RESUMEN
BACKGROUND: CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. METHODS: The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG- isoform, the first described mRNA for CYYR1 locus) or the presence (CAG+ isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. RESULTS: The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG- or CAG+ isoform expression compared to control tissues. CYYR1 CAG- isoform was significantly more expressed than CAG+ isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. CONCLUSION: A new "subtle" splicing isoform (CAG+) of CYYR1 mRNA, the sequence and the expression of this gene were defined in a large series of NE tumors.
Asunto(s)
Empalme Alternativo , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Tumores Neuroendocrinos/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Adulto , Anciano , Alanina/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Codón/genética , Neoplasias del Sistema Digestivo/genética , Evolución Molecular , Femenino , Humanos , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Persona de Mediana Edad , Datos de Secuencia Molecular , Neoplasias/genética , Pan troglodytes/genética , Mutación Puntual , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia , Especificidad de la EspecieRESUMEN
The activation of the insulin-like growth factor 1/IGF1 receptor system (IGF1/IGF1R) is a critical event in the transformation and tumorigenicity processes in a wide variety of human tumors. The IGF1/IGF1R system has been recently studied in carcinoid tumors that often arise in the gastrointestinal tract; these tumors are characterized by hypersecretion of bioamines and neuropeptides, leading to functional tumor disease. Two alternatively spliced IGF1R mRNA transcripts have been described to differ by only three nucleotides (CAG) in the coding sequence, resulting in an amino-acid change from the originally described Thr-Gly to an Arg in the extracellular portion of the receptor beta subunit. In transfected Chinese hamster ovary cells, the form without CAG (CAG-) exhibited an approximate 2-fold increase in IGF1 stimulation of activities required for its mitogenic properties. In this study, we examine the relative expression of the two IGF1R mRNA isoforms by a semiquantitative RT-PCR approach using highly standardized conditions, beta-2 microglobulin (B2M) as a reference gene and gel imaging analysis. We analyzed a large series of human neuroendocrine tumors (32 samples) and 9 normal tissues. A significant higher expression of both isoforms in the tumor samples (approximately 2-fold increase) was found, while a constant CAG+/CAG- IGF1R mRNA isoforms of an approximate 3:1 ratio was observed in all tumoral and normal cell types studied. The phylogenetic study of the IGF1R locus in several species suggests that human IGF1R CAG- mRNA isoform is evolutionarily more recent compared to the IGF1R CAG+ mRNA isoform and it could be used by the splicing apparatus at this intron/exon junction with a lower efficiency. This study highlights the relevance of IGF1R mRNA expression in neuroendocrine tumor cells, and the constant presence of 'subtle' alternative splicing for the IGF1R locus.
Asunto(s)
Empalme Alternativo , Tumores Neuroendocrinos/genética , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Adulto , Anciano , Secuencia de Aminoácidos , Carcinoma de Células de los Islotes Pancreáticos/genética , Carcinoma de Células de los Islotes Pancreáticos/metabolismo , Carcinoma de Células de los Islotes Pancreáticos/patología , Diferenciación Celular , Cartilla de ADN/química , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Isoformas de Proteínas , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Down syndrome critical region gene 1-like 2 (DSCR1L2) belongs to the human DSCR1-like gene family, which also includes DSCR1 and DSCR1L1. Both DSCR1 and DSCR1L1 proteins interact with calcineurin, a calcium/calmodulin-dependent phosphatase. To date, no interactor has been described for DSCR1L2. The aim of this work was to perform a first functional study of DSCR1L2 using yeast two-hybrid analysis conducted on a human heart cDNA library. Here, we report the interaction between DSCR1L2 and the human cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus. This interaction was confirmed by both yeast cotransformation and GST (glutathione-sepharose transferase) fusion protein assay. Moreover, a new DSCR1L2 mRNA isoform, generated by alternative splicing, was identified and cloned in different tissues: it lacks two central exons, encoding the most conserved domains among the DSCR1-like protein family. A quantitative relative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that in heart tissue the normalized expression level ratio for DSCR1L2 and DSCR1L2-E2E5 mRNA isoforms is 3.5:1, respectively. The yeast cotransformation and GST fusion protein assay demonstrated the interaction between this new DSCR1L2 variant and the human cardiac troponin I and the prominent role of DSCR1L2 exon 2 in determining binding between both DSCR1L2 isoforms and TNNI3. These data indicate an entirely new role for a DSCR1-like family gene, suggesting a possible involvement of DSCR1L2 in cardiac contraction.
