Cat3, a third gene locus coding for a tissue-specific catalase in maize: Genetics, intracellular location, and some biochemical properties

Summary A new and unique catalase isozyme, CAT-3, has been found in Zea mays. It is encoded in the Cat3 nuclear structural gene which is distinct from the two previously described catalase structural genes, Cat1 and Cat2. The Cat3 gene is both tissue- and time-dependent in its expression, being expressed primarily in young leaves and in the pericarp of nearly mature kernels. Cell fractionation experiments, utilizing epicotyl (coleoptile+primary leaf) and mesocotyl cells, suggest that CAT-3 is associated with the mitochrondria where it may play a role in the alternate oxidase pathway. CAT-3 was purified and characterized with respect to some of its biochemical properties. While CAT-3 differs in some of its properties from CAT-1 and CAT-2, it is similar to these and to other catalases in most respects.Research supported by National Institutes of Health Grant No. GM 22733 to JGS.Paper No. 5255 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC.     

Genetic and biochemical characterization of a Cat2 catalase null mutant of zea mays

Summary In all maize inbred lines examined to date, the Cat2 gene which codes for the CAT-2 catalase is expressed primarily in the scutellum upon seed imbibition. The activity of CAT-2 increases dramatically during the initial four days after germination and subsequently declines. In contrast, we have recently identified and inbred strain (A16) of maize which does not express the Cat2 gene (i.e., the CAT-2 catalase is undetectable). Electrophoretic and immunological analyses indicate that the CAT-2 protein is not present in either an active or inactive form in line A16. Genetic analysis suggests that the absence of CAT-2 expression in line A16 is due to a null allele at the Cat2 gene locus although the possibility of a mutation at a regulatory locus, closely linked to the structural gene has not been excluded. Two other enzymes involved in H₂O₂ metabolism (superoxide dismutase and peroxidase) were also compared in W64A and A16 with no significant differences being observed. Aminotriazole (AT), a known inhibitor of catalase, has been used to simulate the A16 phenomenon by inhibiting catalase activity in line W64A (which has normal expression of CAT-2). AT, in very low concentrations, effectively inhibits the expression of CAT-2 in the scutellum. This inhibition of catalase by AT does not result in changes of the developmental time-course of superoxide dismutase and peroxidase.Research supported by National Institutes of Health Grant No. GM 22733-05 to J.G.S.Paper No. 6601 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Chromosomal location of the catalase structural genes in Zea mays using B-A translocations

Summary B-A translocations have been used to map the catalase genes, Cat1, Cat2, and Cat3 of Zea mays. Cat1 was found to be on the short arm of chromosome 5, 9.1 map units from brittle endosperm (bt ₁). Cat2 was located on chromosome 1S, while Cat3 was located on the distal half of chromosome 1L. There was no linkage between Cat2 and Cat3. The significance of mapping the catalase structural genes is discussed.This research was supported by Grant No. GM22733 from the National Institute of General Medical Sciences, National Institutes of Health, USPHS to JGS.This is Paper No. 6437 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Summary A cDNA library was constructed from poly(A)⁺RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)⁺-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.Abbreviations poly(A)⁺ polyadenylated - DS sodium dodecyl sulfate - D kilodalton - bp base pairs Supported by Research Grant GM 19807 from the United States Public Health Service and by additional funds from the University of California Research Council.     

In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

Analysis of variants affecting the catalase developmental program in maize scutellum

Summary The catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6–67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6–67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.     

Linkage and expression of the Eg locus controlling inclusion of β-glucuronidase into microsomes

In the mouse, one structural gene codes for the amino acid sequence of the -glucuronidase found in both lysosomes and microsomes. The function of a second, independently segregating locus, Eg, is required for the inclusion of -glucuronidase into microsomes. In microsomes, the enzyme, which contains four subunits, is found in a macromolecular complex with up to four additional protein chains; the attachment of these chains is defective in the Eg ⁰ mutant lacking microsomal glucuronidase. The Eg gene has now been linked with Es-1 (1.1±0.3% recombination) on chromosome 8. The -glucuronidase structural gene Gus is on chromosome 5. Thus the gene responsible for processing the polypeptide chain is not genetically linked to the gene directing the synthesisof the enzyme itself.This work was supported in part by the Training Program in Cancer Research, CAO 5016 16, and by U.S. Public Health Service Grant GM 19521.     

