Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

Malate dehydrogenase (MDH) catalyzes a reversible NAD⁺-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO₂ assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO₂ assimilation/intercellular CO₂ curves at low CO₂, and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.Plant tissues contain multiple isoforms of malate dehydrogenase (l-malate-NAD-oxidoreductase [MDH]; EC 1.1.1.37) that catalyze the interconversion of malate and oxaloacetate (OAA) coupled to reduction or oxidation of the NAD pool. These isoforms are encoded by separate genes in plants and have been shown to possess distinct kinetic properties as well as subcellular targeting and physiological functions (Gietl, 1992). While the MDH reaction is reversible, it strongly favors the reduction of OAA. The direction of the reaction in vivo depends on substrate/product ratios and the NAD redox state, and it can vary even in the same tissue due to prevailing physiological conditions. Isoforms operate in mitochondria, chloroplasts, peroxisomes, and the cytosol, but due to the ready transport and utilization of malate and OAA and the availability of NAD, this reaction can cooperate across compartments and is the basis for malate/OAA shuttling of reducing equivalents in many different metabolic schemes of plant cellular function (Krömer, 1995). It is clear, however, that the exchange through the membranes is strictly controlled, since large redox differences in NAD(H) pools exist between compartments (Igamberdiev and Gardeström, 2003).The mitochondrial MDH (mMDH) is thought to operate in at least three different pathways in plants. First, it is a classical tricarboxylic acid (TCA) cycle enzyme that oxidizes the malate product from the fumarase reaction to OAA for the citrate synthase-dependent condensation with acetyl-CoA to form citrate. Second, it is considered to operate in the reverse direction during the conversion of Gly to Ser by reducing OAA to malate and providing a supply of NAD⁺ for Gly decarboxylase (Journet et al., 1981). Third, in a more specialized pathway in C4 plants, it provides a supply of CO₂ for fixation in bundle sheath chloroplasts by reducing OAA (generated from Asp transported from mesophyll cells) into malate that is then decarboxylated by NAD-malic enzyme (NAD-ME) to CO₂ and pyruvate (Hatch and Osmond, 1976). Plant mitochondria can support TCA cycle activity with malate as the sole substrate due to MDH and NAD-ME, both ubiquitous in plants (Palmer, 1984). OAA is readily transported both into and out of isolated plant mitochondria (Douce and Bonner, 1972), in contrast to mammalian mitochondria, which are essentially impermeable to this organic acid.While these three mMDH schemes and metabolic schemes for other MDH isoforms are plausible, widely accepted, and consistent with a range of biochemical studies, the depletion, removal, and overexpression of specific MDH isoforms in plants have led to surprising insights into MDH roles in vivo. For example, the peroxisomal MDH (PMDH) was until recently generally considered to be involved in the synthesis of NADH for hydroxypyruvate reduction in the photorespiratory cycle and for the oxidation of NADH generated during β-oxidation of fatty acids, but its potential role in the oxidation of malate in the glyoxylate cycle was unclear. However, studies of the double knockout of PMDH in Arabidopsis (Arabidopsis thaliana) showed that while PMDH is essential for β-oxidation, its removal does not impair glyoxylate cycle activity (Pracharoenwattana et al., 2007) and has only a limited impact on hydroxypyruvate reduction (Cousins et al., 2008).Changes in mMDH have been reported both through the study of spontaneous mutants and the expression of antisense constructs. Spontaneous