Multiple soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes)

Maria Regina de Aquino-Silva Maria Luiza B. Schwantes Arno Rudi Schwantes About the authors

Abstracts

A recent locus duplication hypothesis for sMDH-B* was proposed to explain the complex electrophoretic pattern of six bands detected for the soluble form of malate dehydrogenase (MDH, EC 1.1.1.37) in 84% of the Geophagus brasiliensis (Cichlidae, Perciformes) analyzed (AB1B2 individuals). Klebe's serial dilutions were carried out in skeletal muscle extracts. B1 and B2 subunits had the same visual end-points, reflecting a nondivergent pattern for these B-duplicated genes. Since there is no evidence of polyploidy in the Cichlidae family, MDH-B* loci must have evolved from regional gene duplication. Tissue specificities, thermostability and kinetic tests resulted in similar responses from both B-isoforms, in both sMDH phenotypes, suggesting that these more recently duplicated loci underwent the same regulatory gene action. Similar results obtained with the two sMDH phenotypes did not show any indication of a six-banded specimen adaptive advantage in subtropical regions.


A fim de explicar o padrão eletroforético de seis componentes detectado para a malato desidrogenase solúvel (MDH, EC 1.1.1.37) em 84% dos exemplares de G. brasiliensis analisados (Cichlidae, Perciformes), uma duplicação recente no loco sMDH-B* é sugerida. Diluições seriadas de Klebe realizadas com extratos de músculo esquelético mostraram para as subunidades B1 e B2 o mesmo ponto final visual sugerindo um padrão de expressão não divergente para esses genes duplicados. Uma vez que não existe evidência de poliploidia na família Cichlidae, é sugerido que a duplicação no loco sMDH-B* seja resultante de uma duplicação regional. Especificidade tissular, termoestabilidade e propriedades cinéticas mostraram-se similares para as isoformas B, em ambos os fenótipos detectados, sugerindo estarem esses sob a ação do mesmo gene regulador. Os resultados similares obtidos para os fenótipos de três (AB1) e seis (AB1B2) componentes aqui analisados não mostraram nenhum indicativo de vantagem adaptativa deste último sobre o primeiro, em região subtropical.


Multiple soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes)

Maria Regina de Aquino-Silva, Maria Luiza B. Schwantes and Arno Rudi Schwantes

Universidade Federal de São Carlos, Departamento de Genética e Evolução, Caixa Postal 676, 13565-905 São Carlos, SP, Brasil. Send correspondence to M.L.B.S. Fax: +55-16-271-9094.

ABSTRACT

A recent locus duplication hypothesis for sMDH-B* was proposed to explain the complex electrophoretic pattern of six bands detected for the soluble form of malate dehydrogenase (MDH, EC 1.1.1.37) in 84% of the Geophagus brasiliensis (Cichlidae, Perciformes) analyzed (AB1B2 individuals). Klebe's serial dilutions were carried out in skeletal muscle extracts. B1 and B2 subunits had the same visual end-points, reflecting a nondivergent pattern for these B-duplicated genes. Since there is no evidence of polyploidy in the Cichlidae family, MDH-B* loci must have evolved from regional gene duplication. Tissue specificities, thermostability and kinetic tests resulted in similar responses from both B-isoforms, in both sMDH phenotypes, suggesting that these more recently duplicated loci underwent the same regulatory gene action. Similar results obtained with the two sMDH phenotypes did not show any indication of a six-banded specimen adaptive advantage in subtropical regions.

INTRODUCTION

Dimeric enzyme malate dehydrogenase (MDH, EC 1.1.1.37) catalyzes the reversible oxidation of malate to oxaloacetate, and is represented by two forms in vertebrates and invertebrates: mitochondrial (mMDH) and soluble (sMDH). The mitochondrial form acts in the Krebs cycle, and the soluble form is involved in gluconeogenesis, lipogenesis, and the malate-aspartate shuttle during aerobic glycolysis (Zink and Shaw, 1968). Two gene loci, sMDH-A* and sMDH-B*, encode sMDH in most of the fish and amphibian species studied (Bailey et al., 1969, 1970; Whitt, 1970; Wheat et al., 1971; Schwantes and Schwantes, 1977, 1982a,b; De Luca et al., 1983; Coppes et al., 1987a; Fenerich-Verani et al., 1990; Farias and Almeida-Val, 1992; Lin and Somero, 1995a,b; Caraciolo et al., 1996).

Differential expression of orthologous homologues (homologues encoded by a single gene locus common to different species) and paralogous homologues (homologues encoded by different gene loci in a species) of proteins during thermal acclimation or acclimatization has been reported (Hochachka, 1965; Hochachka and Somero, 1973, 1984; Tsukuda and Ohsawa, 1974; Tsugawa, 1976; Schwantes and Schwantes, 1982a,b; Graves and Somero, 1982; De Luca et al., 1983; Coppes et al., 1987a,b,c; Coppes and Somero, 1990; Lin and Somero, 1995a,b; Aquino-Silva et al., 1997). Adaptation to temperature by the two soluble MDH gene loci of teleost fish, where sMDH-A* encodes a thermostable isoform and sMDH-B* encodes a thermolabile isoform, was shown by Schwantes and Schwantes (1982a,b); De Luca et al. (1983); Coppes et al. (1987a); Farias and Almeida-Val (1992); Lin and Somero (1995a,b); Caraciolo et al. (1996); Monteiro et al. (1991, 1998) and Aquino-Silva et al. (1997). Lin and Somero (1995b), comparing the sMDH of Eastern Pacific barracudas from different latitudes, suggested that the variation in the paralogous ratio and the absence of the thermolabile isoform in warm-adapted species may be the result of a specific adaptation to high temperature. However, Farias and Almeida-Val (1992) and Caraciolo et al. (1996) detected both isoforms, with a predominance of the thermostable in studies of the sMDH of Amazon fishes.

Monteiro et al. (1991, 1998) studying the sMDH of 22 subtropical fish species belonging to the orders Characiformes, Siluriformes and Perciformes, showed that most had these paralogous isoforms. However, two of these species showed a much more complex electrophoretic pattern as do ther teleosts (Whitt, 1970; Bailey et al., 1970; Wheat et al., 1971; Farias and Almeida-Val, 1992). To explain the six-banded pattern detected in the characiform Hoplias malabaricus, in addition to sMDH-B*, a recent locus duplication hypothesis for sMDH-A was proposed, since the two homodimers coded by these loci exhibited a non-divergent pattern of expression and thermostability (Monteiro et al., 1991, 1998; Aquino-Silva et al., 1997). The six-banded pattern detected in 87% of Geophagus brasiliensis (Cichlidae) specimens analyzed suggested three hypotheses: a duplication event in processing their sMDH-B locus; the presence of three loci sMDH-A*, sMDH-B1* and sMDH-B2* with a null allele within B2*, and overdominance of variant allele *119 at sMDH-B*. In order to choose which one of these hypotheses best explains the multiplicity detected in the sMDH of G. brasiliensis, the present paper describes tissue specificities, thermostability and kinetic properties of sMDH.

