Ribosomal proteins synthesis and exchange in the absence of 28-S and 18-S ribosomal RNA synthesis in L5178Y cells.

The effect of the adenosine analogue toyocamycin on ribosomal proteins synthesis and assembly within ribosomal particles was investigated in the murine cells, L5178Y. The analogue was used for periods not exceeding 5 h, at a concentration which permits the synthesis of ribosomal precursor RNA but inhibits the maturation process. The following observations were made: 1. Ribosomal proteins, synthesized de novo in the presence of the drug, were associated with toyocamycin-containing 45-S pre-rRNA in preribosomal-like 80-S ribonucleoproteins which accumulated in the nucleolus. Two-dimensional electrophoresis revealed a full protein complement of these particles, although minor discrepancies were observed in the relative proportions of a limited number of polypeptides. 2. In the absence of 28-S and 18-S rRNA formation, a surprisingly high proportion of newly synthesized ribosomal proteins were incorporated into high-salt washed ribosomal subunits. The extent of individual protein exchange as well as their apparent turnover rates were markedly heterogeneous. Most of these exchangeable proteins were shown to be labeled rapidly in ribosomal subunits of normal cells. Some alternative interpretations of these results are discussed.

Influence of toyocamycin on the assembly and processing of preribosomal ribonucleoproteins in the nucleolus of mammalian cells.

The adenosine analogue toyocamycin inhibits the maturation of ribosomal RNA, but permits the synthesis of other RNA species, including 45 S preribosomal RNA. In this work, the dose vs. response analysis of rRNA processing upon toyocamycin treatment of L5178Y cells is studied. It is shown that the latter steps of rRNA processing are more affected than the earlier. The mechanism responsible for the lack of conversion of toyocamycin-containing 45 S RNA into mature rRNA has not yet been elucidated. In order to investigate whether protein factors are involved in this mechanism, the effects of toyocamycin on the ability of preribosomal RNA to bind proteins and on the assembly of nucleolar preribosomes are investigated. The analogue allows the formation and the accumulation, in the nucleolus, of ribonucleoprotein complexes which contain 45 S RNA and newly synthesized proteins, but cannot be converted to mature ribosomal subunits. These complexes are not clearly distinguishable from 80 S particles synthesized in the absence of toyocamycin, with respect to their sedimentation rate in linear sucrose gradients, to their protein/RNA ratio and to their density measured in metrizamide gradients.

Kinetic studies on ribosomal proteins assembly in preribosomal particles and ribosomal subunits of mammalian cells.

Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar peribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process.

Studies on the distribution of ribosomal proteins in mammalian ribosomal subunits.

Murine L5178Y cell ribosomes were dissociated into subunits either with potassium chloride in the presence of puromycin or with the chelating agent EDTA. The proteins of ribosomal subunits obtained by these different methods were compared by means of bidimensional polyacrylamide gel electrophoresis. KCl-derived 60S and 40S subunits were shown to contain 38 and 31 proteins respectively, 3 of them having identical electrophoretic mobilities. Preparations of EDTA-dissociated ribosomal subparticles contained different proportions of these proteins, and 11 major spots were shared between the EDTA-derived large and small ribosomal subunits. Furthermore, 10 proteins absent from subunits treated by high concentrations of KCl were reproducibly found in EDTA-treated ribosomal subparticles.

Characterization of proteins from nucleolar preribosomes of mouse leukemia cells by two-dimensional polyacrylamide gel electrophoresis.

Proteins of isolated 80-S and 60-S nucleolar preribosomal particles were characterized by means of two-dimensional polyacrylamide gel electrophoresis, in the lymphocytic mouse leukemia cells L5178Y. Their identification and metabolic relationships with ribosomal subunit proteins were investigated using co-electrophoresis of unlabeled polysomal proteins with labeled proteins of either nucleolar preribosomes or ribosomal subunits. The large and small ribosomal subunits contain 40 and 31 proteins, respectively. The nucleolar 80-S preribosomes were analysed after 2 and 5 h of incubation with tritiated valine and leucine and were shown to contain about 55 proteins. Most of them were identical to cytoplasmic ribosomal subunit proteins. The nucleolar 60-S preribosomes contain all the proteins which are common to 80-S preribosomes and large ribosomal subunits, and one additional protein (L10). The ribosomal proteins which were absent from nucleolar particles were found to be labeled in the cytoplasmic ribosomes after the same incubation period. Thus, in addition to the association of the bulk of ribosomal proteins with 45-S RNA within the 80-S preribosomes, results indicate that a group of ribosomal proteins and particularly from the small subunits, become associated at later stages of the maturation process of mammalian ribosomes. It was further shown that a set of 10 proteins, different from ribosomal polypeptides, were present in nucleolar preribosomal particles. Several of them were associated with polyribosomes in the cytoplasm, whereas the others were unique to the nucleolus.

