Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of β-glycosidases

Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3 portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA-and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a -glycosidase enzyme family comprising both -glycosidases and phospho--glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these -glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between -glycosidases and cellulases.     

The myrosinase gene family in Arabidopsis thaliana: gene organization,expression and evolution

Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1.) is in Brassicaceae species such as Brassica napus and Sinapis alba encoded by two differentially expressed gene families, MA and MB, consisting of about 4 and 10 genes, respectively. Southern blot analysis showed that Arabidopsis thaliana contains three myrosinase genes. These genes were isolated from a genomic library and two of them, TGG1 and TGG2, were sequenced. They were found to be located in an inverted mode with their 3 ends 4.4 kb apart. Their organization was highly conserved with 12 exons and 11 short introns. Comparison of nucleotide sequences of TGG1 and TGG2 exons revealed an overall 75% similarity. In contrast, the overall nucleotide sequence similarity in introns was only 42%. In intron 1 the unusual 5 splice border GC was used. Phylogenetic analyses using both distance matrix and parsimony programs suggested that the Arabidopsis genes could not be grouped with either MA or MB genes. Consequently, these two gene families arose only after Arabidopsis had diverged from the other Brassicaceae species. In situ hybridization experiments showed that TGG1 and TGG2 expressing cells are present in leaf, sepal, petal, and gynoecium. In developing seeds, a few cells reacting with the TGG1 probe, but not with the TGG2 probe, were found indicating a partly different expression of these genes.     

Characterization of a new myrosinase in Brassica napus

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.     

Temporal,cell-specific,and tissue-preferential expression of myrosinase genes during embryo and seedling development in Sinapis alba

The expression of the genes for two types of myrosinase (EC 3.2.3.1), designated MA and MB, during embryo and seedling development was investigated in Sinapis alba L. by in-situ and RNA slot-blot analyses. The expression of MA and MB genes followed similar temporal profiles during embryogenesis, but MB mRNA was present in considerably higher amounts than MA mRNA. In the embryo, both MA and MB genes are activated in cotyledons and axis. The MB genes are preferentially expressed in the cotyledons whereas MA genes are preferentially expressed in the axis. In the developing seedling, MA mRNA was not present in the organs investigated. By contrast, MB mRNA was found in appreciable amounts in hypocotyls, cotyledons and developing leaves. The MB genes seem to be activated preferentially in tissues undergoing rapid cell division and — or cell expansion.Abbreviations DAP days after pollination - MA, MB A type, B type myrosinases in Sinapis alba Anna-Stina Höglund (Uppsala Genetic Center) is gratefully acknowledged for valuable discussion, Anders Gobl (Department of Immunology, Uppsala University) for kindly advice with the labeling of probes and Qingzhu Zhai (Department of Pharmaceutical Biosciences, Uppsala University) for help with seed harvest. This work was supported by grants from the Swedish Research Council for Forestry and Agriculture.     

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.     

Hexose transporters of tomato: molecular cloning,expression analysis and functional characterization

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H⁺/hexose symporter. The K _m of the symporter for glucose is 45 M.     

红树植物杯萼海桑SAMS基因的克隆与生物信息学分析

红树植物杯萼海桑是最耐盐的红树植物之一。S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthetase,SAMS)是S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)生物合成途径的关键酶。SAMS作为一个逆境胁迫响应蛋白在植物的耐盐调控中发挥着极其重要的作用。本文结合杯萼海桑根的转录组注释,根据编码区序列设计引物,通过PCR克隆杯萼海桑SAMS基因的编码区cDNA,并对其进行生物信息分析,为研究杯萼海桑适应逆境的机制奠定理论基础。结果显示PCR扩增了一个长1 182 bp的基因片段,该片段编码由393个氨基酸组成的S-腺苷甲硫氨酸合成酶。同源性比对及进化树分析显示杯萼海桑的SAMS氨基酸序列进化上相对保守。本研究首次从红树林植物杯萼海桑中克隆S-腺苷甲硫氨酸合成酶基因,并获得其编码区序列,为进一步研究杯萼海桑应对逆境胁迫的分子生物学机制与胁迫相关基因调控网络奠定基础。     

The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassiceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Characterization of a Brassica napus myrosinase expressed and secreted by Pichia pastoris

In Brassica napus three different gene families with different temporal and tissue-specific expression and distribution patterns encode myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1). Myrosinases encoded by the MA gene family are found as free and soluble dimers, while myrosinases encoded by the MB and MC gene families are mainly found in large insoluble complexes associated with myrosinase-binding proteins and myrosinase-associated proteins. These large complexes impede purification and characterization of MB and MC myrosinases from the plant. We used Pichia pastoris to express and secrete functional recombinant MYR1 myrosinase from B. napus to allow further characterization of myrosinase belonging to the MB gene family. The purified recombinant myrosinase hydrolyzes sinigrin with a K(m) of 1.0 mM; the specific activity and calculated k(cat)/K(m) were 175 U/mg and 1.9 x 10(5) s(-1) M(-1), respectively. A novel in-gel staining method for myrosinase activity is presented.     

