Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Purification and characterization of an estrogen-binding peroxidase from human fetuses.

A peroxidase found under two forms with a molecular weight of 220,000 and 170,000 respectively, was purified from human fetuses. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, gel filtration and hydrophobic interaction chromatography. The purification factor approximated 400. These two forms of peroxidase were found to be immunologically identical as shown when utilizing immunodiffusion. They were able to bind estradiol in the presence of H2O2. This bond resisted to denaturation and solvent extraction therefore suggesting a covalent binding of estradiol to the enzyme.     

Inhibition of adenylate cyclase activity of human thyroid membranes by gangliosides

Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.     

鸡胚成纤维细胞cDNA表达文库的构建

鸡胚成纤维细胞(CEF)是研究鸡传染性法氏囊病病毒(IBDV)的主要细胞材料,而构建CEF的cDNA表达文库是筛选IBDV在CEF中的细胞受体,研究细胞嗜性的基础平台。采用Gateway技术构建CEF的表达文库,避免使用限制性内切酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。该技术将CEF的mRNA分离纯化后,以5′端生物素标记的Oligo(dT)primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×106cfu/mL,文库总容量为1.2×107cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×105cfu/mL,文库总容量为5.5×106cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为研究病毒受体和病毒入侵途径,进一步了解IBDV的致病机理奠定了基础。     

Genetics of the peroxidase isoenzymes in Petunia

Summary Two alleles of the structural gene prxA from Petunia, prxA6 and prxA7, could be identified by their differential temporal expression. The alleles prxA6 and prxA7 code for peroxidases with a similar electrophoretic mobility as the products of the previously described alleles prxA1 and prxA5, respectively. The former two alleles differ in that they have a different temporal expression with regard to the temporal expression of the allele prxA2. Crossing experiments indicated that the mutations involved are (cisacting) internal site mutations. In the case of the allele prxA6, the experiments indicated a difference with respect to the allele prxA1 in responsiveness to the action of a trans-acting factor.     

Fine structural localization of peroxidase in the rat thyroid

Summary The fine structural localization of a peroxidase activity in the rat thyroid follicular epithelial cell was studied by histochemistry at electron microscopic level. The reaction product is recognized chiefly in the cisternae of the elements of granular endoplasmic reticulum and of nuclear envelope. Golgi vesicles or apical small vesicles, mitochondria, and dense granules are sometimes positive for this reaction. The relationship between the fine structural localization of peroxidase and the site of the iodination of thyroglobulin is discussed.     

Neuropeptidergic control of cyclic AMP accumulation in human thyroid cell

Somatostatin (SRIF), cholecystokinin (CCK), gastrin and substance P, as single agents, do not influence baseline cellular cAMP levels in human thyroid cultures. SRIF inhibits TSH-induced cAMP accumulation in human thyroid cell, while CCK, gastrin and substance P do not modify cAMP response to TSH. Vasoactive intestinal peptide (VIP) increases cellular cAMP levels in human thyroid cultures and its effect is additive to increases produced by norepinephrine (NE) and isoproterenol (ISO). Neither SRIF nor the other tested peptides influence adrenergic and VIP-ergic cAMP stimulation.     

Discovery of 342 putative new genes from the analysis of 5'-end-sequenced full-length-enriched cDNA human transcripts

In this work we describe the process that, starting with the production of human full-length-enriched cDNA libraries using the CAP-Trapper method, led us to the discovery of 342 putative new human genes. Twenty-three thousand full-length-enriched clones, obtained from various cell lines and tissues in different developmental stages, were 5'-end sequenced, allowing the identification of a pool of 5300 unique cDNAs. By comparing these sequences to various human and vertebrate nucleotide databases we found that about 40% of our clones extended previously annotated 5' ends, 662 clones were likely to represent splice variants of known genes, and finally 342 clones remained unknown, with no or poor functional annotation. cDNA-microarray gene expression analysis showed that 260 of 342 unknown clones are expressed in at least one cell line and/or tissue. Further analysis of their sequences and the corresponding genomic locations allowed us to conclude that most of them represent potential novel genes, with only a small fraction having protein-coding potential.     

Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin

Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57 000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.     

Genetics of the peroxidase isoenzymes in Petunia

Summary Three electrophoretic variants of the peroxidase b isoenzymes in Petunia have been found. The encoding gene prxB is shown to be located on chromosome I by its linkage with the gene Hfl. Analysis of prxB heterozygotes showed a gradual increase of the electrophoretic mobility of all three PRXb allozymes during development and differential expression in enzyme activity of three prxB alleles. The location of prxB on chromosome I was confirmed by an allelic dosage effect in trisomies I, trisomie segregation and the construction of trisomies I with triple-banded PRXb phenotype. From telotrisomic analysis it was concluded that prxB and Hfl are located on the same arm of chromosome I. The unexpected linkage of prxB and Hfl with the gene Fl in one of the crosses was suggested to be caused by a translocation in line SI, involving the gene Fl.     

Genetics of the peroxidase isoenzymes in Petunia

Summary As detected by starch gel electrophoresis, the fast moving anodal group of peroxidase isoenzymes, the PRXa complex, of a Petunia homozygous for the encoding gene can be made up of one to four bands, depending on the tissue sampled, the age of the tissue and of the plant, and the genetic background. Additional evidence is presented showing that the PRXa complex is encoded by one structural gene, prxA, rather than by tandem duplicated genes. On the basis of electrophoretic variation in Petunia hybrida and related species, five prxA alleles were found. A prxA internal site mutation was found recognized by the absence of recombination between the mutation that affected the temporal programme of the gene and the mutation that altered the mobility of the enzyme. By a three-point test, the gene prxA was located on chromosome III and found to be linked to the genes Mf1 and Ht1 in the order prxA-Mf1-Ht1. The construction of a trisomic III triply heterozygous for prxA confirmed the location of prxA.     

Expression of human immunodeficiency virus genes using baculovirus expression system

The structural protein genes of HIV-1 and HIV-2 have been expressed inSpodoptera frugiperda (SF) cells using baculovirus expression system. The noncoding flanking sequences of HIV structural genes were removed and a putative ribosome binding site was placed in front of the open reading frame of each gene by using crossover linker mutagenesis. The coding sequences of thegag, pol, env, andvif proteins were inserted intoAutographa californica nuclear polyhedrosis virus (AcNPV) so that HIV genes were under the control of the AcNPV polyhedrin promoter. All recombinant AcNPV-infected SF cells express high levels of HIV structural proteins. Detailed strategies of recombinant AcNPV construction for high level protein expression are presented.     

Activation volumes for horseradish peroxidase compound ii reactions

The activation volumes for the reactions of horseradish peroxidase compound II with L-tyrosine. 3-iodo-L-tyrosine. p-aminobenzoic acid and ferrocyanide were determined by using a high-pressure stopped-flow technique at 25°C and pH 7. For the tyrosines, the solvent electrostriction accompanying substrate ionization and H⁺ transfer from the substituted phenol to a basic group of the enzyme can account for the observed negative activation volumes. For p-aminobenzoic acid a simple electron transfer without H⁺ transfer appears to occur. The positive activation volume for ferrocyanide may be explained in terms of electron transfer associated with a large change in electrostriction of the inorganic redox couple.     

Covalent attachment of horseradish peroxidase to the outer surface of liposomes

We describe a method by which horseradish peroxidase may be attached covalently to the surface of liposomes under conditions which permit minimal non-covalent association of the enzyme with the lipids. The coupling method adopted does not allow the formation of homopolymers of liposomes or peroxidase. For phosphatidylethanolamine/phosphatidylcholine and stearylamine/phosphatidylcholine vesicles, minimal disruption of vesicular structure is observed, whilst for phosphatidylserine vesicles, the lipid-protein complex appears to form structures much smaller than 25 nm in diameter. Stearylamine/phosphatidylcholine vesicles have been shown to retain entrapped inulin, and activity measurements for the peroxidase suggest that it is located exclusively on the external surface of the liposome membrane. Peroxidase can be localized histochemically which has permitted the morphological study of the coated liposomes and their interactions with cells.