1 Station de Recherches Avicoles, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; 2 Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, Rehovot 76100; 3 Department of Life Sciences, Bar Ilan University, Ramat Gan 52900; and Institute of Horticulture, The Volcani Center, Bet Dagan 50250, Israel
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ABSTRACT |
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The chicken leptin sequence, in
contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA (AF012727). The presence of an extra Cys may
confer a different structure and affect the leptin's biological
activity. To address this, we studied the effects of wild-type and
mutated (C4S) chicken leptins in vitro and in vivo and compared them
with mammalian leptin prepared from ovine leptin cDNA. The prokaryotic
expression vector pMON, encoding full-size A(1) chicken leptin
(AF012727), was mutated using a mutagenesis kit, yielding the C4S
analog. Escherichia coli cells transformed with this vector
overexpressed large amounts of chicken leptin C4S upon induction with
nalidixic acid. The expressed protein, found in the inclusion bodies,
was refolded and purified to homogeneity on a Q-Sepharose column, yielding three electrophoretically pure fractions, eluted from the
column by 100, 125, and 150 mM NaCl, respectively. All three fractions
showed a single band of the expected molecular mass (16 kDa) and were
composed of >95% monomeric protein. Proper refolding was evidenced by
comparing the circular dichroism spectrum of the analog with spectra of
nonmutated chicken and ovine leptins. The biological activity of the
C4S analog was evidenced by its ability to stimulate proliferation of
leptin-sensitive BAF/3 cells transfected with a long form of human
leptin receptor construct similar to its nonmutated counterpart,
indicating that Cys4 plays no role in leptin activity. The in vitro
activity of both wild-type and mutated chicken leptins was ~10-fold
lower than that of ovine leptin. After intravenous or intraperitoneal
injections, C4S analog and the nonmutated chicken and ovine leptins all
lowered the food intake of starved 9-day-old broiler or 5-wk-old layer
male chickens by 11-34%. Monitoring food behavior revealed that
the attenuated food intake resulted not from a decreased number of
approaches to the feeders but from a decrease in the average time spent
eating during each approach.
food intake; unpaired cystein; mutagenesis; hormone
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INTRODUCTION |
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OBESE PROTEIN (OB), also known as leptin, is a 146-amino acid hormone secreted by adipose tissue that serves as a signal acting on the central nervous system to regulate food intake and the body's energy balance (3, 8-10, 13, 21) through specific receptors. This protein, which belongs to the cytokine family (6, 17), exhibits high homology in mammalian species. We recently cloned and sequenced the first nonmammalian leptin, chicken leptin (AF012727; see Ref. 16), and its sequence was confirmed by Ashwell et al. (1). Chicken leptin cDNA exhibits >83% homology to human leptin but has only 145 amino acids (16). More interestingly, in avian species, leptin is expressed in liver and adipose tissue, as evidenced by the presence of leptin mRNA (1, 16). The cDNA encoding chicken leptin was subcloned into a prokaryotic expression vector, expressed as Ala-leptin, and was purified to homogeneity, yielding up to 400 mg protein/10 l of bacterial culture (14). The biological activity of the recombinant chicken leptin was demonstrated by its ability to stimulate the proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct and by its effect on lowering the food intake of starved chickens after intravenous or intraperitoneal injection (14). One unique structural feature of chicken leptin is that, in contrast to mammalian leptins, it contains an unpaired Cys at position 3 of the original cDNA (or position 4 in the Ala-leptin). To test whether this residue is important for biological activity, we mutated it to serine, prepared the C4S analog, and tested its in vitro and in vivo activities. An additional aim of the present work was to compare the in vivo activities of chicken and ovine leptins, as the latter lacks the unpaired Cys at position 3, and to elucidate whether their inhibitory effect on food intake results from a decreased number of approaches to the feeders or from a decrease in the average time spent eating during each approach.
