What the papers say: Molecular karyotypes: Separating chromosomes on gels

For many years, there has been a gap in our capacity to study the structure and organization of chromosomal DNA molecules. The very small genomes of some viruses and bacteriophages (≤ 50,000 bp or 50 kb) are amenable to analysis by conventional gel electrophoresis, while the extremely large DNA molecules (> 100,000 kb) comprising the chromosomes of higher eukaryotes have been analysed under the light microscope, using a range of banding and in situ hybridization techniques. However, intact DNA molecules with sizes between these two extremes have been largely inaccessible experimentally. This gap has recently been bridged with the development of two-dimensional electrophoretic procedures that allow the separation and purification of chromosome-sized DNA molecules ranging from ～ 50 kb to several thousand kb.^1–3 There are currently two variations of the technique in use: pulsed field gradient (PFG) gel electrophoresis^1,2 and orthogonal-field-alteration gel electrophoresis (OFAGE).³ Both are based on a common principle, differing primarily in the geometry of the electrodes. Already, they have been employed to determine the approximate chromosome sizes and numbers for a variety of lower eukaryotes, including yeast and several protozoa.^2–10 These ‘molecular karyotypes’ provide fundamental information about the genomic organization of each organism, and allow very rapid construction of linkage maps. Surprisingly, they have also revealed a remarkable plasticity in the genomes of several lower eukaryotes.     

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.     

Plasmodium falciparum: mapping genes to nine parasite chromosomes

Using pulsed field gel electrophoresis with pulse time of 120 sec, eight chromosomal DNA molecules from clone 7G8 of the Plasmodium falciparum Brazilian isolate IMTM22 were resolved. A ninth chromosomal molecule which did not enter the gel was identified at the slot by hybridization to two DNA probes and by restriction enzyme analysis. Thirteen parasite DNA sequences were mapped to the nine chromosomes, with at least one sequence mapped to each chromosome. The restriction enzyme NotI appeared to produce only one cut in the entire IMTM22 genome.     

Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae

The genome size of Actinobacillus pleuropneumoniae was determined by pulsed field gel electrophoresis of AscI and ApaI digested chromosomal DNA. The genome size of the type strain 4074^T (serotype 1) was determined to be 2404±40 kb. The chromosome sizes for the reference strains of the other serotypes range between 2.3 and 2.4 Mb. The restriction pattern profiles of AscI, ApaI and NheI digested chromosomes showed a high degree of polymorphism among the different serotype reference strains and allowed their discrimination. The analysis of the macrorestriction pattern polymorphism revealed phylogenetic relationships between the different serotype reference strains which reflect to some extent groups of serotypes known to cross-react serologically. In addition, different pulsed fields gel electrophoresis patterns also revealed heterogeneity in the chromosomal structure among different field strains of serotypes 1, 5a, and 5b, while strains of serotype 9 originating from most distant geographical places showed homogeneous ApaI patterns in pulsed field gel electrophoresis.     

Chromosomes of kinetoplastida

We have compared chromosome-sized DNA molecules (molecular karyotypes) of five genera (nine species) of kinetoplastida after cell lysis and deproteinization of DNA in agarose blocks and size fractionation of the intact DNA molecules by pulsed field gradient (PFG) gel electrophoresis. With the possible exception of Trypanosoma vivax and Crithidia fasciculata, all species have at least 20 chromosomes. There are large differences between species in molecular karyotype and in the chromosomal distribution of the genes for alpha- and beta-tubulin, rRNA and the common mini-exon sequence of kinetoplastid mRNAs. In all cases, the rRNA genes are in DNA that is larger than 500 kb. Whereas T. brucei has approximately 100 mini-chromosomes of 50-150 kb, only few are found in T. equiperdum; T. vivax has no DNA smaller than 2000 kb. As all three species exhibit antigenic variation, small chromosomes with telomeric variant surface glycoprotein genes cannot be vital to the mechanism of antigenic variation. The apparent plasticity of kinetoplastid genome composition makes PFG gel electrophoresis a potentially useful tool for taxonomic studies.     

Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes.

