Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor

Insect Biochem. Molec. Biol. Vol.26, No. 2, pp. 127-133, 1996

0965-1748(95)00071-2

Pergamon

Copyright© 1996ElsevierScienceLtd Printedin GreatBritain.All rightsreserved 0965-1748/96 $t5.00 + 0.00

Cloning and Baculovirus Expression of a Desiccation Stress Gene from the Beetle, Tenebrio molitor LAURIE A. GRAHAM,* WILLIAM G. BENDENA,* VIRGINIA K. WALKER*t Received 10 January 1994; revised and accepted 9 August 1995

The eDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph. Insect Coleoptera Thermal hysteresis eDNA Protein expression Hemolymph protein

INTRODUCTION Water shortage is a fundamental problem faced by most terrestrial organisms and some of the physical and behavioral adaptations that permit terrestrial living have been well documented. The regulation and function of proteins expressed during water shortage have only recently been examined. A number of proteins are induced in plants and seeds during or following dehydration and although the function of most of these is unknown, some appear to be involved in the synthesis of low molecular weight solutes such as sorbitol, or in the inhibition of degradative enzymes (reviewed in Tomos, 1992). Many plant desiccation genes are also induced by cold as well as by the plant hormone, abscisic acid, and it has been shown that acclimation t o one stress can increase tolerance to the other (Guy et al., 1992). Interestingly, an Arabidopsis gene induced by desiccation, cold and salt stress, and abscisic acid, was reported to have some similarity to a fish antifreeze protein (Kurkela and Borg-Franck, 1992). Water and osmotic stress proteins have also been found in bacteria. The secreted water stress proteins of the cyanobacterium Nostoc commune have an N-terminal amino acid sequence similar to xylos-

*Department of Biology,Queen's University,Kingston,Ontario,Canada, K7L 3N6. #Author for correspondence.

idase (Hill et al., 1994) while several species of cyanobacteria accumulate proteins following osmotic stress that cross-react with antibodies specific to a plant desiccation stress protein motif (Close and Lammers, 1993). The common yellow mealworm, Tenebrio molitor, is a pest of stored grain products and has a higher resistance to drying than beetles from wetter habitats (Riddle, 1986). Tenebrio withstands dry environments, in part, by regulating the concentration of hemolymph components as hemolymph volume decreases. Tenebrio larvae also possess a rectal complex containing a saturated solution of KC1 which absorbs excess water from faeces and moist air imported through the anus (Tupy and Machin~ 1985). Tenebrio have also adapted to survive sub-zero temperatures by supercooling and the hemolymph contains thermal hysteresis (antifreeze) proteins (Grimstone et al., 1968) which prevent ice crystal formation. Thus, Tenebrio has adapted to both cold and dry periods and hemolymph proteins could play a role in maintaining water balance or preventing cold and desiccation damage. Previously, it was shown that the concentration of an abundant Tenebrio hemolymph protein increased 3-fold following desiccation stress and 2-fold following cold stress (Kroeker and Walker, 1991b). This 28 kDa desiccation stress protein (dsp28) is synthesized by fat body and may be experimentally induced following treatment of pupae with the juvenile hormone analogue, methop-

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rene (Kroeker and Walker, 1991a). Dsp28 was purified, anti-dsp28 antibodies generated and the amino acid composition (Kroeker and Walker, 1991b) and N-terminal protein sequence (Kroeker, 1989) determined. Since the function of dsp28 is unknown, cDNA cloning and sequencing were undertaken to determine if dsp28 shares sequence similarity to any known proteins. In addition, it was important to determine if dsp28 can contribute to hemolymph thermal hysteresis properties since Tenebrio antifreeze proteins have also been shown to be induced by desiccation, and cold (Patterson and Duman, 1978) and since thermal hysteresis activity was previously found associated with dsp28 preparations (Kroeker, 1989). MATERIALS AND METHODS

cDNA library construction Insects were reared as previously described (Kroeker and Walker, 1991b). Total RNA was isolated from final instar larval fat body using guanidinium isothiocyanate and lithium chloride precipitation (Cathala et al., 1983). Poly (A)+ mRNA was selected by oligo(dT) cellulose (type 7, Pharmacia) affinity chromatography (Sambrook et al., 1989). A cDNA library (primary titre of 2.5 x 105 pfu) was generated in )tzap II (Stratagene) as per the manufacturer's instructions with the exception that cDNA was separated from linkers using an Elutip column (Schleicher and Schuell). Isolation and sequencing of dsp28 cDNA Approximately 3 x 105 clones were screened using the ProtoBlot Immunoscreening System (Promega) and 17.6/xg/ml anti-dsp28 IgG. Phage growth and DNA purification followed standard methodologies (Sambrook et al., 1989). Double stranded DNA was sequenced using the dideoxy chain termination method (Sanger et al., 1977) using either the Sequenase DNA sequencing kit (United States Biochemical Corp.) or a 373A automatic DNA sequencer system (Applied Biosystems) with dye terminators. Oligonucleotides were generated for use as primers to obtain full length sequence from both strands [Fig. l(b)]. Northern analysis Larvae were placed in a ventilated chamber in which drierite (W. A. Hammond Co., Xenia, OH) was used to reduce the relative humidity to less than 10%. Control larvae were reared under ambient conditions and provided with wet paper towels 3 times a week. All larvae were fed ample amounts of wheat bran. Poly (A)+ enriched RNA was isolated from pooled insects using the QuickPrep micro mRNA purification kit (Pharmacia). Approximately 5/xg of RNA/sample was resolved by glyoxal-DMSO electrophoresis in a 1.2% agarose gel (Sambrook et al., 1989). The RNA was transferred by capillary action to a Hybond-N membrane (Amersham)

using 20x SSC (Sambrook et al., 1989). 32p-labelled DNA probe was generated from a gel purified PCR fragment using a random-primed oligolabelling kit (Pharmacia). The fragment was synthesized from a dsp28 cDNA template using the primers described below. The membrane was prehybridized in 6x SSPE, 10x Denhardt's, 0.5% SDS and 200/xg/ml salmon sperm DNA for 20 h at 65°C and hybridized in the same solution plus probe for 18 h at 65°C (Sambrook et al., 1989). The membrane was washed 4 x 2 0 rain in 1 x SSPE, 0.5% SDS and 1 x 10 min in 0.1x SSPE, 0.5% SDS at 65°C. The membrane was subsequently hybridized to a labelled PstI/HindIII fragment containing the majority of the Drosophila melanogaster c~-tubulin coding sequence (Mischke and Pardue, 1982). Prehybridization and hybridization were performed as above but at a reduced temperature of 60°C. The membrane was washed 4 x 2 0 min in 2xSSPE, 0.1% SDS and 1 x 10 min in 0.5x SSPE, 0.1% SDS at 60°C. Exposures were taken using preflashed (Sambrook et aI., 1989) Kodak X-OMAT AR film and a Dupont Cronex Lightning Plus intensifying screen at -70°C. An L B K 2222020 Ultroscan XL Laser Densitometer (Pharmacia) was used to quantify band intensities. Cell culture Spodoptera frugiperda IPLB-SF-21 cells were cultured in Grace's medium (Gibco) supplemented with 10% fetal calf serum, 2.6g/1 tryptose (Difco) and 50 ~g/ml gentamicin (Gibco) during transfection and plaque purification. Dsp28 was expressed in Sf21 cells cultured in Sf900II serum free medium (Gibco) supplemented with 50/zg/ml gentamicin (Gibco). All cells were grown at 28 + 0.5°C in stationary monolayer culture. Construction and generation of recombinant transfer vector and baculovirus The dsp28 coding region, including the signal peptide sequence, was amplified using Taq polymerase (Promega) and PCR with synthetic oligonucleotides containing tandem NheI sites. The 5' primer (5'GCTAGCGCTAGCNi'CATGAACAAGTTACTCATC ATAG-3') was constructed to change the -2 nucleotide from C to T (marked with a dot), which has been reported to increase levels of expression of a luciferase gene in baculovirus (Richardson et al., 1992). The 3' primer (5'GCTAGCGCTAGCGTTTAAAAGACGTTTATTTCA TC-3') spanned the polyadenylation signal. The amplified product was digested with NheI (Pharmacia) and subcloned into the NheI site of the transfer vector pETL, which contains the /3-galactosidase (/3-gal) gene as a marker for the detection of recombinant virus (Richardson et al., 1992). The constructs were screened for correct orientation by restriction enzyme digestion and the fidelity of the cDNA and flanking transfer vector sequences was confirmed by sequencing as above. The transfer vector containing dsp28 cDNA was co-trans-

