Association of the transformed glucocorticoid receptor with a cytoskeletal protein complex

In a recent paper we described a system in which glucocorticoid receptors associate with particulate complexes containing tubulin [Cancer Res. 49 (1989) 2222s–2229s]. When L cell cytosol is mixed with a microtubule stabilizing buffer and heated to 37°C, the receptor becomes associated with a complex that can be centrifuged out of solution at 150,000 g. In this work we show that the glucocorticoid receptor—cytoskeletal protein complex forms in a temperature and glutamate-dependent manner. Molybdate does not affect generation of the cytoskeletal protein complex but it inhibits association of the receptor with the complex. This suggests that transformation of the receptor to its DNA-binding form is required for interaction with the cytoskeletal complex. Colchicine has no effect on generation of the particulate complex or on the association of receptor with it, suggesting that formation of the complex does not represent a classic in vitro process of tubulin polymerization.     

Phylogenic relationships of the amino acid sequences of prosome (proteasome, MCP) subunits

Prosomes [or proteasomes, Multi-Catalytic Proteinase (MCP)] are multisubunit protein complexes, found from archaebacteria to man, the structure of which (a 4-layer cylinder) is remarkably conserved. They were first observed as subcomplexes of untranslated mRNP, and then as a multicatalytic proteinase with several proteolytic activities. A number of sequences from subunits of these complexes are now available. Analysis of the sequences shows that these subunits are evolutionarily related, and reveals three highly conserved amino acid stretches. Based on a phylogenic approach, we propose to classify the sequenced subunits into 14 families, which fall into two superfamilies, of the - and -type. These data, together with several recently published observations, suggest that some subunits may be interchangeable within the complexes, which would thus constitute a population of heterogenous particles.     

Characterization of the chromatin structure in the upstream region of the chicken alpha-globin gene domain

The distribution of DNase I hypersensitive sites upstream of the chicken -globin gene cluster was studied. A group of hypersensitive sites with a complex pattern of tissue specificity, including erythroid-specific elements, was found at a distance of 11.5–14.5 kb upstream of the gene, the first gene in the cluster. The observations indicate that this area, located upstream of the block of AT-rich sequences and MAR sites (at –8 kb) and upstream of the site of permanent DNA attachment to the nuclear matrix (–3 kb), still belongs to the domain of the -globin genes.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.     

Characterization of pre-messenger-RNA-containing nuclear ribonucleoprotein particles from avian erythroblasts.

Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule.     

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.