An electrophoretic karyotype of Aspergillus niger

Summary An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomesized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage groupspecific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5–38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.     

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6

Summary Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47° C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome.Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401.The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.     

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi ( sensu lato )

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible. Received: 6 February 1996 / Accepted: 21 January 1997     

Deep sequencing of a candidate region harboring the SOX9 gene for the canine XX disorder of sex development

A disorder of sex development (DSD) in dogs with female sex chromosomes (78, XX), a lack of the SRY gene and the presence of testes or ovotestes is commonly diagnosed in numerous breeds. The molecular background of DSD is not fully recognized but has been linked to the copy number variation in the region harboring the SOX9 gene. We applied a genome‐wide association study and targeted next‐generation sequencing techniques to compare DSD and normal female dogs. The genome‐wide association study did not indicate a significant chromosome region. Targeted next‐generation sequencing of a 1.5‐Mb region on canine chromosome 9 harboring the SOX9 gene revealed two putatively DSD‐associated copy number variations 355 kb upstream and 691 kb downstream of SOX9, four blocks of low polymorphism and two blocks of an elevated heterozygosity. An initial next‐generation sequencing analysis showed an association with two SNPs, but validation in larger cohorts did not confirm this result. We identified a large homologous fragment (over 243.8 kb), named hfMAGI2, located upstream of SOX9, that overlaps a known copy number variation region. It shows a high sequence similarity with the 5′ flanking region of the MAGI2 gene located on canine chromosome 18 that encodes a protein involved in ovary formation during early embryonic development. Our study showed that the identified copy number variation region located upstream of the SOX9 gene contains potential regulatory sequences (long non‐coding RNA and hfMAGI2) and led to the assumption that a multiplication of this element may alter expression of the SOX9 gene, triggering the DSD phenotype.     

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anticompetitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.     

Identification and mapping of regions that confer plasmid functions and of sites for excisive recombination of plasmid pMMC7105

Summary Strain PP808 of Pseudomonas syringae pv. phaseolicola contains pEXC8080 (34.6 kb), the smallest of several plasmids that originated by partial excision of the cryptic plasmid, pMMC7105 (150 kb), from the host chromosome. This excision plasmid is derived entirely of sequences from pMMC7105 and contains a 24 kb region referred to as common DNA, which is present in each of the other excision plasmids. A six enzyme restriction endonuclease map was constructed of pEXC8080. The replication region was mapped by identifying small restriction fragments that conferred replication properties to pMB1 plasmids that otherwise fail to replicate in Pseudomonas. This region is located within the common DNA and is 0.8–3.8 kb in size. Sequences from pEXC8080 failed to stabilize pMB1 derivatives in Pseudomonas in the absence of antibiotic selection, but stability functions were mapped to a region of pMMC7105 that presumably remains integrated in the chromosome of strain PP808. An incompatibility region was mapped to a 7.3 kb region on pEXC8080 that is closely linked to, but not included within, the replication region. The recombination site was mapped to a 1.2 kb region of the fusion fragment that was formed upon excision of pEXC8080. RS-I, a repetitive sequence, found on pMMC7105 was present in the fusion fragment at the site of recombination. RS-I was also mapped to BamHI fragments that recombined upon excision of pEXC8080 and suggest that it provides sites for homologous recombination.     

Single‐copy gene‐based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single‐copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome‐specific gene probes. This single‐copy gene‐based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis‐aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross‐species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.     

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.     

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Summary The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.     

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.     

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi (sensu lato)

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible.     

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

Sizing of theRhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation

The two restriction enzymesAsnI andDraI were found to produce DNA fragment sizes that could be used for mapping theRhodococcus sp. R312 (formerlyBrevibacterium sp. R312) genome by pulsed-field gel electrophoresis.AsnI produced 24 fragments (4 to 727 kb) andDraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on theAsnI andDraI fragments.     

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides

Summary A 1.2 Mb minichromosome resolved by pulsed-field electrophoresis was present in two independent race 3 isolates of Colletotrichum gloeosporioides causing Type B anthracnose specifically on Stylosanthes guianensis cv. Graham in Australia. This chromosome was absent in duplicate isolates representing races 1, 2 and 4 which infect other S. guianensis cultivars. A gene library was prepared specifically from the 1.2 Mb mini-chromosome and ten independent DNA clones unique to this chromosome were identified by differential hybridisation to whole chromosome probes. All of the ten selected probes hybridised only to the 1.2 Mb minichromosome unique to the race 3 isolates but not to any chromosome in isolates of the other races. These ten probes also hybridised only to restriction-digested DNA of race 3 and were thus both chromosome- and strain-specific for Type B C. gloeosporioides. Hybridisation analysis of NotI fragments of the 1.2 Mb minichromosome with these sequences indicated that they were not tightly clustered on the chromosome. These data demonstrate that the variation in the occurrence of the 1.2 Mb minichromosome did not arise by rearrangement of the genome of a progenitor strain but involved either large scale deletion or addition of DNA. The 1.2 Mb minichromosome did not contain a cloned high-copy-number repeat sequence present on all other mini- and maxichromosomes, suggesting addition from a genetically distinct strain. All ten chromosome-specific DNA probes hybridised to a 2.0 Mb chromosome in all races of C. gloeosporioides causing Type A anthracnose on Stylosanthes spp. including S. guianensis. Restriction fragment length polymorphism analysis demonstrated that only 15% of the hybridising restriction fragments of the Type A 2.0 Mb chromosome and the 1.2 Mb Type B race 3 minichromosome were identical. This indicated that it is unlikely that the 1.2 Mb minichromosome of the race 3 Type B pathogen was recently introgressed from-the Type A pathogen.     

