Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

A Two-Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus

Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two-dimensional electrophoretograms were studied with the aid of computer-assisted image analysis (CAIA). A database map of 32P-labeled proteins was constructed; approximately 500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and isoelectric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+-dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC12 cells.     

Inter-strain Variability of Structural Proteins in Tetrahymena*

SYNOPSIS The high molecular weight proteins found in isolated pellicles of Tetrahymena have been compared in several individual strains within the genus using SDS polyacrylamide gel electrophoresis. Three variants of the B-protein of epiplasm (MW 174,000; 155,000; 145,000) and 2 of the C-protein (MW 140,000; 122,000) were found among the strains examined. No variation was observed in the major kinetodesmal fiber protein (MW 250,000). The variation found between strains in the proteins of a structure which is (as far as we know) the same in all strains indicates a disjunction between evolutionary change at the 2 levels of organization. The taxonomic implications of the observed variation in structural proteins in Tetrahymena are discussed.     

Temperature gradient gel electrophoresis: detection of a single base substitution in the cattle β-lactoglobulin gene

An A in equilibrium with G transition in exon III is known to differentiate alleles A and B of the cattle beta-lactoglobulin (BLG) gene. A BLG exon III fragment containing the transition site was amplified by the polymerase chain reaction. Temperature gradient gel electrophoresis (TGGE) was then used to detect this transition and hence to genotype cattle: the AT base-pair in allele A was readily distinguished from the GC base-pair of allele B. TGGE can be used to detect any single base-pair substitution, and thus is a powerful method of detecting genetic variability.     

Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

Type I interferon genes in cattle: restriction fragment length polymorphisms,gene numbers and physical organization on bovine chromosome 8

Multiple, superimposed Type I interferon (IFN) restriction fragments were resolved following 72–92 h of horizontal electrophoresis. Restriction fragment length polymorphisms (RFLPs) for α IFN (IFNA), β IFN (IFNB), ωIFN (IFNW) and trophoblast IFN (IFNT) genes were identified in Hin dill, Eco RI and Taql digestions from 313 cattle. RFLPs with codominant segregation in cattle pedigrees were considered alleles, and 19 distinct polymorphic Type I IFN loci (5 IFNA, 4 IFNB, 8 IFNW and 2 IFNT) were identified. Allele frequencies and observed heterozygosity values were calculated for each locus and several loci were considered highly informative for linkage analysis. Bovine IFN gene numbers (10 IFNA, 6 IFNB, 20 IFNW and 6 IFNT) were estimated from the number of polymorphic loci plus additional monomorphic hybridizing bands present in Eco RI and Hindlll digestions. Physical linkage of the Type I IFN gene families on bovine chromosome 8 was demonstrated by pulsed field gel electrophoresis (PFGE). Hybridization of two or more IFN probes to similarly sized PFGE fragments suggested the tentative gene family order: IFNA/IFNW-IFNT-IFNB. These studies provide a basis for the development of more detailed genetic and physical maps of the bovine Type I IFNs.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L III. Specific changes in protein synthesis and translatable RNA during turion development

Abstract The developmental process leading to the formation of the abscisic acid (ABA) induced turion of Spirodela polyrrhiza was accompanied by a repression of nucleic acid and protein synthesis. DNA synthesis in the developing lurion (induced by 10⁻⁴mol m⁻³ ABA) was inhibited within 3h of ABA addition, followed by a repression of protein synthesis after 24 h, while RNA synthesis was not inhibited until 3 d. The inhibitory effect of ABA on protein synthesis was found to be selective and the synthesis of several novel proteins appeared to be induced. These effects were specific to ABA-sensitive tissue. The relationship between the changes in the protein and mRNA profiles during the development of the turion was investigated. The rapid general inhibition of protein synthesis at early stages of lurion formation could not be accounted for by the level of translatable mRNA, indicating an effect of ABA at the translational level. The specific alteration to the pattern of in vivo labelled proteins could have resulted, however, from control of the level of specific mRNAs for those particular proteins. Only after 3 d in ABA, when the developing primordium is committed to the turion developmental pathway, is there a total inhibition in the production of mRNA leading to the shutdown of all primary processes and the onset of the irreversible events leading to the dormant state.     

Effect of Calcium and Zinc on Subcellular Distribution, Activity and Thermosensitivity of Superoxide Dismutase in Mnium Affine

In the leafstem moss Mnium affine two superoxide dismutase (SOD) isoforms were found in chloroplasts and two in mitochondria. Four other isozymes were probably cytosolic and two of them had high activity and thermostability and were very sensitive to H₂O₂. On the other hand, one of the mitochondrial isoenzymes was very sensitive to high temperature. The activity and thermosensitivity of SOD was considerably dependent on calcium and zinc ions. The effect was different for the individual isoforms and related to their subcellular distribution. Calcium ions predominantly activated and stabilized one cytosolic and the mitochondrial SODs, while zinc ions influenced one chloroplastic and two cytosolic SODs. This revised version was published online in July 2006 with corrections to the Cover Date.     

ADP-ribosylation of nucleolar proteins in HeLa tumor cells

ADP-ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with ³²P-NAD; then the nucleolar proteins were analyzed by 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE-cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP-ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP-ribosylation.