Contour-clamped Homogeneous Electric Field Gel Research Articles

Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa.

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.

Analysis of the electrophoretic karyotype ofFlammulina velutipes

The karyotype ofFlammulina velutipes (Curt.: Fr.) Sing. was analyzed electrophoretically using contourclamped homogeneous electric field gel electrophoresis and hybridization with DNA probes. The chromosomal DNA from the monokaryon (Fv-4K) and the dikaryon (Fv-4) were resolved into six and eight bands, respectively. The sizes of the chromosomes ranged from 1.9 to 6.0 megabase (Mb) pairs. Each of the separated bands of chromosomal DNA was identified by use of five cloned probes. The number of these chromosomes was estimated to be 6 and 12, respectively; and the size of the entire genome was estimated to be about 20.1 and 38.6Mb, respectively. From a comparison of the hybridization patterns, the existence of allelic chromosomes of different sizes was deduced in the Fv-4 strain.

Genetic characteristics of Borrelia coriaceae isolates from the soft tick Ornithodoros coriaceus (Acari: Argasidae).

Two Borrelia isolates (CA434 and CA435) cultured from the soft tick Ornithodoros coriaceus were analyzed by contour-clamped homogeneous electric field gel electrophoresis of unrestricted and ApaI-restricted DNA, standard electrophoresis of BamHI- and HindIII-restricted DNA, Southern hybridization, restriction fragment length polymorphism and sequencing of the 16S rRNA gene, and amplification of the 5S-23S intergenic spacer region. These isolates were compared with Borrelia coriaceae type strain Co53, B. burgdorferi sensu stricto strain CA4, and the relapsing-fever spirochete B. parkeri (undesignated). The 16S rRNA region of CA434 and CA435 differed from that of B. coriaceae type strain Co53 by the presence of 1 base (C) at position 367 (GenBank accession no. U42286). The linear plasmid profile of CA434 was similar to that of Co53, and the ApaI, BamHI, and HindIII restriction fingerprints of the total cellular DNA of CA434 and Co53 were similar. In contrast, CA435 differed somewhat from CA434 and Co53, which demonstrates that B. coriaceae is genetically diverse. Southern hybridization showed that the DNAs of CA434 and CA435 hybridized strongly with the digoxigenin-labeled DNA of Co53. Low homology was found between the DNA of Co53 and that of B. parkeri. The 16S rRNA sequence of B. parkeri was identical to previously published results for B. parkeri strain M3001 (GenBank accession number U42296). CA434 and CA435 represent only the second and third isolates of B. coriaceae obtained from any source since its initial isolation from an O. coriaceus tick in 1985. All three B. coriaceae isolates were derived from adult ticks collected from the same locality in northwestern California. Difficulties encountered in detecting B. coriaceae in, and isolating this spirochete from, the tissues of O. coriaceus are discussed. The lack of concordance between different detection or isolation methods suggests that reliance upon a single technique may grossly underestimate the true prevalence of spirochetal infection in wild-caught O. coriaceus ticks.

Physical characterisation of the plastid DNA in Neospora caninum

The physical characteristics of the plastid DNA in Neospora caninum were investigated using pulsed-field gel electrophoresis and TEM. In a comparison of contour-clamped homogenous electric field and field inversion gel electrophoresis, the latter proved the more successful technique for studying the plastid molecules. In most cases, restriction or modifying enzymes were required to enable the plastid DNA molecules to enter the gel from the well area. The unit length of the plastid of N. caninum is approximately 35 kb; however, there is evidence for the formation of oligomeric molecules, which may migrate as linear molecules in approximate multiples of the unit length. Four different plastid genes encoding the ssrRNA, lsrRNA, rpoC and tufA genes were identified by hybridisation studies of contour-clamped homogenous electric field and field inversion gel electrophoresis gels. Transmission EM was performed on isolated plastid DNA, and circular structures similar in size and appearance to those described in other apicomplexans were observed, with an approximate length of 19 μm. The data presented here conclusively show that the Nc-Liverpool canine strain of N. caninum possesses a plastid DNA, with physical characteristics similar to the plastids found in other apicomplexans.

