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1.
Cell Mol Life Sci ; 62(13): 1409-17, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15924259

ABSTRACT

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.


Subject(s)
Molecular Chaperones/metabolism , RNA/metabolism , Animals , Capsid Proteins/metabolism , Fragile X Mental Retardation Protein , Fragile X Syndrome/metabolism , Gene Products, gag/metabolism , HIV Infections/metabolism , HIV-1 , Humans , Intellectual Disability/metabolism , Nerve Tissue Proteins/metabolism , Prion Diseases/metabolism , Prions/metabolism , Protein Conformation , RNA-Binding Proteins/metabolism , Structure-Activity Relationship , Viral Proteins/metabolism , gag Gene Products, Human Immunodeficiency Virus
3.
Hum Mol Genet ; 10(24): 2803-11, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11734545

ABSTRACT

Fragile X mental retardation 1 protein (FMRP) is the archetype of a class of cytoplasmic mRNA-binding proteins that includes the fragile X-related 1 and 2 proteins (FXR1P and FXR2P). Whereas absence of FMRP is the cause of fragile X syndrome, it is not known if FXR1P and FXR2P are associated with any pathology. It is also still elusive whether these homologous proteins can partially compensate for the absence of FMRP in the case of the fragile X syndrome. FXR1 is widely expressed in mammals and its expression pattern is complex since several mRNA variants and protein isoforms are detected. In mouse, we observed that the highest level of FXR1 is found in the adult testis. This tissue is an exception, since all known FXR1P isoforms, some of which have been considered as tissue specific, are detected in it. In young animals, changes in mRNA-spliced variants and their corresponding protein isoforms occur during spermatogenesis. Using biochemical, immunohistochemical and electron microscopic techniques, we show that FXR1P is associated with microtubule elements. Since the cytoskeletal framework is implicated in cellular plasticity as well as in mRNA transport, we propose new possibilities for the function(s) of the FXR proteins.


Subject(s)
Microtubule-Associated Proteins/biosynthesis , Spermatocytes/metabolism , Aging/metabolism , Animals , Blotting, Northern , Fluorescent Antibody Technique, Indirect , Gene Expression , Immunoblotting , Immunoenzyme Techniques , Liver/metabolism , Male , Mice , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Polymerase Chain Reaction , Polyribosomes , Protein Isoforms , RNA, Messenger , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , Sperm Tail/metabolism , Sperm Tail/ultrastructure , Spermatocytes/cytology , Spermatocytes/ultrastructure , Spermatogenesis , Testis/cytology , Testis/growth & development , Testis/metabolism , Transcription, Genetic
4.
Biochem Cell Biol ; 78(4): 437-45, 2000.
Article in English | MEDLINE | ID: mdl-11012082

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming/metabolism , Cell Transformation, Viral , DNA/biosynthesis , Hot Temperature , S Phase , Simian virus 40/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Antigens, Polyomavirus Transforming/chemistry , Cell Cycle , Cell Nucleus/metabolism , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Epithelial Cells , Heat-Shock Proteins/biosynthesis , Immunoblotting , Kidney/cytology , Mice , Simian virus 40/genetics , Solubility , Tumor Suppressor Protein p53/chemistry
5.
BMC Genet ; 1: 4, 2000.
Article in English | MEDLINE | ID: mdl-11178106

ABSTRACT

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.


Subject(s)
Muscle, Skeletal/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/metabolism , Cell Differentiation/physiology , Cell Line , Cell Nucleus/chemistry , Epitopes/chemistry , Epitopes/immunology , Fragile X Syndrome/metabolism , Immunohistochemistry , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Muscle Development , Muscle, Skeletal/cytology , Myoblasts/chemistry , Myoblasts/cytology , Organ Specificity , Polyribosomes/chemistry , Protein Isoforms/biosynthesis , Protein Isoforms/immunology , Protein Isoforms/metabolism , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/immunology , Rabbits
6.
Biochem Cell Biol ; 77(4): 331-42, 1999.
Article in English | MEDLINE | ID: mdl-10546896

ABSTRACT

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.


