The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Corresponding author: Ching Kung, Laboratory of Molecular Biology, University of Wisconsin-Madison, 1525 Linden Dr., Madison, WI 53706., ckung{at}facstaff.wisc.edu (E-mail)

Communicating editor: S. L. ALLEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca²⁺ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca²⁺ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

THE roles of specific ion channels in signal transduction and the resulting effects on the motility response of Paramecium have been established through the comparison of wild-type cells with several behavioral mutants (SAIMI and KUNG 1987 ). Most of the mutations are at unlinked loci and inherited in a Mendelian fashion (KUNG 1975 ). Electrophysiological analyses show these mutations often result in an alteration or loss of one or more ion currents that depolarize and subsequently repolarize the membrane during an avoidance response (SAIMI and KUNG 1987 ). One class of mutants is called pawns, after the chess piece, because they are unable to reverse their cilia and swim backward (KUNG 1971A , KUNG 1971B ). These mutants fall into three unlinked complementation groups: pwA, pwB, and pwC (CHANG and KUNG 1973 ; CHANG et al. 1974 ). Genetic crosses indicate the mutations behave as recessive Mendelian factors (CHANG and KUNG 1973 ; CHANG et al. 1974 ). The pawn phenotype also correlates with the loss of a depolarization-activated Ca²⁺ current that provides an important and presumably specific localized influx of Ca²⁺ (KUNG and ECKERT 1972 ; OERTEL et al. 1977 ; SATOW and KUNG 1979 ). Even though several pathways for Ca²⁺ entry into the cell have been indicated in recent studies, there appears to be just one that is critical for reversing the ciliary beat (KLAUKE and PLATTNER 1998 ; BLANCHARD et al. 1999 ; IMADA and OOSAWA 1999 ; PRESTON and HAMMOND 1999 ).

Until recently, cloning a gene responsible for even a Mendelian heritable trait in Paramecium appeared to be very difficult, given that the genome is very AT rich (75% in noncoding; 64% in coding; W. J. HAYNES and C. KUNG, unpublished results) and two standard stop codons (TAA and TAG) code for glutamine (CARON and MEYER 1985 ; PREER et al. 1985 ). Fortunately, the amitotic division and high telomerase activity in the large macronucleus allowed Paramecium to be transformed by microinjection of DNA fragments and a method for cloning genes by complementation was developed (HAYNES et al. 1996 ). We found that clonable fragments of total digested DNA that retained the ability to clonally revert the mutant phenotype could be fractionated by gel electrophoresis, allowing us to use very small sublibraries to isolate the complementing plasmids with inserts (HAYNES et al. 1998 ). With the successful development of this technique, several genes that correspond to exocytotic, morphogenetic, and behavioral phenotypes have now been isolated and characterized (SKOURI and COHEN 1997 ; KELLER and COHEN 2000 ).

Pawn mutants were chosen for our first attempts at cloning by complementation because the mutations are recessive and the transformation can be easily assayed using simple behavioral tests (KUNG 1971A ). In addition, some biochemical characteristics of the affected proteins are known from microinjecting crude protein fractions prepared from both wild-type and pawn cells, which temporarily restore backward swimming among all combinations of complementation groups, but not alleles within the same group (HAGA et al. 1982 ). This reversion activity indicates that the effective proteins are enriched in internal microsomal membranes and are shown to be sensitive to both trypsin and N-ethyl maleimide (HAGA et al. 1984 ).

In this article we describe the cloning of the DNA fragment that complements the pawn-B mutations. We also discover that the previously reported "pawn-D" strain (stock d4-662) that appeared to complement pawn-B in genetic crosses (E. AMBERGER, M. A. WALLEN-FRIEDMAN and Y. SAIMI, unpublished results) is apparently a misnomer and actually suffers the retention of an internal eliminated sequence (IES) that disrupts its pwB gene. IESs are transposon-like fragments scattered throughout the micronuclear DNA sequence that are precisely deleted during macronuclear development (STEELE et al. 1994 ). The pwB open reading frame (ORF) has no recognizable match in the current databases. This protein is predicted to have a short hydrophobic domain at the amino terminus and two more closely spaced putative transmembrane domains within the first half of the molecule. The remaining half is hydrophilic especially toward the carboxyl terminus where there is a stretch of poly-asparagine. We discuss the possible roles of such a protein in the regulation and/or structure of the calcium channel.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Stocks and cultures:
Paramecium tetraurelia stock 51s (+/+) (SONNEBORN 1970 ), nd6 (nd6/nd6) (LEFORT-TRAN et al. 1981 ), pawn-A {d4-94} (nd6/nd6, pwA/pwA), pawn-B {d4-95} (+/+, pwB/pwB and nd6/nd6, pwB/pwB), pawn-B {d4-96} (+/+, pwB/pwB and nd6/nd6, pwB/pwB), and pawn-B {d4-662} (nd6/nd6, pwB/pwB; previously pawn-D) (KUNG 1971A , KUNG 1971B ; E. AMBERGER, M. A. WALLEN-FRIEDMAN and Y. SAIMI, unpublished results), were cultured at 20° or 28° in a growth medium of buffered wheat-grass extract inoculated with Enterobacter aerogenes (SONNEBORN 1970 ). Cells with the nd6 mutation are behaviorally wild type but unable to fire their trichocysts. Lacking the discharge reaction, these mutant cells survive the trauma of macronuclear microinjection better than the wild type. Therefore, generally the cell lines used for microinjection were homozygous for the nd6 mutation.

Preparation of total wild-type DNA or micronuclear enriched samples:
Standard molecular biology techniques were used (SAMBROOK et al. 1989 ; AUSUBEL et al. 1993 –99). Total DNA and micronuclear enriched DNA were obtained as previously described (PREER et al. 1992 ; HAYNES et al. 1995 ; LING et al. 1998 ). Total Paramecium DNA samples for microinjection were digested with BclI, BglII, HindIII, or XbaI to completion as judged by gel electrophoresis and the constancy of small fragments over time. Each sample was then bound to Wizard Clean-up DNA purification resin (Promega, Madison, WI), washed in columns, and eluted with twice-distilled water. The DNA samples were then precipitated and resuspended in TE [10 mM Tris(hydroxymethyl)-aminomethane hydrochloride (Tris) and 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0] at >1 µg/µl. Restriction digests were electrophoresed and fractionated using agarose gels (0.5% Sea Plaque GTG; FMC, Rockland, ME), where overloading of the gel was carefully avoided. Fractions were excised from the gel and extracted with agarase (Epicentre Technologies, Madison, WI) or QIAGEN gel extraction columns (QIAGEN, Valencia, CA). This process was repeated until several micrograms of DNA were obtained for each of the fractions. Samples were finally precipitated and resuspended in TE at 1–5 µg/µl for macronuclear injection.

