RESUMO
Cone alpha transducin (GNAT2), known to be expressed in photoreceptors, was found to be transcribed in human fetal cochlea. Due to the unexpected finding of expression of this gene in the inner ear and the success of the candidate gene approach in identifying mutations for a variety of heritable disorders, we investigated the possible role of this gene in Usher syndrome type I and type II. Single-strand conformation polymorphism (SSCP) was used to screen the GNAT2 coding region, as well as splice donor and acceptor sites, for mutations in a total of 140 unrelated patients. Two nucleotide changes leading to two silent amino acid changes and one rare polymorphism were found. In view of these results and those of a previously published Southern blot analysis, it is unlikely that mutations in GNAT2 are a common gene abnormality in Usher syndrome type I or type II.
Assuntos
Cóclea/metabolismo , Surdez/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Transducina/metabolismo , Sequência de Bases , Cóclea/embriologia , Eletroforese em Gel de Poliacrilamida , Idade Gestacional , Humanos , Dados de Sequência Molecular , Mutação/genética , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/metabolismo , Retinose Pigmentar/genética , Transducina/genéticaRESUMO
G-protein-dependent receptor kinases (GRKs) play a key role in the adaptation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid desensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of GRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently cloned human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family.
Assuntos
Cromossomos Humanos Par 13 , Proteínas do Olho , Proteínas Quinases/genética , Células Fotorreceptoras Retinianas Bastonetes , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Mapeamento Cromossômico , DNA Complementar , Éxons , Receptor Quinase 1 Acoplada a Proteína G , Humanos , Células Híbridas , Íntrons , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Dissociation of an activated alpha-subunit from the beta-gamma complex directly regulates secondary messenger proteins. To address the potential role of G proteins expressed in human fetal cochlea, degenerate oligonucleotide primers corresponding to the 3'-end of the conserved region of alpha-subunits were used for polymerase chain reaction amplification of reverse-transcribed total human fetal cochlear mRNAs; GNAZ and GNAQ were isolated. These two G proteins are unique among the G-protein family because they lack a typical pertussis modification site. GNAZ is expressed in high levels in neural tissue while GNAQ is ubiquitously expressed. We characterized GNAZ expression using Northern blots, tissue in-situ hybridization and immunohistochemistry techniques to elucidate the potential role of this protein in inner ear function. Our data suggest that GNAZ may play a role in maintaining the ionic balance of perilymphatic and endolymphatic cochlear fluids.
Assuntos
Cóclea/metabolismo , Proteínas Fetais/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Autorradiografia , Sequência de Bases , Northern Blotting , Clonagem Molecular , Cóclea/embriologia , DNA/química , DNA/genética , Orelha Interna/fisiologia , Proteínas Fetais/metabolismo , Proteínas Fetais/fisiologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Perilinfa/metabolismo , Perilinfa/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sistemas do Segundo Mensageiro , Transcrição Gênica/genéticaRESUMO
We report localization of the human cone transducin (GNAT2) gene using fluorescence in situ hybridization on chromosome 1 in band p13. The recent assignment of a gene for Stargardt disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. We investigated 66 unrelated patients for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. No disease-specific mutations were found, indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt disease.
Assuntos
Cromossomos Humanos Par 1 , Degeneração Macular/genética , Mutação Puntual , Células Fotorreceptoras Retinianas Cones/metabolismo , Transducina/genética , Sequência de Bases , Bandeamento Cromossômico , Mapeamento Cromossômico , Análise Mutacional de DNA , Primers do DNA , Éxons , Ligação Genética , Humanos , Hibridização in Situ Fluorescente , Substâncias Macromoleculares , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Gi alpha proteins, members of the G protein signal transduction family, include a small number of polypeptides: Gi alpha 1 (GNAI1), Gi alpha 2 (GNAI2), and Gi alpha 3 (GNAI3). A cDNA for the human GNAI2 gene has been isolated from a human T-cell library and is mapped by chromosomal in situ hybridization to the short arm of chromosome 3 at 3p21. A related sequence, GNAI2L, is mapped by in situ hybridization to the short arm of chromosome 12 at p12-p13. These mapping results are further supported by amplification of GNAI2-specific sequences in a monochromosomal human/rodent somatic cell hybrid containing only human chromosome 3. Of note, these assignments are to chromosome regions in which other G proteins reside. Localization of GNAI2 to 3p21 is of great interest as this region of the short arm of chromosome 3 is frequently involved in rearrangements in various human tumors.
