Asunto(s)
Aves/genética , Evolución Molecular , Peces/genética , Mamíferos/genética , Filogenia , Factor de Crecimiento Transformador beta/genética , Animales , Teorema de Bayes , Aves/clasificación , Peces/clasificación , Funciones de Verosimilitud , Mamíferos/clasificación , Miostatina , Selección Genética , Análisis de Secuencia de ADNRESUMEN
We describe an efficient in vitro assay to test growth hormone effects on mRNA levels and fatty acid synthase (FAS, EC. 2.3.1.85) activity. Swine adipose tissue explants were long-term cultured in medium containing growth hormone and FAS mRNA levels and enzyme activity were measured. We quantified FAS transcripts by competitive reverse transcriptase PCR (RT-PCR) using total RNA from cultured adipose tissue explants and RT-PCR standard-curves were constructed using a cloned 307 bp segment of native FAS cDNA and a shorter fragment from which a 64 bp (competitor, 243 bp) internal sequence had been deleted. A known amount of competitor was added to each PCR as an internal control and æ-actin transcripts were also measured to correct for differences in total RNA extraction and reverse transcription efficiency. In cultures with added growth hormone FAS mRNA levels decreased 70 percent (p < 0.01) and FAS enzyme activity decreased 22 percent (p < 0.05). These in vitro growth hormone effects were consistent with those observed in vivo, showing that in vitro adipose tissue culture combined with RT-PCR is a useful and accurate tool for studying growth hormone modulation of adipose tissue metabolism. This technique allowed the diagnosis of lower levels of FAS mRNA in the presence of growth hormone and these low levels were associated with decreased FAS activity in the adipose tissue explants.
Asunto(s)
Animales , Ácido Graso Sintasas/genética , Hormona del Crecimiento/farmacología , ARN Mensajero , Porcinos/genética , Tejido Adiposo , Enzimas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Myostatin is a potent growth and differentiation factor involved in skeletal muscle tissue formation in vertebrates. In the present study, temporal and spatial expression patterns of myostatin transcripts were investigated in chicken embryos. Myostatin mRNA was detected by RT-PCR analysis in embryos collected immediately after oviposition (stage HH1) and persisted until the fifth day of incubation (stage HH26). Whole-mount in situ hybridization revealed myostatin to be expressed in the ventral myotomal region of mature somites, thus confirming the importance of myostatin in skeletal muscle tissue formation during avian embryogenesis. A smaller myostatin transcript was also identified. This transcript appears to have resulted from an alternative splicing event from common GT-AG processing sites. Analysis of the amino acid sequence generated from this alternative transcript confirmed the presence of a truncated protein that lacks the C terminal region, including the cysteine domains characteristic of the TGF-beta super family. The temporal and spatial patterns of myostatin expression presented in this study agree with the proposed role of myostatin as modulator of muscle cell proliferation.
Asunto(s)
Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Factor de Crecimiento Transformador beta/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Miostatina , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The nucleotide sequence of the rDNA 18S region isolated from diploid and tetraploid species of the amphibian Odontophrynus americanus was determined and used to predict the secondary structure of the corresponding 18S rRNA molecules. Comparison of the primary and secondary structures for the 2n and 4n species confirmed that these species are very closely related. Only three nucleotide substitutions were observed, accounting for 99% identity between the 18S sequences, whereas several changes were detected by comparison with the Xenopus laevis 18S sequence (96% identity). Most changes were located in highly variable regions of the molecule. A noticeable feature of the Odontophrynus 18S rRNA was the presence of unusual extra sequences in the V2 region, between helices 9 and 11. These extra sequences do not fit the model for secondary structure predicted for vertebrate 18S rRNA.
Asunto(s)
Anuros/genética , ADN Ribosómico/genética , ARN Ribosómico 18S/genética , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ploidias , ARN Ribosómico 18S/química , Homología de Secuencia de Ácido Nucleico , Xenopus laevisRESUMEN
Ribosomal intergenic spacers (IGSs) of Odontophrynus americanus 2n and 4n were cloned, restriction mapped, and partially sequenced. Three distinct regions, namely alpha, beta, and delta, were identified in the IGSs. The alpha and beta regions flanked the 28S and 18S rRNA genes, respectively, conserving an identical restriction pattern at each ploidy level. The delta region, located between alpha and beta, was highly variable in size and restriction pattern, enclosing different BamHI subrepeats (B-SR), 87- to 530-bp-long. Sequence analysis showed that B-SRs were composed mainly of different arrangements of similar blocks of sequences. Another family of repetitive sequences was found in the delta region, clustered inside large BamHI fragments. These subrepeats are 189-bp-long and, although very similar in diploid and tetraploid IGSs, show a pattern of concerted evolution. A hypothetical functional role for the 189-bp repeats is discussed in view of their predicted secondary structure and presence of potential E2 binding sites inside diploid subrepeats. Although the same structural elements were present both in diploid and tetraploid IGSs, the higher level of repeatability of tetraploid IGSs suggests that common ancestor sequences have undergone several rounds of amplification after O. americanus polyploidy.