Directory UMM :Journals:Journal of Insect Physiology:Vol46.Issue7.Jul2000:

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www.elsevier.com/locate/jinsphys

Expression of Drosophila homologue of senescence marker

protein-30 during cold acclimation

Shin G. Goto

Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan

Received 2 August 1999; accepted 30 November 1999

Abstract

Gene expression during cold acclimation at a moderately low temperature (15°C) was studied in Drosophila melanogaster using a subtraction technique. A gene homologous to senescence marker protein-30 (SMP30), which has a Ca2+_{-binding function, was}

up-regulated at the transcription level after acclimation to 15°C. This gene (henceforth referred to as Dca) was also expressed at a higher level in individuals reared at 15°C from the egg stage than in those reared at 25°C. Moreover, DCA mRNA increased at the senescent stage in Drosophila, although SMP30 is reported to decrease at senescent stages in mammals. In situ hybridization to polytene chromosomes revealed that the Dca gene was located at 88D on chromosome 3R. The 59flanking region of this gene had AP-1 (a transcription factor of SMP30) binding sites, stress response element and some other transcription factor binding sites. The function of DCA was discussed in relation to the possible regulation of cytosolic Ca2+_{concentration.}__{2000 Elsevier Science}

Keywords: Drosophila; Cold acclimation; Dca; Senescence marker protein-30; Senescence

1. Introduction

In general, ectothermal organisms, from bacteria to higher plants or vertebrates, become more cold tolerant when maintained at low temperature. In bacteria, plants or fish, this change in cold tolerance is suggested to be associated with changes in fatty acid composition of phospholipids; percentages of saturated fatty acids in phospholipids decrease in individuals reared at low tem-perature, and this change results in the maintenance of membrane functions at cold (Hochachka and Somero, 1984; Hazel and Williams, 1990). However, Ohtsu et al. (1998, 1999) found that Drosophila species exhibit an inverse response; individuals acclimated to low tempera-ture decreased the degree of unsaturation of phospholip-ids. Thus, the relation between the qualitative changes of phospholipids and cold tolerance is still unclear in Drosophila. On the other hand, it is well known in insects that cryoprotectants such as glycerol, sorbitol or trehalose are often accumulated when insects are

main-* Fax:+81-11-706-2225.

E-mail address: [email protected] (S.G. Goto).

tained at low temperatures (reviewed by Storey and Sto-rey, 1991). In addition, novel proteins called ‘cold acclimation proteins’ have been recently reported in some bacteria and plants which were acclimated to low temperature (Neven et al., 1993; Berger et al., 1997; Monroy et al., 1998). Moreover, in many cold-water marine fishes and terrestrial arthropods, antifreeze pro-tein and ice-nucleating propro-teins were reported (reviewed by Lee, 1991). However, there have been no reports of such proteins in Drosophila. Here, I report an attempt to find genes that are specifically expressed during acclimation at moderate cold in Drosophila melanogas-ter using subtraction technique.

2. Materials and methods 2.1. Flies

D. melanogaster Meigen (Canton S strain) was main-tained under laboratory conditions (continuous light at

25°C) on cornmeal–malt medium, and used for

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2.2. Cold acclimation

Flies maintained at 25°C were transferred to vials

(containing food medium) that were cooled to 15°C prior

to experiment, and then maintained at 15°C (continuous

light) for 1 or 2 days. Half lethal temperature (temperature that kills half the population when exposed

for 24 h) was 3.0°C for individuals reared at 25°C to

the 7-day adult stage, and 1.0°C for those acclimated to

15°C for 1 day. When flies were reared at 15°C from

the egg stage to the 14-day adult stage, half lethal tem-perature was ₂3.4°C.

2.3. RNA extraction and mRNA purification

RNA was extracted according to Goto et al. (1998). mRNA was purified from total RNA using BioMag mRNA purification kit (PerSeptive Biosystems) accord-ing to the supplier’s instruction.

2.4. Subtraction and differential screening 2.4.1. cDNA synthesis

ds CDNAs were synthesized from 4 µg of poly(A)+

RNA with oligo (dT) according to Sambrook et al. (1989) and digested with Rsa I.

Henceforth, the cDNA derived from flies acclimated

to 15°C for 1 day and reference cDNA derived from

control flies reared at 25°C to the 7-day adult stage were referred to as ‘tester’ and ‘driver’ respectively. Digested cDNAs were phenol-extracted, ethanol-precipitated and dissolved in 5.5 µl of water.

