Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.

Heat treatment induces dephosphorylation of pRb and dissociation of T-antigen/pRb complex during transforming infection with SV40.

The effects of heat treatment on key cell cycle regulatory molecules is currently unknown. Using primary mouse kidney cell cultures induced to re-enter S-phase by transforming infection with SV40, the effects of thermal treatment on the phosphorylation state of the Retinoblastoma gene product (pRb) and on its ability to bind T-antigen were examined. Time course analysis showed no detectable inhibition of cellular protein and T-antigen synthesis up to 180 min treatment at 42.5 degrees C, while a gradual disappearance of the phosphorylated forms of pRb was observed after 90 min. Dephosphorylation of pRb was shown to correlate with inhibition of SV40-induced DNA synthesis. Also, large T-antigen/pRb protein complex was affected since a gradual dissociation of the hypophosphorylated pRb from large T-antigen was observed concomitant to the inhibition of DNA synthesis. The effects of heat treatment were found to be reversible after shifting the cultures to 37 degrees C. Cells were shown to resume DNA synthesis concomitant to the reappearance of the phosphorylated forms of pRb and rebinding of the hypophosphorylated form to large T-antigen within 6-8 h after recovery at 37 degrees C. In addition, no evidence for a bona fide hsp71/large T-antigen protein complex was seen under the experimental conditions used. The data strongly suggest that inhibition of DNA synthesis by heat treatment might involve either an increase in phosphatase activity or the inhibition of a pRb kinase activity.

One single burst of SV40 gene expression induces cell proliferation in transforming infection of mouse cells.

We have studied SV40 gene expression during transforming infection of Go-arrested primary mouse kidney cell cultures. The results show that both early and late viral genes are simultaneously expressed during a short restricted period of time. Synthesis of T-antigen is also time limited and precedes the first SV40-induced cellular DNA replication and mitosis. Cells in mitosis retained stable T-antigen which was redistributed and transmitted to daughter nuclei. Even though no reinitiation of viral transcription occurred after the first mitosis, several cycles of cellular DNA synthesis and mitosis was observed. These results suggest that over a several day period, stable T-antigen conferred a continuous mitotic stimulus to the infected cells.

Phosphorylation of the retinoblastoma protein is modulated in mouse kidney cells infected with polyomavirus.

Lytic infection with polyomavirus, an oncogenic DNA-containing virus, leads in G0-arrested primary baby mouse kidney (BMK) cell cultures to a mitotic host reaction. In the present work, we examined the expression of the retinoblastoma gene (RB) and of its product (Rb) in virus-infected BMK with the aim of correlating its modulation with the sequential activation of cellular processes leading to the induction of S phase by virus. In contrast to cell cycle-regulated genes whose expression is induced by viral infection, expression of RB is not altered during the transition from G0/G1 to S phase. In BMK cell cultures irreversibly arrested in the G0 phase of the cell cycle, an unphosphorylated species is the only detectable form of the RB protein (Rb). Time course analysis showed that in polyoma-infected cells induced to re-enter the S phase of the cell cycle the appearance of the phosphorylated forms of Rb coincided in time with the accumulation of large T antigen and preceded DNA synthesis. During the late phase of infection, the majority of Rb was present as phosphorylated forms. Ongoing DNA synthesis was not required for the cells to phosphorylate Rb, indicating that this post-translational modification takes place during the activation of the cellular DNA-synthesizing apparatus. Using hamster anti-polyoma tumor serum, it was observed that the underphosphorylated form of Rb co-precipitated with polyoma large T antigen extracted from infected cells late during infection. Our data add more evidence to the proposal that interactions between viral early proteins encoded by DNA tumor viruses and the product of RB may play a pivotal role in the mitogenic effect induced by viral infection.

Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast.

BACKGROUND: The family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied. RESULTS: Using antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism. CONCLUSIONS: The pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s) of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.

