Characterization of DNA Sequences Cloned From Resting Spores of Plasmodiophora brassicae

Forty-four clones containing P. brassicae genomic DNA fragments were isolated from a partial genomic library (pUC118) of the fungus. Eleven DNA fragments were arbitrarily selected from them and partially sequenced. Southern blot analysis was performed using the 11 DNA fragments as probes to estimate the copy number of these DNA fragments in the P. brassicae genome and to see if they hybridized also to genomic DNA of host plant (Chinese cabbage). Five of those (Nos 24, 48, 54, 57, and 108) were of single-copy and hybridized only to DNA bands of P. brassicae. Two fragments (Nos 4 and 15) were found to contain repetitive sequence of/", brassicae. Three fragments (Nos 69,73, and 82) hybridized to DNA bands of both host and P. brassicae while the size and the number of the bands were different between host and P. brassicae. Data base searches revealed that one of the single-copy fragments (No. 48) had high homology with yeast RNA polymerase II gene.     

Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma

In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two -carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.     

Superparasitism in Cotesia glomerata: response of hosts and consequences for parasitoids

Abstract.  1. Superparasitism occurs in Cotesia glomerata (Hymenoptera: Braconidae), a gregarious endoparasitoid of Pieris spp. (Lepidoptera: Pieridae). The response of P. brassicae larvae to superparasitism and the consequences for the parasitoid were examined in order to elucidate the ecological significance of this behaviour.
2. Field surveys of a Swiss population revealed that C. glomerata brood sizes from P. brassicae larvae ranged from three to 158, and both the female ratio and the body weight of emergent wasps correlated negatively with brood size. In the laboratory, single oviposition on P. brassicae larvae did not produce any brood size larger than 62, but brood size increased with superparasitism.
3. Laboratory experiments demonstrated that both naive and experienced female wasps were willing to attack hosts that had been newly parasitised by themselves or conspecifics. Superparasitism reduced survivorship but increased food consumption and weight growth in P. brassicae larvae. Superparasitism lengthened parasitoid development and prolonged the feeding period of host larvae.
4. Despite a trade-off between maximising brood size and optimising the fitness of individual offspring, two or three ovipositions on P. brassicae larvae resulted in a greater dry female mass than did a single oviposition on the host. Thus, superparasitism might be of adaptive significance under certain circumstances, especially when host density is low and unparasitised hosts are rare in a habitat.     

Cytogenetic studies of four South American species of Paullinia L. (Sapindaceae)

Four South American species of Paullinia ( P. elegans , P. meliaefolia , P. pinnata , and P. rhomboidea ) were compared using conventional chromosome staining, C-Giemsa and C-chromomycin A₃/4',6-diamidino-2-phenylindole (C-CMA₃/DAPI) banding, and fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe. All species showed a somatic complement of 2 n  = 24 chromosomes, agreeing with earlier records in some cases, and showing a tendency for the chromosome number to be conserved in this genus. The chromosome number of P. rhomboidea is a new report. The karyotypes differed in chromosome size and degree of karyotype asymmetry. The chromosomal band patterns and location of the 45S rDNA sites are reported for the first time in the genus. Terminal C-CMA₃ bands were associated with the 45S rDNA sites, but varied in number and size between the species. The occurrence of other C-Giemsa bands that were not revealed by CMA₃ suggests that more than one family of repetitive DNA may be involved in karyotype differentiation. The systematic implications of these results on the infrageneric relationships are discussed.   © 2007 The Linnean Society of London . Botanical Journal of the Linnean Society , 2007, 154 , 313–320.     

Estimation of chromosome number and size by pulsed-field gel electrophoresis (PFGE) in medically important Candida species.

The chromosomal DNAs of eight medically important Candida species, C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata, were analysed by pulsed-field gel electrophoresis under various conditions. The corresponding bands in the gels were assigned by three kinds of DNA probe which hybridized to DNA of all the species: rDNA, TUB2 and PEP4. The best conditions for separating the chromosomal DNAs were investigated and the numbers and molecular sizes of the chromosome bands were determined for each species. The chromosomal DNAs of the species were separated into 5-14 bands ranging in size from 0.5 to 4.5 Mb. Based on the quantification of the chromosome band intensities using a laser fluorescent gel scanner, the chromosome numbers were estimated. The apparent average total number of chromosomes per cell was 16 for C. albicans, 16 for C. stellatoidea, 12 for C. tropicalis, 14 for C. parapsilosis, 8 for C. krusei, 8 for C. guilliermondii, 18 for C.kefyr, and 14 for C. glabrata; the total chromosomal DNA size of each species per cell was calculated at about 31 Mb, 33 Mb, 31 Mb, 26 Mb, 20 Mb, 12 Mb, 29 Mb and 14 Mb, respectively.     

