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1.
Sci Rep ; 5: 8484, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25719731

RESUMO

Selective breeding has strongly reduced the genetic diversity in livestock species, and contemporary breeding practices exclude potentially beneficial rare genetic variation from the future gene pool. Here we test whether important traits arising by new mutations can be identified and rescued in highly selected populations. We screened milks from 2.5 million cows to identify an exceptional individual which produced milk with reduced saturated fat content, and improved unsaturated and omega-3 fatty acid concentrations. The milk traits were transmitted dominantly to her offspring, and genetic mapping and genome sequencing revealed a new mutation in a previously unknown splice enhancer of the DGAT1 gene. Homozygous carriers show features of human diarrheal disorders, and may be useful for the development of therapeutic strategies. Our study demonstrates that high-throughput phenotypic screening can uncover rich genetic diversity even in inbred populations, and introduces a novel strategy to develop novel milks with improved nutritional properties.


Assuntos
Diacilglicerol O-Aciltransferase/genética , Leite/metabolismo , Mutação de Sentido Incorreto , Animais , Sequência de Bases , Bovinos/genética , Ácidos Graxos/biossíntese , Feminino , Estudos de Associação Genética , Metabolismo dos Lipídeos/genética , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único
2.
J Proteomics ; 75(14): 4429-35, 2012 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-22554911

RESUMO

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by ß-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and ß-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than ß-oxidation.


Assuntos
Bovinos/metabolismo , Lactação/metabolismo , Fígado/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteoma/metabolismo , Animais , Feminino , Especificidade de Órgãos/fisiologia
3.
Proteomics ; 8(7): 1502-15, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18383006

RESUMO

2-DE and MALDI mass fingerprinting were used to analyse mammary tissue from lactating Friesian cows. The goal was detection of enzymes in metabolic pathways for synthesis of milk molecules including fatty acids and lactose. Of 418 protein spots analysed by PMF, 328 were matched to database sequences, resulting in 215 unique proteins. We detected 11 out of the 15 enzymes in the direct pathways for conversion of glucose to fatty acids, two of the pentose phosphate pathway enzymes and two of the enzymes for lactose synthesis from glucose. We did not detect enzymes that catalyse the first three reactions of glycolysis. Our results are typical of enzyme detection using 2-DE of mammalian tissues. We therefore advocate caution when relating enzyme abundances measured by 2-DE to metabolic output as not all relevant proteins are detected. 2-D DIGE was used to measure interindividual variation in enzyme abundance from eight animals. We extracted relative protein abundances from 2-D DIGE data and used a logratio transformation that is appropriate for compositional data of the kind represented in many proteomics experiments. Coefficients of variation for abundances of detected enzymes were 3-8%. We recommend use of this transformation for DIGE and other compositional data.


Assuntos
Glândulas Mamárias Animais/química , Proteoma/química , Animais , Bovinos , Ciclo do Ácido Cítrico , Eletroforese em Gel Bidimensional , Ácidos Graxos/biossíntese , Feminino , Gluconeogênese , Glucose/metabolismo , Glicólise , Lactação/metabolismo , Glândulas Mamárias Animais/enzimologia , Redes e Vias Metabólicas , Via de Pentose Fosfato , Proteômica/métodos , Ácido Pirúvico/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
J Neurosci ; 24(25): 5816-26, 2004 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-15215304

RESUMO

Glycine receptors (GlyRs) and specific subtypes of GABA(A) receptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP-GTP exchange factor collybistin. We report the characterization of several new variants of collybistin, which are created by alternative splicing of exons encoding an N-terminal src homology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native collybistin isoforms appear to harbor the SH3 domain, this suggests that collybistin activity may be regulated by protein-protein interactions at the SH3 domain. We localized the binding sites for collybistin and the GlyR beta subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for collybistin-gephyrin and GlyR-gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both collybistin-gephyrin interactions and an intact collybistin pleckstrin homology domain. The vital importance of collybistin for inhibitory synaptogenesis is underlined by the discovery of a mutation (G55A) in exon 2 of the human collybistin gene (ARHGEF9) in a patient with clinical symptoms of both hyperekplexia and epilepsy. The clinical manifestation of this collybistin missense mutation may result, at least in part, from mislocalization of gephyrin and a major GABA(A) receptor subtype.


Assuntos
Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/citologia , Proteínas de Transporte/genética , Células Cultivadas , Epilepsia/complicações , Epilepsia/genética , Éxons , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptores da Glicina/metabolismo , Reflexo de Sobressalto , Fatores de Troca de Nucleotídeo Guanina Rho
5.
J Biol Chem ; 278(27): 24688-96, 2003 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-12684523

RESUMO

Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.


Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , Doença dos Neurônios Motores/genética , Receptores da Glicina/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Transporte/metabolismo , Éxons/genética , Variação Genética , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Doença dos Neurônios Motores/metabolismo , Mutação , Ligação Proteica , Isoformas de Proteínas/genética , Receptores da Glicina/genética , Alinhamento de Sequência
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