2.4.2. Adaptor ligation and subtractive hybridization The following procedure was mainly according to

Gurskaya et al. (1996). Oneµl of tester and driver were

diluted with 5µl of water. Then, 2µl of tester or driver

solution was mixed with 2µM Adaptor 1 or 2µM

Adap-tor 2R (Table 1) in 10-µl reactions. After ligation at 16°C

for 18 h with 350 U of T4 DNA ligase (TaKaRa), the

products were added with 1 µl of 0.5 M EDTA, heated

at 70°C for 5 min and stored at 220°C.

For the first round of the subtractive hybridization, 1.5

µl of Rsa I-digested driver was mixed with 1.5 µl of

Adaptor 1-ligated tester or Adaptor 2R-ligated tester: the former and latter mixtures were referred to as ‘forward

A’ and ‘forward B’, respectively. In addition, 1.5µl of

Rsa I-digested tester was mixed with 1.5 µl of Adaptor 1-ligated driver or Adaptor 2R-ligated driver: the former and latter mixtures were referred to as ‘reverse A’ and ‘reverse B’, respectively. These mixtures were added

with 1µl of hybridization buffer (200 mM HEPES–HCl,

pH 7.5/2 M Na/0.08 mM EDTA). After mineral oil was

overlaid on the solutions, DNA was denatured at 98°C

for 1.5 min and annealed at 68°C for 12 h.

For the second round of the subtractive hybridization,

1µl of Rsa I-digested driver or Rsa I-digester tester was

mixed with 1 µl of hybridization buffer and 2 µl of

water. These solutions were overlaid with mineral oil

and heated at 98°C for 1.5 min. Forward A and reverse

A mixtures were mixed with forward B and reverse B, respectively. Then, forward mixture was added with 1

µl of freshly denatured driver, and reverse mixture was

added with 1µl of denatured tester. After incubation at

68°C for 24 h, samples were mixed with 200 µl of

dilution buffer (20 mM HEPES–HCl, pH 8.3/50 mM

NaCl/0.2 mM EDTA, pH 8.0) and stored at ₂20°C.

2.4.3. Selective PCR amplification

Primary PCR used 1µl of the sample and final

concen-tration of 0.2 mM of dNTP, 0.4 µM of PCR primer 1

(Table 1) 1×PCR reaction buffer as formulated by

Clon-tech and 1×Advantage cDNA polymerase mix

(Clontech) in total volume of 25 µl. After incubation at

75°C for 5 min and heating at 94°C for 25 s, 35 cycles of PCR (10 s at 94°C; 30 s at 66°C; 1.5 min at 72°C) were conducted.

Secondary reaction used the same components for the primary reaction, except that the primers were Nested

PCR primer 1 and 2R (Table 1) and the target was 1µl

of 10-fold-diluted primary PCR products. PCR

con-ditions were 15 cycles of 10 s at 94°C, 30 s at 68°C,

and 1.5 min at 72°C. Henceforth, PCR products derived

from the forward mixture and from the reverse one in subtractive hybridization were referred to as ‘forward subtracted cDNAs’ and ‘reverse subtracted cDNAs’, respectively.

2.4.4. Differential screening

Forward subtracted cDNAs were ligated into plasmid using pGEM-T Vector (Promega) according to the sup-plier’s instruction.

cDNA inserts from colonies were amplified with col-ony direct PCR. The reaction used 0.5 U of AmpliTaq

(Perkin Elmer) and final concentration of 1×PCR buffer

II as formulated by Perkin Elmer, 0.5µM of Nested PCR

primer 1 and 2R (Table 1), 0.2 mM of dNTP and 1.5

mM of MgCl2in a total volume of 20µl. Amplification

was performed with 28 cycles of 30 s at 93°C, 30 s at

68°C, and 30 s at 72°C.

PCR products were mixed with the same volume of

0.6 N NAOH. Oneµl of these mixtures were blotted on

to two nylon membranes.

Forward and reverse subtracted cDNAs were digested with Rsa I, EcoT52 I and Sma I, and applied to QIA-quick PCR purification kit (QIAGEN) in order to remove the Adaptor 1 and 2R. DNA was labeled with Digoxig-enin-11dUTP using DIG DNA labeling kit (Boehringer Mannheim).

