Contour-clamped Homogeneous Electric Field Gel Research Articles

Electrophoretic karyotype of Saprolegnia monoica

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.

Electrophoretic karyotype ofSaprolegnia monoica

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.

Stability of karyotype in serial isolates of Candida albicans from neutropenic patients.

Serial isolates of Candida albicans were obtained from 20 patients with leukemia over periods of up to 8 months. The fingerprinting of these isolates by interrepeat PCR and random amplified polymorphic DNA PCR has been described previously (A. van Belkum, W. Melchers, B. E. de Pauw, S. Scherer, W. Quint, and J. F. Meis, J. Infect. Dis. 169:1062-1070, 1994). Contour-clamped homogeneous electric field gel electrophoresis was used to examine the chromosomes of these isolates. When changes in the karyotype were seen in a series of isolates, additional interrepeat PCR and Southern blotting with a repeat DNA sequence from the 27A family were performed. These two genotyping tools were used to determine if karyotypic changes seen in a series of isolates were due to chromosome rearrangements in a single strain or due to colonization with more than one strain. It was determined that changes in karyotype in a series of strains indicated infection by a new strain.

Electrophoretic karyotype of Cercospora kikuchii.

Classical genetic analyses are not possible with the phytopathogenic fungus Cercospora kikuchii since no sexual stage has been identified. To facilitate gene mapping and to develop an understanding of the genome organization of C. kikuchii, an electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis (CHEF). Eight chromosomes, two of which migrate as a doublet, have been separated into seven bands ranging from 2.0 to 5.5 Mb. Using this determination of chromosome number and size, the total genome size of C. kikuchii is estimated to be 28.4 Mb. In addition, genes encoding tubulin, ribosomal DNA, and four previously isolated light-enhanced cDNAs from C. kikuchii were assigned to chromosomes by Southern-hybridization analysis of CHEF blots.

Analysis of Epidemic and Endemic Isolates of Xanthomonas maltophilia by Contour-Clamped Homogeneous Electric Field Gel Electrophoresis

Analysis of Epidemic and Endemic Isolates of Xanthomonas maltophilia by Contour-Clamped Homogeneous Electric Field Gel Electrophoresis

Complementation of a Capsule-Deficient Mutation of Cryptococcus neoformans Restores its Virulence

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.

Physical map of a 2 Mb chromosome of the intestinal protozoan parasiteGiardia duodenalis

The protozoan parasite, Giardia duodenalis, is regarded as the most primitive eukaryote. The two apparently identical nuclei presumably carry the same chromosomes but the number of different chromosomes in the organism is unknown. A genome map of G. duodenalis is required to resolve this issue and mapping studies were initiated using chromosome 5. This chromosome was estimated to be approximately 2 Mb when Giardia chromosomes were separated by contour-clamped homogeneous electric field gel electrophoresis. A plasmid library of chromosome 5-specific DNA sequences was constructed from gel-extracted chromosome 5 and selected probes were used as markers to identify NotI DNA segments derived from chromosome 5. Fifty-nine unique copy markers were used to identify thirteen NotI segments which ranged in size from 47 kb to 400 kb. The sum of the NotI segments was 1.78 Mb which indicated that most, if not all, of the chromosome was accounted for and that chromosome band 5 of the cloned line WB-1B, used in this study comprised only one chromosome type. The NotI segments were ordered on the map by comparison of hybridization patterns of the markers with partial NotI cleavages of whole chromosomes. Chromosome rearrangements occur readily in Giardia, and in two drug-resistant lines selected for resistance to different drugs, partial conservative duplications of chromosome 5 were observed in addition to the original, full length chromosome 5. Both duplications retained the central region of chromosome 5 but were deleted at different termini resulting in one duplication of 1.5 Mb and the other of 1.3 Mb.(ABSTRACT TRUNCATED AT 250 WORDS)

Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.

