摘要:

AbstractThe theory of mass transport coupled to revesible macromolecular interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility‐shift behavior of protein‐DNA complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putative cage effect; (iii) cooperative binding to multiple sites; (iv) formation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooperative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target genes. Favorable comparison of simulated with experimental mobility‐shift behavior indicates that the phenomenological mechanisms, whereby observed mobility‐shift patterns are generated during electrophoresis, are embodies in the theory. These studies have provided guidelines for definitive interpretation of mobility‐shift assays and for the design of experiments to develop a detailed understanding of the particular system under inve

ISSN:0173-0835    

DOI:10.1002/elps.1150190202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractIn this study we show how the use of exon‐primed, intron‐crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine‐specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch,Erythrura gouldiae.A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low‐melting domain. Sequencing of the entire fragment from 19Er. gouldiaerevealed five nucleotide substitution differences within the low‐melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high‐melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these

ISSN:0173-0835    

DOI:10.1002/elps.1150190203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractUtilization of existing isozyme analysis facilities to detect sequence‐tagged microsatellite site (STMS) polymorphism or any simple sequence repeat (SSR) variation is described. Different parameters concerning the difficulties in transferring molecular techniques to less sophisticated laboratory infrastructures (i. e.tropical outstations) are discussed (e.g.reproducibility, efficacy, precision). Nonradioactive STMS analysis is bound to foster collaborative research between “biodiversity” and “biotechnology

ISSN:0173-0835    

DOI:10.1002/elps.1150190204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractOligonucleotide fingerprinting was performed on three beetle species from different families (Coleoptera: Staphylinidae:Aleochara curtula; Silphidae:Nicrophorus vespilloides; Tenebrionidae:Blaps lethifera) to obtain detailed information on parentage in mating systems. We report variations in the number of hybridizing fragments for the different species depending on the combinations of probes and restriction enzymes used. In addition to conventional multilocus fingerprint patterns, we established a single locus system forA. curtula(GTG5;HinfI) and described an oligolocus system inN. vespilloides(GATA4;HaeIII).

ISSN:0173-0835    

DOI:10.1002/elps.1150190205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5–9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base delection. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG‐SSCP). Fluorescent‐labeled PCR primers were used for amplification of specific exons for mutation detection. SG‐SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations. The separation with respect to wild type varied for each mutation examined. Subambient temperature (20°C) was preferable overall for discrimination of these mutations as a group. We intend to use this system to examine a much larger panel of p53 mutation standards that are now under dev

ISSN:0173-0835    

DOI:10.1002/elps.1150190206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrohoresis (CE‐SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG‐SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5′ Primers were labeled with FAM (5‐carboxyfluorescein) and 3′ primers were labeled with JOE (2′,7′‐dimethoxy‐4′,5′‐dichloro‐6‐carboxyfluorescein). CE‐SSCP was performed using the Perkin Elmer ABI PRISM™ 310 Genetic Analyzer with GeneScan™ Software and the Beckman P/ACE™ 5510 CE equipped for laser‐induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild‐type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE‐SSCP measurements were performed at temperatures ranging from 10 to 60°C on a prototype instrument. For mutations measured at ambient temperature (25°C), characteristic shift in direction and magnitude were observed in the migration times to both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitute and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature‐specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE‐SSCP. In addition, by using CE, discrete intra‐strand isoforms could be easily observed at different temperatures. The combination of mutation‐specific temperature profiling and analysis of isoforms by CE‐SSCP should be of help to the diagn

ISSN:0173-0835    

DOI:10.1002/elps.1150190207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractTemporal control over both pH and ionic strength of an electrolyte solution with high accuracy was achieved with a dynamic, computer feedback‐controlled amperometric pH‐stat device consisting of four pH‐regulating electrodes placed in electrolyte reservoirs that are separated by dialysis membranes from a central compartment. Theoretical predictions of the behavior of this arrangement, obtained by computer simulation, were validated by running temporal pH programs such as step functions, oscillations, and linear pH gradients. Deviations from nominal values given by the computer program are within the limits of accuracy of the pH‐measuring electrodes. No volume changes accompany a change of pH or conductivity since ions are forced to leave or enter the central compartment through the membranes by the electrical force applied between the pH‐regulating electrodes. The device is flexible, easy to use and easily miniaturized. We discuss a wide range of possible applications in biochemistry and cell science. These include automated pH adjustment, isoelectric protein separation, amperometric measurement of enzyme kinetics and the response of cell cultures to well‐defined

ISSN:0173-0835    

DOI:10.1002/elps.1150190208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractFor calculating the buffer capacity of ampholytes an application of the stepwise and parallel dissociation models is considered. It is demonstrated that the scheme with stepwise dissociation possesses a “nonadditive” sum,i.e., the resulting buffer capacity of the system exceeds the sum of the separate contributions of the two ionogenic groups. On the other hand, the scheme of parallel dissociation is free from this disadvantage, and thus does not impose any restriction on the ΔpKparam

ISSN:0173-0835    

DOI:10.1002/elps.1150190209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe evolution of an isotachophoresis (ITP) system in acidic or basic pH ranges can be quite different from that predicted by the existing theory. It was found theoretically and proved experimentally that the contribution of hydrogen or hydroxyl ion to conductivity of solution and/or its net charge changes the behavior of the ITP system, creating in the terminating electrolyte an additional zone close to the initial interfaces between electrolytes (leader and terminator). One boundary of the zone, being either sharp or dispersed, moves toward the leader; the other is always sharp and stationary and coincides with initial electrolytes' discontinuity. The latter can be registered in the presence of electroosmotic flow which delivers it to the detection point. In order to describe the dynamics of the ITP system at pH extremes an algorithm of analytical solution was developed, based on the revised Kohlrausch theory. Its predictions coincide well with computer simulations and experimental data. The results presented can help in a correct analysis of ITP data and explain some confusing phenomena which were considered to be artifacts.

ISSN:0173-0835    

DOI:10.1002/elps.1150190210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractNonfluorescing protein bands can be detected by the fluorescence optics of the commerical gel electrophoresis apparatus with automated scanning of the migration path (HPGE‐1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins (“fluorescence reduction”). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of “fluorescence reduction” equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by “fluorescence reduction” provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 μg/lane of the gel tray. A theory of “fluorescence reduction” is presented which accounts for the existence of a linear relationship between

ISSN:0173-0835    

DOI:10.1002/elps.1150190211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY