RESUMEN
This study aimed to elucidate the effects of broiler embryos soaked in ferulic acid (FA) solution on alleviating the negative impact of thermal manipulation (TM) on chicken embryo development and to provide a theoretical and experimental basis for applying TM and FA in the poultry feeding industry. A total of 120 broiler fertilized eggs were randomly divided into three groups: control group, TM group, and comprehensive group (TM + FA), with 40 eggs in each group. The TM group and the comprehensive group from the 7th embryonic age to the 16th embryonic age received TM for ten days, treated with a temperature of 39.5 °C and relative humidity of 65% for 18 h a day. In the comprehensive group, broiler embryos were immersed in FA solution at a concentration of 80 mg/L for 6 min at 16:00 every day from the 6th to the 8th embryo age. They were incubated continuously after being soaked until the chicks hatched. The results showed that the rates of dead embryos and weak chicks in the TM group were significantly higher than those in the control group and comprehensive group. Chick body temperatures of the TM group and comprehensive group were significantly lower than those of the control group. The heart weights of the TM group and comprehensive group were significantly lower than those of the control group, and the leg weights of the TM group were significantly decreased compared with those of the control group and comprehensive group. The SOD activity of serum in the comprehensive group was significantly higher than that in the control group and TM group, while the CAT activity of serum in the comprehensive group and control group was significantly higher than that in the TM group; however, there was no difference between the comprehensive group and control group. The activities of SOD and CAT in the liver were significantly higher than those of the TM group; however, the MDA content of the liver in the comprehensive group and control group was significantly lower than that of the TM group. The gene expression of Nrf2 and SOD in the comprehensive group and TM group was significantly higher than that in the control group; however, there was no significant difference between the comprehensive group and TM group. Soaking broiler embryonic eggs in an FA solution can improve the antioxidant capacity of the liver by upregulating Nrf2-Keap1 signal pathway-related gene expression. FA can effectively alleviate the side effects of TM on chicken embryos and does not impact the effects of TM.
Asunto(s)
Antioxidantes , Pollos , Ácidos Cumáricos , Desarrollo Embrionario , Animales , Ácidos Cumáricos/farmacología , Embrión de Pollo/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/metabolismoRESUMEN
Glucocorticoids (GCs) are an essential mediator hormone that can regulate animal growth, behavior, the phenotype of offspring, and so on, while GCs in poultry are predominantly corticosterones. The biological activity of GCs is mainly regulated by the intracellular metabolic enzymes, including 11ß-hydroxysteroid dehydrogenases 1 (11ß-HSD1), 11ß-hydroxysteroid dehydrogenases 2 (11ß-HSD2), and 20-hydroxysteroid dehydrogenase (20-HSD). To investigate the embryonic mechanisms of phenotypic differences between breeds, we compared the expression of corticosterone metabolic enzyme genes in the yolk-sac membrane and chorioallantoic membrane (CAM). We described the tissue distribution and ontogenic patterns of corticosterone metabolic enzymes during embryonic incubation between Tibetan and broiler chickens. Forty fertilized eggs from Tibetan and broiler chickens were incubated under hypoxic and normoxic conditions, respectively. Real-time fluorescence quantitative PCR was used to examine the expression of 11ß-HSD1/2, and 20-HSD mRNA in embryonic tissues. The results showed that the expression levels of yolk-sac membrane mRNA of 11ß-HSD2 and 20-HSD in Tibetan chickens on E14 (embryonic day of 14) were significantly lower than those of broiler chickens (P < 0.05), and these genes expression of CAM in Tibetan chickens were higher than those of broiler chickens (P < 0.05). In addition, the three genes in the yolk-sac membrane and CAM were followed by a down-regulation on E18 (embryonic day of 18). The 11ß-HSD1 and 11ß-HSD2 genes followed a similar tissue-specific pattern: the expression level was more abundantly in the liver, kidney, and intestine, with relatively lower abundance in the hypothalamus and muscle, and the expression level of 20-HSD genes in all tissues tested was higher. In the liver, 20-HSD of both Tibetan and broiler chickens showed different ontogeny development patterns, and hepatic mRNA expression of 20-HSD in broiler chickens was significantly higher than that of Tibetan chickens of the same age from E14 to E18 (P < 0.05). This study preliminarily revealed the expression levels of cortisol metabolic genes in different tissues during the development process of Tibetan and broiler chicken embryos. It provided essential information for in-depth research of the internal mechanism of maternal GCs programming on offspring.
Asunto(s)
Pollos , Corticosterona , Animales , Embrión de Pollo , Corticosterona/metabolismo , Pollos/genética , Pollos/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Tibet , Glucocorticoides/metabolismo , Hidroxiesteroide Deshidrogenasas/genética , Hidroxiesteroide Deshidrogenasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Expresión GénicaRESUMEN
The objective of this study was to explore the molecular mechanism of male sterility in yak hybrids based on HAT1 and HDAC1. Total RNA was extracted from the testes of adult yaks (n = 11) and sterile cattle-yaks (n = 11) followed by reverse transcription. The coding sequence (CDS) of yak HAT1 and HDAC1 were obtained by conventional polymerase chain reaction (PCR) and gene cloning. The testicular mRNA and protein levels of HAT1 and HDAC1 in yaks and cattle-yaks were detected by quantitative PCR (qPCR) and Western blotting, respectively, and the histone H3 lysine 9 (H3K9) histone acetylation level in the testes of yaks and cattle-yaks was assayed using enzyme linked immunosorbent assay (ELISA). The results showed that the CDS of HAT1 and HDAC1 were 1242 bp and 1449 bp in length, encoding 413 and 482 amino acids, respectively; yaks had a similar mRNA sequence as cattle in both genes. The testicular mRNA and protein levels of HAT1 of cattle-yaks were significantly lower than those of yaks, and the protein level of HDAC1 was significantly higher than that of yaks. ELISA showed that the acetylation level of testicular H3K9 was significantly lower in yak hybrids than that of yaks. The present results suggest that the decreased level of HAT1 and increased level of HDAC1 may result in the decreased H3K9 acetylation in cattle-yaks and might be associated with their sterility.
RESUMEN
Extended lactation is a common phenomenon in lactating yaks under grazing and natural reproduction conditions. To elucidate differences in milk protein compositions and mammary gland functions between yaks of standard lactation (TL yaks) and prolonged lactation (HL yaks), whole milk samples of TL yaks and HL yaks (n = 15 each) were collected from a yak pasture at the northwest highland of China. The iTRAQ technique was used to compare the skim milk proteins in the two yak groups. A total of 202 differentially expressed proteins (DEPs) were revealed, among which 109 proteins were up-regulated and 93 were down-regulated in the milk of HL yaks compared to TL yaks. Caseins including κ-casein, αs1-casein, αs2-casein, and ß-casein were up-regulated in HL yak milk over 1.43-fold. The GO function annotation analysis showed that HL yaks produced milk with characteristics of milk at the degeneration stage, similar to that of dairy cows. KEGG enrichment showed that the metabolic pathways with the most differences are those that involve carbohydrate metabolism and the biosynthesis of amino acids. The present results highlight detailed differences in skim milk proteins produced by HL yaks and TL yaks and suggest that the mammary gland of HL yak is at the degeneration stage.
