Molecular Weight Ladder Research Articles

A novel DNA molecular weight ladder

A novel DNA molecular weight ladder

Experimentally Induced Cryptorchidism Increases Apoptosis in Rat Testis1

Although surgical induction of cryptorchidism in the rat is known to cause infertility due to disruption of spermatogenesis, the exact cellular mechanism responsible for the degenerative changes in cryptorchid testes is unclear. Using a sensitive autoradiographic method for the detection of apoptotic DNA fragmentation, we have investigated the effect of experimentally induced cryptorchidism on apoptotic cell death in testes of immature rats. Bilateral or unilateral cryptorchidism decreased the weight of affected testes within 4 days; these decreases (24-27%) became significant (P < 0.05) at 7 days after the operation. Testes of sham-operated animals contained predominantly high molecular weight DNA (> 15 kb), whereas DNA cleavage into low molecular weight ladders characteristic of apoptosis was increased by induction of bilateral cryptorchidism in a time-dependent manner, i.e., 2.0-, 2.8-, and 4.2-fold (p < 0.05) at 2, 4, and 7 days after operation, respectively. In unilaterally cryptorchid animals, sham-operated testes also contained predominantly high molecular weight DNA, whereas induction of cryptorchidism of the contralateral testes increased DNA cleavage into low molecular weight fragments 3.0-, 2.8-, and 3.9-fold (p < 0.05) at 2, 4, and 7 days after the operation, respectively. In situ analysis of DNA fragmentation in testes of unilaterally cryptorchid rats at 7 days after the operation indicated that germ cells, mainly primary spermatocytes were affected and that the percentage of seminiferous tubules containing labeled cells increased in the operated testis as compared to the contralateral control in the same animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoretic analysis of the lipopolysaccharides ofBacteroides spp.

Lipopolysaccharide (LPS) extracts of reference strains and isolates of Bacteroides spp. prepared by the proteinase K method were resolved by tricine-sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and located by silver staining. A considerable diversity of LPS profiles was evident within the Bacteroides genus although profiles were essentially species-specific, with some minor interstrain variations apparent among isolates of B. uniformis, B. ovatus, B. eggerthii and B. thetaiotaomicron. The LPS of most species consisted of a major rough LPS component of 2-5 kDa and a series of higher molecular weight bands which varied with species. B. vulgatus LPS was distinctive in showing an extensive ladder of multiple repeating oligosaccharide units with molecular weights ranging from 4 to > 17 kDa B. stercoris LPS included a high molecular weight (> 17 kDa) ladder of repeating oligosaccharide units. B. fragilis and B. thetaiotaomicron differed from most other species in producing a short ladder of repeating oligosaccharide units interspersing the rough LPS and a 5.6 kDa (B. fragilis) or 9 kDa (B. thetaiotaomicron) yellow-staining component. The heterogeneity of LPS profiles within the Bacteroides genus may reflect the differences in pathogenicity among the species and prove useful for typing.

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3 % N, N′‐methylenebisacrylamide

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.

Anomalously slow electrophoretic mobilities of DNA restriction fragments in polyacrylamide gels are not eliminated by increasing the gel pore size

The effect of gel pore size on the anomalous mobility of certain curved DNA restriction fragments in polyacrylamide gels was studied by comparing the electrophoretic mobilities of normal and anomalous fragments in gels of varying composition but constant acrylamide concentration. Molecular weight ladders were prepared from two 147 base pair G-C rich restriction fragments, called 12A (anomalous) and 12B (normal), obtained from the MspI digestion of plasmid pBR322. The electrophoretic mobilities of multimers of the two fragments increasingly diverged with increasing molecular weight. The anomalous mobility of fragment 12A was essentially independent of gel pore size, regardless of whether the pore size was varied by increasing the acrylamide concentration at constant cross-linker concentration or by increasing the cross-linker concentration at constant acrylamide concentration. The anomalous mobility of higher multimers of fragment 12A decreased with increasing gel pore size when the pore size was varied by changing the gel concentration. However, when the gel pore size was changed by varying the cross-linker concentration at constant acrylamide concentration, the anomalous mobility of higher multimers of fragment 12A went through a maximum and then decreased as the pore size was increased. Copolymerizing acrylamide with high molecular weight linear polyacrylamides had no effect on the anomalous mobility of the 12A multimer ladder, even though the apparent absolute mobilities of all fragments increased markedly. Only by incorporating charged residues into the gel matrix or by copolymerizing acrylamide with the intercalator ethidium bromide could the difference in mobility between the 12A and 12B multimer ladders be substantially reduced or eliminated. Similar results were observed with a molecular weight ladder containing 78 base pair repeats of the bending locus of kinetoplast DNA. These results suggest that pore size alone is not responsible for the anomalously slow migration of curved DNA molecules in polyacrylamide gels.