Biochemical genetic differentiation between Pomatoschistus marmoratus and P. tortonesei

Several diagnostic genetic markers were identified in Pomatoschistus marmoratus and P. tortonesei using polyacrylamide gel electrophoresis (PAGE) of allozymes. Twenty-one loci were resolved, including the electrophoretic pattern of muscle proteins. The MDH*, PGM-,2*, EST-1,2*, FUM* and PGI-2* loci exhibited different alleles which were fixed for the two species being analysed. Genetic distance, as calculated by Nei's index, showed a value of 0·413. Environmental hypersalinity, could have influenced the geographical distribution of P. tortonesei .     

Biochemical Polymorphism in Yellow Catfish, Mystus nemurus (C&V), from Thailand

Yellow catfish, Mystus nemurus (Cuv. & Val.), is becoming one of the major freshwater species farmed by aquaculturists in Southeast Asia. It was of interest to examine levels of genetic subpopulation differentiation among samples of this species obtained from parts of its range, as well as to compare the genetics of wild and hatchery-bred fish. Horizontal starch gel electrophoresis and histochemical staining techniques were used to examine genetic variation within and among eight wild and one hatchery populations of M. nemurus from northern, northeastern, central and southern Thailand. Four tissues (heart, liver, kidney, and muscle) from individual specimens were used to analyze variations at 23 protein-coding loci. Fifteen of the 23 loci examined (65.22%), namely, ACP*, AAT-1*, EST-1*, EST-2*, GPI*, IDH-1*, IDH-2*, MDH-1*, MDH-2*, MDH-3*, ME*, PGM*, 6PGD*, SOD*, and HB*,were polymorphic at the 0.95 level. Observed heterozygosities ranged from 0.041 to 0.111, with an average of 0.068 ± 0.028. Genetic distances ranged from 0.005 to 0.164. The greatest genetic distance was found between the Chainat and the Suratthani populations (0.164), a level indicative of subspecific differentiation in M. nemurus from within Thailand.     

Protein differences among the Mediterranean species of the genus Spicara

Protein electrophoresis (PAGE) was used to study the three morphologically different species of Spicara (S. flexuosa, S. maena, S. smaris). Of the 28 enzymatic and additional myogenic loci, five monomorphic loci (LDH-1*, G6PD-1*, PGI-1* and two PMMs*) were species-specific markers of S. smaris with respect to S. flexuosa and S. maena. Four of the 28 enzymatic loci were polymorphic (EST-1*, GLDH*, PEPD*, PGI-2*). Discriminating genetic markers were not identified between S. flexuosa and S. maena. Genetic distance (D) as calculated by Nei's index (1978), between S. smaris v. S. maena and S. flexuosa showed a value, respectively of Z) = 0·137 and 0·141. Between S. flexuosa and S. maena the value was Z)=0-006. From the data it can be inferred that S. flexuosa and S. maena are conspecific, despite morphological differences.     

Taxonomic study of the Iberian Cobitis (Osteichthyes, Cobitidae), with description of a new species

Morphometric and electrophoretic variation are analysed among Iberian and African populations of Cobitis (Cobitidae). Three discrete morphotypes are distinguished, showing a remarkable genetic differentiation among them. Each of the three morphotypes is judged to represent a species, one being described as a new species. Both approaches agree on the separate status of the Iberian populations from the Alagón drainage, Tagus basin of west central Spain. This population is described as a new species, Cobitis vettonica sp. nov., a sexually dimorphic spined loach was assigned to the subgenus Iberocobitis Bacescu, 1961. Morphometric and electrophoretic data placed this new species phenetically close to Cobitis maroccana Pellegrin, 1929 and Cobitis pahdica De Buen, 1930, but differs from these species in overall shape, male coloration pattern, shorter barbels and fins, posterior lamina of the cleithrum, seven branched rays on the dorsal fin and in having a unique alleles at sAAT-2*, EST-1* and MPI* loci. The new species range described in this paper is more restricted than that of C. pahdica. Both inhabit the Tagus basin but have not been reported to be sympatric.     

