RESUMEN
Selection for increased growth rate in farm and laboratory animals has been used to develop lines with increased body and muscle weights. However, very little is known about the underlying molecular pathways and how their constitutive genes influence this process. In this study, the differential display-reverse transcription PCR (DDRT-PCR) method was employed to identify longissimus muscle genes that are differentially expressed between a line of pigs selected for increased 200-d weight and a randomly selected control line. A 590-bp DDRT-PCR cDNA product was identified and isolated based on its greater abundance in the longissimus muscle of the select line relative to the control line animals. This DDRT-PCR product has 89% identity to the end of the 3'-untranslated region of the bovine 16-kDa cAMP-regulated phosphoprotein (ARPP-16) cDNA sequence. Reverse transcription PCR (RT-PCR) amplification of the porcine homologue of ARPP-16 and subsequent sequencing established that the DDRT-PCR product corresponds to the 3'-end of the porcine ARPP-16 transcript. Semiquantitative RT-PCR verified that ARPP-16 is up-regulated in the select line and determined that the relative expression level of ARPP16 mRNA is approximately fourfold higher (P < .01) in the select than in the control animals. The deduced amino acid sequence of ARPP-16 is highly homologous to the deduced amino acid sequences of bovine, human, and rat ARPP-16, and RT-PCR with ARPP-16-specific PCR primers indicated that this gene is expressed in many different porcine tissues. The porcine homologue of the 19-kDa cAMP-regulated phosphoprotein (ARPP-19) was also amplified by RT-PCR, cloned, and sequenced. The deduced amino acid sequence of ARPP19 differs from ARPP-16 only by the addition of 16 N-terminal amino acids. In all tissues studied, ARPP-19 mRNA was detected by RT-PCR amplification; however, the relative expression level of ARPP-19 mRNA was not differentially expressed between the select and control line animals (P > .05). The fourfold relative increase in ARPP-16 mRNA expression in the select line animals indicates that this gene may play an important role in the molecular pathway(s) that regulate postnatal skeletal muscle growth in the pig.
Asunto(s)
Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Fosfoproteínas/genética , ARN Mensajero/biosíntesis , Porcinos/crecimiento & desarrollo , Regulación hacia Arriba , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Fosfoproteínas/biosíntesis , Reacción en Cadena de la Polimerasa/veterinaria , RatasRESUMEN
The centromeric region of swine chromosomes is comprised of tandemly repeated, divergent DNA monomer units. Here we report that these divergent DNA monomer sequences are organized into higher-order repeats, analogous to the hierarchical organization of alpha-satellite monomers in human centromeres. In this study, a centromeric cosmid clone was shown to be comprised entirely of a 3.3-kb higher-order repeat, with independent copies of this higher-order repeat more than 99% identical to each other. This higher-order repeat is composed of ten divergent monomer units of approximately 340 bp. The ten monomers are on average 79% identical, and all ten monomers are arranged in the same 5' to 3' orientation. In FISH analysis, a cloned 3.3-kb higher-order repeat hybridized to the centromere of Chromosome (Chr) 9 in metaphase spreads and detected two discrete foci in interphase nuclei, demonstrating that this swine higher-order repeat is chromosome-specific. The Chr 9 centromeric array spanned approximately 2.2 Mb as determined by pulsed-field gel electrophoresis. Moreover, the swine Chr 9 centromere is highly polymorphic, because an EcoRI restriction site polymorphism was detected. Thus, the assembly of divergent satellite sequences into chromosome-specific higher-order repeats appears to be a common organizational feature of both the human and swine centromere and suggests that the evolutionary mechanism(s) that create and maintain higher-order repeats is conserved between their genomes.
Asunto(s)
Centrómero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Porcinos/genética , Animales , Secuencia de Bases , Cromosomas , Clonación Molecular , Cósmidos/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Genoma , Hibridación Fluorescente in Situ , Interfase/genética , Datos de Secuencia Molecular , Mapeo RestrictivoAsunto(s)
Mapeo Cromosómico/veterinaria , Cósmidos/genética , Repeticiones de Microsatélite/genética , Porcinos/genética , Animales , Bandeo Cromosómico/veterinaria , Cartilla de ADN , Hibridación Fluorescente in Situ/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo GenéticoRESUMEN
To facilitate the identification of microsatellite genetic markers from a single swine chromosome, chromosome microisolation and microcloning have been used to generate a swine chromosome 6-specific DNA library. Ten copies of swine chromosome 6 were scraped from metaphase spreads, ligated to custom-prepared adaptors, and amplified by PCR. The purity of the amplified product was verified by fluorescent in situ hybridization. The utility of the chromosome painting probe for heterologous painting was demonstrated and confirmed that swine chromosome 6 is syntenic to human chromosomes 1p and 19q. A small insert genomic library of 1.39 x 10(6) clones was generated from the PCR-amplified chromosome 6 genomic DNA and screened for (GT)n microsatellite genetic markers. Nine (GT)n microsatellite markers were developed and genotyped on a Yorkshire x Meishan swine reference family. All nine markers genetically mapped to chromosome 6, confirming the purity of the microisolation method. The method used here should be adaptable to the microdissection of subchromosomal regions of not only the swine genome but also other livestock genomes.
Asunto(s)
Biblioteca de Genes , Porcinos/genética , Animales , Humanos , Repeticiones de MicrosatéliteRESUMEN
Recent advances in the use of microsatellite markers and the development of comparative gene mapping techniques have made the construction of high resolution genetic maps of livestock species possible. Framework and comprehensive genetic linkage maps of porcine chromosome 6 have resulted from the first international effort to integrate genetic maps from multiple laboratories. Eleven highly polymorphic genetic markers were exchanged and mapped by four independent laboratories on a total of 583 animals derived from four reference populations. The chromosome 6 framework map consists of 10 markers ordered with high local support. The average marker interval of the framework map is 15.1 cM (sex averaged). The framework map is 135, 175 and 109 cM in length (for sex averaged, female and male maps, respectively). The comprehensive map includes a total of 48 type I and type II markers with a sex averaged interval of 3.5 cM and is 166, 196 and 126 cM (for sex averaged, female and male maps, respectively). Additional markers within framework map marker intervals can thus be selected from the comprehensive map for further analysis of quantitive trait loci (QTL) located on chromosome 6. The resulting maps of swine chromosome 6 provide a valuable tool for analysing and locating QTL.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas , Porcinos/genética , Animales , Secuencia de Bases , Femenino , Marcadores Genéticos , Masculino , Repeticiones de Microsatélite , Datos de Secuencia MolecularRESUMEN
The first integrated physical and genetic linkage map encompassing the entire swine chromosome 7 (SSC7) reveals that the porcine MHC (SLA) spans the centromere. A SLA class II antigen gene lies on the q arm, whereas class I and III genes lie on the p arm, suggesting that the presence of a centromere within the SLA does not preclude a functional complex. The SLA appears smaller than other mammalian MHC, as the genetic distance across two class I, three class II, and three class III SLA gene markers is only 1.1 cM. There are significant variations in recombination rates as a function of position along the chromosome, and the SLA lies in the region with the lowest rate. Furthermore, the directed integration approach used in this study was more efficient than previous efforts that emphasized the screening of large insert libraries for random microsatellites.