Each membrane was hybridized with probes derived from forward and reverse subtracted cDNAs, respect-ively. Hybridization and detection was performed using

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Table 1

Sequences of primers and adaptors

Oligonucleotides Sequence

DF-1 59-CTC CAG CAG GAT TTT ACC GGT GCT T-39 DF-2 59-GAC CTC CAA AAG CCA CCG AGG TGA T-39 DF-3 59-AAC AGG GTG CGA GTG ACC TT-39

DF-5 59-TGA AGA TAT CGC CAC TGT CGG-39 DF-6 59-CGA GGT CCA CGT AGT ACA GAC TCT G-39 DF-7 59-CCT CAC ACC TAC ACA TAG CA-39

DF-8 59-GCA ACA CTC GAA TAA GCC A-39

DR-1 59-AGG TGC AGC CGG ATC TGA AGG AAA A-39 DR-2 59-TCA TCG TCC AGT GGG ATG GAG TCT C-39 DR-3 59-CTA CAA TCA GAG CAC CGG CG-39 DR-5 59-CCA CTA ACT ACG AAC ACC GGG TT-39 DR-X 59-AAG GAG TCG GGA CAG GGG CAC AGT T-39 DR-X2 59-TTG CTC GCG CTC TCA AGA-39

DR-X3 59-GTG TGT GAG ATT GCG AAT GG-39 M13-M4 59-GTT TTC CCA GTC ACG AC-39 M13-RV 59-CAG GAA ACA GCT ATG AC-39

Nested PCR primer 1 59-TCG AGC GGC CGC CCG GGC AGG T-39 Nested PCR primer 2R 59-AGC GTG GTC GCG GCC GAG GT-39 PCR primer 1 59-CTA ATA CGA CTC ACT ATA GGG C-39 Adaptor primer 59-CCA TCC TAA TAC GAC TCA CTA TAG GGC-39

Adaptor 1 59-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3939-GGC CCG TCC A-59 Adaptor 2R 59-CTA ATA CGA CTC ACT ATA GGG CAG CGT GGT CGC GGC CGA GGT-3939-GCC GGC TCC A-59 RACE adaptor 59-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3939-PO4-CCC GTC

CA-NH2-39

DIG luminescent detection kit (Boehringer Mannheim) according to the supplier’s instruction.

2.5. Northern hybridization

Poly(A)+ RNAs were electrophoresed in denaturing

gel and transferred to nylon membrane according to Sambrook et al. (1989). Hybridization and detection were made as above. D. melanogaster ras2 gene probe, pUC8-HB-1.2 kb, was used as a control (Bishop and Corces, 1988; Juni et al., 1996).

2.6. RACE

To sequence the full length of cDNA, RACE (Rapid Amplification of cDNA Ends) was performed according to Siebert et al. (1995). ds cDNA was ligated to RACE adaptor (Table 1). The ligation product was diluted to 250-fold with water. Primary reaction for nested PCR

used 1µl of the sample and a final concentration of 0.2

mM of dNTP, 0.2 µM of Adaptor primer, 0.2 µM of

DF-2 primer for 5₉-RACE but DR-2 primer was used

instead of DF-2 for 3₉-RACE (Table 1) and

1×Advantage cDNA polymerase mix (Clontech).

Ampli-fication was performed for 30 s denaturing at 94°C, 5

cycles of 5 s at 94°C and 2 min at 72°C, 5 cycles of 5

s at 94°C and 2 min at 70°C, and 25 cycles of 5 s at

94°C and 2 min at 68°C.

Secondary reaction used the same components for the

primary reaction, except that the primers for 5₉- and 3₉ -RACE were DF-1 and DR-1 (Table 1), respectively, and

the targets were 1 µl of 250-fold-diluted primary

pro-ducts. PCR conditions were as above, except that the final step was 20 cycles.

2.7. DNA extraction

Genome of D. melanogaster was extracted with QIAGEN genomic-tip (QIAGEN) according to sup-plier’s instruction.

2.8. Amplification of 59 flanking region

Genome was digested with Dra I, Sca I, Eco RV, Pvu II and Stu I. Digested products were ligated to RACE adaptor and diluted to 10-fold with water.

Primary reaction for nested PCR used the same components that were used in RACE, except that the

primer was DF-2 (Table 1) and the targets were 1 µl of

the adaptor-ligated genomes. Amplification was

perfor-med awith 30 s denaturing at 94°C, 5 cycles of 5 s at

94°C and 3 min at 72°C, 5 cycles of 5 s at 94°C and 3

min at 70°C, and 25 cycles of 5 s at 94°C and 3 min

at 68°C.

Secondary reaction used the same components that were used in the primary reaction, except that the target

was 1 µl of 100-fold-diluted primary products and the

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same as for the primary reaction, except that the final step was 15 cycles. The amplified fragments were ligated into pGEM-T Vector (Promega).

