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Am J Physiol Regul Integr Comp Physiol 277: R1164-R1170, 1999;
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Vol. 277, Issue 4, R1164-R1170, October 1999

Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study

Gary P. Burness1, Scot C. Leary2, Peter W. Hochachka1, and Christopher D. Moyes2

1 Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4; and 2 Department of Biology, Queens University, Kingston, Ontario, Canada K7L 3N6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The activities of oxidative and glycolytic enzymes show body size-dependent relationships across a wide variety of taxa; however, the mechanistic basis remains unknown. We sampled white epaxial muscle from rainbow trout (Oncorhynchus mykiss) spanning a 100-fold range in body mass. We measured activities of enzymes from aerobic and anaerobic metabolic pathways, RNA [total RNA and mRNA, pyruvate kinase (PK), citrate synthase (CS), and MyoD mRNA], and total DNA. Total RNA and DNA showed a biphasic relationship with body size, with a break point occurring after fish reached 1 yr of age. In contrast, total RNA/total DNA was constant across the entire size range. Neither CS activity nor CS mRNA levels scaled with body mass. PK activity and PK mRNA levels increased in parallel in yearling fish only (r2 = 0.91, P < 0.01). This suggests that although PK expression is transcriptionally regulated in yearlings, the molecular mechanisms regulating expression change with growth and age. This was supported by a positive correlation between MyoD and PK mRNA levels (r2 = 0.17, P < 0.05).

mRNA; regulation; muscle; fish


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALLOMETRIC SCALING relationships are ubiquitous in biology, occurring from ecosystem levels of analysis to individual cells (5, 21, 25). Such relationships also extend to the activities of enzymes in metabolic pathways. When expressed on a per gram of tissue basis, enzymes associated with anaerobic metabolism [e.g., lactate dehydrogenase (LDH), pyruvate kinase (PK), creatine phosphokinase (CPK)] increase with increasing body mass (9, 13, 29-31). In contrast, enzymes associated with aerobic metabolism [e.g., citrate synthase (CS)] decrease with increasing body mass in accordance with the mass-specific decrease in whole animal aerobic metabolism (9, 13, 29-31). The molecular mechanisms that determine changes in metabolic enzyme activity with mass are largely unknown but are expected to be influenced by many factors related to size and growth rates.

Much of the work on the scaling of enzyme expression has focused on fish (29-31). Many species have indeterminate growth, which results in large intraspecific ranges in body size, while avoiding the problems associated with interspecies comparisons. In addition, individuals within a population often grow at different rates in relation to stress, competition, and resource limitations. This makes many species useful for investigation into the molecular mechanisms governing the allometry and ontogeny of enzyme expression. In the only study that has investigated the molecular mechanism regulating changes in enzyme expression with body size, it was found that LDH activity in barred sand bass, Paralabrax nebulifer, did not correlate with LDH mRNA (36). Although LDH activity increased with increasing body mass, LDH mRNA did not. The potential influence of development and growth rates was not considered at that time.

Genes for different enzymes can fine tune their expression rates according to their functional role in a metabolic pathway (14, 18). Consequently, mechanisms governing expression of LDH may differ from those regulating other glycolytic or oxidative enzymes. Regulation of de novo enzyme synthesis can occur transcriptionally or posttranscriptionally (6, 16, 20). These mechanisms can act alone or in combination to regulate the expression of gene products (4). The principal aim of the present study was to determine the regulatory mechanisms that govern changes in various enzyme concentrations with increasing body size. We hypothesized that allometric variation in enzyme concentration would parallel changes in mRNA levels. As an index of concentration, we measured the maximum catalytic activity (Vmax) of enzymes involved in anaerobic (PK, LDH, CPK) and aerobic (CS) ATP production. As different-sized fish in our study may be growing at different rates, we also measured MyoD mRNA, a transcription factor involved in myogenesis. Quantification of PK, CS, and MyoD mRNA levels suggested that regulatory mechanisms governing enzyme concentrations change with fish size, age, and growth rate.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and tissue collection. Rainbow trout (Oncorhynchus mykiss) were collected from a local supplier (Pure Springs Trout Farm, Shannonville, ON). To obtain sufficient size variation, we used yearlings (mass range 19.3-377.3 g) and fish >1 yr of age (mass range 929.4-2,589.9 g). After fish were killed, an ~3-g sample of epaxial white muscle was removed dorsal to the lateral line and was immediately frozen in liquid nitrogen. Sampling position along the body has no effect on enzyme activities (31). Muscle samples were powdered in liquid N2 and stored at -80°C until analysis (within 1 wk). For each assay, all tissues were randomized and treated identically.

