RESUMO
The potential of embryonal day (ED) 14 fetal liver epithelial progenitor (FLEP) cells from Fischer (F)344 rats to repopulate the normal and retrorsine-treated liver was studied throughout a 6-month period in syngeneic dipeptidyl peptidase IV (DPPIV-) mutant F344 rats. In normal liver, FLEP cells formed: 1) hepatocytic clusters ranging in size up to approximately 800 to 1000 cells; 2) bile duct structures connected to pre-existing host bile ducts; and 3) mixed clusters containing both hepatocytes and bile duct epithelial cells. Liver repopulation after 6 months was moderate (5 to 10%). In retrorsine-treated liver, transplanted cells formed large multilobular structures containing both parenchymal and bile duct cells and liver repopulation was extensive (60 to 80%). When the repopulating capacity of ED 14 FLEP cells transplanted into normal liver was compared to adult hepatocytes, three important differences were noted: 1) FLEP cells continued to proliferate at 6 months after transplantation, whereas adult hepatocytes ceased proliferation within the first month; 2) both the number and size of clusters derived from FLEP cells gradually increased throughout time but decreased throughout time with transplanted mature hepatocytes; and 3) FLEP cells differentiated into hepatocytes when engrafted into the liver parenchyma and into bile epithelial cells when engrafted in the vicinity of the host bile ducts, whereas adult hepatocytes did not form bile duct structures. Finally, after transplantation of ED 14 FLEP cells, new clusters of DPPIV+ cells appeared after 4 to 6 months, suggesting reseeding of the liver by transplanted cells. This study represents the first report with an isolated fetal liver epithelial cell fraction in which the cells exhibit properties of tissue-determined stem cells after their transplantation into normal adult liver; namely, bipotency and continued proliferation long after their transplantation.
Assuntos
Fígado/patologia , Fígado/cirurgia , Células-Tronco/fisiologia , Animais , Agregação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Células Epiteliais/citologia , Transplante de Tecido Fetal , Feto , Idade Gestacional , Transplante de Células-Tronco Hematopoéticas , Hepatócitos/patologia , Hepatócitos/transplante , Fígado/efeitos dos fármacos , Fenótipo , Período Pós-Operatório , Alcaloides de Pirrolizidina/farmacologia , Ratos , Ratos Endogâmicos F344 , Valores de Referência , Células-Tronco/patologiaRESUMO
Differentially expressed cDNA clones from fetal rat liver were isolated using suppression subtractive hybridization, combined with an efficient screening strategy. Approximately 30,000 clones were screened, yielding 643 genes whose expression was induced, of which 201 clones were distinct and 68 represented ESTs or newly discovered genes of unknown function. Based on their expression patterns in different organs, fetal liver, liver regeneration models, and gut epithelial progenitor cell lines, the subtracted clones presented in this work were placed into four categories: (1) hepatoblast-specific genes; (2) hematopoietic cell-specific genes; (3) genes expressed in hepatoblasts, in hematopoietic cells, and at varying levels in other tissues; and (4) genes overexpressed in fetal liver, in models of activation of liver progenitor cells, and in epithelial progenitor cell lines. Hepatoblast-specific clones and those representing genes induced during liver regeneration are under further study to define their specific function(s) in liver cell growth control and/or differentiation.
