Contour-clamped Homogeneous Electric Field Gel Research Articles

Molecular Tools for Leveraging the Potential of the Acid-Tolerant Yeast Zygosaccharomyces bailii as Cell Factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed beenused by humans since the previous centuries for biotransformations. Among them, yeasts combine the advantage of aunicellular state with a eukaryotic organization. Moreover, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycete budding yeast, is widely known for its peculiar tolerance to different stresses, among which are organic acids. Moreover, the recent reclassification of the species, including diverse hybrids, is further expanding both fundamental and applied interests. It is therefore reasonable that despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here we describe in detail the methods for determining chromosome number, size, and aneuploidy, transformation, classical target gene disruption or gene integration, and designing of episomal expression plasmids helpful for engineering the yeast Z. bailii .

Characterisation of Non-Saccharomyces Yeasts Using Different Methodologies and Evaluation of their Compatibility with Malolactic Fermentation

Although Saccharomyces cerevisiae is the yeast species predominantly used for alcoholic fermentation, non-Saccharomyces yeast species are also important because they produce secondary metabolites that can contribute to the final flavour and taste of wines. In this study, 37 strains representing seven non-Saccharomyces species were characterised and evaluated for potential use in wine production, as well as for their effects on malolactic fermentation (MLF). Contour-clamped homogeneous electric field (CHEF) gel electrophoresis and matrix-assisted laser desorption ionisation using a time-of-flight mass spectrometer (MALDI-TOF MS) were used to verify species identity and to determine intra-species variation. Extracellular enzyme production, malic acid degradation and the fermentation kinetics of the yeasts were also investigated. CHEF karyotyping and MALDI-TOF MS were useful for identifying and typing Hanseniaspora uvarum, Lachancea thermotolerans, Candida zemplinina (synonym: Starmerella bacillaris) and Torulaspora delbrueckii strains. Only H. uvarum and Metschnikowia pulcherrima strains were found to have β-glucosidase activity. M. pulcherrima strains also had protease activity. Most of the strains showed limited malic acid degradation, and only Schizosaccharomyces pombe and the C. zemplinina strains showed mentionable degradation. In synthetic wine fermentations, C. stellata, C. zemplinina, H. uvarum, M. pulcherrima and Sc. pombe strains were shown to be slow to medium fermenters, whereas L. thermotolerans and T. delbrueckii strains were found to be medium to strong fermenters. The effect of the yeasts on MLF varied, but inhibition was strain dependent.

Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis.

Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb-9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes, and sub-chromosomal DNA fragments, etc. Here, we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes.

Electrophoretic karyotyping of Hypsizygus marmoreus and evaluation of variation among its basidiospores.

The molecular karyotype of Hypsizygus marmoreus was explored by contour-clamped homogeneous electric field gel electrophoresis. Eleven chromosomal bands were separated from the dikaryotic mycelia of H.marmoreus (strain Hm 3-10), and the chromosomes ranged in size from 1.9 to 5.8Mb. The total genome size of the strain was estimated to be 36.3Mb. The chromosome numbers were also confirmed by telomere fingerprinting, and 22 telomeric bands were identified. This result suggests that 11 chromosomes exist in Hm 3-10. The marker sequences for each chromosome were determined and were applied to identify each chromosome. Karyotyping and Southern blot analysis revealed that the size of chromosomes in the basidiospores were greatly different from those of parental dikaryon Hm 3-10 cells.

The Dynamics of Diverse Segmental Amplifications in Populations ofSaccharomyces cerevisiaeAdapting to Strong Selection

Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.

