Evolution of nucleotide substitutions and gene regulation in the amylase multigenes in Drosophila kikkawai and its sibling species

In order to determine evolutionary changes in gene regulation and the nucleotide substitution pattern in a multigene family, the amylase multigenes were characterized in Drosophila kikkawai and its sibling species. The nucleotide substitution pattern was investigated. Drosophila kikkawai has four amylase genes. The Amy1 and Amy2 genes are a head-to-head duplication in the middle of the B arm of the second chromosome, while the Amy3 and Amy4 genes are a tail-to-tail duplication near the centromere of the same chromosome. In the sibling species of D. kikkawai (Drosophila bocki, Drosophila leontia, and Drosophila lini), sequencing of the Amy1, Amy2, Amy3, and Amy4 genes revealed that the Amy1 and Amy2 gene group diverged from Amy3 and Amy4 after duplication. In the Amy1 and Amy2 genes, the divergent evolution occurred in the flanking regions; in contrast, the coding regions have evolved in concerted fashion. The electrophoretic pattern of AMY isozymes was also examined. In D. kikkawai and its siblings, two or three electrophoretically different isozymes are encoded by the Amy1 and Amy2 genes (S isozyme) and by the Amy3 and Amy4 genes (F (M) isozymes). The S and F (M) isozymes show different patterns of band intensity when larvae and flies were fed in different media. Amy1 and Amy2, which encode the S isozyme, are more strikingly regulated than Amy3 and Amy4, which encode the F (M) isozyme. The GC content and codon usage bias were higher for the Amy1 and Amy2 genes than for the Amy3 and Amy4 genes. Although the ratio of synonymous and replacement substitutions within the Amy1 and Amy2 gene group was not significantly different from that within the Amy3 and Amy4 gene group, the synonymous substitution rate in the lineage of Amy1 and Amy2 was lower than that of Amy3 and Amy4. In conclusion, after the first duplication but before speciation of four species, the synonymous substitution rate between the two lineages and the electrophoretic pattern of the isozymes encoded by them changed, although we do not know whether there was any evolutionary relationship between the two.     

In vivo adenosine receptor preconditioning reduces myocardial infarct size via subcellular ERK signaling

The protective effects of adenosine receptor acute preconditioning (PC) are well known; however, the signaling mechanism mediating this effect has not been determined in in vivo models. The purpose of this study was to determine the role of the extracellular signal-regulated kinase (ERK) pathway in mediating adenosine PC in in vivo rat myocardium. Open-chest rats were submitted to 25 min of coronary artery occlusion and 2 h of reperfusion. ERK activation was assessed by measuring total and dually phosphorylated p44/42 ERK isoforms in nuclear and/or myofilament, mitochondrial, cytosolic, and membrane fractions. Adenosine receptor PC with the A1/A2a agonist 1S-[1a,2b,3b,4a(S*)]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-3H-imidazo[4,5-b]pyridyl-3-yl]cyclopentane carboxamide (AMP-579) reduced infarct size from 49 +/- 3% to 29 +/- 3%, an effect that was blocked by the mitogen-activated protein kinase-ERK inhibitor U-0126. ERK isoforms were present in all fractions, with the greatest expression in the cytosolic fraction and the least in the mitochondrial fraction. AMP-579 treatment increased preischemic p44/42 ERK phosphorylation in all fractions 2.7- to 6.9-fold. Reperfusion increased ERK isoform activation in all fractions, but there were no differences between control and AMP-579 hearts. Preischemic increases in phospo-p44/p42 ERK with AMP-579 were blunted by U-0126, although only in mitochondrial and membrane compartments. The PC effects of AMP-579 on infarct size and ERK were blunted by both the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine and, surprisingly, the A2a antagonist ZM-241385. These results indicate that the unique adenosine receptor agonist AMP-579 exerts its beneficial effects in vivo via both A1 and A2a receptor modulation of subcellular ERK isoform signaling.     

Comprehensive characterization of secreted aspartic proteases encoded by a virulence gene family in Candida albicans

Candida albicans is a commensal organism, but causes life-threatening infections in immunocompromised patients. Certain factors such as yeast-hyphae transition and hydrolytic enzymes are suggested as virulence attributes of C. albicans. Among them, 10 types of secreted aspartic protease (SAP) genes have received particular attention as a major virulence gene family. However, their full functional repertoire, including its biochemical properties, remains to be elucidated. Hence, we purified all Sap isozymes using Pichia pastoris and comprehensively determined and compared their biochemical properties. While optimum pH of Sap7 was 6.5 and that of Sap8 was 2.5, presence of other Sap isozymes functioning within a broad range of optimum pH could allow C. albicans to survive and cause infections in various tissues. The substrate specificities of Sap isozymes were analysed by using FRETS-25Xaa libraries. Sap7 and Sap10 showed high substrate specificity, while other Sap isozymes had broad substrate specificities. Principal component analysis revealed that the 10 Sap isozymes were clustered into 3 distinct groups in terms of their substrate specificities. Interestingly, Sap4-6, which are coproduced in the hyphal form, were clustered as the same group, indicating that they may target similar host proteins. These results will lead to further understanding of C. albicans pathogenicity.     

Adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning

The purpose of this study was to determine whether the adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning. Regional stunning was produced by 15 min of coronary artery occlusion and 3 h of reperfusion (RP) in anesthetized open-chest pigs. In acute protection studies, animals were pretreated with saline, low-dose AMP-579 (15 microg/kg iv bolus 10 min before ischemia), or high-dose AMP-579 (50 microg/kg iv at 14 microg/kg bolus + 1.2 microg.kg(-1).min(-1) for 30 min before coronary occlusion). The delayed preconditioning effects of AMP-579 were evaluated 24 h after administration of saline vehicle or high-dose AMP-579 (50 microg/kg iv). Load-insensitive contractility was assessed by measuring regional preload recruitable stroke work (PRSW) and PRSW area. Acute preconditioning with AMP-579 dose dependently improved regional PRSW: 129 +/- 5 and 100 +/- 2% in high- and low-dose AMP-579 groups, respectively, and 78 +/- 5% in the control group at 3 h of RP. Administration of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.7 mg/kg) blocked the acute protective effect of high-dose AMP-579, indicating that these effects are mediated through A1 receptor activation. Delayed preconditioning with AMP-579 significantly increased recovery of PRSW area: 64 +/- 5 vs. 33 +/- 5% in control at 3 h of RP. In isolated perfused rat heart studies, kinetics of the onset and washout of AMP-579 A1 and A2a receptor-mediated effects were distinct compared with those of other adenosine receptor agonists. The unique nature of the adenosine agonist AMP-579 may play a role in its ability to induce delayed preconditioning against in vivo myocardial stunning.     

Multiple molecular forms of the gibberellin-induced alpha-amylase from the aleurone layers of barley seeds

A class of plant growth regulators, gibberellins, induce the synthesis of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in the aleurone layers of barley (Hordeum vulgare L. var. Himalaya) seeds. The purified alpha-amylase is composed of multiple isozymic forms with indistinguishable molecular weights, but different net charges. These alpha-amylase isozymes separate on isoelectric focusing gels into two groups, each containing multiple species. One group has an apparent isoelectric point (pI) of approximately 5.8 (the high pI group). The other group's pI values are around 4.5 (the low pI group). On some gels a small amount of protein focuses between the high and low pI isozymes. These proteins comigrate with the low pI isozymes upon reelectrophoresis. The synthesis of these two groups is temporally regulated. The high pI group is the dominant set of isozymes secreted from embryoless half seeds during the first two days of gibberellin administration. After four days, however, the major isozymes are those of the low pI group. This shift in isozyme pattern is due to a shift in their relative rates of synthesis. Peptide analysis of these two groups of isozymes with Staphylococcus aureus V8 protease and cyanogen bromide shows amino acid sequence differences. However, members within the same group have similar peptide patterns. Both groups of isozymes are synthesized in vitro in a wheat germ extract primed with poly(A)+ RNA isolated from gibberellin-treated aleurone layers. This indicates that the synthesis of the two groups of alpha-amylase isozymes is probably directed by two or more different populations of mature mRNA. A model that explains these observations and the available genetic information is that barley aleurone alpha-amylase isozymes are encoded by at least two sets of structural genes.     

Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with pI values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their pI values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the alpha class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the mu class, whereas GST 8.15 and 5.4 belong to pi class. The N-terminal sequences and pI values of the mu class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the pi class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.     

Isozymes in wheat-barley hybrid derivative lines

Zymogram analysis was used to identify the barley chromosomes that carry the structural genes for particular isozymes. Wheat, barley, and wheatbarley hybrid derivative lines (which contained identified barley chromosomes) were tested by gel electrophoresis for isozymes of particular enzymes. It was found that barley chromosome 4 carries structural genes for acid phosphatase and amylase isozymes, barley chromosome 5 carries genes for phosphoglucose isomerase and malate dehydrogenase isozymes, and that barley chromosome 2 carries a gene for at least one glucose-6-phosphate dehydrogenase protomer. These results reinforce previous conclusions that barley chromosome 4 shows homoeology with wheat chromosome group 4 and that barley chromosome 5 shows homoeology with wheat chromosome group 1.     