Assuntos
Perfilação da Expressão Gênica , Fígado/metabolismo , Animais , Northern Blotting , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Etiquetas de Sequências Expressas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Fígado/citologia , Fígado/embriologia , Regeneração Hepática/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNA , Células-Tronco/citologia , Células-Tronco/metabolismo , Distribuição TecidualRESUMO
To identify cells that have the ability to proliferate and differentiate into all epithelial components of the liver lobule, we isolated fetal liver epithelial cells (FLEC) from ED 14 Fischer (F) 344 rats and transplanted these cells in conjunction with two-thirds partial hepatectomy into the liver of normal and retrorsine (Rs) treated syngeneic dipeptidyl peptidase IV mutant (DPPIV(-)) F344 rats. Using dual label immunohistochemistry/in situ hybridization, three subpopulations of FLEC were identified: cells expressing both alpha-fetoprotein (AFP) and albumin, but not CK-19; cells expressing CK-19, but not AFP or albumin, and cells expressing AFP, albumin, and cytokeratins-19 (CK-19). Proliferation, differentiation, and expansion of transplanted FLEC differed significantly in the two models. In normal liver, 1 to 2 weeks after transplantation, mainly cells with a single phenotype, hepatocytic (expressing AFP and albumin) or bile ductular (expressing only CK-19), had proliferated. In Rs-treated rats, in which the proliferative capacity of endogenous hepatocytes is impaired, transplanted cells showed mainly a dual phenotype (expressing both AFP/albumin and CK-19). One month after transplantation, DPPIV(+) FLEC engrafted into the parenchyma exhibited an hepatocytic phenotype and generated new hepatic cord structures. FLEC, localized in the vicinity of bile ducts, exhibited a biliary epithelial phenotype and formed new bile duct structures or were incorporated into pre-existing bile ducts. In the absence of a proliferative stimulus, ED 14 FLEC did not proliferate or differentiate. Our results demonstrate that 14-day fetal liver contains lineage committed (unipotential) and uncommitted (bipotential) progenitor cells exerting different repopulating capacities, which are affected by the proliferative status of the recipient liver and the host site within the liver where the transplanted cells become engrafted. These findings have important implications in future studies directed toward liver repopulation and ex vivo gene therapy.
Assuntos
Transplante de Células , Transplante de Tecido Fetal , Fígado/citologia , Fígado/cirurgia , Células-Tronco/citologia , Animais , Ductos Biliares/citologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/citologia , Hepatectomia/métodos , Fígado/embriologia , Mitógenos/farmacologia , Período Pós-Operatório , Alcaloides de Pirrolizidina/farmacologia , Ratos , Ratos Endogâmicos F344 , Transplante Isogênico , Tri-Iodotironina/farmacologiaRESUMO
Recently, we reported near-complete repopulation of the rat liver by transplanted hepatocytes using retrorsine (RS), a pyrrolizidine alkaloid that alkylates cellular DNA and blocks proliferation of resident hepatocytes, followed by transplantation of normal hepatocytes in conjunction with two-thirds partial hepatectomy (PH). Because two-thirds PH is not feasible for use in humans, in the present study, we evaluated the ability of thyroid hormone (triiodothyronine [T(3)]), a known hepatic mitogen, to stimulate liver repopulation in the retrorsine model. Because T(3) initiates morphogenesis in amphibians through a process involving both cell proliferation and apoptosis, we also determined whether apoptosis might play a role in the mechanism of hepatocyte proliferation induced by T(3). Following hepatocyte transplantation and repeated injections of T(3), the number of transplanted hepatocytes in the liver of RS-pretreated animals increased progressively to repopulate 60% to 80% of parenchymal cell mass in 60 days. We show further that T(3) treatment augments proliferation of normal hepatocytes, as evidenced by increased histone 3 mRNA and cyclin-dependent kinase 2 (cdk2) expression, and this is followed by apoptosis. These combined effects of T(3) lead to selective proliferation of transplanted hepatocytes in RS-pretreated rats, while endogenous hepatocytes, which are blocked in their proliferative capacity by RS, mainly undergo apoptosis. Thus, T(3) can replace PH in the RS-based rat liver repopulation model and therefore represents a significant advance in developing methods for hepatocyte transplantation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Transplante de Células , Regeneração Hepática/fisiologia , Fígado/citologia , Tri-Iodotironina/fisiologia , Animais , Apoptose , Divisão Celular/fisiologia , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Histonas/genética , Hibridização In Situ , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiologia , Mitógenos/farmacologia , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Alcaloides de Pirrolizidina/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Tri-Iodotironina/farmacologiaRESUMO