Electrophoretic karyotype of Mucor circinelloides

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis was used to separate chromosomal size DNA molecules of two Mucor circinelloides strains. Electrophoretic karyotypes revealed the presence of eight distinct bands for the M. circinelloides f. lusitanicus strain, and four, presumably multiple, bands for the M. circinelloides f. gryseo-cyanus strain. The approximate sizes of the resolved chromosomal DNA bands ranged from 2.3 to 8.1 Mb, giving estimated genome sizes of 38.7 and 32.6 Mb, respectively. Hybridisation techniques were used to assign the leuA gene to a chromosome.

Electrophoretic karyotype of Fusarium solani

The electrophoretic karyotype of Fusarium solani was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Thirteen chromosomal bands were obtained. The size of these bands, based on their migration relative to the chromosomal DNA of Schizosaccharomyces pombe and Saccharomyces cerevisiae, is estimated to be between 0·42 and 6·08 Mb, the total molecular size of the DNA being 39·90 Mb.

Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Electrophoretic Karyotypes of Fusarium spp.

Migheli, Q., Berio, T., and Gullino, M. L. 1993. Electrophoretic karyotypes of Fusarium spp. Experimental Mycology 17, 329-337. The electrophoretic karyotype of 17 antagonistic and pathogenic strains of Fusarium spp. has been established by using contour-clamped homogeneous electric field gel electrophoresis. Intact chromosomal DNA was prepared from fungal protoplasts with standard procedures. Up to 11 distinct chromosomal bands were resolved after 184 h of migration at 50 V. Polymorphic karyotypes were observed in different species of Fusarium, formae speciales of F. oxysporum , and races of F. oxysporum f.sp. dianthi. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, the size of the Fusarium genome was estimated to range from approximately 18.1 to 51.5 Mb. The suitability of electrophoretic karyotyping as a tool for strain characterization, as well as some applications in hybridization analysis of Fusarium spp., is discussed.

Assignment of linkage groups to the electrophoretically-separated chromosomes of the fungus Podospora anserina.

An electrophoretic karyotype of the filamentous fungus Podospora anserina has been obtained using contour-clamped homogeneous electric field gel electrophoresis. Six chromosomal bands were separated with one migrating as a doublet. The size of the chromosomes was estimated to be between 3.8 and 6.0 megabase pairs (mb) using the chromosomes of Schizosaccharomyces pombe as size standards, giving a total genome size of about 34 mb for the P. anserina genome. Homologous probes were used to assign five of the seven linkage groups (LGs) to chromosomal bands on the gel. Analysis of reciprocal translocation strains allowed us to complete the karyotype. In decreasing size order, the P. anserina chromosomes are LG I (6.0 mb); LG II (5.5 mb); LG V (5.1 mb); LG III (4.9 mb); LGs VI and VII (4.3 mb) and LG IV (3.8 mb).

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556.

The URA3 gene, coding for orotidine-5'-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29.3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identities of 81% and 88%, respectively. Using contour-clamped homogenous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.

Use of electrophoretic karyotyping and DNA-DNA hybridization in yeast identification

Contour-clamped homogenous electric field (CHEF) gel electrophoresis was used to separate intact, chromosome-size DNA of different species of Saccharomyces and Zygosaccharomyces. Strains of the same Saccharomyces species had similar electrophoretic karyotypes. However, reproducible differences between individual bands indicated that strain-specific chromosome length polymorphism (CLP) is widely spread in these organisms. Strains of Zygosaccharomyces spp. showed karyotype differences beyond chromosome length polymorphism. A new, DNA-DNA hybridization technique was developed to test conspecificity, using individual chromosomes as templates to prepare randomly primed, radioactive probes. Under our conditions of stringency, species and chromosome-specific hybridization reactions were achieved with these probes. Using isolated chromosomes of Sacch. cerevisiae, Sacch. bayanus, and Zygosacch. rouxii as templates for probe preparation, conspecificity of strains was established, and closely related yeast species were distinguished with high reproducibility. This method is an efficient tool for studying genetic diversity of a given yeast species. Also, it can assist in yeast species identification and taxonomy.

