RESUMEN
The messenger RNAs specifying certain proteins involved in the inflammatory response and certain oncoproteins contain a conserved UA-rich sequence in the 3' untranslated region. This sequence, which is composed of several interspersed repeats of the octanucleotide UUAUUUAU, has been shown to destabilize mRNA in some eukaryotes. However, this effect is not seen when mRNAs are transferred to Xenopus oocytes, which made it possible to separate stability from translational regulation. For interferon, granulocyte-macrophage colony-stimulating factor, and c-fos RNAs, the UA-rich sequence was observed to preclude mRNA translation.
Asunto(s)
Factores Estimulantes de Colonias/genética , Sustancias de Crecimiento/genética , Interferón Tipo I/genética , Biosíntesis de Proteínas , Proto-Oncogenes , Animales , Secuencia de Bases , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Datos de Secuencia Molecular , ARN Mensajero/metabolismoRESUMEN
The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3' untranslated region (3'UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3'UTR containing 8 nucleotides more than the short 3'UTR allows the recovery of an efficiency of translation similar to that of the long 3'UTR. Moreover, the two guanine residues located at the 3' ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3'UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3'UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.
Asunto(s)
Regiones no Traducidas 3' , Precursor de Proteína beta-Amiloide/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Células CHO/metabolismo , Cricetinae , Repeticiones de Dinucleótido , Femenino , Humanos , Datos de Secuencia Molecular , Oocitos/metabolismo , Poli A , ARN Mensajero/metabolismo , Extractos de Tejidos/metabolismo , XenopusRESUMEN
The human c-myc proto-oncogene is transcribed from four alternative promoters (P0, P1, P2, and P3) giving rise to mRNAs having 5' leader sequences of various length. The c-myc P0 mRNA contains three open reading frames (ORFs), the last one encoding c-Myc1 and c-Myc2 proteins generated by alternative translation initiated at CUG and AUG codons. The middle ORF (MYCHEX1) and the 5' ORF (ORF1) code for proteins 188 and 114 amino acids in length, respectively. We and others previously identified an internal ribosome entry site (IRES) in P0 and P2 c-myc mRNAs, promoting the cap-independent translation of c-Myc1 and c-Myc2. Here, we report the presence of a second IRES (named IRES1) promoting the cap-independent translation of MYCHEX1 in c-myc P0 mRNA. Using deletion analysis, we mapped an 80-nt region essential for IRES1 activity. c-myc P0 mRNA is thus the first eukaryotic polycistronic mRNA described for which translation initiation of two different open reading frames (MYCHEX1 and c-Myc1/c-Myc2) involves internal ribosome entry.
Asunto(s)
Genes myc , Biosíntesis de Proteínas , ARN Mensajero/genética , Ribosomas/genética , Regiones no Traducidas 5' , Secuencia de Bases , Codón , Cartilla de ADN , Células HeLa , Humanos , Sistemas de Lectura Abierta , Proto-Oncogenes MasRESUMEN
The turnover of tumor necrosis factor (TNF) mRNA in permanently transfected macrophages of the RAW 264.7 cell line was studied directly (by Northern blot analysis using a probe specific for TNF) and indirectly (through studies of the turnover of various reporter mRNAs, either containing or lacking the TNF 3' untranslated region (UTR)). The TNF mRNA was found to be very unstable in RAW 264.7 cells. Instability appeared to result from two distinguishable nucleolytic processes. The major degradative process involved was not specific for the TNF 3' UTR of reporter mRNAs, and was inhibited by actinomycin D pretreatment. It appeared to be expressed constitutively, in that cell activation by lipopolysaccharide (LPS) did not modify message stability. When cells were treated with actinomycin D, a minor nucleolytic activity was 'uncovered'. This minor activity was noted to increase with time following LPS activation. It also exhibited specificity, in that reporter mRNAs bearing the 3' UTR of TNF were more susceptible to degradation in the presence of actinomycin D than were constructs lacking the 3' UTR of TNF. Thus, TNF mRNA turnover appears complex, and depends upon at least two separable degradative pathways. The TNF 3' UTR apparently contributes only modestly to the instability of this mRNA under normal conditions.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Macrófagos/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genética , Animales , Northern Blotting , Línea Celular , Dactinomicina/farmacología , Cinética , Lipopolisacáridos/farmacología , Activación de Macrófagos/fisiología , Macrófagos/efectos de los fármacos , Ratones , ARN Mensajero/genéticaRESUMEN
