Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary beta heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein.(ABSTRACT TRUNCATED AT 250 WORDS)

REFERENCES

Asai
D. J.
,
Brokaw
C. J.
,
Thompson
W. C.
,
Wilson
L.
(
1982
).
Two different monoclonal antibodies to alpha-tubulin inhibit the bending of reactivated sea urchin spermatozoa.
Cell Motil
2
,
599
614
Asai
D. J.
,
Tang
W.-J. Y.
,
Ching
N. S.
,
Gibbons
I. R.
(
1991
).
Cloning and sequencing of the ATP-binding domains of novel isoforms of sea urchin dynein.
J. Cell Biol
115
,
369
–.
Beckwith
S. M.
,
Asai
D. J.
(
1993
).
Ciliary dynein of Paramecium tetraurelia: Photolytic maps of the three heavy chains.
Cell Motil. Cytoskel
24
,
29
38
Beckwith
S. M.
,
Kandl
K. A.
,
Forney
J. D.
,
Asai
D. J.
(
1992
).
Dynein heavy chain isoforms are encoded by separate genes in Paramecium. Mol. Biol.
Cell
3
,
161
–.
Brokaw
C. J.
(
1991
).
Microtubule sliding in swimming sperm flagella: Direct and indirect measurements on sea urchin and tunicate spermatozoa.
J. Cell Biol
114
,
1201
1215
Chomczynski
P.
,
Sacchi
N.
(
1987
).
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
Cohen
J.
,
Adoutte
A.
,
Grandchamp
S.
,
Houde-Bine
L.-M.
,
Beisson
J.
(
1982
).
Immunocytochemical study of microtubular structures throughout the cell cycle of Paramecium. Biol.
Cell
44
,
35
44
Corthesy-Theulaz
I.
,
Pauloin
A.
,
Pfeffer
S. R.
(
1992
).
Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex.
J. Cell Biol
118
,
1333
1345
Eshel
D.
,
Urrestarazu
L. A.
,
Vissers
S.
,
Jauniaux
J.-C.
,
van Vliet-Reedijk
J. C.
,
Planta
R. J.
,
Gibbons
I. R.
(
1993
).
Cytoplasmic dynein is required for normal nuclear segregation in yeast.
Proc. Nat. Acad. Sci. USA
90
,
11172
11176
Forney
J.
,
Rodkey
K.
(
1992
).
A repetitive DNA sequence in Paramecium macronuclei is related to thesubunit of G proteins.
Nucl. Acids Res
20
,
5397
5402
Gibbons
I. R.
(
1988
).
Dynein ATPases as microtubule motors.
J. Biol. Chem
263
,
15837
15840
Gibbons
I. R.
,
Asai
D. J.
,
Tang
W.-J. Y.
,
Gibbons
B. H.
(
1992
).
A cytoplasmic dynein heavy chain in sea urchin embryos. Biol.
Cell
76
,
303
309
Gibbons
I. R.
,
Gibbons
B. H.
,
Mocz
G.
,
Asai
D. J.
(
1991
).
Multiple nucleotide-binding sites in the sequence of dyneinheavy chain.
Nature
352
,
640
643
Gibbons
I. R.
,
Lee-Eiford
A.
,
Mocz
G.
,
Phillipson
C. A.
,
Tang
W.-J. Y.
,
Gibbons
B. H.
(
1987
).
Photosensitized cleavage of dynein heavy chains: Cleavage at the ‘V1 site’ by irradiation at 365 nm in the presence of ATP and vanadate.
J. Biol. Chem
262
,
2780
2786
Kagami
O.
,
Kamiya
R.
(
1992
).
Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein.
J. Cell Sci
103
,
653
664
King
S. M.
,
Witman
G. B.
(
1988
).
Structure of theand heavy chains of the outer arm dynein from Chlamydomonas flagella. Location of epitopes and protease-sensitive sites.
J. Biol. Chem
263
,
9244
9255
Koonce
M. P.
,
Grissom
P. M.
,
McIntosh
J. R.
(
1992
).
Dynein from Dictyostelium: Primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein.
J. Cell Biol
119
,
1597
1604
Larsen
J.
,
Barakalow
K.
,
Hamasaki
T.
,
Satir
P.
(
1991
).
Structural and functional characterization of paramecium dynein: Initial studies.
J. Protozool
38
,
55
61
Lefebvre
P. A.
,
Rosenbaum
J. L.
(
1986
).
Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration.
Annu. Rev. Cell Biol
2
,
517
546
Li
Y.-Y.
,
Yeh
E.
,
Hays
T.
,
Bloom
K.
(
1993
).
Disruption of mitotic spindle orientation in a yeast dynein mutant.
Proc. Nat. Acad. Sci. USA
90
,
10096
10100
Machemer
H.
,
Ogura
A.
(
1979
).
Ionic conductances of membranes in ciliated and deciliated Paramecium.
J. Physiol
296
,
49
60
Mikami
A.
,
Paschal
B. M.
,
Mazumdar
M.
,
Vallee
R. B.
(
1993
).
Molecular cloning of the retrograde transport motor cytoplasmic dynein (MAP1C).
