RESUMEN
OBJECTIVE: To describe maternal concerns at 2 weeks and mothers' ability to function in various roles at 4 weeks postpartum. DESIGN: Subjects were mailed questionnaires at 2 and 4 weeks postpartum. SETTING: A mid-Atlantic hospital that is a part of an academic health center. PARTICIPANTS: A convenience sample of 100 healthy women who were discharged < or = 48 hours postpartum and met the study criteria. MAIN OUTCOME MEASURES: Instruments included a demographic questionnaire, the Maternal Concerns Questionnaire, and the Inventory of Functional Status After Childbirth. RESULTS: Physiologic concerns during the early postpartum period related primarily to perineal sutures, breast care, body image, and fatigue. More than 25% of the women continued to experience depression at 2 weeks, and 40% had days at 1 month when they never got dressed. Much concern was expressed about the mothering role but little about interaction with the mate. Older maternal age was associated with a longer hospital stay, and the trend suggested improved functioning at 1 month with longer hospitalization. More than half of the participants said they would visit a nurse-run clinic during the 1st postpartum week if given the opportunity. CONCLUSIONS: The results provide descriptive information about effects of early hospital discharge. Mothers discharged in 48 hours or less adjust to motherhood without the education and assessment that was formerly possible with extended hospitalization. The current study reinforces the need for nurses to explore strategies in hospitals or communities to provide early care for postpartum mothers.
Asunto(s)
Adaptación Psicológica , Tiempo de Internación , Enfermería Maternoinfantil , Relaciones Madre-Hijo , Alta del Paciente/normas , Periodo Posparto , Adolescente , Adulto , District of Columbia , Femenino , Humanos , Recién Nacido , Persona de Mediana Edad , Educación del Paciente como Asunto , Rol , Encuestas y CuestionariosRESUMEN
The UvrABC excinuclease is involved in the nucleotide excision repair (NER) pathway. Sequence-dependent differences in repair efficiency have been reported for many different lesions, and it is often suggested that sites with poor repair contribute to the occurrence of mutation hot spots. However, guanine bases modified by N-2-acetylaminofluorence (AAF) within the NarI site (5'-G1G2CG3CC-3') are incised by the UvrABC excinuclease with different efficiencies in a pattern not correlated with the potency of mutation induction. To gain insight into the mechanism of sequence-dependent modulation of NER, we analyzed the formation, the structure and the stability of UvrB-DNA pre-incision complexes formed at all three positions of the AAF-modified NarI site. We show that the efficiency of release of UvrA2 from specific UvrA2B-DNA complexes is sequence-dependent and that the efficiency of incision is inversely related to the stability of the pre-incision complex. We propose that the pre-incision complex, [UvrB-DNA], when formed upon dissociation of UvrA2, undergoes a conformational change (isomerization step) giving rise to an unstable but incision-competent complex that we call [UvrB-DNA]'. The [UvrB-DNA] complex is stable and unable to form an incision-competent complex with UvrC. As the release of UvrA2, this isomerization step is sequence-dependent. Both steps contribute to modulate NER efficiency.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas , Reparación del ADN , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Oligodesoxirribonucleótidos/metabolismo , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/farmacología , Aductos de ADN/metabolismo , Daño del ADN , Huella de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Electroforesis en Gel de Poliacrilamida , Guanina/análogos & derivados , Guanina/metabolismo , Oligodesoxirribonucleótidos/síntesis químicaRESUMEN
Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro. In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG1G2G3-3'). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified. The modified homoduplex was designated SmaI/3. deltaC/O and deltaG/O were weakly recognized by UvrA2B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.
Asunto(s)
Aductos de ADN/metabolismo , ADN Helicasas , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Mutación del Sistema de Lectura , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/farmacología , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Guanina/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , Especificidad por SustratoRESUMEN
Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.
