RESUMEN
DiGeorge syndrome is characterized by cardiovascular, thymus and parathyroid defects and craniofacial anomalies, and is usually caused by a heterozygous deletion of chromosomal region 22q11.2 (del22q11) (ref. 1). A targeted, heterozygous deletion, named Df(16)1, encompassing around 1 megabase of the homologous region in mouse causes cardiovascular abnormalities characteristic of the human disease. Here we have used a combination of chromosome engineering and P1 artificial chromosome transgenesis to localize the haploinsufficient gene in the region, Tbx1. We show that Tbx1, a member of the T-box transcription factor family, is required for normal development of the pharyngeal arch arteries in a gene dosage-dependent manner. Deletion of one copy of Tbx1 affects the development of the fourth pharyngeal arch arteries, whereas homozygous mutation severely disrupts the pharyngeal arch artery system. Our data show that haploinsufficiency of Tbx1 is sufficient to generate at least one important component of the DiGeorge syndrome phenotype in mice, and demonstrate the suitability of the mouse for the genetic dissection of microdeletion syndromes.
Asunto(s)
Aorta Torácica/patología , Síndrome de DiGeorge/patología , Proteínas de Dominio T Box/genética , Animales , Aorta Torácica/embriología , Línea Celular , Síndrome de DiGeorge/embriología , Eliminación de Gen , Marcación de Gen , Haplotipos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis , Proteínas de Dominio T Box/fisiologíaRESUMEN
Deletions or rearrangements of human chromosome 22q11 lead to a variety of related clinical syndromes such as DiGeorge syndrome (DGS) and velo--cardiofacial syndrome (VCFS). In addition, patients with 22q11 deletions have an increased incidence of schizophrenia and several studies have mapped susceptibility loci for schizophrenia to this region. Human molecular genetic studies have so far failed to identify the crucial genes or disruption mechanisms that result in these disorders. We have used gene targeting in the mouse to delete a defined region within the conserved DGS critical region (DGCR) on mouse chromosome 16 to prospectively investigate the role of the mouse DGCR in 22q11 syndromes. The deletion spans a conserved portion ( approximately 150 kb) of the proximal region of the DGCR, containing at least seven genes ( Znf74l, Idd, Tsk1, Tsk2, Es2, Gscl and Ctp ). Mice heterozygous for this deletion display no findings of DGS/VCFS in either inbred or mixed backgrounds. However, heterozygous mice display an increase in prepulse inhibition of the startle response, a manifestation of sensorimotor gating that is reduced in humans with schizophrenia. Homozygous deleted mice die soon after implantation, demonstrating that the deleted region contains genes essential for early post-implantation embryonic development. These results suggest that heterozygous deletion of this portion of the DGCR is sufficient for sensorimotor gating abnormalities, but not sufficient to produce the common features of DGS/VCFS in the mouse.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 22 , Síndrome de DiGeorge/genética , Animales , Secuencia de Bases , Cartilla de ADN , Heterocigoto , Humanos , Masculino , RatonesRESUMEN
We have used a modified direct selection technique to detect transcripts that are both evolutionary conserved and developmentally expressed. The enrichment for homologous mouse cDNAs by use of human genomic DNA as template is shown to be an efficient and rapid approach for generating transcript maps. Deletions of human 22q11 are associated with several clinical syndromes, with overlapping phenotypes, for example, velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS). A large number of transcriptional units exist within the defined critical region, many of which have been identified previously by direct selection. However, no single obvious candidate gene for the VCFS/DGS phenotype has yet been found. Our technique has been applied to the DiGeorge critical region and has resulted in the isolation of a novel candidate gene, Cdc45l2, similar to yeast Cdc45p. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ0223728 and AF0223729.]
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Síndrome de DiGeorge/genética , Evolución Molecular , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Cromosomas Humanos Par 22 , Clonación Molecular , Secuencia Conservada , ADN Complementario , Peces/genética , Expresión Génica , Biblioteca de Genes , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Hibridación de Ácido Nucleico , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Translocación Genética , Levaduras/genéticaRESUMEN
Chromosome deletions leading to the hemizygous loss of groups of contiguous genes are a major cause of human congenital defects. In some syndromes haploinsufficiency of a single gene causes the majority of the syndromal features, whereas other diseases are thought to be the consequences of a combined haploinsufficiency. In the case of the DiGeorge and velocardiofacial syndromes, caused by deletions within 22q11, the genetic analyses have so far failed to implicate a single gene. By virtue of FISH analysis and the creation of a BAC/P1 genomic clone contig we have mapped 19 murine homologues of genes and nine EST groups from the region deleted in DiGeorge syndrome and found them to be linked on mouse chromosome 16. Rearrangements during the divergence of mouse and human have led to differing gene orders in the two species, with implications for the most appropriate means of mimicking particular human deletions. The map confirms and extends previous analyses and the contig resources toward the generation of targeted deletions in the mouse.