MATERIAL AND METHODS

Two hundred and forty-three specimens of G. brasiliensis were collected by throw net from the Monjolinho Reservoir at the Federal University of São Carlos, State of São Paulo, Brazil. Annual temperature at the G. brasiliensis capture site ranged from 14.7 to 21.7°C. White muscle, heart and liver from each individual were dissected immediately after capture and kept at -20°C. A small piece of each tissue was homogenized (w/v) in 50 mM phosphate buffer, pH 7.0, using a Potter-Elvejhem tissue grinder, and then centrifuged at 19,000 g for 30 min at 4°C in a Sorvall RC 5B centrifuge. The resulting crude extracts were used for electrophoretic and spectrophotometric analyses.

Electrophoreses were carried out in horizontal gels containing 14% (w/v) corn starch prepared according to Val et al. (1981), using the pH 6.9 Whitt (1970) buffer system. A voltage gradient of 5 V/cm was applied for 14-17 h at 4°C. After electrophoresis, the starch gels were sliced lengthwise and the lower half incubated in an MDH-staining solution described by Monteiro et al. (1991). Thermal stability of isozymes was tested by subjecting each tissue extract to 58°C for 1, 2, 3, 5, 10, 15 or 20 min. Afterwards, these extracts were cooled on ice, centrifuged at 19,000 g for 30 min at 4°C, and electrophoresed together with the control (kept in ice water). After electrophoresis, the gels were stained as described above. To compare the relative activities of sMDH isozymes, Klebe's method (1975) was applied to the extracts previously submitted to three assay temperatures (10, 20, and 30°C). The term divergent ratio was utilized to indicate a ratio different from 1 for the activity of subunits encoded by duplicate genes within a given tissue. Nomenclature of sMDH gene loci, subunits and iso/allozymes was according to Shaklee et al. (1989).

The effect of temperature on Geophagus sMDH was measured in the direction of oxaloacetate reduction by the change in absorbance at 340 nm in a Beckman 25K spectrophotometer at three different temperatures (10, 20 and 30°C) and two pH regimens: constant-pH (7.0) buffer at each of the three temperatures, and temperature-dependent pH buffer at both 10°C (pH 7.08) and 30°C (pH 6.9). Temperature was controlled using a Lauda K-2/R circulating bath. The assays were carried out in a 50 mM imidazole chloride buffer containing 0.2 mM NADH and different concentrations of oxaloacetate. Oxamate at 10 mM concentration was added to inhibit lactate dehydrogenase activity, which could result from any pyruvate present in the assay medium. All reactions were performed at least in triplicate and initiated by adding 10 ml of enzyme to 1.0 ml of assay medium. Apparent Km values were calculated by the Lineweaver Burk method, using 1/S vs. 1/V plots. When saturation curves were sigmoidal the E-S affinities were determined by the Hill - S0.5 plot.

RESULTS

Tissue extracts sMDH from 243 G. brasiliensis specimens confirmed both patterns obtained previously by Monteiro et al. (1991, 1998) in 55 other specimens of this species. Of these, 202 showed an electrophoretic pattern with six bands, and 41 had three bands (Figure 1). According to the first hypothesis by Monteiro et al. (1998), the event of sMDH-B* duplication could be due to a process not yet completed in 48 individuals of this sampled population (41 detected here and seven in a previous sample). In their second hypothesis, since it was impossible to electrophoretically distinguish between B2-heterozygotes for an enzymatically inactive (i.e. null) allele, 100/Q0 and the B2 100-homozygotes, the calculation 48/298 showed that 16.1% of the individuals would be B2 Q0 homozygotes (q 2 = 0.161), and the null allele would appear in this sample population with a frequency of 0.40. In their third hypothesis, overdominance, 84% of the screened individuals would show a variant allele at MDH-B* - *119. Allele frequencies, assuming Hardy-Weinberg equilibrium and the c2 value obtained (155.85), indicate that these samples are not in equilibrium for this locus. Relative fitness values (WB100 = 0.28, WB 100/119 = 1, WB119 = 0) and selection coefficients (SB100 = 0.72, SB100/119 = 0, SB119 = 1) for the genotypes were utililized in calculating their genetic load (0.42).

Figure 1
- Malate dehydrogenase from skeletal muscle extracts from (1-7 and 9) AB1B2 individuals, and (8 and 10) AB1 phenotypes of Geophagus brasiliensis.

A nondivergent thermostability pattern was observed among paralogous isoforms of G. brasiliensis phenotypes. Thus, thermostability tests showed no difference between products detected in the sMDH-B* region, nor between these and the A* region (Figure 2). Also, Klebe's serial dilutions carried out at three temperatures with skeletal muscle extracts from AB1B2 individuals showed that B1 and B2 subunits (restricted to this tissue) had the same visual end-points, indicating the same molar ratio expected for nondivergent genes. In the skeletal muscle extracts, A and B (B1 and B2) subunits were present in an approximate proportion of 4:1:1. The contribution of these subunits to all those present was Ö(4:1:1), and their contribution to the isozymes present was 2 A:1 B1:1 B2 subunits. In AB1 skeletal muscle extracts sMDH-A* and sMDH-B* did not diverge (1 A:1 B1). AB1 heart and liver extracts showed only A-isoforms and heterodimers. Theoretical levels of B1-isoforms were calculated according to Klebe¢s method (1975). In both tissues, subunits were present in an approximate proportion of 64:1 and their contribution to all the isozymes present was Ö(64:1) or 8 A to 1 B subunits. In AB1B2 heart extracts, sMDH-B2* products were absent, and A and B1 subunits were present in an approximate proportion of 16:1. Their contribution to all the isozymes present was Ö(16:1) or 4 A:1 B1 subunits. In liver extracts, with no sMDH-B2* products, their subunits were present in an approximate proportion of 64:1 and their contribution to all the isozymes present was Ö(64:1) or 8 A:1 B1 subunits. These results showed the nondivergent duplicate sMDH-B2* expression restricted to Geophagus skeletal muscle.

Figure 2
- The effects of heat treatment on MDH from skeletal muscle extract of AB1 (A) and AB1B2 (B) Geophagus brasiliensis. Samples of unfractionated MDH were incubated at 58°C for (1) 1, (2) 2, (3) 3, (4) 5, (5) 10, (6) 15, and (7) 20 min, electrophoresed and stained for residual activity. C, Control.