Assignment of RAPD marker probes designed from 12 linkage groups of Flammulina velutipes to CHEF-separated chromosomal DNAs

Electrophoretic karyotype analyses of Flammulina velutipes FSB and its monokaryotic progeny, omFSB1 and omFSB2, obtained from oidia were performed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. At least 11 chromosome-sized DNA bands (CB 1 through CB 11) for FSB, 6 bands for omFSB1, and 7 bands for omFSB2, respectively, were resolved on a CHEF gel. Southern hybridization analysis on CHEF-separated chromosomal DNA of FSB was carried out using RAPD marker probes prepared from each of the 12 linkage groups. The bands CB 1, 2, and 4 each hybridized to two or three probes for different linkage groups. The bands CB 5 and 6 both hybridized to a common probe. The bands CB 3, 7, 8, and 9 each hybridized to a single specific probe for different linkage groups. The two smallest bands (CB 10 and 11) did not hybridize with any probes.

Assignment of RAPD marker probes designed from 12 linkage groups of <i>Flammulina velutipes</i> to CHEF-separated chromosomal DNAs

Electrophoretic karyotype analyses of Flammulina velutipes FSB and its monokaryotic progeny, omFSB1 and omFSB2, obtained from oidia were performed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. At least 11 chromosome-sized DNA bands (CB 1 through CB 11) for FSB, 6 bands for omFSB1, and 7 bands for omFSB2, respectively, were resolved on a CHEF gel. Southern hybridization analysis on CHEF-separated chromosomal DNA of FSB was carried out using RAPD marker probes prepared from each of the 12 linkage groups. The bands CB 1, 2, and 4 each hybridized to two or three probes for different linkage groups. The bands CB 5 and 6 both hybridized to a common probe. The bands CB 3, 7, 8, and 9 each hybridized to a single specific probe for different linkage groups. The two smallest bands (CB 10 and 11) did not hybridize with any probes.

ORIGINAL RESEARCH: Analysis of an Aspergillus niger glucoamylase strain pedigree using comparative genome hybridization & real-time quantitative polymerase chain reaction

Abstract Microarray-based comparative genome hybridization (CGH) analysis was used to obtain information regarding chromosomal changes that occurred during successive rounds of mutagenesis in a pedigree of Aspergillus niger glucoamylase production strains. This analysis revealed both deletions and amplifications of discrete DNA segments ranging in size from less than one kb to as much as 400 kb. Most importantly, we observed repeated amplification of a 216 kb region that included the glaA (glucoamylase) gene. CHEF (Contour-clamped Homogeneous Electric Field) gel analyses indicated that the amplified DNA segment was translocated nonreciprocally and several chromosomes were rearranged in successive strains of the lineage. The number of glaA gene copies increased from 2 in the original parent strain to approximately 10 in the final strain of the pedigree. Quantitative real-time PCR analyses confirmed the CGH results, leading us to hypothesize that at least some of the incremental improvements in glucoamylase...

Electrophoretic karyotyping and construction of a bacterial artificial chromosome library of the winter mushroom Flammulina velutipes

An electrophoretic karyotype of Korean Flammulina velutipes was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The monokaryotic progenies (4019-20 and 4019-18) and a dikaryotic strain (4019-2018) had 6-8 chromosomes, with sizes ranging from 1.6 to 5.8 megabase (Mb) pairs. Among the 3 strains that were examined, strain 4019-20 resolved into at least 7 chromosomes, with a total genome size of approximately 26.7Mb. We selected several chromosome-specific genes from the cDNA library of F. velutipes using Southern hybridization analysis. In order to determine the whole genome sequence, we constructed a bacterial artificial chromosome (BAC) library of the 4019-20 strain. The BAC library comprised 3840 clones, and the insert size ranged from 60 to 228kb, with an average size of 136kb.

CHEF PROCEDURES: A RAPID HIGH‐TEMPERATURE METHOD FOR SAMPLE PREPARATION, A HIGH VOLTAGE HEPES BUFFER SYSTEM AND THE USE OF NUSIEVE® AGAROSE

ABSTRACT A rapid, high‐temperature method of sample preparation from gram‐negative bacteria for contour‐clamped homogenous electric field (CHEF) electrophoresis is described, which utilizes a diethylpyrocarbonate nuclease inactivation step for preventing latent degradation of DNA. Also described is a simple 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid buffer system that permits high‐voltage CHEF electrophoresis in standard apparatus while preventing the strand scission events associated with tris(hydroxymethyl)aminomethane buffers. Finally, the use of NuSieve agarose in combination with standard analytical agarose was compared to specialty CHEF agarose, demonstrating the potential use of this reagent in CHEF gel separations. PRACTICAL APPLICATIONSContour‐clamped homogenous electric field (CHEF) gel electrophoresis is a widely used technique in epidemiological and comparative genetic studies of microorganisms. Standard techniques work variably well, making options that can be used to “tweak” methodology useful. In this article, we describe a rapid, higher‐temperature variation of sample preparation that yields inherently stable DNA with a more efficient usage of expensive reagents. Also detailed is a simple electrophoresis buffer that eliminates the tris(hydroxymethyl)aminomethane‐mediated strand scission events that have complicated the comparison of bacterial strains in some studies. This buffer permits the use of the higher‐voltage electrophoresis conditions used in many studies without the addition of potentially harmful chemicals. Finally, we demonstrate that the combination of NuSieve (Lonza) branded agarose with standard agarose can be used effectively in CHEF gels, thus expanding analytical options while also providing an alternative to maintaining other expensive specialty agaroses. The methods herein may be applied in whole or in part, allowing their possible inclusion in existing or future protocols as desired.

Sgs1 RecQ Helicase Inhibits Survival of Saccharomyces cerevisiae Cells Lacking Telomerase and Homologous Recombination

In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.

Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels

Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels

Variation in electrophoretic karyotype among Mexican isolates of Mycosphaerella fijiensis

The electrophoretic karyotype of the filamentous fungus Mycosphaerella fijiensis was determined using contour-clamped homogenous electric field (CHEF) gel electrophoresis. Among 10 isolates from different geographical areas, the highest variation in the banding profiles was found in the chromosomes with sizes below 1.5 megabases pairs (Mbp). The minimal genome size was estimated to range from 27 to 35 Mbp. Eight to 13 chromosomal bands, ranging approximately from 0.5 to 8.6 Mbp in size, were separated. These results open perspectives for the physical mapping of specific genes, such as those associated with avirulence, pathogenicity, and fungicide resistance, and for the construction of chromosome-specific libraries.

Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

Electrophoretic molecular karyotype of the dermatophyte Trichophyton rubrum

The electrophoretic karyotype of the dermatophyte Trichophyton rubrum was established using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Five chromosomal bands of approximately 3.0 to 5.8 megabase pairs (Mbp) each were observed and together indicated that 22.05 Mbp of the total genome are organized as chromosomal macromolecules. In addition to establishing the number and size of T. rubrum chromosomes, these results open perspectives for the construction of chromosome-specific libraries and for the physical mapping of genes of interest, thus permitting future gene linkage studies in this pathogen. A detailed understanding of the karyotype and genomic organization of T. rubrum should contribute to further genetic, taxonomic and epidemiological studies of this dermatophyte.

Pisatin demethylase genes are on dispensable chromosomes while genes for pathogenicity on carrot and ripe tomato are on other chromosomes in Nectria haematococca.

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda- isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda-progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda- isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.

A molecular genetic map and electrophoretic karyotype of the plant pathogenic fungus Cochliobolus sativus.