Allozyme variability and genetic divergence of Pacific trout (species Parasalmo) from western Kamchatka

Genetic variation in several populations of Parasalmo (Onchorhyncus) mykiss from Kamchatka was examined on the basis of data obtained by the author and results from literature. Three (sSOD-1*, LDH-C*, and EST-1*) out of 44 protein-coding loci were highly polymorphic. Low-frequency alternative alleles occurred at sAAT-1,2*, LDH-A2*, EST-5*, IDDH-1,2*, and sMDH-1,2*. The results of the present work and comparison with evidence on North American species indicated that, in the Kamchatka part of the range, P. mykiss is represented by several populations carrying unique alleles and forming a genetically independent group. Gene exchange between North American and Western Kamchatka populations is mainly determined by straying in the feeding stock of the American coastal form entering the Sea of Okhotsk. The genetic divergence and mean heterozygosities in the Kamchatka populations were low (D magnitude of 0.0002-0.0275; HS magnitude of 0.011-0.0371). The difference between the Western Kamchatka populations from the North American coastal form was so small (D magnitude of 0.0109-0.0241) that these forms clustered together. Genetic divergence of the Kamchatka populations and the inland North American P. mykiss is an order of magnitude higher (D magnitude of 0.1973-0.2367).     

Protein variation in wild Atlantic salmon, with particular reference to southern Ireland

The extent of genetic variation in wild Atlantic salmon parr, Sulmo salur L., from river systems in Ireland, Iceland and eastern Canada, was investigated using starch gel electrophoresis. Within Ireland, seven polymorphic enzyme loci ( sAAT-4 *, GPI-1 *, IDDH-1 *, IDDH-2 *, IDHP-3 *, MDH-3 * and mMEP-2 *) were screened in nine different rivers and nine tributaries from the River Blackwater. Significant heterogeneity in gene frequencies occurred between riverine samples and between samples from tributaries of the River Blackwater. Variation between tributaries was as great as between rivers elsewhere in the country. Levels of population differentiation were comparable to those found in other regions throughout the range of the species, and temporal stability in gene frequencies was apparent when the results were compared with previously published data. Screening of riverine samples from Iceland and eastern Canada (Newfoundland and New Brunswick) allowed the Irish results to be considered in a broader context. Irish salmon cluster in the western European group, to which may be added Icelandic populations. Salmon from eastern Canada show a high level of genetic distinctiveness from the European group.     

Gene–Centromere Distances of Allozyme loci in Even- and Odd-year Pink Salmon, (Oncorhynchus gorbuscha)

We produced gynogenetic progeny families to estimate gene-centromere (G-C) distances of allozyme loci in even-year and odd-year pink salmon (Oncorhynchus gorbuscha). G-C distances of 37 loci distributed on a chromosome ranged from 1 cM at LDH-A1* to 49 cM at ADA-2*, DIA-2*, and sMDH-B1,2*. The distribution of the G-C distances along the chromosome arm was not even and appears telomeric. Eight loci in even-year and seven in odd-year showed high G-C distances (>45 cM), indicating that one crossover per chromosome arm is usual in pink salmon. Variation was observed in the results from different families; 14 loci out of 21 tested, showed heterogeneity. At mAH-3*, G-C distances from five odd-year families ranged from 6 to 37 cM; the widest range observed in this study. At isoloci such as sMDH-A 1,2* and sMDH-B1,2* the distances from different families were grouped into statistically discrete distributions, suggesting that it may be a reflection polymorphism at both isoloci. It appears G-C distances in salmonid species are well conserved with some minor differences.     

