Fred A. M. Asselbergs

Improved resolution of calf lens β-crystallins

Native crystallins isolated from the calf lens cortex were separated by a modified gel filtration procedure. The constituent polypeptides were then separated by gel electrophoresis. It was found, that, when gel filtration was performed in the presence of 1 m-NaCl or alternatively at room temperature, a 60 000 dalton constituent, consisting mainly of βBp, was released from βH leaving a “core βH” of about 180 000 daltons. The βL fraction was partially resolved into several sizes of protein aggregates.

Translation of mouse mammary tumor virus RNA: precursor polypeptides are phosphorylated during processing.

Abstract The synthesis of viral polypeptides of the mouse mammary tumor virus (MMTV) was studied by pulse-labeling of MMTV-producing cells and by translating MMTV virion RNA in vitro , in Xenopus laevis oocytes. Virus-related polypeptides were detected by means of immunoprecipitation withm monospecific antisera against the major viral proteins gp49 and p24 and analysis of the immunoprecipitates on polyacrylamide gels. In pulse-labeled MMTV-producing cells (Mm5mt/c1), a precursor polypeptide of 73,000 daltons was immunoprecipitated by anti-p24 serum (Pr73 gag ). Pr73 gag co-migrated with the 73,000-dalton glycosylated precursor for the envelope proteins (Pr73 env ) immunoprecipitated by anti-gp49 serum. Pr73 gag was, during chase, converted into a 76,000-dalton polypeptide, also reacting with the anti-p24 serum (Pr76 gag ). After prolonged incubation, the mature internal protein p24 was synthesized. Pulse-labeling with 32 P and subsequent chasing revealed that phosphate was incorporated into Pr76 gag and not into Pr73 gag . Isolated virion 70 S RNA of MMTV, microinjected into Xenopus oocytes, gave rise to synthesis of Pr73 gag , Pr76 gag , and p24, all immunoprecipitated by anti-p24 serum, and the viral core proteins p14 and p10, precipitated by polyvalent anti-MMTV serum. 70 S RNA did not instruct synthesis of the viral envelope glycoproteins.

β-Crystallin synthesis in Xenopus oocytes

Abstract Calf lens polyribosomes were microinjected into oocytes of the frog, Xenopus laevis. These cells have the capacity both to synthesize and to assemble α-crystallin polypeptides into high molecular weight aggregates. In contrast, high molecular weight β-crystallin aggregates were not synthesized. β-Crystallin polypeptides were found only in low molecular weight aggregates. Oocytes also did not synthesize the βB1b-polypeptide, which is thought to be derived from a precursor polypeptide, βB1a, by some post-translational modification. Our results support the hypothesis, that the formation of high molecular weight β-crystallin as well as the formation of βB1b are results of ageing processes in the lens.

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes.

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17–20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3′poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as “old” mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of α-globin in all systems, while the presence of the cap analogue m7G(5′)p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5′ terminal “cap”. Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.

CAP analogues do not inhibit mRNA translation in Xenopus laevis oocytes

m7G5’ppp5’N, the cap of eukaryotic mRNA, is thought to serve a dual function. It protects the mRNA against S-exonucleolytic degradation [ 1,2] and it plays a role during the initiation of protein synthesis (reviewed [3] ). Protection against nucleases has been demonstrated using microinjection into Xenopus

The effect of the messenger RNA concentration on the competitive inhibition of translation by cap-analogues.

Inhibition of translation of several mRNA species in a micrococcal nuclease treated reticulocyte lysate by cap analogues was compared with the competition between two mRNAs. Inhibition characteristics were very similar, only complete mRNA molecules inhibited at concentrations 150 times lower than m7 G5′ppp5′G. The inhibition of mRNA translation by cap analogues could be neutralized by the addition of extra mRNA in a manner predicted from the competitive nature of the inhibition by cap analogues.