Studies on the regeneration-restore and karyotype of protoplast in Metarhizium anisopliae var. majus

Abstract:  The characteristics and regeneration-restore of protoplasts and its karyotype of an insect pathological fungus, Metarhizium anisopliae var. majus were studied. Among the protoplasts, 25.3% were without a nucleus, and 74.7% contained a nucleus. Among the nucleus protoplasts, 53.6% contained a single nucleus. The regeneration-restore of protoplasts was of three distinct shapes. Considering the frequency of regeneration and the growing speed of the colony, 0.7 mol/l glucose was the optimum as osmotic stabilizer of culture medium in the regeneration-restore of the protoplasts. The chromosomal DNA molecules of M. anisopliae var. majus have been separated into seven bands by pulsed-field gel electrophoreses. Using the Schizosaccharomyces pombe chromosomes as size standard, the size of chromosomal DNA was estimated to be 1.1–6.5 Mb and its karyotype exhibited polytypism among strains.     

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.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

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.     

Separation of megabased-sized DNA molecules of Aspergillus niger using pulsed field gel electrophoresis

In this study, the chromosomal DNAs were extracted from Aspergillus niger Z10 wild type strain and these DNAs were separated using the contour clamped homogeneous electric field gel electrophoresis (CHEF) system. This system is laboratory-made and is operated by a computer program. Total DNAs resolved into five distinct chromosomal bands. The size of the chromosomes was estimated as being between 3.3 Mb to 6.4 Mb.     

Genetic characterization of the yeast Pichia anomala (strain K), an antagonist of postharvest diseases of apple

AIMS: To obtain information about the genomic organization of Pichia anomala (strain K) and about its genomic diversity at species and intraspecies level. METHODS AND RESULTS: The PFGE karyotype of strain K was composed of four bands ranging in size from 1.1 to 3.2 Mb. The number of chromosomes was estimated at six since bands 2 and 3 seemed to result from the comigration of two chromosomes with similar size. A comparison of strain K and Hansenulawingeii migration profiles led to the estimate of K strain genome size at 11.7 Mb. Comparison with isogenic strains, resulting from the sporulation of strain K, highlighted some major karyotypic differences. Two segregants (KH6 and KH7) showed supernumerary chromosomes and one (KH9) displayed chromosomal length polymorphism. This genomic instability was confirmed by molecular hybridization with four probes, consisting of URA3, LEU2, PAEXG1 and PAEXG2 genes of P. anomala. URA3 and LEU2 probes showed second hybridization signals on supernumerary chromosomes of strain KH7 and on chromosome 6 of strain K for LEU2 only. Karyotypic comparison of seven non-isogenic P. anomala strains revealed chromosomal length polymorphism, a sign of intraspecies variation. CONCLUSIONS: This work has supplied information about genome size and chromosome number of strain K of P. anomala. The strain seems to be aneuploid because of the presence of supernumerary chromosomes and additional hybridization signals for URA3 and LEU2 probes in the chromosomal profile of some segregants. The work also highlighted genomic diversity within the P. anomala species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results obtained here increase information about the aneuploidy of P. anomala (strain K). Information about the genomic diversity of the segregants will be of great interest for further studies on strain K mode of action. The genome size and chromosomal profile of P. anomala presented here are different from the results obtained elsewhere for Hansenula anomala, while Hansenula is included as a synonym of Pichia. This warrants further studies to investigate this taxonomic relationship.     

Insertion of transformation vector DNA into different chromosomal sites of Dictyostelium discoideum as determined by pulse field electrophoresis

Chromosomes of the cellular slime mold Dictyostelium discoideum were fractionated on three pulse field gel electrophoresis systems (pulse field, orthogonal field and C.H.E.F. (Contour-clamped Homogeneous Electric Fields] into a series of 13 bands ranging from 0.1 Mb to over 2 Mb in size. Since this organism has only seven chromosomes (estimated to be 1-10 Mb), and -90 copies of an 88-kilobase linear ribosomal DNA molecule (14% of genome), it was apparent that not all of these bands were whole chromosomes. However these bands were reproducibly obtained with the cell preparation used. They fell into three categories: i) four large poorly resolved DNA molecules (-2 Mb in size) which represent very large fragments or intact chromosomes, ii) eight faint bands ranging from 0.1 Mb to 2 Mb, iii) a prominent band in the apparent size range of about 0.15 Mb. Cloned Fragment V of an EcoR1 digest of the ribosomal DNA, hybridized to the 0.15 Mb band indicating it contained the linear ribosomal DNA. This chromosomal banding pattern was used to examine the stability and location of vector DNA in 16 transformed strains of D. discoideum. Each transformed strain was initially selected on the basis of G418 resistance with an integrating vector containing pBR322 sequences. Eleven transformants still carried pBR322 sequences after more than 60 generations of growth without selection on G418. All four strains transformed with constructs containing regions of the D. discoideum plasmid Ddp1 had lost their pBR322 insert, indicating that integration of Dictyostelium plasmid DNA into chromosomes leads to instability. Orthogonal field electrophoresis of the eleven strains still carrying pBR322 sequences revealed at least seven different integrating sites for the transforming DNA. We conclude that these vectors have many possible sites of integration in the D. discoideum genome.     

