RESUMEN
The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala(37)-Pro(48)) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala(49)-Pro(57)) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala(37)-Pro(48) stretch and requires the presence of the membrane anchoring domain.
Asunto(s)
Intestino Delgado/metabolismo , Proteínas de la Membrana/genética , Microvellosidades/metabolismo , Complejo Sacarasa-Isomaltasa/genética , Animales , Transporte Biológico/genética , Línea Celular , Centrifugación por Gradiente de Densidad , Perros , Glicosilación , Intestino Delgado/enzimología , Cinética , Metabolismo de los Lípidos , Proteínas de la Membrana/química , Eliminación de Secuencia , Complejo Sacarasa-Isomaltasa/química , TransfecciónAsunto(s)
Daño del ADN , Plásmidos/efectos de los fármacos , Especies Reactivas de Oxígeno/toxicidad , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Agar/métodos , Vectores Genéticos , Indicadores y Reactivos , Datos de Secuencia Molecular , Radioisótopos de Fósforo , Plásmidos/química , Plásmidos/aislamiento & purificación , Técnica de Dilución de RadioisótoposRESUMEN
A 203 bp HpaII fragment (X9) cloned from F9 embryonal carcinoma cell DNA showed multiple bands on Southern hybridization towards mouse DNA digested with various restriction enzymes. No tandem repeats were evident. A 200 bp MspI band corresponded to several copies of the cloned fragment and many elements appeared each associated with a 12-kb PvuII fragment. X9 is therefore a member of a moderately repetitive family probably dispersed in the mouse genome. X9 displayed polymorphisms between different Mus species, whereas multiple hybridizing bands were detected neither in DNA from rodent species outside the Mus genus nor in DNA from man. Therefore, X9 provides a useful probe for studies of murine evolution. X9 contains a TATA box and a CCAAT box as well as several homologies to known transcription factor binding sites. When cloned in front of a reporter gene, it acted as a strong promoter in several different cell types. Most genomic sequences detected by X9 were highly methylated. A single copy appeared hypomethylated in adult mouse tissues, whereas several copies were hypomethylated in F9 and embryonic stem cells. It is speculated that X9 might represent the promoter of a murine gene family whose activity is regulated by DNA methylation.
Asunto(s)
ADN de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Teratoma/genética , Animales , Secuencia de Bases , Southern Blotting , Línea Celular , Cromosomas/ultraestructura , Clonación Molecular , ADN de Neoplasias/aislamiento & purificación , Regulación Neoplásica de la Expresión Génica , Metilación , Ratones , Datos de Secuencia Molecular , Mapeo Restrictivo , Teratoma/patologíaRESUMEN
Singlet molecular oxygen (1O2) was generated in aqueous solution (H2O or D2O) at 37 degrees C by the thermal dissociation of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). Guanosine and deoxyguanosine quench 1O2 with overall quenching rate constants of 6.2 X 10(6) M-1 s-1 and 5.2 X 10(6) M-1 s-1, respectively. Reaction with 1O2 results in the formation of 8-hydroxyguanosine (8-OH-Guo) and 8-hydroxydeoxyguanosine (8-OH-dGuo), respectively, with a yield of 1.5% at 1 mM substrate with an NDPO2 concentration of 40 mM; a corresponding 8-hydroxy derivative is not formed from deoxyadenosine. In D2O the yield of 8-OH-Guo is 1.5-fold that in H2O. Sodium azide suppresses 8-OH-Guo and 8-OH-dGuo production. In contrast, the hydroxyl radical scavengers, tert-butanol, 2-propanol, or sodium formate, do not decrease the production of the 8-OH derivatives. The formation of 8-OH derivatives is significantly increased (2-5-fold) by thiols such as dithiothreitol, glutathione, cysteine, and cysteamine. With use of a plasmid containing a fragment of the mouse metallothionein I promoter (pMTP3') and a novel end-labeling technique, the position of 1O2-induced single-strand breaks in DNA was examined. Strand breaks occur selectively at dGuo; no major differences (hot spots) were observed between individual guanines.