The aim of the present work was to estimate the effect of intracellular glutathione depletion on melanogenesis in human melanoma cells. We determined tyrosine hydroxylation activity, the rate-limiting step of the pathway, and 14C-melanin formation, an assay reflecting the global eumelanogenic pathway. Intracellular glutathione was depleted by treatment with buthionine-S-sulfoximine, a well-known inhibitor of gamma-glutamylcysteine synthetase. The intracellular depletion of glutathione was substantial after 20 h of incubation with 50 microM buthionine-S-sulfoximine, although a significant effect could be observed after 6 h. Tyrosine hydroxylase activity increased in parallel with glutathione depletion, to reach 160% with respect to the control values during 24 h of buthionine-S-sulfoximine treatment. We have found the response to buthionine-S-sulfoximine to be dose dependent and the two different human cell lines HBL and LND1 to have similar, if not identical, responses. 14C-melanin formation assay revealed even greater activation, up to 400% of the control values. This indicates that glutathione depletion may have two distinct effects: first, a direct one on tyrosinase activity and, second, an effect on the promotion of eumelanogenesis. The stimulation of tyrosine hydroxylase can be explained by a possible inactivation of the enzyme by endogenous thiol compounds rather than by a direct effect of buthionine-S-sulfoximine itself on tyrosinase. The data suggest that thiol compounds may play a role for stimulation of melanogenesis by ultraviolet radiation.
Asunto(s)
Glutatión/deficiencia , Melanoma/enzimología , Monofenol Monooxigenasa/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Butionina Sulfoximina , Humanos , Melaninas/biosíntesis , Melanoma/patología , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Compuestos de Sulfhidrilo/farmacología , Factores de Tiempo , Células Tumorales Cultivadas , Tirosina 3-Monooxigenasa/antagonistas & inhibidores , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.
Asunto(s)
Interferón Tipo I/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Secuencia de Bases , Femenino , Humanos , Datos de Secuencia Molecular , Oocitos/metabolismo , Plásmidos , Conejos , Reticulocitos/metabolismo , Transcripción Genética , XenopusRESUMEN
The IFI-56K and IFI-54K human genes are coordinately regulated by interferon, double-stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI-56K and IFI-54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.
Asunto(s)
Cromosomas Humanos Par 10 , Cromosomas Humanos Par 13 , Regulación de la Expresión Génica , Genes , Interferón Tipo I/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Peso MolecularRESUMEN
The early embryonic development of Xenopus is mainly governed by post-transcriptional regulations until the mid-blastula transition. In this report, we present evidence demonstrating that fertilization of Xenopus eggs triggers a complete translational arrest of mRNAs containing UA-rich elements in their 3'-untranslated region. This control is maintained at least until the mid-blastula transition. Neither maturation nor pseudo-fertilization of the egg is sufficient for triggering this control, suggesting that components originating from the male gamete are involved in the mechanism. Moreover, this control is exerted whether the mRNA is polyadenylated or not.
Asunto(s)
Fertilización , Oocitos/fisiología , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/metabolismo , Femenino , Técnicas In Vitro , Cinética , Masculino , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Plásmidos , Progesterona/farmacología , ARN Mensajero/química , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , Espermatozoides/fisiología , Factores de Tiempo , Transcripción Genética , Xenopus laevisRESUMEN
In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.
Asunto(s)
AMP Cíclico/farmacología , Proteínas Quinasas/farmacología , Glándula Tiroides/ultraestructura , Actinas/metabolismo , Animales , Bucladesina/farmacología , Catálisis , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Células Cultivadas , Colforsina/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/ultraestructura , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Perros , Activación Enzimática , Epitelio/ultraestructura , Técnica del Anticuerpo Fluorescente , Microinyecciones , Proteínas Quinasas/metabolismo , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacologíaRESUMEN
2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.
