Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.)

The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences.     

Phylogenetic Relationships and Biochemical Properties of the Duplicated Cytosolic and Mitochondrial Isoforms of Malate Dehydrogenase from a Teleost Fish, Sphyraena idiastes

Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 1.1.1.37; NAD⁺: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis–Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity. Received: 5 April 2001 / Accepted: 28 June 2001     

Cloning and sequence analysis of cDNAs encoding mammalian cytosolic malate dehydrogenase. Comparison of the amino acid sequences of mammalian and bacterial malate dehydrogenase

A cDNA clone, named ppcMDH-1 and covering a part of the coding region for the porcine cytosolic malate dehydrogenase (cMDH) mRNA, was isolated from a porcine liver cDNA library. Subsequently, mouse cMDH cDNA clones were isolated from mouse liver and heart cDNA libraries, using the ppcMDH-1 cDNA as a probe. The longest clone, named pmcMDH-5, was sequenced and the primary structure of the mouse cMDH deduced from its cDNA sequence showed that the mouse cMDH consists of the 334-amino acid residues. When the amino acid sequence of the mouse cMDH was compared with that of the porcine cMDH, they shared a 93% homology. On the other hand, the amino acid sequences of mouse cMDH and mitochondrial MDH (mMDH) showed about 23% overall homology. Surprisingly, comparison of the amino acid sequences among the mammalian and bacterial MDHs revealed that the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and Escherichia coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice.     

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.     

Comparison of properties of malate dehydrogenase isoenzymes from hare and rabbit hearts

The hare heart mitochondrial malate dehydrogenase (mMDH) was established to have a much higher electrophoretic mobility than the corresponding enzyme from the rabbit heart. Differences of kinetic properties of both mMDH and cytoplasmic malate dehydrogenase (cMDH) from these two sources were shown. The hare heart mMDH and cMDH isoenzymes have a higher affinity to malate (in direct reaction) and oxaloacetate and NADH (in reverse reaction), i.e., they have lower K _M values in comparison with the isoenzymes from the rabbit heart. Malate dehydrogenase seems to operate more effectively in the hare heart, which might be important in adaptive and evolutionary aspects.     

Dissociation of mitochondrial malate dehydrogenase into active soluble subunits

Gel exclusion chromatographic studies demonstrate that cytosolic and mitochondrial malate dehydrogenases (cMDH and mMDH) dissociate into subunits in the presence of 0.1% of the non-ionic detergent Triton X-100 (TX-100). The presence of cofactor and catalytically competent cofactor-substrate pairs does not protect mMDH against this dissociation. In contrast, cMDH dimers resist dissociation in the presence of either addition. Since steady state kinetic studies indicate both enzymes are fully active in the presence of 0.1% TX-100, we conclude that quaternary structure is not a kinetically important feature of mMDH structure and cooperativity does not account for mMDH kinetic anomalies. In contrast, cooperativity is a reasonable explanation for cMDH kinetic properties even in the presence of 0.1% TX-100, since this enzyme's subunits associate in the presence of active site ligands. The existence of fully active mMDH subunits raises the possibility that this species rather than the dimer may be a constituent of proposed multi-enzyme complexes of the mitochondrion. Preliminary chromatographic experiments involving gently disrupted mitochondria have found MDH activity in differently sized complexes, all with molecular weights larger than the mMDH dimer but smaller than complexes anticipated for multi-enzyme complexes involving other enzymes and the mMDH dimer.     

Phylogeography and population structure of the common warthog (Phacochoerus africanus) inferred from variation in mitochondrial DNA sequences and microsatellite loci

Global climate fluctuated considerably throughout the Pliocene and Pleistocene, influencing the evolutionary history of a wide range of species. Using both mitochondrial sequences and microsatellites, we have investigated the evolutionary consequences of such environmental fluctuation for the patterns of genetic variation in the common warthog, sampled from 24 localities in Africa. In the sample of 181 individuals, 70 mitochondrial DNA haplotypes were identified and an overall nucleotide diversity of 4.0% was observed. The haplotypes cluster in three well-differentiated clades (estimated net sequence divergence of 3.1-6.6%) corresponding to the geographical origins of individuals (i.e. eastern, western and southern African clades). At the microsatellite loci, high polymorphism was observed both in the number of alleles per locus (6-21), and in the gene diversity (in each population 0.59-0.80). Analysis of population differentiation indicates greater subdivision at the mitochondrial loci (FST=0.85) than at nuclear loci (FST=0.20), but both mitochondrial and nuclear loci support the existence of the three warthog lineages. We interpret our results in terms of the large-scale climatic fluctuations of the Pleistocene.     