Enzyme assays and DNA extraction. Approximately 100 mg of powdered muscle were weighed out in a 4°C cold room and homogenized on ice in 9 vol of homogenization buffer A (50 mM HEPES, 0.05% Triton, 1 mM EDTA, pH 7.4) with a ground glass homogenizer. PK was assayed within 2 h of homogenization. Extracts for other enzymes and DNA were frozen at -80°C until analysis.

Enzyme assays. As an index of enzyme concentration, we measured Vmax, at 25°C, on a 96-well Molecular Devices Spectromax 250 plate spectrophotometer. Uncentrifuged homogenates were used to avoid potential losses in the pellet. All enzymes (except CS) were assayed in 50 mM imidazole (pH 7.4) at 340 nm. CS was assayed in 20 mM Tris (pH 8.0) at 412 nm. Maximum activities were assayed under the following conditions: for PK, 5 mM ADP, 100 mM KCl, 10 mM MgCl2, 0.15 mM NADH, 10 mM fructose 1,6-bisphosphate, 5 mM phospho(enol)pyruvate, and excess LDH; for LDH, 5 mM pyruvate and 0.15 mM NADH; for CPK, 5 mM creatine, 5 mM ATP, 5 mM phospho(enol)pyruvate, 10 mM MgCl2, 0.15 mM NADH, and excess PK and LDH; for CPK, 0.12 mM acetyl-CoA, 0.2 mM 5,5'-dithiobis(2-nitrobenzoic acid), and 0.5 mM oxaloacetate (omitted from control). In preliminary experiments, control reaction rates (reactions omitting substrate) were negligible for enzymes other than CS. All substrates and cofactors were saturating but not inhibitory.

A central assumption in our study is that enzyme activity approximates enzyme concentration ([E]). This conclusion is drawn from a variety of evidence. Changes in Vmax can be due to changes in [E] or differences in the catalytic efficiency (kcat). Systematic changes in kcat with body size are unlikely, given that kcat is phylogenetically quite conserved (32). This suggests that the primary source of variation in values of Vmax is [E]. Although PK activity is covalently regulated in trout liver (17, 22, 33), there is no evidence for this in white muscle (12).

DNA extraction. DNA was extracted from 400 ml of DNA per enzyme extract (containing 40 mg tissue) by phenol-chloroform extraction (19). Samples were digested overnight with 0.1 mg/ml proteinase K. An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added to each digest, and the sample was vortexed and centrifuged for 10 min at 1,700 g. The upper aqueous phase was retained and precipitated with 0.5 vol of ammonium acetate (7.5 M) and 2 vol of ethanol (100%) and then centrifuged for 3 min at 1,700 g. The pellet was washed in 70% ethanol, allowed to air dry, and resuspended in 250 ml of distilled water. DNA purity was assessed at 260/280 nm. Absorbance at 260 nm was used for quantification in triplicate.

RNA extraction. Total RNA was extracted as described previously (3). Briefly, 1 g of powdered tissue was weighed out in a 4°C cold room and immediately homogenized for 10 s in 10 vol of buffer B (4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 15 mM mercaptoethanol) with a Polytron tissue homogenizer. This extract was frozen at -80°C until analysis on the following day. After the extract was thawed, 1 vol of 2 M sodium acetate (pH 4.0), 10 vol of buffer-saturated phenol (pH 4.3), and 2 vol of chloroform-isoamyl alcohol (49:1) were added to each homogenate and mixed thoroughly between steps. Homogenates were left on ice for >= 15 min, then centrifuged for 30 min at 3,000 g. The aqueous phase was retained and mixed with an equal volume of isopropanol and allowed to precipitate at -20°C overnight. After removal from the freezer and centrifugation for 30 min at 3,000 g, the RNA pellet was resuspended in 0.3 ml of guanidine thiocyanate buffer and reprecipitated in 0.3 ml of isopropanol. Samples were centrifuged for 5 min at 12,000 g and resuspended in diethyl pyrocarbonate-treated water. RNA purity was assessed using absorbance at 260/280 nm and quantified in triplicate at 260 nm. In calculations of DNA and RNA per gram of tissue, we considered the proportion of supernatant recovered in the phenol-chloroform extraction (typically 75%). Poly(A)+ (mRNA) was extracted from 140 µg of total RNA with use of streptavidin-coated beads and biotinylated oligo(dT) (CPG).