Mechanism of suppression in Drosophila. V. Localization of the purple mutant of Drosophila melanogaster in the pteridine biosynthetic pathway

The suppressible eye color mutant purple (pr) of Drosophila melanogaster is known to be unable to synthesize a wild-type complement of pteridine eye pigments. This study measures the reduced levels of drosopterins, sepiapterin, and an unidentified presumed pteridine in pr and pr ^bw. Pteridine analyses in double mutants combining pr with one of three other eye color mutants sepia, Henna-recessive³, and prune², suggest that the metabolic block in pr occurs prior to sepiapterin biosynthesis. Measurements of GTP and GTP cyclohydrolase in pr showed wild-type levels and indicate the metabolic block in pr to be at one of the steps converting dihydroneopterin triphosphate to sepiapterin. Quantitation of pteridines in suppressed purple [su(s) ²; pr and pr; su(pr) ^e3] shows restoration of pteridines to wild-type or nearly wild-type levels.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Isozyme variability in species of the genus Drosophila. I. A multiple allelic isozyme system in Drosophila busckii: Inheritance and general considerations

Starch gel electrophoretic analysis of a triallelic leucine aminopeptidase polymorphism in a laboratory population of Drosophila busckii is described. The three alleles involved are expressed without dominance. A series of single-pair matings revealed an excess of heterozygous types in most segregating families, suggesting selection against the homozygous genotypes. A few cases of heterogeneity among progeny extracted from a single family were the result of matings that produced Mendelian ratios. These few cases had no clear genetic explanation, but there is a suggestion of two isoalleles for the electrophoretically intermediate enzyme.The research reported here was begun at the University of Hawaii and completed at the University of Texas and was supported (in part) by Public Health Service Research Grant No. GM 11609 to W. S. Stone and M. R. Wheeler and by Training Grant No. 2 T1-GM-337-06 and GM 00337-07 to R. P. Wagner et al., from the National Institutes of Health.     

Structure and function of the photosynthetic reaction center fromRhodobacter sphaeroides

The three-dimensional structure of the photosynthetic reaction center fromRhodobacter sphaeroides is described. The reaction center is a transmembrane protein that converts light into chemical energy. The protein has three subunits: L, M, and H. The mostly helical L and M subunits provide the scaffolding and the finely tuned environment in which the chromophores carry out electron transfer. The details of the protein-chromophore interactions are from studies of a trigonal crystal form that diffracted to 2.65-Å resolution. Functional studies of the multi-subunit complex by site-specific replacement of key amino acid residues are summarized in the context of the molecular structure.This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38 and by Public Health Service Grant GM36598.     

Generative cells ofLilium longiflorum possess translatable mRNA and functional protein synthesis machinery

Metabolic labelling with [³⁵S]-methionine demonstrated that generative cells ofLilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [³⁵S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

Catalase-1 (CAT-1) and nucleoside diphosphate kinase-1 (NDK-1) play an important role in protecting conidial viability under light stress in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Recently we reported that Catalase-1 (CAT-1) played an important role in protecting conidial viability in Neurospora crassa, and interacted with a light signal transducer, nucleoside diphosphate kinase-1 (NDK-1). To disclose the functional interaction between CAT-1 and NDK-1 at the genetic level, we created CAT-1 and NDK-1 double mutants, cat-1;ndk-1-1 and cat-1;ndk-1-2, by crossing single mutants of cat-1 ^RIP and ndk-1 ^P72H previously isolated in our laboratory. The double mutant strains grew normally, but showed increased CAT-2 activity. In cat-1 ^RIP , NDK activity was increased when dCDP was used as a substrate. ndk-1 ^P72H , cat-1;ndk-1-1, and cat-1;ndk-1-2 were more sensitive to riboflavin than the wild type and cat-1 ^RIP under strong light (100 μE m⁻² s⁻¹). The pull-down experiment suggests that His-tagged NDK-1 is bound to [³²P]NADH. However, his-tagged NDK-1^P72H was not bound to [³²P]NADH. The double mutants showed much lower conidial viability and lost all conidial germination ability much more rapidly than cat-1 ^RIP , when they were cultured under continuous light for more than 2 weeks. These results indicate that the interaction of CAT-1 with NDK-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress including singlet oxygen, and confirm our former conclusion that reactive oxygen species play an important role in light signal transduction via NDK-1 at the genetic level. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic,ontogenetic, and tissue-specific variation of dipeptidases in Drosophila melanogaster

Three dipeptidases in Drosophila melanogaster are under independent genetic control and their structural genes have been localized, Dip-A to 2R and Dip-B and Dip-C to 3R (Voelker and Langley, 1978; Ohnishi and Voelker, 1981). These enzymes were characterized with respect to their substrate specificities, genetic variability (electrophoretic mobility and quantitative activity level), ontogeny (activity and isozyme pattern), and tissue localization. The dipeptide substrate specificities of DIP-A and DIP-B overlap each other considerably, but do not overlap with DIP-C. In natural populations, DIP-B and DIP-C are essentially monomorphic electrophoretically whereas DIP-A is polymorphic for three allozymes. Both DIP-A and DIP-B show quantitative genetic variation of activity level within an allozyme class. All three enzymes are expressed at all stages in the life cycle, but DIP-A and DIP-B activities vary considerably according to developmental stage and sex of adult. The tissue localizations of DIP-A and DIP-B activities show similar patterns and a nearly ubiquitous occurrence of both enzymes, but with particularly high values in larval and adult midguts and in the adult female reproductive system. These results suggest a general metabolic role for the enzymes, such as regulation of the concentrated pools of amino acids and oligopeptides found in Drosophila tissues.This work was supported by Public Health Service Grant GM 11546.Paper No. 7066 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh.     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

