Polyploidization-induced genome variation in triticale.

Polyploidization-induced genome variation in triticale (x Triticosecale Wittmack) was investigated using both AFLP and RFLP analyses. The AFLP analyses were implemented with both EcoRI-MseI (E-M) and PstI-MseI (P-M) primer combinations, which, because of their relative differences in sensitivity to cytosine methylation, primarily amplify repetitive and low-copy sequences, respectively. The results showed that the genomic sequences in triticale involved a great degree of variation including both repetitive and low-copy sequences. The frequency of losing parental bands was much higher than the frequency of gaining novel bands, suggesting that sequence elimination might be a major force causing genome variation in triticale. In all cases, variation in E-M primer-amplified parental bands was more frequent in triticale than that using P-M primers, suggesting that repetitive sequences were more involved in variation than low-copy sequences. The data also showed that the wheat (Triticum spp.) genomes were relatively highly conserved in triticales, especially in octoploid triticales, whereas the rye (Secale cereale L.) genome consistently demonstrated a very high level of genomic sequence variation (68%-72%) regardless of the triticale ploidy levels or primers used. In addition, when a parental AFLP band was present in both wheat and rye, the tendency of the AFLP band to be present in triticale was much higher than when it was present in only one of the progenitors. Furthermore, the cDNA-probed RFLP analyses showed that over 97% of the wheat coding sequences were maintained in triticale, whereas only about 61.6% of the rye coding sequences were maintained, suggesting that the rye genome variation in triticale also involved a high degree of rye coding sequence changes. The data also suggested that concerted evolution might occur in the genomic sequences of triticale. In addition, the observed genome variation in wheat-rye addition lines was similar to that in triticale, suggesting that wheat-rye addition lines can be used to thoroughly study the genome evolution of polyploid triticale.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

Comparison of the protein fractions of finger millet

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has been employed to resolve protein subunits of finger millet (Eleusine coracana) varieties according to their MW. These studies have established that varietal differences exist in the protein composition of finger millet varieties. The MW distribution of the protein subunits in the albumin-globulin, prolamin and glutelin fractions show many differences between the parental and cross-bred varieties and these differences are greater in the albumin-globulin and glutelin fractions than in the prolamin fraction. The amino acid compositions of the protein fractions show some differences between varieties.     

Isolation of the smallest component of silk protein.

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.     

A polymorphism of the complement regulatory protein MCP (membrane cofactor protein or gp45-70)

Membrane cofactor protein (MCP or gp45-70) is a recently described regulatory glycoprotein of the complement system which binds iC3 or C3b and is present on human platelets, T cells, B cells, monocytes, and mononuclear-derived cell lines. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MCP migrates as a doublet with an Mr of the upper band of 63,000 and the lower band of 58,000. The same pattern was found on all cell populations in a given individual and was stable over time. In order to further characterize the two band pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MCP was isolated by affinity chromatography or immunoprecipitation from 72 healthy unrelated donors. All individuals expressed both bands and, based on the densitometric scanning of gels, three patterns were noted: upper band predominant in 65%, approximately equal distribution of upper and lower bands in 29%, and lower band predominant in 6%. These observed phenotypic frequencies fit with expectations based on Hardy-Weinberg equilibrium for a two-allele system. Family studies also support this model as none of the 26 offspring had a phenotype that deviated from the expected, assuming an autosomal codominant model of inheritance. These results are consistent with a simple two-allele system that controls the expression of the two bands of MCP.     

Evidence for the involvement of multiple forms of cytochrome P-450 in the occurrence of sex-related differences of drug metabolism in the rat

Multiple forms of cytochrome P-450 in liver microsomes of untreated male and female rats could be divided into several fractions by the use of ω-amino-n-octyl Seph. 4B and DE-52 columns. Male cytochrome P-450 fractions (I-b - I-e) differed from female fractions (I-b - I-e) with respect to absorption peaks in carbon monoxide difference spectra and 7-prop-oxycoumarin O-depropylation activities. Although male and female I-a fractions showed quite similar protein bands on SDS-polyacrylamide gel electrophoresis, some protein bands in other male fractions (I-b - I-e) were absent in corresponding female fractions. Immunochemical examinations using immunoglobulin G raised to cytochrome P-450 purified from untreated male rats also showed that liver microsomes from male and female rats contain different forms of cytochrome P-450. Based on these results, we propose that sex-related differences of drug metabolizing activities in liver microsomes are caused by multiple forms of cytochrome P-450.     

Microsomal target proteins of metabolically activated aromatic hydrocarbons.

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.     

Constitutive HSP70: oligomerization and its dependence on ATP binding.

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.     

A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis

DNA in a denaturing gradient gel electrophoresis (DGGE) band that could not be sequenced after recovery from the gel was cloned into a TA cloning vector and a library was constructed and then 13 clones randomly picked up from the library was sequenced. Although the excised DNA from the DGGE gel showed a single band, the library consisted of several different sequences phylogenetically. This phenomenon was also observed in several other DGGE bands. Therefore, this suggests that a single DGGE band does not always represent a single bacterial strain and a new bias for quantitative analyses based on band intensities has been identified.     

