RESUMO
Musculoskeletal embryonic nuclear protein 1 (MUSTN1) gene is involved in myogenic fusion and differentiation in rats. We previously showed the differential expression of MUSTN1 in week (W) 2 and W6 breast muscles of Pekin ducks. In this study, we further investigated its molecular characteristics and expression profiles in different tissues at W7 and in breast and leg muscles at W1, W3, W5, W7, and W9. The relationship between muscle development and muscle fiber areas was also investigated. A 358-bp cDNA sequence was obtained. The coding sequence of duck MUSTN1 cDNA encoded a 78-amino acid sequence, which showed high similarity with those of other species (96% similarity with zebra finch and 94% with chicken). In addition, a 6435-bp genomic DNA sequence of MUSTN1 was obtained. In total, 231 transcription factor-binding sites were found in the promoter region, and many of these transcription factors were involved in the regulation of muscle development. MUSTN1 expression in breast muscle increased from W1 to W5 and then decreased at W9. In leg muscle, the expression increased from W1 to W3 and then decreased. The relative growth rates of breast and leg muscle fibers reached their peaks at W3-W5 and W1-W3, respectively. Since the greatest relative growth rates appeared at the highest expression levels of the MUSTN1 gene, it was thought to play roles in duck muscle development. Our findings would be helpful in understanding the molecular characteristics and functions of the MUSTN1 gene in breast muscle development of ducks.
Assuntos
Proteínas Aviárias/genética , Patos/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/metabolismo , Patos/crescimento & desenvolvimento , Evolução Molecular , Perfilação da Expressão Gênica , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Alinhamento de SequênciaRESUMO
Myostatin, encoded by the MSTN gene, is a negative regulator of muscle growth, and its expression level in muscle tissue is closely correlated with muscle growth and satellite cell proliferation. To identify the characteristics of the Pekin duck MSTN gene and the relationship between its polymorphism and breast muscle traits in Pekin duck, cDNA cloning and analysis and the expression pattern in breast muscle development and polymorphism were performed using molecular cloning, quantitative real-time reverse-transcription polymerase chain reaction, and molecular marker technology. The results showed that a 1320-bp sequence, including a 93-bp 5'-UTR, 1128-bp CDS, and 99- bp 3'-UTR, was obtained, and two alternative splicing isoforms were detected. The alternative splicing isoforms encoded 375- and 251-amino acid residues. The amino acid sequence of Pekin duck MSTN was similar to other vertebrates and exhibited the highest similarity to chicken. The expression pattern of MSTN in breast muscle tissue showed a tendency to increase, except for a slight decrease at 6 weeks. Three single nucleotide polymorphisms were found in the Pekin duck MSTN gene by cDNA sequencing from different individuals. The T129C had significant association with breast muscle thickness, and the T952C had significant association with the fossilia ossis mastodi length. This study reveals the molecular characteristics of the Pekin duck MSTN gene and the relationship of its polymorphism with breast muscle traits in Pekin duck. Therefore, it can provide some useful basic understanding of MSTN functions.
Assuntos
Patos/genética , Músculo Esquelético/crescimento & desenvolvimento , Miostatina/genética , Polimorfismo de Nucleotídeo Único , Processamento Alternativo , Sequência de Aminoácidos , Animais , Mama/crescimento & desenvolvimento , Patos/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Miostatina/metabolismo , Filogenia , Alinhamento de Sequência , Vertebrados/genéticaRESUMO
To confirm the entire developmental process and transition point of embryonic Pekin duck pectoral muscle, and to investigate the association between pectoral muscle development and their regulating genes, anatomical and morphological analyses of embryonic Pekin duck skeletal muscles were performed, and the expression patterns of its regulating genes were investigated. The anatomical analysis revealed that body weight increased with age, while increases in pectoral muscle weight nearly ceased after the embryo was 20 days of hatching (E20). The developmental morphological characteristics of Pekin duck pectoral muscle at the embryonic stage showed that E20 was the transition point (from proliferation to fusion) of Pekin duck pectoral muscle. The expression patterns of MRF4, MyoG, and MSTN indicated that E19 or E20 was the fastest point of pectoral muscle development and the crucial transition for Pekin duck pectoral muscle development during the embryonic stage. Together, these findings imply that E20 is the crucial transition point (from proliferation to fusion) of Pekin duck pectoral muscle and that there is no muscle fiber hypertrophy after E20. Results of this study provide further understanding of the developmental process and transition point of Pekin duck pectoral muscle during the embryo stage.