Population structure of the African savannah elephant inferred from mitochondrial control region sequences and nuclear microsatellite loci

Two hundred and thirty-six mitochondrial DNA nucleotide sequences were used in combination with polymorphism at four nuclear microsatellite loci to assess the amount and distribution of genetic variation within and between African savannah elephants. They were sampled from 11 localities in eastern, western and southern Africa. In the total sample, 43 haplotypes were identified and an overall nucleotide diversity of 2.0% was observed. High levels of polymorphism were also observed at the microsatellite loci both at the level of number of alleles and gene diversity. Nine to 14 alleles per locus across populations and 44 alleles in the total sample were found. The gene diversity ranged from 0.51 to 0.72 in the localities studied. An analysis of molecular variance showed significant genetic differentiation between populations within regions and also between regions. The extent of subdivision between populations at the mtDNA control region was approximately twice as high as shown by the microsatellite loci (mtDNA F(ST) = 0.59; microsatellite R(ST) = 0.31). We discuss our results in the light of Pleistocene refugia and attribute the observed pattern to population divergence in allopatry accompanied by a recent population admixture following a recent population expansion.     

Variation in mitochondrial DNA in a cline of allele frequencies and shell phenotype in the dog-whelk Nucella lapillus (L.)

Genetic constitution in the intertidal gastropod Nucella lapillus (L.) influences shell shape, growth rate and physiology. Clinal variation in these traits along a 5 km stretch of coastline in south Devon can be related to environmental variation in temperature and desiccation stress. We have examined mtDNA variation along this shore to investigate whether the cline represents primary or secondary contact. Two distinct mtDNA haplotypes were found which exhibit coincident step clines with karyotypic, allozymic and phenotypic variation and covary with the environmental pressures of temperature and desiccation. These results are interpreted in the context of the wider scale distribution of genetic and phenotypic variation in N. lapillus. It is suggested that the shore studied may represent one of a number of regions of secondary contact within a mosaic hybrid zone in N. lapillus , where coadapted phenotypic variation correlates with habitat and the position of the clines represents an environmental transition.     

Regulation of malate dehydrogenases from neonatal,adolescent, and mature rat brain

Since the malate-aspartate shuttle in brain has been shown to be closely linked to brain energy metabolism and neurotransmitter synthesis, the activity of MDH, one of the enzymes of the malateaspartate shuttle, was studied in cortical non-synaptic mitochondria (mMDH) and cytosol (cMDH) in 1–4 day, 18–20 day and 7–8 week old rats. The mean mMDH activity (nmol/min/mg protein) was 10,517±734 (mean±SEM), 8,882±241 and 10,323±561 and cMDH activity was 2,453±99, 4,673±152 and 6,821±205 in 1–4 day, 18–20 day and 7–8 week old rats, respectively. While cMDH activity increased with age (p<0.0001), mMDH activity showed no change. This study also determined if endogenous compounds, previously shown to alter malate metabolism, affected MDH activities. Lactate inhibited only cMDH activity, by a competitive mechanism. Oxaloacetate inhibited mMDH by partial non-competitive inhibition and cMDH by competitive inhibition. Alpha-ketoglutarate competitively inhibited both enzymes; however, the inhibition of mMDH activity was more pronounced than that of cMDH activity. Citrate inhibited mMDH via an uncompetitive mechanism and cMDH via a noncompetitive mechanism. The mechanisms of inhibition of mMDH and cMDH by each of the effectors were the same over the three ages. The results suggest mMDH and cMDH activities show a dissimilar developmental pattern and may be regulated differently by endogenous effectors. The greater sensitivity of mMDH, compared to cMDH, to certain effectors may be related to the dual role of mMDH in the tricarboxylic acid cycle and the malate-aspartate shuttle.These data were presented in part at the meeting of the Federation of American Societies for Experimental Biology in Atlanta, Georgia, April 1991. This work was performed in partial fulfillment of the requirements for the M.S. Degree in Nutritional Sciences (P.M.)     

Some observations on shell shape variation in Pacific Nucella

This paper considers the patterns of shell shape variation shown by Nucella canalicuata, N. emarginata and N. lamellosa from two areas of the Pacific Northwest: the shores near Friday Harbour on San Juan Island and near Bamfield on the west coast of Vancouver Island. No clear pattern of variation in association with changes in exposure was seen in either N. canaliculata or N. lamellosa . It appears that genetic influences are more important controls of shell shape than environmental selection in both these species. Nucella emarginata shows the nearest approximation to the pattern shown by the Atlantic species, N. lapillus , but only at the exposed end of the wave-action gradient. On those shores, enclaves from the most surf-washed open coast headlands have shells with proportionally larger apertures (and thus a shorter, squatter form) than their equivalents in local shelter. But, unlike in N. lapillus , the trend does not continue onto genuinely sheltered shores. Under these circumstances the species is generally rare and, where enclaves do occur, their shells are of much the same shape (although of a much larger size) as in more exposed situations.     

