Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

一种新的脉冲电泳分子量标记*

在脉冲电泳（ｐｕｌｓｅｄ－ｆｉｅｌｄ　ｇｅｌ　ｅｌｃｔｒｏｐｈｏｒｅｓｉｓ,ＰＦＧＥ）研究中,经常使用的分子量标记有啤酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｅｓｌａｅ）和粟酒裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）的染色体完整ＤＮＡ。其中,啤酒酵母（如菌株ＹＮＮ２９５）有１６条染色体,分子量变化范围为０．２５Ｍｂ～２．２Ｍｂ（ＭｃＣｌｕｓｋｅｙｅｌａｌ,１９９０）,适于作为小于２．２Ｍｂ的染色体ＤＮＡ的分子量标记;粟酒裂殖酵母（如菌株９７２ｈ－）有３条染色体,分子量…     

Electrophoretic karyotype of Saprolegnia monoica

Abstract The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae , Schizosaccharomyces pombe and Hansenula wingei , is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.     

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.     

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.     

The hydrodynamics of DNA electrophoretic stretch and relaxation in a polymer solution

Theories of DNA electrophoretic separations generally treat the DNA as a free draining polymer moving in an electric field at a rate that depends on the effective charge density of the molecule. Separations can occur in sieving media ranging from ultradilute polymer solutions to tightly cross-linked gels. It has recently been shown that DNA is not free-draining when both electric and nonelectric forces simultaneously act on the molecule, as occurs when DNA collides with a polymer during electrophoretic separations. Here we show that a semidilute polymer solution screens the hydrodynamic interaction that results from the application of these forces. Fluorescently labeled DNA tethered at one end in a semidilute solution of hydroxyl-ethyl cellulose stretch more in an electric field than they stretch in free solution, and approach free-draining behavior. The steady stretching behavior is predicted without adjustable parameters by a theory developed by Stigter using a hydrodynamic screening length found from effective medium theory. Data on the relaxation of stretched molecules after the electric field is removed agree with the Rouse model prediction, which neglects hydrodynamic interactions. The slowest relaxation time constant, tau(R), scales with chain length as tau(R) approximately L(1.9+/-0.17) when analyzed by the data collapse method, and as tau(R) approximately L(2.17+/-0.17) when analyzed by multiexponential fit.     

DNA content and structure of (double) minutes of a methotrexate-resistant cell line

We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.     

Adapting to contour-clamped homogeneous electric field minichamber technology the PulseNet protocols to resolve XbaI-DNA fragments of Salmonella serotype Braenderup

We propose cost-effective protocols for preparing and resolving the PulseNet universal DNA size standard in contour-clamped homogeneous electric field (CHEF) minichambers. Intact DNA molecules were prepared with protease-free solutions, and electrophoresis separations of the DNA standards needed 5.5 h, giving band pattern resolutions similar to those attained with the PulseNet protocols standardized in CHEF chambers.     

DNA content and structure of (double) minutes of a methotrexate-resistant cell line

Summary We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.This study was supported in part by grant NKI 84-20 of the Queen Wilhelmina Fund to P.B.     

Restriction analysis of Thiobacillus ferrooxidans DNA using pulsed field electrophoresis]

The DNA of Thiobacillus ferrooxidans digested by any of the five restriction endonucleases (DraI, EcoRI, Eco321, HindIII, XbaI) was studied by electrophoresis in the pulsating differently directed electric fields (PF). The influence of PF conditions on the sized row of the divided DNA fragments was studied. Only the XbaI restriction endonuclease was shown to cleave the Thiobacillus ferrooxidans DNA into a number of fragments permitting one to define the sizes of fragments and genome (no more than 2300 bp). The prospects of using the restriction analysis of Thiobacillus ferrooxidans wild type culture for improving its properties in obtaining heavy metals are discussed.     

Determination of the minimum size of Gallus gallus domesticus chicken microchromosome by a pulse electrophoresis method]

The karyotype of the chicken Gallus gallus domesticus was studied by means of pulsed-field electrophoresis. An electrokaryogram was obtained for the microchromosomal (MI) portion of the chicken genome. Chicken MIs were separated into two fractions. A fraction with a higher mobility included MIs sized 3.4-4.8 Mb; the lower size limit of a less mobile fraction corresponded to MIs of approximately 5 Mb. The smallest MI in the chicken karyotype was estimated at 3.4 +/- 0.25 Mb.     