Subject(s)
Fragile X Syndrome/genetics , Intellectual Disability/genetics , Nerve Tissue Proteins/physiology , RNA-Binding Proteins , Animals , Chromosome Banding , Disease Models, Animal , Fragile X Mental Retardation Protein , Humans , Mice , Nerve Tissue Proteins/genetics
7.
Am J Pathol ; 154(4): 1245-57, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10233862

ABSTRACT

The study of misplaced endometrial cells, which abnormally implant and grow outside the uterine cavity, is of considerable interest for the understanding of the pathophysiology of endometriosis. However, endometriotic cells, particularly epithelial cells, required for primary cell culture are not easily available. We report here the characterization of an endometriotic cell line immortalized after infection of primary endometriotic cell cultures with simian virus 40. Transformed cells express T-antigen, and blot hybridization analysis showed that the viral genome is present as an episome. Cytogenetic analysis revealed a polyploid karyotype with numerical and structural rearrangements involving mainly the same chromosomes (6, 10, 11, 15, and 17). The cell line has been maintained in culture for over 80 passages and was still proliferating without any noticeable change in the biological properties investigated. Transformed endometriotic cells expressed both progesterone and estradiol receptors and were stimulated by these ovarian hormones to secrete monocyte chemotactic protein-1, a factor that may play an important role in the recruitment and activation of peritoneal macrophages. In addition, this response was enhanced in interleukin-1-treated cells. Taken together, these findings support the view that this cell line may be an interesting tool for the study of the pathophysiology of endometriosis.


Subject(s)
Endometriosis/pathology , Endometriosis/virology , Plasmids/genetics , Simian virus 40/genetics , Adult , Antigens, Viral, Tumor/metabolism , Cell Line, Transformed , Chemokine CCL2/metabolism , DNA, Viral/metabolism , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Endometriosis/metabolism , Endometrium/cytology , Endometrium/drug effects , Endometrium/metabolism , Estradiol/pharmacology , Female , Fluorescent Antibody Technique , Humans , Interleukin-1/pharmacology , Karyotyping , Precipitin Tests , Progesterone/pharmacology , Receptors, Estradiol/biosynthesis , Receptors, Progesterone/biosynthesis , Time Factors
8.
Hum Mol Genet ; 7(13): 2121-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9817930

ABSTRACT

The fragile X syndrome results from transcriptional silencing of the FMR1 gene and the absence of its encoded FMRP protein. Two autosomal homologues of the FMR1 gene, FXR1 and FXR2, have been identified and the overall structures of the corresponding proteins are very similar to that of FMRP. Using antibodies raised against FXR1P, we observed that two major protein isoforms of relative MW of 78 and 70 kDa are expressed in different mammalian cell lines and in the majority of mouse tissues. In mammalian cells grown in culture as well as in brain extracts, both P78and P70isoforms are associated with mRNPs within translating polyribosomes, similarly to their closely related FMRP homologues. In muscle tissues as well as in murine myoblastic cell lines induced to differentiate into myotubes, FXR1P78and P70isoforms are replaced by novel unpredicted isoforms of 81-84 kDa and a novel FXR1 exon splice variant was detected in muscle RNA. While P81-84isoforms expressed after fusion into myotubes in murine myoblast cell lines grown in culture are associated with polyribosomes, this is not the case when isolated from muscle tissues since they sediment with lower S values. Immunohistochemical studies showed coexpression of FMRP and FXR1P70and P78in the cytoplasm of brain neurons, while in muscle no FMRP was detected and FXR1P81-84were mainly localized to structures within the muscle contractile bands. The complex expression pattern of FXR1P suggests tissue-specific expression for the various isoforms of FXR1 and the differential expression of FMRP and FXR1Ps suggests that in certain types of cells and tissues, complementary functions may be fulfilled by the various FMRP family members.


Subject(s)
Muscles/metabolism , RNA-Binding Proteins/genetics , 3T3 Cells , Alternative Splicing , Animals , Base Sequence , Brain/metabolism , COS Cells , Cell Line , Chemical Fractionation , Fragile X Mental Retardation Protein , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Gene Expression Regulation, Developmental , HeLa Cells , Humans , Immunoblotting , Immunohistochemistry , Mice , Molecular Sequence Data , Muscle Development , Muscles/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Polyribosomes/metabolism , RNA/genetics , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Sequence Homology, Amino Acid
10.
Neurosci Res ; 31(2): 147-54, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9700720

ABSTRACT

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.


Subject(s)
Carotid Body/metabolism , Receptors, Dopamine D1/analysis , Animals , Base Sequence , Blotting, Northern , Carotid Body/chemistry , Cats , Molecular Sequence Data , RNA, Messenger/analysis , Rabbits , Rats , Rats, Sprague-Dawley , Sequence Homology, Nucleic Acid , Superior Cervical Ganglion/chemistry , Superior Cervical Ganglion/metabolism
11.
Hum Mol Genet ; 6(12): 2051-60, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9328468

ABSTRACT

Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Promoter Regions, Genetic/physiology , RNA-Binding Proteins , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Female , Fibroblasts/metabolism , Fragile X Mental Retardation Protein , Fragile X Syndrome/genetics , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Lymphocyte Activation/genetics , Lymphocytes/metabolism , Male , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Protein Binding/genetics , Regulatory Sequences, Nucleic Acid , Ribonucleoproteins/metabolism , Structure-Activity Relationship
12.
Hum Mol Genet ; 6(9): 1465-72, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9285783

ABSTRACT

The fragile X syndrome results from a transcriptional silencing of the FMR1 gene and the absence of its encoded protein. FMRP is a cytoplasmic RNA-binding protein, whose specific cellular function is still unknown. We present evidence that virtually all detectable cytoplasmic FMRP in mouse NIH 3T3 and human HeLa cells is found strictly in association with mRNA in actively translating polyribosomes. Furthermore, FMRP released from polyribosomes is associated with ribonucleoprotein complexes with sedimentation coefficients of 60-70S and selection on oligo(dT)-cellulose reveals that this association is specific to poly(A)-containing mRNPs. This association with actively translating polyribosomes is not affected by alteration of translational processes induced by serum stimulation and starvation in NIH 3T3 cells, suggesting that FMR1 expression is not cell cycle regulated and that FMRP might have a house-keeping function. FXR2 protein, which is closely related to FMRP, is also detected associated with mRNPs in translating polyribosomes. The results strongly suggest that FMRP might be a mRNA chaperone interacting with mRNP complexes.


Subject(s)
Nerve Tissue Proteins/genetics , Poly A/genetics , Polyribosomes/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , 3T3 Cells/metabolism , Animals , Fragile X Mental Retardation Protein , Fragile X Syndrome/genetics , Gene Expression Regulation, Enzymologic , HeLa Cells/metabolism , Humans , Intellectual Disability/genetics , Mice , Microscopy, Fluorescence , Nerve Tissue Proteins/metabolism , Poly A/metabolism , Polyribosomes/metabolism , Protein Biosynthesis , RNA-Binding Proteins/metabolism
13.
Brain Res ; 760(1-2): 287-9, 1997 Jun 20.
Article in English | MEDLINE | ID: mdl-9237549

ABSTRACT

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.


Subject(s)
Carotid Body/metabolism , Receptors, Dopamine D2/metabolism , Animals , Cats , Corpus Striatum/metabolism , Female , Male , Polymerase Chain Reaction , RNA, Messenger/metabolism , Rabbits , Rats , Rats, Sprague-Dawley , Stereoisomerism
14.
Am J Respir Cell Mol Biol ; 15(3): 374-81, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8810642

ABSTRACT

The two isoforms of dopamine D2 receptor, D2 short (D2s) and D2 long, are generated by alternative splicing of premessenger RNA and differ in the length of their third cytoplasmic loop. Expression of these two isoforms has not yet been studied at the level of the peripheral arterial chemoreflex pathway. Using reverse transcriptase-polymerase chain reaction, we evaluated the relative abundance of dopamine D2 receptor mRNA by amplifying a common segment between both D2 receptor mRNA isoforms which reflects the total D2-receptor mRNA and the two isoforms in the carotid bodies, the petrosal ganglia, and the superior cervical ganglia of 1-, 10-, and 25-day-old and adult rabbits, GH4C1 cell line, which does not express D2 receptors, was used as negative control, whereas GH4C1 19 cell line, which expresses only the D2s mRNA and the striatum of each age, were used as positive controls. Both D2-receptor mRNA isoforms were found to be expressed in all organs studied of newborn and adult rabbits. The expression of total D2 receptor mRNA in 10- and 25-day-old and adult rabbits was found to be 3-, 5-, and 1.5-fold more, respectively, compared with 1-day-old rabbits for all organs studied. Our data suggest that the expression of D2-receptor mRNA is modulated with age at the level of the peripheral arterial chemoreflex pathway.


Subject(s)
Chemoreceptor Cells/physiology , Peripheral Nervous System/chemistry , Receptors, Dopamine D2/genetics , Afferent Pathways , Age Factors , Animals , Carotid Body/chemistry , Chemoreceptor Cells/chemistry , DNA Primers/genetics , Ganglia, Autonomic/chemistry , Isomerism , Neostriatum/chemistry , Peripheral Nervous System/cytology , Peripheral Nervous System/growth & development , Polymerase Chain Reaction , RNA, Messenger/metabolism , Rabbits , Rats , Receptors, Dopamine D2/chemistry , Superior Cervical Ganglion/chemistry
17.
Am J Hum Genet ; 57(5): 1006-18, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7485149