Comparison of Pulsed-Field Gel Electrophoresis with Isoenzyme Profiles as a Typing System for Candida tropicalis

Candida species are important nosocomial pathogens, particularly in immunocompromised and critically ill patients. A variety of methods have been used to differentiate strains, but an optimal system has not been established. We compared methods for typing a panel of nine related isolates of Candida tropicalis from an outbreak of sternal wound infections as well as four unrelated control isolates of this species. (The genetic relationships of the nine isolates in the panel had been confirmed previously by restriction fragment analysis.) Typing was undertaken without knowledge of an isolate's origin. Karyotyping by contour-clamped homogeneous electric field (CHEF) gel electrophoresis failed to distinguish between outbreak and control isolates. However, when chromosome-sized DNA was digested with SfiI, EagI, SacII, or NaeI and the fragments were separated by CHEF electrophoresis, the outbreak isolates were readily identified. The isoenzyme profiles of the outbreak isolates were identical and were distinctly different from those of the control isolates. While both isoenzyme profiles and the modified CHEF procedure were discriminatory, the latter is recommended as a relatively convenient and reproducible technique for comparison of types of C. tropicalis.

Structure and Organization of Amplified DNA on Double Minutes Containing the mdm2 Oncogene

We have been studying a transformed derivative of a mouse fibroblast line (3T3DM) that stably maintains double minute chromosomes (DMs). In this report we describe a comprehensive analysis of the structure of the DMs within this cell line, utilizing a combination of long-range mapping via pulsed-field gel electrophoresis, screening of DM-enriched genomic libraries, and DM sizing using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Our data indicate that the minute particles in these cells exist as a homogeneous population of circular molecules, roughly 4 Mb in size, upon which three genes are amplified. One of these is the mdm2 oncogene, which has also been found to be amplified in a number of human sarcomas. Further, we present evidence that these three genes are arranged as two identical inverted repeat units linked by spacer regions of heterogeneous size. This work has led to the first model for the structure of an entire double minute particle containing an amplified oncogene; this model provides clues to later events occurring in the gene amplification process in tumor cells.

Electrophoretic karyotypes of clinical isolates of Coccidioides immitis

Chromosomes of the fungal respiratory pathogen, Coccidioides immitis, were separated by contour-clamped homogeneous electric field gel electrophoresis. Twelve isolates were examined, the majority of which showed four chromosomes with a range of molecular size from 11.5 to 3.2 Mb. Three isolates (C634, C735, and L) revealed three chromosomal bands under the conditions employed for electrophoretic separation. However, in two of these isolates (C634 and C735), four chromosomes were visible on membrane transfers of pulsed-field gels after Southern hybridization between the chromosomal DNA and selected DNA probes. The probes included a conserved ribosomal gene and three previously described cDNAs isolated from C. immitis expression libraries. The L isolate was determined to have the same genome size as a typical four-chromosome isolate on the basis of microspectrophotometric comparison of fluorescence intensity of the ethidium bromide-stained nuclear DNA. The genome size of C. immitis determined by microspectrophotometry was approximately 28.2 +/- 2.6 Mb. The calculated genome size based on addition of the average molecular weights of chromosomal bands separated by contour-clamped homogeneous electric field gel electrophoresis was approximately equal to the estimate derived from the spectrophotometric analyses. This is the first report of the electrophoretic karyotype of C. immitis.

Separation of chromosomes of Phytophthora species using CHEF gel electrophoresis

A broadly applicable technique for separation of chromosomes of Phytophthora species is desirable as a genetic tool for estimating genome size, determining chromosome number, and localizing specific genes on chromosomes. Chromosomal DNAs from seven different Phytophthora species were electrophoresed using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Ramped switching intervals of 3 to 45 min at 50 V for 7 days (14°C) allowed resolution of 13 bands for Phytophthora megasperma isolate 63, in agreement with the chromosome number reported from cytological studies. At 45 V, at least six chromosomes were resolved for P. cactorum, four to five for P. boehmeriae, seven to nine for P. megasperma isolate 77, and three to four for P. capsici. Some separation but poor resolution was observed for chromosomes of P. palmivora and P. parasitica. Chromosomes of P. infestans failed to separate, migrating as a single high molecular weight band. Substantial variation in band intensity was observed within isolates of all species. Specific hybridization to chromosomes on CHEF gels was obtained with the seven species using a ribosomal DNA probe from Pythium paroecandrum.