Preparation of RNA from Paramecium:
Cells were harvested and washed in Dryl's solution (DRYL 1959 ). Approximately 250-µl aliquots of cells (2 x 10⁵ cells) were lysed in 750 µl of TRI-REAGENT (Molecular Research Center, Cincinnati). The protocol for RNA isolation was provided by the company, including the following additional steps. Prior to the addition of chloroform to separate the aqueous and organic phases the samples were centrifuged and the supernatant transferred to a fresh tube. Isopropanol (1/10 volume) was added to the aqueous phase and the tubes were centrifuged to precipitate some of the contaminating DNA. In addition, a salt precipitation was also done to eliminate any potential contaminating proteoglycans.

Microinjection:
A total of 5–10 pl of DNA solutions at various concentrations was injected into the macronucleus of each recipient cell as previously described (HAYNES et al. 1995 ). All injected plasmids were linearized with either SfiI (pPXV plasmids) or XmnI or ScaI (pBluescript plasmids). Each plasmid DNA sample used for injection was first cleaned up with a resin column as described. The DNA samples were then precipitated and resuspended in TE. Recipients were cells from young clones only four to six fissions after the last autogamy. Each DNA sample was injected into at least six cells. The descendants of the individual recipients were cultured as individual clones.

Behavioral assay:
The pawn mutant cells injected with wild-type total DNA or plasmids carrying inserts were cultured for four to seven fissions before their behavior was tested. Cells were placed in an adaptation solution [4 mM KCl, 1 mM CaCl₂, 1 mM N-(2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (HEPES), 0.01 mM EDTA, pH 7.2] for 10 min and then individually transferred into a K⁺-test solution (30 mM KCl, 1 mM CaCl₂, 1 mM HEPES, 0.01 mM EDTA, pH 7.2) where the duration of continuous backward swimming of each cell was monitored using a stereomicroscope and a stopwatch (KUNG 1975 ).

Electrophysiology:
The techniques used to record Ca²⁺ currents from Paramecium with a two-electrode voltage clamp have been described previously (PRESTON et al. 1992 ). Cells were cultured for 5–7 days before testing. Briefly, membrane potential was clamped using intracellular capillary microelectrodes filled with 3 M CsCl. Cells were incubated in adaptation solution for 10 min before being placed into a standard solution of 10 mM tetraethylammonium (TEA) chloride, 0.25 mM Ca(OH)₂, 0.75 mM CaCl₂, 0.01 mM EDTA, and 1 mM HEPES, pH 7.2 for electrical recording. Currents were elicited by stepwise increases in membrane potential from -40 mV and were filtered at 2 kHz. Leakage currents were estimated from the membrane response to small (3–12 mV) hyperpolarizing steps from rest and were subtracted from active membrane responses prior to analysis.

Cloning of the transforming fragment:
A gel-purified fraction of Paramecium DNA, digested with BglII, which transformed the behavior of clonal descendants from the originally injected pawn cells, was incubated with BamHI methylase and cloned into the BamHI site of pBluescript II KS^- (Stratagene, La Jolla, CA). The ligations were sabotaged with BamHI before transforming Epicurian Coli Electroporation-Competent Sure Cells (Stratagene). Plasmid DNA from groups of bacterial colonies resistant to ampicillin were prepared and microinjected into Paramecium. As previously described, duplicate plates and nitrocellulose lifts were used to sort the colonies until an individual colony was isolated (HAYNES et al. 1998 ).

PCR of micro- and macronuclear DNA and RT-PCR from total RNA:
All PCR and reverse transcriptase (RT)-PCR were performed in a programmable thermal controller 100 (MJ Research Inc., Watertown, MA) using either Taq DNA polymerase (Promega, Madison, WI) or Advantage cDNA polymerase mix (Clontech Laboratories, Inc., Palo Alto, CA) for PCR and SuperScript preamplification system (Life Technologies, Inc., Rockville, MD) for first-strand cDNA synthesis using a variety oligonucleotide primers (Operon Technologies Inc., Alameda, CA).

Sequencing subcloned PCR and RT-PCR products:
The original complementing 7.0-kb insert and a variety of subcloned PCR products were amplified and tagged for autosequencing using the ABI PRISM Big Dye Terminator cycle sequencing kit (PE Biosystems). The reactions were then sequenced (U.W. Biotech Center, Madison, WI) on a PE Biosystems 377XL automated DNA sequencing instrument, using a 5% Long Ranger (FMC) acrylamide gel. Data were analyzed using the SeqMan II program (DNAStar, Madison, WI).

Analysis of total RNA by Northern blot:
Total RNA of wild-type 51s, pwB d4-95, pwB d4-96, and pwB d4-662 were qualitatively screened in 1% agarose gels stained with ethidium bromide and quantified by spectrophotometry. RNA molecular standards at 3 µg/lane (0.16–1.77 kb and/or 0.24–9.5 kb; Gibco-BRL Life Technologies, Gaithersburg, MD) and samples of 25–30 µg/lane were each prepared in loading buffer and dye, electrophoresed in 1.2% formaldehyde denatured agarose gels, blotted onto nitrocellulose transfer membranes (Schleicher & Schuell, Keene, NH), and hybridized with specified probe(s) according to the standard protocols (SAMBROOK et al. 1989 ). The cloned DNA probes, including the pwB ORF and the 5' and 3' flanking regions, the calmodulin (CAM) ORF and the Ase I fragment from the ß-tubulin ORF, were labeled using the Rediprime random prime labeling system with [-³²P]dCTP as directed by the manufacturer (Amersham Life Science Inc., Arlington Heights, IL). The radioactive signals were recorded on Phospho-Imager cassettes and then digitized and analyzed using the ImageQuant 1.2 program (Molecular Dynamics, Sunnyvale, CA).

