scholarly journals A Drosophila ESC-E(Z) Protein Complex Is Distinct from Other Polycomb Group Complexes and Contains Covalently Modified ESC

2000 ◽  
Vol 20 (9) ◽  
pp. 3069-3078 ◽  
Author(s):  
Joyce Ng ◽  
Craig M. Hart ◽  
Kelly Morgan ◽  
Jeffrey A. Simon
Keyword(s):  
Germ Line ◽  
Gel Filtration ◽  
Developmental Time ◽  
Polycomb Group ◽  
Sex Combs ◽  
Sex Comb ◽  
Z Protein ◽  
Polycomb Group Complexes

ABSTRACT The extra sex combs (ESC) and Enhancer of zeste [E(Z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence that ESC is phosphorylated in vivo and that this modified ESC is preferentially associated in the complex with E(Z). Modified ESC accumulates between 2 and 6 h of embryogenesis, which is the developmental time whenesc function is first required. We find that mutations inE(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants that express ESC proteins bearing site-directed mutations that disrupt ESC-E(Z) binding in vitro. These mutant ESC proteins fail to provideesc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that ESC phosphorylation normally occurs after assembly into ESC-E(Z) complexes and that it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression.

scholarly journals Structure of a Polycomb Response Element and In Vitro Binding of Polycomb Group Complexes Containing GAGA Factor

2000 ◽  
Vol 20 (9) ◽  
pp. 3187-3197 ◽  
Author(s):  
Béatrice Horard ◽  
Christophe Tatout ◽  
Sylvain Poux ◽  
Vincenzo Pirrotta
Keyword(s):  
Polycomb Group ◽  
Gaga Factor ◽  
Consensus Sequences ◽  
Polycomb Response Element ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Polycomb Response Elements ◽  
Polycomb Group Complexes

ABSTRACT Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

scholarly journals Characterization of Interactions between the Mammalian Polycomb-Group Proteins Enx1/EZH2 and EED Suggests the Existence of Different Mammalian Polycomb-Group Protein Complexes

1998 ◽  
Vol 18 (6) ◽  
pp. 3586-3595 ◽  
Author(s):  
Richard G. A. B. Sewalt ◽  
Johan van der Vlag ◽  
Marco J. Gunster ◽  
Karien M. Hamer ◽  
Jan L. den Blaauwen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Complexes ◽  
Expression Patterns ◽  
Polycomb Group ◽  
Polycomb Group Protein ◽  
Sex Combs ◽  
Group Protein ◽  
Nuclear Domains ◽  
Z Protein

ABSTRACT In Drosophila melanogaster, thePolycomb-group (PcG) andtrithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxGgene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog ofeed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs(esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.

scholarly journals The Drosophila esc and E(z) Proteins Are Direct Partners in Polycomb Group-Mediated Repression

1998 ◽  
Vol 18 (5) ◽  
pp. 2825-2834 ◽  
Author(s):  
Clark A. Jones ◽  
Joyce Ng ◽  
Aidan J. Peterson ◽  
Kelly Morgan ◽  
Jeffrey Simon ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Molecular Data ◽  
Polycomb Group ◽  
Physical Interaction ◽  
Binding Assays ◽  
Amino Acid Region ◽  
Z Protein ◽  
In Vitro Interaction

ABSTRACT The extra sex combs (esc) and Enhancer of zeste [E(z)] proteins are members of the Drosophila Polycomb group (Pc-G) of transcriptional repressors. Here we present evidence for direct physical interaction between the esc and E(z) proteins using yeast two-hybrid and in vitro binding assays. In addition, coimmunoprecipitation from embryo extracts demonstrates association of esc and E(z) in vivo. We have delimited the esc-binding domain of E(z) to an N-terminal 33-amino-acid region. Furthermore, we demonstrate that site-directed mutations in the esc protein previously shown to impair esc function in vivo disrupt esc-E(z) interactions in vitro. We also show an in vitro interaction between the heed and EZH1 proteins, which are human homologs of esc and E(z), respectively. These results suggest that the esc-E(z) molecular partnership has been conserved in evolution. Previous studies suggested that esc is primarily involved in the early stages of Pc-G-mediated silencing during embryogenesis. However, E(z) is continuously required in order to maintain chromosome binding by other Pc-G proteins. In light of these earlier observations and the molecular data presented here, we discuss how esc-E(z) protein complexes may contribute to transcriptional silencing by the Pc-G.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Biochemical and pharmacokinetic characterisation of two PEGylated variants of dipetarudin