A molecular genetic map was constructed and an electrophoretic karyotype was resolved for Cochliobolus sativus, the causal agent of spot blotch of barley and wheat. The genetic map consists of 27 linkage groups with 97 amplified fragment length polymorphism (AFLP) markers, 31 restriction fragment length polymorphism (RFLP) markers, two polymerase chain reaction amplified markers, the mating type locus (CsMAT), and a gene (VHv1) conditioning high virulence on barley cv. Bowman. These linkage groups covered a map distance of 849 cM. The virulence gene VHv1 cosegregated with six AFLP markers and was mapped on one of the major linkage groups. Fifteen chromosome-sized DNAs were resolved in C. sativus isolates ND93-1 and ND9OPr with contour-clamped homogeneous electric field (CHEF) electrophoresis combined with telomere probe analysis of comigrating chromosome-sized DNAs. The chromosome sizes ranged from 1.25 to 3.80 Mbp, and the genome size of the fungus was estimated to be approximately 33 Mbp. By hybridizing genetically mapped RFLP and AFLP markers to CHEF blots, 25 of the 27 linkage groups were assigned to specific chromosomes. The barley-specific virulence locus VHv1 was localized on a chromosome of 2.80 Mbp from isolate ND9OPr in the CHEF gel. The total map length of the fungus was estimated to be at least 1,329 cM based on the map distance covered by the linked markers and the estimated gaps. Therefore, the physical to genetic distance ratio is approximately 25 kb/cM. Construction of a high-resolution map around target loci will facilitate the cloning of the genes conferring virulence and other characters in C. sativus by a map-based cloning strategy.

Mexican isolates of Leptosphaeria maculans belong to the aggressive strain of the fungus1

Black leg of crucifers, caused by Leptosphaeria maculans (Desm.) Ces. and de Not., is the most important disease of broccoli and cauliflower in central Mexico, causing up to 70% yield loss in cauliflower crops. Isolates recovered from broccoli crops at Aguascalientes and Zacatecas, central Mexico, had the morphological characteristics of the aggressive strain of the fungus. Most of the isolates were pathogenic to cauliflower and canola, but two isolates had a low aggressivity on these hosts. Electrophoretic karyotypes of the Mexican isolates were generated by contour-clamped homogeneous electric field gel electrophoresis. The karyotypes were characteristic of isolates belonging to the aggressive strain ofL. maculans, with 9–12 chromosomes distributed in three size ranges, namely 2.3–1.9, 1.6–1.2, and 0.9–0.8 (minichromosome) megabases. Overall, minimum genome size ranged from 16.1 to 23.0 megabases. We have shown that the LMR1 repetitive element, which is specific to aggressive isolates ofL. maculans, was present in all the Mexican isolates, including the two isolates with low aggressivity on cauliflower and canola, confirming that these belong to the aggressive strain of the fungus.

Electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum and chromosomal location of several xylanolytic genes.

The electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum has been resolved. Using contour-clamped homogeneous electric field gel electrophoresis, five chromosomal bands were separated, with estimated sizes of 7.1, 5.2, 3.7, 2.9 and 2.3 Mbp, giving a total genome size of 21.2 Mbp. To our knowledge, this is the smallest Penicillium genome determined so far. By Southern blots and using homologous probes, the chromosomal location of five xylanolytic genes from P. purpurogenum was determined: axeI (acetyl xylan esterase I), xynB (endoxylanase B) and abf1 (arabinofuranosidase 1) in chromosome I, xynA (endoxylanase A) in chromosome II, and axeII (acetyl xylan esterase II) in chromosome III. This is the first study where the location of xylanase genes in a Penicillium genome has been established.

Electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum and chromosomal location of several xylanolytic genes

The electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum has been resolved. Using contour-clamped homogeneous electric field gel electrophoresis, five chromosomal bands were separated, with estimated sizes of 7.1, 5.2, 3.7, 2.9 and 2.3 Mbp, giving a total genome size of 21.2 Mbp. To our knowledge, this is the smallest Penicillium genome determined so far. By Southern blots and using homologous probes, the chromosomal location of five xylanolytic genes from P. purpurogenum was determined: axeI (acetyl xylan esterase I), xynB (endoxylanase B) and abf1 (arabinofuranosidase 1) in chromosome I, xynA (endoxylanase A) in chromosome II, and axeII (acetyl xylan esterase II) in chromosome III. This is the first study where the location of xylanase genes in a Penicillium genome has been established.