null mutants of mMDH1 in soybean (Glycine max) are linked to a yellow foliage phenotype and are associated with the removal of two of the three mMDH isoforms (Imsande et al., 2001). Expression of an antisense fragment of mMDH in tomato (Solanum lycopersicum), driven by the 35S promoter, lowered mMDH protein in mitochondria, decreased total cellular MDH by approximately 60%, but had a positive impact on photosynthetic activity, CO₂ assimilation rate, and total plant dry matter in long-day-grown plants (Nunes-Nesi et al., 2005). A range of carbohydrates also accumulated in the tomato antisense plants, as did redox-related compounds such as ascorbate. The increase in ascorbate content may be linked to the enhancement of photosynthesis, as ascorbate feeding to leaves can also increase photosynthetic performance (Nunes-Nesi et al., 2005). This link is not absolute, however, given that short-day-grown antisense tomato plants had stunted growth, which was potentially due to impaired photosynthesis, but still had elevated levels of ascorbate due to a higher ratio of reduction of the ascorbate pool compared with the wild type (Nunes-Nesi et al., 2008). Analysis of roots from these antisense tomato plants revealed a negative impact of mMDH loss, leading to a lower root dry weight and lower root respiratory rate (van der Merwe et al., 2009). This implies a distinct impact of mMDH loss on roots and shoots. Overexpression of cytosolic MDH led to a 4-fold elevation of root organic acids in alfalfa (Medicago sativa) plants and high rates of organic acid exudation that increased aluminum tolerance through metal chelation in the soil (Tesfaye et al., 2001). These studies imply that there is a complex form of functional redundancy between MDH isoforms in different compartments, allowing MDH in separate locations to maintain specific pathways via malate/OAA shuttling, or that a range of redox requirements that have been linked to MDH in accepted metabolic schemes are incorrect and other reactions couple NAD/NADH pool homeostasis. In addition, these studies clearly show that changes in the amount of MDH isoforms can alter metabolic flux into a range of organic acids and have far-reaching effects on plant growth and development.To better understand the importance of the mMDH and to determine if plants are viable without any mMDH isoforms due either to the role of NAD-ME and/or malate/OAA shuttling to other compartments, we have constructed and analyzed mMDH mutants in Arabidopsis. A major and a minor MDH isoform exist in Arabidopsis mitochondria, evidenced by differing levels of gene expression and differing protein abundance (Lee et al., 2008). We hypothesized that if mMDH works in concert with other MDH isoforms and is responsible for the reduction of OAA to malate for export from the mitochondrion, then if we remove mMDH, not only would the loss of extramitochondrial malate and the slowing of Gly decarboxylation limit photorespiratory carbon flux, but oxidation of NADH remaining in the mitochondrion could lead to elevated leaf respiration and alteration in plant growth. We found that not only did mutants have low photorespiratory flux, but they also increased respiration and had slow growth due to lowered net CO₂ assimilation. The previously established correlation between mMDH abundance, photosynthetic performance, and foliar ascorbate levels was also investigated. Elevated levels of the metabolite were found in Arabidopsis, consolidating the work done in tomato (Nunes-Nesi et al., 2005). Proteomic analyses, followed by immunodetection studies, unearthed altered abundance of the terminal enzyme of the ascorbate biosynthetic pathway, galactono-1,4-lactone dehydrogenase (GLDH), as a mechanistic element in the phenomenon linked directly to mitochondrial function.     

Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

Summary The ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.     

Isozyme expression in F1 hybrids between carp and goldfish

Interspecific genetic differences in malate dehydrogenase (MDH), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and esterase (EST) isozymes in carp (Cyprinus carpio) and goldfish (Carassius auratus) were used to examine the allelic expressions in the hybrid between these species. A unique liver SOD and muscle LDH phenotype unambiguously identifies all presumed hybrid individuals. There was no evidence of F₂ or backcross phenotypes in hybrid individuals. Liver MDH and EST phenotypes in hybrids show a preferential expression of goldfish isozymes. Variation in the levels of carp liver MDH isozymes may result from the polymorphism of a regulatory mutation affecting isozyme expression, leading to gene silencing after duplication.This work was supported through NSERC (Canada) grants to James P. Bogart and John F. Leatherland.     

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     

Linkage of genes for PHI,halothane sensitivity,A-O inhibition,H red blood cell antigens and 6-PGD variants in pigs*

Data from one apparent crossover between S and H, two between PHI and HAL on one side and S on the other, and one between PHI on one side and HAL, S and H on the other, indicate a gene order in pigs of Phi-Hal-S-H-Pgd for genes for PHI, halothane sensitivity, inhibition of expression of A and O, H red blood cell antigens and 6-PGD types. Rasmusen et al. (1980) provided data for a gene order in pigs ofPhi-Hal-H-Pgd for genes for phosphohexose isomerase (PHI) isozyme variants, halothane sensitivity (HAL), H red cell antigens and 6-phosphogluconate dehydrogenase (6-PGD) variants, and suggested that there might be a locus for a gene for inhibition of expression of A and O separate from the locus for H. This is contrary to an earlier proposal by Rasmusen (1972) that the H-system genotype directly influences expression of A and O. Imlah (1980) suggested that the recessive gene for halothane sensitivity has a suppressant effect on the expression of A and O. Andresen (1981) proposed that the locus for inhibition of A and O (for which Rasmusen, 1964, proposed the symbol S) was between the loci for HAL and H types. Data presented in Table 1, which includes haplotypes for three recombinant offspring described by Rasmusen et al. (1980) (883-1, 233-3 and 3864-1) as well as one other recombinant (296-2) provide evidence for the gene order for five genes proposed by Andresen. Types for 6-PGD are listed for all pigs, although they do not provide evidence for gene order in these cases. Male 883-1 (Table 1, and Rasmusen et al., 1980, Table 5) provided the original evidence for recombination between S and H. His phenotype, as well as his genotype as revealed by progeny test (Rasmusen et al., 1980, Table 6) indicated that recombination had occurred between the genes for PHI, HAL and S and the gene for H type in his dam, so that the S locus mapped between H and the loci for the other three traits. The phenotype of one of his sons (233-3, Table 1, and Rasmusen et al., 1980, Table 6) indicated that there had been a recombination between genes for PHI and HAL types on one side and S and H types on the other, providing evidence that the S locus was separate from PHI and HAL as well as H. Another pig listed in Table 1,3864-1, was also described by Rasmusen et al. (1980, Table 9) as a recombinant. This pig provides evidence for recombination between PHI on one side and HAL, S and H on the other, establishing a gene order of Phi-Hal-S-H-Pgd. The last pig listed in Table 1,296-2, is a recombinant comparable to 233-3. The H type of his dam provides markers indicating the recombination was between PHI and HAL on one side and S and H on the other, although the unusual expression of HAL phenotype in both parents of 296-2 makes her haplotypes somewhat uncertain. (Recombination may have been between PHI and HAL rather than as indicated in Table 1.) In spite of incomplete penetrance for HAL (Ollivier et al., 1975; Smith & Bampton, 1977) which makes haplotypes for HAL questionable in some cases, the other genetic markers available are useful to show that recombination has taken place. Without considering the results of halothane testing, if the apparent recombinants are accepted as being as indicated, the order of the genes at the other four loci seems established. Alleles for S types appear to be separable by recombination from those for PHI and H, and the S locus appears to be between the loci for PHI and H. For the five loci, data obtained thus far are cohsistent with a gene order of Phi-Hal-S-H-Pgd.     

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Résumé

À l'inverse de la situation des vertébrés et de la majorité des insectes, chez qui un gène unique code pour l'acétylcholinestérase (AChE), quatre gènes d'AChE ont été clones et caractérisés chez Caenorhabditis elegans. Le gène ace-1 (localisé sur le chromosome X) et le gène ace-2 (chromosome I) assurent respectivement l'expression de l'AChE dans les tissus musculaire (ace-1) et nerveux (ace-2). Les gènes ace-x et ace-y ne sont séparés que de quelques centaines de paires de bases sur le chromosome II et leur rôle est pour l'instant inconnu.     

