The kinetics for imino hydrogen exchange, at individual base pairs in the DNA sequence corresponding to the lactose operon operator ofEscherichia coli, has been examined by NMR saturation recovery measurements as a function of temperature. Three 17-base-pair subsections of the kac operator DNA were chemically synthesized for these studies. The results support our previous observations in the 36-base-pair complete lac operator DNA fragment that has been used in our previous NMR studies. The results indicate faster opening kinetics at a GTG/CAC that is also the site of operator mutations leading to the highest level of constitutive f8-galactosidase synthesis. The GTG/CAC sequence occurs frequently and often symmetrically in prokaryotic and eukaryotic DNA sites where one anticipates specific protein interaction for gene regulation or recombination.Diffraction studies (1-5) of double helical DNA have yielded a diversity of geometries that vary from uniform B-form DNA (6). In the double helical dodecamer, solved to atomic resolution, there are variations from B-form seen in the structural parameters along the double helical structure (7-9). Since the regular structure of DNA is sequence dependent (10, 11) and varies under physiological conditions (12, 13), these variations can be a part of what proteins recognize. The potential biological significance of this type of heterogeneity is exemplified by the recent interest in Z-DNA (3, 14-16). We report here the possibility of a local structural variation in the lactose operon operator DNA of Escherichia coli detected by a series of kinetic experiments.In our NMR studies of the interaction between lac repressor and lac operator (17), we used a 36-base-pair (bp) DNA fragment containing the lac operator sequence derived from a chemically synthesized sequence cloned in tandem on a plasmid (18). The imino proton region (19) of the NMR spectrum of the 36-bp lac operator fragment shows a nonsequential loss of resonance intensity occurs as the temperature is elevated (20); i.e., melting does not only start from the ends of the double helix. To simplify the examination of this DNA sequence, three 17-bp subsections were synthesized (Fig. 1).The imino proton resonances of the subsections were assigned by nuclear Overhauser enhancement (NOE) and their exchange kinetics were examined by saturation recovery methods developed to probe the structure and dynamics of tR.NA in solution (20,21). Similar kinds of observations have been applied to short DNA fragments obtained from both restriction endonuclease digestion (22,23) and chemical synthesis (24-28).We report here the saturation recovery rates for the resolved imino protons in the lac operator DNA sequence as a function of temperature. There is an interesting dynamic heterogeneity with a maximal opening rate centered about a GTG/CAC sequence that appears frequently and often symmetrically in a number of other prokaryotic and eukaryotic systems where a specific interaction occurs between protein and DNA. Our suggestion is that...
A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.
Raman spectra from three subfragments of the Escherichia coli lactose promoter region were obtained in 0.1 M NaCl. The three DNAs are 21, 40, and 62 bp in length. The 21 and 62 bp DNAs contain the binding site for the catabolite gene activator protein (CAP). The 40 bp DNA contains the binding site for the lac repressor. A quantitative analysis of Raman band characteristics indicates an overall B-type conformation for these gene regulatory sites. Bands which correspond to A-family (807 cm-1) and B-family (834 cm-1) deoxyribose phosphate vibrations have the same intensities as bands found in heterogeneous DNAs. The spectra of the 21 bp CAP site have, however, a small band at 867 cm-1 and several other small differences similar to some characteristics observed in C-DNA spectra. Several dG nucleosides in the CAP site appear to be altered from the conventional C2'-endo/anti conformation. At 45 degrees C, well below the melting region of these DNAs, small changes occur in the spectra of the 40 bp lac repressor site which are not observed in the other DNAs. A weak band occurs at 705 cm-1, and intensity changes are observed at 497, 682, and 792 cm-1. The changes suggest that the conformations of several dG nucleosides are altered and that a small region may exist with characteristics of an A-family backbone. This conformational change at 45 degrees C coincides with previous NMR observations indicating an enhanced imino proton exchange rate at a GTG sequence within the lac operator site.
To investigate contemporary national trends in the diagnosis and management of varicoceles, and identify the most important predictors of varicocele repair.DESIGN: Population-based retrospective cohort. MATERIALS AND METHODS: The MarketScan Commercial Claims and Encounters database was queried using relevant CPT, ICD9, and HCPCS codes to identify all 18-45 year old men diagnosed with a varicocele during 2009-2015. Differences in age, clinical characteristics, geographic distribution, and medical management between men who did and did not undergo varicocele repair during the study period were compared using unpaired ttests and Chi-squared tests for continuous and categorical variables, respectively. Multivariable logistic regression analysis was used to evaluate age, geographic location, and use of semen analysis, hormone analyses, and ultrasound, as predictors of varicocele repair. SAS v.9.4 was used for all statistical analyses. Significance was set at p<0.05.RESULTS: Of the 21,195 men were diagnosed with a varicocele between 2009-2015, 8231 (39%) underwent surgical repair, using either an open (82%), microsurgical (11%), or laparoscopic approach (7%). Men who underwent varicocele repair were more likely to have a diagnosis of male infertility compared to those who did not undergo repair (15 vs. 8%), and were more likely to have complete semen analyses (36 vs. 12%) and serum testosterone evaluation (43 vs. 19%). Use of gonadotropin therapy, selective estrogen receptor modulators, and aromatase inhibitors was also more common among men who underwent varicocele repair. In multivariable regression models, the strongest predictors of varicocele repair were semen analysis evaluation (OR 3.13, 95% CI 2.9-3.38), age 18-25 (OR 2.65, 95% CI (2.37-2.96), and serum testosterone evaluation (OR 2.64, 95% CI 2.46-2.84).CONCLUSIONS: Male hypogonadism is emerging as an important and independent predictor of varicocelectomy among U.S. men. The most common approach to varicocele repair remains open ligation, followed by microsurgery, and then laparoscopy.
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