RESUMEN
Polycomb group (PcG) protein-mediated histone methylation (H3K27me3) controls the correct spatiotemporal expression of numerous developmental regulators in Arabidopsis. Epigenetic silencing of the stem cell factor gene WUSCHEL (WUS) in floral meristems (FMs) depends on H3K27me3 deposition by PcG proteins. However, the role of H3K27me3 in silencing of other meristematic regulator and pluripotency genes during FM determinacy has not yet been studied. To this end, we report the genome-wide dynamics of H3K27me3 levels during FM arrest and the consequences of strongly depleted PcG activity on early flower morphogenesis including enlarged and indeterminate FMs. Strong depletion of H3K27me3 levels results in misexpression of the FM identity gene AGL24, which partially causes floral reversion leading to ap1-like flowers and indeterminate FMs ectopically expressing WUS and SHOOT MERISTEMLESS (STM). Loss of STM can rescue supernumerary floral organs and FM indeterminacy in H3K27me3-deficient flowers, indicating that the hyperactivity of the FMs is at least partially a result of ectopic STM expression. Nonetheless, WUS remained essential for the FM activity. Our results demonstrate that PcG proteins promote FM determinacy at multiple levels of the floral gene regulatory network, silencing initially floral regulators such as AGL24 that promotes FM indeterminacy and, subsequently, meristematic pluripotency genes such as WUS and STM during FM arrest.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/genética , Meristema/genética , Meristema/metabolismoRESUMEN
The objective of this study was to explore the molecular mechanism for male sterility of yak hybrids based on two demethylases. Total RNA was extracted from the testes of adult yaks (n = 10) and yak hybrids (cattle-yaks, n = 10). The coding sequences (CDS) of two lysine demethylases (KDMs), KDM1A and KDM4B, were cloned by RT-PCR. The levels of KDM1A and KDM4B in yaks and cattle-yaks testes were detected using Real-time PCR and Western blotting for mRNA and protein, respectively. In addition, the histone methylation modifications of H3K36me3 and H3K27me3 were compared between testes of yaks and cattle-yaks using ELISA. The CDS of KDM1A and KDM4B were obtained from yak testes. The results showed that the CDS of KDM1A exhibited two variants: variant 1 has a CDS of 2622 bp, encoding 873 amino acids, while variant 2 has a CDS of 2562 bp, encoding 853 amino acids. The CDS of the KDM4B gene was 3351 bp in length, encoding 1116 amino acids. The mRNA and protein expression of KDM1A and KDM4B, as well as the level of H3K36me3, were dramatically decreased in the testes of cattle-yaks compared with yaks. The present results suggest that the male sterility of cattle-yaks might be associated with reduced histone methylation modifications.
RESUMEN
Plant leaf margins produce small outgrowths or teeth causing serration in a regular arrangement, which is specified by auxin maxima. In Arabidopsis, the spatiotemporal pattern of auxin dependents on both, the transcription factor CUC2 and the signal peptide EPFL2, a ligand of the growth-promoting receptor kinase ERECTA (ER). Ectopic expression of CUC2 can have contrary effects on leaf growth. Ubiquitous expressed CUC2 suppresses growth in the whole leaf, whereas cuc2-1D mutants have enlarged leaves, through ER-dependent cell proliferation in the teeth. Here we investigated the growth dynamics of cuc2-1D leaves and the growth restricting the function of CUC2 using the ubiquitous inducible CUC2-GR transgene. In time courses, we dissected the serration promoting the function of CUC2 in the leaf margin and ectopic growth inhibition by CUC2 in the leaf plate. We found that CUC2 limits growth rather by cell cycle inhibition than by cell size control. Furthermore, endogenous CUC2 was rapidly induced by CUC2-GR indicating a possible auto-inducible feedback. In contrast, EPFL2 was quickly decreased by transient CUC2 induction but increased in cuc2-3 mutant leaves suggesting that CUC2 can also counteract the EPFL2-ER pathway. Therefore, tooth growth promotion and growth inhibition by CUC2 involve partially the same mechanism but in contrary ways.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hojas de la Planta/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Expresión Génica Ectópica/genética , Expresión Génica Ectópica/fisiología , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/genéticaRESUMEN
This study investigated the variations of the nucleotide sequences and ovarian expression levels of genes related to follicular development and atresia in prolific Jintang black goats and nonprolific Tibetan goats. Eight genes, FSHB, LHB, FSHR, LHCGR, ESR2, B4GANT2, BCL2 and BAX, were examined using reverse transcription-polymerase chain reaction and quantitative real-time PCR. The results showed that the nucleotide and deduced amino acid sequences of the LHB and BAX genes were not different, but there was one base change in the FSHR genes between the two breeds. There was one base change in the FSHB gene, which resulted in one amino acid substitution; there were nine base changes in the LHCGR gene, which resulted in five amino acid substitutions; and there were six base changes in the B4GANT2 gene, which resulted in four amino acid substitutions. The expression levels of the FSHR, LHCGR, ESR2, B4GANT2, BCL2 and BAX genes in the ovaries were not different between the two breeds. The plasma concentrations of FSH were not different, but the plasma concentrations of LH, P4 and E2 were lower in prolific Jintang black goats than in nonprolific Tibetan goats (P Ë 0.05) at 40 hr after removal of the Controlled Internal Drug Release Devices. These results provide some foundations elucidating the endocrine and molecular mechanisms controlling ovulation rate in goats, but these need to be further verified.