Expression of dopamine D1-receptor mRNA in the carotid body of adult rabbits, cats and rats.

Dopamine is a major neurotransmitter in the carotid body of several animal species and its functional role at the level of peripheral arterial chemoreflex pathway is attributed to the presence of the dopamine D2-receptors. We present evidence that the dopamine D1-receptor mRNA is also expressed in the carotid body of adult rabbits, cats and rats. A DNA fragment of 611 bp of this receptor was first isolated from rabbit. The nucleic acid sequence of this fragment was found to be 84.5% identical to that of rat. This specific 611 bp fragment was used as a probe to detect, either by Northern analysis or by the reverse transcription-polymerase chain reaction, the dopamine D1-receptor mRNA. The results revealed the presence of dopamine D1-receptor transcript in the carotid body as well as in the petrosal ganglion and the superior cervical ganglion from the three animal models studied here. The physiological significance of dopamine D1-receptor expression in the carotid body is discussed.

Expression of dopamine D2 receptor mRNA isoforms in the carotid body of rat, cat and rabbit.

Using the Reverse transcription-Polymerase chain reaction, we detected dopamine D2 receptor mRNA short and long isoforms in the adult carotid body of rats, cats, and rabbits. For these animals, the relative short/long ratios were 0.60, 0.65 and 0.57, respectively. Our results suggest that the variety of dopamine effects on carotid chemoreceptor activity, that has been related to species differences, may not be dependent on the expression levels of the dopamine D2 receptor mRNA isoforms in the studied species.

Disordered RNA chaperone proteins: from functions to disease.

RNA chaperones are ubiquitous proteins that play pivotal roles in cellular RNA metabolism and RNA virus replication. Here we propose that they act by organizing complex and highly dynamic networks of RNA-RNA, RNA-protein and protein-protein interactions. How this is achieved and how their malfunction may lead to disease will be discussed through the examples of human immunodeficiency virus type 1 nucleocapsid protein (NCp7), the fragile X mental retardation protein and the prion protein.

UV crosslinking of RNA to nylon membrane enhances hybridization signals.

An improvement in the detection by nucleic acid hybridization of size-fractionated RNA immobilized to nylon-based membranes is described. Electrophoretic transfer of RNA to nylon membranes permits a quantitative determination of different RNA transcripts on the same membrane after sequential hybridization using different 32P-labeled DNA probes. UV crosslinking of the RNA to the nylon membrane increased the intensity of the radioactive signals. Using the method reported here, increased signals of between 10 and 40 fold were observed, depending on the species of transcript tested. Moderately abundant as well as rare transcripts can easily be detected in as little as 5 micrograms total cellular RNA.

Biology of the fragile X mental retardation protein, an RNA-binding protein.

The fragile X syndrome, an X-linked disease, is the most frequent cause of inherited mental retardation. The syndrome results from the absence of expression of the FMR1 gene (fragile mental retardation 1) owing to the expansion of a CGG trinucleotide repeat located in the 5' untranslated region of the gene and the subsequent methylation of its CpG island. The FMR1 gene product (FMRP) is a cytoplasmic protein that contains two KH domains and one RGG box, characteristics of RNA-binding proteins. FMRP is associated with mRNP complexes containing poly(A)+mRNA within actively translating polyribosomes and contains nuclear localization and export signals making it a putative transporter (chaperone) of mRNA from the nucleus to the cytoplasm. FMRP is the archetype of a novel family of cytoplasmic RNA-binding proteins that includes FXR1P and FXR2P. Both of these proteins are very similar in overall structure to FMRP and are also associated with cytoplasmic mRNPs. Members of the FMR family are widely expressed in mouse and human tissues, albeit at various levels, and seem to play a subtle choreography of expression. FMRP is most abundant in neurons and is absent in muscle. FXR1P is strongly expressed in muscle and low levels are detected in neurons. The complex expression patterns of the FMR1 gene family in different cells and tissues suggest that independent, however similar, functions for each of the three FMR-related proteins might be expected in the selection and metabolism of tissue-specific classes of mRNA. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated as well as the putative role(s) of FXR1P and FXR2P as compensatory molecules.

