Hung Fan

Regulation of protein synthesis in mammalian cells: II. Inhibition of protein synthesis at the level of initiation during mitosis☆☆☆

Abstract The decline in the rate of protein synthesis during mitosis in eukaryotic cells was studied using Chinese hamster ovary cells arrested in metaphase with Colcemid. The rate of protein synthesis in mitotic cells is approximately 30% of the interphase rate. Ribosomes become attached to messenger RNA and initiate polypeptide synthesis at a reduced rate. After initiation of polypeptide synthesis, ribosomes are translated at a normal rate. Reducing the rate of translation with low levels of cycloheximide mobilizes a large proportion of monomeric ribosomes into active polyribosomes. The amount of messenger RNA associated with polyribosomes is also increased in mitotic cells treated with cycloheximide. It appears that the reduced protein synthesis during mitosis results from a lowered rate of attachment of ribosomes to messenger RNA and initiation of polypeptide synthesis.

RNA metabolism of murine leukemia virus: Detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA☆

Abstract Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells. Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.

Regulation of synthesis and processing of nucleolar components in metaphase-arrested cells☆

Abstract As cells enter mitosis, nucleolar RNA synthesis ceases and nucleolar structure disappears. However, the ribosomal RNA precursors (45 and 32 s RNA) persist in nearly interphase amounts. Nucleolar synthesis and processing co-ordinately cease as HeLa and Chinese hamster ovary cells enter metaphase; 45 and 32 s RNA are stable in metaphase-arrested cells. Processing of the rRNA intermediates resumes as cells leave mitosis. 45 and 32 s RNA are associated with the chromosome fraction of metaphase-arrested cells. Ribonucleoprotein particles containing 45 and 32 s RNA, which resemble interphase subnucleolar particles, are obtained by gentle disruption of the chromosome fraction. The state of association of the ribonucleoprotein particles during metaphase is different from interphase since they are liberated by ionic conditions that have little effect on interphase nucleoli. Nucleolar RNA synthesis and processing resume within 90 minutes of release from metaphase arrest. This resumption is not dependent on the synthesis of new proteins.

Further Characterization of Virus-like 30S (VL30) RNA of Mice: Initiation of Reverse Transcription and Intracellular Synthesis

We have studied the virus-like 30S (VL30) RNA sequences of mice. Previous work has shown that these sequences are coded in the mouse genome, expressed in some normal cells and released as pseudotypic particles from cells producing murine C-type retroviruses. VL30 sequences have some similarities to standard retrovirus RNA, but differences also exist. To further assess the similarities and differences, several aspects of VL30-specific metabolism were investigated. We studied the initiation of VL30-specific DNA synthesis during an endogenous reverse transcriptase reaction. Short initial VL30-specific cDNA transcripts were covalently attached to RNA as measured by equilibrium banding in caesium sulphate density gradients. Therefore, reverse transcription of VL30-specific cDNA is initiated by an RNA primer. The intracellular synthesis of VL30 RNA was investigated by pulse labelling uninfected JLS-V9 cells with 3H-uridine. Hybridization of the pulse-labelled nuclear RNA indicated that the major VL30-specific RNA evident after a 15 min label was the same size as the mature VL30 RNA. Thus, VL30 RNA is apparently not synthesized via a higher mol. wt. precursor. Both of these results demonstrate similarity of VL30 RNA sequences to standard retroviruses. One unique feature of VL30 RNA was detected. JLS-V9 cells contained both the monomeric VL30 RNA and a hydrogen-bonded 38S form which yielded the monomer when denatured. This contrasts with standard murine leukaemia virus which is only found as a monomer within cells.

Synthesis by reverse transcriptase of DNA complementary to globin messenger RNA.

RNA tumor viruses contain a DNA polymerase that can synthesize a faithful DNA copy of viral RNA (1,5,23,25,26,31,32). This enzyme is easily released and purified from virions and can utilize a wide variety of polymers as templates (6,7,13,14,18,35,38). In order for a template to be copied, a primer or initiator that binds to the template by hydrogen bonds is required (3). The 3′-OH end of the primer is then covalently attached to the newly synthesized DNA (29). When the 60–70S tumor viral RNA is transcribed, the primer is apparently a short polyribonucleotide that is found attached to the DNA product (10,18,35,37).