Ultrathin-layer Isoelectric Focusing Research Articles

Procena uniformnosti jednog lota hibrida kukuruza primenom UPOV morfoloških I proteinskih markera

Monitoring genetic uniformity and identity is very important in maize breeding and seed production. Due to practical reasons, molecular and biochemical methods for the determination of genetic purity and uniformity should reliably reflect genetic differences associated with traditional morphological traits, even when they are not agriculturally important. A comparison of genetic purity and uniformity of one maize hybrid lot, based on morphological UPOV markers and the UTLIEF (Ultrathin-layer isoelectric focusing) method is performed in this research. Tested hybrid samples expressed uniform protein markers profiles, but on the other hand, unsatisfactory uniformity of morphological markers in the field, indicating some laches in seed production. Although the UTLIEF method, standardized by ISTA rules, provides enough accurate distinction between F1 seed and self-pollinated maternal seed, a "clean" isozyme or protein profile will not necessarily correlate with morphological homogeneity. It is most likely that the non-uniformity of the tested hybrid lot originates from the non-uniformity of one of the parental lines. Therefore, to establish where the laches occurred, it is necessary to perform post-control tests with reference samples of maize hybrid and parental lines, as well as, the insight into their official descriptions according to the UPOV descriptor. Thus, it is very important to require the deposition of referent samples of hybrids and varieties whose seed production is allowed in Serbia, as well as to have harmonised protocols for conduction of laboratory and field post-control tests.

Varietal identification of Thai hot chilli using seed protein analysis by ultrathin layer isoelectric focusing technique

Varietal identification of Thai hot chilli using seed protein analysis by ultrathin layer isoelectric focusing technique

Varietal verification of Thai rice using ultrathin layer isoelectric focusing of seed proteins

Varietal verification of Thai rice using ultrathin layer isoelectric focusing of seed proteins

Genetic purity testing of pumpkin hybrid seed by ultrathin-layer isoelectric focusing

Ultrathin-layer isoelectric focusing of seed proteins was used to test the genetic purity of pumpkin hybrid seeds. In the pH range of 5-8, one male marker band and two female marker bands were found in the male and female parental line, respectively. Based on the male marker band and female marker bands, the genetic purity of F1 seeds can easily be determined in the laboratory.

Application of ultrathin-layer isoelectric focusing for genetic purity testing of F1 hybrid seeds in napa cabbage

Application of ultrathin-layer isoelectric focusing for genetic purity testing of F1 hybrid seeds in napa cabbage

Variation in seed protein within and among species of Cucurbita by ultrathin-layer isoelectric focusing

Variation in seed protein within and among species of Cucurbita by ultrathin-layer isoelectric focusing

Identification and purity testing of maize hybrids with one parent in common by ultrathin-layer isoelectric focusing of seed salt-soluble proteins

The objective of this study was to discriminate 11 maize hybrids with one parent in common using ultrathin-layer isoelectric focusing (UTLIEF) of seed salt-soluble proteins in 0.02% NaCl solution in the laboratory. Thirty-two protein bands were found in the pH range of 5.0-7.0, 62.5% of which were polymorphic. Six hybrids with the same male parent and 5 hybrids with the same female parent were differentiated by their patterns and numbers of polymorphic protein bands. The result showed that UTLIEF has the characteristics of high resolution to discriminate close genetic relationships. Meanwhile, the genetic purity of 11 hybrids were tested, and the purities for different hybrids were as follows: Shenyu10, 96%; Shenyu13, 96.7%; Shenyu16, 98%; Shenyu17, 99.3%; Shenyu18, 96%; Shenyu23, 96.7%; Shenyu21, 95.3%; Shenyu22, 97.3%; Shenyu29, 98.7%; Shenyu30, 100%; and Shenyu31, 98%.

Prolamin extraction from high polyphenol seeds of sorghum (Sorghum spp.) and species discrimination and varieties identification by ultrathin-layer isoelectric focusing

