Electrophoretic Analysis Of Polypeptides Research Articles

Impact of iron deficiency and iron re‐supply during the early stages of vegetative development in maize (Zea mays L.)

ABSTRACTThe consequences of iron deficiency and iron re‐supply were evaluated during the early stages of growth and development of young maize plantlets grown hydroponically in the absence of iron. Various parameters, such as fresh and dry weights, and the concentration of chlorophylls, iron, copper, manganese, calcium, magnesium and potassium in leaves, were measured at various times during the first 15 d of culture. Ten‐day‐old maize plantlets grown without iron displayed severe alterations, with a 50% decrease in iron and chlorophyll concentrations in leaves, and serious impairments in mitochondria and chloroplast ultrastructure. In contrast, neither leaf nor root growth, nor other mineral concentrations other than iron were significantly affected at this stage of development. In an attempt to characterize proteins potentially involved in iron nutrition or the adaptative response to iron starvation, comparative 2D‐gel electrophoretic analysis of polypeptides was carried out on soluble and membrane fractions prepared from leaves and roots of iron‐deficient and iron‐sufficient 10‐d‐old maize plantlets. Two polypeptides (11 and 17 kDa, pI of about 6.8) from the microsomal fraction of leaves were found to be repressed under iron‐deficient conditions. Some other polypeptides were found to he induced in microsomal fractions either from roots or leaves. Significant variations in the concentration of most of these polypeptides were observed from one experiment to another. It can be concluded from this study that, at this early stage of maize vegetative growth and development, molecular variations induced by iron deficiency do not affect major house‐keeping proteins, but probably affect very specific events depending on low abundance proteins.

The Effect of Stratification and Thidiazuron Treatment on Germination and Protein Synthesis of Pyrus serotina Rehd cv. Niauli

Breakage of seed dormancy in Pyrus serotina Rhed cv. Niauli is achieved by cold treatment. The chilling time necessary for germination of Pyrus serotina Rhed cv. Niauli seeds was determined, and synthesis of polypeptides during stratification and thidiazuron (TDZ) treatments was investigated. The chilling time required was about 21 d. The longer the chilling time, the higher the germination percentage. The effect of 200 μM TDZ on seed germination was equivalent to 14-21 d of chilling, and TDZ could be used as a substitute for cold treatment. Use of in vivo35 S-methionine label and two-dimensional gel electrophoretic analysis of polypeptides revealed that the patterns of untreated (dormant) and chilled embryonic axes differed significantly in their polypeptide profiles. The difference became conspicuous after prolonged chilling exposure. During 28 d of stratification followed by 5 d of incubation at 25 °C, changes were noted in 35 soluble polypeptides (ESPs) and 38 pellet polypeptides (EPPs) from the embryonic axis. The changes included increase, decrease or newly induced synthesis. Common patterns of changes in soluble and pellet polypeptides of embryonic axes, can be broken down into four categories. The possible functions and roles of these four categories of polypeptides in breaking seed dormancy and germination are discussed. Although the magnitude of polypeptide changes are not as extensive in seeds receiving TDZ treatment as after stratification, similar polypeptide changes were co-induced by the two treatments.

Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum.

Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.

Expression of avian sarcoma virus genes in tumor cells derived from different periods of tumor growth

Avian sarcoma virus (ASV)-induced tumor cells that are derived from regressing neoplasms contain about 75% less pp60 src kinase activity than do progressively-growing tumor cells. Paradoxically, the polyadenylated RNAs extracted from ‘regressor’ cells found in regressing neoplasms are much more productive than those from progressively-growing tumors in a reticulocyte lysate in vitro translation system, as determined both by time course and concentration curve studies. Electrophoretic analysis of polypeptides corresponding to known ASV proteins further suggests that the translation product from the mRNAs of regressing tumor cells contains much more viral protein than does that derived from progressively-growing sarcoma cells. The identity of two of these viral proteins was confirmed by immunoprecipitation of the respective translation products, using a tumor-bearing rabbit serum with specificity for pp60 src and Pr76 gag precursor polypeptides, and showed that much more of pp60 src and Pr76 gag were produced by the mRNAs from regressor than from progressor cells. We further found that ASV genes including genomic and subgenomic species (8.6, 5.0 and 3.2 kb) were present in greater amounts in regressing as opposed to progressively-growing tumor cells. These results support the notion that viral RNAs are accumulated in ‘regressor’ tumor cells.

ACTION OF 6‐METHYL PURINE ON RNA SYNTHESIS: INCORPORATION OF THE ADENINE ANALOGUE INTO CODING SEQUENCES AND POLYADENYLATE TRACTS OF MESSENGER RNA IN CHLORELLA

Summary6‐methyl purine has been tritiated and, by two‐dimensional paper chromatography, an impurity and two degradation products formed during tritiation have been removed. The authentic 6‐methyl purine has been identified by n.m.r. spectroscopy.Chlorella 211‐8p grown autotrophically in the presence of 1 mM tritiated 6‐methyl purine incorporates radioactivity into RNA. Electrophoretic analysis shows that 45% of the incorporated radioactivity is in RNA fragments with less than 90 bases. There is only trace incorporation into 25s, 18s and 5s rRNA but tRNA accommodates 8% of incorporated label.The bulk of radioactivity found in completed RNA is present in a polydisperse size range of molecules which are identified as mRNA. They are recovered as poly(A)+ RNA by oligo (dT) cellulose binding and are of an appropriate size for a messenger function, since the message which contributes the greatest weight to the total population is of 0·65 × 106mol. wt and could accommodate 677 codons, while electrophoretic analysis of polypeptides from Chlorella shows the size which contributes the greatest weight to be 5 × 104 mol. wt, equivalent to 450 amino acids. This correlates well with the capacity of the predominant message, allowing for the presence of a poly(A) tract.6‐methyl purine is incorporated without modification, since two‐dimensional thin layer chromatography of ribosides derived from mRNA showed that radioactivity co‐chromatographed exclusively with authentic 6‐methyl purine riboside.Poly(A) tracts, prepared from mRNA by digestion of the coding sequences, can be separated from the resulting mononucleotides by electrophoresis. Accurate location of the mononucleotides and the selection of authentic poly(A) segments with oligo (dT) cellulose establishes that tracts of 100 to 200 adenine residues contribute the bulk of poly(A) and so corrects an earlier report that tracts in Chlorella are unusually small. The established sizes of poly(A) tracts and complete messenger molecules indicate a predicted distribution of adenine bases between tract and coding sequences which is close to the observed values of 21 and 89% seen in digests of isolated mRNA. A similarity with poly(A) metabolism in mammalian cells is detected by analysis of the number of tracts of each size, which shows that the smaller sizes are numerically predominant.6‐methyl purine is recovered from both poly(A) tract and coding sequences of mRNA after a 1 h period of autotrophic growth with the inhibitor. At the end of this period one in every three adenines in the tract sequences and one in every 10 adenines in the coding sequences has been substituted by 6‐methyl purine. The incidence in messengers being synthesized in the presence of 6‐methyl purine is probably higher since messengers persisting from before exposure to the inhibitor will give a lower average incidence in the total messenger population.Using minimum estimates of 6‐methyl purine substitution it is calculated that coding sequences for a protein of 40000 mol. wt have only a 1 in 5·3 × 1011probability of containing zero 6‐methyl purine and 99·94% of such messages will contain 12 or more substitutions in the coding sequence. It is concluded that there is negligible formation of normal mRNA by Chlorella 211‐8p in the presence of 1 mM 6‐methyl purine.