Constructing green fluorescent protein-pwB fusion plasmids:
Fusion constructs were made using the recently available green fluorescent protein (GFP) ORF designed for expression in Paramecium (HAUSER et al. 2000 ). The pwB ORF alone was cloned with a 5' primer carrying a Spe I linker and a 3' primer carrying a Kpn I linker into the multiple cloning site of pPXV (see HAYNES et al. 1995 ). Fusion constructs were then made by linking the GFP ORF to the 3' end of the putative pwB ORF in pPXV-pwB. The primers used allowed us to subclone the GFP ORF into the Kpn I site in pPXV-pwB with or without a stop codon (TGA) between the two frames.

Monitoring of GFP-fusion fluorescence:
Transformants were trapped within a small amount of fluid beneath silicon oil (see HAYNES et al. 1995 ) and analyzed with a Nikon Diaphot 300 inverted microscope using appropriate objectives and a High Q Endow GFP longpass emission filter set (Chroma Technology Corp., Brattleboro, VT). Images were projected for 0.3–2 sec onto a CH250 slow scan, cooled CCD camera (Photometrics Ltd., Tucson, AZ). Digitized 16-bit images at 0.5, 0.25, or 0.1 µm/pixels were collected using Metamorph imaging software (Universal Imaging Corp., Westchester, PA). In some cases images were rescaled to a 256 grayscale using National Institutes of Health (NIH) image software. Shadowing and sharpening convolutions (or masks) were provided by NIH Image software package. Deciliation was done by placing cells into a solution containing 5 mM NiCl, 1 mM CaCl₂, 1 mM MgCl₂, 2 mM KCl, and 1 mM HEPES, pH 7.2 for 5 min. Cells were then transferred to the adaptation solution used in the behavioral tests (see above). Fixation of cells was done as recommended by Clontech for retaining the GFP fluorescent signal. Cells were washed in phosphate buffered saline (PBS; 10 mM [PO₄], 150 mM [NaCl], pH 7.2) and then fixed with 4% paraformaldehyde in PBS for 1 hr at 25°.

Sequence comparison and secondary structure prediction:
The protein or nucleotide sequence from the expected ORF was used to search for homologues in the most recent databases employing several different algorithms (BLAST, BLASTP, BEAUTY, BLITZ, FASTA, FASTA-SWAP, FASTA-PAT, MPSRCH, PROPSEARCH). Additional searches were done with the percentages of amino acids using the program PROPSEARCH (European Molecular Biology Laboratory, Heidelberg, Germany). Periodically, CD-ROM recorded databases (DNAStar) were also searched. A statistical analysis of the protein was performed using the SAPS program (Stanford University). The simplified 11-letter amino acid alphabet used by the SAPs program to analyze the protein for repeats is as follows: i, LVIF; +, KR; -, ED; s, AG; o, ST; n, NQ; a, YW; p, P; h, H; m, M; and c, C. Secondary structure was analyzedusing methods available in the programs PROTEAN (DNAStar), PHD (European Molecular Biology Laboratory), PSA (Biomolecular Engineering Research Center, Boston), PSSP (Baylor College of Medicine), and COILS (ISREC). Potential signal sequences and domains were searched for using PROSITE (University of Geneva), PSORT (National Institute for Basic Biology, Japan), and BLOCKS (Fred Hutchinson Cancer Research Center).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of the pwB transforming fragment:
Agarose gel electrophoresis was used to separate BglII digested total wild-type DNA into four size fractions (<3.0; 3–5.5; 5.5–8.5; >8.5 kb). Only the gel-eluted DNA from the 5.5- to 8.5-kb fraction retained the ability to complement pawn-B cells by microinjection. This fraction was further separated into three fractions, each covering ~1 kb, and the strongest activity was followed to fragments that were 6.5–7.5 kb in length. These fragments were incubated with BamHI methylase and ligated into BamHI linearized pBluescript II KS^- (Stratagene). Ligation reactions were sabotaged with BamHI and then electroporated into Escherichia coli (Epicurian Coli Electroporation-Competent Sure Cells; Stratagene) and kept as frozen stocks in Luria broth (LB) with 15% gylcerol and 50 µg/ml ampicillin at -80°. The number of colony-forming units was determined and ~500 resistant colonies were grown on each of 10 individual plates under selective conditions. Nitrocellulose membrane (Protran BA85, Schleicher & Schuell, Keene, NH) was used to lift colonies from each of the 10 plates to a second replica plate and also into 10 separate liquid LB cultures. Only one of the minipreps of plasmid DNA made from these liquid cultures transformed injected pawn-B Paramecium. A second nitrocellulose lift from the plate containing the transforming colony was cut into eight sectors and minipreparations of plasmid DNA from each sector were injected. Sixty-four colonies were then hand picked from one transforming sector and placed on a fresh LB selective master plate in an 8 x 8 array. Plasmid preparations from rows and columns were injected and a single transforming colony was found containing a plasmid with a 7.0-kb insert, called pPwnB. This plasmid, when injected in a dilution series, complemented the pawn-B mutant phenotype to wild type at a level similar to the assumed number of copies in the digests of Paramecium DNA originally injected (data not shown; see HAYNES et al. 1998 ). All available pawn-B cells (stocks d4-95 and d4-96) injected were transformed and the resulting backward swimming behavior was not detectably different from the wild type (see HAYNES et al. 1998 ). We injected the plasmid into mutant cells from two additional separate complementation groups, pwA (stock d4-94) and pwD (d4-662). While the insert had no effect on the behavioral phenotype of pawn-A, "pawn-D" (d4-662) cells were transformed as effectively as pawn-B, suggesting that d4-662 is a pawn-B allele.