Chromosome mapping in Cryptosporidium parvum and establishment of a long-range restriction map for chromosome VI

We used contour-clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvum and establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvum was found to consist of partially co-migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvum chromosomes or isolated chromosome VI using gene-specific probes and an oligonucleotide specific for C. parvum telomeres allowed the development of a long-range restriction map of chromosome VI.

Comparison of two isolates of Encephalitozoon hellem and E. intestinalis (microspora) by pulsed field gel electrophoresis

Summary Contour-clamped homogeneous electric field gel electrophoresis was used to separate chromosome-sized DNA from two strains each of Encephalitozoon hellem and Encephalitozoon intestinalis isolated in vitro from AIDS patients. The two isolates of E. hellem differed significantly in molecular karyotype (EhD with 8 bands from 207 to 281 kb, and EhC with 12 bands from 175 to 315 kb), indicating a marked genetic polymorphism. The two E. intestinalis isolates displayed the same molecular karyotype with 10 bands comprised of between 182 and 279 kb. Of microsporidia so far investigated, species of Encephalitozoon have the smallest nuclear genomes (2.3–2.9 Mb).

Chromosome mapping in Cryptosporidium parvum and establishment of a long-range restriction map for chromosome VI.

We used contour-clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvum and establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvum was found to consist of partially co-migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvum chromosomes or isolated chromosome VI using gene-specific probes and an oligonucleotide specific for C. parvum telomeres allowed the development of a long-range restriction map of chromosome VI.

Electrophoretic karyotype of environmental isolates ofParacoccidioides brasiliensis

Five of the 12 environmental isolates of the dimorphic fungus Paracoccidioides brasiliensis known to date, were analysed by contour-clamped homogeneous electric field gel electrophoresis (CHEF). The electrophoretic pattern was shown to consist of five bands, with molecular size ranging from 3.2 to 10 Mb, a model quite similar to the one found in the clinical isolates previously tested and used here as controls. However, one of the bands in the environmental isolates had a lesser weight (7.2 Mb), than the one corresponding to the clinical counterparts (8.8 Mb). This resulted in a smaller genome, approximately 29.7 Mb. The small differences that were found indicate the presence of chromosome polymorphism in this fungus.

Chromosomal DNA patterns and gene stability of Pichia pastoris.

We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains. Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient host for heterologous gene expressions. We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth. No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation.

Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Outbreak of Candida albicans fungaemia in a neonatal intensive care unit.

During a 4-month period, 9 premature infants hospitalized in a neonatal intensive care unit (NICU) developed Candida albicans fungaemia. All 9 infants received antifungal agents. Fluconazole was administered in 7 patients and successfully eradicated this organism in 6 with no adverse effects. For epidemiological investigation, 64 environmental specimens and hand-washings of all 54 staff members involved in the NICU were examined for the presence of this organism. No C. albicans could be identified from environmental sources, while the hand-washing of 1 nurse was C. albicans-positive. Two genotyping methods, including electrophoretic karyotyping using contour-clamped homogeneous electric field gel electrophoresis and polymerase chain reaction-based direct sequencing of rRNA gene, were used in the analysis of the isolates recovered from blood cultures of the infants, the hand-washing of the nurse and 7 control isolates. Both methods yielded comparable results and revealed that all 13 isolates from infected infants as well as the isolate from hand washing of the nurse were of the same genotype while the control isolates were distinct. These results suggest that the outbreak of C. albicans fungaemia was caused by a particular strain and possibly via cross-infection. In addition, we showed that fluconazole seemed to be safe and effective in treating C. albicans fungaemia in neonates, although the data were limited.

An electrophoretic molecular karyotype of a clinical isolate of Aspergillus fumigatus and localization of the MDR-like genes AfuMDR1 and AfuMDR2

The molecular karyotype of a clinical isolate of Aspergillus fumigatus (10AF/86/10) was determined by contour-clamped homogeneous electric field gel electrophoresis. Five chromosomal bands were resolved by this method. The resolved chromosomes ranged in size from 1.7 to 4.8 Mb, and together constituted a total genomic size of at least 15.8 Mb. Southern analysis of the separated chromosomes located the position of two MDR-like genes, AfuMDR1 and AfuMDR2, on chromosomes III and IV, respectively. The methods described herein may enable the application of molecular karyotyping of A. fumigatus in epidemiologic surveillance studies.

