Showing papers by "Michael Bachmann published in 1987"

Transport of mRNA from nucleus to cytoplasm

Abstract: Publisher Summary Transport of mRNP (messenger ribonucleoprotein) from nucleus to cytoplasm plays an important role in gene expression in eukaryotic cells. This chapter focuses on energy-(ATP)-dependent mRNP transport. Nucleocytoplasmic transport of ribosomal RNA can also be induced by ATP, but also occurs by varying [Ca 2+ ]:[Mg 2+ ]. Release of ribosomal RNPs seems to be accompanied by an expansion of the nucleus. Nucleocytoplasmic transport of mRNA seems to be also distinct from the export of tRNA or the exchange of snRNPs and proteins across the nuclear envelope. Nucleocytoplasmic transport of tRNA seems to involve a facilitated diffusion mechanism, showing saturability and sequence specificity; apparently, it does not depend on ATP. In contrast to the transport of mRNPs through the nuclear pore, which appears strictly vectorial, snRNPs can shuttle between nuclear and cytoplasmic compartments. The nuclear uptake of at least some kinds of U-snRNAs in oocytes seems to depend on their association with proteins stockpiled in the cytoplasm. In contrast to the mRNA export, the import of most proteins into the nucleus seems to be energy-independent, although in some cases nucleotides promote this process. The accumulation of karyophilic proteins in the nucleus may be mediated by specific signal sequences recognizing the intranuclear binding sites of these proteins. However, some proteins seem to migrate into the nucleus via a transport mechanism.

Cytochalasin B selectively releases ovalbumin mRNA precursors but not the mature ovalbumin mRNA from hen oviduct nuclear matrix.

Abstract: Hen oviduct nuclear matrix-bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP-dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP, but inhibited the ATP-induced detachment of mature mRNA. RNA release was also achieved with a monoclonal antibody against actin but not with monoclonal antibodies against tubulin and intermediate filaments. These results suggest that actin-containing filaments are involved in the restriction of immature messengers to the cell nucleus.

Permeability measurements with closed vesicles from rat liver nuclear envelopes.

Abstract: Closed nuclear envelope ghosts in the physiological orientation were prepared from rat liver and nuclei as previously described. Here we report transport measurements of various proteins and ribonucleic acids across the envelope of these vesicles. Histones were accumulated rapidly in the ghosts, in contrast to other, nonnuclear, proteins. Triton X-100 removal of the external nuclear membrane from loaded vesicles, as well as comparative studies with open vesicles, excluded the effects of external adsorption. The exchange rate of histones across the nuclear envelope is strongly depressed in the presence of GTP and GDP. The vesicles contain the translocation mechanism for poly(A)-containing RNA. The translocation of poly(A), messenger RNA, and ribosomal RNA was investigated after entrapment of these nucleic acids during the preparation of vesicles. Our data show that the complete export of only poly(A)-containing RNA from the vesicles is enhanced in the presence of 2 mM ATP. This RNA, as well as poly(A), is transported unidirectionally.

Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.

Abstract: 45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Muller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.

Deoxyspergualin, a potent antitumor agent: further studies on the cytobiological mode of action.

Abstract: Under otherwise identical conditions, deoxyspergualin preferentially inhibits the growth of the T-cell leukemia line L5178y; an effective dose for a 50% inhibition (ED50) of 0.0007μM was determined. A much weaker cytostatic activity was found for murine lymphocytes (ED50: approximately 25 μM) and for CV-1 monkey kidney cells (ED50: 16.3 μM). Deoxyspergualin causes biphasic and differential effects on DNA metabolism of murine T and B lymphocytes. At lower concentrations (0.3 - 5 μM) the [3H]TdR incorporation into nonactivated or lipopolysaccharide-activated lymphocytes is significantly stimulated by the compounds; this effect was not observed with lymphocyte cultures stimulated with concanavalin A. This change of TdR incorporation rates was found to parallel with the variations of DNA polymerase α activity. Deoxyspergualin causes an additive effect together with bleomycin and a significant synergistic cytostatic effect in combination with avarol and avarone. Moreover, it is reported that deoxyspergualin causes neither a selective inhibitory effect on DNA-, RNA- or protein synthesis nor an alteration of the intracellular distribution pattern of the Ro and La antigens. However, detailed enzymic studies revealed that deoxyspergualin reduces DNA polymerase α but not β activity in lymphocytes at the ED50 concentration of this compound. These results support previous documentations that deoxyspergualin is of potential clinical usefulness (a) in treatment of certain tumors and (b) in organ transplantation.

Age-correlated changes in the lupus erythematosus antigens Ro, La, Sm and RNP of the thymus gland.

Abstract: The concentrations of the Sm, RNP, La and Ro antigens of thymus glands from rats were determined depending on the developmental stage of the animals. It was found that lupus antigens strongly decrease after birth. Parallel with this change, the activities of the enzymes DNA polymerase alpha and terminal nucleotidyl transferase in the thymus glands drop during maturation and ageing. These biochemical analyses were supported by immunofluorescence studies using human thymus glands. Moreover, it is documented that a redistribution of Sm and Ro occurs during development. Focusing on Sm, fetal thymus glands contain this antigen predominantly in the cytoplasm, while in immature, mature or old animals Sm is found almost exclusively in the nucleus. From these data we conclude that the amounts of the lupus antigens are additional parameters for the age-correlated function of thymocytes.