Mutations affecting phenol oxidase activity inDrosophila: quicksilver andtyrosinase-1

The complex enzyme phenol oxidase plays a major role in sclerotization and melanization of cuticle in insects. Production of active enzyme from the inactive proenzyme involves at least six protein components inDrosophila. We examine here the biochemical phenotype of two loci that affect phenol oxidase activity—quicksilver (qs; 1–39.5) andtyrosinase-1 (tyr-1; 2–54.5). Three mutations isolated by different procedures in three different laboratories are alleles at thequicksilver locus. The effects of these mutations have been monitored by means of enzyme assaysin vitro and in polyacrylamide gels and by measurement of catecholamine pool sizes. The activity of all three active enzyme components (A1, A2, and A3) is reduced inqs mutants. The activated enzyme of oneqs allele is thermolabile, while its activator is normal. Deletion and genetic mapping placetyr-1 nearpurple (pr; 2–54.5). Enzyme activity is reduced to 10% of normal but is not thermolabile and the activator is normal. The activity of all three A components is reduced. The diphenol oxidase activity in double mutant combinations shows that these mutations andDox-A2 (Pentzet al., 1986) affect this enzyme in different ways.B.C.B. was supported by National Institutes of Health Research Grant GM31217 and E.S.P. was supported by National Institutes of Health Research Grant GM19242 to T.R.F.W.     

TheN haplotype of the murineβ-glucuronidase gene is altered in both its systemic regulation and its response to androgen induction

A new haplotype of the -glucuronidase gene complex, [Gus]^N, has been characterized following its transfer from the PAC/Cr strain to the standard strain C57BL/6J. TheN haplotype contains a novel structural gene allele which encodes an allozyme differing from all previously characterized allozymes in both size and charge. Altered systemic regulation is exhibited by the [Gus]^N haplotype. Multiple tissues contain levels of GUS protein that are 60±15% those found in the standardB haplotype. The regulatory mechanism for reduction is complex, involving tissue-specific changes in both enzyme synthesis and enzyme turnover. The changes in GUS protein synthesis do not result from changes in GUS mRNA levels. Instead, the amount of mature enzyme formed per mRNA molecule, or translational yield, is altered. These regulatory changes parallel those seen in other systemic regulatory variants of GUS which are also altered in translational yield. A commonality of mechanism among systemic regulatory variants of this gene is suggested. TheN haplotype is also exceptional in the nature of its response to androgenic induction in kidney proximal tubule epithelial cells. The time course for GUS induction consists of a lag period followed by a progressive increase in mRNA, rate of enzyme synthesis, and enzyme activity. For the [Gus]^N haplotype the lag is of an exceptionally short duration and the plateau is of a greater magnitude than for any haplotype previously described.This work was supported by United States Health Service Research Grant GM 31656.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

Characterization of the βγ-crystallin domains of βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima

βγ-CAT is a naturally existing 72-kDa complex of a non-lens βγ-crystallin (α-subunit, CAT-α) and a trefoil factor (β-subunit, CAT-β) that contains a non-covalently linked form of αβ₂ and was isolated from the skin secretions of the toad Bombina maxima. The N-terminal region of CAT-α (CAT-αN, residues 1–170) contains two βγ-crystallin domains while the C-terminal region (CAT-αC) has sequence homology to the membrane insertion domain of the Clostridium perfringens epsilon toxin. To examine the biochemical characteristics of the βγ-crystallin domains of βγ-CAT, CAT-αN, CAT-αC and CAT-β were expressed in Escherichia coli. Co-immunoprecipitation of the naturally assembled βγ-CAT confirmed that the CAT-α and CAT-β complex always exists. Furthermore, recombinant CAT-β bound recombinant CAT-αN. Ca²⁺-binding motifs were identified in CAT-αN, and recombinant CAT-αN was able to bind the calcium probe terbium. However, the conformation of CAT-αN was not significantly altered upon Ca²⁺ binding. βγ-CAT possesses strong hemolytic activity toward human erythrocytes, and treatment of erythrocytes with βγ-CAT resulted in a rapid Ca²⁺ influx, eventually leading to hemolysis. However, in the absence of extracellular Ca²⁺, no significant hemolysis was detected, even though the binding and oligomerization of βγ-CAT in the erythrocyte membrane was observed. Our data demonstrate the binding of CAT-β (a trefoil factor) to CAT-αN (βγ-crystallin domains) and provide a basis for the formation of a βγ-crystallin and trefoil factor complex in vivo. Furthermore, the βγ-crystallin domains of βγ-CAT are able to bind Ca²⁺, and βγ-CAT-induced hemolysis is Ca²⁺ dependent.