Chromosomal assignment of gene sequences coding for protein disulphide isomerase (PDI) in wheat

 Three different probes, obtained by PCR amplification and labelling of different segments of a PDI cDNA clone from common wheat, were used to identify and assign to wheat chromosomes the gene sequences coding for protein disulphide isomerase (PDI). One of these probes, containing the whole coding region except for a short segment coding for the C-terminal sequence, displayed defined and specific RFLP patterns. PDI gene sequences were consequently assigned to wheat chromosome arms 4BS, 4DS, 4AL and 1BS by Southern hybridisation of EcoRI- HindIII- and BamHI-digested total DNA of nulli-tetrasomic and di-telosomic lines of Chinese Spring. This probe was also employed for assessing the restriction fragment length polymorphism in several hexaploid and tetraploid cultivated wheats. These showed considerable conservation at PDI loci; in fact polymorphism was only observed for the chromosome 1B fragment. Received: 7 July 1998 / Accepted: 14 August 1998     

New member of the protein disulfide isomerase (PDI) family identified in Amblyomma variegatum tick

Ticks belonging to arthropoda are blood feeding, geographically widespread ectoparasites of mammals, reptiles and birds. Their saliva contains active substances that protect them from host immune attack and allow for transmission of various pathogens during the feeding process. Characterization of tick saliva components can therefore contribute to the development of effective methods for the control of tick-borne diseases.

Here we describe the identification and basic characterization of a gene encoding a 55 kDa protein found in the salivary glands (SG) of Amblyomma variegatum tick. Based on the primary structure and homology to the family of protein disulfide isomerases (PDI; EC 5.3.4.1) the gene was named AvPDI. The 1461 nt long AvPDI open reading frame codes for a 487 amino acid protein. In vitro expressed AvPDI was exclusively localized in the endoplasmic reticulum. RT-PCR and Western blot analysis revealed that AvPDI expression is not restricted to the SG of the tick. More detailed analysis on tissue slides from SG detected an AvPDI specific signal in granular cells of the acini type II and III. Finally, reductase activity of AvPDI was confirmed in an insulin assay. The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.     

Evolutionary features of chondriome divergence in Triticum (wheat) and Aegilops shown by RFLP analysis of mitochondrial DNAs

The first comprehensive analysis was made of restriction fragment length polymorphism (RFLP) of the mitochondrial (mt) DNA of two related genera, Triticum (wheat) and Aegilops. This led to clarification of the nature of mtDNA variability and the inference of the phylogeny of the mitochondrial genomes (=chondriome). Forty-six alloplasmic lines and one euplasmic line of common wheat (2n = 42, genomes AABBDD) carrying plasmons (cytoplasmic genomes) of 47 accessions belonging to 33 species were used. This consisted of nearly all the Triticum and Aegilops species. RFLP analysis, carried out with seven mitochondrial gene probes (7.0 kb in total) in combination with three restriction endonucleases, found marked variation: Of the 168 bands detected, 165 were variable (98.2%), indicative that there is extremely high mtDNA variability in these genera. This high variability is attributed to the variation present in the intergenic regions. Most of the variation was between chondriomes of different plasmon types; only 8 bands (4.8%) between those of the same plasmon types were variable, evidence of clear chondriome divergence between different plasmon types. The first comprehensive phylogenetic trees of the chondriome were constructed on the basis of genetic distances. All but 1 of the polyploids had chondriomes closely related to those of 1 putative parent, indicative of uniparental chondriome transmission at the time of polyploid formation. The chondriome showed parallel evolutionary divergence to the plastome (chloroplast genome). Use of a minimum set of 3 mtDNA probe-enzyme combinations is proposed for tentative plasmon type identification and the screening of new plasmon types in those genera. Received: 20 March 1999 / Accepted: 22 June 1999     

Comparative RFLP mapping of the chlorotoluron resistance gene (Su1) in cultivated wheat (Triticum aestivum) and wild wheat (Triticum dicoccoides)