TENEBRIO DESICCATION STRESS GENE

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119 37

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CAC H

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179 57

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299 97

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GAT D

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GTG V

AAG K

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CTT L

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TTC F

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ACC T

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ACC T

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ATT I

419 137

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TTG GAA GAG L E E

TCA AAT S N

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~TG G A C L D

479 157

AAC N

TTT GCG AAA AAA F A K K

CAT AAA H K

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ACC T

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AAA K

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TGT GTT C V

CAA ACA Q T

CGA R

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~CC 599 A / T 197

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AAC GTC GCT CTT N V A L

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TTC GAT F D

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GCT A

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TAG AAT ATA •

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ATT

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780

FIGURE 1. (a) Complete cDNA sequence and conceptual translation including polymorphisms. The most 5' nucleotide of four cDNA clones are shown by arrows. The hydrophobic core region of the signal peptide is shown in bold italics. The Nterminal sequence of mature dsp28 determined by Edman degradation is underlined with two undetermined residues denoted by asterisks. An N-linked glycosylation site is shown in bold and underlined, the polyadenylation signal is double-underlined and the termination codon is marked by a triangle. (GenBank accession #U41298.)

(b) EcoRI

SalI/AceI

A ~ I HaeII

SpeI

0

100 200 300 400 500 600 ATG - - [ F _ / _ / ~ / / / / / / / / / / / / / / / / / / / ]

XhoI 700

800bp 3--

...............................................................~....... ~ .................................................................................. T3 ...............................................................~................. I'7 ..................................................................................

FIGURE 1. (b) Schematic representation of dsp28 cDNA, partial restriction map and sequencing strategy. The location of sequencing primers is represented by short lines with dashed lines representing the direction and extent of sequence determined. The coding region is hatched while vector sequences are denoted by a thin line. The cDNA was directionally inserted into the vector pBluescript SK (-) using EcoRUXhoI sites.

fected with H R 5 - A c M N P V D N A into Sf21 cells using lipofectin (Gibco) (Groebe et al., 1990). After 5 days incubation, the viral supernatant was harvested and plated onto fresh cells. Viral plaques were screened for/3-gal production by overlaying freshly infected monolayers with Graces plus 1% Sea Plaque low melting point agarose

(FMC) containing 100/~g/m] Bluogal (Gibco). After three plaque screening rounds, the virus was judged to be plaque pure and the viral supematant of five isolates was examined for dsp28 production by S D S - P A G E (Laemmli, 1970) and Western blotting analysis. Viral stocks were titred by end point dilution (O'Reilly et al., 1992).

LAURIEA. GRAHAMet al.