After quantification, RNA samples were denatured by glyoxal-DMSO treatment for 1 h at 50°C (2). Samples were frozen overnight at -20°C, thawed, and fractionated on a single 1.4% agarose gel running at 0.4 V/cm for 4 h. RNA was capillary transferred overnight with 20× saline-sodium citrate (SSC) onto an uncharged Duralon nylon membrane. The membrane was rinsed with 2× SSC and allowed to air dry. RNA was fixed to the membrane by ultraviolet cross-linking.

For detection of PK mRNA levels, the membrane was prehybridized in 25 mM KH2PO4 (pH 7.4), 5× SSC, 5× Denhardt's reagent (20 mg/ml Ficoll 400, 20 mg/ml polyvinylpyrrolidine, 20 mg/ml BSA), and 50% formamide for 1-6 h at 42°C. After prehybridization, the solution was replaced with a hybridization solution [25 mM KH2PO4 (pH 7.4), 5× SSC, 5× Denhardt's reagent, 50% formamide, and 25 g/l dextran sulfate] and allowed to hybridize with radiolabeled PK cDNA for 16-24 h at 42°C. For detection of CS mRNA levels, the membrane was prehybridized and hybridized at 42°C in 6× SSC, 5× Denhardt's reagent, and 0.5% SDS with time lines identical to those described for PK. After both hybridizations, the membrane was washed at 42°C, twice for 15 min with 1× SSC-0.1% SDS and twice for 15 min in 0.25% SSC-0.1% SDS. For determination of MyoD mRNA levels, the membrane was hybridized using QuickHyb (Stratagene) for 4 h at 68°C and washed twice at room temperature in 1× SSC-0.1% SDS and once for 15 min at 60°C with 0.25× SSC-0.1% SDS. Band intensities were quantified using a PhosphorImager and ImageQuant Software (Molecular Dynamics). All mRNA levels are expressed in relative units.

A cDNA probe for PK was constructed as described previously (3). The CS probe was amplified via PCR from first-strand cDNA prepared from total RNA of rat gastrocnemius at 57.5°C with 5'-GAAACATC(A/G)GTTCTTGATCC-3' and 5'-GTGTATTCCAGATGTAGTC(A/T)CGTAA-3'. Primers for CS were designed on the basis of consensus sequence from pig and rabbit (1). A 765-bp product was cloned into pCRII (Invitrogen), transfected into One-shot cells (Invitrogen), and found to be 90% identical to human CS mRNA (MOBIX, McMaster University). The trout MyoD cDNA probe (provided by Dr. Rescan, Institut National de la Recherche Agronomique) included the coding region with 30-bp 5'-untranslated region (23). All radiolabeled probes were prepared as described by Battersby and Moyes (3).

Statistical analyses. Enzyme and body mass data were log base 10 transformed; molecular data remain untransformed. Regression lines were fit using least squares linear regression. Spurious correlations can result when two variables that each correlate with a third (e.g., body mass) are compared. To avoid this, multiple regressions were performed with body mass included as one of the independent terms. To compare the slopes and intercepts among simple linear regressions, an analysis of covariance was performed. All tests were two-tailed, and statistical significance was claimed at P < 0.05. Analyses were performed using Systat 5.2.1 (35).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Allometry of enzyme expression. Figure 1 shows the ranges of maximum enzyme activities and presents regression equations for each enzyme on body mass. Larger fish demonstrated higher PK, LDH, and CPK activities than smaller fish (Fig. 1, A-C). After controlling for body mass through multiple regression, individuals with relatively high PK activities had relatively high LDH activities (P < 0.001). However, individuals with relatively high glycolytic capacities, as indexed by PK or LDH activity, did not necessarily have high CPK activity (P > 0.10).


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  		Fig. 1.   Scaling of aerobic and anaerobic enzyme activity from trout white muscle on body mass. Maximum catalytic activities were assayed spectrophotometrically at 25°C in presence of saturating substrates and cofactors. PK, pyruvate kinase; LDH, lactate dehydrogenase; CPK, creatine phosphokinase; CS, citrate synthase. Regression equations are in following form: Y = aXb, where Y is enzyme activity and X is mass (both are linear). open circle , Fish >1 yr old; , yearlings.

