RESUMEN
We have examined the ability of proliferating myoblasts and post-mitotic, differentiated myotubes to produce retroviral vector using hybrid adeno-retroviral vectors as templates. We show that production of retroviral vector from myoblasts peaks 48 h after adenoviral infection at 4.8 x 10(4) cfu/ml and is scarcely detectable by 96 h. Both fully and partially differentiated myotubes were able to generate a sustained increase in the levels of retroviral vector compared with myoblasts peaking 48 h at 1.4 x 10(5) cfu/ml and 1.8 x 10(5) cfu/ml, respectively. Addition of the cell cycle inhibitor aphidicolin (5 microg/ml) had no effect on the production of retroviral vector from fully differentiated myotubes, but resulted in an 80% increase in vector production from partially differentiated myotubes. Thus indicating that retroviral vector production is more efficient in post-mitotic myotubes and is independent of muscle cell cycle progression.
Asunto(s)
Adenoviridae/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Fibras Musculares Esqueléticas/virología , Distrofia Muscular de Duchenne/terapia , Retroviridae/genética , Células 3T3 , Animales , Antivirales/farmacología , Afidicolina/farmacología , Diferenciación Celular , Línea Celular , Expresión Génica , Ingeniería Genética/métodos , Vectores Genéticos/farmacología , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratones , Microscopía Fluorescente , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/ultraestructuraRESUMEN
The complete sequence of the polycystic kidney disease gene (PKD1) and its transcript have been described. The predicted protein is not a member of a previously described gene family, but contains several structural motifs that are present in proteins of known function. Most of these domains are present in the extracellular parts of proteins involved in interactions with other proteins and carbohydrates. The PKD1 gene product also contains potential transmembrane sequences. The molecule is likely to be involved in cell-cell or cell-matrix interactions, which is consistent with the different manifestations of polycystic kidney disease.
Asunto(s)
Genes , Proteínas/genética , Humanos , Riñón Poliquístico Autosómico Dominante/genética , Proteínas/fisiología , Relación Estructura-Actividad , Canales Catiónicos TRPPRESUMEN
The major locus for autosomal dominant polycystic kidney disease (PKD1) is located in a gene-rich region on chromosome 16p13.3. Recently the identification of the gene responsible for PKD1 has been described. While searching for candidate genes in this region, we isolated a new member of the cyclin family. We have characterized the transcript by sequencing, determination of the exon intron boundaries, and Northern blot analysis. Cyclin F is related to A- and B-type cyclins by sequence, but its function is unknown.
Asunto(s)
Ciclinas/genética , Riñón Poliquístico Autosómico Dominante/genética , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 16 , Ciclinas/química , ADN Complementario , Humanos , Datos de Secuencia Molecular , Proteínas/química , Canales Catiónicos TRPPRESUMEN
A female patient with Turner syndrome and the karyotype mos45,X/46,X,r(Y)/46,XY is described. Physical mapping of the ring chromosome by Y-specific single-copy and moderately repeated DNA sequences as molecular probes showed that, in addition to the heterochromatic part of Yq, a considerable portion of the Yp has also been lost in the course of the rearrangement. Thus, molecular findings provide independent support that this structurally abnormal sex chromosome is a ring Y and agree with the generally accepted model of ring formation requiring breaks in both chromosome arms. Clinical consequences of Y chromosome mosaicism in patients with Turner syndrome are discussed.
Asunto(s)
Cromosomas en Anillo , Síndrome de Turner/genética , Cromosoma Y/ultraestructura , Southern Blotting , Niño , Bandeo Cromosómico , ADN/análisis , Femenino , Reordenamiento Génico , Marcadores Genéticos , Humanos , MosaicismoRESUMEN
Autosomal dominant polycystic kidney disease (PKD1) is linked to the alpha-globin locus near the telomere of chromosome 16p. We established the existence of a conserved linkage group in mouse by mapping conserved sequences and cDNAs from the region surrounding the PKD1 gene in the mouse genome. Results obtained with the BXD recombinant strain system and somatic cell hybrids show the homologous region to be located on mouse chromosome 17 near the globin pseudogene Hba-ps4, an unprocessed alpha-like globin gene. The markers we mapped are widely distributed over the region known to contain the PKD1 gene, and it is therefore likely that the mouse homologue of PKD1 is also located on mouse chromosome 17.
Asunto(s)
Cromosomas Humanos Par 16 , Ligamiento Genético/genética , Globinas/genética , Enfermedades Renales Poliquísticas/genética , Homología de Secuencia de Ácido Nucleico , Animales , Southern Blotting , Marcadores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Polimorfismo de Longitud del Fragmento de Restricción , Seudogenes/genéticaRESUMEN
A young male with a karyotype of 46,X,+ mar is described. Physical mapping of the marker chromosome by using Y-specific single-copy or moderately repeated DNA sequences as molecular probes showed that, in addition to the heterochromatic part of the Yq, a considerable portion of the euchromatin in both Yp and Yq had been lost. These findings suggest that the marker chromosome is a ring Y, for the generally accepted model of ring formation implies breakages in both chromosome arms. The clinical features of the patient correlated well with the phenotypic changes expected from the loss of genetic material from the Y.
Asunto(s)
Cromosomas en Anillo , Aberraciones Cromosómicas Sexuales/genética , Cromosoma Y , Adolescente , Southern Blotting , Mapeo Cromosómico , Sondas de ADN , Femenino , Humanos , Cariotipificación , Masculino , Linaje , FenotipoRESUMEN
Using Southern hybridization with the DNA probe pY3.4, we were not able to detect fetal DNA in blood of 36 pregnant women carrying male fetuses. Gestational ages ranged from 8-40 weeks of pregnancy. Using the same DNA probe, we were able to detect the male-specific signal in experimental dilution series down to 1/5000 on autoradiograms. We conclude that the ratio of fetal DNA in maternal circulation, in contrast to previous estimations, must be lower than 1/5000.
Asunto(s)
ADN/sangre , Desarrollo Embrionario y Fetal , Edad Gestacional , Embarazo/sangre , Autorradiografía , Southern Blotting , Sondas de ADN , Femenino , Humanos , Hibridación Genética , Masculino , Biología Molecular , Reacción en Cadena de la Polimerasa , Embarazo/genética , Diagnóstico Prenatal/métodos , Factores SexualesRESUMEN
We investigated the risk of maternal contamination in antenatal DNA diagnosis after second- and third-trimester transabdominal placental biopsy. For this purpose, we compared the restriction fragment length polymorphism (RFLP) patterns of 11 chorionic villus DNA samples after late chorionic villus sampling (CVS) with those of the corresponding maternal DNA. Ten of 11 aspirated tissue samples were not separated from maternal contamination before DNA extraction. All 11 mother-embryo pairs were informative for analysis of maternal contamination, ie, the mother showed one RFLP allele not present in the embryo. In none of the 11 cases did the fetal DNA show maternal contamination after molecular hybridization, although ten samples were contaminated with maternal tissue macroscopically and microscopically. Despite some maternal tissue admixture, the risk of contamination seems to be lower in the second- and third-trimester CVS than in first-trimester CVS, based on previous reports and our own experiences. This is most likely due to the anatomically closer contact of villi and decidua in the first trimester of pregnancy.