RESUMEN
bicaudal was the first Drosophila mutation identified as producing mirror-image pattern duplications along the anteroposterior axis of the embryo. However the mutation has been little studied due to its low penetrance and suppressibility. We undertook cloning of the bicaudal locus together with studies of the mutation's effects on key elements of the posterior embryonic patterning pathway. Our mapping studies place the bicaudal mutation within a approximately 2 kb region, 3' to the protein coding sequence of the Drosophila homolog of beta NAC, a subunit of Nascent polypeptide Associated Complex (NAC). Genomic DNA encoding beta NAC completely rescues the bicaudal phenotype. The lethal phenotype of Enhancer of Bicaudal, E(Bic), a mutation hypothesized to affect the bicaudal locus, is also completely rescued by the beta NAC locus. We further demonstrate that the E(Bic) mutation is caused by a P element insertion into the transcribed region of the beta NAC gene. NAC is among the first ribosome-associated entities to bind the nascent polypeptide after peptide bond formation. In contrast to other bicaudal-embryo-producing mutations, bicaudal leads to ectopic translation of mRNA for the posterior determinant nanos, without affecting the localization of mRNA for its upstream regulator, oskar, in the embryo. These findings suggest that repression of nanos mRNA translation occurs on the ribosome and involves a role for beta NAC.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas de Insectos/genética , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Clonación Molecular , Secuencia de Consenso , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Morfogénesis , Biosíntesis de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genética , Transactivadores/química , Transactivadores/genéticaRESUMEN
We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenosina Trifosfatasas/farmacología , Proteínas Bacterianas/farmacología , Benzopirenos/farmacología , ADN Helicasas , Reparación del ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/farmacología , Endodesoxirribonucleasas , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Daño del ADN , ADN Bacteriano/efectos de los fármacos , Desoxirribonucleasas/metabolismo , Isomerismo , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis , TransfecciónRESUMEN
The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.