In contrast to anaerobic scaling patterns, CS activity, an index of capacity for aerobic metabolism, showed no relationship with body mass (Fig. 1D). Visual inspection of the CS data suggested a difference in the scaling relationships between yearlings and fish >1 yr of age. When the five older fish were excluded from analysis of CS, there was a negative relationship between enzyme activity and body size (r2 = 0.48, P = 0.001; Table 1). Exclusion of the older fish had little effect on the relationships of the other enzymes with body mass (Table 1).

                              
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  		Table 1.   Allometric relationships and descriptive statistics for enzyme activities in white epaxial muscle of <1-yr-old rainbow trout

We present enzyme activities as units per gram of tissue wet weight. The water content of the tissues was not constant across individual fish, inasmuch as smaller fish had a 3.5% higher muscle water content than larger fish (on the basis of a random 12 samples). The equation for conversion is as follows: U/g dry wt = U/g wet wt × 100/[3.87 (log mass) + 12.9]. Expression of activity per unit dry weight does not change correlation coefficients or statistical probabilities; consequently, we present data in the standard units per gram of wet weight.

Allometry of DNA, RNA, and mRNA levels. When the molecular data were plotted against body mass, there was a break point between yearlings and fish >1 yr of age. Total DNA per gram of tissue (Fig. 2A) and total RNA per g of tissue (Fig. 2B) increased with increasing body mass up to ~400 g. This relationship was not maintained with further increases in mass. After statistically controlling for the common influence of body mass on DNA and RNA, there was a positive correlation between the amount of DNA and RNA per gram of tissue (P < 0.001).


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  		Fig. 2.   Scaling of nucleic acid levels from trout white muscle on body mass. Regression lines were fitted for yearlings only. Nucleic acid levels were determined spectrophotometrically. Y, nucleic acid level; X, log (mass). open circle , Fish >1 yr old; , yearlings.

The RNA pool is typically dominated by ribosomal and transfer RNAs, with mRNA representing a relatively small percentage of total RNA. Total mRNA represented an increasing percentage of total RNA with increasing body size (r2 = 0.17, P = 0.048, n = 23); however, this relationship was weak and heavily influenced by two points. A distribution-free Spearman rank correlation was not significant (rs = 0.31, P > 0.10, n = 23), indicating that total mRNA was a relatively constant 1.34 ± 0.21% of total RNA regardless of fish size. The ratio of total RNA to DNA, a common index of growth rate (10), also represented a relatively constant value over a large range of body mass (r2 = 0.05, P = 0.32, n = 24).

To determine the molecular mechanism underlying changes in enzyme activity with body mass, we quantified mRNA levels for PK and CS. As was the case for total RNA per gram, PK mRNA increased with body mass up to ~400 g and then declined in the older, larger fish (Fig. 3A). There was also a general increase in the levels of PK mRNA with increasing total RNA concentrations, regardless of fish age (r2 = 0.37, P = 0.002, n = 24). This relationship held even after statistically controlling for the common influence of body mass (P < 0.001).


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  		Fig. 3.   A: relationship between relative PK mRNA levels and body mass. Y, PK mRNA; X, log(mass). B: relationship between PK activity and relative PK mRNA levels. Regression lines were fit for yearlings only. open circle , Fish >1 yr old; , yearlings.

To determine whether there was evidence for transcriptional regulation of PK expression, we analyzed all fish together and compared enzyme activity with transcript levels. PK mRNA was a significant predictor of PK activity, with 85% of the variance in PK activity attributable to the combined effects of PK mRNA and body mass (P < 0.001, n = 24). The strength of the overall relationship was, however, based primarily on the influence of the yearling fish (Fig. 3B). When yearlings (<400 g) were analyzed separately, PK mRNA explained a striking 89.3% of the variation in PK activity (Fig. 3B; n = 19), whereas body mass became an insignificant predictor (P = 0.18). These data support the hypothesis that PK activity is regulated at the mRNA level in yearlings but not in older fish.

To test the hypothesis that PK expression is influenced partly by growth rate, we quantified MyoD mRNA levels. MyoD is a transcription factor involved in myogenesis. There was a significant positive correlation between PK mRNA and MyoD mRNA levels (Fig. 4; P < 0.05).


