites are high A + T regions with transcription starts frequently observed close to the telomeres. From this data, we designed a convenient primer in the putative short (25 nt) 5'-nontranslated region of the mRNA for RT-PCR experiments to definitely solve the hypothesis of the second intron.
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ShUEV Is a Structural and Functional Homologue of UEV Family Proteins
The full-length ShUEV sequence was compared to some E2 and UEV proteins taken from public databases and the alignment is shown in figure 2
. Except for the extensions of the UEV proteins of human and Drosophila, all UEVs are of a very similar size (140 aa). The sequence of St. histriomuscorum is readily aligned with UEV proteins. By comparison, the alignment with other E2s is limited to the central domain. As shown in figure 2
, this conserved core between E2 and UEV is about 80 amino acids long.
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ShUEV Expression Is Specific to Growing Cells
In the absence of food, St. histriomuscorum form resting dedifferentiated cysts which can transform back to the vegetative form when food is restored. As previously observed (Baroin-Tourancheau et al. 1999
), croc-1like transcripts are found in cysts and in vegetative cells, but the initially reported small difference in size between cyst and vegetative transcripts is not confirmed here: the same length (about 600 nt) is observed on Northern blots in both stages (fig. 5A
). We note that a faint signal corresponding to a 3-kb band can be observed in cysts several weeks old (fig. 5A
), but not in newly formed cysts (fig. 5B
). As the cells undergo profound morphological transformations and metabolic changes during the encystment-excystment cycle, we have also examined on Northern blots the expression of ShUEV in three different physiological states, namely, starved cells, cysts, and excysting cells. As shown in figure 5B
, expression of ShUEV is drastically diminished in starved cells. The down-regulation appears to be rapid and can be observed in prestarved cells (i.e., fed cells collected in the absence of food, data not shown). In contrast, there is no apparent alteration in the accumulation of transcripts during the process of excystment.
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Discussion |
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Along with the sequence of Paramecium (Okano et al. 1996
), ShUEV is the second available ciliate member of the E2 gene family, and it encodes the first inactive E2 protein isolated in protists to be positioned in the Ub-conjugating tree. Our analysis re-examines the data previously obtained by Keeling et al. (1996)
and brings new information. E2 enzymes are encoded by a multigenic family, and the intermingling of protist and nonprotist species in the global topology of the tree supports an early origin of the different E2 copies in the evolutionary history of the eukaryotes. The molecular tree shows several robust monophyletic clusters, one of which contains all known UEVs, including ShUEV. It thus confirms that UEVs constitute a coherent family as proposed in several recent studies (Sancho et al. 1998
; Xiao et al. 1998
). Moreover, the sequence from a ciliated protist in the UEV cluster implies that genes encoding UEV are ancient, not specific to the multicellular lineages, and may have originated early in the eukaryotes. This point is also illustrated by the fact that the E2 sequence of Paramecium, the other ciliate representative of the data set, branches off in a distinct group of sequences in contradiction with the monophyly of the ciliates. The duplication event leading to these different subfamilies thus precedes the divergence of the group of ciliates from the other eukaryotes. It also confirms that the E2 family is multigenic in ciliates. Homologous E2 copies of St. histriomuscorum (or of other ciliate representatives) therefore likely exist in other well-defined clusters. In St. histriomuscorum, the large distance between the isolated UEV gene and its expected distant E2 counterpart(s) is suggested by the fact that Southern blot hybridization at high stringency revealed only the croc-1like gene at 800 bp. This is also true of the Northern blots as the positive expression is associated with a single type of transcript (600 bp). The faint band corresponding to a much larger transcript could also result from an E2 distant member, although the reason for its absence in newly formed cysts is not clear.
At the functional level, the deep separation of the UEV from the other members of the E2 family suggests an early and specific functional assignment to the E2 variant subfamily. Evidence exists for the functional conservation from yeast to humans. Indeed, a conserved core functional structure between mammalian and yeast proteins has been highlighted by showing that the human CROC-1B core domain (after N-terminal truncation) and hMMS2 complement the yeast mms2 (Xiao et al. 1998
). Conversely, yeast MMS2 is able to mediate the activation of human c-fos promoter (Xiao et al. 1998
). Given the large genetic distance between protists and multicellular organisms, the existence of a ciliate homologue (as distantly related to other UEV members as its yeast homologue) could extend this functional conservation to many protistan phyla and throughout the entire eukaryotic kingdom. Our results showing that ShUEV corrects the DNA repair defect of S. cerevisiae mms2 null mutant cells bring strong support to a conserved mode of action of all UEV family proteins during evolution.