ABSTRACT

The fragile X syndrome is the second leading cause of mental retardation after Down syndrome. Fragile X premutations are not associated with any clinical phenotype but are at high risk of expanding to full mutations causing the disease when they are transmitted by a carrier woman. There is no reliable estimate of the prevalence of women who are carriers of fragile X premutations. We have screened 10,624 unselected women by Southern blot for the presence of FMR1 premutation alleles and have confirmed their size by PCR analysis. We found 41 carriers of alleles with 55-101 CGG repeats, a prevalence of 1/259 women (95% confidence interval 1/373-1/198). Thirty percent of these alleles carry an inferred haplotype that corresponds to the most frequent haplotype found in fragile X males and may indeed constitute premutations associated with a significant risk of expansion on transmission by carrier women. We identified another inferred haplotype that is rare in both normal and fragile X chromosomes but that is present on 13 (57%) of 23 chromosomes carrying FMR1 alleles with 53-64 CGG repeats. This suggests either (1) that this haplotype may be stable or (2) that the associated premutation-size alleles have not yet reached equilibrium in this population and that the incidence of fragile X syndrome may increase in the future.


Subject(s)
Fragile X Syndrome/genetics , Heterozygote , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Alleles , Base Sequence , Female , Fragile X Mental Retardation Protein , Fragile X Syndrome/epidemiology , France/ethnology , Genetic Linkage , Genetic Testing , Genetics, Population , Humans , Molecular Sequence Data , Prevalence , Quebec , Repetitive Sequences, Nucleic Acid
18.
Hum Mol Genet ; 4(5): 783-9, 1995 May.
Article in English | MEDLINE | ID: mdl-7633436

ABSTRACT

The fragile X syndrome is an X-linked inherited disease and is the result of transcriptional inactivation of the FMR1 gene and the absence of its encoded FMR protein (FMRP). Using a specific monoclonal antibody directed against human FMRP, we have studied the steady-state levels of its murine homolog in several tissues and organs of adult and young mice. In immunoblot analyses, the antibody recognizes a heterogeneous subset of proteins with apparent molecular weights ranging from 80 to 70 kDa. These proteins are detected in all the 27 tissues tested; however, the relative proportion of each polypeptide recognized varies between tissues, and a significantly higher expression is observed in young animals. Northern blot analysis of RNA extracted from selected tissues from adult mouse shows that these tissues express the major 4.8 kb mRNA, although at different levels, and contain several additional shorter transcripts, particularly in muscular tissues. We also report that expression of the FMR1 gene is modulated in proliferating and quiescent primary mouse kidney cell cultures with an inverse relationship between levels of FMR1 mRNA and of its encoded proteins. This suggests that FMRPs are highly stable in quiescent cells and that FMR1 expression is likely post-transcriptionally controlled. Our results document the widespread expression of the FMR1 gene, and suggest that it is controlled by different mechanisms implicated in cell growth and differentiation.


Subject(s)
Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins , Animals , Antibodies, Monoclonal , Cells, Cultured , Female , Fragile X Mental Retardation Protein , Fragile X Syndrome/genetics , Gene Expression , Humans , Kidney/metabolism , Male , Mice , Nerve Tissue Proteins/immunology , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue Distribution
19.
Oncogene ; 10(2): 359-67, 1995 Jan 19.
Article in English | MEDLINE | ID: mdl-7838534

ABSTRACT

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.


Subject(s)
Antigens, Viral, Tumor/metabolism , Cell Transformation, Viral , Hot Temperature , Retinoblastoma Protein/metabolism , Animals , Cells, Cultured , DNA/biosynthesis , Heat-Shock Proteins/metabolism , Mice , Phosphorylation , S Phase , Time Factors
20.
Biochem Cell Biol ; 72(5-6): 239-43, 1994.
Article in English | MEDLINE | ID: mdl-7840944

ABSTRACT

The major advantages of the horseradish peroxidase chemiluminescence (HRP-CL) immunodetection method in Western blot analysis are its high sensitivity, nonradioactive detection, economy of the primary antibody, and speed of detecting the signal. However, we observed a strong and reproducible signal that was detected regardless of the primary antibody and of the cell type used. This signal, present at 56-54 kilodaltons (kDa), is generated in absence of any primary antibody and seems to be an intrinsic reaction of the HRP-labeled second antibody anti-immunoglobulin with an unidentified cellular protein. The use of dry milk throughout all the steps of the procedure abolishes this signal. For those interested in one of the numerous proteins migrating at or close to 56-54 kDa, the question therefore arises as to which signals generated by the HRP-CL in this region are bona fide and which are non grata pseudo signals.


Subject(s)
Horseradish Peroxidase , Immunoblotting , Luminescent Measurements , Proteins/analysis , Tumor Suppressor Protein p53/analysis , Animals , Blotting, Western , Cells, Cultured , Immunoenzyme Techniques , Kidney/chemistry , Mice , Molecular Weight , Resting Phase, Cell Cycle
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