Asunto(s)
Atresia Folicular/genética , Expresión Génica , Enfermedades de las Cabras/genética , Folículo Ovárico/metabolismo , Animales , Femenino , Enfermedades de las Cabras/metabolismo , Cabras , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Análisis de Secuencia de ADN/veterinaria , Especificidad de la EspecieRESUMEN
The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.
Asunto(s)
Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Miogenina/análisis , Cadenas Pesadas de Miosina/análisis , Miostatina/análisis , Conejos/crecimiento & desarrollo , Animales , Femenino , Expresión Génica , Ganado , Masculino , Músculo Esquelético/química , Miogenina/genética , Miogenina/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miostatina/genética , Miostatina/metabolismo , Conejos/genética , Conejos/metabolismo , Especificidad de la EspecieRESUMEN
The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.
Asunto(s)
Bovinos/genética , L-Lactato Deshidrogenasa/genética , Polimorfismo Genético , Alelos , Animales , Cruzamiento , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Frecuencia de los Genes/genética , Genotipo , Punto Isoeléctrico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Subunidades de Proteína/genética , Análisis de Secuencia de ADN , Electricidad EstáticaRESUMEN
The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl2 at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions.
Asunto(s)
Enfermedades de los Bovinos/parasitología , Dípteros/enzimología , L-Lactato Deshidrogenasa/aislamiento & purificación , Miasis/veterinaria , Animales , Bovinos , China , Cromatografía de Afinidad/veterinaria , Cromatografía en Gel/veterinaria , Dípteros/clasificación , Dípteros/genética , Electroforesis en Gel de Poliacrilamida/veterinaria , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Larva/clasificación , Larva/enzimología , Larva/genética , Cloruro de Mercurio/farmacología , Peso Molecular , Miasis/parasitología , NAD/metabolismo , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Piruvatos/metabolismo , TemperaturaRESUMEN
Myostatin (MSTN) is a negative regulator of skeletal muscle growth. The objective of the present study was to express yak (Bos grunniens) recombinant MSTN protein in E. coli and study its characteristics of immunogenicity. cDNA encoding yak MSTN mature peptide was amplified by reverse-transcription PCR, and cloned into pET28a(+) vector and expressed in E. coli. The expressed recombinant MSTN was purified by affinity chromatography and used to prepare rabbit anti yak MSTN antibody. The results showed that yak MSTN mature peptide gene contained 330 bp nucleotides coding 109 amino acids. Content of the target protein accounted for 21% of the total expression products when MSTN-pET28a(+)-BL21(DE3) bacterium was incubated in LB medium with 0.1 mM IPTG for 6 hours. The molecular weight of the purified yak MSTN recombinant protein was 16.5 kDa, exhibiting excellent immunogenicity as shown by ELISA. The obtained recombinant MSTN of yak is suitable for further analysis of yak MSTN functions.
Asunto(s)
Miostatina/inmunología , Miostatina/metabolismo , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Bovinos , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Femenino , Masculino , Miostatina/química , Miostatina/genética , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.