Chlorotoluron is a selective phenylurea herbicide widely used for broad-leaved and annual grass weed control in cereals. Variation in the response to chlorotoluron (CT) was found in both hexaploid bread wheat (Triticum aestivum L.) and wild tetraploid wheat (Triticum dicoccoides KöRN.). Here, we describe the comparative mapping of the CT resistance gene (Su1) on chromosome 6B in bread and wild wheat using RFLP markers. In bread wheat, mapping was based on 58 F₄ single-seed descent (SSD) plants of the cross between a genotype sensitive to chlorotoluron, ‘Chinese Spring’ (CS), and a resistant derivative, the single chromosome substitution line, CS (‘Cappele-Desprez’ 6B) [CS (CAP6B). In T dicoccoides, mapping was based on 37 F₂ plants obtained from the cross between the CT-susceptible accession B-7 and the resistant accession B-35. Nine RFLP probes spanning the centromere were chosen for mapping. In bread wheat Su1 was found to be linked to α-Amy-1 (9.84 cM) and Xpsr371 (5.2 cM), both on the long arm of 6B, and Nor2 (2.74 cM) on the short arm. In wild wheat the most probable linkage map was Nor2-Xpsr312-Su1-Pgk2, and the genetic distances between the genes were 24.8cM, 5.3cM, and 6.8cM, respectively. These results along with other published map data indicate that the linear order of the genes is similar to that found in T. aestivum. The results of this study also show that the Su1 gene for differential response to chlorotoluron has evolved prior to the domestication of cultivated wheat and not in response to the development and use of chemicals.     

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a′_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.     

Hessian fly-resistance gene transferred from chromosome 4Mv of Aegilops ventricosa to Triticum aestivum

 A new Hessian fly (Mayetiola destructor) resistance gene from Aegilops ventricosa and its transfer to hexaploid wheat is described. The 4D(4M^v) substitution line H-93-33 derived from the cross [(Triticum turgidum H-1-1×Aegilops ventricosa no. 11)×Triticum aestivum H-10-15] was highly resistant to the Spanish population tested. Resistance seemed to be inherited as a single dominant factor in the F₂ generation resulting from a cross of H-93-33 with its susceptible parent (H-10-15). Resistance in Ae. venticosa no. 10 was located on chromosome 4M^v using M^v wheat/Ae. ventricosa addition lines. The resistance gene transferred from Ae. ventricosa no. 11 to H-93-33 (H27) is allelic with respect to that of Ae. ventricosa no. 10 and is non-allelic with respect to the genes H3 and H6 from Monon and Caldwell respectively. The assignment of H27 gene to chromosome 4M^v is further supported by its linkage to a gene encoding isozyme Acph-M^v1, previously located on chromosome 4M^v in the line H-93-33. A new marker from homoeologous chromosome group 4 (Amp-M^v2) present in H-93-33 and the 4M^v addition line is described. Received: 12 October 1996 / Accepted: 22 November 1996     

蛋白质二硫键异构酶的结构及抑制剂研究进展

蛋白质二硫键异构酶(PDI)可催化二硫键的形成、断裂和重排,并促进蛋白质折叠,对稳定蛋白质的三维结构至关重要. PDI的表达或酶活性的失调与一系列疾病如癌症、神经退行性疾病、血栓形成等密切相关.本文综述了PDI结构、与疾病的关系及其抑制剂的研究进展,并指出目前PDI抑制剂存在的问题及未来发展方向,以期为PDI抑制剂的进一步研究提供参考.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Co-expression of human protein disulfide isomerase (hPDI) enhances secretion of bovine follicle-stimulating hormone (bFSH) in Pichia pastoris

Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a superovulation treatment for cows. To increase the production of recombinant bFSH, human protein disulfide isomerase (hPDI) was expressed simultaneously in engineered Pichia strains. The secretion characteristics of bFSH with or without hPDI were examined. The co-expression of bFSH and hPDI is increased to 1.56 mg/l of heterodimer in the culture medium, which is 6-fold higher when compared with the control strain carrying the bFSH gene only. These results may be generally applicable to increase the expression of other glycoprotein hormones in yeast.     