2.9. Sequencing

Plasmids were purified using QIAprep Spin Miniprep kit (QIAGEN). The sequence was obtained from a 373A DNA sequencer (PE Applied Biosystems) using Dye Primer and Dye Terminator Cycle Sequencing FS Ready Reaction kit (PE Applied Biosystems).

2.10. In situ hybridization

In situ hybridization to salivary gland chromosomes was performed using DIG-labeled probe according to the method of Langer-Safer et al. (1982) and Engels et al. (1986).

3. Results

3.1. Selective PCR amplification and differential screening

After the selective PCR amplification, forward sub-tracted cDNAs showed six major and some minor bands in agarose–gel electrophoresis. For differential screen-ing, 72 colonies were amplified and blotted onto the membranes. Among them, 12 clones showed stronger signals when detected using forward subtracted cDNAs as probes than when detected using reverse subtracted cDNAs as probes (data not shown). According to their length and restriction sites, these 12 clones were subdiv-ided into six groups and these groups coincsubdiv-ided with the major six bands observed in the electrophoresis of for-ward subtracted cDNAs, respectively (data not shown). 3.2. Northern hybridization analysis

Northern hybridization was performed using the above six fragments as probes. It appeared that a gene was clearly up-regulated in flies acclimated to 15°C for 1 day (Fig. 1), while the remaining five showed only slight differences between acclimated and control flies (data not shown). In Northern hybridization using this positive fragment as a probe, two bands of approx. 1.6 and 1.1 kb in length, were observed, but the signal of the 1.6 kb band was very weak or sometimes undetect-able. The major signal became weaker in flies acclimated

to 15°C for 2 days than in those acclimated for 1 day,

but still stronger than in control flies (Fig. 1). Hence-forth, this positive gene was referred to as Dca (Drosophila cold acclimation gene).

Fig. 1. Northern hybridization analysis using Dca (upper panel) and

ras2 probe (lower panel) in control flies (reared at 25°C to the 7-day adult stage; lane 1), and those acclimated to 15°C for 1 day (lane 2) and 2 days (lane 3). Positions and sizes of the marker are indicated at the right.

3.3. In situ hybridization analysis

In order to confirm the location of Dca gene, in situ hybridization to salivary gland chromosomes was perfor-med. The probe hybridized at 88D on chromosome 3R (Fig. 2).

3.4. RACE

The Dca fragment was sequenced using M13-M4, M13-RV, Nested PCR primer 1 and 2R. After the sequencing, DF-1, DF-2, DR-1 and DR-2 primers were designed and used for 5₉- and 3₉-RACE reactions (Table

1). Single and double bands were amplified in 5₉- and

3₉-RACE reactions, respectively. These PCR products

were subcloned and sequenced using DF-1, DF-5, DR-1, DR-3, M13-M4 and M13-RV primers (Table 1). For

59-RACE products, 10 independent clones were

sequenced in order to obtain the full sequence at the 59

end of mRNA. Sequences of both long and short 39

-RACE products were identical except that long product had two inserted sequences (Fig. 3). The long cDNA

(combination of 59-RACE and long 39-RACE products)

Fig. 2. In situ hybridization to salivary gland chromosomes of D.

melanogaster using Dca probe. Arrow head indicates band 88D on

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Fig. 3. Partial sequences of predicted cDNA (combination of 59-RACE and short 39-RACE products; upper row) and long 39-RACE product (lower row). The gaps are indicated by bars. Box indicates the sequence of DF-1 primer which extends over exons (see Fig. 4). Nucleotides located at 59and 39end of the intron (indicated by bold) follow Chambon’s rule. Numbers on the left side are from the first base of predicted cDNA (see Fig. 4).

has a shorter open reading frame (ORF) than the short

cDNA (combination of a 59-RACE and short 39-RACE

products) because the insertion had a terminator codon. In addition, both ends of the insertion followed

Cham-bon’s rule (Fig. 3); the 5₉ end of the intron has GT and

the 3₉end of the intron has AG (Breathnach and

Cham-bon, 1981). Therefore, it is considered that the longer cDNA is hnRNA, and therefore one of the insertions is, at least, an intron (Fig. 3). Because the DF-1 primer

extended over two exons, 5₉-RACE using this primer did

not amplify hnRNA. Thus, translatable mRNA would be

the short cDNA (combination of 59-RACE and short 39

-RACE products) with a length of 1022 bp and ORF of 909 bp, which encodes 303 amino acids (Fig. 4; the

nucleotide sequence was available from

DDBJ/GenBank/EMBL under accession number

AB029490). The length of this short cDNA corresponds to the major band observed in Northern hybridization. The molecular weight of the deduced polypeptide was

33.3 kDa and the estimated pI was 6.21. In the 3₉

untranslated region, two RNA instability motifs

(ATTTA) were observed (Fig. 4). Other consensus motifs were searched by PROSITE (Bairoch et al., 1997) and SignalP (Nielsen et al., 1997) database; there are several motifs concerning post-translational modifi-cation, such as casein kinase II phosphorylation, protein kinase C phosphorylation, N-myristoylation and N-gly-cosylation sites. There is no signal peptide in the deduced amino acid. The analysis by PSORT II (Horton and Nakai, 1997) predicted that the protein is mainly located in the cytosol.