准噶尔雅罗鱼组织同工酶表达特性

使用不连续PAGE法,分析了10尾准噶尔雅罗鱼(Leuciscus merzbacheri)眼睛、鳃、皮、背部肌肉、鳍和肝胰脏6种组织的10种同工酶(LDH,CCO,EST,CAT,POD,ME,MDH,G6PD,GDH,ADH)的差异表达,并对部分同工酶基因位点及表达酶谱表型进行了分析,以期为其种质资源保护和开发以及遗传育种等方面的研究提供基础资料。结果显示,10种同工酶中9种在6种组织中出现了明显的组织差异性,仅CCO在6种组织中的差异性较小。在对准噶尔雅罗鱼的10种同工酶的遗传多样性分析中,共记录到了21个基因位点,其中Est-1、Me-B、s-MDH、G6pd-A、G6pd-B和Adh-A为多态性基因位点。多态位点比例为:P=6/21=28.57%。     

The biochemical differentiation of Enterobacter sakazakii genotypes

Background  

Enterobacter sakazakii is an emergent pathogen that has been associated with neonatal infections through contaminated powdered infant milk formula. The species was defined by Farmer et al. (1980) who described 15 biogroups according to the biochemical characterization of 57 strains. This present study compares genotypes (DNA cluster groups based on partial 16S rDNA sequence analysis) with the biochemical traits for 189 E. sakazakii strains.     

Organization of genes in the four minute region of the Escherichia coli chromosome: Evidence that rpsB and tsf are Co-transcribed

Summary Restriction endonuclease-generated DNA fragments of polC-9 (Friesen et al. 1976) have been cloned on plasmid vehicles. Expression of genes carried by these plasmids was determined either by genetic complementation of the appropriate mutants, or in ultraviolet-irradiated cells. On the basis of these experiments we have inferred the following gene order in the four minute region of the Escherichia coli chromosome: tonA-dapD4-dapD2-rpsB-tsf-22 kilodalton protein — fir 27,000-firA-dnaE. We suggest that rpsB and tsf are in one tranccriptional unit, with rpsB being promoter-proximal. We also suggest the possible position of the promoter for dnaE.     

Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12

Summary The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.     

On the identity of dnaP and dnaF genes of Bacillus subtilis

Summary The dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.Abbreviations and symbols HPUra 6-(p-hydroxyphenylazo)-uracil; mic, minimum inhibitory concentration - MMS methyl-methanesufonate - Pol I Pol II and Pol III: DNA polymerase I, II and III respectively - PCMB parachloro-mercuri-benzoate     

Comparative analysis of malate dehydrogenases of Drosophila melanogaster

The malate dehydrogenases of D. melanogaster have been resolved into a cytoplasmic form (cMDH) and a mitochondrial matrix form (mMDH). Flies homozygous for allozyme variants exhibit isozymes of cMDH detected by starch gel electrophoresis and acrylamide gel isoelectric focusing. The basis of these isozymes was investigated, and the results suggest either conformational or epigenetic modification of isozymes. The probable structural gene for cMDH (Mdh-1) has been mapped genetically by allozyme variants to II-35 ± 3 and cytologically by monitoring gene dosage in segmental aneuploids to between 28D and 29F on II-L of the Drosophila salivary gland chromosome map. The structural gene for mMDH is neither identical to nor in the near chromosomal proximity of Mdh-1. Nevertheless, the two enzymes exhibit markedly similar properties with respect to (1) catalytic activity, (2) pH optima, (3) pH optimum shift in response to different ionic environments, and (4) molecular weight as determined by sucrose density gradient sedimentation.This project was supported by NIH postdoctoral research fellowship No. 6-FO2-GM-49, 633-01 from the National Institute of General Medical Sciences.     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

Evolution of the Vertebrate Cytosolic Malate Dehydrogenase Gene Family: Duplication and Divergence in Actinopterygian Fish