2009 ◽  
Vol 102 (09) ◽  
pp. 454-459 ◽  
Author(s):  
Anne Koehler ◽  
Goetz Nowak ◽  
Mercedes López
Keyword(s):  
Gel Filtration ◽  
Pharmacokinetic Profile ◽  
Peripheral Compartment ◽  
Therapeutic Application ◽  
Similar Activity ◽  
Elimination Half ◽  
K 12 ◽  
Extravascular Compartment

SummaryDipetarudin was coupled to polyethylene glycol (PEG)-5000 residues in order to improve its pharmacokinetic profile and to enhance its anticoagulant efficacy. The resulting compounds, mono-and di-PEGylated dipetarudin were purified by gel filtration. Mono-PEGylated dipetarudin exhibited similar activity like its non-conjugated equivalent both in vitro and in vivo. However, di-PEGylated dipetarudin showed longer distribution and elimination half-lives and higher area under the time-concentration curve in comparison with the unmodified inhibitor which may be attributed to decreased renal clearance. Futhermore, ratio k 12/k 21 decreased when the number of PEG chains coupled to dipetarudin increased. It means that the intercompartment transfer of dipetarudin, characterised by a fast distribution and a high retention in the peripheral compartment, is reverted by coupling to PEG. Thus, the transfer of mono-PEGylated dipetarudin between these compartments is similar in both senses and the transfer of di-PEGylated dipetarudin is slower from vascular to extravascular compartment than vice versa. Our results show that di-PEGylated dipetarudin produces a better and longer anticoagulant effect than unmodified dipetarudin which is a desirable attribute for future therapeutic application.

Age-dependent changes in volume and haemoglobin content of erythrocytes in the carp (Cyprinus carpio L.)

1989 ◽  
Vol 141 (1) ◽  
pp. 133-149 ◽  
Author(s):  
W. Speckner ◽  
J. F. Schindler ◽  
C. Albers
Keyword(s):  
Gel Filtration ◽  
Linear Increase ◽  
Human Erythrocytes ◽  
Main Peak ◽  
Red Cell ◽  
Human Haemoglobin ◽  
Cell Fractions ◽  
Haemoglobin Content

Carp erythrocytes were fractionated by angle-head centrifugation which yielded fractions with a linear increase in density. Haematological examinations revealed that the heavier red blood cells of carp had greater volumes (MCV), more haemoglobin (MCH) and higher haemoglobin concentrations (MCHC) than light ones. The same experiments with human red cell fractions yielded a decrease in MCV, constant MCH and an increase in MCHC. Haemoglobin content in individual erythrocytes was also determined by scanning stage absorbance cytophotometry to establish the frequency distribution of the cellular haemoglobin contents. In carp, the distribution was symmetrical with the means increasing with density. No such change with cell density was found in human erythrocytes. Both carp and human erythrocytes incorporated [2-14C]glycine in vitro. After gel filtration, radioactivity was detected in carp, but not in human, haemoglobin fractions. 14C was found in all three haemoglobin fractions, obtained by isoelectric focusing, and was present in the haem and in the globin. [2-14C]glycine-labelled erythrocytes were reinjected into chronically cannulated carp and followed in vivo for several months. With time, the main peak of scintillation counts shifted from red cell fractions of low to high density. This is considered as evidence that density and age of red cells in carp are positively correlated and that erythrocytes can synthesize haemoglobin while circulating in the peripheral blood.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

HEMODIALYSIS OF THE RAT

1966 ◽  
Vol 44 (5) ◽  
pp. 849-859 ◽  
Author(s):  
Sumner M. Robinson ◽  
David A. Hurwitz ◽  
Robert Louis-Ferdinand ◽  
William F. Blatt
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Low Molecular Weight

A technique is described for hemodialysis of either anesthetized or non-restrained rats. In the apparatus the dialysis plates of an autoanalyzer system are used with only minor modification. The efficiency of this method has been evaluated with regard to the clearance of saccharides, both in vitro and in vivo, as well as the extraction of nitrogenous low molecular weight moieties from circulating blood. Approximately 50% of the dialyzable material was obtained in a 1-hour dialysis. Further fractionation of the dialyzate was accomplished by gel filtration (Sephadex G-25).

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