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Evidence for a myosin gene in Tetrahymena
Cell Biology
International
Reports,
Vol. 13, No. 7, July
643
1989
EVIDENCE FOR A MYOSIN GENE IN TETRARYMENA E Jane National Research
Mitchell
and Luis
H Martin.
Research Council of Canada, Biotechnology Institute, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2 Canada.
INTRODUCTION The isolation of an actin gene from Tetrahvmena and Pearlman, 1986, Hirono et al, 1987) and (Cupples biochemical evidence for actin (Mitchell and Zimmerman, over has laid to rest the controversy the 1985) actin in these cells (Mitchell et al, existence of Immunofluorescence studies (Hirino et al, 1985). 1987) cell division, have implicated roles for actin in and exocytosis. The primary structure of endocytosis as derived from its gene sequence Tetrahvmena actin Pearlman, Hirono et al, (Cupples and 1986, 1978) it is the most divergent of all actins indicated that discovered to date. Therefore, the question exists not Tetrahvmena actin whether or interacts with myosin in some or all of the cellular functions in which it is involved. Myosin possesses APTase activity and is the molecular motor which interacts with actin filaments to produce movement (ref. Warrick and Spudich, 1987, for review). We therefore sought evidence for myosin in Tetrahvmena and we report here our preliminary evidence for a myosin gene. MATERIALSANDMETRODS Total genomic DNA from Tetrahvmena thermoohila SB255 (obtained from B Amos) was isolated and digested with endonucleases essentially as described (Maniatis et al, 1982). DNA 9 DNA hybridisation followed the method of Southern (Southern, 1975) except that Hybond-N membrane (Amersham) was used. The myosin gene probes used were: 1) a 688 bp Pst I/Bst EII cDNA fragment which encodes Acanthamoeba myosin Sl residues 99-244 (a kind gift from JA Hammer III); 2) a 2.4 kb Hind III/Bgl II cDNA fragment encoding most of the yeast Saccharomvces cerevisiae myosin Sl region (a kind gift from E Orr); and 3) a 2.5 kb Eco RI cDNA construct encoding the nematode Caenorhabditis eleaans myosin Sl (Mitchell et al, 1989). Endonuclease treated and 32P end-labelled EDNA was used for molecular size markers (Fig. lA,B & C; lane 1). Eco RI digested nematode genomic DNA was also electrophoresed (Fig. lA, B & C; lane 2).
0309-I 6511891070643-41$03.00/O
@ 1989 Academic
Press Ltd.
644
Cell Biology
Fig,
1
International
A
Reports,
B
Vol. 73, No. 7, July 1989
C
123451234512345 kb 23.1 9,4 6,6 4,4
2.3 2,o
Fig 1. Southern blot analysis of genomic DNA from DNA was hybridised with myosin gene probes Tetrahvmena. (as described in text) from: A) Acanthamoeba; B) yeast; Samples electrophoresed in each of A, B, C C) nematode. are: lane 1, molecular weight markers; lane 2, nematode DNA digested with Eco RI; Tetrahvmena DNA digested with: 3); Bam HI (lane 4); Hind III (lane 5). Eco RI (lane
RESULTS AWD DISCUSSION A similar pattern of hybridisation to Tetrahvmena genomic DNA was observed with each of the myosin gene probes, Acanthamoeba (Fig. lA), yeast (Fig. 1B) and nematode (Fig. 1C). Eco RI digestion of Tetrahvmena DNA yields three bands of approximately 18, 6.6, and 3.7 kb (Fig. lA,B &C; lane 3). The largest band is the weakest one and may represent a partial digestion product. Hind III digestion produces a strongly hybridising band at 5.2 kb as well as two other fainter bands at 5.6 and 7.2 kb (Fig. lA, B & C; lane 5). The strong signal at 5.2 kb may be due to the presence of co-migrating myosin gene fragments. Bam HI cut infrequently resulting in unresolved high molecular weight bands (Fig. lA, B & C; lane 4). The strongest signals seen are with the nematode myosin probe l nematode DNA hybridisation (Fig. lC, lane 2). It is
Cell Biology
International
Reports,
645
Vol. 13, No. 7, July 1989
DNA combinations give noteworthy that all other probe l that intensities suggesting roughly equivalent lower, four species are equally the myosin Sl genes for the than two of more the presence Moreover, homologous. digest of hybridising fragments in the Hind III that the myosin gene Tetrahvmena genomic DNA indicates may be distributed over a larger distance than that While these accounted for by the coding sequence alone. of a myosin gene, the in favour strongly argue data myosin gene and sequencing of a Tetrahvmena cloning which we are presently undertaking will define its exact It will be of particular interest: a) to define nature. unlike the actin gene (Cupples and Pearlman, if indeed, present; and b) 1986, Hirono et al, 1987), introns are the divergence of myosin proteins, if terms of in conventional double-headed molecule Tetrahvmena has the known as myosin or the small monomeric rodless molecule I (Korn and Hammer, 1988). REFERENCES Cupples, C.G. characterization thermophila.