Identification by microinjection of the ORF:
Previous injections showed that HindIII digested wild-type (51s) Paramecium DNA transformed pawn-B mutant cells (HAYNES et al. 1996 ). After the pPwnB insert was digested with HindIII, the resulting fragments still transformed injected pawn-B cells (Fig 1A). Upon subcloning the five HindIII fragments from the 7.0-kb insert, only the 2.9-kb fragment had the ability to transform (Fig 1A). In addition to the subcloned 2.9-kb fragment, the entire 7.0-kb 51s macronuclear DNA insert was sequenced. While a sequence containing two domains with homology to receptor kinases in plants was recognized, the transformation did not require this upstream ORF to be intact (Fig 1A, middle arrow, ORF 2; BLASTP bit scores between 60 and 90; expect values from 10^-7 to 10^-18). An ORF further upstream translated in the opposite direction was also recognized by BLASTP to have homology to myosins and some endosomal proteins (Fig 1A, left arrow, ORF 3; bit score between 50 and 60; expect values from 10^-5 to 10^-7), but unlike the kinase sequence, randomization of this matching sequence occasionally showed some homology to myosins (data not shown). Several subcloned PCR products and restriction fragments from within the 2.9-kb sequence had the ability to transform pawn-B cells only when an 800-bp region was included (ORF 3, thick gray arrow in Fig 1A). The smallest macronuclear DNA fragment injected that retained the ability to transform the phenotype of pawn-B cells was generated with primers A and B (Fig 1A and Fig B). Injection of this fragment resulted in a transformation that was not significantly different from that seen with the entire 7.0-kb fragment.

View larger version (41K): In this window In a new window Download PPT slide   Figure 1. (A) A graphic representing the subcloned 7.0-kb macronuclear fragment. The six HindIII sites (vertical bars) in the original 7.0 kb with the subcloned 2.9-kb fragment displaced downward are shown. The probe for the Northern hybridization is shown as a gray box. The three ORFs are represented as arrows pointing in the direction of possible translation. ORF 1 has homology to endosomal proteins and myosins. ORF 2 translates starting with a hydrophobic (signal?) sequence (open), followed by a cysteine-rich sequence (three stars), and then a probable kinase domain (hatched). An intron (thick line) connects to a second domain with five repeats of 25 amino acids (open arrow with narrow lines). ORF 3 has one known intron (thick black line) and is the sequence required for transforming pwB (thick gray arrow). The dashed line represents the location of the smallest tested fragment and its sequence is described in Fig 1B. (B) The nucleotide sequence and protein translation of the pwB ORF. The location of the intron (3) and IESs (4 and 5) and the sites of the mutations in pwB d4-96 (1), pwB d4-95 (2), and pwB d4-662 (which retains IES 427 with a single point mutation) are marked and their sequence is presented below. The inserted adenosine in the frameshift construct is shown (*). The primer sequences are labeled A–F and each sequence is in boldface. Arrows represent whether the primers were sense or antisense. Primers A and B were used to PCR the smallest tested genomic fragment. The boldface sequences below and above arrows C and D are theprimer sequences used to amplify the longest product tested from first-strand cDNAs generated with the primer sequence above arrow F. Primer pair E and F was used to successfully RT-PCR probable start and stop codons.

Sequence comparison of PCR products from wild-type and mutant stocks:
In addition to the originally cloned 7.0-kb fragment, a variety of primers were used to generate PCR products from total DNA and micronuclear enriched DNA isolated from wild-type stock 51s and pawn-B stocks d4-95, d4-96, and d4-662. Subcloned and sequenced PCR products generated with primers covering the entire 2.9-kb fragment (Fig 1A) confirmed that there was only a single nucleotide substitution in the d4-96 stock (G 003 A) and the d4-95 stock (G 173 C), while the d4-662 stock contained a 44-bp insertion (Fig 1B). Although many other primers were used to amplify, subclone, and sequence from both the 7.0- and 2.9-kb regions, we did not see any further confirmed variations in the sequence. A comparison of the subclones and sequences from PCR products generated with various primers using micronuclear enriched DNA from 51s, d4-95, and d4-96 as template revealed two IESs within the same sequence (IES 426 and 657; Fig 1B). These products confirmed the d4-95 and 96 mutations and also showed that the 44-bp insertion in d4-662 corresponded to IES 427, with the exception of a single point mutation (C to T) four bases inside the 5' end of the IES (Fig 1B). In addition, several PCR products generated from 51s total DNA were found to have an insertion of nine bases (AATAACAAT) at position 658. This insertion sequence corresponds precisely to the last nine bases of IES 658 and is likely the result of an alternative excision (Fig 1B).

Analysis of total RNA by Northern blot:
Separate lanes on a single blot reveal that signals (Fig 2) are present in total RNA from pwB (d4-96), pwB (d4-95), and wild type (51s) of about the same size but no signal was detectable from pwB (d4-662) when the blot was hybridized with a 980-bp probe generated by PCR (see Fig 1A). Two additional probes generated by PCR of the cloned ß-tubulin ORF and calmodulin ORF were then hybridized to the same blot to verify that the RNA in each lane was generally not degraded and that the loading among lanes was qualitatively equivalent (Fig 2). Several additional Northerns were done using a variety of probes from the 2.9-kb sequence and these consistently showed that 51s, d4-95, and d4-96 produced an RNA from the pwB ORF, but not d4-662 (data not shown).

View larger version (56K): In this window In a new window Download PPT slide   Figure 2. Northern hybridization of total RNA. A single nitrocellulose blot with total RNA prepared from cell cultures of d4-96, d4-95, d4-662, and 51s stocks was probed with a random primed probe (see Fig 1A) that included the pwB ORF and was exposed for 96 hr (left). The band seen is ~1000 bases calibrated on the ethidium staining of an RNA ladder. Random primed probes of the internal Ase I fragment of ß-tubulin ORF and the entire calmodulin ORF hybridized to the same blot show a qualitative similarity between the four separate samples of total RNA (lanes 1–4) without apparent degradation (right).