To compare the effect of temperature-pH on the sMDH phenotypes of Geophagus, we examined the responses of their apparent Km of oxaloacetate (oxa) to different temperatures under two pH regimens, both in an imidazole buffer system with temperature-dependent pH (10 and 30°C) and constant pH (10, 20 and 30°C). Skeletal muscle extracts, which had the lowest A subunit proportion among the tissues analyzed, showed hyperbolic kinetics for both phenotypes, pH regimens and the three temperatures. Figure 3 and Table I illustrate the variation in AB1 and AB1B2 Geophagus apparent Km/S0.5 for oxaloacetate as a function of assay temperature and pH regimen. AB1 Km values were smaller for muscle extracts under both pH regimens (4-19 mmol/lwith temperature-dependent pH and 9-18 mmol/l with constant pH) than for heart (16-68 mm/l and 16-30 mm/l) and liver (47-54 mm/l and 16-28 mm/l) extracts. Also, AB1B2 Km values were smaller for muscle extracts under both pH regimens (15-19.6 mmol/lwith temperature-dependent pH and 8.5-25 mmol/l with constant pH) than for heart (13-48 mm/l and 23-30 mm/l) and liver (36-50 mm/l and 21-33 mm/l) extracts. In their physiological temperature range (14.7-21.7°C) and temperature-dependent pH, Km/S0.5 values for white muscle, heart and liver from individuals of both phenotypes were maintained within the range of approximately 9-23 mM/l, 20-35 mM/l, and 32-44 mM/l, respectively (Figure 3).

Figure 3
- The effect of temperature on the

DISCUSSION

The first hypothesis proposed by Monteiro et al. (1991, 1998) best explains the complex electrophoretic pattern of six bands detected in 84% of the studied G. brasiliensis (Cichlidae, Perciformes). Consequently, a duplication event probably occurred at MDH-B* of these individuals but not in the other 16%. Studies of five Amazonian cichlid species have also shown the occurrence of the six-banded rather than the regular three-banded electrophoretic pattern for sMDH (Farias and Almeida-Val, 1992). The activity of three gene loci for sMDH and the occurrence of a gene duplication in the sMDH-B* locus in an ancestor of the Amazonian cichlids were suggested as an explanation. Based upon the staining pattern of electrophoretic gels, rather than measurements of their enzyme activity, the second hypothesis postulated that the sMDH of G. brasiliensis could be encoded at three loci, MDH-A*, B1* and B2*, with a null allele at B2* - the B2*Q0. Electrophoretic studies of salmonid fishes have shown that about two-thirds of their duplicate genes by genome duplications have become silenced through the fixation of alleles that produce no detectable enzyme product (i.e., null alleles) (reviewed by Li, 1982). Studies with eukaryotes have shown that the amount of enzyme activity could be of adaptive significance (McDonald and Ayala, 1978; Allendorf et al., 1983). According to Ferguson et al. (1988), it is possible that organisms with reduced enzyme activity (i.e., those with null alleles) have reduced fitness depending upon the importance of the enzyme function in metabolism and the position of the enzyme in the biochemical pathway. The low frequency of null alleles in diploid species has been attributed to their mildly deleterious effects in the heterozygous state (Voelker et al., 1980; Langley et al., 1981; Allendorf et al., 1982; Burkhart et al., 1984). Even though several null alleles have been reported in polyploid fish (Engel et al., 1973; Wright et al., 1975; Klar and Stalnaker, 1979), there is little direct evidence of their deleterious effect on fitness. Thus, reduction in enzyme activity with a possible decrease in the reduction of oxaloacetate to malate could be considered a deleterious effect of the null allele at sMDH-B2* in G. brasiliensis, on the oxidative metabolism of carbohydrates and therefore fitness. But the existence of this null allele at a frequency of 0.36 (Monteiro et al., 1991, 1998) or 0.40 (obtained here after increasing the sample number) could hardly be associated with possible deleterious effects of this allele such as reduced viability of its carriers in the population. According to Ferris and Whitt (1979), if a locus product is not essential, it could be silenced through formation and fixation of a null allele. At the same time, Zouros et al. (1982) hypothesized that loss of gene expression does not occur in cases in which the duplication event is followed by divergence of either the regulation of expression or the enzymatic properties of the duplicate loci (or both). This seems to be the case for G. brasiliensis, since their sMDH-B* loci are divergently expressed in heart and liver extracts. The third hypothesis is that of overdominance. In this case, its sMDH could be encoded at two loci, as in most diploid species, with a fast anodal variant allele at sMDH-B* occurring in this population sample at a frequency of 0.42. According to Berger (1971), in overdominance involving the best heterozygote adaptation, the allelic distribution inside the population would be a function of selective values of each genotype, and the heterozygote would present the highest adaptive value. Zouros and Foltz (1987) showed that the adaptive superiority of heterozygotes is neither the most common nor the fundamental mechanism in the maintenance of genetic variation. According to these authors, biological factors are important for the maintenance of overdominance. In our results the relatively high segregational load obtained (0.42) indicates its improbable occurrence in nature. Considering the second and third hypotheses formulated to explain the complex sMDH pattern of Geophagus, no differences were observed when E-S affinity of AB1 and AB1B2 muscle extracts were compared with each other, or with H. malabaricus. Also, regarding the second hypothesis (null allele), no difference in MDH activity between the two phenotypes or among them and H. malabaricus or other vertebrates was detected. This fact, together with the high frequency of this putative null allele in our sample, should render this hypothesis unlikely.

Adaptation to temperature by the two soluble MDH gene loci of teleost fish where A is the thermostable isoform and B the thermolabile (Schwantes and Schwantes, 1982a,b; De Luca et al., 1983; Coppes et al., 1987a; Farias and Almeida-Val, 1992; Lin and Somero, 1995a,b, and Caraciolo et al., 1996; Aquino-Silva et al., 1997) was not shown here in thermostability tests with G. brasiliensis tissues.

Three categories describe the types of duplicated gene expression (Ferris and Whitt, 1979): nondivergent, unidirectionally divergent and bidirectionally divergent. In the first, duplicate genes are expressed equally in all tissues in which the enzyme is present. This nondivergent pattern was observed here in the expression of B-duplicated genes. This pattern, according to Farias and Almeida-Val (1992), was also observed in sMDH-B1* and B2* products of Amazon cichlid fishes indicating that these genes probably undergo the same regulatory gene action and that the duplication event occurred recently, after the divergence of sMDH-A* and B*. Also, for sMDH from H. malabaricus, we (Aquino-Silva et al., 1997) reported, in addition to sMDH-B*, a recent locus duplication in the sMDH-A* and a nondivergent pattern for the expression of these A-duplicated genes.

