Mutations in the ArsA ATPase that restore interaction with the ArsD metallochaperone

The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA–ArsD interface that affect their interactions.     

The isozymic similarity of indoleacetic Acid oxidase to peroxidase in birch and horseradish

The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found.     

Peroxidase Isozymes from Meloidogyne spp. and Their Origin

Two peroxidase isozymes (Ef 0.43 and 0.53) were detected by electrophoretic analysis in homogenates of Meloidogyne arenaria, M. hapla, M. javanica, and M. incognita females reared on tomato. No peroxidase isozymes were detected electrophoretically in homogenates of adult males, preparasitic larvae, or eggs. Peroxidase isozymes from females reared on bean, eggplant, or tobacco differed from those from females reared on tomato. Bean and eggplant populations had a single peroxidase isozyme each, respectively Ef 0.21 and 0.28. No peroxidase isozymes were detected in tobacco populations under the conditions used, although total activity assays did reveal low levels of peroxidase activity in homogenates of tobacco populations. The peroxidase isozymes detected in females reared on tomato or bean appear similar to the peroxidase isozymes present in root-knot galls, adjacent ungalled roots, and roots from uninoculated plants of the corresponding hosts. The probability is discussed that most of the peroxittase activity associated with Meloidogyne spp. females is of host origin.     

Specific aspects of detection of peroxidase isozymes and their genetic control in barley

Identical specimens were separated by electrophoresis in two gels to detect and fix peroxidase isozymes. Both gels were stained by Coomassie brilliant blue for detecting proteins. One gel was previously incubated for detecting peroxidase activity. The differences in electrophoretic patterns between the gels indicate the zones of peroxidase activity. It has been shown that locus Prx 6H, controlling a low-mobility grain peroxidase (PRX 6H), is localized to barley chromosome 6. Two loci, Alb 4H and Alb 7H, controlling the biosyntheses of water-soluble proteins of barley endosperm, were localized to chromosomes 4 and 7. It has been demonstrated that barley species is polymorphic at multiple molecular forms of peroxidase.     

Enzyme activity and electrophoretic pattern of isoenzymes of peroxidase,esterase and alkaline and acid phosphatase in relation to flowering inAmaranthus viridis L. - a quantitative SD plant

Amaranthus viridis is a quantitative SD plant in which inflorescence primordia are initiated under both 24- and 8-h photoperiods after 12 and 10 days, when 8 and 7 leaves are differentiated, respectively. Photoperiod plays a non-determinate role, whereas the maturity of plants linked with the attainment of minimum leaf number is significant and of primary importance in floral induction. This is further confirmed by the more or less identical nature of changes in the total enzyme activity and isoenzyme patterns of peroxidase, esterase and alkaline and acid phosphatase under the two photoperiods. These changes occur once the minimum vegetative growth has been achieved prior to the reproductive transformation, irrespective of the photoperiod, pointing to the activation of a general common pathway of events leading to floral induction.     

The relative specificity of mutagens for the induction of forward mutations inCorynebacterium

Auxotrophic mutants ofCorynebacterium sp. VÚA 9366 used for industrial biosynthesis of some amino acids were induced by UV-light and nitrogen mustard. The mutants were isolated by means of the penicillin technique. It was assumed when comparing mutation spectra after the treatment with both mutagens that the mutant cell gives origin to a clone of genetically identical cells; it was hence possible to express numbers of mutants as corresponding numbers of mutations. Requirements for vitamins, components of nucleic acids and amino acids were studied. It was found that mutations causing requirements for arginine which can be substituted by citrulline or ornithine, requirement for valineisoleucine and components of nucleic acids are significantly more frequent after treatment with UV-light. On the other hand, mutations leading to the requirement for arginine which can be substituted by citrulline were more frequent after treatment with nitrogen mustard.     

Mutations in mice that influence natural killer (NK) cell activity

A series of mutations in mice was tested for splenic NK-cell activity against YAC-1 target cells. Mutations at six loci that reduce NK-cell activity in the homozygous state were identified, including beige (bg), hairless (hr), motheaten (me), obese (ob), steel (Sl) and, to a lesser extent, dominant spotting (W). Motheaten mice displayed the most profound NK-cell deficiency, with NK-cell activity virtually absent. Two mutations, nude (nu) and lymphoproliferation (Ipr), produced elevated NK-cell-mediated lysis. The double homozygous recessivenu/nu bg/bg nude-beige mouse was viable and NK-cell-deficient, with activity slightly higher than that of +/?bg/bg beige littermate controls. Pigmentation mutants related to beige, including pale ears (ep), pearl (pe), and ruby eyes (ru ^2J ) did not dramatically influence NK-cell levels. Unlike the obese gene, other mutations leading to obesity, diabetes (db) and yellow (Asuy), did not impair NK-cell function. The possible site of gene action of these mutants in the NK-cell pathway is discussed.     

