Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.

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UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1048-1061.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1048-1061

Author(s):

I J Lee ◽

L Tung ◽

D A Bumcrot ◽

E S Weinberg

Keyword(s):

Binding Site ◽

Sea Urchin ◽

Transcriptional Start Site ◽

Sodium Dodecyl ◽

Nuclear Extract ◽

Dnase I ◽

Histone H4 ◽

Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

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Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

Infection and Immunity ◽

10.1128/iai.66.9.4374-4381.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4374-4381 ◽

Cited By ~ 58

Author(s):

John C. McMichael ◽

Michael J. Fiske ◽

Ross A. Fredenburg ◽

Deb N. Chakravarti ◽

Karl R. VanDerMeid ◽

...

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Bacterial Attachment ◽

Vaccine Candidates ◽

Isolation And Characterization ◽

Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

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Characterization of proteins from the cytoskeleton of Giardia lamblia

Journal of Cell Science ◽

10.1242/jcs.59.1.81 ◽

1983 ◽

Vol 59 (1) ◽

pp. 81-103 ◽

Cited By ~ 1

Author(s):

R. Crossley ◽

D.V. Holberton

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulphate ◽

Giardia Lamblia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Molecular Size ◽

Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

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Stimulation of interleukin-6 and prostaglandin E2 secretion from peritoneal macrophages by polymers of albumin

Blood ◽

10.1182/blood.v82.9.2853.2853 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2853-2864 ◽

Cited By ~ 16

Author(s):

E Shacter ◽

GK Arzadon ◽

JA Williams

Keyword(s):

Prostaglandin E2 ◽

Interleukin 6 ◽

Acute Infection ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Polymyxin B ◽

Peritoneal Macrophages ◽

Stimulatory Activity

Abstract Interleukin-6 (IL-6) is a multifunctional cytokine that is elevated in vivo during acute infection, chronic inflammation, and some hematopoietic malignancies. To understand how IL-6 becomes elevated in vivo, it is important to identify factors that can stimulate its secretion from effector cells. We found that commercial preparations of bovine serum albumin (BSA) stimulated murine macrophages to secrete high levels of IL-6. In fact, BSA was at least as potent as bacterial lipopolysaccharide (LPS) in stimulating IL-6 production. Stimulation was clearly visible at concentrations as low as 20 micrograms/mL and reached saturation at 0.5 to 1 mg/mL albumin, at which concentration 1.1 x 10(6) oil-elicited macrophages produced 6,000 +/- 700 B9 units of IL-6 in an overnight incubation. Prostaglandin E2 production was induced by the same concentrations of BSA. Both resident and oil- elicited peritoneal cells were responsive to the albumin. The stimulatory activity did not derive from contamination of the protein with Escherichia coli LPS; when compared directly with LPS, the response to BSA was more rapid, had a higher amplitude, and was not inhibitable by polymyxin B. In addition, macrophages isolated from C3H/HeJ mice, which have an inherited defect in their ability to respond to LPS, secreted IL-6 in response to BSA but not to LPS. The stimulatory activity was stable to heat, mild acid, and reduction/alkylation and copurified with albumin on Cibachron Blue agarose (Sigma, St Louis, MO) and anti-albumin immunoaffinity chromatography. Comparison of different sources and preparations of albumin showed differences in the levels of IL-6-inducing activity; three different lots of commercial fatty acid-free BSA and one lot of polymer-enhanced BSA stimulated IL-6 secretion by more than 100-fold over basal levels whereas other preparations showed more limited activity. A sample of BSA that was active in vitro caused a marked elevation of IL-6 when injected into BALB/c mice, thus demonstrating inflammatory activity in vivo. When the albumin preparations were fractionated by ion exchange and gel filtration chromatography and then analyzed by sodium dodecyl sulfate-gel electrophoresis and Western blot immunoassay, it was found that the IL-6-inducing activity resided in high molecular weight polymers of albumin. The ability of albumin polymers to stimulate IL-6 production represents a novel mechanism for modulation of this cytokine.

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Biosynthesis and regulation of rat α1-inhibitor3, a negative acute-phase reactant of the macroglobulin family

Biochemical Journal ◽

10.1042/bj2450493 ◽

1987 ◽

Vol 245 (2) ◽

pp. 493-500 ◽

Cited By ~ 14

Author(s):

T Geiger ◽

Y Lamri ◽

T A Tran-Thi ◽

F Gauthier ◽

G Feldmann ◽

...