Among categories which describe the types of gene expression utilizing Klebe's method (1975) at three different temperatures, the sMDH-A* and both duplicate B* locus products of G. brasiliensis might be referred to as a unidirectionally divergent pattern, which includes most of the divergent patterns observed (Ferris and Whitt, 1979, Schwantes and Schwantes, 1982a). These vary from a nondivergent ratio in AB1 skeletal muscle samples to a divergent ratio of eight in AB1B2 liver samples. The bidirectionally divergent category, where B-isoforms predominate in skeletal muscle and A-isoforms in liver (Bailey et al., 1969, 1970; Clayton et al., 1973; Aspinwall, 1974; Fisher et al., 1980; De Luca et al., 1983; Coppes et al., 1987a; Monteiro et al., 1991), was not detected in G. brasiliensis.

Comparative studies of orthologous of several enzymes from animals adapted to different temperatures have revealed strong conservation of certain kinetic properties at the physiological temperatures of the species (Somero, 1978, 1986; Somero et al., 1983; Hochachka and Somero, 1984; Coppes and Somero, 1990). Although Km values tend to be strongly perturbed by temperature, highly similar values are found at normal body temperatures among species having widely different average body temperatures. According to Hochachka and Somero (1984), the biological significance of this marked conservation of Km values for A4-LDH is indicated by similar piruvate concentrations among different vertebrates. If this biological significance is valid for unfractionated sMDH, then, in the physiological temperature range of G. brasilensis analyzed here, oxaloacetate concentrations in their cellular microenvironment must be around the Km values obtained. Then, oxa concentration in AB1 muscles, where a nondivergent ratio of subunits was detected (1 A:1 B), would be 1.8-2.3 times smaller than heart extracts (8 A:1 B) and 2.3 less than liver (8 A:1 B) extracts. For AB1B2 muscles with a subunit ratio of 2 (2 A:1 B1:1 B2), oxa concentrations would be 1.3-1.4 times smaller than heart extracts (4 A:1 B1), and 1.6-2.0 less than liver extracts (8 A:1 B1).

Conservation of Km values is interpreted as reflecting selection for retention of catalytic and regulatory capacities of enzymes and is manifested by adaptations to pressure, osmotic conditions and pH as well as to temperature (Hochachka and Somero, 1984). According to Reeves (1977), pH values of blood and cytosol decrease with rising temperatures with an approximate slope of 0.017 pH units per degree C rise in temperature. For this reason, Hochachka and Somero (1984) suggested that the pH-temperature relationship leads to short-term and evolutionary changes in body temperature. In non-physiological conditions of constant pH, the variation in Km of piruvate among interspecific homologues of A4-LDH is approximately 10-fold (Somero, 1981). In contrast to these data, our results obtained with unfractionated muscle, heart and liver sMDH under three temperature assays and two pH regimens showed that in the adaptive temperature range of Geophagus, the variation in Km under conditions of constant pH imidazole buffer was smaller (approximately two-fold) than under temperature-dependent pH buffer (five-fold). Similar results were obtained for the characiform H. malabaricus (Aquino-Silva et al., 1997).

However, according to Lin and Somero (1995b), while A4-LDH conservation of Km at physiological temperatures was achieved by an evolutionary change in the amino acid sequence of orthologous homologues, that of the cytosolic form of MDH may also be achieved by altering the ratio of paralogous thermostable and thermolabile isozymes. As thermostable sMDH differs from thermolabile in having a higher Km of oxaloacetate (Bailey et al., 1970; De Luca et al., 1983; Lin and Somero, 1995b; Aquino-Silva,M.R., Schwantes, M.L.B. and Schwantes, A.R., unpublished results), the contribution of this high-Km thermostable isoform predominated when present in approximately two-fold excess relative to the thermolabile isoform. In agreement with these authors, estimation of the ratio of both isoforms in unfractionated muscle, heart and liver homogenates of AB1 Geophagus individuals by Klebe's (1975) method showed that in the last one, where Km values were the highest, contributions of non-thermodivergent A and B subunits to the isozymes present were 8:1. Also, the highest Km values for AB1B2 individuals were obtained in liver extracts, and A and B1 subunit contributions to the isozymes present were 8 A:1 B1:1 B2. On the other hand, muscle extracts showed the smallest Km values and a ratio of subunits varying from 1 (non-divergent - 1A:1 B1) to 2 (2 A:1 B1:1 B2).

Several investigators consider cichlids highly specialized fishes having an extremely rapid speciation rate (Lowe-McConnell, 1969; Thompson, 1981; Kornfield, 1982). Feldberg and Bertollo (1985), working with ten neotropical Cichlidae species, G. brasilensis among them, suggested that the chromosomal evolution of this group was more conservative than divergent, at least with respect to its chromosome number, maintaining a diploid number of 48 as the most frequent. Therefore, between the different processes which lead to gene duplication, polyploidization (duplication of the entire genome), and in tandem duplication (duplication of a single gene locus or several gene loci in a group in linkage), these chromosomal studies permit us to discard the possibility of the first as a cause of the G. brasilensis sMDH-B* duplication. According to Ohno (1970), one major consequence of duplication in tandem is the occurrence of only one structural gene duplication, without duplication of the regulatory gene locus which controls its synthesis. Tissue specificities and thermostability and kinetic tests resulted in similar responses from both B-isoforms, in both sMDH phenotypes, suggesting that these more recently duplicated loci undergo the same regulatory gene action. Farias and Almeida-Val (1992) suggested that the duplicated gene in the Amazon cichlid species could be maintained by heterosis, which is an adaptive characteristic of these species in tropical regions. However, similar results obtained between three- and six-banded specimens studied here did not show any indication of an adaptive advantage in subtropical regions.

ACKNOWLEDGMENTS

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The authors wish to thank Dr. Paula Ann Matvienko-Sikar, who critically reviewed this manuscript. Publication supported by FAPESP.

RESUMO

A fim de explicar o padrão eletroforético de seis componentes detectado para a malato desidrogenase solúvel (MDH, EC 1.1.1.37) em 84% dos exemplares de G. brasiliensis analisados (Cichlidae, Perciformes), uma duplicação recente no loco sMDH-B* é sugerida. Diluições seriadas de Klebe realizadas com extratos de músculo esquelético mostraram para as subunidades B1 e B2 o mesmo ponto final visual sugerindo um padrão de expressão não divergente para esses genes duplicados. Uma vez que não existe evidência de poliploidia na família Cichlidae, é sugerido que a duplicação no loco sMDH-B* seja resultante de uma duplicação regional. Especificidade tissular, termoestabilidade e propriedades cinéticas mostraram-se similares para as isoformas B, em ambos os fenótipos detectados, sugerindo estarem esses sob a ação do mesmo gene regulador. Os resultados similares obtidos para os fenótipos de três (AB1) e seis (AB1B2) componentes aqui analisados não mostraram nenhum indicativo de vantagem adaptativa deste último sobre o primeiro, em região subtropical.