Asunto(s)
Secuencia de Bases , Aductos de ADN , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Aductos de ADN/química , Aductos de ADN/genética , Reparación del ADN/genética , Escherichia coli/enzimología , Guanina/química , Humanos , Datos de Secuencia Molecular , Polidesoxirribonucleótidos/síntesis química , Polidesoxirribonucleótidos/genéticaRESUMEN
(A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees. The UvrB-DNA complex is then recognized by UvrC and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy. Both methods reveal that the mutant UvrB is unable to bend DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas , ADN/metabolismo , Proteínas de Escherichia coli , Mutación/fisiología , Conformación de Ácido Nucleico , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , ADN/química , ADN/efectos de la radiación , ADN/ultraestructura , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Microscopía Electrónica/métodos , Espectrofotometría Ultravioleta/métodos , Rayos UltravioletaRESUMEN
We have tested in vitro the activity of T4 endonuclease VII on three different double-stranded oligonucleotides bearing a single N-2-acetylaminofluorene (AAF) adduct covalently bound to each of the three guanine residues located within the NarI site (G1G2CG3CC), a strong frameshift mutation hot spot in E. coli. With the oligonucleotides modified at G2 and G3 a specific cleavage pattern with T4 endonuclease VII was observed in the complementary strand while no cleavage was found in the adduct-bearing strand. On the other hand, when G1 was modified, only a very faint cleavage band was observed (< 1%). These differences in nicking among the three AAF-modified DNA substrates are discussed in terms of the polymorphic nature in adduct-induced DNA structures as previously shown. This "non-physiological" activity of a DNA resolvase is discussed in terms of a potential role for such enzymes in the induction of frameshift mutations.
Asunto(s)
2-Acetilaminofluoreno/metabolismo , ADN/genética , Endodesoxirribonucleasas/metabolismo , Mutación del Sistema de Lectura/genética , 2-Acetilaminofluoreno/farmacología , Bacteriófago T4/enzimología , Secuencia de Bases , ADN/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismoRESUMEN
The interaction between UvrABC excinuclease from Escherichia coli and ultraviolet light-(UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence of effector ATP. The change was specific for UV-damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV-damaged DNA. The structural modification of DNA, promoted by the (UvrA2-UvrB) complex, probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.
Asunto(s)
Daño del ADN , ADN Helicasas , Reparación del ADN/fisiología , ADN Viral/efectos de la radiación , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago T7/química , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Sustancias Macromoleculares , Análisis Espectral/métodos , Rayos UltravioletaRESUMEN
(A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli. The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.
Asunto(s)
Daño del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Oligodesoxirribonucleótidos/síntesis química , Composición de Base , Secuencia de Bases , ADN/química , Desoxirribonucleasa I , Cinética , Datos de Secuencia Molecular , Mapeo Nucleótido , Unión Proteica , Especificidad por SustratoRESUMEN
We have determined the kinetics of initiation of transcription of the wild-type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M-1 and 1.7 x 10(-2) s-1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild-type sequence with an up-mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (-0.05), leading to a 12-fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Metiltransferasas/genética , Regiones Promotoras Genéticas , Transcripción Genética , Secuencia de Bases , ADN-Topoisomerasas de Tipo I/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Datos de Secuencia Molecular , Mutación , O(6)-Metilguanina-ADN Metiltransferasa , Sondas de Oligonucleótidos , Plásmidos , Unión ProteicaRESUMEN
Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.
Asunto(s)
Reparación del ADN , Amplificación de Genes , Genes Bacterianos , Plásmidos , Respuesta SOS en Genética , Alelos , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/efectos de la radiación , Expresión Génica , Transformación Bacteriana , Rayos Ultravioleta , beta-Lactamasas/metabolismoRESUMEN
In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped-flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 microliters), and the short dead time (approximately 0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3-fold. Nevertheless, it does not change significantly the product KBk2, which is indicative of promoter strength and shows that uvrA is an 'association-limited' promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.