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Protein production and partial purification of baculovirus expressed dsp28 Cells were infected at a multiplicity of infection of 0.2. Maximal dsp28 production (approx 10 mg/1) with minimal contamination of other proteins was judged to occur between 3 and 4 days post-infection by SDS-PAGE. The supernatant was centrifuged at 1000 g for 10 min and then at 12,000g for 30 min to remove cells and particulates. Low molecular weight contaminants were removed using a G75 (Pharmacia) gel filtration column eluted with 20 mM Tris-HC1 pH 7.5 and dsp28, monitored by SDSPAGE, eluted in the void volume. Pooled dsp28 containing fractions were loaded onto a Mono-Q anion exchange column (Pharmacia) and developed with a 0-1 M NaC1 gradient in the same buffer. Dsp28 eluted at about 250 mM NaC1 and was concentrated using a Centricon30 microconcentrator (Amicon). Purification of native protein Dsp28 was purified by electroelution from nondenaturing-PAGE followed by Mono-Q ion exchange chromatography as previously described (Kroeker and Walker, 1991b). Dsp28 was concentrated using a Centricon-30 microconcentrator (Amicon) or by lyophilization. Low molecular weight thermal hysteresis proteins were concentrated by lyophilization. Western blotting, amino-terminal sequencing and glycoprotein detection Samples were resolved by 12% SDS-PAGE and transferred overnight at 5 mA to a nitrocellulose membrane (BioRad) using Towbin buffer (Towbin et al., 1979). For N-terminal sequencing, methanol was omitted and Immobilon-P membrane (Millipore) was used. Western blots were blocked with 5% skim milk powder (Carnation) plus 0.2% Tween 20 (Bio-Rad). Detection was performed as in cDNA library screening (above) using anti-dsp28 IgG at a concentration of 2 ng/ml. Glycoproteins were detected using the DIG glycan detection kit (Boehringer Mannheim, Germany). Positive and negative controls were included (data not shown). Thermal hysteresis All thermal hysteresis measurements were done on 510 nl samples using a nanoliter osmometer (Clifton Technical Physics, Hartford, NY) with a cooling step rate of 0.01 C°/30 s. Protein concentrations were estimated by the BCA protein assay (Pierce, Rockford, IL) using bovine serum albumin as a standard. RESULTS

Dsp28 cDNA and sequence analysis Nine anti-dsp28 antibody positive clones (700-800 bp in length) were isolated from the Tenebrio larval fat-body cDNA library and the four longest clones were sequenced in their entirety [Fig. l(b)]. The translated

sequence of the cDNA clones [Fig. l(a)] closely matched that of the N-terminal sequence obtained from purified dsp28 (Kroeker, 1989). The longest clone contained a start codon preceded by an eight base pair sequence that conforms well to a eukaryotic ribosome binding site (Staden, 1984). The first 20 amino acids represent a secretory signal peptide with a core of 12 hydrophobic amino acids. The N-terminal amino acid of the mature protein is found at a location predicted to be a signal peptide cleavage site (von Heijne, 1986), so it is unlikely that further N-terminal processing occurs after cleavage of the signal peptide. The remaining sequence encodes a 205 amino acid polypeptide with a predicted molecular mass of 23 kDa, one potential N-linked and a number of potential Olinked glycosylation sites. Thus, glycosylation may account for the difference between the predicted and apparent (28 kDa) molecular mass of the protein. The sequence of 4 different clones revealed two polymorphisms (two clones/polymorphism), resulting in a conservative amino acid substitution at one site and a silent substitution at the other [Fig. l(a)]. Secondary structure algorithms (Gascuel and Golmard, 1988) estimate that dsp28 contains 36% a-helix, 18% /3-pleated sheet and 46% random coil. In addition, the clones contained 72 bp of 3' untranslated sequence with a consensus polyadenylation site [Fig. l(a)]. Dsp28 does not show significant sequence similarity as determined by blastN of blastP (Altschul et al., 1990) to any amino acid or nucleotide sequence in the National Center for Biotechnology Information non-redundant sequence databases (June, 1995). Detailed comparisons between dsp28 and a variety of desiccation and cold stress proteins, antifreeze proteins, and other insect hemolymph proteins also failed to reveal any significant similarities.