REFERENCES

Allendorf, F.W., Knudsen, K.L. and Phelps, S.R. (1982). Identification of a gene regulating the tissue expression of a phosphoglucomutase locus in rainbow trout. Genetics 102: 259-268.

Allendorf, F.W., Knudsen, K.L. and Leary, R.F. (1983). Adaptive significance of differences in the tissue-specific expression of a phosphoglucomutase gene in rainbow trout. Proc. Natl. Acad. Sci. USA 80: 1397-1405.

Aquino-Silva, M.R., Schwantes, M.L.B. and Schwantes, A.R. (1997). The multiple soluble malate dehydrogenase of Hoplias malabaricus (Characiformes). Exp. Biol. Online 2: 18.

Aspinwal, N. (1974). Genetic analysis of duplicate malate dehydrogenase in the pink salmon, Oncorhynchus gorbuscha. Genetics 76: 65-72.

Bailey, G.J., Cocks, G.T. and Wilson, A.C. (1969). Gene duplication in fishes: malate dehydrogenase of salmon and trout. Biochem. Biophys. Res. Comm. 34: 605-612.

Bailey, G.S., Wilson, A.C., Halver, J.E. and Johnson, C.L. (1970). Multiple forms of supernatant malate dehydrogenase in salmonid fishes: biochemical, immunological and genetic studies. J. Biol. Chem. 245: 5927-5940.

Berger, F.M. (1971). A temporal survey of allelic variation in natural and laboratory populations of Drosophila melanogaster. Genetics 67: 121-136.

Burkhart, B., Dickson, B., Montgomery, E., Langley, C.H. and Voelker, R.A. (1984). Characterization of allozyme null and low activity alleles from two natural populations of Drosophila melanogaster. Genetics 107: 295-305.

Caraciolo, M.C., Val, A.L. and Almeida-Val, V.M.F. (1996). Malate dehydrogenase polymorphism in Amazon curimatids (Teleostei: Curimatidae): Evidence of an ancient mutational event. Braz. J. Genet. 19: 57-64.

Clayton, J.W., Harris, R.E.K. and Tretiak, D.N. (1973). Identification of supernatant and mitochondrial isozymes of malate dehydrogenase on electropherograms applied to the taxonomic discrimination of walleye (Stizolection vitreum vitreum), sanger (S. canadense) and suspected interspecific hybrid fishes. J. Fish Res. Board. Can. 30: 927-938.

Coppes, Z.L. and Somero, G.N. (1990). Temperature-adaptive differences between the M4 lactate dehydrogenases of stenothermal and eurythermal sciaenid fishes. J. Exp. Zool. 254: 127-131.

Coppes, Z.L., Schwantes, M.L.B. and Schwantes, A.R. (1987a). Adaptive features of enzymes from family Sciaenidae (Perciformes). I. Studies on soluble malate dehydrogenase (s-MDH) and creatine kinase (CK) of fishes from the South Cost of Uruguay. Comp. Biochem. Physiol. 88B: 203-210.

Coppes, Z.L., Schwantes, M.L.B. and Schwantes, A.R. (1987b). Adaptive features of enzymes from family Sciaenidae (Perciformes). II. Studies on phosphoglucose isomerase (PGI) of fishes from the South Cost of Uruguay. Comp. Biochem. Physiol. 88B: 211-218.

Coppes, Z.L., Schwantes, M.L.B. and Schwantes, A.R. (1987c). Adaptive features of enzymes from family Sciaenidae (Perciformes). III. Studies on lactate dehydrogenase (LDH) of fishes from the South Cost of Uruguay. Comp. Biochem. Physiol. 88B: 1005-1012.

De Luca, P.H., Schwantes, M.L.B. and Schwantes, A.R. (1983). Adaptive features of ectothermic enzymes. IV. Studies on malate dehydrogenase of Astyanax fasciatus (Characidae) from Lobo Reservoir (São Carlos, São Paulo, Brazil). Comp. Biochem. Physiol. 47B: 315-324.

Engel, W., Schmidtke, J., Vogel, W. and Wolf, U. (1973). Genetic polymorphism of lactate dehydrogenase isoenzymes in the carp (Cyprinus carpio) apparently due to a null allele. Biochem. Genet. 8: 281-289.

Farias, I.P. and Almeida-Val, V.M.F. (1992). Malate dehydrogenase (sMDH) in Amazon cichlid fishes: evolutionary features. Comp. Biochem. Physiol. 103B: 939-943.

Feldberg, E. and Bertollo, L.A.C. (1985). Nucleolar organizing regions in some species of neotropical cichlid fish (Pisces, Perciformes). Caryologia 38: 319-324.

Fenerich-Verani, N., Schwantes, M.L.B. and Schwantes, A.R. (1990). Patterns of gene expression during Prochilodus scrofa (Characiformes: Prochilodontidae) embryogenesis - II. Soluble malate dehydrogenase. Comp. Biochem. Physiol. 97B: 247-255.

Ferguson, M.M., Knudsen, K.L., Danzmann, R.G. and Allendorf, F.W. (1988). Developmental rate and viability of rainbow trout with a null allele at a lactate dehydrogenase locus. Biochem. Genet. 26: 177-189.

Ferris, S.D. and Whitt, G.S. (1979). Evolution of the differential regulation of duplicate genes after polyploidization. J. Mol. Evol. 12: 267-317.

Fisher, S.F., Shaklee, J.B., Ferris, S.D. and Whitt, G.S. (1980). Evolution of five multilocus isozyme systems in the chordates. Genetics 52/53: 73-85.

Graves, J.E. and Somero, G.N. (1982). Electrophoretic and functional enzymic evolution in four species of eastern Pacific barracudas from different thermal environments. Evolution 36: 97-106.

Hochachka, P.W. (1965). Isoenzymes in metabolic adaptation of a poikilotherm: Subunit relationships in lactic dehydrogenase of goldfish. Arch. Biochem. Biophys. 111: 96-103.

Hochachka, P.W. and Somero, G.N. (1973). Strategies of Biochemical Adaptation. W.B. Saunders Co., Philadelphia.

Hochachka, P.W. and Somero, G.N. (1984). Biochemical Adaptation. Princeton, University Press, New Jersey.

Klar, G.T. and Stalnaker, C.B. (1979). Electrophoretic variation in muscle lactate dehydrogenase in Snake Valley cuthroat trout. Comp. Biochem. Physiol. 64B: 391-394.

