Translation of beta-tubulin mRNA in vitro generates multiple molecular forms.

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

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RNA turnover in Trypanosoma brucei.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1242 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1242-1249 ◽

Cited By ~ 57

Author(s):

B Ehlers ◽

J Czichos ◽

P Overath

Keyword(s):

Protein Synthesis ◽

Trypanosoma Brucei ◽

Half Life ◽

De Novo ◽

State Level ◽

Surface Glycoprotein ◽

Beta Tubulin ◽

Fold Reduction ◽

Tubulin Mrna

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

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Multiple Molecular forms of Factor VIII Antigen in Normal Individuals and von Willebrand’s Disease Patients

10.1055/s-0039-1682471 ◽

1977 ◽

Cited By ~ 1

Author(s):

T. Zimmerman ◽

J. Kimball ◽

T. Edgington ◽

C. Abildgaard

Keyword(s):

Factor Viii ◽

Molecular Forms ◽

Type I ◽

Type Ii ◽

Von Willebrand's Disease ◽

Von Willebrand’S Disease ◽

Multiple Molecular Forms ◽

Normal Individuals ◽

Factor Viii Antigen

Factor VIII antigen is present in normal individuals in multiple molecular forms which can be separated according to size. The smaller forms have little or no ristocetin co-factor activity. In order to evaluate the possibility that Factor VIII antigen forms of large size may be an artifact of in vitro aggregation, we have ultracentrifuged plasma on a 20% sucrose cushion at 37 C for 10 min at 254,000 xg (peak). The rate of clearing of Factor VIII antigen was compared to that of other plasma proteins. The results indicate that Factor VIII-related antigen forms of high S exist even when plasma is maintained at physiological temperature and analyzed with minimal delay, suggesting that these larger molecular forms also exist as such in vivo.In von Willebrand’s disease (vWd) all forms of Factor VIII antigen may be present but decreased in quantity (Type I) or large forms may be missing with smaller forms present in normal or increased quantities (Type II). Factor VIII antigen was isolated from plasma of three patients with Type I and three patients with Type II vWd by counter immunoelectrophoresis. The Factor VIII antigen was then reduced and electrophoresed on SDS-containing Polyacrylamide gels. The presence of carbohydrate was evaluated by staining with perchloric acid-Schiff’s reagent (PAS). The 210,000 MW Factor VIII antigen subunit from each patient was PAS-positive. Though subtle changes in carbohydrate content or composition could not be evaluated by this technique, a total defect of glycosylation is unlikely in this sample of vWd patients.

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Multiple molecular forms of avian aldolases. III. Tissue distribution and properties of the predominant isozymes from chicken (Gallus domesticus)

Canadian Journal of Biochemistry ◽

10.1139/o69-190 ◽

1969 ◽

Vol 47 (12) ◽

pp. 1187-1194 ◽

Cited By ~ 4

Author(s):

Ronald R. Marquardt

Keyword(s):

Tissue Distribution ◽

Heart Tissue ◽

Isozyme Pattern ◽

Gallus Domesticus ◽

Molecular Forms ◽

Molecular Weights ◽

Aldolase C ◽

Multiple Molecular Forms ◽

Aldolase A

The distribution of chicken (Gallus domesticus) tissue (muscle, oviduct, heart, brain, kidney, and liver) aldolase isozymes was established electrophoretically. Each tissue, being composed of varying proportions of six isozymes, has a distinct isozyme pattern. Brain, oviduct, and heart tissue possess five isozymes of aldolase: 1, 2, 3, 4, and 5. Leg and breast muscle of adults possess aldolase 5. Kidney and liver predominantly possess aldolase 6 with traces of isozymes 1–5. Aldolase 1 corresponds to aldolase C of mammals, 5 to aldolase A, and 6 to aldolase B.Antibodies to 5 react with 2, 3, and 4, but not with 1 and 6. Aldolase 5 hybridizes in vitro with 1 to form 2, 3, and 4. Aldolase 6 also hybridizes in vitro with 1 to form three intermediate hybrids, but these hybrids have not been detected in tissues. These observations imply that aldolase 1 (C) and 5 (A) are frequently synthesized simultaneously in the cell with the formation of the 2, 3, and 4 hybrids. Aldolase 6 (B), in contrast, may not be accompanied by the synthesis of other aldolases in the same cell. Pure preparations of aldolases 1, 5, and 6 were found to have similar molecular weights but different enzymatic properties.

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