Prevalence of CYP2C9 and VKORC1 alleles in the Argentine population and implications for prescribing dosages of anticoagulants

Dicumarinic oral anticoagulants have a narrow therapeutic range and a great individual variability in response, which makes calculation of the correct dose difficult and critical. Genetic factors involved in this variability include polymorphisms of genes that encode the metabolic enzyme CYP2C9 and the target enzyme vitamin K epoxide reductase complex 1 (VKORC1); these polymorphisms can be associated with reduced enzymatic expression. We examined the frequency of the most relevant variants encoding CYP2C9 (alleles *1, *2 and *3) and VKORC1 (SNP -1639A>G) in the Argentinian population. Molecular typing was performed by PCR-RFLP on a randomly selected sample of 101 healthy volunteers from the Hospital Italiano de Buenos Aires gene bank. Fifty-seven subjects were identified as homozygous for CYP2C9*1 and 14 for *2, while 24 and 5 were heterozygous for *2 and *3 alleles; one individual was a composite heterozygote (*2/*3). When we examined VKORC1, 21 subjects were AA homozygous, 60 were AG heterozygotes and 20 were GG homozygotes. This is the first analysis of genotypic frequencies for CYP2C9 and VKORC1 performed in an Argentinian population. These allele prevalences are similar to what is known for Caucasian population, reflecting the European ancestor of our patient population, coming mostly from Buenos Aires city and surroundings. Knowledge of this prevalence information is instrumental for cost-effective pharmacogenomic testing in patients undergoing oral anticoagulation treatment.     

Tissue-specific esterases in the xiphophorine fish Platypoecilus maculatus,Xiphophorus helleri,and their hybrid

Tissue-specific esterases of the xiphophorine fishes Platypoecilus maculatus (platyfish), Xiphophorus helleri (swordtail), and their F₁ hybrid have been analyzed using disc electrophoresis. Seven esterase zones (resolved into a maximum of nine bands) exist in these fishes, and these have been classified by employing specific inhibitors. Five of the seven zones, EST-1, EST-2, EST-5, EST-6, and EST-7, appeared to be carboxylesterases; while the two remaining zones, EST-3 and EST-4, were classified as cholinesterases. In the liver of the platyfish, all seven esterase zones were detected, while the liver of the swordtail exhibited only five esterase zones. EST-1 and EST-3 were lacking in the liver tissue of the swordtail. All seven esterase loci were expressed in the liver tissue of the F₁ hybrid. The reciprocal crosses gave the same results. In the fin, skin, skeletal muscle, and eye tissues from all three genotypes, three major esterase zones, EST-2, EST-5, and EST-7, were detected. In addition, EST-1 was frequently detected in all these tissues of the platyfish and the F₁, but was lacking in the swordtail. Serum from three genotypes showed one prominent esterase zone, EST-5; however, trace activity of EST-2 and EST-7 zones could also be detected. It seems that in all tissues of the F₁ hybrid there is expression of all the esterase genes from the platyfish. The results of the present study are discussed in comparison to those from other studies on teleost esterases.This research was supported by grants from the Sonderforschungsbereich 103 Zellenergetik und Zelldifferenzierung (Marburg). M. R. A. is a Richard-Merton Guest Professor supported by the Deutsche Forschungsgemeinschaft.     

Allozyme variation of nonspecific esterases in Russian sturgeon (Acipenser güldenstädti Brandt

Electrophoresis in 7% polyacrylamide gel was used to analyze nonspecific esterases from different organs and tissues of Russian sturgeon. The basic enzymatic activity was observed the four zymogram zones. In sixteen cases the patterns of isozyme distribution in these zones allowed to consider them as allele systems (14 polymorphic and 2 monomorphic) corresponding to the four structural genes. The observed genotype frequencies of the nine of these systems (EST-1* of brain and intestine; EST-2* of serum and intestine; EST-3* of spleen and kidney; and EST-4* of spleen, brain, and heart) were largely concordant with Hardy-Weinberg proportions for codominant disomic inheritance. Genetic control of other polymorphic systems is unknown, but, high enzymatic activity of these esterases, sufficient for qualitative electrophretic detection, permits utilization of these polymorphisms for phenotypic monitoring of Russian sturgeon populations.     