The seeds of sorghum (Sorghum spp.) are rich in polyphenols, which cause protein precipitation and coloration of extracts. Because of this precipitation, proteins in seeds of sorghum cannot be effectively extracted using common methods. Here we report two modified methods for the seed protein extraction. The result showed that the method in which the samples were pretreated by polyvinylpyrrolidone (PVPP) before protein extraction (modified method 2) got more prolamine content and can be used for later ultrathin layer isoelectric focusing electrophoresis (UTLIEF) analysis. Seed prolamin variation between two sorghum (Sorghum spp.) species included twelve sorghum varieties and eight sudangrass varieties were analyzed by UTLIEF. The result showed that obvious polymorphisms within and among varieties can be revealed through electrophoresis of sorghum single seed sample. Meanwhile, polymorphisms within species and varieties are concealed when sample was bulk which contains more than 100 seeds. By means of visual evaluation of the presence or absence of the special region bands of electrophogram, sorghum and sudangrass can be discriminated from each other. Furthermore, 10 of 12 sorghum varieties or lines and six of eight sudangrass varieties can be identified successfully according to the unique banding patterns in the discriminated regions which Rf was 0.314 to 0.332 and 0.605 to 0.826, respectively. Key word: Sorghum, species discrimination, variety identification, ultrathin layer isoelectric focusing electrophoresis.

Logo Genetic purity testing of two-line hybrid rice seeds by ultrathin-layer isoelectric focusing of proteins

To test for inbreds among F1 seeds of two-line hybrid rice (Oryza sativa L.) in the laboratory, ultrathin-layer isoelectric focusing (UTLIEF) of seed proteins was used. One female marker band in the photo-thermo-sensitive genic male sterile rice (cv. Peiai 64S) and one common male marker band in the four restorer lines (cv. G67, Peifu, I9-1 and Minkezhan) were found in the pH range of 5-8. Embryoless half-seeds contained the same male marker proteins as whole seeds and could be used for UTLIEF to test inbreds in the laboratory. The corresponding embryo-containing half-seeds could germinate to form seedlings on vermiculite and seedlings could be transplanted into the field for grow-out tests. The genetic purity determined by UTLIEF of proteins from embryoless half-seeds was confirmed by grow-out tests of the corresponding embryo-containing half-seeds. This work shows that UTLIEF of seed proteins could replace grow-out tests to determine F1 genetic purity of two-line hybrid rice. Using half-seeds, the comparison between laboratory and field tests was more objective and accurate than using whole seeds.

Identification of varieties and testing of hybrid purity of rice by ultrathin-layer isoelectric focusing of seed protein

Variety characterization and purity are among the most important requirements of a rice seed lot for sale. Therefore, variety identification, especially through examination of seeds, becomes increasingly important in terms of seed quality control and protection of breeders’ rights. Ultrathin-layer isoelectric focusing (UTLIEF) of seed protein is considered a convenient, quick, cheap, and reliable laboratory method and has been included in the International Seed Testing Association rules for variety verification (ISTA 1999). The cost per kernel of maize purity testing is US$0.14, $0.49, and $1.87 when analyzed by UTLIEF, simplesequence repeat (SSR) DNA markers, and isozymes, respectively (LUFA Augustenberg, unpubl. data). So far, UTLIEF has not been used in variety testing of rice. The objective of this study was to identify 20 rice varieties (Fig. 1) collected by the Seed Testing and Applied Botany, State Institute for Agricultural Testing and Research, Augustenberg, Germany, from different countries by UTLIEF.

Molecular cloning, characterization, and mutagenesis of a pel gene from Pseudomonas syringae pv. lachyrmans encoding a member of the Erwinia chrysanthemi pelADE family of pectate lyases.

The pelS gene from Pseudomonas syringae pv. lachrymans 859 was cloned by heterologous expression in nonpectolytic P. syringae pv. syringae BUVS1, using genomic DNA libraries constructed with two novel broad-host-range cosmid vectors, pCPP34 and pCPP47. Screening of P. syringae pv. syringae transconjugants for the ability to pit pectate media at pH 6.0 and 8.5 yielded several overlapping clones of the same DNA region. Ultrathin-layer isoelectric focusing gels, activity-stained with diagnostically buffered substrate overlays, revealed that this region encoded a single pectate lyase (PelS) with a pI of 9.4. pelS was subcloned from cosmid pCPP5020 and sequenced, revealing it to encode a member of the Erwinia chrysanthemi PelADE family, with highest similarity to Pseudomonas viridiflava PelV. A pelS probe hybridized at high stringency in DNA gel blots with total DNA from P. syringae pv. lachrymans strains 859 and Pla5, P. syringae pv. tabaci, P. syringae pv. phaseolicola, P. syringae pv. glycinea, P. fluorescens (marginalis), P. viridiflava, and Xanthomonas campestris pv. campestris, but not with P. syringae pv. pisi, P. syringae pv. syringae, P. syringae pv. tomato, P. syringae pv. papulans, E. chrysanthemi, or Ralstonia (Pseudomonas or Burkholderia) solanacearum. The PelS sequence revealed an N-terminal signal peptide, whose processing in Escherichia coli was confirmed by protein sequence analysis. PelS was similar to E. chrysanthemi PelE in its substrate preference and ability to reduce the viscosity of pectate and to macerate potato tuber tissue. A pelS:: omega Kmr mutation was marker-exchanged into P. syringae pv. lachrymans Pla5, pelS was also subcloned into the broad-host-range expression vector pML122 under control of the vector nptII promoter, and then transformed into P. syringae pv. lachrymans Pla5 to produce a strain overproducing PelS. Necrotic lesions developed in cotyledons following inoculation with all of the P. syringae pv. lachrymans Pla5 derivatives, regardless of their Pel phenotype. However, only cotyledons infected with pelS+ strains showed evidence of maceration and yielded Pel activity upon extraction. In contrast, pelS+ P. syringae pv. syringae BUVS1(pCPP5020) produced no symptoms in cucumber cotyledons. Thus, PelS in P. syringae pv. lachrymans appears to alter the final symptoms in infected cucumber cotyledons without contributing to pathogenicity or altering host range.

Two dimensional electrophoresis and immunoblotting for the study of ovine whey protein polymorphism.

Different electrophoretic techniques have been applied to the study of ovine whey protein polymorphism. Isoelectric focusing (IEF) over the pH gradient 3.5-9.5 was a suitable method for resolving both genetic variants of ovine beta-lactoglobulin (beta-lg); using this type of IEF, ovine beta-lg A seemed to be defined by a major and a minor 'satellite' band. Approximate isoelectric point, relative molecular mass, amino acid composition and N-terminal sequence of this minor band were determined. A good separation of ovine beta-lg was achieved using ultrathin layer isoelectric focusing (UTLIEF) over the pH gradient 2.5-7.0. However, addition of urea in UTLIEF gels led to some differences in the patterns of the ovine whey proteins when compared with those obtained by IEF on gels not containing urea. Two dimensional electrophoretic techniques provided a considerable separation of ovine whey proteins. Immunoblotting applied to a two dimensional separation permitted the identification of the bands belonging to beta-lg and alpha-lactalbumin fractions.

A simplified method for typing haemoglobin using ultrathin-layer isoelectric focusing

Haemoglobin alleles were identified by isoelectric focusing using ultrathin-layer polyacrylamide gels, containing pH 3.0-10.0 ampholytes. The run parameters were chosen to co-ordinate with other isoelectric focusing methods currently used by the authors' laboratory. The method devised separated the A, F, S, and C alleles.

A method for simultaneous determination of group-specific component (GC) and alpha 2HS-glycoprotein (AHSG) by ultrathin-layer isoelectric focusing and immunoblotting.

A method for simultaneous determination of group-specific component and alpha 2-HS-glycoprotein by ultrathin-layer isoelectric focusing and immunoblotting is described. The advantages of this combined technique are the saving of chemicals and time. The band patterns of both marker proteins were always reproducible.

A rapid and economical method for hybrid purity testing of tomato (Lycopersicon esculentum L.) F1 hybrids using ultrathin‐layer isoelectric focusing of alcohol dehydrogenase variants from seeds

In the cultivated tomato (Lycopersicon esculentum L.), two electrophoretic variants of the enzyme alcohol dehydrogenase (ADH) encoded by the alleles Adh-1+ and Adh-1(1) are found. A rapid and economic method for testing the hybrid purity of tomato F1 seeds, based on the expected presence of Adh-1 alleles, was developed. The method is based on the analysis of the ADH variants by ultrathin-layer isoelectric focusing, pH range 3-10, of crude extracts from imbibed seeds, followed by enzyme activity staining. The isoelectric points (pI's) of the ADH variants were estimated to be 5.5 and 5.7 for Adh-1+ and Adh-1(1), respectively. Using the procedure described and a newly developed sample applicator strip, it is possible for one person to routinely analyze 1152 seeds per day using only a single electrophoresis unit. An investigation of a large number of inbred lines, both experimental and commercial hybrids, together with open-pollinated varieties, showed the potential of the method. Among F1 hybrids, a higher frequency of the Adh-1(1) allele was found than among open pollinated varieties, suggesting that F1 hybrid breeding has resulted in a higher frequency of Adh-1(1) alleles by selection of linked genes.