Chromosome topology and genome size of selected actinomycetes species

Information about the genome organization of actinomycetes species is restricted to a few genera: Corynebacterium, Mycobacterium, Rhodococcus, Saccharopolyspora and Streptomyces. Streptomyces species and Saccharopolyspora erythraea were shown to contain a single linear 8 Mb chromosome. In contrast, the Corynebacterium, Mycobacterium and Rhodococcus species studied were demonstrated to possess a smaller (3 Mb–6.5 Mb) single circular chromosome. To investigate whether linear chromosome topology and genome sizes above 7 Mb are unique features of Streptomyces and S. erythraea we have started to investigate the chromosome topology, the genome size and the status of accessory elements of additional actinomycetes species: Actinoplanes philippinensis, Amycolatopsis orientalis, Micromonospora chalcea, Nocardia asteroides, Rhodococcus opacus and Streptoverticillium abikoense. Our data which are based on PFGE experiments clearly suggest that large genome sizes and chromosome linearity are seen in mycelium forming actinomycetes genera. In addition we have identified large linear plasmids in Nocardia asteroides, Streptoverticillium abikoense and Rhodococcus opacus.     

Cloning and mapping of the genetic determinants for microcin C51 production and immunity

Microcin C51 is a small peptide antibiotic produced by Escherichia coli cells harbouring the 38 kb low copy number plasmid pC51, which codes for microcin production and immunity. The genetic determinants for microcin synthesis and immunity were cloned into the vectors pBR325, pUC19 and pACYC184. Physical and phenotypic analysis of deletion derivatives and mutant plasmids bearing insertions of transposon Tn5 showed that a DNA fragment of about 5 kb is required for microcin C51 synthesis and expression of complete immunity to microcin. Partial immunity can be provided by a 2 kb DNA fragment. Mutant plasmids were tested for their ability to complement Mic^– mutations. Results of these experiments indicate that at least three plasmid genes are required for microcin production. The host OmpR function is also necessary for microcin C51 synthesis.     

Single-site manipulation of tomato chromosomes in vitro and in vivo using Cre-lox site-specific recombination

With the aim of developing new techniques for physical and functional genome analysis, we have introduced the Cre-lox site-specific recombination system into the cultivated tomato (Lycopersicon esculentum). Local transposition of a Ds(lox) transposable element from a T-DNA(lox) on the long arm of chromosome 6 was used to position pairs of lox sites on different closely linked loci. In vitro Cre-lox recombination between chromosomal lox sites and synthetic lox oligonucleotides cleaved the 750 Mb tomato genome with 34 pb specificity to release unique 65 kb and 130 kb fragments of chromosome 6. Parallel in vitro experiments on Saccharomyces cerevisiae chromosomes show the efficiency of cleavage to be 50% per chromosomal lox site at maximum. By expressing the Cre recombinase in tomato under control of a constitutive CaMV 35S promoter, efficient and specific somatic and germinal in planta inversion of the 130 kb fragment is demonstrated. The combined use of in vitro and in vivo recombination on genetically mapped lox sites will provide new possibilities for long range restriction mapping and in vivo manipulation of selected tomato genome segments.     

Organization and copy number of initiator tRNA genes in slow- and fast-growing mycobacteria

We have previously reported the isolation and characterization of a functional initiator tRNA gene,metA, and a second initiator tRNA-like sequence,metB, fromMycobacterium tuberculosis. Here we describe the fine mapping of the initiator tRNA gene locus of the avirulent (H37Ra) and virulent (H37Rv) strains ofM. tuberculosis. The genomic blot analyses show that the 1.7 kb (harbouringmetE) and the 6.0 kb BamHI (harbouringmetA) fragments are linked. Further, sequencing of a portion of the 6.0kb fragment, in conjunction with the sequence of the 1.7 kb fragment confirmed the presence of an IS6110 element in the vicinity ofmetB. The IS element is flanked by inverted (28 bp, with 3 contiguous mismatches in the middle) and direct (3 bp) repeats considered to be the hallmarks of IS6110 integration sites. The organization of the initiator tRNA gene locus is identical in both the H37Ra and H37Rv strains and they carry a single copy of the functional initiator tRNA gene. Interestingly, the fast growingMycobacterium smegmatis also bears a single initiator tRNA gene. This finding is significant in view of the qualitative differences in total tRNA pools and the copy number of rRNA genes in the fast- and slow-growing mycobacteria. Finally, we discuss hypotheses related to the origin ofmetB inM. tuberculosis.