It is now clear that yeast and mammalian UEVs act as positive regulators of a specific E2 enzyme UBC13, through heterodimer formation leading to a unique polyubiquitin chain assembly process (Hofmann and Pickart 1999
). The fact that expression of ShUEV complements the yeast mms2 mutant indicates that ShUEV must interact with yeast UBC13 in the host cell. On the other hand, despite massive overexpression of ShUEV, it does not restore mms2 mutants to the wild type level, which is in contrast to the overexpression of human (Xiao et al. 1998
) and mouse (Franko, Ashley, and Xiao 2001
) UEV genes in yeast cells. Although the compromised activity of ShUEV in heterologous cells may be attributed to several reasons, we consider a reduced level of interaction between ShUEV and yeast UBC13 to be the most likely scenario. The crystal structure of hMMS2-hUBC13 complex indicates that the two proteins interact through several hydrophobic bonds involved in a number of amino acid residues (Moraes et al. 2001). ShUEV contains up to 15 nonconserved residues that are identical among yeast, human, and mouse UEVs, some of which may contribute to the interface. In St. histriomuscorum, we do not know yet if ShUEV interacts with a UBC13-like partner; however, it is striking to note that UEV and UBC13 clusters are by far the best-resolved parts of the tree. All known UBC13 homologues belong to a solid branch with which a protistan representative (L. major) is strongly affiliated. This raises the possibility that in this highly conserved E2 subfamily, ciliate homologues (notably St. histriomuscorum) exist and that the two deeply separated families (UBC13 and UEV) coevolved to maintain the functional association to each other.
Human cells contain at least two UEV family genes and three proteins (hMMS2, CROC-1A, and CROC-1B) and they appear to have distinct cellular activities. Whereas CROC-1 is implicated in differentiation and proliferation (Rothofsky and Lin 1997
; Ma et al. 1998
; Sancho et al. 1998
; Deng et al. 2000
), hMMS2 appears to be specific for DNA postreplication repair (Li et al. 2001
). Although our complementation data imply that ShUEV is able to support UBC13-mediated Lys63 polyubiquitination in yeast cells and plays a role in maintaining genome integrity, we do not know to what extent the ubiquitination process is similar between yeast and St. histriomuscorum. Nevertheless, it is interesting to note that there is a differential expression of ShUEV in dividing (vegetative) versus nondividing (starved) population. In hypotrichs, starvation is accompanied by a general decrease of transcriptional activity, leading to a differential down-regulation of many transcripts depending on their relative stability (Brandt and Klein 1995
). The observed rapid down-regulation of ShUEV is therefore not a general feature of the mRNA population and implies that this transcript is rapidly destroyed in nongrowing cells. Its up-regulation in cysts, excysting cells and vegetative cells agrees with our recent proposal that many transcripts in cysts are stored to act later during excystment and vegetative growth of the cells (Villalobo et al. 2001
). This differential pattern of expression could suggest a contribution of the ciliate UEV gene in the regulatory processes involved in the control of cell cycle and cellular growth. Experimental work aimed to disrupt the gene function in St. histriomuscorum is under way.
The nucleotide sequence accession numbers are AF139024 and AF382210.
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Acknowledgements |
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Footnotes |
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Keywords: ciliates
cyst
CROC-1
excystment
Sterkiella histriomuscorum
ubiquitin-conjugating enzyme
1 Present address: Departamento de Microbiologia, Facultad de Biologia, Universidad de Sevilla, Spain.
Address for correspondence and reprints: Anne Baroin-Tourancheau, Laboratoire de Biologie Cellulaire 4, (UPRES-A 8080), Bâtiment 444, Université Paris-Sud, 91405 Orsay Cedex, France. anne.baroin{at}bc4.u-psud.fr
.
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