RT-PCR products generated from total RNA:
In addition to oligo(dT) primers, several sequence-specific primers (including primer F, Fig 1B) were used to generate first-strand cDNA pools from several independent purifications of 51s total RNA. RT-PCR using first-strand cDNA pools generated with antisense [or oligo(dT)] primers (Fig 1B, primers B or F) resulted in large amounts of product shorter than the macronuclear-size DNA and miniscule amounts of macronuclear-size DNA. When first-strand cDNA pools were generated using sense primers (Fig 1B, primer C) only miniscule amounts of macronuclear-size DNA fragments were produced. Sequencing showed that the longer products were identical to PCR products from the macronuclear DNA and that the shorter products had the exact same sequence except an internal 29-bp sequence is missing (Fig 1B). A comparison of this 29-base sequence with the 63 previously described Paramecium introns showed that they both have a relatively high A-T content (80%), are short (26 ± 2.5 bp), and the excised sequences begin with a GT and, in almost all known introns, end with an AG (from GenBank and Fig 1B). First-strand cDNA generated with oligo(dT) or various sequence-specific primers (e.g., primers B or F, Fig 1B) was successfully amplified (e.g., primers E and F or C and D) containing the 795-bp ORF. Two types of RT-PCR products, one with and one without the variable 9 bases previously seen in PCR products of the macronuclear DNA, were found (see Fig 1B; 3' end of IES 658). Thus, the putative alternative excision of 9 bases from the 3' end of IES 658 apparently does not prevent the transcription of the RNA in wild type. While RT-PCR from d4-95 and d4-96 RNA consistently contained cDNA with the intron removed, reactions with d4-662 RNA resulted in only genomic size products containing the intron (data not shown).

The putative translation product:
There is only one significantly long ORF found in this sequence after the removal of the intron sequence and its translation is shown in Fig 1B. The putative translation does not appear to have primary sequence homology to any known or hypothetical protein in the databases (see MATERIALS AND METHODS). The protein would have three relatively hydrophobic domains toward the 5' end and a relatively long hydrophilic domain at the 3' end (Fig 3). The internal two hydrophobic domains are predicted to be possible transmembrane domains by several of the algorithms available (see MATERIALS AND METHODS). The mutation found in pwB d4-96 would result in the possible substitution of an isoleucine codon in place of a methionine, eliminating a possible translation signal sequence (see Fig 3). The mutation in d4-95 is a surprisingly conservative alanine for a glycine substitution at the beginning of the first of two closely spaced stretches of hydrophobic residues that could be transmembrane domains (Fig 3). This suggests that the orientation of the amino acid sequence, specifically around this glycine side chain, is critical for function. Only two cysteines are found on the molecule and since previous biochemical data showed that the pwB curing protein had a sensitivity to N-ethylmaleimide, they may interact with each other or with a closely associated protein (C's in Fig 3). The SAPS program revealed a nine-amino-acid repeat (iisiiiioo) of unknown significance at the amino terminus of the first putative transmembrane domain and again at the carboxyl terminus of the second transmembrane domain (open bars, Fig 3; see MATERIALS AND METHODS). The remainder of the molecule is relatively hydrophilic particularly in the portion that contains the variable poly-asparagine sequence (hatched bar, Fig 3).

View larger version (19K): In this window In a new window Download PPT slide   Figure 3. A Kyte-Doolittle hydrophilicity plot (averaged over nine amino acids). The positions and the deduced amino acid substitutions that were found in the three independently isolated pawn mutant alleles are indicated. The poly-asparagine sequence is represented by the hatched bar. The two open bars represent the nine-amino-acid repeats (iisiiioo; see MATERIALS AND METHODS). The only two cysteine residues found are labeled C.

Verification of a translated product:
Several injection experiments were done to test whether the transformation by this sequence was due to a translated product. In addition to the original pPwnB, several constructs were injected and their effect on the cells' behavior is outlined in Fig 4. In all cases where transformation was observed, the behavioral response was not significantly different from wild-type cells (nd6 or 51s). Control injections of empty pBluescript (without an insert) had no influence on the behavioral response of any of the injected cells. The first construct (plasmid 1; pBs-pwB⁺-genomic, Fig 4A) was the smallest transforming macronuclear DNA fragment cloned and tested from 51s, as described above (Fig 1B). Injection of this linearized plasmid into d4-95, d4-96, and d4-662 resulted in transformation of descendants in all injected clonal cell lines, while even a 5 to 10 times higher concentration of the plasmid had no noticeable effect on the response of wild-type or pwA cells (Fig 4B). An identical construct containing the pwB (d4-95) point mutation [plasmid 2, pBs-pwB (d4-95)-genomic] had no effect on any of the pawn-B cells injected (Fig 4A and Fig B). In addition, the presence or absence of the nine alternatively excised bases of IES 658 had no discernible effect on the transformation (data not shown).

View larger version (23K): In this window In a new window Download PPT slide   Figure 4. (A) Diagrammatic representations of injected plasmids. Plasmids are referred to by their number in the text. Solid black lines represent the vector sequence (either pBluescript or pPXV). Light stippled bars represent the pwB ORF with a black bar showing the location of the intron if present. The position of the point mutation in d4-95 is represented by the black dot (plasmid 2). Open bars represent the GFP ORF cloned from pPXV-GFP (HAUSER et al. 2000 ). Open or gray bars represent 5' or 3' UTR from the pwB gene or the Paramecium calmodulin gene, respectively. Open arrows on the ends represent telomere sequences in the pPXV plasmids. A stop was introduced between the two ORFs on plasmid 6 (TGAg). (B) The behavior of transformants clonally descended from the cells injected with various plasmids. The behavior of the uninjected or untransformed pawn cells (-) is to swim only forward when placed in the K+-test solution (see MATERIALS AND METHODS). The response of the wild-type and all of the transformed pawn cells was to swim backward for 20 seconds (+). None of the plasmids when injected into wild-type and pwA cells elicited a discernible affect on their behavior.

To test if the ORF alone was responsible or capable of transforming the cells we placed the pwB coding sequence, without the 5' or 3' untranslated regions (UTRs), into an expression construct shown to express and translate a variety of ORFs in Paramecium (Fig 4A; plasmid 3 with 5' and 3' UTRs from the Paramecium calmodulin gene flanked by Tetrahymena telomeres; see HAYNES et al. 1995 , HAYNES et al. 1996 ; HAUSER et al. 2000 ). Control injections of empty pPXV (without insert) or pPXV-GFP had no effect on behavior (plasmid 5, Fig 4A and Fig B), while pPXV-pwB⁺-cDNA (plasmid 3, Fig 4A) transformed the response of all pawn-B cells injected. This same plasmid (3) failed to affect the behavior of the wild-type (data not shown) or pwA cells (Fig 4B). Injection of a frameshifted ORF that had the nucleotide A inserted between the first and second possible methionine codons [ Fig 4A, plasmid 4, pPXV-pwB⁺-cDNA (frameshift) and Fig 1B between nucleotide 6 and 7] resulted in a complete loss of the ability of the plasmid to transform the response of any cells injected (Fig 4B). Since a single nucleotide insertion is not likely to abolish a function specific to the RNA, but will shift the ORF, it seems likely that it is the translation of the putative protein product (Fig 3) that results in the phenotypic transformation and the translation begins at the first methionine codon.