R.E. (1986) Isolation and of the actin gene from Tetrahymena Proc. Natl. Acad. Sci. USA 83: 5160-5164
Hirano,
Endoh,H., Okada,N., Numata, 0. (1987) Tetrahymena actin. Cloning of the Tetrahymena actin gene of its gene product. J.Mol. Biol
M.,
Watanabe,Y. sequencing identification 181-192.
and Pearlman,
and and and 194:
Mitchell,
E.J. and Zimmerman, A.M. (1985) Biochemical evidence for the presence of an actin orotein in Tetrahymena pyriformic. J. Cell Sci. 73: 2791297.
Mitchell, Actin Cell
E.J. Zimmerman, and tubulin in Biol. 63: 389-396.
S. and Zimmerman, Tetrahymena. Can.
Hirano, M., Nakamura, M., Tsunemoto, M., -- _ Ohba,H., Numata, 0. and Watanabe-Y. (1978) actin actin
537-545. Warrick,
localization and possible in Tetrahymena cel1s.J.
H.M. and Spudich,
and function 379-421.
Maniatis,
T.,
in
cell
Fritsch,
Molecular Cloning: Harbor Laboratory
J.A.
motility.
a Press,
E.F.
A.M. (1985 1 J. Biochem
Yasuda,
T.
I
Tetrahymena biological roles of Biochem.(Tokyo) 102:
(1987) Myosin structure Ann. Rev. Cell Biol. 3:
and
Sambrook,J.
(1982)
Laboratory Manual (Cold Spring Cold Spring Harbor, N.Y. )
646
Cell Biology
international
Reports,
(1975) Detection Southern, E.M. DNA fragments separated among J. Mol. Biol. 94: 503-517. Mitchell,
E.J.
(1989)
J.
Mol.
Korn, E.D. and Hammer, J.A. cells. Ann. Rev. Biophys.
Paper
received
15.05.89
Vol. 73, No. 7, July 1989
of specific sequences by gel electrophoresis.
Biol.(in
Press).
(1988) Myosins Biophys. Chem.
Paper
accepted
of non-muscle 17: 23-45.
26.05.89.
International
Reports,
Vol. 13, No. 7, July
643
1989
EVIDENCE FOR A MYOSIN GENE IN TETRARYMENA E Jane National Research
Mitchell
and Luis
H Martin.
Research Council of Canada, Biotechnology Institute, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2 Canada.
INTRODUCTION The isolation of an actin gene from Tetrahvmena and Pearlman, 1986, Hirono et al, 1987) and (Cupples biochemical evidence for actin (Mitchell and Zimmerman, over has laid to rest the controversy the 1985) actin in these cells (Mitchell et al, existence of Immunofluorescence studies (Hirino et al, 1985). 1987) cell division, have implicated roles for actin in and exocytosis. The primary structure of endocytosis as derived from its gene sequence Tetrahvmena actin Pearlman, Hirono et al, (Cupples and 1986, 1978) it is the most divergent of all actins indicated that discovered to date. Therefore, the question exists not Tetrahvmena actin whether or interacts with myosin in some or all of the cellular functions in which it is involved. Myosin possesses APTase activity and is the molecular motor which interacts with actin filaments to produce movement (ref. Warrick and Spudich, 1987, for review). We therefore sought evidence for myosin in Tetrahvmena and we report here our preliminary evidence for a myosin gene. MATERIALSANDMETRODS Total genomic DNA from Tetrahvmena thermoohila SB255 (obtained from B Amos) was isolated and digested with endonucleases essentially as described (Maniatis et al, 1982). DNA 9 DNA hybridisation followed the method of Southern (Southern, 1975) except that Hybond-N membrane (Amersham) was used. The myosin gene probes used were: 1) a 688 bp Pst I/Bst EII cDNA fragment which encodes Acanthamoeba myosin Sl residues 99-244 (a kind gift from JA Hammer III); 2) a 2.4 kb Hind III/Bgl II cDNA fragment encoding most of the yeast Saccharomvces cerevisiae myosin Sl region (a kind gift from E Orr); and 3) a 2.5 kb Eco RI cDNA construct encoding the nematode Caenorhabditis eleaans myosin Sl (Mitchell et al, 1989). Endonuclease treated and 32P end-labelled EDNA was used for molecular size markers (Fig. lA,B & C; lane 1). Eco RI digested nematode genomic DNA was also electrophoresed (Fig. lA, B & C; lane 2).