A cereal cyst nematode (Heterodera avenae Woll.) resistance gene transferred from Aegilops triuncialis to hexaploid wheat

 The cereal cyst nematode (Heterodera avenae) is an important root parasite of common wheat. A high level of resistance was transferred to wheat from Aegilops triuncialis (TR lines) using the cross [(T. turgidum×Ae. triuncialis)×T. aestivum]. Low fertility (3–5 viable kernels per plant) was observed during the process but the surviving hybrid plants were highly vigorous. To obtain stable resistant lines further crosses to T. aestivum were performed. The resistance in TR lines seems to be transferred from the C genome of Ae. triuncialis (genomes CCUU). Ae. triuncialis was highly resistant to the two Spanish populations of H. avenae tested, as well as to four French races and two Swedish populations. The histological analysis showed a hypersensitive reaction in the roots of a resistant TR line inoculated with the Ha71 pathotype of H. avenae, whereas well-formed syncytia were observed in the roots of the susceptible control. Resistance to the H. avenae Ha71 pathotype seemed to be inherited as determined by a single dominant factor in the crosses between resistant TR lines and susceptible cultivars. Received: 11 November 1997 / Accepted: 9 December 1997     

Variation in the distribution of a genome-specific DNA sequence on chromosomes reveals evolutionary relationships in the Triticum and Aegilops complex

 The present study analyzed the distribution pattern of the Ae. speltoides–derived repetitive clone pGc1R-1 in the Triticum/Aegilops complex. Fluorescence in situ hybridization analysis showed that clone pGc1R-1 is a S-genome-specific repetitive sequence that hybridized to the S-genome of three species in the section Sitopsis, Aegilops speltoides (S), Ae. longissima (S^l), and Ae. sharonensis (S^sh), but not to Ae. bicornis (S^b) and Ae. searsii (S^s), nor to any other diploid Aegilops species. This clone also hybridized to the very closely related G-genome of T. timopheevii subsp. armeniacum and T. timopheevii ssp. timopheevii, but not to the B-genome of T. turgidum and T. aestivum. Hybridization also was observed in the polyploid Aegilops species, Ae. kotschyi (U^kS^k), Ae. peregrina (U^pS^p), and Ae. vavilovii (X^vaD^vaS^va). Large inter- and intraspecific variations were observed. Our results confirm that the S genome is related more to the S^l and S^sh genomes than to the S^b and S^s genomes; there is a greater affinity between the G and S genomes than between the B and S genomes. Mechanisms to account for the variation in the FISH pattern with different genomes include sequence amplification and deletion. Variation in the distribution of this genome-specific DNA sequence, pGc1R-1, on chromosomes can be used to reveal evolutionary relationships in the Triticum and Aegilops complex. Received April 10, 2002; accepted July 12, 2002 Published online: November 28, 2002 Address of the authors: Peng Zhang, Bernd Friebe (e-mail: friebe@ksu.edu), Bikram S. Gill, Wheat Genetics Resource Center, Department of Plant Pathology, 4024 Throckmorton, Plant Sciences Center, Kansas State University, Manhattan, KS 66506-5502, USA.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

节节麦细胞不同分裂时期和阶段的G—带核型及其变动性分析

采用改良的ASG法获得了中期和3个染色体凝缩程度不同的早中期阶段（分别称为早中期Ⅰ、Ⅱ、Ⅲ）染色体的G-带,并进行了G-带核型和变动性分析。所分析的分裂时期和阶段,每条染色体的全长显示出了密切邻近的多重的带纹,带纹细窄、大小较相近,带间区小,带纹分布较密集而均匀。随着有丝分裂进程推进,染色体的带纹数目减少,早中期Ⅰ、Ⅱ、Ⅲ于中期单倍染色体组的G-带带纹总数分别减少41%、36%、28%,而染色体组的绝对长度分别缩短43%、37%、27%,带数减少幅度与染色体长度缩短的幅度几乎相等。早中期Ⅰ至早中期Ⅱ、Ⅲ和早中期Ⅱ至早中期Ⅲ的带纹减少幅度与染色体长度缩短幅度也基本一致。染色体组中各染色体之间带纹减少和染色体缩短的比例不尽相同,有一定的变幅。早中期Ⅰ、Ⅱ、Ⅲ和中期染色体组中每单位绝对长度的带数（带/μm）分别为2.19、2.22、2.32和2.29,差异不大。对节节麦G-带的特性等问题进行了讨论。     