Restoration of the voltage-dependent Ca²⁺ current by pawn-B ORF:
The inability of pawn mutants to swim backward is due to the loss of an inward transient Ca²⁺ current. Examination of the clonal descendants of pawn-B cells injected with plasmid pPXV-pwB-cDNA (800 copies/pl) using a two-electrode voltage clamp clearly shows that the inward Ca²⁺ current had been restored (Fig 5A). A comparison of the magnitude of the peak current plotted against membrane potential indicated that there is no significant difference in the magnitude of the current and its voltage sensitivity between wild-type and transformed cells injected with the plasmids containing the cDNA insert (mean ± standard error; Fig 5B). Peak currents from cells injected with plasmids containing the 7.0-kb or 1.1-kb genomic inserts were not significantly different (data not shown).

View larger version (20K): In this window In a new window Download PPT slide   Figure 5. Ca2+ currents in wild-type and mutant paramecia examined under a two-electrode voltage clamp. (A) Currents elicited from wild-type (top current trace), pawn-B (middle trace), and transformed pawn-B injected with pPXV-pwB-cDNA (plasmid 3, lower trace) in Ca2+-TEA solution. Currents were elicited by 40-msec voltage steps to -10 mV from -40 mV (voltage trace). The voltage-activated Ca2+ current is apparent as an inward peak during step depolarization in the wild type and in transformants, but not in pawn-B mutant cells. Capacitive transients have been suppressed. (B) Peak inward Ca2+ currents (Ipeak) are plotted against the membrane potential at which they were elicited (Vm). Points are means ± SD determinations from 6 wild-type (solid circles), 4 pwB mutants (solid squares), or 6 cells descended (10 to 15 fissions after injection) from pwB injected with the plasmid pPXV-pwB-cDNA (open squares).

Expression of GFP fusion constructs:
Microinjection of the PXV-pwB-GFP fusion plasmids 6 and 7 (Fig 4A) resulted in a behavioral transformation of all pwB mutants injected (Fig 4B), but the examination of the fluorescent signal from trapped living cells revealed that only descendants from cells injected without the stop codon (TGA) between the two open reading frames (plasmid 7) effectively expressed the GFP (Fig 6A and Fig B). The same results were seen when the two plasmids were injected into wild-type cells (data not shown). At higher magnification (x100) a phase micrograph shows the cilia and cortical ridges of a living cell expressing the pwB-fusion protein (Fig 7A). The corresponding fluorescent signal (Fig 7B) shows a pattern of hollow rings (1 µm in diameter) that reflect a displacement from or interaction with other structures (arrows with dashed lines) as well as signals that appear round and filled (2 µm in diameter; arrows with solid lines). Focusing toward the middle of the cell (Fig 7C), a series of vertical vesicle-like structures reach toward the surface at about the same frequency as the cortical ridges. These structures do not correspond to trichocysts seen in phase micrographs (data not shown) but connect both horizontally and vertically to other vesicular-like signals. Surprisingly, there is no apparent signal from the plasma membrane or cilia when examined at a number of focal planes and at a variety of magnifications (Fig 7C arrow with dashed line and data not shown). These signals clearly differ from cells expressing just GFP alone (plasmid 5) where the more even signal highlights a sharp boundary at the edge of the cell (Fig 7D) and emphasizes the lack of a uniform plasma membrane signal in previous micrographs (Fig 7B and Fig C). These fluorescent micrographs (Fig 7) were of living cells partially flattened within a narrow space of fluid. Since there was no obvious ciliary signal, cells were deciliated to allow for clear resolution of internal and external surfaces near the plasma membrane of the cell. After deciliation a remarkably bright signal can be visualized in the living cell along the membrane of the nucleus (solid arrow), as well as near the cortex below the plasma membrane (dashed arrow, Fig 8A). Raising the focal plane to the surface of the cell reveals the oral cavity (solid arrow) and a reticular pattern (dashed arrow, Fig 8B). Upon fixation, which cross-links proteins without eliminating GFP fluorescence, most of the vesicular signal is lost whereas the reticular signal near the cortex, as seen in the deciliated cell (Fig 8B), is clearly retained (data not shown).

View larger version (67K): In this window In a new window Download PPT slide   Figure 6. Injection of the fusion construct pPXV-pwB-GFP with (plasmid 6 in Fig 4A) or without (plasmid 7) a stop between the two ORFs. (A) Phase micrograph of a pwB (d4-95) descendant cell 3 days after injection with pPXV-pwB-w/stop(TGA)-GFP (plasmid 6; 6000 copies/pl) on the right and a descendant cell injected with pPXV-pwB-GFP (plasmid 7; 2000 copies/pl) on the left. Bar, 20 µm or 40 pixels. (B) The fluorescence from the same two cells shows GFP expression in the descendant after injection with plasmid 7, but no clear GFP signal is seen in the descendant injected with plasmid 6. Bar, 20 µm or 40 pixels.

View larger version (170K): In this window In a new window Download PPT slide   Figure 7. Cells transfomed with pPXV-pwB-GFP (plasmid 7) at high magnification. (A) A phase micrograph of the anterior end of a living Paramecium (see MATERIALS AND METHODS). Bar, 4 µm or 40 pixels. (B) Fluorescent signal from the same cell focused just below the cortex after applying a shadowing mask (see MATERIALS AND METHODS). Dashed arrows indicate nonfluorescent circular structures (1 µm in diameter). Solid arrows indicate circular filled structures (2 µm in diameter) that appear to be horizontially connected, Bar, 4 µm or 40 pixels. (C) A focal plane in middle of the same cell after a shadowing convolution (see MATERIALS AND METHODS). Solid arrow indicates vertical vesicular-like structures that appear to branch out toward the surface of the cell. Dashed arrow indicates that there is no sharp demarcation between the inside and outside of the cell. No ciliary signal can be observed. Bar, 4 µm or 40 pixels. (D) Fluorescence micrograph of a cell injected with pPXV-GFP only. A homogenous labeling of the cytosolic compartment is seen along with a sharp boundary between the inside and the outside of the cell (solid arrow). Bar, 4 µm or 40 pixels.