Assuntos
Patos/embriologia , Perfilação da Expressão Gênica/veterinária , Músculos Peitorais/embriologia , Animais , Peso Corporal , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Regulação Miogênica/biossíntese , Fatores de Regulação Miogênica/genética , Miogenina/biossíntese , Miogenina/genética , Miostatina/biossíntese , Miostatina/genética , Músculos Peitorais/anatomia & histologia , Músculos Peitorais/crescimento & desenvolvimento , RNA Mensageiro/biossínteseRESUMO
We have treated 9 patients with sickle cell anemia (SS) with hydroxyurea (HU). All 9 patients carried 4 alpha-globin genes and the beta s-globin haplotypes 19/19 (Benin/Benin), except for 1 who had haplotype 19 together with type 3 (Benin/Senegal). Six patients received HU for 10 months and were again treated with the drug for 5 months after an interval of 1 year. One patient was given HU for 22 consecutive months. A record was kept of hematological and biochemical data, Hb F and G gamma levels, as well as possible clinical complications. Our data show that HU generally improves the hematological and biochemical values and the level of Hb F, and reduces painful crises in some patients. However, although the clinical symptoms improved in some patients during HU therapy, the older patients did not observe any changes in their general condition; the same is the case for the patient with haplotype 19/3. One patient also experienced life-threatening liver sequestration during treatment. We conclude that the selection of patients who may benefit from HU therapy needs further evaluation.
Assuntos
Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/uso terapêutico , Hidroxiureia/uso terapêutico , Adulto , Antidrepanocíticos/administração & dosagem , Antidrepanocíticos/efeitos adversos , Esquema de Medicação , Feminino , Hemoglobina Fetal/análise , Humanos , Hidroxiureia/administração & dosagem , Hidroxiureia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Países BaixosRESUMO
We have identified and quantitated the different types of mRNA in single BFU-E derived colonies from Hb S and Hb Atlanta [beta 75 (E19)Leu-->Pro] heterozygotes and observed that the normal and mutated mRNAs were present in equal quantities. Similar studies for the different protein products gave less accurate data because high performance liquid chromatography methods were not sensitive enough for the analysis of a single colony, and as many as five colonies needed to be combined. The level of Hb S (approximately 40%) was the same as in red cell lysates of the Hb S heterozygote, while that of the unstable beta-Atlanta chain was lower than expected from the values observed in red cells. Similar studies for a carrier of the stable Hb Costa Rica [beta 77(EF1) His-->Arg] which was reported to be the result of a somatic cell mutation (1) gave quite different data. Dot-blot analysis with 32P-labeled probes and allele specific amplification methodology identified numerous colonies with beta A-mRNA only, while 12-15% of the colonies contained both beta A- and beta-Costa Rica-mRNA. This limited distribution of the beta-Costa Rica-mRNA was confirmed by hemoglobin analysis with anion exchange high performance liquid chromatography. These results are considered to provide additional and convincing evidence for a somatic mosaicism for the CAC-->CGC mutation at codon 77 of the beta gene which occurred during the development of the embryo and results in the presence of only some 6-8% of the abnormal Hb Costa Rica in the circulating red cells.
Assuntos
Células Precursoras Eritroides/metabolismo , Globinas/genética , Hemoglobinas Anormais/genética , RNA Mensageiro/genética , Células Precursoras Eritroides/citologia , Humanos , MutaçãoRESUMO
We have identified a minor hemoglobin component (approximately 5%) in the blood of a healthy Costa Rican female, but not in her mother and two brothers (father not studied), that has an His --> Arg replacement at position beta 77 (Hb Costa Rica). No other amino acid replacements were observed and no beta- or gamma-chain-like peptides were present. Hb Costa Rica has abnormal stability. Sequence analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the beta gene failed to identify a CAC --> CGC (His --> Arg) mutation. The same was the case when cDNA was sequenced, indicating that a beta-Costa Rica-mRNA could not be detected with this procedure. Gene mapping of genomic DNA with Bg/II, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities of the beta chain variants Hb J-Iran and Hb Fukuyama with related mutations at beta 77 vary between 30% and 45% in heterozygotes, whereas that of Hb F-Kennestone with the same His --> Arg mutation but in the G gamma-globin gene, is a high 40%-45% (as percentage of total G gamma) in a heterozygous newborn. These different observations exclude a heterozygosity of the A --> G mutation at codon beta 77, as well as a deletion comparable to that of Hbs Lepore or Kenya, or a beta-globin gene duplication, and point to a nontraditional inheritance of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with 32P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A --> G mutation apparently occurred in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica over the red cells supports this possibility.