The computer assisted homology analyses revealed that the Dca shared similarity with the senescence

marker protein-30 (SMP30) which has been cloned in rat, mouse and human in nucleic acid (52-1–55.3%) and amino acid sequences (33.4–34.8%; Fig. 5). Therefore, it is concluded that the gene, Dca, is the Drosophila homologue of SMP30.

3.5. Sequencing of the 5₉ flanking region of Dca gene To identify potential cis-regulator elements of Dca,

the 5₉ flanking region of Dca gene was amplified by

PCR. All PCR products showed a single band in electro-phoresis, indicating that the Dca gene is present as a single copy in the genome of D. melanogaster. PCR pro-ducts derived from Dra I- and Sca I-digested genomes show approx. 0.6 and 1.5 kb in length, respectively. These fragments were cloned and sequenced with the primer walking method using DF-6, DF-7, DF-8, DF-9, DF-N, DR-5, DR-X, DR-X2, DR-X3, M4, M13-RV primers (Table 1). Fig. 6 shows the sequence of the

5₉ flanking region of Dca (the nucleotide sequence was

available from DDBJ/GenBank/EMBL under accession

number AB029491). The presence of an intron at the 5₉

flanking of the initiator codon was suggested. The exon–

intron junction also followed Chambon’s rule

(Breathnach and Chambon, 1981). Since the ATG initiator codon is located at the beginning of the second

exon, the first exon represents a 5₉ untranslated

sequence. In the proximal promoter region, both the TATA sequence and the CAAT box were located. AP-1 (a transcription factor of SMP30) binding site, STRE (stress response element) and some other putative tran-scription factor binding sites were detected by TFSE-ARCH (Heinemeyer et al., 1998) (Fig. 6).

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Fig. 4. Nucleotide (upper row) and deduced amino acid (lower row) sequences of the predicted cDNA. The putative RNA instability signals are indicated by rectangles. The termination codon (TAG) is indicated by *. Arrows indicate primers and their directions. The nucleotide sequence is available from DDBJ.GenBank/EMBL under accession number AB029490.

3.6. Accumulation of DCA mRNA in flies reared to a senescent stage and at low temperature

To investigate whether Dca changes expression according to aging or not, Northern hybridization was performed. It was observed that this gene was up-regu-lated in senescent (49–53 days after eclosion) flies in comparison to young (7 days after eclosion) ones. In addition, the expression level was higher in flies reared at 15°C from the egg stage to the 14-day adult stage than in control flies (reared at 25°C to the 7-day adult stage) (Fig. 7).

4. Discussion

A gene encoding a Drosophila homologue of the sen-escence marker protein-30 (Dca) was up-regulated after acclimation to 15°C. The Dca gene is present as a single copy in the genome, located at position 88D on chromo-some 3R, and has chromo-some introns. From the deduced amino acid sequence, DCA protein is estimated to have a mol-ecular weight of 33.3 kDa, pI of 6.2, and to be present in the cytosol.

The mammalian gene, SMP30, has also been reported to be preferentially expressed in the cytosol of hepato-cytes and renal tubular epithelia (Fujita et al., 1992a,b).

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Fig. 5. Alignment of amino acid sequences of DCA (upper row) and rat SMP30 (lower row). Accession numbers in DDBJ/GenBank/EMBL of DCA and rat SMP30 are AB029490 and X69021, respectively. Asterisks (*) indicate identity to the DCA sequence. Crosses (+) indi-cate the amino acids showing the same property. Bars indiindi-cate the alignment gaps.

The expression of this gene is maintained at a high level throughout the tissue maturing process (Fujita et al., 1996), but decreases during senescent stages in both sexes (Fujita and Maruyama, 1991; Fujita et al.,

1992a,b). This protein has also been reported as a Ca2+

-binding protein regucalcin (RC) by Shimokawa and

Yamaguchi (1993) and is suggested to activate (Ca2+_–

Mg2+_{)-ATPase which acts in the removal of Ca}2+ _from

cytosol across plasma membranes (Takahashi and Yam-aguchi 1993a,b, 1994; Fujita et al., 1998). It is therefore possible that DCA plays some role in the maintenance of cytosolic Ca2+ _level.