Recently, we described a new strategy for hepatocyte transplantation, using retrorsine/partial hepatectomy (PH) in a DPPIV- mutant Fischer rat model. Treatment of rats with retrorsine, a pyrrolizidine alkaloid, blocks endogenous hepatocytes from proliferating, so that after exposure to this agent coupled with PH and hepatocyte transplantation, transplanted hepatocytes selectively repopulate the liver. In the present study, we determined whether this method of cell transplantation can restore biosynthetic and physiological function in the liver by transplanting normal hepatocytes into rats genetically deficient in albumin synthesis, the Nagase analbuminic rat (NAR). After hepatocyte transplantation, albumin mRNA and protein were identified in the liver by in situ hybridization and immunohistochemistry, respectively, and serum albumin levels were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and enzyme-linked immunosorbent assay (ELISA) methods. At 1 month posttransplantation, large clusters of cells expressing albumin mRNA and protein were identified in the liver, representing approximately 50% of hepatocytes for albumin mRNA and approximately 61% for protein. At 2 months' posttransplantation, cells expressing albumin mRNA represented approximately 77% of hepatocyte mass, and cells expressing albumin protein represented approximately 81% of total hepatocyte mass. Hepatocyte-transplanted NAR also exhibited normal or near-normal serum albumin levels (3.0 +/- 0.2 g/dL). High levels of serum albumin were sustained for the 2-month duration of experiments. These results demonstrate the ability of this protocol for hepatocyte transplantation to restore a major biosynthetic and physiological function of the liver, and suggest its potential use as a method to treat genetic-based or acquired liver diseases.
Assuntos
Acetilglucosaminidase/deficiência , Transplante de Células , Fígado/metabolismo , Albumina Sérica/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Separação Celular , Dieta , Hibridização In Situ , Fígado/patologia , Masculino , Alcaloides de Pirrolizidina/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Albumina Sérica/biossíntese , Albumina Sérica/deficiênciaRESUMO
Recently, we described a new model for hepatocyte transplantation with nearly total replacement of the liver by exogenous hepatocytes (E. Laconi et al., Am. J. Pathol., 153: 319-329, 1998). The model is based on the mitoinhibitory effect of the pyrrolizidine alkaloid retrorsine on hepatocytes in the resident liver while transplanted hepatocytes proliferate. In this study, we exploit this novel approach to address the important and controversial issue of whether hepatocytes, when proliferating extensively, undergo dedifferentiation and give rise to foci of undifferentiated hepatocytes. Genetically marked hepatocytes (isolated from normal Dipeptidyl peptidase IV+ Fischer 344 rats) were delivered intraportally (2 x 10(6) cells) into the liver of retrorsine-treated Dipeptidyl peptidase IV- mutant Fischer 344 rats in conjunction with partial hepatectomy. Transplanted hepatocytes were detected histochemically or immunohistochemically, and cell proliferation was studied by in situ hybridization for histone-3 mRNA. Expression of alpha-fetoprotein (AFP) mRNA, a marker of hepatocyte dedifferentiation, was also revealed by in situ hybridization. One day after partial hepatectomy and hepatocyte transplantation, endogenous hepatocytes and oval cells expanding in the liver expressed histone-3 mRNA (cells had entered S phase); 2 days later, transplanted hepatocytes and nonparenchymal cells also expressed histone-3 mRNA. Although the majority of endogenous hepatocytes did not divide and became arrested as quiescent megalocytes, the exogenous hepatocytes, as well as newly formed small hepatocytes, most probably derived from liver progenitor cells, underwent extensive proliferation. After 7-14 days, the nonparenchymal cells stopped proliferating, but transplanted hepatocytes and small endogenous hepatocytes continued to proliferate for 1 month, forming foci of dividing parenchymal cells. Although many of the hepatocytes in clusters were in S phase (histone-3 mRNA positive), none expressed AFP mRNA. In contrast, high expression of AFP mRNA was observed in proliferating oval and transitional cells, forming duct-like structures of cytokeratin-19-positive cells. From these studies, we conclude that hepatocyte proliferation in the adult liver is not associated with dedifferentiation.