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MATERIALS AND METHODS |
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Materials
Ovine (fraction SP) and chicken leptins were prepared in our laboratory as described previously (5, 14). Molecular weight markers for gel electrophoresis, RPMI 1640 medium, and nalidixic acid were obtained from Sigma Chemical (St. Louis, MO). SDS-PAGE reagents were purchased from Bio-Rad Laboratories (Richmond, CA). FCS was purchased from Labotal (Jerusalem, Israel), and a Superdex 75 HR 10/30 column and Q-Sepharose (fast flow) were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden). All other chemicals were of analytical grade.Construction of Chicken Leptin Analog C4S Expression Vector
The chicken leptin expression vector (14) was modified with the Stratagene Quickchange mutagenesis kit (La Jolla, CA) according to the manufacturer's directions using two complementary primers: the sense primer 5'-GGCAGTGCCGTCCCAAATCTTCCAGGATGAC-3' (the mutated base is underlined) and the antisense primer 5'-GTCATCCTGGAAGAATTGGGACGGCACTGCC-3'. The procedure included 12 PCR cycles as specified by the manufacturer's manual and the use of Pfu polymerase enzyme for the reaction. The template used for mutant construction was wild-type chicken leptin in the prokaryotic expression vector pMON (14). The mutated construct was then digested with Dpn I restriction enzyme, which is specific to methylated and hemimethylated DNA (target sequence: 5'-Gm6ATC-3'), to digest the template and to select for mutation-containing synthesized DNA. The vector was then transfected into XL1 competent cells. Two colonies were sequenced and confirmed to contain the mutation without any undesired misincorporation of nucleotides.Expression, Refolding, and Purification of Chicken Leptin Analog C4S
A preliminary experiment was performed with four clones in a 250-ml flask containing 30 ml terrific broth medium (18). Four hours after nalidixic acid induction, Escherichia coli MON105 cells transformed with the expression plasmids containing C4S chicken leptin analog cDNA expressed it (~16 kDa) as a major cell protein. One of the colonies expressing the analog was selected for large-scale preparation. Expression and refolding were carried out as described previously for the unmodified chicken leptin (14).Determination of Purity and Monomer Content of the Isolated Protein
SDS-PAGE was carried out according to Laemmli (11) in a 15% polyacrylamide gel under reducing conditions. Gels were stained with Coomassie brilliant blue R. HPLC gel-filtration chromatography was performed on a Superdex 75 HR 10/30 column with 0.2-ml aliquots of Q-Sepharose column-eluted fractions using 25 mM Tris · HCl buffer, pH 8, containing 150 mM NaCl. Freeze-dried samples were dissolved in H2O.Circular Dichroism Spectra
The circular dichroism (CD) spectra in degrees were measured with an AVIV model 62A DS CD spectrometer (Aviv Associates, Lakewood, NJ) with a 0.020-cm rectangular QS Hellma cuvette. The spectrometer was calibrated with camphorsulfonic acid (19). Lyophilized protein samples were dissolved in 55-65 mM sodium carbonate buffer, pH 7.5, at concentrations of 0.55-0.75 mg/ml and were cleared by centrifugation at 18,000 g for 10 min. The CD measurements were performed at 25.0°C, as controlled by thermoelectric Peltier elements with an accuracy of 0.1°C. The CD spectra were measured in five repetitions, resulting in an averaged spectrum for each protein. SD of the averaged CD signal at 222 nm was in the 0.5-2.0% range. For the secondary structure determination, the CD data were expressed in degrees per square centimeter per decimole per mean residue of the molecular weight, which was calculated to be 109.7 for ovine Ala-leptin and 109.5 for chicken Ala-leptin and its C4S analog, resulting in respective molecular masses of 16,124, 15,984 and 15,968 kDa. The protein concentration of the cleared solutions was determined using a buret method in five repetitions at different dilutions for each protein and with bovine pancreatic RNase A as a reference (protein concentration E = 0.715 ml/mg at 277 nm). The SD of the concentration determination was in the 1.6-7.1% range.BAF/3 Cell Proliferation Bioassay
