Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Genomic characterization of <Emphasis Type="Italic">Oryza</Emphasis> species-specific CACTA-like transposon element and its application for genomic fingerprinting of rice varieties

A repeated DNA fragment (pKRD) was isolated from the genomic library of weedy rice in Korea. The pKRD showed significant homology to Em/Spm CACTA-like transposon in whole genome sequences of rice released in the Blast rice sequence database of NCBI and was closely related to the TNP2 transposase group, including a TNP-like transposable element of rice. A Southern hybridization experiment demonstrated that the pKRD sequence is unique to the Oryza genome. The 126 sequences homologous to pKRD were evenly distributed in all 12 different chromosomes in rice genomes with multiple copy numbers. Different copy numbers ranging from 1,500 to 4,500 corresponding to rice species were detected by slot blot hybridization. In a DNA fingerprinting experiment, a pKRD probe was assessed to be the potential molecular marker for studying evolution and divergence, biodiversity and phylogenic analysis of rice species.     

Detecting specific repeated sequences in large,complex genomes by using representative difference analysis and double-probe verification

We present a modification of the representative difference analysis (RDA) technique used to target AT-rich repeated sequences, such as transposable elements, with a double-probe verification system. RDA is a subtractive/amplification PCR-based technology used to identify specific sequences that are different between 2 related genomes.Vsp I restriction enzyme was used to target AT-rich sequences. RDA products were cloned with a high efficiency. Double-probe verification is based on reverse dot-blot of cloned RDA products and uses a positive and a negative probe. We tested thisVsp I-modified RDA on different combinations of bread wheat (Triticum aestivum) and relatives.Triticeae members have large, complex genomes with various ploidy levels. RDA experiments were performed with single or bulked DNA. Reverse dot-blot double-probe verification detected specific repeated sequences quickly and efficiently. Together, the 2 systems provide a powerful tool for obtaining specific transposable elements and repeated sequences that are different between related genomes, regardless of genome size and ploidy.     

Chromosomal location and distribution of As51 satellite DNA in five species of the genus Astyanax (Teleostei, Characidae, Incertae sedis)

Constitutive heterochromatin makes up a substantial portion of the genome of eukaryotes and is composed mainly of satellite DNA repeating sequences in tandem. Some satellite DNAs may have been derived from transposable elements. These repetitive sequences represent a highly dynamic component of rapid evolution in genomes. Among the genus Astyanax , the As51 satellite DNA is found in species that have large distal heterochromatic blocks, which may be considered as derived from a transposable DNA element. In the present study, As51 satellite DNA was mapped through in situ fluorescent hybridization in the chromosomes of five species of the genus. The possible roles of this type of saltatory DNA type in the genome of the species are discussed, along with its use for the phylogenetic grouping of the genus Astyanax , together with other shared chromosomal characters. However, the number of As51 clusters is presented as a homoplastic characteristic, thereby indicating evident genomic diversification of species with this type of DNA.     

Novel non-autonomous transposable elements on W chromosome of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex chromosomes of the silkworm Bombyx mori are designated ZW(XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in the WGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.     

Development and Characterization of a Genetic Linkage Map of Cryptococcus neoformans var. neoformans Using Amplified Fragment Length Polymorphisms and Other Markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.     

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: Flanking sequences are mobile elements

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30 (101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to the chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.We dedicate this paper to Professor W. Beermann at the occasion of his 60th birthday     

Repeat-induced point mutation and the population structure of transposable elements in Microbotryum violaceum

Repeat-induced point mutation (RIP) is a genome defense in fungi that hypermutates repetitive DNA and is suggested to limit the accumulation of transposable elements. The genome of Microbotryum violaceum has a high density of transposable elements compared to other fungi, but there is also evidence of RIP activity. This is the first report of RIP in a basidiomycete and was obtained by sequencing multiple copies of the integrase gene of a copia-type transposable element and the helicase gene of a Helitron-type element. In M. violaceum, the targets for RIP mutations are the cytosine residues of TCG trinucleotide combinations. Although RIP is a linkage-dependent process that tends to increase the variation among repetitive sequences, a chromosome-specific substructuring was observed in the transposable element population. The observed chromosome-specific patterns are not consistent with RIP, but rather suggest an effect of gene conversion, which is also a linkage-dependent process but results in a homogenization of repeated sequences. Particular sequences were found more widely distributed within the genome than expected by chance and may reflect the recently active variants. Therefore, sequence variation of transposable elements in M. violaceum appears to be driven by selection for transposition ability in combination with the context-specific forces of the RIP and gene conversion.     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels     