View larger version (81K): In this window In a new window Download PPT slide   Figure 8. (A) Fluorescence from within a living deciliated cell injected with pPXV-pwB-GFP. Solid arrow points to the anterior edge of the macronucleus, which shows a striking membrane-associated signal. Dashed arrow points to the cortical signal seen in the cell. Bar, 10 µm or 40 pixels. (B) Fluorescence from the same cell focused just below the surface of the cell. Solid arrow indicates the oral cavity. Dashed line indicates a clear reticular signal seen across this focal plane of the cell. Bar, 10 µm or 40 pixels.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this report, we have used the method for sorting Paramecium total DNA by microinjecting sublibraries (HAYNES et al. 1998 ) to isolate an ORF from a 7.0-kb BglII macronuclear fragment that transforms the mutant pawn-B but not pawn-A. We have established, using a variety of molecular techniques, that the transformation corresponds with the translation of a single ORF. RT-PCR and Northern blots revealed that RNA was transcribed from the ORF sequence and a 29-bp intron was removed. Injections of smaller genomic fragments, cDNA, frameshifted cDNA, and GFP-fusion constructs indicated that the ORF is not only transcribed but also has been translated in association with the transformation. The transformation fully restored wild-type behavior within 24 hr when pawn-B cells were injected with an estimated 1000 copies of the genomic copy (plasmid 1, Fig 4A; data not shown). In addition, sequencing of an internal 2.9-kb HindIII fragment revealed single point mutations within this same ORF in each of the two previously characterized pwB alleles (d4-95 and d4-96). The unexpected transformation of d4-662, which was previously thought to be a mutation at an additional pawn locus, led to the discovery of an insertion within this same ORF. Sequencing of wild-type micronuclear DNA of the 2.9-kb fragment showed that the insertion in d4-662 is actually an IES with a single point mutation that apparently inhibits excision of the IES during macronuclear development.

As in the case of pawn-A and previous studies on pawn-B (OERTEL et al. 1977 ; SATOW and KUNG 1979 ), the electrophysiological data and behavioral response of swimming backward correlate with the presence or absence of a voltage-dependent Ca²⁺ current. The results presented here demonstrate that the translation of the expected ORF also correlates with a voltage-dependent Ca²⁺ current. However, the conceptual translation of the pwB gene does not resemble any known pore-forming subunit of a Ca²⁺ channel. Instead, this protein would appear to have two putative membrane-spanning domains and play the role of a direct or indirect regulator for the production, localization, maintenance, or activity of a Ca²⁺ channel.

Although the lack of clear homologs in the current database makes the interpretation of function difficult, the three pwB mutants do provide useful information about certain parts of the protein. The d4-96 mutation (ATG to ATA) indicates that the start of the ORF is with the first of two methionines since this mutation is at the very 3' end of the primer sequence that generated the longest cDNA. In addition, injection of the ORF with the d4-96 mutation does not transform (data not shown). Furthermore, a single base insertion between these two methionine codons abolished transformation (plasmid 4, Fig 4A) and the translation of the GFP fusion protein was prevented by a stop in the expected frame (plasmid 6, Fig 4A). We have previously shown that the calmodulin promoter effectively expresses ORFs with methionines downstream or even out of frame from the original wild-type calmodulin methionine (HAUSER et al. 2000 ; W. J. HAYNES, unpublished data). Therefore the distance between the promoter and the second methionine (in the pwB ORF) would probably not be a factor in preventing expression. This indicates that the amino-terminal hydrophobic domain is translated and serves as a signal peptide or plays a critical role in protein assembly or function (Fig 4).

The d4-95 G-to-C transversion substitutes a glycine for an alanine and indicates a critical residue in the first putative transmembrane domain. Since the substitution would only decrease the hydrophilicity (0.4 for Gly and -1.8 for Ala) this residue is not simply contributing to a transmembrane domain. Even the smallest possible increase in this residue's side chain obliterates the activity of the pwB protein and thus indicates that the flexibility or length afforded by the glycine is crucial.

The d4-662 point mutation in IES 427 appears to inhibit its excision during macronuclear development. The retention of the 44-bp sequence (Fig 1B) shifts the frame, replacing the last 125 amino acid residues of the hydrophilic carboxyl terminus with a different sequence of 51 residues (data not shown). Though one might expect an altered or extended RNA molecule, the lack of a Northern signal and our inability to generate cDNAs from the d4-662 strain suggest that the transcripts are not present at a detectable level. It seems likely that the altered transcript is selectively degraded and the locus is silenced. In effect, this mutant is a knockout that can provide a null background to study the function and localization of wild type or mutant pawn-B proteins expressed from a plasmid.

The importance of the fifth nucleotide of the 8-bp inverted repeat at the 5' junction of the excised sequence was recently suggested by the discovery of the AIM-1 mutant (MAYER et al. 1998 ). The excision of IES 2591 from the ORF of the immobilization surface antigen-A was inhibited by a single point mutation at the fifth nucleotide within the 5' 8-bp inverted repeat, even though an internal sequence within that same IES was still recognized and spliced (MAYER et al. 1998 ). The pawn strain d4-662 has a slightly different 8-bp repeat sequence, but it has the exact same single point mutation of C to T at, again, the fifth nucleotide in the 5' inverted repeat. This mutation also apparently results in the sequence being retained in the macronucleus, but in this case there is no cryptic internal sequence removed from the IES. However, the importance of any particular nucleotide in the fifth position may be dependent on the surrounding sequence since we have recently discovered another IES with a T in this same position (TATTTGTG; K.-Y. LING and C. KUNG, unpublished results), which appears to be effectively spliced during development.

The presence of the mutation in the IES does not, by itself, explain the fact that crosses between d4-95 and d4-662 result in F₁ heterozygotes that swim backward, which led to their initial assignment to separate loci (E. AMBERGER, M. A. WALLEN-FRIEDMAN and Y. SAIMI, unpublished results). While a hypothesis that the presence of the wild-type IES affects the ability of the mutant IES to excise (DUHARCOURT et al. 1998 ; MAYER and FORNEY 1999 ) could be proposed, the additional complexity of transcriptional silencing shown in the Northern blot (Fig 2) makes it difficult to speculate as to the precise mechanism without further investigation.