The proliferation rate of leptin-sensitive BAF/3 1442-Cl 4 cells transfected with the long form of human leptin receptor was used as an in vitro leptin bioassay (5, 14). In stationary cultures, these cells were grown in RPMI 1640 medium supplemented with 5% FCS and 0.1 ng human interleukin-3 (IL-3)/ml at 37°C in 95% air-5% CO2. Before the experiment, the cells were collected by centrifugation, washed two times, and resuspended in the same medium without IL-3. The cells were then diluted to 50,000 cells/ml and divided into 24-well plates at 1 ml/well. No or various concentrations of ovine, chicken, or chicken leptin C4S analog were added, and the cells were grown for an additional 72 h. Cell growth was determined by counting the cells with a Coulter counter (Coulter Electronics, Hialeah, FL), and the number of doublings was calculated according to the following equation: log (no. of cells in wells treated with leptin/no. of cells in wells without leptin)/log 2. Ovine leptin (leptin-SP), which is as active as human or mouse leptin in this bioassay (5), was used as a control.Animal Experiments
To avoid any misleading conclusions concerning a general effect of leptin on food intake in chickens, we used two different strains characterized by slow (layer) or fast (broiler) growth. Both chicken strains were used in three different protocols. In protocol A, 9-day-old broiler (Shaver) male chickens (body weight ~100 g) were used. They were housed in individual cages in a temperature-, humidity-, and light-controlled room with free access to H2O and a regular diet (3,100 kcal, 19% protein). To synchronize feeding behavior and to create a high-appetite state, chickens were accustomed to daily 2-h fasting periods (0900-1100) for 3 days before each assay. On the day of the experiment, chickens that had been fasted for 90 min received a single intravenous (0.1 ml in wing vein) injection of 1 mg chicken leptin or its C4S analog per kilogram body weight. They were refed 30 min later, and food intake was measured manually every 30 min for 5 h. In protocol B, 5-wk-old semiheavy layer genotype, ISA Brown male chickens (body weight ~600 g) were housed under the same conditions except that each individual cage was placed on an electronic balance linked to a computer for continuous recording of food intake, namely by recording throughout the experiment in real time (at 1-s intervals), as described previously (21). Chickens were divided into two groups of nine chickens each, and to synchronize feeding behavior and to create a high-appetite state, chickens were accustomed to daily 2-h fasting periods (0900-1100) for 3 days before each assay, as described in protocol A. On day 1, the two groups received an intraperitoneal injection of isotonic saline solution (placebo), and food intake was monitored continuously for 24 h using a computerized system. On day 2, each group received 1 mg of chicken or ovine leptin per kilogram. On day 3, the groups that had received ovine leptin received chicken leptin and vice versa, resulting in 18 chickens used for each treatment. In an additional experiment carried out in the same way, food intake was also continuously monitored for 24 h, and we report values obtained for the first 2.5-10 h of treatment. Feeding activity and time spent eating were measured using constantly connected electronic balances. The number of approaches was estimated by counting the number of uninterrupted eating periods lasting >5 s (21). In protocol C, two groups of 5-wk-old layer chickens (n = 3) were accustomed to fasting and refeeding as described above. On day 1 of the experiment, both groups received a placebo, and food intake was continuously measured after the treatment. On day 2, each group received 1 mg of either wild-type or mutant chicken leptin per kilogram, respectively, and cumulative food intake was measured continuously using the computerized system.Statistical Analysis
Statistical analyses were performed with Statistical Analysis System (SAS) software (SAS Institute, Cary, NC) using the Student-Newman-Keuls test for variables. The results obtained according to protocol C were also analyzed by paired t-test. ![]() |
RESULTS |
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Purification of Chicken Leptin Analog C4S