Northern analysis Dsp28 cDNA hybridizes to a 1 kb mRNA (Fig. 2), which is consistent with the predicted size if the 5' untranslated region is relatively short. Characterization of poly (A)+ enriched RNA from larvae subjected to desiccation showed that levels of dsp28 mRNA in larvae increased 2.7-fold following 2 weeks of desiccation and 4.7-fold following 4 weeks of desiccation (Fig. 2) relative to c~-tubulin mRNA levels. This is similar to the 2and 3-fold increase observed in dsp28 protein levels following 2 and 4 weeks of desiccation respectively (Kroeker and Walker, 1991b). Expression in a bacuIovirus vector Dsp28 cDNA was cloned into the baculovirus transfer vector pETL and recombinant AcNPV isolated following transfection. These recombinant virus produced a 28 kDa protein which cross-reacted with anti-dsp28 IgG, whereas mock (uninfected) or wild-type infected cells did not (Fig. 3). Slight migration differences between native and baculovirus expressed dsp28 were occasionally observed. Dsp28 is the predominant protein secreted by infected

TENEBRIO DESICCATION STRESS GENE

(a)

131

(a) kb

!

2

3

4

5

6

7

8

9

4,40 --

M

2

t

3

4

5

6

7

kD~ 97 66

2.37 - -

45 1.35 --

31

4--

22 0.24 --

(b)

FIGURE 2. (a) Northern blot analysis of dsp28 mRNA levels in desiccated and control larvae. Each sample consists of eight pooled insects. 5/zg of poly (A)÷ enriched RNA was isolated from larvae following no desiccation (1-3), 2 weeks of desiccation (4-6), and 4 weeks of desiccation. Samples were electrophoresed, blotted and hybridized to dsp28 cDNA as described in Materials and Methods. (b) The blot was hybridized to Drosophila melanogaster a-tubulin.

FIGURE 3. (a) SDS-PAGE analysis of secreted and intracellular proteins produced by baculovirus infected cells. Media removed from cells 3.5 days post infection was concentrated 7-fold using a Centricon-30 microconcentrator (Amicon) and 7.5/zl of concentrated media from mock (2) (uninfected control), wild type (3) and recombinant (4) infected cells, as well as 2.5 Ixl of the cell pellet from mock (5), wild type (6) and recombinant (7) infected cells was separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. An arrow indicates the position of dsp28.0.5/zl of Tenebrio hemolymph (1) and a marker lane (M) are included for comparison.

(b) 1

2

3

4

5

6

kDa

(c)

97-6645

Weeks of Desiccation FIGURE 2. (c) Histogram showing dsp28 mRNA levels following desiccation stress treatment. Dsp28 mRNA levels in each sample were normalized relative to a-tubulin and the mean and standard error for each treatment was then determined. The values were normalized to set the mean control value at one. Dsp28 mRNA levels following 4 weeks of desiccation were significantly different from controls as determined using the Student's t-test.

cells [Fig. 3(a)]. E d m a n degradation and sequence analysis showed that the N-terminal sequence of the baculovirus expressed protein (10 aa) corresponded exactly to the c D N A derived sequence and to the sequence of the native protein, confirming the identity of the protein and indicating correct N-terminal processing. Staining for glycoproteins demonstrated that both r e c o m b i n a n t and native dsp28 are glycosylated (Fig. 4). Small migration differences seen in n o n - d e n a t u r i n g P A G E (data not shown) and elution from the M o n o - Q c o l u m n at different salt concentrations (250 vs 320 nM) indicate, hbwever, that there could be some differences in glycolytic processing in the two forms.

--

31--

22--

14-FIGURE 3. (b) Western blot of baculovirus produced and Tenebrio produced dsp28 using anti-dsp28 IgG. 7.5 p,1 of media from two plaque purified recombinant (1 and 2), mock (3) and wild rype (4) infected cells, as well as 1 p,g of dsp28 purified from Tenebrio hemolymph (5) and baculovirus infected cells (6) were treated as described in Materials and Methods.

