scholarly journals Protein changes during the asexual cycle of Neurospora crassa.

1985 ◽  
Vol 5 (4) ◽  
pp. 839-848 ◽  
Author(s):  
V Berlin ◽  
C Yanofsky
Keyword(s):  
Two Dimensional ◽  
Protein Profiles ◽  
Wild Type ◽  
Two Dimensional Gel Electrophoresis ◽  
Large Numbers ◽  
Aerial Hyphae ◽  
Reticulocyte Lysate ◽  
Steady State Levels ◽  
Asexual Cycle

A method for synchronizing conidiation and isolating large numbers of cells at discrete stages of conidia development is described. Using two-dimensional gel electrophoresis, we analyzed the protein profiles of mycelia, aerial hyphae, and conidia and observed that the concentration of 14 polypeptides increase and 38 decrease during the asexual cycle. Twelve polypeptides were present in extracts of aerial hyphae or conidia, but not mycelia, suggesting that they may be conidiation specific. The protein profiles of mutants defective in conidiation were also analyzed. Differences were detected in the two-dimensional profiles of protein extracts from fluffy and wild-type aerial hyphae. Polyadenylated RNA isolated from wild-type mycelia and conidiating cultures was translated in vitro in a rabbit reticulocyte lysate. Differences were detected in the polypeptide products specified by the two RNA populations, suggesting that changes in steady-state levels of polyadenylated RNAs also occur during conidiation.

Protein changes during the asexual cycle of Neurospora crassa

1985 ◽  
Vol 5 (4) ◽  
pp. 839-848
Author(s):  
V Berlin ◽  
C Yanofsky
Keyword(s):  
Two Dimensional ◽  
Protein Profiles ◽  
Wild Type ◽  
Two Dimensional Gel Electrophoresis ◽  
Large Numbers ◽  
Aerial Hyphae ◽  
Reticulocyte Lysate ◽  
Steady State Levels ◽  
Asexual Cycle

A method for synchronizing conidiation and isolating large numbers of cells at discrete stages of conidia development is described. Using two-dimensional gel electrophoresis, we analyzed the protein profiles of mycelia, aerial hyphae, and conidia and observed that the concentration of 14 polypeptides increase and 38 decrease during the asexual cycle. Twelve polypeptides were present in extracts of aerial hyphae or conidia, but not mycelia, suggesting that they may be conidiation specific. The protein profiles of mutants defective in conidiation were also analyzed. Differences were detected in the two-dimensional profiles of protein extracts from fluffy and wild-type aerial hyphae. Polyadenylated RNA isolated from wild-type mycelia and conidiating cultures was translated in vitro in a rabbit reticulocyte lysate. Differences were detected in the polypeptide products specified by the two RNA populations, suggesting that changes in steady-state levels of polyadenylated RNAs also occur during conidiation.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides

1993 ◽  
Vol 106 (1) ◽  
pp. 209-218 ◽  
Author(s):  
S.W. James ◽  
C.D. Silflow ◽  
P. Stroom ◽  
P.A. Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Resistance Mutation ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Two Dimensional ◽  
Wild Type ◽  
Tubulin Gene ◽  
Alpha Tubulin ◽  
Tubulin Genes

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

The Culture of Small Aggregates of Amphibian Embryonic Cells in vitro

Development ◽  
1963 ◽  
Vol 11 (1) ◽  
pp. 135-154
Author(s):  
K. W. Jones ◽  
T. R. Elsdale
Keyword(s):  
Pigment Cells ◽  
Two Dimensional ◽  
Embryonic Cells ◽  
Common Procedure ◽  
Large Numbers ◽  
Organ Type ◽  
Typical Cell ◽  
Primary Explant ◽  
Migration Of Cells

A Common procedure in amphibian embryology has been to remove portions from embryos and culture these under conditions in which the large numbers of cells retain a close-knit association, favourable to the differentiation of primitive organs in the explant. It has not, in general, been the aim to employ the primary explant as a source of a two-dimensional outgrowth of cells on the substrate, as in typical cell culture procedures. Because of their inherent migratory tendencies, however, outgrowths of pigment cells are readily obtained from explants of the amphibian neural crest, and these have stimulated the interest of a number of investigators (see Wilde, 1961). Holtfreter (1938, 1946) and Finnegan (1953) have also observed the migration of cells from explants of Urodele embryos. Several investigators have employed cell cultures as opposed to organ type cultures in induction studies, Niu & Twitty (1953), Niu (1958), Barth & Barth (1959) and Becker, Tiedemann & Tiedemann (1959).

scholarly journals Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

2020 ◽  
Vol 58 (1) ◽  
pp. 21-33
Author(s):  
Pingping Shen ◽  
Johnny Dang ◽  
Zerui Wang ◽  
Weiguanliu Zhang ◽  
Jue Yuan ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Wild Type ◽  
Gpi Anchor ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Weights ◽  
Prion Propagation

AbstractAlteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

scholarly journals Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases.

