The Drosophila kelch gene produces a single transcript with a UGA stop codon separating two open reading frames (ORF1 and ORF2). From the transcript, 76 kDa ORF1 and 160 kDa full-length (ORF1 + ORF2) proteins are made. The expression of these two proteins is regulated in a tissue-specific manner causing the ratio of full-length to ORF1 protein to vary in different tissues. The only detected defect for kelch mutants is female sterility, and kelch protein is localized to the ovarian ring canals. kelch mutant ring canals are disorganized and have partly occluded lumens, causing a failure to transport cytoplasm. ORF1 and full-length kelch proteins co-sediment with ring canals suggesting that both proteins are found in the ring canals. Transgenetic analysis reveals that ORF1 kelch protein is sufficient to rescue ring canal morphology and fertility. In addition, we have mutated the UGA stop codon to a UAA stop codon and to three sense codons that allow constitutive readthrough. Analysis of these mutants reveals that a full-length kelch protein can partially compensate for the loss of endogenous kelch, but the residue included at the stop codon is critical for function. Finally, these studies suggest that the mechanism of stop codon suppression of kelch is by tRNA suppression.

REFERENCES

Atkins
J.
,
Weiss
R.
,
Gesteland
R.
(
1990
).
Ribosome gymnastics-Degree of difficulty 9.5, style 10.0.
Cell
62
,
413
423
Bardwell
V.
,
Treisman
R.
(
1994
)
The POZ domain: a conserved protein-protein interaction motif.
Genes Dev
8
,
1664
1677
Baron
C.
,
Heider
J.
,
Böck
A.
(
1993
)
Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA.
Proc. Nat. Acad. Sci. USA
90
,
4181
4185
Bergstrom
D.
,
Merli
C.
,
Cygan
J.
,
Shelby
R.
,
Blackman
R.
(
1995
)
Regulatory autonomy and molecular characterization of the Drosophila out at first gene.
Genetics
139
,
1331
1346
Berry
M.
,
Banu
L.
,
Larsen
P.
(
1991
)
Type I iodothyronine deiodinase is a selenocysteine-containing enzyme.
Nature
349
,
438
440
Berry
M.
,
Harney
J.
,
Ohama
T.
,
Hatfield
D.
(
1994
)
Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations.
Nucleic Acids Res
22
,
3753
3759
Berry
M.
,
Larsen
P.
(
1993
)
Recognition of UGA as a selenocysteine codon in eukaryotes: a review of recent progress.
Biochem. Soc. Trans
21
,
827
832
Berry
M. J.
,
Banu
L.
,
Chen
Y.
,
Mandel
S. J.
,
Kieffer
J. D.
,
Harney
J. W.
,
Larsen
P. R.
(
1991
)
Recognition of UGA as a selenocysteine codon in Type I deiodinase requires sequences in the 3untranslated region.
Nature
353
,
273
276
Böck
A.
,
Forchhammer
K.
,
Heider
J.
,
Baron
C.
(
1991
)
Selenoprotein synthesis: an expansion of the genetic code.
Trends Biochem. Sci
16
,
463
467
Brown
C.
,
Stockwell
P.
,
Trotman
C.
,
Tate
W.
(
1990
)
Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes.
Nucleic Acids Res
18
,
6339
6345
Brown
N. H.
,
Kafatos
F. C.
(
1988
)
Functional cDNA libraries from Drosophilaembryos.
J. Mol. Biol
203
,
425
437
Bullitt
E. S. A.
,
DeRosier
D. J.
,
Coluccio
L. M.
,
Tilney
L. G.
(
1988
)
Three-dimensional reconstruction of an actin bundle.
J. Cell Biol
107
,
597
611
Chambers
I.
,
Frampton
J.
,
Goldfarb
P.
,
Affara
N.
,
McBain
W.
,
Harrison
P.
(
1986
)
The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the ‘termination’ codon, TGA.
EMBO J
5
,
1221
1227
Chardin
P.
,
Courtois
G.
,
Mattei
M.-G.
,
Gisselbrecht
S.
(
1991
)
The KUP gene, located of human chromosome 14, encodes a protein with two distant zinc fingers.
Nucleic Acids Res
19
,
1431
1436
Chen
W.
,
Zollman
S.
,
Couderc
J.-L.
,
Laski
F.
(
1995
)
The BTB domain of bric à brac mediates dimerization in vitro.
Mol. Cell. Biol
15
,
3424
3429
Comer
A.
,
Searles
L.
,
Kalfayan
L.
(
1992
)
Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression.
Gene
118
,
171
179
Curtis
D.
,
Lehmann
R.
,
Zamore
P.
(
1995
)
Translational regulation in development.
Cell
81
,
171
178
Davey
J.
,
Becker
K.
,
Schneider
M.
,
Germain
D. S.
,
Galton
V.
(
1995
)
Cloning of a cDNA for the type II iodothyronine deiodinase.
J. Biol. Chem
270
,
26786
26789
DiBello
P. R.
,
Withers
D. A.
,
Bayer
C. A.
,
Fristrom
J. W.
,
Guild
G. M.
(
1991
)
The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers.