Klebe, R.J. (1975). A simple method for the quantification of isozyme patterns. Biochem. Genet. 13: 805-812.

Kornfield I.L., Smith, D.C. and Gagnon, P.S. (1982). The cichlid fish of Cuatro Ciénegas, Mexico: direct evidence of conspecificity among distinct tropic morphs. Evolution 36: 658-664.

Langley, C.H., Volker, R.A., Leigh-Brown, A.J., Ohnishi, S., Dickson, B. and Montgomery, E. (1981). Null allele frequencies at allozyme loci in natural populations of Drosophila melanogaster. Genetics 99: 151-156.

Li, W. (1982). Evolutionary change of duplicate genes. In: Current Topics in Biological and Medical Research (Rattazi, M.C. and Scandalios, G.S., eds.). Alan R. Liss, New York.

Lin, J.J. and Somero, G.N. (1995a). Temperature-dependent changes in expression of thermostable and thermolabile isozymes of cytosolic malate dehydrogenase in the eurythermal goby fish Gillchthys mirabilis. Physiol. Zool. 68: 114-128.

Lin, J.J. and Somero, G.N. (1995b). Thermal adaptation of cytoplasmic malate dehydrogenase of Eastern Pacific barracuda (Sphyraena ssp): The role of differential isoenzyme expression. J. Exp. Biol. 198: 551-560.

Lowe-McConnell, R.H. (1969). Speciation in tropical freshwater fishes. Biol. J. Linn. Soc. 1: 51-75.

McDonald, J.F. and Ayala, F.J. (1978). Gene regulation in adaptive evolution. Can. J. Genet. Cytol. 20: 159-175.

Monteiro, M.C., Schwantes, M.L.B. and Schwantes, A.R. (1991). Malate dehydrogenase in subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes. I. Duplicate gene expression and polymorphism. Comp. Biochem. Physiol. 100B: 381-390.

Monteiro, M.C., Schwantes, M.L.B., Schwantes, A.R. and Aquino-Silva, M.R. (1998). Thermal stability of soluble malate dehydrogenase isozymes of subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes. Braz. J. Genet. 21: 191-199.

Ohno, S. (1970). Evolution by Gene Duplication. Springer-Verlag, New York.

Reeves, R.B. (1977). The interaction of body temperature and acid-base balance in ectothermic vertebrates. Ann. Rev. Physiol. 39: 559-586.

Schwantes, M.L.B. and Schwantes, A.R. (1977). Electrophoretic studies on polyploid amphibians. III. Lack of locus duplication evidence through tetraploidization. Comp. Biochem. Physiol. 57B: 341-351.

Schwantes, M.L.B. and Schwantes, A.R. (1982a). Adaptive features of ectothermic enzymes. I. Temperature effects on the malate dehydrogenase from a temperate fish, Leiostomus xanthurus. Comp. Biochem. Physiol. 72B: 49-58.

Schwantes, M.L.B. and Schwantes, A.R. (1982b). Adaptive features of ectothermic enzymes. II. The effects of acclimation temperature on the malate dehydrogenase of the spot, Leiostomus xanthurus. Comp. Biochem. Physiol. 72B: 59-64.

Shaklee, J.B., Allendorf, F.W., Morizot, D.C.F. and Whitt, G.S. (1989). Genetic nomenclature for protein-coding loci in fish: Proposed Guidelines. Transac. Am. Fish Soc. 118: 218-227.

Somero, G.N. (1978). Temperature adaptation of enzymes: Biological optimization through structure-function compromises. Ann. Rev. Ecol. Syst. 9: 1-29.

Somero, G.N. (1981). pH-temperature interaction on proteins. Principles of optimal pH and buffer system design. Mar. Biol. Lett. 2: 163-178.

Somero, G.N. (1986). Protein adaptation and biogeography: Threshold effects on molecular evolution. Trends Ecol. Evol. 1: 124-127.

Somero, G.N., Siebenaller, J.F. and Hochachka. P.W. (1983). Biochemical and physiological adaptations of deep-sea animals. In: The Sea (Rowe, G., ed). Wiley, New York.

Thompson, K.W. (1981). Karyotypes of six species of African Cichlidae (Pisces, Perciformes). Experientia 37: 351-352.

Tsugawa, K. (1976). Direct adaptation of cells to temperature: similar changes of LDH isozyme pattern by in vitro and in situ adaptation in Xenopus laevis. Comp. Biochem. Physiol. 55B: 259-261.

Tsukuda, H. and Ohsawa, W. (1974). Effect of temperature acclimation on the isozyme pattern of liver lactate dehydrogenase in the goldfish, Carassius auratus (L.). Annot. Zool. JPN. 182: 59-68.

Val, A.L., Schwantes, A.R., Schwantes, M.L.B. and De Luca, P.H. (1981). Amido hidrolisado de milho como suporte eletroforético. Ciênc. Cult. 33: 992-996.

Voelker, R.A., Langely, C.H., Leigh-Brown, A.J.J., Ohnishi, S., Montgomery, E. and Smith, S.C. (1980). Enzyme null alleles in natural populations of Drosophila melanogaster. Frequencies in a North Carolina populations. Proc. Natl. Acad. Sci. USA 77: 1091-1101.

Wheat, T.E., Childers, W.F., Miller, E.T. and Whitt, G.S. (1971). Genetic and in vitro molecular hybridization of malate dehydrogenase isozymes in interspecific bass (Micropterus) hybrids. Anim. Blood Groups Biochem. Genet. 2: 3-14.

Whitt, G.S. (1970). Genetic variation of supernatant and mitochondrial malate dehydrogenase isozymes in the Teleosts Fundulus heteroclitus. Experientia 26: 734-736.

Wright, J.E., Heckman, J.R. and Atherton, L.M. (1975). Genetic and developmental analyses of LDH isozymes in trout. In: Isozymes: Developmental Biology (Markert, C.L., ed.). Academic Press, New York.

Zink, M.W. and Shaw, D.A . (1968). Regulation of malic isozymes and malic dehydrogenase in Neurospora crassa. Can. J. Microbiol. 14: 907-912.

Zouros, E. and Foltz, D.W. (1987). The use of allelic isozyme variation for the study of heterosis. In: Isozymes: Currents Topics in Biological and Medical Research (Ratazzi, M.C., Scandalios, J.G. and Whitt, G.S., eds.). Alan R. Liss, New York.

Zouros, E., Loukas, M., Economopoulos, A. and Mazomenos, B. (1982). Selection at the alcohol dehydrogenase locus of the olive fruit fly Dacus oleae under artificial rearing. Heredity 48: 169-185.