Rapid identification of Vibrio anguillarum by Colony hybridization

A 562 base pair fragment of DNA from a serotype A strain of Vibrio anguillarum was cloned into pUC9 and used as a hybridization probe for the rapid identification of Vibrio anguillarum by colony hybridization. The probe was tested on nine different fish pathogens, 15 Vibrio isolates, 2 organisms closely related to Vibrio, and 9 serotypes of V. anguillarum. The probe hybridized only with the DNA of V. anguillarum serotypes A and H. The sequence of the 562 nucleotides have been determined. This probe allows rapid, reliable, and specific detection of V. anguillarum in freshwater ayu, Plecoglossus altivelis.     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

Restriction fragment length polymorphisms distinguish ectomycorrhizal fungi

Basidiomycetous fungi, two saprophytes and three mycorrhizal, were used to assess the specificity of DNA hybridization for distinguishing genera from one another. Interspecific comparisons were done with several isolates of mycorrhizal fungi,Laccaria bicolor andL. laccata, collected from diverse geographical sites. The DNAs were digested with four restriction nucleases and separated by gel electrophoresis into patterns of DNA fragments called restriction fragment length polymorphisms (RFLPs). The RFLPs were hybridized with a radioactively-labeled DNA probe encoding Basidiomycetous ribosomal RNA genes. The five genera were discernable using both unprobed and probed RFLPs. Hybridization of probe DNA with RFLPs was isolate-specific for all nine Laccaria isolates examined. The reclassification of aL. bicolor isolate is supported, demonstrating that hybridization of RFLPs offers an additional tool for taxonomy of ectomycorrhizal fungi. The method may have field application for distinguishing known isolates if their DNA fingerprints are previously ascertained and are distinct from RFLPs of indigenous organisms.     

The Prevalence of Sequences Homologous to IS256 in Clinical Enterococcal Isolates

Using dot blot hybridization techniques and an internal IS256 probe, we screened 103 clinical enterococcal isolates for the presence of sequences homologous to IS256. Most screened isolates exhibited resistance to one or more antimicrobial agents. Overall, hybridization to the internal IS256 probe was demonstrable in 88/103 (85%) isolates, 49/53 (92%) gentamicin-resistant isolates hybridized with the IS256 probe. In addition, 34/45 (76%) gentamicin-susceptible, aph2″(-) strains possessed sequences homologous to IS256. Southern hybridization experiments indicated that IS256 was frequently present in multiple copies in gentamicin-susceptible strains. These results suggest that IS256 is highly prevalent in clinical enterococcal isolates and that we may anticipate the emergence of novel, IS256-based composite mobile elements.     

Molecular characterization of sylvatic isolates of Trichinella spiralis

Genetic relationships of 20 Trichinella isolates from Indiana wildlife were assessed and compared to Trichinella isolated from an infected swine herd. Trichinella larvae were isolated from coyotes, mink, raccoons, and red foxes. The larvae were maintained and amplified in white mice (ICR) and wild mice (Peromyscus leucopus). Differences in phenotypic characters of sylvatic isolates in the 2 laboratory hosts included an approximately 10-30-fold increase in parasite fecundity in wild mice. DNA for each isolate was extracted from Trichinella larvae and analyzed by dot-blot hybridization using a repetitive DNA probe pBP2 that recognizes DNA sequences specific for swine Trichinella. The probe hybridized only to Trichinella from swine and a single coyote isolate. Restriction endonucleases were used to digest DNA and the resulting fragments were separated by gel electrophoresis. Based on the presence of repetitive DNA sequences in the Trichinella genome, distinctive banding patterns were seen among the isolates. Trichinella isolated from swine had a pattern distinct from all sylvatic isolates except 1 from a coyote. Because this coyote was from the same general locality as the swine Trichinella outbreak, it was concluded that the isolate represents transmission of swine trichinellosis to the wildlife population. Further analysis using the enzyme Cla I identified unique banding patterns for wild isolates, suggesting that the sylvatic group is a genetically heterogeneous complex.     