Assuntos
Transplante de Células , Regeneração Hepática , Transplante de Fígado , Alcaloides de Pirrolizidina/farmacologia , alfa-Fetoproteínas/metabolismo , Animais , Diferenciação Celular , Divisão Celular , Dipeptidil Peptidase 4/genética , Hepatectomia , Histonas/metabolismo , Filamentos Intermediários/metabolismo , Mutação , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , alfa-Fetoproteínas/análiseRESUMO
Genetically marked hepatocytes from dipeptidyl peptidase (DPP) IV+ Fischer 344 rats were transplanted into the liver of DPPIV- mutant Fischer 344 rats after a combined treatment with retrorsine, a pyrrolizidine alkaloid that blocks the hepatocyte cell cycle, and two-thirds partial hepatectomy. In female rats, clusters of proliferated DPPIV+ hepatocytes containing 20 to 50 cells/cluster, mostly derived from single transplanted cells, were evident at 2 weeks, increasing in size to hundreds of cells per cluster at 1 month and 1000 to several thousand cells per cluster at 2 months, representing 40 to 60% of total hepatocyte mass. This level of hepatocyte replacement remained constant for up to 1 year, the duration of experiments conducted. In male rats, liver replacement occurred more rapidly and was more extensive, with transplanted hepatocytes representing 10 to 15% of hepatocyte mass at 2 weeks, 40 to 50% at 1 month, 90 to 95% at 2 months, 98% at 4 months, and 99% at 9 months. Transplanted hepatocytes were integrated into the parenchymal plates, exhibited unique hepatic biochemical functions, and fully reconstituted a normal hepatic lobular structure. The extensive proliferation of transplanted cells in this setting of persistent inhibition of resident hepatocytes represents a new general model to study basic aspects of liver repopulation with potential applications in chronic liver disease and ex vivo gene therapy.
Assuntos
Transplante de Células/métodos , Transplante de Fígado/métodos , Fígado/citologia , Alcaloides de Pirrolizidina/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Divisão Celular , Dipeptidil Peptidase 4/metabolismo , Feminino , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Hepatectomia , Queratinas/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Mutantes , Albumina Sérica/metabolismo , Fatores Sexuais , Fatores de TempoRESUMO
The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.
Assuntos
Fígado/patologia , Pâncreas/patologia , Células-Tronco/patologia , Animais , Diferenciação Celular , Transplante de Células , Epitélio/patologia , Masculino , Transplante de Pâncreas , Ratos , Ratos Endogâmicos F344RESUMO
The pattern of mRNA expression for liver-specific proteins and liver-enriched transcription factors was studied in two models of facultative gut epithelial progenitor cells activation: D-galactosamine (GalN)-induced liver injury and dietary copper depletion leading to pancreatic acinar atrophy. After 5 weeks of copper deficiency (CuD), pancreatic acini of Fischer 344 rats underwent atrophy, associated with intense proliferation of small duct-like cells with oval-shaped nuclei. These cells resemble morphologically epithelial progenitor cells of the liver that proliferate after GalN administration. Activated pancreatic epithelial cells express mRNAs for liver-specific genes normally expressed in fetal liver, including alpha-fetoprotein, albumin, alpha-1 antitrypsin, glucose-6-phosphatase, and others, but not genes that are turned on after birth such as serine dehydratase, tyrosine aminotransferase, and multidrug resistance gene-1b. They express mRNAs for liver-enriched transcription factors including HNF-1 alpha, HNF-3 beta and gamma, HNF-4, and members of the CCAAT-enhancer binding protein (C/EBP) family. The only mRNA for a liver-enriched transcription factor not detected in the pancreas of CuD animals was HNF-3 alpha. Expression of HNF-3 alpha, beta, and gamma, and C/EBP-beta mRNA was highly activated in proliferating liver epithelial cells on days 2 and 3 after GalN injury. Increased expression of C/EBP-delta was observed first in the liver on day 1 after GalN administration and in the pancreas at 4 weeks after initiating CuD. We suggest that C/EBP-delta could be involved in the initial activation of epithelial progenitor cells and that HNF-3 alpha, beta, and gamma, and C/EBP-beta might participate in their maturation. We conclude further that pancreatic epithelial progenitor cells undertake differentiation through the hepatocyte lineage but cannot complete the differentiation program within the pancreatic milieu.