The protein contained in the inclusion bodies prepared from 2.5 liters of induced cells was refolded in 4.5 M urea according to the protocol described for wild-type chicken leptin (14). The refolded protein was purified on a Q-Sepharose column by stepwise elution with increasing concentrations of NaCl in Tris · HCl buffer, pH 9 (Fig. 1), as described previously for the wild-type hormone (14). After washing the column with 50 mM NaCl, four fractions were eluted with, respectively, 100, 125, 150, and 500 mM NaCl in the same buffer. The fractions were pooled, dialyzed against 0.2% NaHCO3 (5 × 5 liters), and lyophilized. Fractions eluted with 100, 125, and 150 mM NaCl contained >95% monomeric leptin, and the fraction eluted with 500 mM NaCl consisted of a mixture of oligomers, dimers, and monomers (Fig. 2). All four fractions yielded only one band of ~15-16 kDa after 15% SDS-PAGE performed in the presence of a reducing agent (Fig. 3). Inclusion bodies prepared from 2.5 liters of fermentation culture yielded 40 mg leptin in the 100 mM NaCl eluate, 18 mg in the 125 mM NaCl eluate, 14 mg in the 150 mM NaCl eluate, and 45 mg in the 500 mM NaCl eluate. Because the mutation did not change the expected molar extinction at 280 nm, the value calculated for unmodified chicken leptin (2,394 M/cm
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Figure 4 shows the CD spectra of chicken,
C4S analog, and ovine leptins at neutral pH. All of the spectra
resembled the spectrum of -helix with its characteristic two minima
at 208 and 222 nm and positive band at ~191 nm (20). The
measured spectra had minima at ~209 and 220 nm. The CD spectra of the
leptins enabled a calculation of their secondary structure according to
the procedure and computer program CONTIN (20). The
program determines
-helix,
-strands, and
-turns as percentage
of amino acid residues involved in these ordered forms. The constraint
that the sum of all elements of the secondary structure in a protein
must be equal to unity resulted in a remainder that might be
interpreted as random coil content (20). In the present
study, for calculations by the CONTIN program, a set of standard CD
spectra of 17 proteins was employed. The results are shown in Table
1. A high
-helix content, no
-strands, and ~10% content of
-turns were clearly
characteristic for all the proteins, as expected from the structural
analysis of human leptin (22).
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Biological Activity In Vitro
The biological activity of the 100 mM NaCl-eluted fraction of chicken leptin analog C4S was compared with that of unmodified leptin and ovine leptin in a BAF/3 proliferation bioassay (Fig. 5). The results were analyzed by PRISM software (7), according to a nonlinear regression sigmoidal dose-response curve. In all analyses, the degree of match for the nonlinear correlation was very high (R2 > 0.97). EC50 for ovine leptin, chicken leptin, and chicken leptin analog C4S (100 mM NaCl-eluted fraction) were, respectively, 2.80 × 10
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Biological Activity In Vivo
Experiment 1: Effect of chicken and ovine leptin on food intake in
5-wk-old layer chickens.
Five-week-old layer chickens were divided into two groups
(n = 9) and were treated with saline (placebo) on
day 1, according to protocol B. After 90 min of
fasting, saline was injected intraperitoneally, and animals were refed
30 min later. Food intake was continuously measured 60 min after the
injection, as described in MATERIALS AND METHODS. On
day 2, one group was treated with chicken leptin and the
other with ovine leptin (both at 1 mg/kg), and, on day 3,
the group that had received chicken leptin was treated with ovine
leptin and vice versa. Because there was no significant effect for
giving ovine before chicken leptin or vice versa (data not shown), the
results for each treatment (saline, chicken, or ovine leptin) were
pooled, providing 18 animals for each treatment. The data presented in
Fig. 6 (mean ± SE) show food intake
every 60 min, chosen from the computerized data. No statistically
significant difference was observed within the first 60 min. In
contrast, both hormones significantly reduced (P < 0.05 to P < 0.001) cumulative food intake during the
next 2-8 h of feeding (Fig. 6). It should be noted that chicken
and ovine leptin had comparable effects, with a mean reduction of
~20-31% compared with the placebo. The maximum effect was
observed after 4 h of refeeding, with a 31.5 and 24.6% reduction
in cumulative food intake after chicken and ovine leptin
administration, respectively. The cumulative food intake 4 h after
the treatment was as follows: controls 14.6 ± 0.67 g;
chicken leptin 10.0 ± 0.88 g; and ovine leptin 11.0 ± 0.66 g (mean ± SE, P < 0.001).