Thermal hysteresis analysis Both native and baculovirus expressed dsp28 were examined for thermal hysteresis properties. W h e n native dsp28 was excised and eluted from n o n d e n a t u r i n g P A G E gels, several small proteins ( 1 1 - 1 4 kDa) were recovered (Fig. 5) which were not apparent after Coomassie Blue staining (Kroeker and Walker, 1991b). These proteins did not bind to the c o l u m n during M o n o - Q anion exchange

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LAURIE A. GRAHAMet al.

(a)

(b) 1

2

3

1

2

3

kDa 97-

teresis (Table 1). The non-binding fraction from the Mono-Q column, however, showed significant freezing point depression. The addition of dsp28 to this fraction containing these 11-14 kDa proteins did not enhance the thermal hysteresis observed (Table 1).

66DISCUSSION 45

31-

22-

14FIGURE4. Glycoprotein staining of baculovirus and Tenebrio produced dsp28. (a) 1/zg of dsp28 purified from Tenebrio hemolymph (1) and the supernatant of baculovirus infected cells (2) as well as 1/4/xl hemolymphfrom a Tenebrio larva were resolved by 12% SDSPAGE and stained with Coomassie Brilliant Blue R-250. (b) Duplicate gel stained for glycoproteins as described in Materials and Methods. M

1

2

3

kD

46

31)

21.5

Dsp28 is a T e n e b r i o hemolymph protein induced by desiccation, cold and methoprene (Kroeker and Walker, 1991a,b), indicating that it may play a role during certain stresses and/or during development. We have now cloned the cDNA encoding this protein to examine its properties and regulation. Dsp28 is a novel stress protein since it does not share sequence similarity to other known proteins. Dsp28 mRNA levels are induced 4.7-fold following 4 weeks of desiccation, indicating that the increase in protein levels is mediated by an increase in dsp28 mRNA transcript levels. The slow induction of dsp28 mRNA following desiccation stress may reflect the fact that T e n e b rio has a .very low rate of water loss under desiccating conditions (Riddle, 1986). Desiccation, cold and juvenile hormone also induce increased thermal hysteresis (freezing point depression) activity in T e n e b r i o hemolymph (Patterson and Duman, 1978; Xu e t al., 1992). However, the experiments presented here indicate that dsp28 neither contributes to this activity directly, nor does it interact with antifreeze proteins to increase their activity, as does a D e n d r o i d e s beetle thermal hysteresis activator protein (Wu and Duman, 1991). Although dsp28 does not prevent ice crystal growth, it likely has an important function during both cold and desiccation stress given that it represents 17% of the total soluble protein in the hemolymph of desiccated animals (Kroeker and Walker, 1991b). Surprisingly, it is not associated with the water absorbing rectal complex (data not shown). Some plant desiccation stress genes are also induced by cold acclimation and by a plant stress horTABLE 1. Thermal hysteresis properties of the low molecular weight Tenebrio antifreeze proteins (AFP) in the presence or absence of dsp28

14.3 Sample 6.5

AFP

3.4

Tenebrio dsp28

FIGURE 5. SDS-PAGE of Tenebrio hemolymph proteins excised from a native gel and separated by anion exchange chromatography. 3/xl of material eluted from the dsp28 band from native PAGE (1) as well as 15 tzl of the Mono-Qvoid volume (2) and 15/~1 of eluant from the dsp28 peak (3) were resolved by 15% SDS-PAGE and silver stained (Bio-Rad, Silver Stain Plus). chromatography and did not absorb appreciably at a wavelength of 280 nm, while dsp28 eluted at about 320 mM NaC1 or KC1 (data not shown). Neither baculovirus expressed nor native dsp28 displayed thermal hys-

Baculovirus expressed dsp28 AFP + Buffer 1:1 AFP + Tenebrio dsp28 1:1 AFP + Buffer 1:1 AFP + Tenebrio dsp28 1:1

Conc. (/zg//xl) 0.17 0.13/4.5 4.4 0.09 0.07 + 0.09 0.15 0.15+2.3

Thermal hysteresis _+SEM (C°) 0.23 _+0.02 N=I0 0/0 N=3 0 N= 3 0.11 +0.01 N=8 0.11 _+0.01 N=8 0.23 _+_0.02 N=6 0.22_+0.02 N= 10