Assuntos
Fígado/metabolismo , Pâncreas/metabolismo , Fatores de Transcrição/biossíntese , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Divisão Celular/efeitos dos fármacos , Cobre/efeitos adversos , Cobre/deficiência , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Epitélio/química , Epitélio/efeitos dos fármacos , Galactosamina , Fígado/citologia , Masculino , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Pâncreas/citologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Fatores de Transcrição/genéticaRESUMO
Activation of liver progenitor cells was studied in rat liver induced to regenerate after carbon tetrachloride (CCl4) or D-galactosamine (GalN) injury. A change in the concentration of histone-3 mRNA was used as a marker for cell proliferation and the fetal form of alpha-fetoprotein (AFP) mRNA as a marker for fetal hepatoblasts. gamma-Glutamyltranspeptidase (GGT) and glutathione-S-transferase P were used as markers for activation of putative liver progenitor cells. After CCl4 administration, the proliferative response was high but confined primarily to parenchymal cells. No changes in the relative expression of albumin, glutathione-S-transferase P or insulin-like growth factor-II were observed. On the other hand, the level of AFP mRNA was increased modestly and predominantly in the nonparenchymal cell (NPC) fraction. After GalN administration, proliferation of NPC began within 24 hr, primarily in the portal area around the bile ducts. Activated cells were bile "duct-like" in appearance, had scant cytoplasm, and a pale, oval-shaped nucleus. On Day 2, they formed rows and clusters, expanding from the portal zone and invading the parenchyma, as well as proliferating in regions of focal necrosis. On Days 3 and 5, NPC expressing histone-3 mRNA expanded further, forming pseudoducts and islet-like structures (NPC structures) throughout the hepatic lobule. Proliferating NPC were positive for GGT. Some GGT-positive cells on Days 3 and 5 were also positive for fetal AFP mRNA. Expression of fetal AFP mRNA lagged behind that of GGT by 24 hr, was highest on Day 5, and then declined. Expression of albumin mRNA and glucose 6-phosphatase decreased during the first 48 hr after GalN administration and then resumed. These findings indicate that after GalN injury, the liver responds with activation of putative progenitor cells that proliferate and then differentiate through the hepatocyte lineage, whereas the regenerative response after CCl4 administration is primarily through proliferation of preexisting hepatocytes.
Assuntos
Regeneração Hepática , Fígado/citologia , Albuminas/genética , Animais , Intoxicação por Tetracloreto de Carbono/metabolismo , Intoxicação por Tetracloreto de Carbono/patologia , Galactosamina/toxicidade , Expressão Gênica , Glutationa Transferase/genética , Histonas/genética , Hibridização In Situ , Fator de Crescimento Insulin-Like II/genética , Masculino , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Células-Tronco , alfa-Fetoproteínas/genéticaRESUMO
Rat liver regeneration was studied from 24 hours to 8 days after a single intraperitoneal injection of D-galactosamine (GalN). Morphological changes in the liver were analyzed in parallel with sequential changes in expression of histone-3 mRNA (a marker of cell proliferation), fetal alpha-fetoprotein (AFP) mRNA and gamma-glutamyl transpeptidase (GGT) (markers of fetal hepatocytes), and albumin mRNA and glucose-6-phosphatase (G6Pase) (markers of adult hepatocytes). Proliferation of nonparenchymal epithelial cells (NPC), detected in situ by [3H]thymidine labeling or histone-3 mRNA expression, began after 24 hours primarily in the portal area around the bile ducts. After 2 days, histone-3 labelling intensity increased in rows and clusters of NPC which expanded from the portal zone and invaded into the parenchyma. On days 3 and 5, NPC expressing his-3 mRNA expanded further, forming pseudo-ducts and islet-like structures (NPC structures). Proliferating NPC were positive for GGT. Some GGT positive cells were also positive for the fetal form of AFP mRNA, which lagged behind GGT by 24 hours and peaked on day 5. On day 3, some cells with the appearance of NPC expressed albumin mRNA. Double label in situ hybridization for fetal AFP and albumin mRNAs and dual histochemistry for GGT and G6Pase showed simultaneous expression of these markers in NPC on day 5. Other cells expressing fetal AFP mRNA or GGT on day 5 had a morphological appearance between NPC and hepatocytes (transitional cells). Proliferation of hepatocytes began on day 2, reached maximum on day 5 and then declined. Proliferating hepatocytes did not express fetal AFP mRNA or GGT. These findings indicate that after GalN injury, the liver responds by activation of progenitor cells that proliferate and then differentiate into mature hepatocytes. Adult hepatocytes can also proliferate after GAlN injury, but these hepatocytes do not undergo dedifferentiation/redifferentiation during regeneration of the hepatic lobule.