Between rat and mouse zoo-FISH reveals 49 chromosomal segments that have been conserved in evolution

Mouse single chromosome paints were applied to rat prophase/prometaphase chromosomes to detect homologous chromosome regions. The analysis revealed 49 rat chromosomal regions ranging in size from whole chromosomes down to small bands near the limit of detection with this method, which was estimated to be 2-3 Mb. When all the painted regions were taken into account, the whole rat genome was covered with mouse-homologous regions, with the exception of small segments near the centromeres and the short arms of Chromosomes (Chrs) 3, 11, 12, and 13. These regions were shown to contain high levels of rat-specific repetitive DNA. The number of conserved segments between rat and mouse detected by our high-resolution zoo-FISH method was significantly higher than that reported in previous studies.     

Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva)

Summary Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.     

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     

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).     

An electrophoretic karyotype forPhytophthora megasperma

Chromosomal DNA has been prepared from mycelial spheroplasts ofPhytophthora megasperma (isolate 63, chromosome numbern = 13–14)(E. M. Hansen, C. M. Brasier, D. S. Shaw, and P. B. Hamm, 1986.Trans Brit. Mycol. Soc.87, 557–573) and resolved by a pulsed field gel electrophoresis system which uses contour-clamped homogeneous electric fields. Nine chromosomal DNA bands were separated, the smallest being about 1.4 Mb in size: several larger DNAs were unresolved under all conditions tested. The estimated size was based on migration rates relative to those of chromosomal DNA ofSaccharomyces cerevisiae, Schizosaccharomyces pombe, and aNeurospora crassa translocation strain which has a minichromosome.     

家蚕病原球孢白僵菌的原生质体再生回复及核型分析

家蚕病原球孢白僵菌（Beauveriabassiana）原生质体的分离制备、性状及再生回复,并用脉冲凝胶电泳（PFGE）技术分析了其核型。以6mg／mLDriselase为酶解液,0.7mol／LNaCl液（pH5.8）为渗透压稳定剂,在30℃下轻轻振荡处理幼嫩菌丝1．5h,是原生质体分离的适宜条件。原生质体的无核率为26．5％,有核率为73.5％,其中单核率为53.5％。再生回复的形式可观察到三种,其培养基的渗透压稳定剂以0.7mol／L葡萄糖较为适当。球孢白僵菌至少具有6条染色体,估算大小为2.5～6.6Mb,核型大小为26.5Mb。     

Extrachromosomal circular DNA derived from tandemly repeated genomic sequences in plants

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It was found in various non-plant organisms from yeast to humans. EccDNA is heterogeneous in size and contains sequences derived primarily from repetitive chromosomal DNA. Here, we report the occurrence of eccDNA in small and large genome plant species, as identified using two-dimensional gel electrophoresis. We show that eccDNA is readily detected in both Arabidopsis thaliana and Brachycome dichromosomatica , reflecting a normal phenomenon that occurs in wild-type plants. The size of plant eccDNA ranges from > 2 kb to < 20 kb, which is similar to the sizes found in other organisms. These DNA molecules correspond to 5S ribosomal DNA (rDNA), non-coding chromosomal high-copy tandem repeats and telomeric DNA of both species. Circular multimers of the repeating unit of 5S rDNA were identified in both species. In addition, similar multimers were also demonstrated with the B. dichromosomatica repetitive element Bdm29. Such circular multimers of tandem repeats were found in animal models, suggesting a common mechanism for eccDNA formation among eukaryotes. This mechanism may involve looping-out via intrachromosomal homologous recombination. The implications of these results on genome plasticity and evolutionary processes are discussed.     

Resistance of kale varieties to attack by Mamestra brassicae

1 The objectives of this work were to study the resistance of six kale ( Brassica oleracea acephala group) varieties to cabbage moth Mamestra brassicae (L.) expressed as antibiosis and to determine the effect of plant age on larval survival and development.
2 The influence of plant age on resistance was determined using leaves from seedlings and from mature plants. Survival and development of M. brassicae larvae and feeding rates were determined in laboratory bioassays.
3 Leaves from seedlings were more suitable than those of mature plants for establishing differences in resistance. There were significant differences between kale varieties in larval survival, growth rate, leaf feeding, and time to pupation but not pupal weight. The varieties MBG-BRS0031, MBG-BRS0351, and MBG-BRS0287 reduced survival of M. brassicae larvae. Larvae that fed on MBG-BRS0060 were the heaviest and took the longest time to pupation. MBG-BRS0031 was consumed significantly less by larvae than were all the other varieties examined. Leaves from mature plants of MBG-BRS0142 and MBG-BRS0170 were defoliated significantly less than those of other varieties.
4 In conclusion, the variety MBG-BRS0031 may be a promising source of resistance to M. brassicae . Leaf antibiotic resistance was shown to play a role in defense against M. brassicae attack but it is not the only possible mechanism of resistance.