RESUMEN
Phosphate transport protein (PTP) is a mitochondrial inner membrane protein responsible for the translocation of inorganic phosphate into the mitochondrial matrix. A full length cDNA clone encoding the PTP was isolated from the spruce budworm, Choristoneura fumiferana. The deduced amino acid sequence of the longest ORF of CfPTP cDNA showed high similarity with the amino acid sequences of PTPs cloned from several species. Phylogenetic tree analysis indicated that CfPTP occupied an intermediate position between vertebrates on the one side and yeast and nematodes on the other side. Studies on the developmental expression of CfPTP mRNA showed that higher levels of mRNA were present during the feeding and growing stages than during molting periods.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Mitocondrias , Mariposas Nocturnas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/biosíntesis , Clonación Molecular , ADN Complementario/genética , Evolución Molecular , Expresión Génica , Genes de Insecto , Larva/metabolismo , Proteínas de la Membrana/biosíntesis , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/metabolismo , Proteínas de Unión a Fosfato , Fosfatos/metabolismo , Filogenia , Pupa/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Cloning and characterization of a cDNA of the spruce budworm, Choristoneura fumiferana, that showed high amino acid similarity with the deduced amino acid sequences of E75 cDNAs cloned from Manduca sexta, Galleria melonella, and Drosophila melanogaster are described. Initially, a cDNA fragment and then a full length cDNA were cloned from C. fumiferana. The longest open reading frame of this cDNA had 690 codons and its deduced amino acid sequence had all five domains typical of a steroid hormone nuclear receptor. The deduced amino acid sequence of this cDNA showed the highest identity with the deduced amino acid sequence of E75A cDNAs cloned from M. sexta, G. melonella, and D. melanogaster, and is therefore named Choristoneura hormone receptor 75A (CHR75A). The CHR75A cDNA probe detected a 2.6 kb mRNA that was abundant at the time of the ecdysteroid peaks during molting in the embryonic, larval and pupal stages. In the sixth instar larvae, CHR75 mRNA was detected in the epidermis, fat body and midgut, and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR75 mRNA was induced in ecdysone treated CF-203 cells and in the midgut, fat body and epidermis of larvae that were fed the non-steroidal ecdysteroid agonist, RH-5992. In vitro transcription and translation of the CHR75A cDNA yielded a 79 kDa protein that bound to the retinoic acid receptor related orphan receptor response element (RORE).
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Genes de Insecto , Proteínas de Insectos , Mariposas Nocturnas/genética , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Proteínas de Unión al ADN/biosíntesis , Ecdisona/agonistas , Ecdisona/fisiología , Ecdisterona/farmacología , Epidermis/crecimiento & desarrollo , Epidermis/metabolismo , Cuerpo Adiposo/crecimiento & desarrollo , Cuerpo Adiposo/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hemolinfa/química , Hidrazinas/farmacología , Mucosa Intestinal/metabolismo , Intestinos/crecimiento & desarrollo , Larva , Datos de Secuencia Molecular , Mariposas Nocturnas/embriología , Mariposas Nocturnas/crecimiento & desarrollo , Especificidad de Órganos , Unión Proteica , Pupa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Esteroides/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Choristoneura hormone receptor 3 (CHR3) is a 20E (20-hydroxyecdysone)-induced delayed early gene that is homologous to Manduca hormone receptor 3 (MHR3), Drosophila hormone receptor 3 (DHR3), and Galleria hormone receptor 3 (GHR3). We recently cloned and characterized a cDNA that was copied from the 4.5 kb CHR3B mRNA. To isolate additional CHR3 isoforms, the Choristoneura fumiferana embryonic cDNA library was screened using CHR3B cDNA as a probe. Characterization and partial sequencing of 16 clones showed that one of them differed from the CHR3B in two regions. This cDNA (CHR3C) was completely sequenced; the sequence analysis showed that the longest open reading frame had 651 codons. The deduced amino acid sequence of this open reading frame contained all five domains that are typical for a steroid hormone nuclear receptor. The nucleotide sequence of CHR3C cDNA is identical to the nucleotide sequence of CHR3B cDNA except for two major differences in the A/B and D-domains. The CHR3C specific probes detected two mRNAs 5.4 kb (CHR3C), and 6.4 kb (CHR3D), which were present in the pupal stage. The CHR3C and CHR3D mRNAs are induced by the stable ecdysteroid analog RH-5992. The CHR3C protein also binds to the response element of the retinoic acid receptor-related orphan receptor.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Insectos , Mariposas Nocturnas/genética , Receptores de Péptidos de Invertebrados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Hidrazinas/farmacología , Hormonas Juveniles/farmacología , Datos de Secuencia Molecular , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Pupa , ARN Mensajero , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos de Invertebrados/metabolismo , Transactivadores/metabolismoRESUMEN
Degenerate oligonucleotides and cDNA converted from Choristoneura fumiferana embryonic RNA were used in a polymerase chain reaction (PCR) procedure to isolate a 683 bp cDNA fragment. Comparison of the deduced amino acid sequence of this cDNA fragment showed that it was a region of an MHR3-like gene from C. fumeferana; we therefore named it Choristoneura hormone receptor 3 (CHR3). This CHR3 cDNA fragment was used as a probe to screen a C. fumiferana embryonic cDNA library. Twenty clones were isolated and two overlapping clones were sequenced. The longest open reading frame of CHR3 cDNA codes for 546 amino acids. The deduced amino acid sequence of this open reading frame contained all five regions typical of a steroid hormone nuclear receptor. The C domain showed the highest identity to Manduca hormone receptor 3 (MHR3), Drosophila hormone receptor 3 (DHR3) and Galleria hormone receptor 3 (GHR3). The A/B, D and E domains also showed significant amino acid similarity with MHR3, DHR3 and GHR3. The 683 bp CHR3 cDNA probe detected two mRNAs of 3.8 and 4.5 kb present during the ecdysteroid peaks for embryonic, larval, pupal and adult molts but were not detected during the intermolt periods. In sixth instar larvae, the 3.8 and 4.5 kb mRNA were detected in the epidermis, fat body and midgut tissues and the maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR3 mRNA was induced in 20-hydroxyecdysone treated CF-203 cells as well as in the midgut, fat body and epidermis of larvae that were fed the non-steroidal molting hormone agonist, RH-5992. In vitro transcription and translation of the CHR3 cDNA yielded a 61 kDa protein that bound to the retinoid related orphan receptor response element.