A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Isolation of alpha-amino adipic acid from mature dermal collagen and elastin. Evidence for an oxidative pathway in the maturation of collagen and elastin.

Human serum alpha-2-macroglobulin has been found to be a major cadmium-binding protein $\text{in vitro}$. Serum and alpha-2-macroglobulin equilibrated with cadmium at the 0.20 ppm level were chromatographed over Sephadex and agarose gels to separate and estimate the molecular weights of the proteins. Alpha-2-macroglobulin was found to fragment into reproducible fragments when chromatographed on agarose gels showing different metal-binding fractions for cadmium and endogenous zinc. The distribution of cadmium on serum protein chromatograms was correlated with alpha-2-macroglobulin chromatograms. Cadmium was bound to fractions with molecular weights as high as 800,000 daltons with an affinity greater than that observed for serum albumin.     

Distribution of transcribable DNA sequences in mouse liver hepatoma chromatin

Summary Shearing in either a Virtis small or large volume capacity cup under defined conditions gave chromatin fragment size distribution upon agarose exclusion column chromatography (50 × 10⁶) that varied inversely with shear time. The large- and small-sized chromatin fragments possessed high template capacity while the intermediate fragments (the majority) had relatively low template capacity; the differences in template capacity of these size groups varying inversely with the shear time. The protein/DNA and RNA/DNA ratios remained relatively constant in all size groups.     

Effect of the electric field on the apparent mobility of large DNA fragments in agarose gels

The electrophoresis of a series of DNA fragments ranging in size from 0.5 to 12 kilobase pairs, has been studied as a function of agarose gel concentration and electric field strength. The apparent mobility of all fragments decreased with decreasing electric field strength and with increasing gel concentration. When extrapolated to zero electric field strength and zero agarose concentration, the apparent mobility of all DNA fragments extrapolated to a common value (2.0 ± 0.1) × 10^?4 cm²/V s. The square roots of the retardation coefficients of the various fragments were found to be linearly related to the root-mean-square radii of gyration of the fragments, as predicted by pore-size distribution theory. As predicted by reptation theory, the molecular weights of the various fragments were found to be linearly related to the reciprocal of the apparent mobilities. An equation is given for estimating the apparent pore size of agarose gels between 0.25 and 1.5% in concentration.     

Adenosine deaminase from pig thyroid gland purification and some properties

Adenosine deaminase was isolated from the pig thyroid gland and purified over 900-fold using DEAE Sephadex A-50 column chromatography, Sephadex G-100 gel filtration and DEAE Sephadex A-50 rechromatography. The enzyme was specific towards adenosine. The Michaelis constant based on the Lineweaver-Burk plot was 5 × 10^?5M. The optimum pH was about 7.0, and molecular weight 44 700.     

Purification and Some Properties of Acid-stable α-Amylases from Shochu koji (Aspergillus kawachii)

Aspergillus kawachii α-amylase [EC 3.2.1.1] I and II were purified from shochu koji extract by DEAE Bio-Gel A ion exchange chromatography, Sephacryl S-300 gel chromatography (pH 3.6), coamino dodecyl agarose column chromatography and Sephacryl S-200 gel chromatography. By gel chromatography on a Sephacryl S-300 column, the molecular weights of the purified α-amylase I and II were estimated to be 104,000 and 66,000, respectively. The isoelectric points of α-amylase I and II were 4.25 and 4.20, respectively. The optimal pH range of α-amylase I was 4.0 to 5.0, and the optimum pH of α-amylase II was 5.0. The optimum temperatures of both α-amylases were around 70°C at pH 5.0. Both α-amylases were stable from pH 2.5 to 6.0 and up to 55°C, retaining more than 90% of the original activities. Heavy metal ions such as Hg^{2 +} and Pb^{2 +} were potent inhibitors for both α-amylases.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

The migration behavior of proparathyroid hormone, parathyroid hormone, and their peptide fragments during gel filtration

The migration behavior of bovine proparathyroid and parathyroid hormones, as well as several hormonal peptide fragments, has been analyzed by gel filtration on thin-layer plates and by column chromatography. The two hormones migrated appreciably faster than was expected for their molecular weights. Their migration rates decreased with increasing pH and approached values more characteristic of their molecular weights at pH 11.0. Migration rates were the same over a concentration range of 2 × 10^?6–0.9 × 10^?3m. These results indicate that parathyroid hormone does not exist in solution as an aggregate at the concentrations used in these experiments. These studies suggest that the two hormones are asymmetrical, unfolded monomers in acid solution which become more folded or globular at alkaline pH values.     