0309-I 6511891070643-41$03.00/O
@ 1989 Academic
Press Ltd.
644
Cell Biology
Fig,
1
International
A
Reports,
B
Vol. 73, No. 7, July 1989
C
123451234512345 kb 23.1 9,4 6,6 4,4
2.3 2,o
Fig 1. Southern blot analysis of genomic DNA from DNA was hybridised with myosin gene probes Tetrahvmena. (as described in text) from: A) Acanthamoeba; B) yeast; Samples electrophoresed in each of A, B, C C) nematode. are: lane 1, molecular weight markers; lane 2, nematode DNA digested with Eco RI; Tetrahvmena DNA digested with: 3); Bam HI (lane 4); Hind III (lane 5). Eco RI (lane
RESULTS AWD DISCUSSION A similar pattern of hybridisation to Tetrahvmena genomic DNA was observed with each of the myosin gene probes, Acanthamoeba (Fig. lA), yeast (Fig. 1B) and nematode (Fig. 1C). Eco RI digestion of Tetrahvmena DNA yields three bands of approximately 18, 6.6, and 3.7 kb (Fig. lA,B &C; lane 3). The largest band is the weakest one and may represent a partial digestion product. Hind III digestion produces a strongly hybridising band at 5.2 kb as well as two other fainter bands at 5.6 and 7.2 kb (Fig. lA, B & C; lane 5). The strong signal at 5.2 kb may be due to the presence of co-migrating myosin gene fragments. Bam HI cut infrequently resulting in unresolved high molecular weight bands (Fig. lA, B & C; lane 4). The strongest signals seen are with the nematode myosin probe l nematode DNA hybridisation (Fig. lC, lane 2). It is
Cell Biology
International
Reports,
645
Vol. 13, No. 7, July 1989
DNA combinations give noteworthy that all other probe l that intensities suggesting roughly equivalent lower, four species are equally the myosin Sl genes for the than two of more the presence Moreover, homologous. digest of hybridising fragments in the Hind III that the myosin gene Tetrahvmena genomic DNA indicates may be distributed over a larger distance than that While these accounted for by the coding sequence alone. of a myosin gene, the in favour strongly argue data myosin gene and sequencing of a Tetrahvmena cloning which we are presently undertaking will define its exact It will be of particular interest: a) to define nature. unlike the actin gene (Cupples and Pearlman, if indeed, present; and b) 1986, Hirono et al, 1987), introns are the divergence of myosin proteins, if terms of in conventional double-headed molecule Tetrahvmena has the known as myosin or the small monomeric rodless molecule I (Korn and Hammer, 1988). REFERENCES Cupples, C.G. characterization thermophila.
R.E. (1986) Isolation and of the actin gene from Tetrahymena Proc. Natl. Acad. Sci. USA 83: 5160-5164
Hirano,
Endoh,H., Okada,N., Numata, 0. (1987) Tetrahymena actin. Cloning of the Tetrahymena actin gene of its gene product. J.Mol. Biol
M.,
Watanabe,Y. sequencing identification 181-192.
and Pearlman,
and and and 194:
Mitchell,
E.J. and Zimmerman, A.M. (1985) Biochemical evidence for the presence of an actin orotein in Tetrahymena pyriformic. J. Cell Sci. 73: 2791297.
Mitchell, Actin Cell
E.J. Zimmerman, and tubulin in Biol. 63: 389-396.
S. and Zimmerman, Tetrahymena. Can.
Hirano, M., Nakamura, M., Tsunemoto, M., -- _ Ohba,H., Numata, 0. and Watanabe-Y. (1978) actin actin
537-545. Warrick,
localization and possible in Tetrahymena cel1s.J.
H.M. and Spudich,
and function 379-421.
Maniatis,
T.,
in
cell
Fritsch,
Molecular Cloning: Harbor Laboratory
J.A.
motility.
a Press,
E.F.
A.M. (1985 1 J. Biochem
Yasuda,
T.
I
Tetrahymena biological roles of Biochem.(Tokyo) 102:
(1987) Myosin structure Ann. Rev. Cell Biol. 3:
and
Sambrook,J.
(1982)
Laboratory Manual (Cold Spring Cold Spring Harbor, N.Y. )
646
Cell Biology
international
Reports,
(1975) Detection Southern, E.M. DNA fragments separated among J. Mol. Biol. 94: 503-517. Mitchell,
E.J.
(1989)
J.
Mol.
Korn, E.D. and Hammer, J.A. cells. Ann. Rev. Biophys.
Paper
received
15.05.89
Vol. 73, No. 7, July 1989
of specific sequences by gel electrophoresis.
Biol.(in
Press).
(1988) Myosins Biophys. Chem.
Paper
accepted
of non-muscle 17: 23-45.
26.05.89.