Assuntos
Galactosamina/farmacologia , Regeneração Hepática/fisiologia , Fígado/citologia , Fígado/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Albuminas/análise , Albuminas/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , DNA/metabolismo , Galactosamina/efeitos adversos , Glucose-6-Fosfatase/análise , Glucose-6-Fosfatase/genética , Histocitoquímica , Hibridização In Situ , Fígado/química , Masculino , Modelos Biológicos , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Células-Tronco/química , Timidina/metabolismo , Fatores de Tempo , Trítio , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/genética , gama-Glutamiltransferase/análise , gama-Glutamiltransferase/genéticaRESUMO
Ribosomal protein L32 of Saccharomyces cerevisiae regulates the splicing of its own transcript (1, 2) apparently by interacting with a structure composed largely of the 5' exon. However, even in strains overproducing L32 mRNA, e.g. from a cDNA copy of the gene, little accumulation of L32 is observed after a brief pulse label. When the 5' leader of the RPL32 mRNA is replaced by an exogenous leader, the amount of pulse-labeled L32 increases severalfold, suggesting that L32 regulates the translation of its own mRNA, acting through sequences in the 5' region. This conclusion was confirmed by the observation that in cells carrying a chimeric gene in which the L32 leader is fused to LacZ coding sequences, the presence of a second gene that overexpresses L32 itself reduces the level of beta-galactosidase by 50%, in spite of a doubling of L32-lacZ fusion mRNA, presumably due to stabilization of the message. Mutations within the 5' leader that abolish the regulation of splicing also abolish the regulation of translation, suggesting that the regulation of translation by L32 involves a structure similar to that proposed for the regulation of splicing. In cells overproducing L32-mRNA about half the excess mRNA was found in ribonucleoproteins of < 25 S, unassociated with ribosomal particles. Much of the rest was found in ribonucleoproteins of 80-120 S.
Assuntos
Regulação Fúngica da Expressão Gênica , Biossíntese de Proteínas , Splicing de RNA , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Composição de Bases , Sequência de Bases , Clonagem Molecular , Éxons , Genes Fúngicos , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Polirribossomos/metabolismo , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
Ribosomal protein S7 of Saccharomyces cerevisiae is encoded by two genes RPS7A and RPS7B. The sequence of each copy was determined; their coding regions differ in only 14 nucleotides, none of which leads to changes in the amino acid sequence. The predicted protein consists of 261 amino acids, making it the largest protein of the 40 S ribosomal subunit. It is highly basic near the NH2 terminus, as are most ribosomal proteins. Protein S7 is homologous to both human and rat ribosomal protein S4. RPS7A and RPS7B contain introns of 257 and 269 nucleotides, respectively, located 11 nucleotides beyond the initiator AUG. The splicing of the introns is efficient. Either RPS7A or RPS7B will support growth. However, deletion of both genes is lethal. RPS7A maps distal to CDC11 on chromosome X, and RPS7B maps distal to CUP1 on chromosome VIII.