Asunto(s)
Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , Troponina I/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adulto , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , Glutatión/metabolismo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/química , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Levaduras/citologíaRESUMEN
BACKGROUND: The ADARB1 gene is located in 21q22.3 region, previously linked to familial bipolar disorder, and its product has a documented action in the editing of the pre-mRNA of glutamate receptor B subunit. Dysfunction of glutamatergic neurotransmission could play an important role in the pathophysiology of bipolar disorder (BD). Glutamate excitatory neurotransmission regulation is a possible mechanism of the initial effect of anticonvulsants in regulating mood. METHODS: To investigate the hypothesis of an involvement of ADARB1 gene in the BD, the ADARB1 cDNA has been cloned and sequenced in seven selected bipolar I disorder patients with evidence of familiarity of mood disorders. A detailed investigation of the gene nucleotide sequence in the open reading frame has been performed. RESULTS: No alteration in the sequence of the ADARB1 gene cDNA was found in any patient, except a common neutral polymorphism in three out of seven patients. CONCLUSIONS: Mutations in ADARB1 gene are not commonly associated with bipolar I disorder, therefore other genes in the 21q22 region could be associated with bipolar illness in some families, likely in the context of a multifactorial transmission model.
Asunto(s)
Adenosina Desaminasa/genética , Trastorno Bipolar/genética , Predisposición Genética a la Enfermedad/genética , Adolescente , Adulto , Anticonvulsivantes/uso terapéutico , Trastorno Bipolar/diagnóstico , Trastorno Bipolar/tratamiento farmacológico , Trastorno Bipolar/fisiopatología , Cromosomas Humanos Par 21 , Análisis Mutacional de ADN , Femenino , Ácido Glutámico/fisiología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo Genético/genética , Precursores del ARN/genética , Proteínas de Unión al ARN , Receptores de Glutamato/genética , Análisis de Secuencia de ADN , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
The amino acid sequence of gene products is routinely deduced from the nucleotide sequence of the relative cloned cDNA, according to the rules for recognition of start codon (first-AUG rule, optimal sequence context) and the genetic code. From this prediction stem most subsequent types of product analysis, although all standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. Revision by bioinformatics and cloning methods of 109 known genes located on human chromosome 21 (HC 21) shows that 60 mRNAs lack any in-frame stop upstream of the first-AUG, and that in five cases (DSCR1, KIAA0184, KIAA0539, SON, and TFF3) the coding region at the 5' end was incompletely characterized in the original descriptions. We describe the respective consequences for genomic annotation, domain and ortholog identification, and functional experiments design. We have also analyzed the sequences of 13,124 human mRNAs (RefSeq databank), discovering that in 6448 cases (49%), an in-frame stop codon is present upstream of the initiation codon, while in the other 6676 mRNAs (51%), identification of additional bases at the mRNA 5' region could well reveal some new upstream in-frame AUG codons in the optimal context. Proportionally to the HC 21 data, about 550 known human genes might thus be affected by this 5' end mRNA artifact.
Asunto(s)
Codón Iniciador/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Cromosomas Humanos Par 21/genética , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mucinas/genética , Proteínas Musculares/genética , Proteínas Nucleares , Péptidos , Proteínas/genética , Reproducibilidad de los Resultados , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Homología de Secuencia de Aminoácido , Factor Trefoil-3RESUMEN
Few cases of large-scale segmental paralogy have been reported in the human genome. We have identified a large (approximately 500 kb) segment on human chromosome (HC) 21 (21q22) that is triplicated on HC 1 (1p35) and HC 6 (6p12-21). We also identified a new member of CLIC (Chloride Intracellular Channel) family on 21q, namely CLIC6. All three segments appear to include three functional members of three different gene families: DSCR1-like (Down Syndrome Candidate Region 1-like), CLIC, and AML/Runt (Acute Myeloid Leukemia/Runt). Molecular evolution analysis shows a common evolutionary origin for the triplicated regions. This finding of a further large-scale genomic triplication that went undetected at previously systematic automated searches provides a new model for gene divergence study and underlines the need for new tools to effectively detect inter-chromosomal similarity. An algorithm to overcome current limitations is proposed.
Asunto(s)
Cromosomas Humanos Par 1 , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 6 , Duplicación de Gen , Genoma Humano , Secuencia de Aminoácidos , Canales de Cloruro/genética , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Fosfoproteínas/genética , FilogeniaRESUMEN
A novel human gene has been identified by in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.1), with no coding region predicted in any previous analysis. Brain-derived DNA complementary to RNA (cDNA) sequencing predicts a 154-amino acid product with no similarity to any known protein. The gene has been named cysteine and tyrosine-rich protein 1 gene (symbol cysteine and tyrosine-rich 1, CYYR1). The CYYR1 messenger RNA was found by Northern blot analysis in a broad range of tissues (two transcripts of 3.4 and 2.2 kb). The gene consists of four exons and spans about 107 kb, including a very large intron of 85.8 kb. Analysis of expressed sequence tags shows high CYYR1 expression in cells belonging to the amine precursor uptake and decarboxylation system. We also cloned the cDNA of the murine ortholog Cyyr1, which was mapped by a radiation hybrid panel on chromosome 16 within the region corresponding to that containing the respective human homolog on chromosome 21. Sequence and phylogenetic analysis led to identification of several genes encoding CYYR1 homologous proteins. The most prominent feature identified in the protein family is a central, unique cysteine and tyrosine-rich domain, which is strongly conserved from lower vertebrates (fishes) to humans but is absent in bacteria and invertebrates.