In contrast to mammalian SMP30, expression of the Dca gene did not decrease but increased at the senescent stage in Drosophila. In senescent Drosophila, an

increase in ATP-dependent Ca2+ _{uptake has been}

reported (Shi et al., 1994), suggesting a link between the

up-regulation of Dca and Ca2+ _uptake.

The transcriptional regulation of SMP30 in rat is assumed to be mainly dependent on AP-1 transcription

factor which binds to the sequence located at the 59

flanking region of the SMP30 gene (Murata and Yamag-uchi 1998a,b, 1999). It is reported that DNA binding activity of AP-1 was significantly lower in old individ-uals than in young ones (Helenius et al., 1996; Pahlavani and Harris, 1996; Tumer et al., 1997). Therefore, it is considered that the decrease of SMP30 production dur-ing senescent stages is due to a decrease in the DNA binding activity of 1. The Dca gene also has an

AP-1 binding site at the 5₉ flanking region, suggesting a

possibility that it is controlled by an AP-1 transcrip-tion factor.

The present study revealed that the distal region of the Dca gene contains STRE (stress response element) which has previously been reported only in yeast. In yeast, STREs control transcription of a large number of genes including stress proteins (Moskvina et al., 1998)

and STRE-directed transcription is stimulated not only by heat but also by several other moderate stresses (Varela et al., 1995).

In plants, it has been reported that environmental stresses commonly lead to rapid transient elevations in cytosolic free Ca2+_{concentration (Bush, 1995; Knight et} al., 1991). These cellular Ca2+_{signals lead ultimately to} the increased expression of stress-responsive genes (Knight et al. 1996, 1997). Knight et al. (1998) suggested in Arabidopsis that altered Ca2+

concentration encodes a ‘memory’ of previous stress encounters and may be involved in acclimation to environmental stresses. In addition, Perotti et al. (1990) reported that cold-shock resulted in endonuclease activation and apoptosis: the

prolonged elevation of cytosolic free Ca2+ _induces

apoptosis by stimulating Ca2+_/Mg2+_-dependent

endonu-cleases and modulating Ca2+_{/calmodulin-dependent}

enzymatic activities (McConkey et al., 1989; Gaido and Cidlowski, 1991; Nicotera et al., 1994; Knight et al., 1996). It has also been suggested that SMP30 regulates

Ca2+_-pumping _activity _and _contributes _to _rescue

apoptosis (Fujita et al., 1998). It may be, therefore, that the increased level of DCA plays a role in maintaining Ca2+

levels at low temperatures, and enhances cold toler-ance in individuals maintained at low temperature in Drosophila.

It is noticeable that DCA and SMP30 mRNA sequences have a rapid mRNA degradation signal,

ATTTA, in the 3₉ untranslated region. It has been

reported that this sequence mediates selective degra-dation of mRNAs and is found in proto-oncogenes, a variety of cytokines and lymphokines, and heat-shock proteins (Shaw and Kamen, 1986; Ryseck et al., 1988; Sakai et al., 1989; Yost et al., 1990; Gillis and Malter, 1991). The expression of these genes is critically and immediately regulated under specific stimuli/stresses at the level of mRNA degradation.

Acknowledgements

I thank Drs M.T. Kimura and K.M. Yoshida for their guidance and invaluable advice in this study. The con-tinuous encouragement and support of the members of my laboratory are gratefully acknowledged. This study was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scien-tists.

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Fig. 6. Nucleotide sequence of 59flanking region, first exon, first intron and second exon of Dca. Exon sequences are shown in black boxes. The translation initiator codon is located in the second exon (indicated by arrow head). Nucleotides located at the 59and 39end of intron (indicated by bold) follow Chambon’s rule. Predicted TATA sequence and CAAT box sequences are indicated by rectangles. Other putative AP-1, STRE, HFH-2 and NIT2 binding sites, and GATA box are also indicated by rectangles. Arrows represent primers and their directions. The nucleotide sequence is available from DDBJ/GenBank/EMBL under accession number AB029491.

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Fig. 7. Northern hybridization analysis using Dca (upper panel) and

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S.G. Goto / Journal of Insect Physiology 46 (2000) 1111–1120

has a shorter open reading frame (ORF) than the short

cDNA (combination of a 5

9 -RACE and short 3

9 -RACE

products) because the insertion had a terminator codon.