Asunto(s)
Nucleótidos de Adenina/farmacología , Antivirales/farmacología , Oligorribonucleótidos/farmacología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Interferones/farmacología , Mengovirus/efectos de los fármacos , Microinyecciones , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Interferon-induced 2-5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'-non-coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2-5A synthetase was clearly observed in the cytoplasm and 10-30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Interferón Tipo I/farmacología , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/análisis , Sistema Libre de Células , Citoplasma/análisis , ADN/análisis , ADN Recombinante , Inducción Enzimática/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Microinyecciones , Oocitos/metabolismo , Empalme del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Xenopus laevisRESUMEN
We have investigated the relationship in human cultured normal and malignant melanocytes between the accumulation of mRNAs encoding tyrosinase and tyrosinase-related protein-1 (TRP-1), the activity of tyrosinase and the presence of melanin. Tyrosinase mRNA correlates with tyrosinase activity and with the presence of pheomelanin, eumelanin or both melanin types. In contrast TRP-1 mRNA is only detectable in cells containing eumelanin, which suggests a role for TRP-1 in the eumelanin synthesis pathway.
Asunto(s)
Melaninas/biosíntesis , Melanocitos/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana , Oxidorreductasas , Proteínas/metabolismo , Secuencia de Bases , Células Cultivadas , ADN de Cadena Simple , Humanos , Melaninas/metabolismo , Melanocitos/enzimología , Datos de Secuencia Molecular , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Proteínas/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Several messenger RNAs which are transiently expressed contain a conserved uridine-adenosine-rich sequence in their 3' untranslated region. Many of these mRNas encode cytokines, growth factors or oncoproteins. This UA-rich sequence is composed of several interpsersed repeats of the octanucleotide UUAUUUAU and plays a key role in the post-transcriptional regulation of these mRNAs. Known as instability determinants, these UA-rich elements can also strongly affect mRNA translational efficiency. In this report, we review the data which illustrate this translational regulation and give insight the underlying mechanism.
Asunto(s)
Secuencia de Bases/genética , Citocinas/biosíntesis , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Adenosina/genética , Animales , Secuencia de Bases/fisiología , Citocinas/fisiología , Humanos , Secuencias Reguladoras de Ácidos Nucleicos , Secuencias Repetitivas de Ácidos Nucleicos , Uridina/genéticaRESUMEN
Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.
Asunto(s)
Bombyx/metabolismo , Fibroínas/biosíntesis , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Reticulocitos/metabolismo , Animales , Sistema Libre de Células , Electroforesis en Gel de Poliacrilamida , Técnicas In Vitro , Extensión de la Cadena Peptídica de Translación , ConejosRESUMEN
The nested reverse transcriptase polymerase chain reaction (RT-PCR) provides a powerful tool for detection of minimal residual disease in CML. The RT-PCR used in the present study for detection of the major bcr-abl fusion gene, the hallmark and presumably the cause of CML, was optimized by: (a) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells; (b) using a specific abl primer in this reverse transcriptase reaction, and (c) reamplifying 10% of the RT-PCR product in a nested amplification. This optimized RT-PCR permitted to detect up to 1 copy of RNA bcr-abl synthesized in vitro, mixed with yeast RNA in a quantity equivalent to 10(8) white blood cells (WBC). Using the highly sensitive RT-PCR, a systematic study of the possible expression of bcr-abl RNA in WBC of healthy adults, children and umbilical cord blood (UCB) revealed the presence of bcr-abl transcripts in blood cells of 22/73 adults, 1/22 children but not in 22 samples of UCB. The comparison of these three groups indicated a significant tendency for the anomaly to increase in frequency with age.