Asunto(s)
Proteínas de Ciclo Celular , Mapeo Cromosómico , Síndrome de DiGeorge/genética , Animales , Bacteriófago P1/genética , Mapeo Cromosómico/métodos , Cromosomas Artificiales de Levadura/química , Cromosomas Artificiales de Levadura/genética , Cromosomas Bacterianos/genética , Cromosomas Humanos Par 22/genética , Clonación Molecular , Chaperonas de Histonas , Humanos , Ratones , Ratones Endogámicos , Proteínas Nucleares/genética , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaRESUMEN
A Drosophila-related expressed sequence tag (DRES) with sequence similarity to the peanut gene has previously been localized to human chromosome 22q11. We have isolated the cDNA corresponding to this DRES and show that it is a novel member of the family of septin genes, which encode proteins with GTPase activity thought to interact during cytokinesis. The predicted protein has P-loop nucleotide binding and GTPase motifs. The gene, which we call PNUTL1, maps to the region of 22q11.2 frequently deleted in DiGeorge and velo-cardio-facial syndromes and is particularly highly expressed in the brain. The mouse homologue, Pnutl1, maps to MMU16 adding to the growing number of genes from the DiGeorge syndrome region that map to this chromosome.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromosomas Humanos Par 22/genética , Síndrome de DiGeorge/genética , Proteínas de Drosophila , Proteínas de Microfilamentos , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , GTP Fosfohidrolasas/genética , Expresión Génica , Genes de Insecto , Humanos , Hibridación Fluorescente in Situ , Proteínas de Insectos/genética , Ratones , Datos de Secuencia Molecular , Embarazo , Septinas , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Most cases of DiGeorge syndrome (DGS) and related abnormalities are associated with deletions within 22q11. Shortest region of deletion overlap (SRO) mapping previously identified a critical region (the DGCR) of 500 kb, which was presumed to contain a gene or genes of major effect in the haploinsufficiency syndromes. The DGCR also contains sequences disrupted by a balanced translocation that is associated with DGS--the ADU breakpoint. We have cloned sequences at the breakpoint and screened for novel genes in its vicinity. A series of alternatively spliced transcripts expressed during human and murine embryogenesis, but with no obvious protein encoding potential, were identified. The gene encoding these RNAs has been named DGCR5 and it is disrupted by the patient ADU breakpoint. DGCR5 is distinct from the DGCR3 open reading frame (ORF) previously shown to be interrupted by the ADU translocation, although DGCR3 is embedded within a DGCR5 intron and in the same (predicted) transcriptional orientation. No mutations of DGCR5 have yet been detected. By analogy to other loci encoding conserved, nontranslated RNAs, it is possible that DGCR5 originates from a cis-acting transcriptional control element in the vicinity of the ADU/VDU breakpoint. Disruption of such an element would result in altered transcription of neighboring genes secondary to a position effect, a hypothesis in keeping with recent refinement of the SRO placing the ADU breakpoint outside the DGCR.
Asunto(s)
Síndrome de DiGeorge/genética , ARN/genética , Translocación Genética , Animales , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 22/genética , Cartilla de ADN/genética , ADN Complementario/genética , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
We have established a high-resolution genetic map of the region surrounding the Fused locus as a first step towards the molecular identification and analysis of this gene. The candidate region has been covered to a large extent by YAC and P1 contigs, and has been partly characterized by pulsed-field gel analysis.
Asunto(s)
Mapeo Cromosómico , Cromosomas , Ratones/genética , Animales , Bacteriófago P1 , Secuencia de Bases , Cromosomas Artificiales de Levadura , Clonación Molecular , ADN/genética , Marcadores Genéticos , Vectores Genéticos , Genoma , Impresión Genómica , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Fenotipo , Recombinación GenéticaAsunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 16 , Hominidae/genética , Ratones/genética , Proteínas Represoras/genética , Esclerosis Tuberosa/genética , Animales , Southern Blotting , Biblioteca de Genes , Variación Genética , Humanos , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Polimorfismo Genético , Mapeo Restrictivo , Teratoma/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
On analysis of 46 Geneticin-resistant (GtR) cell lines, derived by electroporation of mouse embryonic stem (ES) cells with a promoterless neo vector, we observed that in two independently derived cell lines, the vector had integrated into the same locus. The sequence flanking the vector integration site in both cell lines was cloned and sequenced. The vector had integrated into a 3 to 6-bp region in both cell lines. No homology is observed between the integration site sequence and the vector sequence.