1988 ◽  
Vol 8 (2) ◽  
pp. 993-995 ◽  
Author(s):  
V K Pathak ◽  
D Schindler ◽  
J W Hershey
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Mutant Form ◽  
Two Dimensional Gel Electrophoresis ◽  
Reticulocyte Lysate

The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.

scholarly journals Integrity of mitochondria in a mammalian cell mutant defective in mitochondrial protein synthesis.

1981 ◽  
Vol 90 (1) ◽  
pp. 108-115 ◽  
Author(s):  
K G Burnett ◽  
I E Scheffler
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Two Dimensional ◽  
Mitochondrial Protein Synthesis ◽  
Two Dimensional Gel Electrophoresis ◽  
Translational Machinery ◽  
Sedimentation Behavior ◽  
Rrna Species

A defect in mitochondrial protein synthesis has previously been identified in the respiration-deficient Chinese hamster lung fibroblast mutant V79-G7. The present work extends the characterization of this mutant. A more sensitive analysis has shown that mutant mitochondria synthesize all mitochondrially encoded peptides, but in significantly reduced amounts. This difference is also seen when isolated mitochondria are tested for in vitro protein synthesis. To distinguish between a defect in the translational machinery and a defect in the transcription of mitochondrial DNA, we investigated the synthesis of the 16S and 12S mitochondrial rRNA species and found them to be made in normal amounts in G7 mitochondria. These rRNA species appear to be assembled into subunits whose sedimentation behavior is virtually indistinguishable from that of the wild-type subunits. We also examined the consequences of the defect in mitochondrial protein synthesis on mutant cells and their mitochondria-utilizing techniques of electron microscopy, two-dimensional gel electrophoresis and immunochemical analysis. G7 mitochondria have a characteristic ultrastructure distinguished by predominantly tubular cristae, but the overall biochemical composition of mitochondrial membrane and matrix fractions appears essentially unaltered except for the absence of a few characteristic peptides. Specifically, we identify the absence of two mitochondrially encoded subunits of cytochrome c oxidase on two-dimensional gels and demonstrate a drastic reduction of both cytoplasmically and mitochondrially synthesized subunits of enzyme in immunoprecipitates of G7 mitochondria.

scholarly journals Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

1981 ◽  
Vol 91 (2) ◽  
pp. 352-360 ◽  
Author(s):  
TW McKeithan ◽  
JL Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Cold Temperature ◽  
Two Dimensional ◽  
Multiple Forms ◽  
Cytoplasmic Fraction ◽  
Two Dimensional Gel Electrophoresis ◽  
Brain Tubulin ◽  
Β Tubulin

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.

scholarly journals Allorestricted cytotoxic T cells. Large numbers of allo-H-2Kb-restricted antihapten and antiviral cytotoxic T cell populations clonally develop in vitro from murine splenic precursor T cells.

1985 ◽  
Vol 162 (2) ◽  
pp. 592-606 ◽  
Author(s):  
J Reimann ◽  
D Kabelitz ◽  
K Heeg ◽  
H Wagner
Keyword(s):  
T Cells ◽  
High Probability ◽  
Cytotoxic T Lymphocyte ◽  
Cytotoxic T Cells ◽  
Large Fraction ◽  
Cytotoxic T Cell ◽  
Wild Type ◽  
Large Numbers ◽  
Simplex Virus

Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.

scholarly journals Heterogeneity of guinea-pig caseins synthesized and sequestered by cell-free protein-synthesizing systems

1981 ◽  
Vol 196 (2) ◽  
pp. 567-574 ◽  
Author(s):  
J C Pascall ◽  
A P Boulton ◽  
D Parker ◽  
L Hall ◽  
R K Craig
Keyword(s):  
Dna Sequences ◽  
Milk Proteins ◽  
Two Dimensions ◽  
Two Dimensional Gel Electrophoresis ◽  
Free System ◽  
Free Protein ◽  
Mrna Species ◽  
Reticulocyte Lysate ◽  
Alpha Lactalbumin

1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

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