The sizes of large DNA fragments produced from genomes of members of the Mycoplasmataceae by digestion with restriction endonucleases having infrequent (1 to 3) cleavage sites within the genome were estimated from their mobility in contour-clamped homogeneous electric field (CHEF) agarose gel electrophoresis by comparison with yeast chromosomal DNA markers. The estimates of total genome size for 7 strains of 6 species ranged from approximately 900 kilo base pairs (kb) for Ureaplasma urealyticum 960T to 1330 kb for M. mycoides subsp. mycoides, GC-1176. The values derived from this new method are considerably higher than those of approximately 500 Mdaltons or 750 kb previously reported for genome sizes in members of the Mycoplasmataceae.     

The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci.

The arrangement of tubulin genes in the genome of the protozoan parasite Leishmania major was studied by genomic Southern blot analysis and mapping of genes to chromosomes fractionated by pulsed field gradient gel (PFG) electrophoresis. alpha-tubulin genes exist as a tandem array of 2.4 kb PstI fragments. beta-tubulin genes are found as a tandem array of 3.9 kb AvaI or PvuI fragments, but additional genes are also found on other genomic DNA fragments. Chromosome-sized DNA molecules released from promastigotes of L. major were fractionated into at least 17 chromosome bands of approximate size 400-4000 kb by PFG gel electrophoresis. Some bands may be present in non-equimolar amounts suggesting that there may be more than 17 chromosomes. All alpha-tubulin genes were localized to a single band (chromosome 7). beta-tubulin genes were localized to four bands (chromosomes 6, 10, 16 and 17). This shows that the alpha- and beta- tubulin gene families are unlinked in L. major. There is a single chromosomal locus for the alpha-tubulin tandem array whereas beta-tubulin genes exist both as a tandem array and as dispersed genes at four chromosomal loci.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.     

Preliminary genomic characterization of microsporidian Nosema bombycis

In order to characterize the genome of Nosema bombycis, the techniques of karyotyping, pulsed field gel electrophoresis, and polymerase chain reaction were applied. Nosema genomic DNA moved as 23 kb fragment on a standard agarose gel. The karyotype showed four chromosomes, the molecular karyotyping by pulsed field gel electrophoresis also showed four chromosomes. Arbitrarily primed polymerase chain reaction (PCR) with various primers showed amplification products of sizes ranging from 1.6 to 0.15 kb. Polymerase chain reaction with specific primer showed an amplification product of approximately 315 nucleotides. The DNA hybridizations are discussed. This is the first report of its kind on microsporidian Nosema bombycis. The current data can play a major role in elucidating the molecular biology of this parasite. This revised version was published online in July 2006 with corrections to the Cover Date.     

Macrorestriction mapping of COL4A1 and COL4A2 collagen genes on human chromosome 13q34

The genes for the alpha-1 and alpha-2 chains of type IV collagen (COL4A1 and COL4A2) map to the same chromosomal band (13q34) and have a high degree of nucleotide homology. We have used pulsed field gel electrophoresis and cloned COL4A1 and COL4A2 DNA fragments as molecular probes to construct a 1200-kb macrorestriction map which encompasses both genes. The two genes are located within a 340-kb region with the 3' end of COL4A2 and the 5' region of COL4A1 separated by at least 100 kb but not more than 160 kb. These genes, therefore, are two members of a gene cluster on chromosome 13q34.     

粟酒裂殖酵母染色体DNA的脉冲电泳分析

采用等高锁状均质电场（CHEF）凝胶电泳技术,对来自中国微生物菌种保藏委员会普通微生物中心的粟酒裂殖酵母（Schizosaccharomycespombe）菌株AS2.214进行染色体DNA分析。CHEF电泳结果显示菌株AS2.214的4条染色体DNA的分子大小分别约为5900kb、3200kb、2500kb和550kb,基因组大小约为12000kb。供试菌株AS2.214第Ⅰ条染色体DNA为5900kb与已研究报道的菌株972h和HM248（5700kb）的基本一致,第Ⅱ条染色体DNA（3200kb）和第皿条染色体DNA（2500kb）,分别较上述2个菌株约小1400kb和1000kb,第Ⅳ条染色体DNA的分子大小与部分非整倍体菌株HM248的极微染色体Ch16DNA相近（500kb）。研究结果表明粟酒裂殖酵母菌株间染色体DNA长度存在显著差异,菌株AS2.214可能是三倍体减数分裂所产生的稳定的部分非整倍体。     

Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae.