Electrophoretic karyotype of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> and its variation among monokaryotic progenies

 The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0 Mb or more. Received: October 11, 2001 / Accepted: November 11, 2002 Acknowledgments We thank Professor T. Morinaga, Hiroshima Prefectural University, and Dr. T. Arima for their technical advice regarding CHEF gel electrophoresis. Correspondence to:E. Tanesaka     

A 12 megabase restriction map at the cystic fibrosis locus.

We have constructed a physical map of the chromosomal region containing the cystic fibrosis locus using seven DNA markers and pulsed-field gel electrophoresis methods. The map includes cleavage sites for 8 rare-cutting restriction enzymes and spans over 12 megabases (Mb) of DNA, with one unlinked probe covering an additional 5 Mb. To our knowledge, this is the largest segment of human DNA which has been restriction-mapped to date. We can identify thirteen putative HTF islands spaced at intervals of 0.3-3.2 Mb. The region between loci D7S8 and MET, where the CF gene lies, includes 1.4-1.9 Mb of DNA.     

Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.     

Isolation of giant DNA fragments from flow-sorted human chromosomes

We have established a method using a conventional cell sorter equipped with a single argon laser to sort intact human chromosomes that can be used as a source for the production of giant DNA fragments. Various improvements were made to both the equipment and sorting method to enhance the sorting resolution and avoid destruction of chromosomal DNA. Using this improved method chromosomes 21 and 22 were sorted from the B-lymphoblastoid line GM00130B, digested with the rare cutting restriction endonuclease NotI, and analyzed by pulsed field gel electrophoresis followed by Southern hybridization using the Alu repetitive sequence as a probe. More than 25 discrete NotI giant DNA fragments ranging from 50 kb to longer than 2.5 Mb were separated and the size distribution pattern was unique for each chromosome, indicating successful sorting of intact chromosomes. The cumulative size of these Alu-positive NotI DNA fragments were 22.7 Mb and 25.5 Mb for chromosomes 21 and 22, respectively. These values are 47% and 49% of the estimated size of chromosomes 21 (48 Mb) and 22 (52 Mb).     

Pulsed field gel electrophoresis: Theory,instruments and application

Pulsed field gel electrophoresis (PFGE) is a technique for the fractionation of high-molecular-weight DNA ranging from 10 kb to 10 Mb by electrophoresis in agarose gel with an electric field that alternates (pulsates) in two directions. This technology plays a key role in modern genomics, as it allows manipulations with DNA of whole chromosomes or their large fragments. In this review, we discuss (1) the theory behind PFGE; (2) different instruments based on the principle of pulsed field, as well as their advantages and limitations; (3) factors affecting the DNA mobility in PFGE gel; and (4) practical applications of the technique.     

Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.

Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given.     

Estimation of the Minimal Size of Chicken Gallus gallus domesticusMicrochromosomes via Pulsed-Field Electrophoresis

The karyotype of the chicken Gallus gallus domesticuswas studied by means of pulsed-field electrophoresis. An electrokaryogram was obtained for the microchromosomal (MI) portion of the chicken genome. Chicken MIs were separated into two fractions. A fraction with a higher mobility included MIs sized 3.4–4.8 Mb; the lower size limit of a less mobile fraction corresponded to MIs of approximately 5 Mb. The smallest MI in the chicken karyotype was estimated at 3.4 ±0.25 Mb.     

Transposon-mediated restriction mapping of the Bacillus subtilis chromosome

Analysis of chromosomal DNA depends upon a knowledge of the locations of restriction sites over several thousand kilobases (kb). However determination of even a subset of these sites can be time-consuming, and it can be difficult to link genetic and physical maps. We describe here a significant improvement which can be used in concert with genetically mapped chromosomal insertions. The circular chromosome of Bacillus subtilis 168 was physically examined on contour-clamped homogeneous electric field (CHEF) gels using the restriction enzyme NotI. Restriction mapping of the 4.7-megabase (Mb) DNA was accomplished using a novel technique involving the transposon Tn917, which linked the genetic and physical maps and also significantly increased the rate at which this was performed. The DNA of 54 strains which contained Tn917 at genetically determined locations was cleaved with NotI and used to determine the approximate positions of 31 restriction fragments with sizes between 45 kb and 290 kb, totalling 3589 kb. This information should greatly assist in the construction of a more detailed map using standard methodology.