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Columbidae/genética , L-Lactato Deshidrogenasa/genética , Mamíferos/genética , Testículo/enzimología , Empalme Alternativo , Animales , Western Blotting , Columbidae/metabolismo , Electroforesis en Gel de Agar , Exones , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Masculino , Mamíferos/metabolismoRESUMEN
This study investigates the molecular mechanism by which yaks (Bos grunniens) adapt to hypoxia based on lactate dehydrogenase (LDH). Three LDH1 variants of the yak were revealed in tissue extracts by electrophoresis, including LDH1-F, LDH1-M, and LDH1-S. Kinetic analysis using purified LDH1 variants showed that the yak LDH1-M variant exhibited a similar K (m) (NADH) and the same mobility on a gel as bovine LDH1, and the LDH1-F variant showed significant differences in K (m) values for NADH or pyruvate from the other two variants of yak LDH1 and bovine LDH1. Among the three muscles assayed, yak longissimus dorsi showed the highest LDH activity and the lowest malate dehydrogenase (MDH) activity; heart muscle was exactly the opposite. Our results suggest that the three LDH1 variants might play an important role in the adaptation to hypoxia.
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Adaptación Biológica/genética , Altitud , Bovinos/genética , Variación Genética , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Animales , Bovinos/metabolismo , Femenino , Hipoxia/enzimología , Hipoxia/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Malato Deshidrogenasa/metabolismo , Masculino , Especificidad de ÓrganosRESUMEN
The genotypes and protein polymorphisms of milk epithelial mucin (MUC1) were analyzed by touch-down PCR and SDS-PAGE respectively using blood samples and milk collected from 50 lactating yaks. A total of seven alleles were revealed, namely A, B, C, D, E, F, and G, and the corresponding number of repetitive units of variable number tandem repeat (VNTR) in MUC1 gene were 21, 20, 19, 18, 17, 16, and 15, respectively. Fifteen genotypes of MUC1 were observed in yaks. The genotypes of MUC1 gene matched completely to the phenotypes of milk MUC1 in each individual. This study demonstrated that the yak MUC1 exhibits abundant polymorphisms in both its gene and protein, and the polymorphisms are due to the expression of VNTR in MUC1 gene. The possible cluster of the VNTR was also discussed in different ruminants.
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Bovinos/genética , Repeticiones de Minisatélite , Mucina-1/genética , Polimorfismo Genético , Alelos , Secuencia de Aminoácidos , Animales , Bovinos/metabolismo , Leche/química , Datos de Secuencia Molecular , Mucina-1/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.
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Bovinos/genética , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/aislamiento & purificación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Cinética , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Ácido Pirúvico/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration in yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformational changes between the two LDH variants.
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L-Lactato Deshidrogenasa/genética , Animales , Bovinos , Clonación Molecular , ADN Complementario , Isoenzimas/genética , Modelos Moleculares , Polimorfismo Genético , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lactate dehydrogenase C (LDH-C) has been reported to play a role in the energy metabolism of mammal spermatozoa. However, the functions and expression patterns of LDH-C still remain unclear. In order to elucidate the functions and expression patterns of LDH-C, we cloned the cDNA of yak LDH-C. Total RNA was extracted from yak testes and reverse transcribed and amplified by PCR. The full length open reading frame (ORF) of LDH-C and its five splice variants were obtained. The full length ORF contained 999 bp encoding a 332-amino-acid protein that showed 100% identity with bovine LDH-C. Compared with the full length ORF of LDH-C, the five variants used the same start codon as the full length ORF and encoded 5 putative proteins. In detail, variants 1 (missing the coding sequence of exon 6 and 7) and 2 (missing the coding sequence of exon 7) bear the entire nicotinamide-adenine dinucleotide (NAD) binding domain and an active site. Variants 3 (missing the first 42 nuleotides of exon 4) and 4 (missing the coding sequences of exons 5, 6 and 7) lack part of the NAD binding domain but contained the entire active site. Variant 5 (missing the coding sequence of exons 4 and 7) lacks a large part of the NAD binding domain and the entire active site. Native polyacrylamide gel electrophoresis was performed to determine if the splice variants can be translated into proteins. However, native PAGE detected no specific bands from yak testes and bovine spermatozoa. This study suggests the alternative splicing of LDH-C is ubiquitous in bovine testes and might be involved in regulation of LDH-C expression. The findings also help to elucidate the functions of LDH-C.