In addition, both ends of the insertion followed

Cham-bon’s rule (Fig. 3); the 5

₉

end of the intron has GT and

the 3

₉

end of the intron has AG (Breathnach and

Cham-bon, 1981). Therefore, it is considered that the longer

cDNA is hnRNA, and therefore one of the insertions

is, at least, an intron (Fig. 3). Because the DF-1 primer

extended over two exons, 5

₉

-RACE using this primer did

not amplify hnRNA. Thus, translatable mRNA would be

the short cDNA (combination of 5

9 -RACE and short 3

9 -RACE products) with a length of 1022 bp and ORF of

909 bp, which encodes 303 amino acids (Fig. 4; the

nucleotide

sequence

was

available

from

DDBJ/GenBank/EMBL

under

accession

number

AB029490). The length of this short cDNA corresponds

to the major band observed in Northern hybridization.

The molecular weight of the deduced polypeptide was

33.3 kDa and the estimated pI was 6.21. In the 3

₉

untranslated

region,

two

RNA

instability

motifs

(ATTTA) were observed (Fig. 4). Other consensus

motifs were searched by PROSITE (Bairoch et al., 1997)

and SignalP (Nielsen et al., 1997) database; there are

several motifs concerning post-translational

modifi-cation, such as casein kinase II phosphorylation, protein

kinase C phosphorylation, N-myristoylation and

N-gly-cosylation sites. There is no signal peptide in the

deduced amino acid. The analysis by PSORT II (Horton

and Nakai, 1997) predicted that the protein is mainly

located in the cytosol.

The computer assisted homology analyses revealed

that the Dca shared similarity with the senescence

marker protein-30 (SMP30) which has been cloned in

rat, mouse and human in nucleic acid (52-1–55.3%) and

amino acid sequences (33.4–34.8%; Fig. 5). Therefore,

it is concluded that the gene, Dca, is the Drosophila

homologue of SMP30.

3.5. Sequencing of the 5

₉

flanking region of Dca gene

To identify potential cis-regulator elements of Dca,

the 5

₉

flanking region of Dca gene was amplified by

PCR. All PCR products showed a single band in

electro-phoresis, indicating that the Dca gene is present as a

single copy in the genome of D. melanogaster. PCR

pro-ducts derived from Dra I- and Sca I-digested genomes

show approx. 0.6 and 1.5 kb in length, respectively.

These fragments were cloned and sequenced with the

primer walking method using DF-6, DF-7, DF-8, DF-9,

DF-N, DR-5, DR-X, DR-X2, DR-X3, M4,

M13-RV primers (Table 1). Fig. 6 shows the sequence of the

5 ₉

flanking region of Dca (the nucleotide sequence was

available from DDBJ/GenBank/EMBL under accession

number AB029491). The presence of an intron at the 5

₉

flanking of the initiator codon was suggested. The exon–

intron

junction

also

followed

Chambon’s

rule

(Breathnach and Chambon, 1981). Since the ATG

initiator codon is located at the beginning of the second

exon, the first exon represents a 5

₉

untranslated

sequence. In the proximal promoter region, both the

TATA sequence and the CAAT box were located.

AP-1 (a transcription factor of SMP30) binding site, STRE

(stress response element) and some other putative

tran-scription factor binding sites were detected by

TFSE-ARCH (Heinemeyer et al., 1998) (Fig. 6).

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3.6. Accumulation of DCA mRNA in flies reared to a

senescent stage and at low temperature

To investigate whether Dca changes expression

according to aging or not, Northern hybridization was

performed. It was observed that this gene was

up-regu-lated in senescent (49–53 days after eclosion) flies in

comparison to young (7 days after eclosion) ones. In

addition, the expression level was higher in flies reared

at 15°C from the egg stage to the 14-day adult stage than

in control flies (reared at 25°C to the 7-day adult stage)

(Fig. 7).

4. Discussion

A gene encoding a Drosophila homologue of the

sen-escence marker protein-30 (Dca) was up-regulated after

acclimation to 15°C. The Dca gene is present as a single

copy in the genome, located at position 88D on

chromo-some 3R, and has chromo-some introns. From the deduced amino

acid sequence, DCA protein is estimated to have a

mol-ecular weight of 33.3 kDa, pI of 6.2, and to be present

in the cytosol.

The mammalian gene, SMP30, has also been reported

to be preferentially expressed in the cytosol of

hepato-cytes and renal tubular epithelia (Fujita et al., 1992a,b).