草鱼同工酶发育遗传学研究——Ⅱ.早期发育过程中的同工酶分析

采用淀粉凝胶电泳技术研究了草鱼早期发育过程中(受精后0—200小时)6种同工酶系统(LDH、MDH、GDH、ADH、IDH、EST)的表达谱式。除了ADH以外,其余5种同工酶系统均具有明显的发育变化谱式。根据早期发育过程中同工酶的变化谱式及其组织分布,草鱼的同工酶可分为三大类型:(1)在未受精卵及早期发育过程中一直存在,并常有较广泛的组织分布;(2)未受精卵及早期发育过程中均不存在,一般仅分布于少数几种组织中;(3)未受精卵及胚胎发育早期不存在,直到早期发育过程中某一特定时期才开始出现。     

A gene modifying mitochondrial malate dehydrogenase isozymes inSorghum (Gramineae)

Malate dehydrogenase (MDH) isozymes extracted from dark-grown seedlings of Sorghum species are encoded by at least two genes with their products localized in the mitochondria (mt) and one gene with its products localized in the cytosol. In homozygous genotypes, the three mt-MDH isozymes represent two homodimers and an intergenic heterodimer. For some plants of S. virgatum and S. aethiopicum, the three mt-MDH isozymes migrate about 3 mm faster (more anodally) when electrophoresed on starch gels. The F1's of plants with normal and fast mt-MDHs had normal migration; the F2's segregate 3:1 for normal to fast migration. It is suggested that a single gene, Mmm (mt-MDH modifier), controls this modification of normal migration and that fast migration occurs when the recessive allele (mmm-m) is homozygous. The designation, Mmm, is borrowed from Zea mays, in which a similar gene has been described.     

Analysis of neuraminidase isozyme phenotypes in mammalian tissues: an electrophoretic approach

A simple cellulose acetate electrophoretic method for visualizing mammalian neuraminidase isozymes has been developed. Application of the method with rat and mouse liver extracts reveals the presence of two distinct isozymes in each species. Each isozyme exhibits tremendous variation in activity between inbred strains. The two isozymes vary independently of one another suggesting that their activities are controlled by different genes. The neuraminidase phenotypes detected in these inbred strains via electrophoresis are consistent with published accounts of neuraminidase phenotypes determined fluorometrically in whole liver homogenates, but also indicate the presence of a second isozyme not perceived by this other procedure.     

Isozyme variations inAegilops andTriticum. III. Chromosomal basis of the esterase isozyme production in different organs of Chinese spring wheat

Developmental changes of esterase isozymes from the germination to the heading stage of normal and aneuploid lines of common wheat,Triticum aestivum cv. Chinese Spring were studied. A total of twenty major isozymes (Bands 1E to 20E) were observed, some of which were further separated to two to three closely located bands. Among these bands, 1E, 2E, 3E, 5E, 7E, 11E, 14E and 16E were found to be leaf-specific isozymes and 9E, 10E, 13E, 15E, 17E and 18E were seed-specific. Leaf-specific isozyme bands 1E, 2E and 5E are controlled by genes on three homoeologous chromosomes group 6, leaf-specific bands 7E, 11E, 14E and 16E and seed-specific bands 9E, 10E, 13E, 15E, 17E and 18E are under control of genes on homoeologous chromosomes of group 3. On the other hand, two bands, 19′E and 19″E are controlled by genes on chromosomes of homoeologous group 2 in roots of seedlings 10 days old. The present investigation showed that the genes for esterase production located on chromosome 6B had large effects in mature leaves, but chromosomes 6A and 6D had little effect on the esterase isozymes in homoeologous group 6. Genes located on chromosomes 3A, 3B and 3D have a large function in germinating seed; however, chromosomes 3B had little effect on the esterase isozymes in the mature leaf. Present findings confirmed that the chromosomes of the A, B and D genomes have different functions in the production of proteins or enzymes. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 401.     

