Gene, 121 (1992) 343-346 0 1992 Elsevier Science Publishers
GENE
B.V. All rights reserved.
343
0378-l 119/92/$05.00
06765
Structure of the Drosophila (Recombinant
DNA;
insertional
Manabu Takahisa, Tadashi Miyake
mutagenesis;
Shin Togashi,
melanogaster
gene encoding cyclin A
cell cycle; mechanosensory
Ryu Ueda, Misa Mikuni,
organ; fruit fly; transcription)
Setsu Tsurumura,
Kanae Kondo
and
Laboratory of Cell Biology, Mitsubishi Kasei institute of Life Sciences, I I Minamiooya, Machida-shi, Tokyo 194. Japan Received
by Y. Sakaki:
17 April 1992; Revised/Accepted:
26 June/29
June 1992; Received
at publishers:
30 July 1992
SUMMARY
A P element (PE)-induced Drosophila melunogaster mutation, hari, affects the formation of the bristle mechanosensory organ in the adult fly. In this mutation, the site of PE insertion is in the first intron of the gene (CycA) encoding cyclin A (CycA). In order to analyze the hari mutant at the molecular level, we cloned and sequenced the cDNA and genomic DNA encoding CycA. CycA has seven exons and six introns, and its transcription unit spans 6 kb. All exon-intron junctions are compatible with the GT/AG consensus. Results of primer extension analysis and RNase protection assay indicate that CycA has major and minor transcription start points (tsp). To our knowledge, this is the first report on the CycA genomic sequence from a multicellular organism.
INTRODUCTION
The mitotic cell cycle in eukaryotes is thought to be controlled by a highly conserved mechanism involving cdc2 protein kinases and associated proteins such as homologous CycA and CycB (Nurse, 1990; Draetta,1990). Activation of CycA-cdc2 kinase complexes occurs earlier than that of CycB-cdc2 kinase complexes, and degradation of CycA occurs earlier than that of CycB (Minshull et al., 1990). At the G2-M transition, CycB is necessary for cdc2 kinase activation and for entry into mitosis (Minshull et al., 1989; Solomon et al., 1990). In addition, recent work implicates CycA but not CycB at the onset of DNA replica-
Correspondence to: Dr. M. Takahisa, Laboratory of Cell Biology, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan. Abbreviations:
Tel. (81-427)24-6249;
aa, amino acid(s); bp, base pan(s);
gene (DNA)
encoding
Introduction;
kb, kilobase
otide(s);
Fax (81-427)29-1252.
CycA;
PE, P (transposon)
CycA, cyclin A; Cyc.4,
CycB, cyclin B; D., Drosophila; hari, see or 1000 bp; N, any nucleoside; element; tsp, transcription
nt, nucle-
start point(s).
tion during the S phase of mammalian cells (Girard et al., 1991). In D. melanogaster, CycA and CycB have distinct roles of fundamental importance for the control of mitosis (Lehner and O’Farrell, 1989; 1990; Whitfield et al., 1990). However, spatial and temporal regulation of the cell cycle during embryonic development in different tissues is poorly understood. In this context, the mechanosensory organ known as the bristle represents a unique model system. An adult bristle consists of four cells designated as shaft, socket, neuron, and glia. In the process of individual bristle formation, a progenitor cell is determined and divides twice, giving rise to these four differentiated cells. Using PE-mediated insertional mutagenesis, we isolated a novel D. melunoguster CycA mutation with abnormal adult bristles. We called this mutation hari, a word for acupuncture needle in Japanese. In hari homozygotes, bristle shafts and/or sockets are frequently lost. In some cases, the shafts are bipolar and lack a socket, or they extend inside the epidermis. These defects originate from abnormal divisions of the progenitor cells of adult bristles. By contrast, the division of epidermal cells occurs normally. Genetic analyses suggest that the hari allele is a loss-of-function muta-