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Experiment 2: Effect of chicken leptin C4S on broiler (9-day-old)
and layer (5-wk-old) chickens.
Two different chicken strains [broilers (9 days old) and layer
chickens (5 wk old)] were treated with either wild-type chicken leptin
or its mutated (C4S) analog. In broiler chickens (protocol A), immediately after food was restored, 120-min control-fasted animals began to eat regularly at varying rates throughout the 5-h
observation period (Fig. 8A).
After intravenous injection of chicken leptin or its C4S analog,
cumulative food intake decreased after 30 min, but the difference was
not statistically significant. However, during the next 4.5 h,
wild-type and mutant chicken leptin significantly (P < 0.05 to P < 0.001) reduced cumulative food intake
(Fig. 8A). Inhibition varied between 11 and 24% for
wild-type leptin and between 19 and 34% for mutant leptin. C4S leptin
was slightly more efficient than the wild type, but the effect was not
statistically significant. After 2 h of treatment, wild-type and
C4S leptins inhibited food intake by 23 and 34%, respectively, compared with the controls. This result indicates that Cys4 in the
wild-type leptin is not required for biological activity.
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DISCUSSION |
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Chicken leptin is characterized by the presence of an extra
unpaired Cys at position 3 of the mature protein or position 4 in the
Ala-leptin produced by us (14). To demonstrate the
putative role of this Cys, wild-type chicken leptin cDNA was mutated
(Cys4 to Ser), and the mutant leptin mass was produced in the same way as the wild-type hormone with a yield of ~120 mg/2.5 l fermentation culture. The yield of mutated chicken leptin was similar to that of the
unmodified leptin. CD spectra (Fig. 2 and Table 1) indicated proper
refolding. The studied leptins did not show -strand content but had
a notable amount of
-turns. According to the Protein Data Bank,
human leptin (PDB code: 1ax8) contains 61.0%
-helix, 11.0%
-turns, and no
-strands, as deduced from X-ray data with 2.40 Å resolution (22). Our data on the secondary structure of
leptins correlated with the PDB data for human leptin, albeit with a
higher
-helix content. The difference in
-helix content, up to
20% between human and ovine or C4S leptins and 10% between human and
chicken leptins, could result from both low resolution of the X-ray
data and errors in the CD spectra determinations. Therefore, at
present, there is no significant evidence that the secondary structures
of the leptins studied in the present work and human leptin differ. In
any case, even if such differences do exist, their extent may not
exceed 10% of
-helix and ~3% of
-turns. Evidently, chicken
leptin, its C4S analog, and ovine leptin do not contain
-strands,
and the 2% in chicken leptin is likely to have resulted from a
determination error.
The effect of mutated leptin was compared with that of wild-type chicken leptin both in vitro and in vivo. In addition, ovine and chicken leptins were compared because ovine leptin lacks a Cys at position 3. In BAF/3 cells stably transfected with the long form of human leptin receptor, wild-type and mutant chicken leptins exhibited equivalent or similar effects on cell proliferation, whereas ovine leptin was ~10-fold more efficient, in agreement with our former result (14). This indicates that, at least for the purpose of cell proliferation, the Cys3 (or Cys4 in Ala-leptin) in chicken leptin plays no role. Furthermore, the differences between ovine and chicken leptin cannot be attributed to improper refolding of the recombinant proteins but rather to differences in their primary structure, which likely affect their affinity toward human leptin receptor. Because the receptor in BAF/3 cells is a human leptin receptor, this hypothesis can only be confirmed once the chicken leptin receptor has been identified and characterized.