Extent of genetic variability of endosperm esterases in Triticum aestivum L. 2n=6x=42

Summary Genetic variability of endosperm esterase has been studied in 42 cultivars of Triticum aestivum L. 2n=6x=42. Different techniques, including sequential electrophoresis and electrofocusing, have been used with various substrates and esterase inhibitors. The electrophoretic patterns in each cultivar are described. Chromosomal location using the nullitetrasomic and ditelosomic lines of Chinese Spring was carried out in order to relate and/or locate the esterase genes to specific chromosomes. Most of the esterase isozymes located were in the long arm of the chromosomes of the homoeology group 3; but we have found six located in the short arms, five of them in the chromosome 3AS and one in the 3DS. This location increases the number of esterase genes described, because no esterase genes had been described so far in short arms of chromosomes of the homoeology group 3. The genetic control is discussed and, according to our results, between 12 and 15 loci, organized in five compound loci, control the endosperm esterases in wheat. Also one modifier gene modifying the mobility of two esterase bands and present in all the cultivars studied is postulated.This work was supported by a personal grant (L. Rebordinos) from the P.F.P.I. and by an institutional grant from the C.A.I.C.Y.T. (PB85-0153)     

Developmental specificity and evolution of the acid phosphatase isozymes of Triticum aestivum and its progenitor species

The tissue and developmental specificities of the acid phosphatase (ACPH) isozymes of Triticum aestivum and its progenitor species T. turgidum and T. tauschii have been determined and compared using the zymogram technique. Tissue and/or developmental variation in relative staining intensity, suggestive of variation in the quantity of active enzyme present, was observed for each of the seven major isozymes expressed. Isozymes homologous to each of the major isozymes of the hexaploid were detected in one or the other of the progenitor species. No difference in the pattern of developmental or tissue specificity was observed between the species for any isozyme. However, ACPH-4, encoded by Acph4, a structural gene linked to chromosome 4A, differs in electrophoretic mobility between T. aestivum and T. turgidum, indicating that divergence has occurred between these species at the Acph4 locus since the origin of the hexaploid. The molecular weight of each of five ACPH isozymes of the hexaploid was determined to be approximately 58,000. This finding, plus the results of the developmental study and the earlier demonstration that the structural genes for six isozymes (including four of those whose molecular weight was determined) are linked to homoeologous chromosomes, provides evidence in support of the suggestion that the ACPH structural genes of hexaploid wheat are homoeologously related.Technical article No. 12233 of the Texas Agricultural Experiment Station. Adapted from a dissertation submitted to the Graduate College, Texas A&M University, by M. A. T. in partial fulfillment of the requirements for the Ph.D. in genetics.     

The genetical control and tissue-specificity of esterase isozymes in hexaploid wheat

A comparative genetic analysis of esterase (E.C.3.1.1.1) isozymes of wheat cultivar Chinese Spring in endosperm, embryo, coleoptile, leaf and root tissues revealed eight sets of isozymes characterised by different tissue specificities, pI ranges and the chromosomal locations of their controlling genes. This data was considered together with previously published work, resulting in a proposed rationalization of nine sets of wheat esterase isozymes. Although this classification included two sets of isozymes controlled by genes on the short arms of homoeologous group 3 chromosomes and three sets on the long arms of the same chromosomes, for which no recombination evidence of genetic distinctness has been obtained among either group, it is argued that the different characteristics of the various sets warrant retention of separate set nomenclatures. Previously unreported esterase genes includeEst-9, a low pI, monomeric, embryo-specific group with controlling genes on chromosomes 3BS and 3DS and two further members ofEs-1,Est-H1 inHordeum vulgare andEst-S ^l1 inAegilops longissima.     

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.     

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).     

Rat liver aldehyde dehydrogenase. II. Isolation and characterization of four inducible isozymes

The purification and properties of 4 inducible cytosolic rat liver aldehyde dehydrogenase isozymes are described. Based on their behavior during purification and their properties, the activities can be grouped into 2 classes. The isozyme inducible in normal liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the tumor-specific isozyme found in hepatocellular carcinomas have apparent molecular weights of 110,000, prefer NADP+ as coenzyme, and preferentially oxidize benzaldehyde-like aromatic aldehydes, but not phenylacetaldehyde. They also have identical pH profiles and responses to effectors. These isozymes differ slightly in isoelectric point and thermal stability. The normal liver phenobarbital-inducible isozyme and the isozyme appearing during the promotion phase of hepatocarcinogenesis appear to be identical. Both have apparent molecular weights of 165,000, are NAD-specific and prefer aliphatic aldehydes. They can oxidize phenylacetaldehyde, but not benzaldehyde-like aromatic aldehydes. They also have identical pH and thermal stability profiles and responses to effectors. While the 4 inducible isozymes share identical subunit molecular weights (54,000) with the normal liver millimolar Km aldehyde dehydrogenases, they are distinctly different enzymatic species. The interrelationships of the various normal liver and inducible rat liver aldehyde dehydrogenases are discussed.