Keyword(s):

Acute Phase ◽

Smooth Endoplasmic Reticulum ◽

Gradient Centrifugation ◽

Primary Cultures ◽

Acute Phase Reactant ◽

Translation System ◽

Side Chains ◽

Phase Reactant

The biosynthesis of rat alpha 1-inhibitor3, a negative acute-phase reactant specifically found in rodents, was studied in vitro in a cell-free translation system from rabbit reticulocytes, in rat hepatocyte primary cultures and in vivo by immunocytochemistry using normal and turpentine-injected rats. By sucrose-gradient centrifugation and subsequent translation of the fractionated RNA in vitro it was found that the mRNA coding for alpha 1-inhibitor3 exhibited a size of about 28S. For the alpha 1-inhibitor3 translated in vitro an apparent Mr of 155,000 was determined. A continuous decrease in the level of alpha 1-inhibitor3 in serum during experimental inflammation induced by turpentine injection was demonstrated by means of quantitative ‘rocket’ immunoelectrophoresis. This result agrees with the observation by immunocytochemistry of a drastic decrease in alpha 1-inhibitor3 levels in hepatocytes 24 h after turpentine injection. At that time alpha 1-inhibitor3 is mainly located in the Golgi apparatus, whereas it is also present in the membranes of the rough and smooth endoplasmic reticulum when normal liver is used. All hepatocytes, but no other hepatic cells, contain alpha 1-inhibitor3. When hepatocyte primary cultures were labelled with [35S]methionine and alpha 1-inhibitor3 was immunoprecipitated from the hepatocyte medium and the supernatant of homogenized cells, two different forms of alpha 1-inhibitor3 were found. The intracellular form of alpha 1-inhibitor3, with an apparent Mr of 173,000, is characterized by oligosaccharide side chains of the high-mannose type. The form of alpha 1-inhibitor3 in the medium exhibited an Mr of 186,000 and carried carbohydrate side chains of the complex type. After labelling hepatocytes with radioactive sugars, [3H]mannose was found in both forms of alpha 1-inhibitor3, whereas [3H]fucose and [3H]galactose were incorporated only into the form found in the medium. In the presence of tunicamycin an unglycosylated alpha 1-inhibitor3 with an apparent Mr of 154,000 was found in cells and in the medium. In a pulse-chase experiment it was shown that inhibition of glycosylation by tunicamycin resulted in a marked delay of secretion of alpha 1-inhibitor3. Thus the oligosaccharide side chains of alpha 1-inhibitor3 play an important role during its transport into the medium.

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UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1048 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1048-1061 ◽

Cited By ~ 14

Author(s):

I J Lee ◽

L Tung ◽

D A Bumcrot ◽

E S Weinberg

Keyword(s):

Binding Site ◽

Sea Urchin ◽

Transcriptional Start Site ◽

Sodium Dodecyl ◽

Nuclear Extract ◽

Dnase I ◽

Histone H4 ◽

Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

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PRELIMINARY CHARACTERIZATION OF AN ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

Acta Endocrinologica ◽

10.1530/acta.0.0620153 ◽

1969 ◽

Vol 62 (1) ◽

pp. 153-164 ◽

Cited By ~ 49

Author(s):

Olav Unhjem ◽

Kjell J. Tveter ◽

Asbjørn Aakvaag

Keyword(s):

Proteolytic Enzymes ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Ventral Prostate ◽

Macromolecular Complex ◽

Sucrose Density Gradient Centrifugation

ABSTRACT Following administration of (1,2-3H)-testosterone to castrated rats or incubation of prostatic tissue with the same steroid, a gel filtration technique has been used for the isolation of a soluble steroid-macromolecular complex from the tissues. Subsequent steroid analyses revealed that 5α-androstan-17β-ol-3-one was the major component associated with the macromolecules both in the in vivo and by in vitro experiments. The complex is destroyed by proteolytic enzymes like trypsin and pronase, but is unaffected by DNase and RNase. The complex is excluded from G-200 as well as P-300 gel beds. By sucrose density gradient centrifugation two macromolecular components were found associated with radioactivity. The largest component had a sedimentation coefficient of 9.3 S and probably corresponds to the macromolecular complex demonstrated by gel filtration, whereas the smaller component had a sedimentation coefficient of 4.5 S and might represent an association of steroids with serum albumin.

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The polypeptide components of scintillons, the bioluminescence organelles of the dinoflagellate Gonyaulax polyedra

Biochemistry and Cell Biology ◽

10.1139/o93-028 ◽

1993 ◽

Vol 71 (3-4) ◽

pp. 176-182 ◽

Cited By ~ 24

Author(s):

Michel Desjardins ◽

David Morse

Keyword(s):

Density Gradient ◽

Density Gradient Centrifugation ◽

Binding Protein ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Close Packing ◽

Gonyaulax Polyedra

Scintillons, the bioluminescence organelles of Gonyaulax polyedra, were purified by isopycnic density gradient centrifugation until only low levels of contaminating chloroplasts and mitochondria were detected by fluorescence and electron microscopy. Purified scintillons catalyzed the luminescent reaction with kinetics identical to those observed during the bioluminescence flash in vivo. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the organelles appeared to contain only two proteins. These proteins were identified as luciferase (135 kilodaltons) and luciferin-binding protein (75 kilodaltons) based on their size and their known functions in the bioluminescence reaction in vitro. The staining of luciferin-binding protein by Coomassie blue was 2.4 ± 0.3 (n = 19) times greater than the luciferase, suggesting that there are four binding protein monomers for every luciferase monomer. A model is proposed for the close packing of the two proteins inside the scintillons.Key words: luciferase, luciferin-binding protein, density gradient centrifugation, dinoflagellate.

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Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3994 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3994-4006 ◽

Cited By ~ 24

Author(s):

J Drawbridge ◽

J L Grainger ◽

M M Winkler

Keyword(s):

Sea Urchin ◽

Messenger Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Embryonic Cells ◽

Filtration Method

Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.

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