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2?±?151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8?±?8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

Isolation and characterization of a gene associated with sulfate assimilation in Sinorhizobium fredii WGF03

Sulfur is an essential element for rhizobia, such as sulfated modified Nod factors and nitrogenase. To investigate the role of sulfur metabolism in Rhizobium-Soybean symbiosis, a transponson random insertional mutants’ library was constructed and a sulfur assimilation-related gene was isolated and characterized. A mutant strain unable to utilized sulfate was screened from 11,000 random insertional mutants of Sinorhizobium fredii WGF03. Sequencing analysis showed that a sulfate assimilation-related gene (cysDN) was inserted by the Tn transponson. Mutants inactivated in cysD and cysN (SMcysDF and SMcysNF) were constructed by homologous recombination using the suicide plasmid pK18mob. The mutants SMcysDF and SMcysNF could no longer utilize sulfate as sulfur source. Phenotype analysis revealed that mutation of cysDN had multiple effects on S. fredii WGF03. Root hair deformation assay showed that the activity of Nod factors secreted by mutants SMcysDR and SMcysNR elicited minimal hair initiation only. Soybean plant tests indicated that the mutant strains delayed 1–2 days to nodulate and exhibited lower nodulation efficiency and symbiotic efficiency than the wild-type strain. The complementary strain of cysD and cysN (HcysDF and HcysNF) could restore the nodulation efficiency.     

Peroxidase isozymes of first internodes of sorghum: tissue and intracellular localization and multiple peaks of activity isolated by gel filtration chromatography

Electrophoretic analyses using Sepraphore III strips indicate the presence of a minimum of five bands of peroxidase activity detectable with o-dianisidine and H₂O₂ in extracts from first internodes of Sorghum vulgare var. Wheatland milo. Three of these isozymes were anodic and two were cathodic forms at pH 8.3. The relative amounts of these forms are compared in zero time and incubated excised internodes, stelar and cortical tissues of internodes, and in other parts of the plant. Localization of these isozymes with respect to walls and cytoplasm was characterized by differential centrifugation after grinding of the internodes and by an in situ extraction of walls by centrifugation after vacuum infiltration. Using the latter in situ method, 32% of the total activity of the fast moving cathodic form was exchanged from the wall after infiltration with 50 mm CaCl₂. Only trace amounts of the other isozymes were localized in the walls of the cortex. The isozymes were eluted as two peaks from columns of Sephadex G-100 and three peaks from Agarose A-15m. Although such groupings may be due to asymmetric molecules and ionic interactions as well as to molecular weight differences, they may indicate associations with complexes or membranes of different cytoplasmic constituents.     

Isozyme Profile During Somatic Embryogenesis and In Vitro Flowering of Bambusa vulgaris

Peroxidase and esterase isozymes were investigated during plant regeneration via somatic embryogenesis in Bambusa vulgaris, The transition of non-embryogenic calli to embryogenic calli, somatic embryo development, germination and subsequent flowering of somatic embryo derived shoots were associated with selective expression or repression of isoforms of peroxidase and esterase. Non-embryogenic callus showed six peroxidase and four esterase bands. During somatic embryogenesis and germination of somatic embryos, some bands were suppressed and new isoforms of peroxidase and esterase appeared. During flowering, in addition to four peroxidase bands, a new unique esterase band ‘a’ appeared. Each developmental stage was thus associated with a definite isozyme profile.     

Interaction of the opaque-2 gene with starch-forming mutant genes on the synthesis of zein in maize endosperm

The combination of opaque-2 with starch-modified or starch-deficient mutants produced a cumulative and synergistic effect, respectively, in regulating zein synthesis. The double mutant, brittle-2 opaque-2, which almost completely prevented the synthesis of Z1 and Z2, had high RNase activity. The possible involvement of RNase in effecting zein synthesis is discussed.     

Analysis of human erythrocyte glucose 6-phosphate dehydrogenase isozymes by isoelectric focusing in polyacrylamide gel

The method of isoelectric focusing in polyacrylamide gel was used to separate G6PD isozymes in crude hemolysates of human, rabbit, and rat erythrocytes. G6PD (B) from erythrocytes of a normal human male donor revealed six bands of activity. Their mean isoelectric points, using pH 3–10 and 5–8 range empholytes, were pI 7.04 for band I, pI 6.60 for band II, pI 6.37 for band III, pI 6.11 for band IV, pI 5.94 for band V, pI 5.79 for band VI. G6PD from rabbit and rat erythrocytes revealed completely different multiple band patterns. The method of isoelectric focusing in polyacrylamide gel is presented as a new way of detecting G6PD isozyme patterns.     

Electrophoretic analysis of plasma membrane proteins of wild-type and differentiation-deficient mutant strains ofPodospora anserina

In the fungusPodospora anserina, themodC mutations inhibit the sexual female organ differentiation. Previous results have suggested that the plasma membrane of this mutant may be altered. Proteins solubilized from highly purified plasmalemma from a wild-type and threemodC mutant strains were studied by one- and two-dimensional electrophoresis. Isoelectric focusing revealed in the wild-type strain about 80 polypeptide spots whose molecular weight ranged from 15,000 to 80,000 daltons; 65% of the spots were between 20,000 and 60,000 daltons, and 60% were in the 6.5–7.5 pH zone. The only difference inprotein noted between wild-type and the threemodC mutants was that the threemodC mutants lacked a plasmalemma protein of M_r=42,000 and of pI=6.7. These results suggest a close relationship between plasma membrane proteins and differentiation in this eukaryotic organism.     

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

Electrophoretic patterns and thermostability of some proteins from heat-hardened wheat

Heat hardening of wheat (Triticum aesticum L.) resulted in increased heat stability in leaf extracts of fraction I protein, malate dehydrogenase and peroxidase. Polyacrylamide disk gel electrophoretic atterns of fraction 1 protein, malate dehydrogenase and peroxidase did not change after the heat hardening. The individual isozymes of malate dehydrogenase and peroxidase were found to possess the different heat stabilities. After heat hardening only some of the isozymes had an increased heat stability.