Asunto(s)
Proteínas Bacterianas/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Fluorometría/instrumentación , Regulación de la Expresión Génica , Plásmidos , Regiones Promotoras Genéticas , Serina Endopeptidasas , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Cinética , Proteínas Represoras/farmacología , Transcripción GenéticaRESUMEN
A comparative study of the interaction of the LexA repressor of Escherichia coli and of its amino-terminal DNA binding domain to the uvrA operator has been undertaken. Most of the binding constants are determined from competition experiments with RNA polymerase by measuring the time-course of the abortive initiation transcriptional activity. The presence of repressor increases the lag time, tau, without affecting the final maximum activity. The inhibition of transcription by LexA, at least in the case of the uvrA gene, is thus a transient, time-dependent phenomenon, because once the RNA polymerase is engaged in a stable "open" complex, it is quasi-irreversibly trapped in this state. A study of the binding constants as a function of ionic strength suggests the formation of 5.5(+/- 1) salt bridges between the uvrA operator and a LexA dimer. Surprisingly, the binding affinity of the amino-terminal domain was only about one order of magnitude smaller than that of the entire LexA repressor. The determination of the binding constant of the RNA polymerase to the "closed" uvrA promoter (KB approximately 1 X 10(7) to 2 X 10(7) M-1) allowed us to determine theoretical repression curves for the two repressor species. These calculations show that the binding constant found for LexA is sufficiently high to account for substantial or complete repression, and that of the amino-terminal domain is sufficiently low to account for partial or nearly full induction. Under solvent conditions used by others for the determination of binding constants to other SOS operators by DNAase I footprinting, the uvrA operator turns out to be a rather weak one (K approximately 3 X 10(7) M-1), being comparable with that of the uvrB gene. The uvrA promoter is "association-limited" with a KB X k2 product fitting very nicely the homology score for the promoter of 55.
Asunto(s)
Proteínas Bacterianas/genética , ADN Bacteriano/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Serina Endopeptidasas , Factores de Transcripción/genética , Sitios de Unión , Desoxirribonucleasa I , Cinética , Transcripción GenéticaRESUMEN
Supercoiling of DNA is now known to have considerable effects on transcription in bacteria. By abortive initiation reaction (6) we have determined the binding constant KB and the forward rate of isomerization k2 as a function of temperature, pH and buffer for the tet promoter in a supercoiled plasmid. If the activation energy of isomerization is very similar to that obtained previously under the same conditions on a linearized plasmid (6) (respectively 21 +/- 5 kcal/mole and 13 +/- 5 kcal/mole) the supercoiling introduces very important and not well understood changes in the thermodynamic parameters of the association polymerase - promoter. Using the technique of superhelical DNA relaxation by eukaryotic topoisomerase I, we have determined the specific unwinding by RNA polymerase of the tet promoter of pBR322 (430 degrees). This unwinding differs only slightly from the mean value (470 degrees) obtained for all the promoters of pBR322.
Asunto(s)
ADN Superhelicoidal/genética , Escherichia coli/genética , Operón , Plásmidos , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Cinética , Espectrometría de Fluorescencia , Moldes Genéticos , TermodinámicaRESUMEN
In order to follow the kinetics of the initiation of transcription by the E. coli RNA polymerase, we have used the procedure of abortive initiation as described by Mc Clure (1980) (7). In place of radioactive labeling we have taken advantage of a fluorescent probe (UTP gamma ANS) to obtain fast and accurate determinations of the rate of transcription and to deduce from kinetic equations both the binding constant (KB) and the rate of isomerization (k2) which characterize the classical two-step model. This analysis was applied to the tet promoter of pBR322 in a linearized plasmid DNA and was studied in function of temperature (from 25 degrees C to 37 degrees C) and of pH (from 6 to 8.3). The association is entropy driven (delta H degrees = 29 Kcal/mole and delta S degrees = 130 e.u.). The activation energy of isomerization is 13 Kcal/mole. Both k2 and k-2 are increasing with pH. The insensitivity to pH of the KBK2 product could be tentatively explained in terms of the processive aspect of the polymerase binding to its specific site.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Operón , Plásmidos , Transcripción Genética , Cinética , Modelos Genéticos , TemperaturaRESUMEN
Core particle DNA unfolding and refolding are followed by stopped-flow circular dichroism technique. When core particles are dissociated in the stopped-flow cuvette, the high CD deviation corresponding to the dissociated state is reached in the first millisecond, which means that the dissociation process is completed within the dead time of the apparatus which is approximately 1 ms. The same conclusion can be drawn when core particles are reassociated, since the low CD value, typical of the associated state, is immediately reached. Similarly histone release from chromatin is a very fast process. We also include some points of discussion about core particle assembly process.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Histonas/metabolismo , Animales , Dicroismo Circular , Cinética , Conformación de Ácido Nucleico , Desnaturalización Proteica , Factores de TiempoRESUMEN
Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.