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  		Fig. 4.   Relationship between relative PK mRNA and MyoD mRNA levels. open circle , Fish >1 yr old; , yearlings.

To determine whether the regulatory mechanisms governing mitochondrial enzyme activity are the same as those of cytosolic enzymes, we quantified CS mRNA levels. In contrast to the PK mRNA levels, total variation in CS mRNA was much more modest. As was the case with PK mRNA, there was a positive correlation between CS mRNA level and total RNA concentrations (r2 = 0.39, P = 0.001, n = 24). There was, however, no relationship between CS mRNA and body mass when all fish were analyzed together (r2 = 0.09, P = 0.153, n = 24) or when only the yearlings were considered (Fig. 5A). There was also no relationship between CS mRNA and CS activity when all fish were compared together (r2 = 0.01, P = 0.643, n = 24) or when only the yearlings were considered (Fig. 5B). After statistically controlling for the influence of body mass, CS mRNA was still not a significant predictor of CS activity for either group of fish (P > 0.10). A lack of relationship between mRNA level and CS expression may be a consequence of the small amount of variation in each variable with increasing body mass.


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  		Fig. 5.   A: relationship between relative CS mRNA levels and body mass. B: relationship between CS activity and relative CS mRNA levels. Regressions were performed on yearlings only. open circle , Fish >1 yr old; , yearlings.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Different scaling relationships between aerobic and anaerobic enzymes have been reported for a variety of taxa, including fish (29, 30, 31), mammals (9, 13), and reptiles (11). In this study the expected positive relationship was found between body mass and muscle activities of PK, LDH, and CPK. Negative scaling of CS was observed only when the older fish were excluded from the analysis. At the level of enzyme activity, the regulatory consequences or benefits of reciprocal trends in glycolytic and oxidative enzymes are likely related to swimming performance. A decrease in CS activity per gram of muscle with increasing body size follows the same general pattern as aerobic metabolism and scales with an exponent similar to that of sustained swimming speed (31). Increased glycolytic enzyme activity with increasing body size is hypothesized to provide the power required for large fish to perform the same relative burst swimming velocities (number of body lengths per second) as smaller fish (29-31).

The mechanism by which basal levels of glycolytic and oxidative enzymes are established is largely unknown. Most glycolytic enzymes and the GLUT-1 glucose transporter share sensitivity to the transcription factor HIF-1 (28). Similarly, a large number of respiratory enzymes are under control of NRF-1, NRF-2, and other families of transcription factors (18, 24). No factor has been shown to reciprocally regulate glycolytic and respiratory genes. Sharing sensitivity to a single transcription factor helps defend stoichiometries within pathways during periods of rapid response to environmental/physiological stressors and may also contribute to the allometric trends. However, each enzyme may also have a unique pattern of reliance on transcriptional, translational, and posttranslational regulation. This complexity was evident in the present study at several levels.

The principal aim of the present study was to examine the regulatory basis of differences in bioenergetic enzyme activity with body size. Previous studies, which focused on the regulation of LDH activities, suggest that transcriptional regulation accounts for many of the differences in LDH activities between populations and physiological states. Interpopulation differences in LDH-B levels in another fish, Fundulus heteroclitus, are achieved through such mechanisms (7, 8, 26). However, regulatory mechanisms in F. heteroclitus change with acclimation temperature and population of origin (27). In the scorpionfish Scopaena guttata, a 3-mo fast results in a decrease in LDH activity and LDH mRNA (37), suggesting transcriptional regulation. In contrast, Yang and Somero (36) report that although LDH activity increases with increasing body mass in barred sand bass, LDH mRNA does not. We had hoped to better understand the nature of allometric variation in enzymes by extending the analysis to other enzymes.

In the present study, PK mRNA was a good predictor of PK activity, consistent with transcriptional regulation. However, this relationship was evident only in small fish (Fig. 3B). In contrast, there was no relationship between CS mRNA and CS activity, even with large fish excluded from the analysis (Fig. 5B). Collectively, these observations suggest that mRNA levels do not consistently reflect enzyme levels, even with genes thought to be transcriptionally regulated in other species (1). Furthermore, the peculiar biphasic pattern emerging in many parameters suggests that simple patterns of allometric variation in enzyme activity can arise through complex regulatory strategies influenced by size and growth rate.