(Received January 28, 1998)

  • Allendorf, F.W., Knudsen, K.L. and Phelps, S.R. (1982). Identification of a gene regulating the tissue expression of a phosphoglucomutase locus in rainbow trout. Genetics 102: 259-268.
  • Allendorf, F.W., Knudsen, K.L. and Leary, R.F. (1983). Adaptive significance of differences in the tissue-specific expression of a phosphoglucomutase gene in rainbow trout. Proc. Natl. Acad. Sci. USA 80: 1397-1405.
  • Aquino-Silva, M.R., Schwantes, M.L.B. and Schwantes, A.R. (1997). The multiple soluble malate dehydrogenase of Hoplias malabaricus (Characiformes). Exp. Biol. Online 2: 18.
  • Aspinwal, N. (1974). Genetic analysis of duplicate malate dehydrogenase in the pink salmon, Oncorhynchus gorbuscha Genetics 76: 65-72.
  • Bailey, G.J., Cocks, G.T. and Wilson, A.C. (1969). Gene duplication in fishes: malate dehydrogenase of salmon and trout. Biochem. Biophys. Res. Comm. 34: 605-612.
  • Bailey, G.S., Wilson, A.C., Halver, J.E. and Johnson, C.L. (1970). Multiple forms of supernatant malate dehydrogenase in salmonid fishes: biochemical, immunological and genetic studies. J. Biol. Chem. 245: 5927-5940.
  • Berger, F.M. (1971). A temporal survey of allelic variation in natural and laboratory populations of Drosophila melanogaster Genetics 67: 121-136.
  • Burkhart, B., Dickson, B., Montgomery, E., Langley, C.H. and Voelker, R.A. (1984). Characterization of allozyme null and low activity alleles from two natural populations of Drosophila melanogaster Genetics 107: 295-305.
  • Caraciolo, M.C., Val, A.L. and Almeida-Val, V.M.F. (1996). Malate dehydrogenase polymorphism in Amazon curimatids (Teleostei: Curimatidae): Evidence of an ancient mutational event. Braz. J. Genet. 19: 57-64.
  • Clayton, J.W., Harris, R.E.K. and Tretiak, D.N. (1973). Identification of supernatant and mitochondrial isozymes of malate dehydrogenase on electropherograms applied to the taxonomic discrimination of walleye (Stizolection vitreum vitreum), sanger (S. canadense) and suspected interspecific hybrid fishes. J. Fish Res. Board. Can. 30: 927-938.
  • Coppes, Z.L. and Somero, G.N. (1990). Temperature-adaptive differences between the M4 lactate dehydrogenases of stenothermal and eurythermal sciaenid fishes. J. Exp. Zool. 254: 127-131.
  • Coppes, Z.L., Schwantes, M.L.B. and Schwantes, A.R. (1987a). Adaptive features of enzymes from family Sciaenidae (Perciformes). I. Studies on soluble malate dehydrogenase (s-MDH) and creatine kinase (CK) of fishes from the South Cost of Uruguay. Comp. Biochem. Physiol. 88B: 203-210.
  • Coppes, Z.L., Schwantes, M.L.B. and Schwantes, A.R. (1987b). Adaptive features of enzymes from family Sciaenidae (Perciformes). II. Studies on phosphoglucose isomerase (PGI) of fishes from the South Cost of Uruguay. Comp. Biochem. Physiol. 88B: 211-218.
  • Coppes, Z.L., Schwantes, M.L.B. and Schwantes, A.R. (1987c). Adaptive features of enzymes from family Sciaenidae (Perciformes). III. Studies on lactate dehydrogenase (LDH) of fishes from the South Cost of Uruguay. Comp. Biochem. Physiol. 88B: 1005-1012.
  • De Luca, P.H., Schwantes, M.L.B. and Schwantes, A.R. (1983). Adaptive features of ectothermic enzymes. IV. Studies on malate dehydrogenase of Astyanax fasciatus (Characidae) from Lobo Reservoir (Săo Carlos, Săo Paulo, Brazil). Comp. Biochem. Physiol. 47B: 315-324.
  • Engel, W., Schmidtke, J., Vogel, W. and Wolf, U. (1973). Genetic polymorphism of lactate dehydrogenase isoenzymes in the carp (Cyprinus carpio) apparently due to a null allele. Biochem. Genet. 8: 281-289.
  • Farias, I.P. and Almeida-Val, V.M.F. (1992). Malate dehydrogenase (sMDH) in Amazon cichlid fishes: evolutionary features. Comp. Biochem. Physiol. 103B: 939-943.
  • Feldberg, E. and Bertollo, L.A.C. (1985). Nucleolar organizing regions in some species of neotropical cichlid fish (Pisces, Perciformes). Caryologia 38: 319-324.
  • Fenerich-Verani, N., Schwantes, M.L.B. and Schwantes, A.R. (1990). Patterns of gene expression during Prochilodus scrofa (Characiformes: Prochilodontidae) embryogenesis - II. Soluble malate dehydrogenase. Comp. Biochem. Physiol. 97B: 247-255.
  • Ferguson, M.M., Knudsen, K.L., Danzmann, R.G. and Allendorf, F.W. (1988). Developmental rate and viability of rainbow trout with a null allele at a lactate dehydrogenase locus. Biochem. Genet. 26: 177-189.
  • Ferris, S.D. and Whitt, G.S. (1979). Evolution of the differential regulation of duplicate genes after polyploidization. J. Mol. Evol. 12: 267-317.
  • Fisher, S.F., Shaklee, J.B., Ferris, S.D. and Whitt, G.S. (1980). Evolution of five multilocus isozyme systems in the chordates. Genetics 52/53: 73-85.
  • Graves, J.E. and Somero, G.N. (1982). Electrophoretic and functional enzymic evolution in four species of eastern Pacific barracudas from different thermal environments. Evolution 36: 97-106.
  • Hochachka, P.W. (1965). Isoenzymes in metabolic adaptation of a poikilotherm: Subunit relationships in lactic dehydrogenase of goldfish. Arch. Biochem. Biophys. 111: 96-103.
  • Hochachka, P.W. and Somero, G.N. (1973). Strategies of Biochemical Adaptation. W.B. Saunders Co., Philadelphia.
  • Hochachka, P.W. and Somero, G.N. (1984). Biochemical Adaptation. Princeton, University Press, New Jersey.
  • Klar, G.T. and Stalnaker, C.B. (1979). Electrophoretic variation in muscle lactate dehydrogenase in Snake Valley cuthroat trout. Comp. Biochem. Physiol. 64B: 391-394.
  • Klebe, R.J. (1975). A simple method for the quantification of isozyme patterns. Biochem. Genet. 13: 805-812.
  • Kornfield I.L., Smith, D.C. and Gagnon, P.S. (1982). The cichlid fish of Cuatro Ciénegas, Mexico: direct evidence of conspecificity among distinct tropic morphs. Evolution 36: 658-664.