344 AAGCTTAGAACTARATAAATATG~A~T~ATGC~TT
-201
CGGCAAAAATAAAAATTAACGTTATACACATTTTTAAATTTGCGC CAGTCCTAAAAAA CAAACTTCTG ~T~GCWICACTGT~GG~~T~T~GAT~GA~ Tam reotlr (+I1 GCCACGACCTATCGATAGCTGGACAGTGTCATCTC~CGACGTGTTGACCGGG mm motif +1 AAAAAATCAAAAGAAGCCAG TTTC,TAAAATACTTI\ATATTAGCTAAGTCCGTGCGAGC p
-141 -81
-21
r
~ ~
40 100 160 220 280 340 400 3 460 23 520 43 580
. r MAS
c FQIHQDMSNKENPGIKIPAG c "KNTKQPLAVlGGKAEKNAL GCGGAGB APRANFAVLNGNNNVPRPAG CGCCGCGGGCCAATTTCC,CCGTGCTCAATGGCAACA4CA ~GTAAG?ACCGTTGCCGATTTCCCGTACATCCAGTGCTG~ CTTTTTATATTTTTATGGTGGTGGGATCGTCAWlCCACfG
646: 65 700 750
ATCTCCTAGTGGTGGTGGTGGTTTTCTCTCCCTTACCTCTCCTTT TCACTTCTCCATCTTACGTTACTCTCTTCTTAGAACGACC~TGCACTTACTTGCGCCTA ACCTA~CCTTAACTAACCTAG~GA~CAGGCCTTGC~TTACGCGCC~TTTT~
820 880 940
v
K
CATGCGTATAGGTGCACACATACACACACACACGAGCAATGG~ GCGCAACTTAAAGTTGGATCGATTTATGGGGGGGGATAATTTTCTTTTTTGCT TACTATATTAAGGATGCATATGT-G~T~ATCTTTGTGTTTTTA~TT~~A~~~~C CACTGCAATATTACCT~TATA4ARTTATTATTATG QVFRDVRNL ~TTTGTC~CTTCGTC~C~TTCCTTCCAG~?~CG~ NVDENVEY GA x K S NV VP r QFKTFSVYEDNNDTQVAPSG AATTCAAGACCTTCTCCGTGTATGAG(;ACAACAnCGATA KSLASLVDKENHDVKF
GENE
B.V. All rights reserved.
343
0378-l 119/92/$05.00
06765
Structure of the Drosophila (Recombinant
DNA;
insertional
Manabu Takahisa, Tadashi Miyake
mutagenesis;
Shin Togashi,
melanogaster
gene encoding cyclin A
cell cycle; mechanosensory
Ryu Ueda, Misa Mikuni,
organ; fruit fly; transcription)
Setsu Tsurumura,
Kanae Kondo
and
Laboratory of Cell Biology, Mitsubishi Kasei institute of Life Sciences, I I Minamiooya, Machida-shi, Tokyo 194. Japan Received
by Y. Sakaki:
17 April 1992; Revised/Accepted:
26 June/29
June 1992; Received
at publishers:
30 July 1992
SUMMARY
A P element (PE)-induced Drosophila melunogaster mutation, hari, affects the formation of the bristle mechanosensory organ in the adult fly. In this mutation, the site of PE insertion is in the first intron of the gene (CycA) encoding cyclin A (CycA). In order to analyze the hari mutant at the molecular level, we cloned and sequenced the cDNA and genomic DNA encoding CycA. CycA has seven exons and six introns, and its transcription unit spans 6 kb. All exon-intron junctions are compatible with the GT/AG consensus. Results of primer extension analysis and RNase protection assay indicate that CycA has major and minor transcription start points (tsp). To our knowledge, this is the first report on the CycA genomic sequence from a multicellular organism.
INTRODUCTION
The mitotic cell cycle in eukaryotes is thought to be controlled by a highly conserved mechanism involving cdc2 protein kinases and associated proteins such as homologous CycA and CycB (Nurse, 1990; Draetta,1990). Activation of CycA-cdc2 kinase complexes occurs earlier than that of CycB-cdc2 kinase complexes, and degradation of CycA occurs earlier than that of CycB (Minshull et al., 1990). At the G2-M transition, CycB is necessary for cdc2 kinase activation and for entry into mitosis (Minshull et al., 1989; Solomon et al., 1990). In addition, recent work implicates CycA but not CycB at the onset of DNA replica-
Correspondence to: Dr. M. Takahisa, Laboratory of Cell Biology, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan. Abbreviations:
Tel. (81-427)24-6249;
aa, amino acid(s); bp, base pan(s);
gene (DNA)
encoding
Introduction;
kb, kilobase
otide(s);
Fax (81-427)29-1252.