Amplification of CFTR Exon 9 Sequences to Multiple Locations in the Human Genome

Cloning and characterization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene led to the identification and isolation of cDNA and genomic sequences that cross-hybridized to the first nucleotide binding fold of CFTR. DNA sequence analysis of these clones showed that the cross-hybridizing sequences corresponded to CFTR exon 9 and its flanking introns, juxtapositioned with two segments of LINE1 sequences. The CFTR sequence appeared to have been transcribed from the opposite direction of the gene, reversely transcribed, and co-integrated with the L1 sequences into a chromosome location distinct from that of the CFTR locus. Based on hybridization intensity and complexity of the restriction fragments, it was estimated that there were at least 10 copies of the “amplified” CFTR exon 9 sequences in the human genome. Furthermore, when DNA segments adjacent to the insertion site were used in genomic DNA blot hybridization analysis, multiple copies were also detected. The overall similarity between these CFTR exon 9-related sequences suggested that they were derived from a single retrotransposition event and subsequent sequence amplification. The amplification unit appeared to be greater than 30 kb. Physical mapping studies includingin situhybridization to human metaphase chromosomes showed that multiple copies of these amplified sequences (with and without the CFTR exon 9 insertion) were dispersed throughout the genome. These findings provide insight into the structure and evolution of the human genome.     

Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 by segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.     

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were charcterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant-93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serorype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.     

Discrimination between closely related Triticeae species using genomic DNA as a probe

Summary Labelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the genera Hordeum and Secale. Discrimination was possible both by Southern hybridization to size-fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum), genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and the labelled probe, and between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species-specific sequences would remain as sites for probe hybridization. These species-specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species-specific sequences). Consequently, rapid nonradioactive methods detected probe hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.     

Isolation and sequence analysis ofCaenorhabditis briggsae repetitive elements related to theCaenorhabditis elegans transposon Tc1

Summary We have identified two repetitive element families in the genome of the nematodeCaenorhabditis briggsae with extensive sequence identity to theCaenorhabditis elegans transposable element Tc1. Five members each of the TCb1 (previously known as Barney) and TCb2 families were isolated by hybridization to a Tc1 probe. Tc1-hybridizing repetitive elements were grouped into either the TCb1 or TCb2 family based on cross-hybridization intensities among theC. briggsae elements. The genomic copy number of the TCb1 family is 15 and the TCb2 family copy number is 33 in theC. briggsae strain G16. The two transposable element families show numerous genomic hybridization pattern differences between twoC. briggsae strains, suggestive of transpositional activity. Two members of the TCb1 family, TCb1#5 and TCb1#10, were sequenced. Each of these two elements had suffered an independent single large deletion. TCb1#5 had a 627-bp internal deletion and TCb1#10 had lost 316 bp of one end. The two sequenced TCb1 elements were highly conserved over the sequences they shared. A 1616-bp composite TCb1 element was constructed from TCb1#5 and TCb1#10. The composite TCb1 element has 80-bp terminal inverted repeats with three nucleotide mismatches and two open reading frames (ORFs) on opposite strands. TCb1 and the 1610-bp Tc1 share 58% overall nucleotide sequence identity, and the greatest similarity occurs in their ORF1 and inverted repeat termini.     

Genetic relatedness of Bradyrhizobium japonicum field isolates as revealed by repeated sequences and various other characteristics.

Forty-nine isolates of Bradyrhizobium japonicum indigenous to a field where soybeans were grown for 45 years without inoculation were characterized by using four DNA hybridization probes from B. japonicum. nifDK-specific hybridization clearly divided the isolates into two divergent groups. Diversity in repeated-sequence (RS)-specific hybridization was observed; 44 isolates derived from 41 nodules were divided into 33 different RS fingerprint groups. Cluster analysis showed that the RS fingerprints were correlated with the nif and hup genotypes. We found multiple bands of RS-specific hybridization for two isolates that differed from the patterns of the other isolates. These results suggest that RS fingerprinting is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.