Asunto(s)
Bovinos/genética , Regulación Enzimológica de la Expresión Génica , L-Lactato Deshidrogenasa/genética , Empalme del ARN/genética , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Variación Genética , Isoenzimas/genética , Masculino , Datos de Secuencia MolecularRESUMEN
GLUT8 is a newly identified member of the facilitative glucose transporter family, which characteristically exhibits high-affinity glucose transport activity. The expression of GLUT8 has been shown to depend on gonadotropin secretion in human testes and to be regulated by insulin in the blastocyst. To characterize GLUT8 and investigate its role in normal mammary gland function, we cloned and sequenced the full-length cDNA of bovine GLUT8. The 2073-base-pair cDNA sequence is predicted to encode a protein of 478 amino acids, with a molecular weight of approximately 51 kDa. The deduced amino acid sequence of bovine GLUT8 is 90%, 84%, 84% and 58% identical to human, mouse, rat and chicken GLUT8, and is 26%, 27% and 24% identical to bovine GLUT1, GLUT3 and GLUT4, respectively. Bovine GLUT8 retains the characteristic structural features of GLUT8 proteins previously identified from other species including membrane spanning helices, glucose transporter motifs, an N-linked glycosylation site on loop 9 and a putative dileucine internalization motif. The major in vitro transcription and translation product of bovine GLUT8 cDNA migrated at an apparent molecular weight of 38 kDa similar to the sizes reported for GLUT8 from other mammalian species. In the presence of canine microsomal membranes, the translation product increased to 40 kDa suggesting glycosylation. Transient transfection studies using a FLAG epitope tagged construct in COS-7 cells revealed that bovine GLUT8 is localized to the cytoplasm in non-stimulated conditions. A 2.1-kb GLUT8 mRNA transcript was detected at high levels in bovine testes, at moderate levels in lactating bovine mammary gland, lung, kidney, spleen, intestine and skeletal muscle, and at low levels in bovine liver. GLUT8 mRNA expression in bovine mammary gland increased about 10-fold (P<0.001) during late pregnancy and early lactation, similar to the pattern of change in GLUT1 mRNA and more dramatic than the increase seen in mouse mammary gland. These results suggest that GLUT8 expression may be regulated by lactogenic hormones and that GLUT8 may play a role in glucose uptake in the lactating mammary gland.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Glándulas Mamarias Animales/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Northern Blotting , Western Blotting , Células COS , Bovinos , Chlorocebus aethiops , Clonación Molecular , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa , Glicosilación , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/metabolismo , Embarazo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Testículo/metabolismo , TransfecciónRESUMEN
The ubiquitously expressed transcription factor Oct-1, a member of the POU domain factors, is involved in the regulation of expression of many tissue-specific and house-keeping genes. Multiple alternatively spliced isoforms of Oct-1 have been identified in human and mouse cells. The expression patterns of these isoforms and the analysis of their genomic organization and structure have suggested that the structural variation of Oct-1 isoforms may be important in conferring target and tissue specificity to its transcriptional activity. In this study, we have cloned and sequenced a new mouse Oct-1 isoform, named mOct-1Z. This novel isoform differs markedly at the C-terminus from the previously identified Oct-1 isoforms A, B, and C. It is generated by alternative splicing from the Oct-1 gene and its transcript exhibits a frameshift followed by an early stop codon, thus, its predicted protein has a distinct, much shorter C-terminal tail. However, this truncated isoform could still effectively bind to a consensus Oct-1 motif oligonucleotide and, like Oct-1B, activated the basal promoter activity of the mouse beta-casein gene. Oct-1Z is another ubiquitously expressed Oct-1 isoform, its transcript being detected in all mouse tissues examined, including the mammary gland, liver, lung, kidney, spleen, small intestine mucosa, uterus, and ovary.