Assuntos
Genes Fúngicos , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , DNA Fúngico/genética , Humanos , Dados de Sequência Molecular , Splicing de RNA , RNA Fúngico/genética , Ratos , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Cycloheximide (Cyh), administered at a dose of 5 mg/kg body wt blocks protein synthesis in normal rat liver (NRL) and regenerating rat liver (RRL). The rate of synthesis of 45S pre-rRNA in RRL, studied after RNA labelling in vivo is activated 2.8 times. Pre-r RNA synthesis in RRL is more sensitive to the stopped translation, but never falls down to the level in NRL. The major contribution to the rDNA transcription activation in RRL comes from the 20-fold increase in the number of pol I molecules engaged in the transcription, the elongation rate being 1.4-fold accelerated. Cyh quenches partially the enhanced rDNA transcription in RRL: the number of pol I molecules and their elongation rate are about 1.7-fold and 1.5-fold higher, respectively, than the corresponding values in NRL after Cyh treatment. The results show that two different mechanisms control the number and the rate of initiation and elongation of RNA polymerase I in rat liver; one of them depends on continuous protein synthesis and can be inactivated by Cyh, the other is Cyh resistant.
Assuntos
Cicloeximida/farmacologia , DNA Polimerase I/biossíntese , Regeneração Hepática/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Precursores de RNA/biossíntese , RNA Ribossômico/biossíntese , Animais , Regeneração Hepática/fisiologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , RatosRESUMO
The yeast ribosomal protein gene RPL32 of Saccharomyces cerevisiae is of particular interest for two reasons: 1) it is adjacent to another ribosomal protein gene, RP29, whose divergent transcription may be driven from the same control sequences, and 2) it appears that the splicing of its transcript is regulated by the product of the gene, ribosomal protein in L32. RPL32 has been analyzed in detail. It is essential for cell growth. Its sequence predicts L32 to be a protein of 105 amino acids, somewhat basic near the NH2 terminus, rather acidic near the COOH terminus, and homologous to ribosomal protein L30 of mammals. The reading frame has been confirmed by partial NH2-terminal analysis of L32. The nucleotide sequence also predicts an intron of 230 nucleotides, which begins with the unusual sequence GTCAGT and ends 40 nucleotides downstream of the consensus sequence TAC-TAAC. The intron has been confirmed by determination of the sequence of a cDNA clone. Transcription initiates 58 nucleotides upstream of the AUG initiation codon, and the polyadenylation site occurs 100 nucleotides downstream of the termination codon. Regulation of the transcription of ribosomal protein genes has been linked to two related consensus sequences. Analysis of the intergenic region between RP29 and RPL32 reveals three copies of these sequences. A deletion removing all three sequences reduces synthesis of a L32-LacZ fusion protein by more than 90%. Some residual activity, however, remains.
Assuntos
Genes Fúngicos , Genes , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , PlasmídeosRESUMO
The rates of synthesis and degradation of ribosomal proteins, prelabelled with [14C]bicarbonate, were determined as an index of the rate of ribosome turnover in regenerating rat liver. The half-life of ribosomes is about 178 and 75 hr in regenerating and normal liver, respectively. The comparison of turnover rates of ribosomal proteins with the corrected values of rRNA, based on re-utilization of nucleotides, suggests that ribosomes are metabolized as a unit in vivo. There is at least 70% overestimation for ribosome half-life when orotate-labelled RNA is used for turnover determinations. The absolute rate of synthesis is estimated as 3925 and 1081 ribosomes/min per cell in 24 hr regenerating and normal rat liver, respectively.
Assuntos
Hepatectomia , Regeneração Hepática , Fígado/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Meia-Vida , Cinética , Masculino , RNA Ribossômico/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.