Acidic extracellular environment induces only a subset of heat-shock proteins in primary mouse kidney cell cultures.

Exposure of primary mouse kidney cell cultures to acidic medium (pH 5.5) induced the expression of a 70 kilodalton (kDa) protein. This protein was identified as the major inducible heat-shock protein 70 (hsp70) by immunoprecipitation with anti-hsp70 serum and Northern blot analysis with a hsp70 cDNA probe. Maximum induction of the 70-kDa protein at pH 5.5 after 240 min was about 30% of that observed after 60 min of thermal treatment at 43 degrees C. In addition, there was an apparent induction of the glucose-regulated proteins (GRPs) of 76-78 and 98-100 kDa, but not of the other hsps. This subset induction of the heat-shock response by acidic medium suggests that different mechanisms are responsible for the induction of the various families of hsps.

Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40.

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5 degrees C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.

A procedure for Northern blot analysis of native RNA.

We describe a modification of the Northern technique that allows the detection of RNA either native and/or containing hidden breaks. We found that the highest sensitivity of the hybridization signals was obtained after denaturation of the RNA in the gel prior to its transfer onto a nylon membrane (GeneScreen) followed by uv irradiation. The sensitivity of the method using native RNA was found to be equivalent to that obtained with denatured RNA.

Release of RNA--DNA-protein complex during differentiation of the water mould Allomyces arbuscula.

A correlation between release of a nucleic acid protein complex into the external medium and sporangial differentiation was found. Conditions preventing differentiation also stopped the release. Lethal lysis is not involved in this release process. Extracellular nucleic acids are very heterogenous, consisting of nucleotides as well as acid-precipitable nucleic acids. RNA was found to be associated with proteins and a hetero-duplex RNA--DNA associated with this nucleoprotein. Some speculations are presented about possible correlation between the release of nucleoprotein complexes and the intracellular events of differentiation.

A 105 000 dalton antigen of transformed mouse cells is a stress protein.

Antisera prepared in mice against syngeneic spontaneously transformed AL/N cells (anti-TAL/N serum) identified a number of protein antigens synthesized by simian virus 40 (SV40) transformed cells, among which was a protein with a molecular mass of 105 000 daltons (p105). Of these transformed cell antigens which were immunogenic in a syngeneic system, only p105 was detected in primary mouse kidney cell cultures. p105 isolated from normal and transformed mouse cells was demonstrated to be identical by two-dimensional gel analysis. Relatively small amounts of p105 were synthesized in quiescent primary cultures, while the protein was actively synthesized in SV40-infected as well as in proliferating mouse kidney cells, and its synthesis in quiescent cells could be induced by subjecting the cultures to glucose starvation or heat-shock treatment. Immunofluorescent staining and cellular fractionation showed that p105 is normally localized to cytoplasmic structures. The results suggest that the expression of p105 is intimately associated with the metabolic state of the cell.

Cyclosporin A prevents induction of the interleukin 2 receptor gene in cultured murine thymocytes.

Blast formation and mitotic activation of G0-arrested mouse thymocytes were triggered by the addition of concanavalin A plus interleukin 2 (IL-2) to the culture medium. When added alone, Con A induces within 6 hr a complex reprogramming ("priming") that comprises the activation of the IL-2 receptor gene. The primed thymocytes are competent to interact with IL-2 and to respond to its growth-promoting effect, which corresponds to blast formation and mitotic activation. Cyclosporin A, an immunosuppressive cyclic peptide of fungal origin, prevents in T lymphocytes the activation of a set(s) of genes encoding lymphokines and the IL-2 receptor but does not affect their expression once they have been activated. The biomedical implications of these observations are discussed.