Allozyme Variation of Nonspecific Esterases in Russian Sturgeon Acipenser güldenstädti Brandt

Electrophoresis in 7% polyacrylamide gel was used to analyze nonspecific esterases from different organs and tissues of Russian sturgeon. The basic enzymatic activity was observed in four zymogram zones. In sixteen cases the patterns of isozyme distribution in these zones allowed to consider them as allele systems (14 polymorphic and 2 monomorphic) corresponding to the four structural genes. The observed genotype frequencies of the nine of these systems (EST-1* of brain and intestine; EST-2* of serum and intestine; EST-3* of spleen and kidney; and EST-4* of spleen, brain, and heart) were largely concordant with Hardy–Weinberg proportions for codominant disomic inheritance. Genetic control of other polymorphic systems is unknown, but, high enzymatic activity of these esterases, sufficient for qualitative electrophretic detection, permits utilization of these polymorphisms for phenotypic monitoring of Russian sturgeon populations.     

Developmental genetics in larvae,pupae and adults ofSepedon fuscipennis fuscipennis [Dipt.: Sciomyzidae]

An analysis of 28 enzyme loci throughout developmental stages ofSepedon fuscipennis fuscipennis Loew indicated that 16 were polymorphic and 4 were monomorphic in all stages. Nine loci were differentially expressed among the stages: EST-1, EST-2, MDH-2, MDH-3 and PGI-1 occurred only in larvae, AK-3 mainly in pupae, and AK-1, AO and HK-1 only in adults. The average heterozygosity ofS. f. fuscipennis was 0.146 (±0.028) across all stages.      

Genetic variation at isozyme loci in blue whiting from the north-east Atlantic

Genetic variation in blue whiting Micromesistius poutassou was investigated by starch gel electrophoresis of enzymes in tissue extracts. A total of 130 specimens from the spawning areas west of the British Isles were collected from trawl catches in 1990 ( n = 30) and 1992 ( n = 100). In 25 tissue enzyme loci screened for genetic variants in the 1990 sample, polymorphisms (0.95 criterion) were found at IDDH-2 *, IDHP-2 * and PGM-1 *, giving a frequency of polymorphic loci of P _0.95= 0.12. In pooled samples the average heterozygosity per locus was estimated at H _e =0.043 and the effective number of alleles per locus at 2.20, 1.45 and 1.53, respectively, for IDDH-2 *, IDHP-2 * and PGM-1 *.     

Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.     

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.     

NADP-dependent isocitrate dehydrogenase of Astyanax scabripinnis (Pisces, Characidae) from three altitudes at Grande Stream, Campos do Jord?o, SP.

Electrokinetic, thermic, and kinetic properties of products of NADP-dependent isocitrate dehydrogenase (IDHP; EC 1.1.1.42) loci of Astyanax scabripinnis (Pisces, Characidae) collected at three different altitudes (700 m, 1,800 m, and 1,920 m) of Grande Stream at Campos do Jord?o, State of S?o Paulo, Brazil, were analyzed. Two IDHP bidirectionally divergent loci, a single skeletal muscle, the IDHP-A*, and a single liver IDHP-B*, both polymorphic, were detected in the three different altitude populations. The variant allele *128 at the IDHP-A* locus, had its highest frequency detected in the 1,920 m population (0.494). Among the nine variant alleles detected at the IDHP-B* locus (*37, *57, *69, *79, *85, *114, *119, *124, and *140), the *37 and 79 were detected only in 1,800 m population. Chi-square values showed that only the 700 m population is not in Hardy-Weinberg equilibrium for the IDHP-A* locus, while for the IDHP-B* locus, no population is. Homogeneity Chi2 test indicated that the populations are significantly different in their A and B phenotype frequencies. Wright's FST mean value (0.036 and 0.32, IDHP-A* and IDHP-B*, respectively) was 0.178 for the three altitude populations which means that 82% of total genetic diversity was found among individuals of each one of the populations. Stability at environmental temperatures (16 degrees to 21 degrees C), and apparent Km and Vmax values of each A-phenotype skeletal muscle crude extract suggest different roles of A-isoforms during the increased lipogenesis that occurs in fish at low temperatures.     