Genetics
129
,
385
397
Eichinger
L.
,
Bomblies
L.
,
Vandekerckhove
J.
,
Schleicher
M.
,
Gettemans
J.
(
1996
)
A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.
EMBO J
15
,
5547
5556
Evan
G.I.
,
Lewis
G.K.
,
Ramsay
G.
,
Bishop
J.M.
(
1985
)
Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol
5
,
3610
3616
Feng
Y.-X.
,
Copeland
T.
,
Oroszlan
S.
,
Rein
A.
,
Levin
J.
(
1990
)
Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.
Proc. Nat. Acad. Sci. USA
87
,
8860
8863
Feng
Y.-X.
,
Hatfield
D.
,
Rein
A.
,
Levin
J.
(
1989
)
Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA.
J. Virol
63
,
2405
2410
Feng
Y.-X.
,
Levin
J.
,
Hatfield
D.
,
Schaeffer
T.
,
Gorelick
R.
,
Rein
A.
(
1989
)
Suppression of UAA and UGA termination codons of mutant murine leukemia viruses.
J.Virol
63
,
2870
2873
Forchhammer
K.
,
Leinfelder
W.
,
Böck
A.
(
1989
)
Identification of a novel translation factor necessary for the incorporation of selenocysteine into protein.
Nature
342
,
453
456
Harrison
S. D.
,
Travers
A. A.
(
1990
)
The tramtrack gene encodes a Drosophila finger protein that interacts with the ftz transcriptional regulatory region and shows a novel embryonic expression pattern.
EMBO J
9
,
207
216
Higuchi
M.
,
Single
F.
,
Köhler
M.
,
Sommer
B.
,
Sprengel
R.
,
Seeburg
P.
(
1993
)
RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency.
Cell
75
,
1361
1370
Hill
K. E.
,
Lloyd
R. S.
,
Yang
J.-G.
,
Read
R.
,
Burk
R. F.
(
1991
)
The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame.
J. Biol. Chem
266
,
10050
10053
Horton
R.
,
Hunt
H.
,
Ho
S.
,
Pullen
J.
,
Pease
L.
(
1989
)
Engineering hybrid gene without the use of restriction enzymes: gene splicing by overlap extension.
Gene
77
,
61
68
Huang
W.
,
Ao
S.-Z.
,
Casjens
S.
,
Orlandi
R.
,
Zeikus
R.
,
Weiss
R.
,
Winge
D.
,
Fang
M.
(
1988
)
A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60.
Science
239
,
1005
1012
Hubert
N.
,
Walczak
R.
,
Carbon
P.
,
Krol
A.
(
1996
)
A protein binds the selenocysteine insertion element in the 3-UTR of mammalian selenoprotein mRNAs.
Nucleic Acids Res
24
,
464
469
Karimpour
I.
,
Cutler
M.
,
Shih
D.
,
Smith
J.
,
Kleene
K.
(
1992
)
Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: Identification of three in-phase TGA selenocysteine codons.
DNA Cell Biol
11
,
693
699
Koonin
E. V.
,
Senkevich
T. G.
,
Chernos
V. I.
(
1992
)
Protein sequence motifs: A family of DNA virus genes that consists of fused portions of unrelated cellular genes.
Trends Biochem. Sci
17
,
213
214
Mahowald
A.
,
Goralski
T.
,
Caulton
J.
(
1983
)
In vitro activation of Drosophila eggs.
Dev. Biol
98
,
437
445
Matsufuji
S.
,
Matsufuji
T.
,
Miyazaki
Y.
,
Murakami
Y.
,
Atkins
J.
,
Gesteland
R.
,
Hayashi
S.
(
1995
)
Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme.
Cell
80
,
51
60
McCaughan
K.
,
Brown
C.
,
Dalphin
M.
,
Berry
M.
,
Tate
W.
(
1995
)
Translational termination efficiency in mammals is influenced by the base following the stop codon.
Proc. Nat. Acad. Sci. USA
92
,
5431
5435
Melcher
T.
,
Maas
S.
,
Herb
A.
,
Sprengel
R.
,
Seeburg
P.
,
Higuchi
M.
(
1996
)
A mammalian RNA editing enzyme.
Nature
379
,
460
464
Owen
C.
,
DeRosier
D.
(
1993
)
A 13-Å map of the actin-scruin filament from the Limulus acrosomal process.
J. Cell Biol
123
,
337
344
Pirrotta
V.
(
1988
)
Vectors for P-mediated transformation in Drosophila.
Biotechnology
10
,
437
456
Robinson
D.
,
Cant
K.
,
Cooley
L.
(
1994
)
Morphogenesis of Drosophila ovarian ring canals.
Development
120
,
2015
2025
Rocher
C.
,
Lalanne
J.-L.
,
Chaudiere
J.
(
1992
)
Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.
Eur. J. Biochem
205
,
955
960
Rodesch
C.
,
Geyer
P. K.
,
Patton
J. S.
,
Bae
E.
,
Nagoshi
R. N.
(
1995
)
Developmental analysis of the ovarian tumor gene during Drosophila oogenesis.
