RESUMEN
Archaebacterial plasma membranes contain an ATPase acting in vivo as a delta mu H(+)-driven ATP synthase. While functional features and their general structural design are resembling F-type ATPases, primary sequences of the two large polypeptides from the catalytic part are closely related to V-type ATPases from eucaryotic vacuolar membranes. The chimeric nature of archaebacterial ATPase from Sulfolobus was investigated in terms of nucleotide interactions and related to specific sequence parameters in a comparison to well known F- and V-type ATPases. The study disclosed a general difference of F- and V-type ATPases at one class of the nucleotide binding sites.
Asunto(s)
Archaea/enzimología , ATPasas de Translocación de Protón/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Quimera , Datos de Secuencia Molecular , Conformación Proteica , ATPasas de Translocación de Protón/metabolismo , Alineación de SecuenciaRESUMEN
Cricket- or B-type allatostatins [W(X(6))W-amides] inhibit the biosynthesis of juvenile hormones in vitro in crickets. Peptides of this family are present also in other insects where they may bare different functions. Here we report the identification of a partial sequence of the B-type preproallatostatin from Gryllus bimaculatus. By PCR screening of a random primer cDNA library and by RACE, a 535bp 3'cDNA sequence was obtained which encodes a putative translation product of 85 amino acids, containing three copies of Grybi-AST B1 and one copy each of Grybi-AST B2, Grybi-AST B3, and Grybi-AST B6. The last represents a novel member of this peptide family. By means of one-step RT-PCR, RNA dot blot, and RT in situ PCR analyses the mRNA expression of the gene in the central nervous system and the digestive tract of female adult crickets was demonstrated. The results confirm that the B-type allatostatins of G. bimaculatus are brain-gut peptides.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Gryllidae/genética , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Clonación Molecular , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/metabolismo , Biblioteca de Genes , Gryllidae/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
The gene encoding allatostatins (AST) of the FGLamide family from the cricket Gryllus bimaculatus is expressed in the brain. The mRNA, which contains four polyadenylation signals, encodes a hormone precursor that is split into at least 14 putative hormones. Five of them have been previously found in the cricket, six to seven others, or their close homologues, are known from other insects. Hormone AST 2 contains an internal cleavage site and may exist in a shorter version 2b. The hormones AST 3 and 4 are identical. The cDNA sequence revealed that a single point mutation and a single deletion eliminated an additional hormone between AST 12 and 13. The deduced hormone precursor is very similar to that in cockroaches, but is different from a shorter precursor in locusts, indicating that the gene evolved very fast in the latter. Regions conserved between cockroaches and crickets include parts of the acidic spacers that separate clusters of hormones, suggesting that these spacers may have additional functions.
Asunto(s)
Gryllidae/genética , Neuropéptidos/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario/genética , Femenino , Masculino , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/análisis , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Allatotropin (AT) is a 13-residue amidated neuropeptide, first isolated from pharate adult heads of the tobacco hornworm, Manduca sexta (Manse-AT), which strongly stimulates the biosynthesis of juvenile hormones (JH) in the corpora allata (CA) of adult moths. In Spodoptera frugiperda, a cDNA that encodes 134 amino acids, including an AT peptide, has been cloned. The S. frugiperda allatotropin mature peptide (Spofr-AT) [GFKNVEMMTARGFa] is identical to that isolated from M. sexta. The basic organization of the Spofr-AT precursor is similar to that of Agrius convolvuli, M. sexta, Pseudaletia unipuncta, and Bombyx mori with 83-93% amino acid sequence identity. The Spofr-AT gene is expressed in at least three mRNA isoforms with 134, 171 and 200 amino acids, differing from each other by alternative splicing. All allatostatins (AS) have an inhibitory action on the JH biosynthesis in the CA. A cDNA that encodes 125 amino acid residues including one copy of the Manse-AS peptide has been cloned from S. frugiperda (Spofr-AS; QVRFRQCYFNPISCF). The basic organization of the Spofr-AS precursor is similar to that of P. unipuncta with 85% amino acid sequence identity. Using one step RT-PCR for semi-quantification of the gene expression, we showed that the three mRNAs of the Spofr-AT gene and the Spofr-AS gene are expressed in brains of last instar larvae, prepupae, pupae, and adults of both sexes of S. frugiperda with variable intensity.
Asunto(s)
ADN Complementario/aislamiento & purificación , Hormonas de Insectos/genética , Manduca/genética , Neuropéptidos/genética , Precursores de Proteínas/genética , Spodoptera/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , ADN Complementario/genética , Hormonas de Insectos/química , Manduca/enzimología , Datos de Secuencia Molecular , Neuropéptidos/química , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spodoptera/enzimologíaRESUMEN
The allatostatin (AST) type A gene of the cricket Gryllus bimaculatus encodes a hormone precursor including at least 14 putative peptides with a common C-terminus Y/FXFGL/Iamide. By RT-PCR we have analyzed the expression of the allatostatin precursor in various tissues of 0-21 days old adult virgin and mated females. In 3-day-old virgin females, the gene is strongly expressed in the brain (oesophageal ganglion), the suboesophageal ganglion and the caecum, but to a lower extent in other parts of the digestive tract (ileum, midgut, colon), and in various other tissues such as the fat body, ovaries and female accessory reproductive glands. In the brain and ovaries of virgin females, the AST expression is rather constant throughout adult life, whereas in brains of mated animals, expression is low until day 7, but increases sharply from day 8 onwards to reach values triple those before day 7. In ovaries of mated animals AST gene expression is also age-dependent, with high expression rates during the first 4 days after imaginable moult, a second but smaller peak from day 15 to 21, and very low values in between. In the fat body of virgin crickets allatostatin expression is high during the first 9 days after ecdysis and declines thereafter, whereas in mated animals two peak values, day 1 and day 6, are observed, and a third peak in older animals.
Asunto(s)
Gryllidae/genética , Neuropéptidos/genética , Envejecimiento/genética , Animales , Encéfalo/metabolismo , Cuerpo Adiposo/metabolismo , Femenino , Perfilación de la Expresión Génica , Ovario/metabolismo , Factores de TiempoAsunto(s)
Adenosina Trifosfatasas/metabolismo , Archaea/enzimología , Membrana Celular/enzimología , Sulfolobus/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Archaea/genética , Bovinos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Sulfolobus/genéticaRESUMEN
The sulfakinins are multifunctional insect neuropeptides displaying sequence similarities with the gastrin/ cholecystokinin (CCK) peptide family. In vertebrates, the peptides gastrin and CCK are involved in the regulation of digestion and food-intake. In this study sulfakinin cDNA was cloned and sequenced from the Mediterranean field cricket Gryllus bimaculatus. The cDNA encodes two peptides flanked by endoproteolytic processing sites, designated GrybiSKI (QSDDYGHMRFG) and GrybiSKII (EPFDDYGHMRFG). The peptides include the characteristic amino acid Tyr, which is potentially sulphated, and a Gly, as a recognition site for amidation yeilding the common C-terminal amino acid sequence of the sulfakinin peptide family. RT-PCR studies indicate an expression of the gene restricted to the brain, with a constant level of expression throughout the last larval stage, but showing an age-dependent decrease of expression in adult females.