Electrophoretic Karyotype of Clinical Isolates ofParacoccidioides brasiliensis

Chromosomal DNA molecules were obtained from eight clinical isolates ofParacoccidioides brasiliensisduring the early stages of the yeast to mycelial transformation process and were separated by contour-clamped homogeneous electric field gel electrophoresis. In seven of the isolates, it was possible to separate five bands, albeit with two different migration patterns (A and B). In the remaining isolate only four bands were observed (pattern C). The isolated chromosomes showed molecular sizes ranging from 3.2 to 10 Mb according toSchizosaccharomyces pombemolecular size standards. The three different patterns observed had bands of 3.2, 3.8, 4.1, 5.2, 6.7, 7.2, 8.8, and 10 Mb. These data imply that at least 30 Mb of the genome are organized as chromosomal macromolecules. Due to the fact thatP. brasiliensisspheroplasts are difficult to obtain, technical details are provided.

Genome Organization inBeauveria bassiana:Electrophoretic Karyotype, Gene Mapping, and Telomeric Fingerprint

Viaud, M., Couteaudier, Y., Levis, C., and Riba, G. 1996. Genome organization inBeauveria bassiana:Electrophoretic karyotype, gene mapping, and telomeric fingerprint.Fungal Genetics and Biology20,175–183. Using telomeric fingerprint and pulsed-field gel electrophoresis, we have analyzed the chromosomal organization among nine entomogenousBeauveria bassianaisolates. A heterologous telomeric probe established that the minimum number of chromosomes was between seven and eight among isolates. Chromosomal DNA was electrophoresed by using contour-clamped homogenous electric field (CHEF) gel electrophoresis. Five to eight chromosomal bands were separated among isolates. Substantial variation in band intensity suggested the presence of some chromosome doublets. The deduced karyotypes were in agreement with the minimum chromosome number reported from telomeric fingerprints. The minimum total genome size was estimated to range from 34.3 to 44.1 Mb. Significant chromosome length polymorphism was observed in karyotypes in the speciesB. bassianaexcept within isolates from one insect,Ostrinia nubilalis.Specific hybridization to chromosomes on CHEF gels was obtained by using homologous genes for proteinase 1, 28S rDNA, β-tubulin, histone 4, nitrate reductase, and the mitochondrial small rDNA. The mitochondrial DNA had a slow mobility in comparison to its circular size, probably because of the presence of very long linear molecules. Comparison of karyotype profiles following Southern analysis demonstrated a very flexible chromosomal organization among isolates from different origins and has confirmed the similarities withinO. nubilalisisolates.

Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning, sequencing and chromosomal localization of its TRP1 gene.

The genome of the amylolytic yeast strain Lipomyces starkeyi NCYC 1436 was analysed using contour-clamped homogeneous electric field gel electrophoresis (CHEF). The banding pattern under a variety of running conditions indicating the presence of 11 different chromosome-sized DNA molecules. The sizes of these chromosome bands were determined by comparison with chromosomes from standard strains of Schizosaccharomyces pombe and Saccharomyces cerevisiae. The chromosomal bands were estimated to be within the range 0.7-2.8 Mb, with the genome (excluding mitochondrial DNA) estimated at 15 Mb. The molecular cloning of the TRP1 gene, isolated from a genomic library of this strain, is also reported: the gene was present on a 6.5-kb Sau3A DNA fragment, and complemented the trpC gene of E. coli. The DNA sequence was determined (EMBL accession No. Z68292) and compared to other tryptophan biosynthetic genes encoding N-(5'-phosphoribosyl) anthranilate isomerase (PRAI) activity. The gene was also used as a probe in hybridization studies, and by this means, its chromosomal location was identified.

The second capsule gene of cryptococcus neoformans, CAP64, is essential for virulence.

The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, (AP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.