Abstract A general correlation between neural expression and negative charge in isozymes suggests charge represents an adaptation to the neural environment. Interestingly, a notable exception exists in teleost fish. Two cytosolic malate dehydrogenase (MDH) isozymes have different spatial expression patterns in certain fishes: one is expressed in all tissues and the second is expressed primarily in the eye and skeletal muscle. While the neural MDH isozyme is negatively charged, the difference in charge between the two isozymes is not as pronounced as that observed in other gene families (e.g., triosephosphate isomerase and lactate dehydrogenase). Most tetrapods express a single cytosolic MDH isozyme, and it has been demonstrated recently that the pair of isozymes found in teleosts results from a gene duplication sometime after the separation of teleosts and tetrapods, although the exact timing of this duplication has not been inferred. Phylogenetic analyses suggest that the duplication of teleost isozymes occurred during the radiation of actinopterygian fish, consistent with the timing of duplication at other loci. Using inferred amino acid sequences, we examine the pattern of change following the duplication and across the rest of the MDH gene tree. Comparison between the MDH gene family and another gene family that shows a larger charge differential among members (triosephosphate isomerase) indicates that the smaller charge difference between MDH isozymes is best explained by greater constraint on amino acid change directly following the duplication, not greater constraint across the entire gene tree. This difference in constraint might result from the wider pattern of expression of the “neural” MDH isozyme.     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide     

Expression pattern of mouse sFRP-1 and mWnt-8 gene during heart morphogenesis

The Wnt genes encode a large family of secreted proteins that play a key role in embryonic development and tissue differentiation in many species (Rijsewijk et al., 1987 and Nusse and Varmus, 1992). Genetic and biochemical studies have suggested that the frizzled proteins are cell surface receptors for Wnts (Vinson et al., 1989, Chan et al., 1992, Bhanot et al., 1996 and Wang et al., 1996). In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified (Finch et al., 1997, Melkonyan et al., 1997 and Rattner et al., 1997). One of these proteins, FrzA, the bovine counterpart of the murine sFRP-1 (93% identity) is involved in vascular cell growth control, binds Wg in vitro and antagonizes Xwnt-8 and hWnt-2 signaling in Xenopus embryos (Xu et al., 1998 and Duplàa et al., 1999). In this study, we report that sFRP-1 is expressed in the heart and in the visceral yolk sac during mouse development, and that sFRP-1 and mWnt-8 display overlapping expression patterns during heart morphogenesis. From 8.5 to 12.5 d.p.c., sFRP-1 is expressed in cardiomyocytes together with mWnt-8 but neither in the pericardium nor in the endocardium; at 17.5 d.p.c., they are no longer present in the heart. In mouse adult tissues, while sFRP-1 is highly detected in the aortic endothelium and media and in cardiomyocytes, mWnt-8 is not detected in these areas. Immunoprecipitation experiments demonstrates that FrzA binds to mWnt-8 in cell culture experiments.     

Mapping of the drug resistance genes carried by the r-determinant of the R100.1 plasmid

Summary We have cloned the EcoRI fragments of pLC1, a circular DNA element found in an Escherichia coli dnaA _ts strain integratively suppressed by R100.1 (Chandler et al., 1977a), using the plasmid vector pCR1. All the resistance genes known to be present on the r-determinant of R100.1 were found to be present on pLC1. The isolation of pCR1 derivatives carrying various EcoRI fragments of either pLC1 or R100.1 has allowed a more precise mapping of the position of the resistance genes on the R100.1 molecule.     

Tissue specific expression of the acetylcholinesterase gene in Drosophila melanogaster

Summary Acetylcholinesterase (AChE) is mainly membrane bound in the central nervous system (CNS) of larvae and in the head and thorax of adults of Drosophila melanogaster; it is mostly soluble in the larval carcass, the adult abdomen, similar to that of the embryos (Zador et al. 1986). The enzyme shows the same number of isozymes (four or five) in larvae and adults as in the head of the fly or in embryos (Zador et al. 1986). In the Df(3R)GE26/MKRS stock both the membrane bound and the soluble enzyme are at about half normal levels while in the Df(3R)Ace ^HD1/MKRS stock this is true only for the membrane bound AChE. Therefore the effect of the above deficiencies in larvae and adults is consistent with that in embryos (Zador et al. 1986). In heat-sensitive combinations of certain Ace mutant alleles both the membrane bound and the soluble enzyme has reduced activity.Abbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - BAP 1,5-bis(allyldimethylammonium-phenyl)-pentan-3-one dibromide - CNS central nervous system