Asunto(s)
Genes abl , ARN Mensajero/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Células Sanguíneas/metabolismo , Células Sanguíneas/ultraestructura , Niño , Preescolar , Expresión Génica , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Cromosoma Filadelfia , Reacción en Cadena de la Polimerasa , ARN Mensajero/sangre , Valores de ReferenciaRESUMEN
Tumor necrosis factor (TNF)-a gene expression can be induced primarily in cells of the monocyte-macrophage lineage by a variety of inducers, including lipopolysaccharides (LPS), phorbol esters, ultraviolet (UV) light, and viruses. In this paper, we analyzed the regulatory mechanisms of TNF-alpha production induced by infection with the Sendai" virus in RAW 264.7 macrophages. We show that in these cells TNF-a synthesis results mainly from TNF-alpha mRNA translational activation. Using CAT reporter genes, we identified the UA- rich (UAR) sequences localized in the TNF-alpha mRNA 3' untranslated region (UTR) as the main sequence involved in this regulation. This sequence has been previously shown to be the essential regulatory element involved in LPS- induced translational activation of TNF mRNA. Activation of TNF gene expression by viral infection presents other similarities with those induced by LPS. First, TNF production in response to viral infection is inhibited by the protein-tyrosine kinase inhibitor herbimycin A as it is in response to LPS. More specifically, we show here that TNF mRNA translational activation induced by viral infection or by LPS is inhibited by pretreating the cells with herbimycin A. Second, TNF production in response to viruses is tissue-specific and is abrogated in RAW 264.7x NIH3T3 hybrid cells, which lack the ability to produce TNF in response to LPS, as a consequence of a defect in the LPS signaling pathway. However, viral infection induces TNF production in LPS- unresponsive C3H/HeJ mouse-derived peritoneal macro phages indicating that viruses and LPS signaling pathways differ for at least one intermediate which is the product of the Lps gene. Finally, we show that this regulatory mechanism can be triggered by different classes of viruses.
Asunto(s)
Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Macrófagos/virología , Respirovirus/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Células 3T3 , Animales , Benzoquinonas , Cloranfenicol O-Acetiltransferasa/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Genes Reporteros , Lactamas Macrocíclicas , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C3H , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , ARN Mensajero/química , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Rifabutina/análogos & derivados , Transducción de Señal , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Macrophage activation by lipopolysaccharide (LPS) results in the translational activation of tumor necrosis factor (TNF) mRNA. The initial phase of macrophage activation is followed by a refractory state called LPS tolerance characterized by an impaired TNF production in response to a secondary LPS challenge. LPS-tolerant macrophages contain high amounts of TNF mRNA, suggesting a translational regulation of TNF biosynthesis. The induction of LPS tolerance was studied in RAW 264.7 macrophages stably transfected with a chloramphenicol acetyl-transferase (CAT) reporter gene construct driven by a constitutive cytomegalovirus promoter and containing the 3' untranslated region of the murine TNF gene. We found that primary stimulation of transfected cells by LPS (1 ng/ml, 12 hr) resulted in a marked suppression (80%) of CAT accumulation in response to a secondary LPS challenge (1 microgram/ml, 6 hr). In contrast, the accumulation of CAT mRNA was not influenced by LPS tolerance. Using the same CAT reporter, we observed that the serine/threonine phosphatases 1 and 2A inhibitor okadaic acid induced TNF mRNA translation and that this activation was not inhibited by LPS-tolerance. In conclusion, these data indicate that deficient production of TNF in LPS-tolerant macrophages in response to a second LPS challenge is characterized by a defective translation of TNF mRNA. However, this hyporesponsiveness to LPS is specific, since translation of TNF mRNA induced by okadaic acid is not inhibited in LPS-tolerant macrophages.
Asunto(s)
Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Citomegalovirus/genética , Tolerancia a Medicamentos , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , Regulación de la Expresión Génica , Genes Reporteros , Cinética , Activación de Macrófagos , Ratones , Ácido Ocadaico , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The kindling model of epilepsy is associated with long-lasting changes in type II calmodulin kinase (CaM kinase) activity and immunoreactivity. In order to determine the mechanism of these alterations, we measured gene expression of CaM kinase using in situ hybridization in septally kindled rat brains and paired controls using a 35S-labeled riboprobe for the beta subunit of the enzyme. We found CaM kinase mRNA concentrated in the hippocampus and other limbic structures. Kindling decreased hippocampal CaM kinase mRNA by 30% in CA1, 34% in CA2, 35% in CA3 41% in CA4, and 29% in the dentate gyrus. Hybridization was also decreased by 21% in the cerebral cortex but not in the lateral septum. These changes are similar in distribution and direction to those previously measured by immunohistochemistry. These data suggest that altered CaM kinase activity and immunoreactivity associated with kindling reflect long-lasting alterations in gene expression of this important synaptic protein, and provide further evidence for its possible importance in the kindling phenomenon.