The second IES in the ORF (IES 658) is the second example of an alternatively spliced IES (DUBRANA et al. 1997 ) but the first known example within an ORF. The fact that there was no significant difference in transformation between the two cDNAs indicates that the additional asparagines inserted into the poly-asparagine sequence do not significantly affect the protein function. Since hydrophilic poly-aspargine residues are predicted to be highly flexible these residues might simply serve as a loop that allows other neighboring residues to have intra- or perhaps intermolecular interactions. While the exact frequency of each alternative transcript is not known, each form was readily cloned from PCR or RT-PCR products. We do not know if there are additional splice variants, but mutagenesis of this region should help decide what effect additional insertions, deletions, or frameshifts would have on this region of the molecule. It would also be interesting to see if either form can bias the developmental excision in trans.

Since the overexpressed GFP-fusion protein transformed the injected cells, functional protein must be present. The fluorescent signal highlights a complex array of internal vesicles and membranes that are very close to the surface of the cell and that are not seen with the expression of GFP alone (Fig 7 and Fig 8). In addition, even in this highly overexpressed condition there is no clear GFP signal from the ciliary or plasma membrane, while signals appear near the cell surface and around the macronuclear membrane. There are fluorescent vertical structures that extend up toward the surface of the cell, which do not appear to generally correspond to trichocysts seen with phase micrographs. The horizontally connected pattern could be a reflection of the structure of endoplasmic reticulum found beneath the surface of Paramecium (EHRET and MCARDLE 1974 ) or an interaction with cytoskeletal structures that are similar to the infraciliary lattice (ALLEN et al. 1998 ). One speculation is that the signal might be related to an unknown network of vesicles not unlike t-tubules in muscle cells (PARTON et al. 1997 ; DOHM and DUDECK 1998; SOELLER and CANNELL 1999 ). In addition to alveolar sacs, a system of vesicular structures has been described associated with cilia, kinetodesmal fibrils, mitochondria, and the parasomal sacs (PATTERSON 1978 ). The description of these structures included a sac-like structure in association with the cilia, referred to as a cilisac, which could be similar to the rounded fluorescent structures seen in Fig 7B (PATTERSON 1978 ). On the other hand, a connection to the infraciliary lattice might be interesting since there is a connection between the movement of basal bodies and the contraction of centrin, the major component of the infraciliary lattice, in the flagella of Chlamydomonas (HAYASHI et al. 1998 ). While there was no significant difference in the ability of the fusion constructs to transform, it is probable that overexpression also leads to some mislocalized or even degraded protein. Further investigations will be done when antibodies are available. In addition, efforts to co-localize the protein with other known molecular structures by dual labeling with antibodies or using differently colored GFPs are planned.

Unlike the pwB ORF, an ORF immediately upstream has two homologous domains that are easily identified. This ORF is so extraordinarily close to the pwB ORF that the two protein products may or may not have a functional or evolutionary connection. The 5' end of this upstream gene is a cysteine-rich sequence (open box with three stars, Fig 1A) and finishes with a domain that has homology to the transmembrane receptor serine/threonine kinase family commonly found to be involved with mating responses in plants (cross-hatched, Fig 1A, VALON et al. 1993 ; SUZUKI et al. 1999 ). However, as previously found in one of the homologous receptor kinases in Arabidopsis, there has been a substitution of the primary catalytic amino acid residue (VALON et al. 1993 ). We have observed a signal on Northern blots with probes made from this upstream ORF and have cloned and sequenced cDNAs that have an intron removed (data not shown). The intron provides a connection in frame to a second type of domain toward the 3' end of this upstream gene. The second domain has five repeats of a 25-amino-acid residue sequence (stippled vertical hatched arrow, Fig 1A) previously found to be attached to the 5' end of an Arabidopsis phosphatidylinositol-4-phosphate 5-kinase (SATTERLEE and SUSSMAN 1997 ). While these repeats are not found in homologous PI-4-P 5 kinases, they are found in other organisms as independent molecules (SATTERLEE and SUSSMAN 1998 ). The ORF of this upstream gene abruptly ends, without a characteristic transmembrane domain seen in the other receptor kinases, at a TGA stop codon that is only 36 nucleotides upstream of the pwB starting methionine. In fact, the removal of the TGA would not only translate a transmembrane domain and make the kinase more homologous to the plant receptor kinases, but also would connect in frame with the pwB ORF. However, within the resolution of our Northern blots and RT-PCR experiments we did not detect a longer species of RNA containing both ORFs and none of our many sequenced PCR products showed any variation in the TGA. Therefore since RT-PCR products from both the upstream kinase and the pwB ORF have introns removed, there are only a few possible interpretations. One possibility is that the two ORFs are expressed as two distinct transcripts. In this case the proximity of the two genes is puzzling, since promoters, the transcriptional termination sequence, the polyadenylation site, and possibly other regulatory functions will have to be compressed within the 36-bp sequence. It is possible that there are longer transcripts that include the pwB ORF that cannot easily be detected. The low level could reflect a low rate of transcription, a lack of stability, or perhaps some unknown splicing mechanism. This latter possibility indicates that perhaps the pwB ORF represents an alternatively expressed domain. This is actually reminiscent of the alternative splicing at the locus of the S receptor kinases in plants (HATAKEYAMA et al. 1998 ). Nonetheless, the data presented here strongly indicate that the pwB ORF produces a separate translatable gene product that transforms pawn-B cells. Further experiments are needed to clarify the connection between signal transduction and motile behavior in which the pawn-B protein plays a crucial role.

	FOOTNOTES

¹ These authors contributed equally to this article.

	ACKNOWLEDGMENTS

We thank Roland Kissmehl and Lynn Haynes for critical comments on the manuscript, as well as G. G. Borisy and S. Limbach for the generous use of the Nikon fluorescence microscope and technical assistance. We also thank the other members of our lab who indirectly contributed to this work. This research was funded by National Institutes of Health grants GM-22714 (C.K.), GM-36386 (Y.S.), and GM-51498 (R.R.P.).

Manuscript received January 21, 2000; Accepted for publication March 30, 2000.

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