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg²⁺-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg²⁺-independent.     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.     

Effects of Calcium on Template Activity and Size of Pea Chromatin Fragments

Pea chromatin was fractionated into template active and inactivefragments by cleavage with DNase II followed by fractionationwith Bio-Gel A 5 m column chromatography. When the chromatin fragments were chromatographed in the presenceof Ca²⁺ or EGTA, the elution patterns suggested that Ca²⁺ assembledmoderately digested chromatin fractions. Moreover, total templateactivity of chromatin fractions was reduced by Ca²⁺ and theactive fraction was eluted at different positions under thechelated and non-chelated conditions. (Received February 12, 1986; Accepted May 28, 1986)     

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×10⁶ to 14×10⁶ dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l0⁶ dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.     

The chromosome of bacteriophage T5 : II. Arrangement of the single-stranded DNA fragments in the T5 and T5st(O) chromosomes

Denatured bacteriophage T5 DNA contains a large number of single-stranded DNA fragments which have been separated by agarose gel electrophoresis and classified as “major” or “minor” species on the basis of their relative abundances (Hayward & Smith, 1972). For further study of these fragments we have centrifuged denatured T5 DNA in CsCl density-gradients in the presence of poly(G). Gel electrophoretic analysis of fractions from these gradients shows that the 37.0 and 13.9 million major fragments of T5⁺ DNA and the 35.3 and 17.2 million of T5st(O) DNA are found in the high buoyant density regions. The other fragments vary in the extent of their interactions with poly(G) and a minor fragment, which has anomalous electrophoretic properties, exhibits the strongest poly(G) interaction.     

Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.

Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage.     

Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

The genome of the cellular slime mold Dictyostelium discoideum has been analyzed by limit digestion with EcoR1 restriction endonuclease. Approximately 15% of the nuclear genome is cleaved into nine discrete fragments as analyzed by agarose gel electrophoresis. These fragments appear to be derived from two nuclear buoyant density satellites, one of which contains sequences coding for ribosomal RNA. The bulk of the nuclear DNA is digested into approximately 7000 fragments with a mean molecular weight of 4 × 10⁶ to 5 × 10⁶. The mitochondrial DNA is digested into four fragments. One of the nuclear bands has been cloned in Escherichia coli using plasmid pSC101 carrying tetracyline resistance. Analysis by renaturation kinetics indicates that it is repeated approximately 200 times per haploid genome and that it is not internally repeated.     

Specific Origin in SV40 DNA Replication

THE oncogenic virus SV40 contains a covalently closed circular DNA molecule of 3 × 10⁶ molecular weight¹. In infected permissive cells, SV40 DNA replicates through a Cairns type intermediate² with the parental strands forming a partially twisted, covalently closed molecule³. We have used a specific bacterial restriction endonuclease⁴ to analyse SV40 DNA replication. The restriction endonuclease of H. influenzae makes double-strand breaks in DNA at specific hexanucleotide sequences^{5, 6} and splits SV40 DNA into eleven fragments, separable by Polyacrylamide gel electrophoresis, ranging in molecular weight from 6.5 × 10⁵ (about 20% of the molecule) to 7.4 × 10⁴ (about 2.5% of the molecule). The largest eight fragments are present in the digest in amounts equimolar with the starting DNA⁴. Therefore, by digesting labelled replicating SV40 DNA and newly completed DNA and measuring the relative yield of each fragment, we could determine whether a particular region of the DNA is synthesized first or last and also estimate the time needed to replicate one molecule completely.     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli.

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.     

Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.