Separation of the Polypeptides of Chlamydia and Its Cell Walls by Polyacrylamide Gel Electrophoresis

The polypeptide composition of Chlamydia was examined by acrylamide gel electrophoresis. When the polypeptide patterns of purified infectious elementary bodies (EB) of C. psittaci meningopneumonitis strain, 6BC strain, and C. trachomatis T'ang strain were compared, no significant differences were observed. The polypeptide patterns of whole EB and reticulate bodies (RB) appeared to overlap, but differences were found. In EB cell walls, nine main and several minor bands of polypeptides were observed in gels containing sodium lauryl sulfate, and the eighth main band from the top of the gel stained positive with periodic acid-Schiff reagent. On the other hand, the polypeptides in bands 3, 6, and 8 in EB cell walls were missing or minor in RB cell walls, and the ninth band was clearly stained by PAS. Band 8 was also stained slightly. Purified subunits, which occur as a lattice structure on the inside layer of EB cell walls but are largely missing in RB cell walls, contained bands 4, 6, and 8, and band 8 was PAS positive. These results indicate that significant polypeptide synthesis or reorganization in the cell walls occurs during the growth cycle.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

Genes controlling prolamin biosynthesis, Pro1 and Pro2, in foxtail millet, Setaria italica (L.) Beauv

Variation and genetic control of seed protein in foxtail millet (Setaria italica) were studied using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Variation in the electrophoregram of the total seed protein were detected in the range between 20 and 30 kDa which is derived from the polymorphism of five prolamin bands. The segregation for each of the bands in F2 seeds showed that these bands are governed by seven alleles at two loci, Pro1 and Pro2, which are not linked to one another. Among 271 local cultivars examined, eight out of ten possible genotypes were observed. With its level of diversity comparable to that of isozymes, the alleles conferring prolamin polymorphism are useful genetic markers.     

Characterisation of rat and human tissue alkaline phosphatase isoforms by high-performance liquid chromatography and agarose gel electrophoresis

Alkaline phosphatase (ALP) exists as several isoenzymes and many isoforms present in tissues and serum. The objective of this study was to separate tissue ALP forms in rats and humans and characterise their properties. The materials for the investigation were intestinal, bone, and liver tissue of rats and commercially available human preparations of tissue ALP. Two methods of separation were used: high-performance liquid chromatography (HPLC) and agarose gel electrophoresis. Using HPLC in the rat tissues, two ALP isoforms in the intestine, one in the bone, and three in the liver were identified. In humans three intestinal, two bone, and one liver isoform were resolved. Electrophoresis showed two ALP activity bands in rat intestine, one wide band in the bone, and three bands in the liver. ALP of human tissues was visualised as a single wide band, with a different mobility observed for each organ. In both species the presence of a form with properties characteristic of the bone isoform of the tissue-nonspecific isoenzyme was observed in the intestine. HPLC offers a higher resolution than electrophoresis with respect to tissue ALP fractions in rats and in humans, but electrophoresis visualises high-molecular-mass insoluble enzyme forms.     

Multiple forms of human renin. Purification and characterization.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.     

Genome merger: from sequence rearrangements in triticale to their elimination in wheat–rye addition lines

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat–rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat–rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale × wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat–rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat–rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.     

Comparison of Staphylococcus spp. cellular and extracellular proteins by SDS-PAGE

In this study, a total of fifteen staphylococcal strains belonging to different species were characterized by whole-cell and extracellular protein profiles using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are presented as dendrograms after quantitative analysis of the band patterns with a computer program. Visual inspection of protein bands and cluster analysis of protein patterns of 15 strains representing 10 Staphylococcus species showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans and other species of Staphylococcus; however, the differences were insufficient for reliable differentiation of Staphylococcus species by the SDS-PAGE method.     

A Biolchemical and Cytochemical Study of Peroxidase Activity in Roots of Pisum sativum: I. A COMPARISON OF DAB-PEROXIDASE AND GUAIACOL-PEROXIDASE WITH PARTICULAR EMPHASIS ON THE PROPERTIES OF CELL WALL ACTIVITY

Peroxidase activity in roots of Pisum sativum has been examinedusing both guaiacol and 3,3'-diaminobenzidine (DAB) as hydrogendonors. Biochemically, differences were observed between thetwo donors with respect to the pH optimum (6–9 and 4–0,respectively), and in response to added NaCl (guaiacol-peroxidasewas unaffected while the DAB-peroxidase was markedly inhibited).Both reactions showed highest specific activity in a high speedsupernatant fraction, and, of nine anionic bands demonstratedby gel electrophoresis with DAB, only six were visible withguaiacol. Histochemically, similar staining patterns were observedwith both donors. Cell wall fractions prepared by bead filtration contained 2%and 3.5% of the total peroxidase and acid phosphatase activitiesrespectively. 50% and 27% of these activities were ionicallybound, as indicated by salt treatment In addition, washing withsalt solutions produced a marked stimulation of peroxidase activityat high salt concentrations: this affect was not observed withthe supernatant peroxidase or with cell wall acid phosphatase.Possible functions of cell wall peroxidase are discussed     

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.     

Genetic aspects of wheat gliadin proteins

Inheritance of gliadin components unique to three different varieties of common wheat (Triticum aestivum L.) was studied in F₁ and F₂ seeds of intervarietal crosses using protein patterns obtained by polyacrylamide gel electrophoresis in aluminum lactate buffer (pH 3.2). The patterns of F₁ seeds of the crosses Cheyenne × Justin and INIA 66R × Justin evidenced all the bands present in the patterns of the parents; band intensities reflected gene dosage levels dependent on whether the contributing parent was maternal or paternal in accordance with the triploid nature of endosperm tissue. Most of the gliadin components examined segregated in accordance with control by a single dominant gene, but in two instances single bands in the one-dimensional electrophoretic patterns segregated in the F₂ as expected if controlled by two genes. A method of two-dimensional electrophoresis was developed that resolved these apparently single bands into two components each, which could segregate independently. Linkage analysis provided evidence of codominant alleles and closely linked genes coding for gliadin protein components in both coupling and repulsion situations. The gliadin protein components seem to be coded for by clusters of genes located on chromosomes of homoeologous groups 1 and 6 in hexaploid wheats.Reference to a company or product name does not imply approval by the U.S. Department of Agriculture to the exclusion of others which may also be suitable.