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2^t and T2, and was considered as part of the same allele at the Glu-D ^t 1 locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2^t and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an ω-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of ω-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F₂ grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii (Glu-D ^t 1 and Gli-D ^t 1) segregated as alleles of the Glu-D1 and Gli-D1 loci of bread wheat. A new locus encoding the ω-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively. Received: 13 October 2000 / Accepted: 18 April 2001     

Comparative analyses of RFLP diversity in landraces of Triticum aestivum and collections of T. tauschii from China and Southwest Asia

 Chinese accessions of Triticum tauschii and T. aestivum L. from the Sichuan white (SW), Yunnan hulled (YH), Tibetan weedrace (TW), and Xinjiang rice (XR) wheat groups were subjected to RFLP analysis. T. tauschii and landraces of T. aestivum from countries in Southwest Asia were also evaluated. For T. tauschii, a west to east gradient was apparent where the Chinese accessions exhibited less diversity than those from Southwest Asia. Compared to the Southwest Asian gene pool, the Chinese T. tauschii was highly homogeneous giving a low frequency of polymorphic bands (16%) and banding patterns (1.33 per probe) with 75 RFLP probe-HindIII combinations. Accessions of T. tauschii from Afghanistan and Pakistan were genetically more similar to the Chinese T. tauschii than those from Iran. Of 368 bands found for 39 Chinese hexaploid wheat accessions with 63 RFLP probe-HindIII combinations, 28.3% were polymorphic with an average of 2.6 banding patterns per probe and 5.0 bands per genotype. The individual Chinese landrace wheat groups revealed less variation than those from Afghanistan, Iran, and Turkey. When classified into country based groups, however, the diversity level over all Chinese landraces was greater than that of some Southwest Asian landraces, especially those from Afghanistan and Iran . The XR wheat group was genetically distinct from the other three Chinese landrace groups and was more related to the Southwest Asian landraces. The TW group was genetically similar to, but more diverse than, the SW and YH groups. The Chinese landraces had a higher degree of genetic relatedness to the Southwest Asian T. tauschii, particularly to accessions from Iran, rather than to the Chinese T. tauschii. ‘Chinese Spring’ was most related to ‘Chengdu-guang-tou’, a cultivar from the SW wheat group. Received: 13 May 1997 / Accepted: 19 September 1997     

RAPDs as molecular markers for the detection of Aegilops markgrafii chromatin in addition and euploid introgression lines of hexaploid wheat

 Aegilops markgrafii contains resistance genes to powdery mildew, leaf rust and stripe rust, and also has high crude protein and lysine contents, which can be useful for wheat improvement. These important traits are localized on different chromosomes. Disomic Triticum aestivum-Ae. markgrafii addition lines and euploid introgression lines showing leaf-rust and powdery mildew resistance were screened with RAPDs to detect chromosome-specific markers which can accelerate the breeding process. RAPD markers for all six available disomic addition lines were obtained. The additional chromosomes B, C, D, E, F and G were identified by three, three, three, two, one and seven primers, respectively. All three chromosome-B-specific RAPD markers demonstrated the presence of alien chromatin in the leaf-rust-resistant 42-chromosome introgression lines as well as in the segregating progeny. The three chromosome-C-identifying primers also demonstrated the presence of that chromosome in powdery mildew-resistant euploid introgression lines. The substitution lines (5A)5C and (5D)5C with different genetic backgrounds for both parents, in comparison to the lines mentioned above, showed the chromosome C-specific band with only two of the three primers. The chromosome F-specific primer and a primer evident on all the Ae. markgrafii chromosomes analysed did not generate the expected fragments on the chromosome F_del addition line, indicating that the markers are located on the deleted part of chromosome F. Received: 20 August 1996 / Accepted 17 January 1997