Assuntos
Genes Fúngicos , Genes Reguladores , Genes , Splicing de RNA , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Biossíntese de Proteínas , Transcrição GênicaRESUMO
The absolute amounts of precursor to ribosomal RNA (pre-rRNA) and ribosomal RNA (rRNA) in isolated rat brain neuronal and oligodendroglial nuclei were determined. The amount of the major pre-rRNA and rRNA species in neuronal nuclei was about twofold higher than in oligodendroglial nuclei. The relative rate of pre-rRNA synthesis in vivo was 2.3- to 2.7-fold higher in neuronal as compared with oligodendroglial nuclei. This corresponds to a 2.7-fold higher activity of the "template-bound" RNA polymerase I in isolated neuronal nuclei, whereas the activity of the "free" enzyme in both neuronal and glial nuclei was almost identical. The higher transcription rates of rRNA genes correlated with the markedly more prominent fibrillar component in neuronal nucleoli. The turnover times of the major pre-rRNA and rRNA species in neuronal and oligodendroglial nuclei were similar, except for 45S pre-rRNA, which turned over at an approximately 1.5-fold slower rate in neuronal nuclei. The relative rates of processing of pre-rRNA and of nucleocytoplasmic transport of rRNA in neuronal cells were approximately 2.7-fold higher than in oligodendroglial cells and corresponded to the differences in rRNA gene transcription rates. The established ribosome formation features correlated with an abundant (neurons) or exceedingly scarce (oligodendrocytes) nucleolar granular component. The turnover rate of cytoplasmic ribosomes in rat brain neurons was twofold slower than in oligodendrocytes, largely because of the about fivefold higher amount of ribosomes in the cytoplasm of neurons. We conclude that ribosome formation and turnover in neuronal and oligodendroglial cells are adapted to the protein synthetic levels in these two types of brain cells.
Assuntos
Encéfalo/citologia , Nucléolo Celular/ultraestrutura , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Oligodendroglia/ultraestrutura , Ribossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Microscopia Eletrônica , Neurônios/metabolismo , Precursores de Ácido Nucleico/biossíntese , Oligodendroglia/metabolismo , RNA Polimerase I/metabolismo , Precursores de RNA , RNA Ribossômico/biossíntese , Ratos , Transcrição GênicaRESUMO
The changes in the specific radioactivities of the pool of total acid-soluble uridine nucleotides and of uridine and cytidine components of total cellular and nuclear RNA were monitored in regenerating rat liver for 12 days after partial hepatectomy. Evidence is presented for the re-utilization of pyrimidine nucleotides derived from cytoplasmic RNA degradation for the synthesis of new RNA. The extent of recycling was assessed and the true rate of rRNA turnover determined more accurately. The reutilization of the uridine components of RNA was 7.0%/day during the proliferative and 3.2%/day during the post-proliferative phase, whereas that of the cytidine nucleotides was more pronounced (9.6%/day and 18.1%/day respectively). The results reveal the existence of partial compartmentalization of pyrimidine ribonucleoside triphosphate pools in the nucleus and cytoplasm of rat liver cells.
Assuntos
Regeneração Hepática , Nucleotídeos de Pirimidina/metabolismo , Animais , Fracionamento Celular , Núcleo Celular/metabolismo , Cinética , Fígado/metabolismo , Masculino , RNA/metabolismo , Ratos , Ratos Endogâmicos , Uridina Monofosfato/metabolismoRESUMO
The ribosome formation in four experimental groups: normal, adrenalectomized, partially hepatectomized and adrenalectomized - partially hepatectomized rats was studied. Ribosomal RNA was labelled for different intervals and the transfer of the radioactivity from 45 S pre-rRNA through the nucleolar pre-rRNA and rRNA pools into cytoplasmic 28S and 18S rRNA was followed. The results show that there are at least two ways of positive control of rRNA synthesis, one of them being glucocorticoid-dependent. The acceleration of the pre-rRNA processing through the shortest maturation pathway in regenerating liver is reduced in the absence of the hormone. Glucocorticoids do not influence nucleo-cytoplasmic rRNA transport.