[Antigenicity of ribosomal proteins from a malignant mouse cell line. Preliminary results]. / Antigénicité des protéines ribosomiques d'une lignée cellulaire maligne chez la souris, résultats préliminaires.

An original immunological two-dimensional technique with two different gels permits the protein analysis of 80 S cytoplasmic ribosomal murine particles and of their 60 S and 40 S subunits. The diagrams isolate and characterize a definite number of specific polypeptidic structures from each ribosomal particle.

Characterization of viral RNA in cells transformed by various isolates of Moloney murine sarcoma virus.

Intracellular polyadenylated viral RNA from cells infected by five different isolates of Moloney murine sarcoma virus (Mo-MuSV) was analysed by gel transfer hybridization. Genomic sizes of 4.6 kilobases (kb) for the ml-MuSV isolate, 5.2 kb for the m3- and 124-MuSV, 6.1 kb for the HTl-MuSV and 6.7 kb for the 78-Al-MuSV were determined. With the exception of the ml strain, subgenomic RNA species were found in cells infected by the various isolates. However, no common subgenomic RNA containing v-mos sequences could be characterized. Each transformed cell line expressed a different set of viral RNA species in terms of size and structure.

Comparative immunochemical study of ribosomal proteins from various mammalian cells by two-dimensional immunoelectrophoresis.

Proteins isolated from ribosomal subunits of various mammalian cels were analysed comparatively by two different methods: a two-dimensional polyacrylamide gel electrophoresis system and a recently described two-dimensional immunoelectrophoresis technique. For this purpose, antisera were raised in rabbits against the total mixture of ribosomal proteins from murine cells. These sera were characterized by ring-test, double immunodiffusion and two-dimensional immunoelectrophoresis. They were shown to contain antibodies to a large number of ribosomal proteins. Immunoelectrophoretic analysis of 60S and 40S subunit proteins from rabbit, lamb, canine and human cells using anti-murine sera revealed a striking conservation of their antigenic properties. These results corroborated those obtained by two-dimensional polyacrylamide gel electrophoresis.

Study of the 78A1 isolate of Moloney murine sarcoma virus. I. Molecular cloning and characterization.

The 78A1 isolate of Moloney murine sarcoma virus (78A1 Mo-MuSV) was cloned from a genomic library obtained from virus producer rat cells, in the lambda vector L47. Among the recombinants hybridizing with a probe specific for the v-mos sequences, we recovered a recombinant which contained leukaemia virus (MuLV) sequences and was able to transform both mouse and rat cells in transfection experiments. The cloned provirus could be rescued by both Mo-MuLV ecotropic and amphotropic viruses in mouse cells, but only with the amphotropic helper virus in rat cells. Comparative restriction mapping indicates that the 78A1 provirus is 200 bp longer than the HT1 provirus. The difference lies in the gag-pol junction region of Mo-MuSV. Other minor differences were found in the gag region, whereas the restriction patterns of the 3' parts of the proviruses were identical.

Study of the 78A1 isolate of Moloney murine sarcoma virus. II. Haematopoietic tropism and tumourigenicity.

A new isolate of Moloney murine sarcoma virus (Mo-MuSV), designated 78A1, has been molecularly cloned. The cloned genome, found to be larger than that of other known isolates of the same virus is close in size to that of the myeloproliferative sarcoma virus (MPSV), also a derivative of the original Mo-MuSV/Moloney murine leukaemia virus (Mo-MuLV) complex. Until now, MPSV was the only Mo-MuSV isolate known to be capable of inducing a myeloproliferative disease associated with a tumoural syndrome when injected intravenously into sensitive mice. We compared the biological activity of our cloned virus isolate (78A1) and that of another cloned Mo-MuSV virus (HT1) whose genome is slightly smaller than that of 78A1. The helper virus (Mo-MuLV) associated with the Mo-MuSV isolates was also injected alone as control. After injection into sensitive mice only the isolate 78A1, as well as MPSV caused a tumoural syndrome invading spleen, liver and other haematopoietic organs, and the appearance of granulo-macrophage precursors not requiring exogenous stimulating factors for their proliferation and differentiation. The 78A1 virus has a longer latency period (3 months) than MPSV (several days) and does not induce a typical myeloproliferative disease.