A Genetic Map of Gibberella fujikuroi Mating Population A (Fusarium moniliforme)

Abstract We constructed a recombination-based map of the fungal plant pathogen Gibberella fujikuroi mating population A (asexual stage Fusarium moniliforme). The map is based on the segregation of 142 restriction fragment length polymorphism (RFLP) markers, two auxotrophic genes (arg1, nic1), mating type (matA+ / matA−), female sterility (ste1), spore-killer (Sk), and a gene governing the production of the mycotoxin fumonisin B1 (fum1) among 121 random ascospore progeny from a single cross. We identified 12 linkage groups corresponding to the 12 chromosome-sized DNAs previously observed in contour-clamped homogeneous electric field (CHEF) gels. Linkage groups and chromosomes were correlated via Southern blots between appropriate RFLP markers and the CHEF gels. Eleven of the 12 chromosomes are meiotically stable, but the 12th (and smallest) is subject to deletions in 3% (4/121) of the progeny. Positive chiasma interference occurred on five of the 12 chromosomes, and nine of the 12 chromosomes averaged more than one crossover per chromosome. The average kb/cM ratio in this cross is ~32.

Karyotyping by PFGE of clinical isolates ofSporothrix schenckii

From October 1991 to December 1992 we had eight patients with sporotrichosis at Tsukuba University Hospital in Japan. With 8 strains isolated from these patients, PFGE (pulsed-field gel electrophoresis) analyses were carried out to examine whether the karyotype of S. schenckii is distinguished by our method and whether this molecular approach is a useful means of biotyping of S. schenckii strains. Chromosomes were separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The strains had six to eight chromosomes and a total genome size was approx. 28 Mbp. Although these karyotypes of all the isolates looked closely similar to each other, they were grouped into three types.

Karyotyping by PFGE of clinical isolates of Sporothrix schenckii

From October 1991 to December 1992 we had eight patients with sporotrichosis at Tsukuba University Hospital in Japan. With 8 strains isolated from these patients, PFGE (pulsed-field gel electrophoresis) analyses were carried out to examine whether the karyotype of S. schenckii is distinguished by our method and whether this molecular approach is a useful means of biotyping of S. schenckii strains. Chromosomes were separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The strains had six to eight chromosomes and a total genome size was approx. 28 Mbp. Although these karyotypes of all the isolates looked closely similar to each other, they were grouped into three types.

A 6-Mb Yeast Artificial Chromosome Contig and Long-Range Physical Map Encompassing the Region on Chromosome 12q15 Frequently Rearranged in a Variety of Benign Solid Tumors

Cytogenetic analysis of a variety of benign solid tumors, among which uterine leiomyoma, lipoma, pleomorphic salivary gland adenoma, and pulmonary chondroid hamartoma, has indicated that these tumors often display chromosome breakpoints in region q13–q15 of chromosome 12. In previous studies, we have reported that these breakpoints map between locus D12S8 and theCHOPgene, the latter of which has been shown to be consistently rearranged in myxoid liposarcomas with t(12;16)(q13′1). Here, we report directional chromosome walking studies starting from D12S8 and resulting in the construction of a YAC contig of about 6 Mb. This YAC contig, whose orientation on chromosome 12 was determined by double-color fluorescencein situhybridization (FISH) analysis, has at least double coverage and consists of 75 overlapping YAC clones, all isolated from CEPH YAC libraries. Their insert sizes were estimated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomal localization and chimerism of the YACs were investigated by FISH analysis. Chimerism of YAC clones was independently determined by restriction mapping. On the basis of YAC end-derived DNA markers and sequence-tagged sites (STSs), with an average spacing of approximately 70 kb, as well as restriction enzyme analysis, a long-range physical map was established for the 6-Mb DNA region of chromosome 12 covered by the YAC contig. Within the YAC contig, the relative positions of various known genes, an expressed sequence-tagged site, and a number of CEPH/Généthon polymorphic markers were determined. The latter data allow full integration of our mapping data with those obtained by CEPH/Généthon as well as those reported at the Second International Workshop on Human Chromosome 12 Mapping. Finally, this YAC contig constitutes the basis for the construction of a transcriptional map of this region and is likely to facilitate identification of genes involved in the formation of various benign solid tumor types.