Plasminogen Polymorphism in Libyans: Description of a New Rare Variant

The genetic polymorphism of human plasminogen (PLG) was investigated in Libyans using wide-scale ultrathin-layer polyacrylamide isoelectric focusing with subsequent immunoblotting. The 2 common alleles, PLG*A and PLG*B, and 4 previously reported rare ones, PLG*A3, PLG*M4, PLG*B1 and PLG*B2, were observed. In addition, a new intermediate rare allele designated PLG*MTripoli (PLG*MT) was encountered. The estimated allele frequencies for the genes PLG*A, PLG*B, PLG*A3, PLG*MT, PLG*M4, PLG*B1 and PLG*B2 were 0.6409, 0.3091, 0.0182, 0.0045, 0.0091, 0.0045 and 0.0136, respectively. The isolated probability of exclusion in cases of disputed paternity among Libyans is 23.3%.

Caractérisation des variants génétiques de l'albumine humaine par focalisation isoélectrique

Normal human serum albumin and bisalbuminic fractions from genetic variants of european origin have been studied by ultrathin-layer isoelectric focusing performed on whole sera or after purification of albumin fractions by affinity chromatography on Blue-Trisacryl. Narrow range ampholytes giving pH gradient between 5 and 8, together with the use of 8 M urea, provided suitable patterns allowing to discriminate the various allotypes. In these conditions, both normal albumin and heterozygous variants were microheterogeneous with several main bands; in the case of normal albumin, four major bands were found, while variants exhibited additional bands differing in number and pI. The position of additional bands comparatively to that of normal albumin was consistent with the electrophoretic behavior of variants. Fast moving allotypes exhibited additional bands with more anodal pIs, whereas slow moving variants were characterized by cathodal additional bands. The number and position of these bands allowed to characterize some variants, when they failed to distinguish between some others, identical patterns being correlated to identical mutations arising at different locations on the albumin molecule. These observations indicate that isoelectric focusing could allow a clear distinction of albumin variants, provided that their mutation were different, or could confirm the occurrence of identical mutation when IEF patterns are indistinguishable. In addition to electrophoretic mobilities at various pH, analytical isoelectric focusing could be a useful technique employed as a second step in the identification of allotypes prior to the determination of the structural change characterizing the variant.

Inbred testing of tomato (Lycopersicon esculentum L.) F1 varieties by ultrathin‐layer isoelectric focusing of seed protein

To test seed lots of tomato F1 hybrid varieties for the presence of undesirable inbred seeds by electrophoresis, a method has been developed on the basis of ultrathin-layer isoelectric focusing. The method is based on the genetic variation of the seed protein PRS-1 which could be visualized by isoelectric focusing of a 5 mM NaCl-soluble seed protein extract in a pH 6-9 gel followed by protein staining. Two genetic variants of the PRS-1 protein, PRS-1+ and PRS-1(1), were found among open-pollinated varieties, as well as among F1 hybrid varieties. The isoelectric points (pI) of the PRS-1 proteins are 7.1 and 6.1 for PRS-1+ and PRS-1(1), respectively. The PRS-1 protein is unique to seed tissue and is located primarily in the embryo. A genetic 1:2:1 segregation of the gene Prs-1 among several F2 populations shows monogenic inheritance. Analysis of commercial F1 hybrid varieties from several seed companies indicated that the Prs-1(1) allele, in contrast to the Prs-1+ allele, is primarily present in gene pools of "Money-maker type" tomatoes. The described method is generally applicable to all tomato F1 varieties that are heterozygous for the gene Prs-1. With the described method one person can routinely analyze more than 768 seeds per day.

Multiple charge isomers of human recombinant interleukin-1β

Human interleukin-1β (rhuIL-1β), obtained by DNA recombinant technology, was radiolabelled. Its isoelectric properties were determined by various analytical techniques such as high-voltage ultrathin layer isoelectric focusing (IEF) and chromatofocusing. The rhuIL- 1β molecule had a molecular mass of 18 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. When examined by IEF on a polyacrylamide gel of 1 mm thickness in the pH range of 3.5–9.5, it was resolved into two broad bands appearing in the pH range of 6.2–5.8 and 5.5–5.2. Each of the two bands was further resolved into multiple bands when electrofocused on (i) a thinner gel of 0.5 mm thickness and (ii) a narrower pH range of 5–8. Upon chromatofocusing in a liquid column, it was possible to isolate various charged components of rhuIL-1β. However, all these components reacted to the antiserum to rhuIL-1β and displayed a molecular mass of 18 kDa suggesting the charge heterogeneity of rhuIL-1β.

Ultrathin-layer isoelectric focusing for bovine serum transferrin phenotyping

We successfully removed sialyl residues from the Tf molecule by neuraminidase treatment and succeeded in phenotyping cattle serum using ultrathin-layer isoelectric focusing