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S.G. Goto / Journal of Insect Physiology 46 (2000) 1111–1120

The expression of this gene is maintained at a high level

throughout the tissue maturing process (Fujita et al.,

1996), but decreases during senescent stages in both

sexes (Fujita and Maruyama, 1991; Fujita et al.,

1992a,b). This protein has also been reported as a Ca

-binding protein regucalcin (RC) by Shimokawa and

Yamaguchi (1993) and is suggested to activate (Ca

–

Mg

)-ATPase which acts in the removal of Ca

from

cytosol across plasma membranes (Takahashi and

Yam-aguchi 1993a,b, 1994; Fujita et al., 1998). It is therefore

possible that DCA plays some role in the maintenance

of cytosolic Ca

level.

In contrast to mammalian SMP30, expression of the

Dca gene did not decrease but increased at the senescent

stage in Drosophila. In senescent Drosophila, an

increase in ATP-dependent Ca

uptake has been

reported (Shi et al., 1994), suggesting a link between the

up-regulation of Dca and Ca

uptake.

The transcriptional regulation of SMP30 in rat is

assumed to be mainly dependent on AP-1 transcription

factor which binds to the sequence located at the 5

9 flanking region of the SMP30 gene (Murata and

Yamag-uchi 1998a,b, 1999). It is reported that DNA binding

activity of AP-1 was significantly lower in old

individ-uals than in young ones (Helenius et al., 1996; Pahlavani

and Harris, 1996; Tumer et al., 1997). Therefore, it is

considered that the decrease of SMP30 production

dur-ing senescent stages is due to a decrease in the DNA

binding activity of 1. The Dca gene also has an

AP-1 binding site at the 5

₉

flanking region, suggesting a

possibility that it is controlled by an AP-1

transcrip-tion factor.

The present study revealed that the distal region of

the Dca gene contains STRE (stress response element)

which has previously been reported only in yeast. In

yeast, STREs control transcription of a large number of

genes including stress proteins (Moskvina et al., 1998)

and STRE-directed transcription is stimulated not only

by heat but also by several other moderate stresses

(Varela et al., 1995).

In plants, it has been reported that environmental

stresses commonly lead to rapid transient elevations in

cytosolic free Ca

concentration (Bush, 1995; Knight et

al., 1991). These cellular Ca

signals lead ultimately to

the increased expression of stress-responsive genes

(Knight et al. 1996, 1997). Knight et al. (1998) suggested

in Arabidopsis that altered Ca

concentration encodes

a ‘memory’ of previous stress encounters and may be

involved in acclimation to environmental stresses. In

addition, Perotti et al. (1990) reported that cold-shock

resulted in endonuclease activation and apoptosis: the

prolonged elevation of cytosolic free Ca

induces

apoptosis by stimulating Ca

/Mg

-dependent

endonu-cleases

and

modulating

Ca

/calmodulin-dependent

enzymatic activities (McConkey et al., 1989; Gaido and

Cidlowski, 1991; Nicotera et al., 1994; Knight et al.,

1996). It has also been suggested that SMP30 regulates

Ca

-pumping

activity

and

contributes

to

rescue

apoptosis (Fujita et al., 1998). It may be, therefore, that

the increased level of DCA plays a role in maintaining

Ca

levels at low temperatures, and enhances cold

toler-ance in individuals maintained at low temperature in

Drosophila.

It is noticeable that DCA and SMP30 mRNA

sequences have a rapid mRNA degradation signal,

ATTTA, in the 3

₉

untranslated region. It has been

reported that this sequence mediates selective

degra-dation of mRNAs and is found in proto-oncogenes, a

variety of cytokines and lymphokines, and heat-shock

proteins (Shaw and Kamen, 1986; Ryseck et al., 1988;

Sakai et al., 1989; Yost et al., 1990; Gillis and Malter,

1991). The expression of these genes is critically and

immediately regulated under specific stimuli/stresses at

the level of mRNA degradation.

Acknowledgements

I thank Drs M.T. Kimura and K.M. Yoshida for their

guidance and invaluable advice in this study. The

con-tinuous encouragement and support of the members of

my laboratory are gratefully acknowledged. This study

was supported by Research Fellowships of the Japan

Society for the Promotion of Science for Young

Scien-tists.

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Fig. 7. Northern hybridization analysis using Dca (upper panel) and ras2 probe (lower panel). Lane 1: control fly (reared at 25°C to the 7-day adult stage), lane 2: senescent flies (reared at 25°C to the 49–53-day adult stage), lane 3: flies reared at 15°C from the eggs to 14-day adult stage. Positions and sizes of the marker are indicated at the right.

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