The role of Cys4 in chicken Ala-leptin was also indirectly investigated
in vivo by comparing the effects of ovine leptin, chicken leptin, and
C4S analog of the latter on reducing in food ingestion capacity. In
5-wk-old layer-genotype chickens, a single intraperitoneal injection of
ovine or chicken leptin exerted a significant reduction in food intake
in animals being refed after 2 h of fasting (Figs. 6 and
7A). The effect lasted >5 h but disappeared after 9-10
h (Fig. 6). Moreover, both ovine and chicken leptins exerted similar
effects on feeding behavior, with a similar impact on time spent eating
but with no effect on the number of approaches to the feeders compared
with placebo-administered controls (Table 2). Because the total
cumulative food intake (Fig. 7A) and the accumulated time
spent eating were reduced by both chicken and ovine leptin treatments
(Fig. 7B), whereas the number of approaches was not affected
by the hormonal treatment (Table 2), we conclude that the reduced feed
intake resulted mainly from the decreased average meal duration during
each approach and not from the change in meal frequency. This is
somewhat different from rodents, in which leptin injection inhibited
food intake by reducing the meal size without affecting meal duration
or meal frequency (4). Hence, chicken sensitivity toward
leptin is clearly demonstrated. In wild-type mice, the effect of leptin
lasted longer (>12 h) but was maintained by daily injections
(8-10), with a significant reduction of 35% in food
intake after 4 days of treatment (10 mg leptin · kg1 · day
1). These data show that
ovine and chicken leptin are equipotent in reducing food intake and
hint at similar affinities toward chicken leptin receptor, although
this has yet to be demonstrated. As already shown, the ovine and
chicken leptins exhibited different biological activities toward human
leptin receptor in a bioassay based on cell proliferation. In addition,
this result indicates, at least indirectly, that the presence of Cys4
has no effect. Treatment of two chicken strains differing in their food
ingestion capacity further supported this hypothesis, despite the fact
that at the same age broiler chickens ingest at least four times more food than layer chickens.
However, it should be noted that, when mouse leptin was injected
directly in the chicken central nervous system, no alteration in food
intake was observed (2). This observation raises the question if the difference between the present and other
(2) results originates from a different way of application
or is related to a different source of leptin. The
difference between the central and peripheral effects of leptin on food
intake may be partially explained by the absence of leptin receptor in
chicken brain or by the notion that mouse leptin does not bind to
chicken leptin receptor. Another possibility is that the infusion
period was too short. For instance, in ewes given
intracerebroventricular infusion of ovine leptin, a considerable
decrease of activity could be seen only after 72 h (D. H. Keisler
and A. Gertler, unpublished data). Moreover, we also suggest
that the peripheral leptin injection may allow leptin to bind to
binding protein and then act centrally. In addition, a specific
peripheral effect of leptin on liver, stomach, and -cells of the
pancreas and others may also play a role in regulating food intake.
Indirect evidence also suggests that food intake control in the central
nervous system of chickens is slightly different from that in mammals.
For instance, electrocoagulation of the ventromedian hypothalamus in
chicken or goose (12, 15) induces moderate
hyperphagia but not as strongly as in mammals.
In conclusion, the present results indicate that Cys3 in chicken leptin (Cys4 in Ala-leptin) is not crucial for its biological effect in vitro or in vivo. Moreover, the discrepancy between the efficiencies of ovine and chicken leptin in vitro and in vivo suggests that the chicken leptin receptor recognizes both molecules but human receptors discriminate between them. Confirmation of this hypothesis awaits the identification of the chicken leptin receptor.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Sergei Y. Venyaminov (Mayo Foundation, Rochester, MN) and Dr. Norma J. Greenfield (UMDNJ-Robert Wood Johnson Medical School, NJ) for the CONTIN program used to calculate the secondary structure from the far ultraviolet CD spectra. We also acknowledge Claude Bouchot for technical assistance and animal care.
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FOOTNOTES |
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* S. Dridi and N. Raver contributed equally to this work.
This research was partially supported by a grant from Chief Scientist of the Israeli Ministry of Agriculture awarded to A. Gertler.
Address for reprint requests and other correspondence: M. Taouis, SRA, INRA, 37380 Nouzilly, France (E-mail: taouis{at}tours.inra.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 2 December 1999; accepted in final form 8 February 2000.
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