Cloning of differential expression fragments in cauliflower after Xanthomonas campestris inoculation

A near isogenic line (NIL) of Brassica oleracea var. botrytis with resistant and susceptible lines C712 and C731, was used in this study. More than 100 differentially expressed cDNA fragments were obtained from black rot resistant cauliflower plants obtained using cDNA-amplified fragment length polymorphism (AFLP) after infection with the pathogen. Thirteen of these fragments were cloned and subjected to reverse Northern blot analysis using both infected and control cDNA pools. Two positive clones, M2 and M6, were isolated. Northern dot blot and Northern blot analyses showed that M2 was constitutively expressed, whereas M6 contained a gene that was differentially expressed during pathogen infection. Moreover, M6 cDNA fragment was also highly expressed 16–24 h after H₂O₂ treatment. Southern blots showed that M6 is a single copy gene in the cauliflower genome, and encodes a protein with 84 % homology to gene on Arabidopsis chromosome 1. The deduced M6 protein has 91 % positive homology with the Arabidopsis 2A6 protein, which regulates ethylene synthesis; 76 % homology with a 1-aminocyclopropane-1-carboxylate oxidase (ACO), the last enzyme in ethylene synthesis; and 70 % homology with an ethylene induced DNA binding factor. These results suggest that M6 gene fragment is a new H₂O₂ downstream defense related gene fragment and can be induced by Xanthomonas campestris pv. campestris and H₂O₂.     

Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) inArabidopsis thaliana L.: identification of three ERDs as HSP cognate genes

InArabidopsis thaliana L., accumulation of abscisic acid (ABA) began to increase 2 h after plants had been subjected to dehydration stress and reached maximum levels after 10h. Differential hybridization was used to isolate 26Arabidopsis cDNAs with gene expression induced by a 1 h dehydration treatment. The cDNA clones were classified into 16 groups based on Southern blot hybridization, and named ERD (early-responsive todehydration) clones. Partial sequencing of the cDNA clones revealed that three ERDs were identical to those of HSP cognates (Athsp70-1, Athsp81-2, and ubiquitin extension protein). Dehydration stress strongly induced the expression of genes for the three ERDs, while application of ABA, which is known to act as a signal transmitter in dehydration-stressed plants, did not significantly affect the ERD gene expression. This result suggests that these HSP cognates are preferentially responsive to dehydration stress inA. thaliana, and that signaling pathways for the expression of these genes under conditions of dehydration stress are not mainly mediated by ABA. We also discuss the possible functions of these three ERD gene products against dehydration stress.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Molecular cloning and characterization of a novel Dehydrogenase/reductase (SDR family) member 1 genea from human fetal brain

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family of NAD(P)(H)-dependent oxidoreductase. They are defined by distinct, common sequence motifs and show a wide range of substrate specialisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human SDR-type dehydrogenase/reductase gene named Dehydrogenase/reductase (SDR family) member 1 (DHRS1). The DHRS1 cDNA is 1411 base pair in length, encoding a 314-amino-acid polypeptide which has a SDR motif. Northern blot reveals two bands, of about 0.9 and 1.4 kb in size. These two forms are expressed in many tissues. The DHRS1 gene is localized on chromosome 14q21.3. It has 9 exons and spans 9.2 kb of the genomic DNA.     

Classification and expression of a family of cyclin gene homologues in Brassica napus

In order to investigate the role of cell division in plant development, we isolated several plant genes which encode homologues of animal and yeast cell cycle regulators known as cyclins.Through the use of degenerate primers and the polymerase chain reaction (PCR) we isolated a Brassica sequence which showed homology to the cyclin box functional domain found within cyclin proteins. Southern blot analysis indicated that Brassica napus has a large number of genes containing cyclin box-related sequences. This was further supported by the isolation of cyclin box sequences from six different genomic clones. In addition, we have isolated two different cyclin cDNA clones, BnCYC1 and BnCYC2, from a Brassica napus shoot apical cDNA library. Both of the cDNA clones contain a destruction box regulatory domain similar to animal mitotic cyclins.Northern blot analysis using BnCYC2 shows mRNA levels which correlate well with the level of cell division in various tissues. Messenger RNA abundance was highest in 1–3 mm leaves, root tips and shoot apices. The mRNA detected using BnCYC1 was restricted to young leaves and the shoot apex, suggesting divergent, organ-specific roles for cyclin family members. The results demonstrate that the plant cyclin gene family is more extensive than previously demonstrated and consists of genes expressed in all dividing tissues as well as a subset of developmentally specific members.     

Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins

Summary cDNA clones were isolated from tissue specific cDNA libraries of barley and maize using as a probe the cDNA of the maize gene C1, a regulator of anthocyanin gene expression. C1-related homology for all of the four cDNAs characterized by sequence analysis is restricted to the N-terminal 120 amino acids of the putative proteins. This region shows striking homology to the N-proximal domain of the myb oncoproteins from vertebrates and invertebrates. Within the myb proto-oncogene family this part of the respective gene products functions as a DNA binding domain. Acidic domains are present in the C-proximal protein segments. Conservation of these sequences, together with the genetically defined regulator function of the C1 gene product, suggest that myb-related plant genes code for trans-acting factors which regulate gene expression in a given biosynthetic pathway.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.