Genetics
141
,
191
202
Salvatore
D.
,
Low
S.
,
Berry
M.
,
Maia
A.
,
Harney
J.
,
Croteau
W.
,
Germain
D. S.
,
Larsen
P.
(
1995
)
Type 3 iodothyronine deiodinase: Cloning, in vitro expression and functional analysis of the placental selenoenzyme.
J. Clin. Invest
96
,
2421
2430
Schmid
M. F.
,
Agris
J. M.
,
Jakana
J.
,
Matsudaira
P.
,
Chiu
W.
(
1994
)
Three-dimensional structure of a single filament in the Limulus acrosomal bundle: Scruin binds to homologous helix-loop-beta motifs in actin.
J. Cell Biol
124
,
341
350
Schupbach
T.
,
Wieschaus
E.
(
1991
)
Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology.
Genetics
129
,
1119
1136
Senkevich
T. G.
,
Muravnik
G. L.
,
Pozdnyakov
S. G.
,
Chizhikov
V. E.
,
Ryazankina
O. I.
,
Shchelkunov
S. N.
,
Koonin
E. V.
,
Chernos
V. I.
(
1993
)
Nucleotide sequence of Xho I O fragment of ectromelia virus DNA reveals significant differences from vaccinia virus.
Virus Res
30
,
73
88
Serano
T. L.
,
Cheung
H.-K.
,
Frank
L. H.
,
Cohen
R. S.
(
1994
)
P element transformation vectors for studying Drosophila melanogaster oogenesis and early embryogenesis.
Gene
138
,
181
186
Shen
Q.
,
McQuilkin
P.
,
Newburger
P.
(
1995
)
RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA.
J. Biol. Chem
270
,
30448
30452
Singleton
K.
,
Woodruff
R.
(
1994
)
The osmolarity of adult Drosophila hemolymph and its effect on oocyte-nurse cell electrical polarity.
Dev. Biol
161
,
154
167
Sommer
B.
,
Köhler
M.
,
Sprengel
R.
,
Seeburg
P.
(
1991
)
RNA editing in brain controls a determinant of ion flow in glutamate-gated channels.
Cell
67
,
11
19
Steinhauer
W.
,
Walsh
R.
,
Kalfayan
L.
(
1989
)
Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for the ovarian tumor protein.
Mol. Cell. Biol
9
,
5726
5732
Theurkauf
W.
,
Smiley
S.
,
Wong
M.
,
Alberts
B.
(
1992
)
Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.
Development
115
,
923
936
Tilney
L. G.
(
1975
)
Actin filaments in the acrosomal reaction of Limulus sperm.
J. Cell Biol
64
,
289
310
Tilney
L. G.
,
Tilney
M. S.
,
Guild
G. M.
(
1996
)
Formation of actin filament bundles in the ring canals of developing Drosophila follicles.
J. Cell Biol
133
,
61
74
Valle
R.
,
Morch
M.-D.
(
1988
)
Stop making sense.
FEBS Lett
235
,
1
15
Varkey
J.
,
Muhlrad
P.
,
Minniti
A.
,
Do
B.
,
Ward
S.
(
1995
)
The Caenorhabditis elegans spe-26 gene is necessary to form spermatids and encodes a protein similar to the actin-associated proteins kelch and scruin.
Genes Dev
9
,
1074
1086
Vendeland
S.
,
Beilstein
M.
,
Yeh
J.-Y.
,
Ream
W.
,
Whanger
P.
(
1995
)
Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium.
Proc. Nat. Acad. Sci. USA
92
,
8749
8753
von Bulow
M.
,
Heid
H.
,
Hess
H.
,
Franke
W.
(
1995
)
Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton.
Exp. Cell Res
219
,
407
413
Way
M.
,
Sanders
M.
,
Chafel
M.
,
Tu
Y.-H.
,
Knight
A.
,
Matsudaira
P.
(
1995
)
-scruin, a homologue of the actin crosslinking protein scruin, is localized to the acrosomal vesicle of Limulus sperm.
J. Cell Sci
108
,
3155
3162
Way
M.
,
Sanders
M.
,
Garcia
C.
,
Sakai
J.
,
Matsudaira
P.
(
1995
)
Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.
J. Cell Biol
128
,
51
60
Xue
F.
,
Cooley
L.
(
1993
)
kelch encodes a component of intercellular bridges in Drosophila egg chambers.
Cell
72
,
681
693
Yamada
K.
(
1995
)
A new translational elongation factor for selenocysteyl-tRNA in eucaryotes.
FEBS Lett
377
,
313
317
Yue
L.
,
Spradling
A.
(
1992
)
hu-li tai shao, a gene required for ring canal formation during Drosophila oogenesis, encodes a homolog of adducin.
Genes Dev
6
,
2443
2454
Zollman
S.
,
Gödt
D.
,
Prive
G.
,
Couderc
J.-L.
,
Laski
F.
(
1994
)
The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila.
Proc. Nat. Acad. Sci. USA
91
,
10717
10721
This content is only available via PDF.