Microgeographic variation in allozyme frequencies in relation to the degree of exposure to wave action in the dogwhelk Nucella lapillus (L.) (Prosobranchia: Muricacea)

Nucella lapillus from 15 sites 50 m to 21 km apart in S. Devon, S. W. England were analysed for allozyme variation at eight soluble enzyme loci. Sites were classified as either Exposed or Sheltered and a hierarchical analysis of allozyme variation carried out using Wright's Fixation Index, F_ST. Whelks from Sheltered sites segregated for alleles at the Est-3, Lap-2, Pep-2 and Mdh-1 loci which were virtually absent from exposed sites resulting in high F_ST values (0.289–0.506) when all samples were included. Consequently mean heterozygosity of the Sheltered samples was roughly twice that of the Exposed. Between Sheltered sites the frequencies of these 'additional' alleles varied substantially, even on a microgeographic scale and were found to be highly correlated with exposure. Within individuals the presence of the 'sheltered' alleles was correlated with the possession of the 'sheltered' shell shape as indicated by length divided by aperture height. This must necessarily be the case, however, if both are related independently to exposure. Although these correlations imply the action of selection there is evidence within the data for stochastic factors affecting the pattern observed. Complicating the picture further is the correspondence observed in the distribution of the 'sheltered' alleles with the known distribution of chromosomal translocations. Phenotypic associations within individuals also support the hypothesis that variation at the Est-3, Lap-2, Mdh-1 and Pet-2 loci is related to variation in chromosome number. However, the nature of the relations of karyotype and allozyme variation with shell shape and exposure remain speculative.     

The influence of recombination on SNP diversity in chickens

The association between recombination rate and diversity, but not divergence is considered to be driven mainly by natural selection: fixation of positively selected variants and associated hitchhiking effects and/or background selection eliminating deleterious alleles. In the present study, we investigated the relationship between recombination rate, SNP diversity and interspecies divergence for 29 loci in chickens. We found that recombination rate is positively correlated with nucleotide diversity but is not correlated with interspecies divergence. It appears that variation in recombination rate explains over 30% of the variation in levels of diversity among 29 loci. Our data suggested that natural selection is a main factor in shaping SNP diversity in chickens. Since SNP diversity is significantly lower at Z-linked than at autosomal loci, we argued that genetic hitchhiking might be more important than background selection in producing the observed correlation.     

The invasive Korea and Japan types of Varroa destructor, ectoparasitic mites of the Western honeybee (Apis mellifera), are two partly isolated clones

Varroa destructor, now a major pest of the Western honeybee, Apis mellifera, switched from its original host, the Eastern honeybee, A. cerana, ca. 50 years ago. So far, only two out of several known mitochondrial haplotypes of V. destructor have been found to be capable of reproducing on A. mellifera (Korea and Japan). These haplotypes are associated in almost complete cytonuclear disequilibrium to diagnostic alleles at 11 microsatellite loci. By contrast, microsatellite polymorphism within each type is virtually absent, because of a severe bottleneck at the time of host change. Accordingly, 12 mitochondrial sequences of 5185 nucleotides displayed 0.40% of nucleotide divergence between haplotypes and no intra haplotype variation. Hence, each type has a quasi-clonal structure. The nascent intratype variability is subsequent to the clone formation 50 years ago: in both types the variant alleles differ from the most common by one (in 10 cases), two (five cases) or three (one case) repeated motifs. In addition to individuals of the two 'pure' types, five F1 hybrids and 19 recombinant individuals (Japan alleles introgressed into the Korea genetic background) were detected. The existence of F1 and recombinant individuals in admixed populations requires that double infestations of honeybee cells occur in a high proportion but the persistence of pure types suggests a post-zygotic isolation between the two clones.     

Resistance to shell breaking in two intertidal snails

The ability of shells to withstand shell breaking forces has been examined in two intertidal prosobranchs, Nucella lapillus and Littorina littorea , using four methods: measuring shell strength on a compressive testing machine, measuring the shell to body mass ratio, measuring the shell thickness and measuring the ability of crabs to break shells in aquarium experiments. Nucella lapillus consistently showed a relationship between shell vulnerability and environmental variables: the shells were easier to break at sites where rock and boulder movement was the least. Although some between-site differences were found in L. littorea shells, these were less than in N. lapillus and did not relate to environment variables: the shells were easier to break at sites where exposure to wave action was the least. Although some between-site differences were found in L. littorea shells , these were less than in N. lapillus and did not relate to environmental factors. However, both species appear to grow into a size refuge in which they are secure from predation by shore crabs at the sites where these crabs are commonest.     