CycA;
PE, P (transposon)
CycA, cyclin A; Cyc.4,
CycB, cyclin B; D., Drosophila; hari, see or 1000 bp; N, any nucleoside; element; tsp, transcription
nt, nucle-
start point(s).
tion during the S phase of mammalian cells (Girard et al., 1991). In D. melanogaster, CycA and CycB have distinct roles of fundamental importance for the control of mitosis (Lehner and O’Farrell, 1989; 1990; Whitfield et al., 1990). However, spatial and temporal regulation of the cell cycle during embryonic development in different tissues is poorly understood. In this context, the mechanosensory organ known as the bristle represents a unique model system. An adult bristle consists of four cells designated as shaft, socket, neuron, and glia. In the process of individual bristle formation, a progenitor cell is determined and divides twice, giving rise to these four differentiated cells. Using PE-mediated insertional mutagenesis, we isolated a novel D. melunoguster CycA mutation with abnormal adult bristles. We called this mutation hari, a word for acupuncture needle in Japanese. In hari homozygotes, bristle shafts and/or sockets are frequently lost. In some cases, the shafts are bipolar and lack a socket, or they extend inside the epidermis. These defects originate from abnormal divisions of the progenitor cells of adult bristles. By contrast, the division of epidermal cells occurs normally. Genetic analyses suggest that the hari allele is a loss-of-function muta-
344 AAGCTTAGAACTARATAAATATG~A~T~ATGC~TT
-201
CGGCAAAAATAAAAATTAACGTTATACACATTTTTAAATTTGCGC CAGTCCTAAAAAA CAAACTTCTG ~T~GCWICACTGT~GG~~T~T~GAT~GA~ Tam reotlr (+I1 GCCACGACCTATCGATAGCTGGACAGTGTCATCTC~CGACGTGTTGACCGGG mm motif +1 AAAAAATCAAAAGAAGCCAG TTTC,TAAAATACTTI\ATATTAGCTAAGTCCGTGCGAGC p
-141 -81
-21
r
~ ~
40 100 160 220 280 340 400 3 460 23 520 43 580
. r MAS
c FQIHQDMSNKENPGIKIPAG c "KNTKQPLAVlGGKAEKNAL GCGGAGB APRANFAVLNGNNNVPRPAG CGCCGCGGGCCAATTTCC,CCGTGCTCAATGGCAACA4CA ~GTAAG?ACCGTTGCCGATTTCCCGTACATCCAGTGCTG~ CTTTTTATATTTTTATGGTGGTGGGATCGTCAWlCCACfG
646: 65 700 750
ATCTCCTAGTGGTGGTGGTGGTTTTCTCTCCCTTACCTCTCCTTT TCACTTCTCCATCTTACGTTACTCTCTTCTTAGAACGACC~TGCACTTACTTGCGCCTA ACCTA~CCTTAACTAACCTAG~GA~CAGGCCTTGC~TTACGCGCC~TTTT~
820 880 940
v
K
CATGCGTATAGGTGCACACATACACACACACACGAGCAATGG~ GCGCAACTTAAAGTTGGATCGATTTATGGGGGGGGATAATTTTCTTTTTTGCT TACTATATTAAGGATGCATATGT-G~T~ATCTTTGTGTTTTTA~TT~~A~~~~C CACTGCAATATTACCT~TATA4ARTTATTATTATG QVFRDVRNL ~TTTGTC~CTTCGTC~C~TTCCTTCCAG~?~CG~ NVDENVEY GA x K S NV VP r QFKTFSVYEDNNDTQVAPSG AATTCAAGACCTTCTCCGTGTATGAG(;ACAACAnCGATA KSLASLVDKENHDVKF