TENEBRIO DESICCATION STRESS GENE

mone, abscisic acid (Tomos, 1992). Therefore, desiccation stress proteins from a variety of organisms could function similarly by protecting cells from desiccation and cold stress. In beetles, osmotic balance could be lost during cold exposure when the metabolic rate presumably decreases, or during desiccation as water loss concentrates hemolymph components (Riddle, 1986). A stress protein that blocks passive ion channels may reduce damaging shifts in intracellular ion pools. Interestingly, fish antifreeze proteins have been reported to reduce ion leakage from pig oocytes during cold stress (Rubinsky et al., 1992). Another possible function could involve the sequestering of ions or other solutes as hemolymph volume decreases to assist in maintaining osmotic balance and to store ions for later use. The brine shrimp desiccation stress protein, artemin, has limited homology to ferritin, indicating that it may store iron during dehydration (De Graaf et al., 1990). Although dsp28 may contribute to the sequestration of ions or to the protection of ion channels, it is also important to note that beetles have high concentrations of amino acids within their hemolymph (Florkin and Jeuniaux, 1974). This opens up the possibility that increases in osmolarity could be countered by converting amino acids into protein, with the additional benefit of producing a water molecule for each amino acid incorporated. A protein serving this function could also have fortuitously evolved other physiological functions associated with desiccation. Because baculovirus produced dsp28 appears to be correctly post-translationally modified, it should prove useful in future studies aimed at testing these hypotheses. REFERENCES Altschul S. F., Gish W., Miller W., Myers E. W. and Lipman D. J. (1990) Basic local alignment search tool. J. Molec. Biol. 215, 403-4 10. Cathala G., Savouret J., Mendez B., West B. L., Karin M., Martial J. A. and Baxter J. D. (1983) Laboratory methods: a method for isolation of intact, translationally active ribonucleic acid. DNA 2, 329-335. Close T. J. and Lammers P. J. (1993) An osmotic stress protein of cyanobacteria is immunologically related to plant dehydrins. Plant Physiol. 101, 773-779. De Graaf J., Amons R. and Moller W. (1990) The primary structure of artemin from Artemia cysts. Eur. J. Biochem. 193, 737-750. Florkin M. and Jeuniaux C. (1974) Hemolymph: composition. In The Physiology of Insecta (edited by Rockstein M.), 2nd Ed., Vol. 5, pp. 255-307. Academic Press, New York. Gascuel O. and Golmard J. L. (1988) A simple method for predicting the secondary structure of globular proteins: Implications and accuracy. Cabios 4, 357-365. Grimstone A. V., Mullinger A. M. and Ramsey J. A. (1968) Further studies on the rectal complex of the mealworm Tenebrio molitor, L. (Coleoptera, Tenebrionidae). Phil. Trans. R. Soc. Lond. B 253, 343-382. Groebe D. R., Chung A. E. and Ho C. (1990) Cationic lipid-mediated co-transfection of insect cells. Nucl. Acids Res. 18, 4033. Guy C., Haskell D., Neven L., Klein P. and Smelser C. (1992) Hydration-state responsive proteins link cold and drought stress in spinach. Planta 188, 265 270. von Heijne G. (1986) A new method for predicting signal sequence cleavage sites. Nucl. Acids Res. 14, 4683-4690.

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Acknowledgements--We would like to thank Ernest Kroeker for dsp28 antibodies and N-terminal sequence, Chris Richardson for providing pETL, and much needed advice, and Bernie Ducker and Peter Davies for assistance with thermal hysteresis readings. Richard Casselman and Eric Carstens are much appreciated for their work in obtaining recombinant virus. This work was supported by an NSERC Graduate Scholarship to L. G. and by Insect Biotech Canada, a Network of Centres of Excellence funded by the Government of Canada, and the Natural Sciences and Engineering Research Council (Canada).