  • Langley, C.H., Volker, R.A., Leigh-Brown, A.J., Ohnishi, S., Dickson, B. and Montgomery, E. (1981). Null allele frequencies at allozyme loci in natural populations of Drosophila melanogaster. Genetics 99: 151-156.
  • Li, W. (1982). Evolutionary change of duplicate genes. In: Current Topics in Biological and Medical Research (Rattazi, M.C. and Scandalios, G.S., eds.). Alan R. Liss, New York.
  • Lin, J.J. and Somero, G.N. (1995a). Temperature-dependent changes in expression of thermostable and thermolabile isozymes of cytosolic malate dehydrogenase in the eurythermal goby fish Gillchthys mirabilis. Physiol. Zool. 68: 114-128.
  • Lin, J.J. and Somero, G.N. (1995b). Thermal adaptation of cytoplasmic malate dehydrogenase of Eastern Pacific barracuda (Sphyraena ssp): The role of differential isoenzyme expression. J. Exp. Biol. 198: 551-560.
  • Lowe-McConnell, R.H. (1969). Speciation in tropical freshwater fishes. Biol. J. Linn. Soc. 1: 51-75.
  • McDonald, J.F. and Ayala, F.J. (1978). Gene regulation in adaptive evolution. Can. J. Genet. Cytol. 20: 159-175.
  • Monteiro, M.C., Schwantes, M.L.B. and Schwantes, A.R. (1991). Malate dehydrogenase in subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes. I. Duplicate gene expression and polymorphism. Comp. Biochem. Physiol. 100B: 381-390.
  • Monteiro, M.C., Schwantes, M.L.B., Schwantes, A.R. and Aquino-Silva, M.R. (1998). Thermal stability of soluble malate dehydrogenase isozymes of subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes. Braz. J. Genet. 21: 191-199.
  • Ohno, S. (1970). Evolution by Gene Duplication Springer-Verlag, New York.
  • Reeves, R.B. (1977). The interaction of body temperature and acid-base balance in ectothermic vertebrates. Ann. Rev. Physiol. 39: 559-586.
  • Schwantes, M.L.B. and Schwantes, A.R. (1977). Electrophoretic studies on polyploid amphibians. III. Lack of locus duplication evidence through tetraploidization. Comp. Biochem. Physiol. 57B: 341-351.
  • Schwantes, M.L.B and Schwantes, A.R. (1982a). Adaptive features of ectothermic enzymes. I. Temperature effects on the malate dehydrogenase from a temperate fish, Leiostomus xanthurus Comp. Biochem. Physiol. 72B: 49-58.
  • Schwantes, M.L.B. and Schwantes, A.R. (1982b). Adaptive features of ectothermic enzymes. II. The effects of acclimation temperature on the malate dehydrogenase of the spot, Leiostomus xanthurus. Comp. Biochem. Physiol. 72B: 59-64.
  • Shaklee, J.B., Allendorf, F.W., Morizot, D.C.F. and Whitt, G.S. (1989). Genetic nomenclature for protein-coding loci in fish: Proposed Guidelines. Transac. Am. Fish Soc. 118: 218-227.
  • Somero, G.N. (1978). Temperature adaptation of enzymes: Biological optimization through structure-function compromises. Ann. Rev. Ecol. Syst. 9: 1-29.
  • Somero, G.N. (1981). pH-temperature interaction on proteins. Principles of optimal pH and buffer system design. Mar. Biol. Lett. 2: 163-178.
  • Somero, G.N. (1986). Protein adaptation and biogeography: Threshold effects on molecular evolution. Trends Ecol. Evol. 1: 124-127.
  • Somero, G.N., Siebenaller, J.F. and Hochachka. P.W. (1983). Biochemical and physiological adaptations of deep-sea animals. In: The Sea (Rowe, G., ed). Wiley, New York.
  • Thompson, K.W. (1981). Karyotypes of six species of African Cichlidae (Pisces, Perciformes). Experientia 37: 351-352.
  • Tsugawa, K. (1976). Direct adaptation of cells to temperature: similar changes of LDH isozyme pattern by in vitro and in situ adaptation in Xenopus laevis Comp. Biochem. Physiol. 55B: 259-261.
  • Tsukuda, H. and Ohsawa, W. (1974). Effect of temperature acclimation on the isozyme pattern of liver lactate dehydrogenase in the goldfish, Carassius auratus (L.). Annot. Zool. JPN. 182: 59-68.
  • Val, A.L., Schwantes, A.R., Schwantes, M.L.B. and De Luca, P.H. (1981). Amido hidrolisado de milho como suporte eletroforético. Cięnc. Cult. 33: 992-996.
  • Voelker, R.A., Langely, C.H., Leigh-Brown, A.J.J., Ohnishi, S., Montgomery, E. and Smith, S.C. (1980). Enzyme null alleles in natural populations of Drosophila melanogaster Frequencies in a North Carolina populations. Proc. Natl. Acad. Sci. USA 77: 1091-1101.
  • Wheat, T.E., Childers, W.F., Miller, E.T. and Whitt, G.S. (1971). Genetic and in vitro molecular hybridization of malate dehydrogenase isozymes in interspecific bass (Micropterus) hybrids. Anim. Blood Groups Biochem. Genet. 2: 3-14.
  • Whitt, G.S. (1970). Genetic variation of supernatant and mitochondrial malate dehydrogenase isozymes in the Teleosts Fundulus heteroclitus. Experientia 26: 734-736.
  • Wright, J.E., Heckman, J.R. and Atherton, L.M. (1975). Genetic and developmental analyses of LDH isozymes in trout. In: Isozymes: Developmental Biology (Markert, C.L., ed.). Academic Press, New York.
  • Zink, M.W. and Shaw, D.A . (1968). Regulation of malic isozymes and malic dehydrogenase in Neurospora crassa Can. J. Microbiol. 14: 907-912.
  • Zouros, E. and Foltz, D.W. (1987). The use of allelic isozyme variation for the study of heterosis. In: Isozymes: Currents Topics in Biological and Medical Research (Ratazzi, M.C., Scandalios, J.G. and Whitt, G.S., eds.). Alan R. Liss, New York.
  • Zouros, E., Loukas, M., Economopoulos, A. and Mazomenos, B. (1982). Selection at the alcohol dehydrogenase locus of the olive fruit fly Dacus oleae under artificial rearing. Heredity 48: 169-185.

Publication Dates

  • Publication in this collection
    01 Mar 1999
  • Date of issue
    Dec 1998

History

  • Received
    28 Jan 1998
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