Neuron
10
,
787
796
Mitchell
D. R.
,
Brown
K. S.
(
1993
).
Sequence analysis of two dynein heavy chains from Chlamydomonas. Mol. Biol.
Cell
4
,
47
–.
Neely
M. D.
,
Erickson
H. P.
,
Boekelheide
K.
(
1990
).
HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits.
J. Biol. Chem
265
,
8691
8698
Ogawa
K.
(
1991
).
Four ATP-binding sites in the midregion of theheavy chain of dynein.
Nature
352
,
643
645
Pallini
V.
,
Mencarelli
C.
,
Bracci
L.
,
Contorni
M.
,
Ruggiero
P.
,
Tiezzi
A.
,
Manetti
R.
(
1983
).
Cytoplasmic nucleoside-triphosphatases similar to axonemal dynein occur widely in different cell types.
J. Submicrosc. Cytol
15
,
229
235
Paschal
B. M.
,
Schpetner
H. S.
,
Vallee
R. B.
(
1987
).
MAP1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties.
J. Cell Biol
105
,
1273
1282
Pfarr
C. M.
,
Coue
M.
,
Grissom
P. M.
,
Hays
T. S.
,
Porter
M. E.
,
McIntosh
J. R.
(
1990
).
Cytoplasmic dynein is localized to kinetochores during mitosis.
Nature
345
,
263
265
Piperno
G.
(
1984
).
Monoclonal antibodies to dynein subunits reveal the existence of cytoplasmic antigens in sea urchin egg.
J. Cell Biol
98
,
1842
1850
Piperno
G.
,
Ramanis
Z.
,
Smith
E. F.
,
Sale
W. S.
(
1990
).
Three distinct inner dynein arms in Chlamydomonas flagella: Molecular composition and location in the axoneme.
J. Cell Biol
110
,
379
389
Porter
M.
,
Knott
J.
,
Gardner
L.
,
Farlow
S.
,
Myster
S.
,
Mansanares
K.
(
1992
).
Characterization of the dynein gene family in Chlamydomonas reinhardtii. Mol. Biol.
Cell
3
,
161
–.
Satir
P.
(
1968
).
Studies on cilia III. Further studies on the cilium tip and a ‘sliding filament’ model of ciliary motility.
J. Cell Biol
39
,
77
94
Schnapp
B. J.
,
Reese
T. S.
(
1989
).
Dynein is the motor for retrograde axonal transport of organelles.
Proc. Nat. Acad. Sci. USA
86
,
1548
1552
Schroeder
C. C.
,
Fok
A. K.
,
Allen
R. D.
(
1990
).
Vesicle transport alongmicrotubule ribbons and isolation of cytoplasmic dynein from Paramecium.
J. Cell Biol
111
,
2553
2562
Schroer
T. A.
,
Steuer
E. R.
,
Sheetz
M. P.
(
1989
).
Cytoplasmic dynein is a minus end-directed motor for membranous organelles.
Cell
56
,
937
946
Shpetner
H. S.
,
Paschal
B. M.
,
Vallee
R. B.
(
1988
).
Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP1C).
J. Cell Biol
107
,
1001
1009
Steuer
E. R.
,
Wordeman
L.
,
Schroer
T. A.
,
Sheetz
M. P.
(
1990
).
Localization of cytoplasmic dynein to mitotic spindles and kinetochores.
Nature
345
,
266
268
Travis
S. M.
,
Nelson
D. L.
(
1988
).
Purification and properties of dyneins from Paramecium cilia.
Biochim. Biophys. Acta
966
,
73
83
Vaisberg
E. A.
,
Koonce
M. P.
,
McIntosh
J. R.
(
1993
).
Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.
J. Cell Biol
123
,
849
858
Vallee
R. B.
(
1993
).
Molecular analysis of the microtubule motor dynein.
Proc. Nat. Acad. Sci. USA
90
,
8769
8772
Verde
F.
,
Berrez
J.-M.
,
Antony
C.
,
Karsenti
E.
(
1991
).
Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: Requirement for phosphorylated factors and cytoplasmic dynein.
J. Cell Biol
112
,
1177
1187
Wilkerson
C. G.
,
King
S. M.
,
Witman
G. B.
(
1992
).
Sequence analysis of multiple dynein heavy chain genes from Chlamydomonas reinhardtii. Mol. Biol.
Cell
3
,
161
–.
Wilkerson
C. G.
,
King
S. M.
,
Witman
G. B.
(
1993
).
Sequence analysis of thedynein heavy chain from the Chlamydomonas outer arm. Mol. Biol.
Cell
4
,
47
–.
Witman
G. B.
(
1992
).
Axonemal dyneins.
Curr. Opin. Cell Biol
4
,
74
79
Wordeman
L.
,
Steuer
E. R.
,
Sheetz
M. P.
,
Mitchison
T.
(
1991
).
Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes.
J. Cell Biol
114
,
285
294
Zhang
Z.
,
Tanaka
Y.
,
Nonaka
S.
,
Aizawa
H.
,
Kawasaki
H.
,
Nakata
T.
,
Hirokawa
N.
(
1993
).
The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.
Proc. Nat. Acad. Sci. USA
90
,
7928
7932
This content is only available via PDF.