Polymorphic shell banding in the dog-whelk, Nucella lapillus (Mollusca)

The shell of the Common dog-whelk (Nucella lapillus (L.)) is white and unbanded at most places around the British Isles. However, high frequencies of banding occur on the Buchan coast, around Anglesey and the Menai Straits, on the Cower Peninsula, around the Devon–Somerset border in the Bristol Channel, and especially on the north Cornish coast (reaching a peak between Newquay and Padstow). The frequency of banding is significantly less in older than younger whelks in the same locality, and this change is uncorrelated with the selection against shell shape variation that takes place on exposed shores. It is concluded that banding is a pleiotropic manifestation of physiological variation, and that a study of such variation in different morphs could indicate the importance of different physiological stresses at different stages of the life history of N. lapillus.     

DNA variation in a conifer,Cryptomeria japonica (Cupressaceae sensu lato)

We investigated the nucleotide variation of a conifer, Cryptomeria japonica, and the divergence between this species and its closest relative, Taxodium distichum, at seven nuclear loci (Acl5, Chi1, Ferr, GapC, HemA, Lcyb, and Pat). Samples of C. japonica were collected from three areas, Kantou-Toukai, Hokuriku, and Iwate. No apparent geographic differentiation was found among these samples. However, the frequency spectrum of the nucleotide polymorphism revealed excesses of intermediate-frequency variants, which suggests that the population was not panmictic and a constant size in the past. The average nucleotide diversity, pi, for silent sites was 0.00383. However, values of pi for silent sites vary among loci. Comparisons of polymorphism to divergence among loci (the HKA test) showed that the polymorphism at the Acl5 locus was significantly lower. We also observed a nearly significant excess of replacement polymorphisms at the Lcyb locus. These results suggested possibilities of natural selection acting at some of the loci. Intragenic recombination was detected only once at the Chi1 locus and was not detected at the other loci. The low level of population recombination rate, 4Nr, seemed to be due to both low level of recombination, r, and small population size, N.     

Chromosomal polymorphism in the Atlantic dog-whelk, Nucella lapillus (Gastropoda: Muricidae): nomenclature, variation and biogeography

Nucella lapillus appears to be unique within the Muricidae, and indeed the Mollusca, in exhibiting a Robertsonian chromosomal polymorphism. The recorded diploid values range from 2 n  = 26 to 2 n  = 36, putatively brought about by fusion of smaller chromosomes to form five pairs of large metacentrics in the 2 n  = 26 form. In this study, the karyotypes of the numerically extreme forms (2 n  = 26 and 2 n  = 36) have been illustrated and described in detail, and a new scheme of nomenclature is proposed for the chromosomal rearrangements observed in N. lapillus . Chromosome numbers and karyotypes were recorded and analysed from 70 sites throughout its wide geographical range, mainly from around the UK coasts but also from sites in Norway, France, Spain, Portugal and the USA, showing remarkable variation in both diploid number and karyotypic configuration. The results provide new evidence that seven chromosomes in the 2 n  = 26 form can be involved in the Robertsonian rearrangements, but the maximum recorded diploid number remains 2 n  = 36. Inversions were confirmed in three chromosomes and one population was found to exhibit aneuploidy. Possible explanations for the geographical trends in karyotypic variation are discussed, but despite the advances in the karyology of N. lapillus , a simple solution to this enigmatic phenomenon remains elusive.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 195–210.     

Extremely low levels of genetic polymorphism in endosymbionts (Buchnera) of aphids (Pemphigus)

Molecular evolutionary studies have suggested that vertically transmitted endosymbionts are subject to accumulation of deleterious mutations through genetic drift. Predictions of this hypothesis for patterns of intraspecific polymorphism were borne out in the single relevant study available, on the symbiont Buchnera aphidicola of Uroleucon ambrosiae. In order to examine the generality of this result, we surveyed DNA sequence variation in Buchnera of the distantly related aphid, Pemphigus obesinymphae. In contrast to Uroleucon species, Pemphigus species have complex life cycles with few dispersal stages. Despite these differences, P. obesinymphae showed patterns of variation at two Buchnera loci and one mitochondrial locus that were remarkably similar to those reported previously for Buchnera of U. ambrosiae. In the western US, Buchnera was nearly monomorphic, and in the eastern US, synonymous divergence ranged from 0.08 to 0.16%. Most polymorphisms involved rare alleles, consistent with a recent range of ancestral polymorphism, probably due to demographic fluctuations in aphid populations. These results support the generality of small effective population size in Buchnera and their aphid hosts.