To explore the interaction of growth rate and body size further, we quantified MyoD mRNA levels. MyoD is a transcription factor involved in myogenesis (i.e., conversion of undifferentiated myoblasts into mature muscle). As such, it is expected to be a good indicator of mass-specific growth rate. A positive (although weak) relationship between PK mRNA and MyoD mRNA levels lends support to the hypothesis that body size and growth rate influenced the allometric relationships.

Does transcriptional regulation account for PK activity? There was a clear break point at ~400 g for many parameters. This corresponds to the boundary between yearlings and fish >1 yr of age. The 20-g fish were similar in age to those weighing 400 g but were presumably growing more slowly. Within this cohort, linear relationships were seen in all the enzymes as well as RNA per gram, DNA per gram, and PK mRNA per gram.

To determine the potential mechanism through which enzyme activity was regulated, we focused on yearling fish only and expressed all parameters as a percentage of maximum values (Fig. 6). Calculation of PK mRNA per gram depends on total RNA per gram, which increased slightly with body mass (Figs. 2B and 6). Because these fish were of a similar age, they achieved different sizes by demonstrating different mass-specific growth rates. Total RNA levels have been shown to correlate with levels of protein synthesis occurring in a cell (reviewed in Ref. 10). Although total RNA per gram increased with body mass, it did so with a slope significantly shallower than that of PK mRNA per gram (Fig. 6; P < 0.001). The increase in PK mRNA per gram results primarily from changing PK mRNA/RNA levels with body mass, as neither the slopes (P = 0.414) nor intercepts (P = 0.541) differ between the two variables (Fig. 6).


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  		Fig. 6.   Regulation of PK expression in fish white muscle. Data are for yearlings only and represent a summary of Figs. 2 and 3. Each dependent variable is expressed as percentage of maximum. See DISCUSSION for statistics. Total RNA/g muscle scaled with a shallower slope than either PK mRNA or PK mRNA/total RNA. Increases in PK mRNA/g with body mass were due primarily to changes in PK mRNA/total RNA levels.

Although regression analysis supports the role of transcriptional regulation in the smaller fish, PK mRNA was not a good predictor of PK activity in the five largest fish. Specifically, although these fish had the expected allometric pattern in PK activity, they exhibited lower levels of RNA per gram and PK mRNA per gram than would be expected from extrapolating the relationship shown for smaller fish (Figs. 2B and 3A). The lower levels of PK mRNA in larger fish may reflect lower levels of enzyme degradation. Rates of protein synthesis are known to decline with age/size (15), consistent with declines in mass-specific growth rate and/or lower protein turnover. This does not preclude a ubiquitous dependency on transcriptional regulation across body mass but simply suggests that less new PK mRNA may be required to obtain or maintain high enzyme activities in more slowly growing fish.

Caveats on growth vs. size. Previous workers have warned of the confounding effects of body size and age on condition indexes (34). Because white muscle is composed of fused myoblasts, DNA per gram is a function of the number of cells that fused to form the fiber (number of nuclei per gram) and the ultrastructural modifications appropriate to the fiber type (fiber geometry).

DNA per gram was a good predictor of RNA per gram. Total RNA levels (expressed per DNA or per gram of tissue) are frequently used as indexes of growth or condition (10). Although the fish in our study were of different sizes and different ages and possibly growing at different rates, the ratio of RNA to DNA was invariant. DNA per gram and RNA per gram demonstrated a biphasic pattern, maximal with fish of ~400 g. In contrast, no relationship between RNA levels and body mass was seen in barred sand bass (36). Although the allometric patterns in enzyme activities were similar to those in other studies, it is impossible to determine the impact of our use of captive reared, genetically homogeneous fish.


    ACKNOWLEDGEMENTS

This work was supported through Natural Science and Engineering Research grants to C. D. Moyes and P. W. Hochachka and an Atlantic Salmon Federation grant to S. C. Leary. G. P. Burness and S. C. Leary were supported by Natural Science and Engineering Research postgraduate scholarships.


    FOOTNOTES

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.

Address for reprint requests and other correspondence: G. P. Burness, Dept. of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 (E-mail: burness{at}zoology.ubc.ca).

Received 29 September 1998; accepted in final form 26 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Compar Physiol 277(4):R1164-R1170
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