Genetic population structure of Norwegian brown trout

Biochemical genetic variation in populations of anadromous and resident brown trout, Salmo trutta L., was studied. Altogether 50 Norwegian populations were screened for 32 enzyme loci. Genetic polymorphism was found at the following 11 loci: AAT-4 * (E.C. 2.6.1.1), CK-1 * (E.C. 2.7.3.2), G3PDH-2 * (E.C. 1.1.1.8), IDHP-2 * (E.C. 1.1.1.42), LDH-5 * (E.C. 1.1.1.27), MDH-2 * (E.C. 1.1.1.37), MDH-3/4 * (E.C. 1.1.1.37), MEP-2 * (E.C. 1.1.1.40), GPI-2 * (E.C. 5.3.1.9). GPI-5 * (E.C. 5.3.1.9) and PGM-1 * (E.C. 5.4.2.2), giving an overall polymorphism of 34%, ranging from 3.7 to 29.6% among individual populations. The average calculated heterozygosity ranged from 1.4 to 10.2% among populations. Genetic heterogeneity was observed among anadromous populations, and significant differences in allelic frequencies were found between anadromous populations in neighbouring watercourses, among resident populations and between anadromous and resident populations inhabiting the same watercourses. Significant heterogeneity was also found among 12 populations from Lake Mjøsa, with a major division between the western and eastern populations of the lake. Differences in allelic frequencies were found between wild stocks and their hatchery derivatives, and between different hatchery derivatives originating from the same wild population. In some cases release of hatchery populations into wild stocks may have influenced the genetic characteristics of wild stocks. The data support the hypothesis of eastern as well as western postglacial colonization lines for Norwegian brown trout.     

Genetic protein variation and natural selection in Atlantic salmon (Salmo salav, L.) parr

The principal focus of this study was an examination of the relationship between diallelic variation at the MEP-2 * locus and growth and survival in Atlantic salmon parr. In addition, patterns of growth and survival among genotypes at the AAT-I *, IDDH-I * and ZDHP-2 * loci were also examined. Significant differences in growth were found among MEP-2 * genotypes, with * 100/ 100 homozygotes largest in one of two independent comparisons and the * 125/I25 homozygotes largest in the other. No growth differences were observed among genotypes at any other locus. Although no significant differences were found in survival among genotypes at any locus, results of joint analysis of the patterns of growth and survival among MEP-2 * genotypes were consistent with a model of positive size-selective mortality. The demonstration of growth differences among MEP-2 * genotypes in parr and smolts may indicate an association between selection on this locus in fresh water and previously reported differences in sea-age at maturity among MEP-2 * genotypes in Atlantic salmon.     

Genetic divergence at the NAD +-dependent malic enzyme locus in Atlantic salmon from Europe and North America

The distribution of genetic variation for NAD+ malic enzyme ( ME* ) polymorphism in the Atlantic salmon was assessed in both anadromous and resident populations. The analysis revealed a major allelic divergence between North America and Europe. The *80 variant occurred in 39 of 40 North American samples, ranging in frequency from 0 to 0.515. In contrast, it was detected in only three fish from two of the 35 European locations analysed. Eleven fish from two rivers in north-west France had the * 110 variant. While heterogeneity among North American populations was significant, no regional differentiation was apparent and anadromous and resident salmon were not found to differ.     

Genetic relationships between Amerindian populations of Argentina

A total of 495 individuals from five different Argentinian tribes was examined for variation in 23 blood group and protein genetic systems, and the results were integrated with previous data on some of these systems. These tribes generally present RH * R1, PGM1 * 1, and ACP * A frequencies lower and RH * R2, ESD * 1, and GLO * 1 prevalences higher than those observed in other South American Indian groups. Earlier studies with mitochondrial DNA showed that haplogroup A was present in low frequencies in these tribes, but haplogroup B showed a high prevalence among the Mataco. Average heterozygosities are very similar in the five tribes, while estimates of non-Indian ancestry are generally low. Both the blood group and protein, as well as the mtDNA data sets, divide the five tribes into two groups, and the relationships obtained with the blood group and protein systems are exactly those expected on the basis of geography and language. However, the topology obtained with the mtDNA results was different, possibly due to sampling effects or diverse patterns of exchange between the groups related to sex.