To elucidate yeast chromosome structure and behavior, we examined the breakage of entangled chromosomes in DNA topoisomerase II mutants by hybridization to chromosomal DNA resolved by pulsed-field gel electrophoresis. Our study reveals that large and small chromosomes differ in the nature and distribution of their intertwinings. Probes to large chromosomes (450 kb or larger) detect chromosome breakage, but probes to small chromosomes (380 kb or smaller) reveal no breakage products. Examination of chromosomes with one small arm and one large arm suggests that the two arms behave independently. The acrocentric chromosome XIV breaks only on the long arm, and its preferred region of breakage is approximately 200 kb from the centromere. When the centromere of chromosome XIV is relocated, the preferred region of breakage shifts accordingly. These results suggest that large chromosomes break because they have long arms and small chromosomes do not break because they have small arms. Indeed, a small metacentric chromosome can be made to break if it is rearranged to form a telocentric chromosome with one long arm or a ring with an "infinitely" long arm. These results suggest a model of chromosomal intertwining in which the length of the chromosome arm prevents intertwinings from passively resolving off the end of the arm during chromosome segregation.     

Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.     

Resistance to antimycin A in yeast by amplification of ADH4 on a linear, 42 kb palindromic plasmid

A yeast strain lacking a functional copy of ADH1 has been isolated that is resistant to antimycin A because of the presence of multiple copies of a nuclear gene, ADH4. The amplified copies of ADH4 exist on linear molecules 42 kb in length, which can be separated from chromosomal DNA by orthogonal-field-alternation gel electrophoresis. These amplified molecules are palindromes that reanneal rapidly after denaturation to form linear, snap-back molecules 21 kb in length. The amplified ADH4 sequences are bounded by telomere-homologous sequences. The chromosomal copy of ADH4 is the most distal marker on the left arm of chromosome VII, and the amplified ADH4-containing molecules appear to contain two copies of the region extending from ADH4 to the telomere.     

Dynamics of DNA molecules in gel studied by fluorescence microscopy

The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy. Driven by an electric field, molecules hooked around isolated obstacles and became extended. By analyzing molecular images, we identified the reptation tube and primitive chain. When the field was turned off, the molecules relaxed. The relaxation time tau1 and primitive chain length at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory. Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis.     

Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes

Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed.     

Rapid pulsed field separation of DNA molecules up to 250 kb.

Pulsed field gel electrophoresis (PFGE) is capable of resolving a wide size range of DNA molecules which would all co-migrate in conventional agarose gels. We describe pulsed field gel conditions which permit DNA fragments of up to 250 kilobases (kb) to be separated in only 3.5 h. The separations, which employ commercially available gel boxes, are achieved using conditions which deviate significantly from traditional pulsed field conditions. PFGE separations have been thought to require reorientation angles greater than 90 degrees to be effective. However, reorientation angles of 90 degrees and even less will resolve DNA fragments a few hundred kb and smaller approximately 5 x faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90 degrees reorientation angles is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120 degrees. With DNA fragments of several hundred kb and smaller, higher field strengths may be used, resulting in still greater increases in separation speed. The conditions described allow DNA from large insert bacterial clones, such as those using cosmid, Fosmid, P1, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) vectors, to be prepared, digested and analyzed on gels within a single working day.     

Rearrangements of yeast chromosomes revealed by pulsed field gel electrophoresis

Electrophoretic karyotypes of yeast Saccharomyces cerevisiae integrant strains containing the pYF91 plasmid integrated into the chromosomes I, III, VI, IX, XI were studied. A possibility was demonstrated of visual identification of the chimaeric chromosomes via the molecular weight increase by 13200 bp (the plasmid size) determined by pulsed field gel electrophoresis. Several gamma-rays induced rearrangements of the yeast chimaeric chromosome I causing instability in hybrids were also studied. The deletions induced in the I chromosome were analysed and their size estimated. The technique of pulsed field gel electrophoresis is recommended for determination of insertions and deletions in the chromosomes of yeast Saccharomyces cerevisiae.