RESUMEN
Two previously healthy, immunocompetent men had persistent Rochalimaea henselae bacteremia with clinical relapses after courses of antibiotics to which the isolates were ultimately demonstrated susceptible in vitro. Both had sustained tick bites prior to their illnesses, thus demonstrating an association not previously identified, although suspected. The first patient had relapsing fever, constitutional symptoms, and an episode of aseptic meningitis despite therapy with amoxicillin, then with doxycycline, and then with ceftriaxone. Thereafter, he spontaneously became asymptomatic during a span of 2 months of persistent bacteremia. Finally, after 2 weeks of therapy with ceftriaxone plus gentamicin, followed by 4 weeks of therapy with oral ciprofloxacin, his bacteremia was cured. The second man had relapsing fever and constitutional symptoms after courses of tetracycline, then of chloramphenicol, and then of doxycycline. He became permanently asymptomatic after serial 2-week courses of chloramphenicol and erythromycin. The greater efficacy of lysis-centrifugation blood cultures in the recovery of R. henselae was noted.
Asunto(s)
Inmunocompetencia , Infecciones por Rickettsiaceae/microbiología , Rickettsieae/aislamiento & purificación , Adulto , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Vectores Arácnidos , Secuencia de Bases , ADN Bacteriano/análisis , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/química , Recurrencia , Mapeo Restrictivo , Infecciones por Rickettsiaceae/tratamiento farmacológico , Infecciones por Rickettsiaceae/etiología , Rickettsieae/efectos de los fármacos , Rickettsieae/genética , GarrapatasRESUMEN
DNA transfected into mammalian cells is subject to the high mutation frequency of approximately 1% per gene. We present data bearing on the derivation of the two main classes of mutations detected, base substitutions and deletions. The DNA sequence change is reported for nearly 100 independent base substitution mutations that occurred in shuttle vectors as a result of passage in simian cells. All of the mutations occur at G:C base pairs and involve either transition to A:T or transversion to T:A. To identify possible mutational intermediates, various topological forms of the vector DNA were introduced separately. Supercoiled and relaxed DNA are mutated at equal frequencies. However, linearized DNA leads to a greatly elevated frequency of deletions. Nicked and gapped templates stimulate both deletions and base substitutions. We discuss a model involving intracellular degradation of the transfected DNA which explains these observations.
Asunto(s)
ADN/genética , Mutación , Transfección , Animales , Secuencia de Bases , Células Cultivadas , Deleción Cromosómica , Vectores Genéticos , Mamíferos , Modelos Genéticos , PlásmidosRESUMEN
Papovavirus-based shuttle vectors containing the bacterial lacI gene were used to show that a mutation frequency in the range of 1% occurs in lacI when such vectors are transfected into COS7 and CV-1 simian cells, NIH 3T3, 3T6, L, and C127 mouse cells, and human 293 and HeLa cells. This frequency is approximately four orders of magnitude higher than the spontaneous mutation frequency in either mammalian or bacterial cells. The mutations are predominantly base substitutions and deletions and also include insertions from the mammalian genome. Time course experiments argue that mutagenesis occurs soon after arrival of the DNA into the nucleus. However, replication of the vector is not required since mutations occur even when the vector lacks all viral sequences. The high mutation frequency appears to be the characteristic outcome of transfection of DNA into mammalian cells.
Asunto(s)
ADN Viral/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Mutación , Transfección , Animales , División Celular , Cromatografía por Intercambio Iónico , Enzimas de Restricción del ADN/metabolismo , Elementos Transponibles de ADN , Desoxirribonucleasa EcoRI , Haplorrinos , Humanos , Cinética , Ratones , Plásmidos , Virus 40 de los Simios/genéticaRESUMEN
We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/genética , Animales , Línea Celular , Mapeo Cromosómico , Cricetinae , Cricetulus , Enzimas de Restricción del ADN , Femenino , Amplificación de Genes , Genes , Vectores Genéticos , Ovario , FenotipoRESUMEN
In eukaryotic cells, each chromosome is divided into several thousand tandemly arranged replicons, each synthesized at a characteristic time during the S period. Although as yet no eukaryotic DNA sequence known for certain to be a eukaryotic chromosomal replication origin has been isolated, electron microscopic and biochemical evidence suggests that eukaryotic DNA synthesis is initiated at specific sites. We have attempted to establish a system in which functional chromosomal origins could be identified in vivo before their isolation by molecular cloning. For this purpose, we have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains 1,000 copies of a 135-kilobase (kb) early-replicating sequence, and which includes the gene for dihydrofolate reductase (DHFR). We have shown that initiation of DNA synthesis within each repeated unit (amplicon) is restricted to a small subset of restriction fragments at the beginning of the S period, suggesting that these fragments contain or flank origins of DNA synthesis. Here we report molecular cloning and restriction mapping experiments showing that all of these early-labelled fragments (ELFs) are derived from a single locus within each repeated unit. This result implies that synthesis of each amplicon initiates from a single origin of replication at the onset of S, and that an amplicon is formally equivalent to a replicon.