Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.     

Identification and differentiation of Heterotardigrada and Eutardigrada species by riboprinting

In the last decades, the number of known tardigrade species has considerably increased to more than 960 species with new ones being discovered every year. However, the study of tardigrade species presents a general problem which is frequently encountered during the work with invertebrates: small size and remarkable degrees of phenotypic plasticity may sometimes not permit a definite identification of the species. In this investigation we have used riboprinting, a tool to study rDNA sequence variation, in order to distinguish tardigrade species from each other. The method combines a restriction site variation approach of ribotyping with amplified DNAs. In eight investigated species of heterotardigrades and eutardigrades we have amplified the genes for the small subunit ribosomal RNA (SSU; 18S) and subsequently sequenced the genes. Virtual riboprints were used for identification of restriction sites from ten already published 18S rDNA sequences and seven new 18S rDNA sequences. On the basis of the obtained sequences, diagnostic restriction fragment patterns can be predicted with only 11 restriction enzymes. The virtual digestion confirmed the obtained restriction fragment patterns and restriction sites of all amplified and digested tardigrade DNAs. We show that the variation in positions and number of restriction sites obtained by standard restriction fragment analysis on agarose gels can be used successfully for taxonomic identification at different taxonomic levels. The simple restriction fragment analysis provides a fast and convenient method of molecular barcoding for species identification in tardigrades.     

Application of partial restriction procedure in both shotgun and non-random strategies for nucleotide sequencing

Partial digestion of a target DNA fragment with 4-bp-recognition restriction enzymes followed by a forced ligation to an M13 vector was employed for the construction of a subfragment library. The library can be used for either shotgun or non-random nucleotide sequencing. Application of the partial digests generated with the 4-bp recognition restriction enzymes instead of DNase I in the improved non-random strategy for nucleotide sequencing (Li and Wu, 1987) made the procedure as easy as that of the random strategy. The library can also be used in shotgun nucleotide sequencing directly, and few self-ligated subfragments were found. The usefulness of this procedure was demonstrated by the sequencing of a goat 6.5-kb EcoRI fragment, which is located 5' to the globin gene.     

Amplified DNA of the Novikoff hepatoma nucleolus is arranged in a 7.3-kilobase tandem repeat

We have reported previously the cloning and characterization of a nucleolar-localized 5.8-kilobase (kb) EcoRI fragment that is approximately 50-fold more highly reiterated in Novikoff hepatoma tumor cells than in normal rat liver [Parker, D. L., Busch, H., & Rothblum, L. I. (1981) Biochemistry 20, 762]. In the present study, the arrangement of these 5.8-kb EcoRI segments within the Novikoff hepatoma genome was investigated. Through the use of "indirect" restriction site mapping, partial restriction enzyme digestions, and molecular cloning, we have determined that the 5.8-kb EcoRI fragment and a 1.5-kb EcoRI fragment together constitute a 7.3-kb unit. The 7.3-kb unit is present in the hepatoma genome as a tandem repeat and constitutes the unit of the DNA that has been amplified. Studies on the arrangement of homologous sequences in the normal rat genome indicate that the amplified DNA may have been derived by a rearrangement and amplification of the nontranscribed spacer of the ribosomal DNA (rDNA) repeat.     

A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample

Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.     

Intervening sequences in the ribosomal RNA genes of Ascaris lumbricoides: DNA sequences at junctions and genomic organization

An rDNA size class in the genome of the nematode Ascaris lumbricoides is described which is interrupted by a 4.5-kb long intervening sequence located in the 26S coding region. This molecular form occurs in approximately 15 copies per haploid genome and amounts to approximately 5% of the total nuclear rDNA. Intervening sequences are present only in the 8.8-kb rDNA, but not in the 8.4-kb rDNA repeating units of A. lumbricoides. Cloning of the interrupted rDNA units revealed, in addition to the main 4.5-kb insertion, shorter intervening sequences of 4-kb and 119-bp length. Both shorter rDNA forms are present in the single copy range of the haploid genome. Sequence analyses of the intervening sequence/rDNA junctions show an identical right-hand junction for all of the three different rDNA forms. The two shorter intervening sequences are a coterminal subset of the right-hand end of the main 4.5-kb insertion, whereas all three insertions have a different left-hand junction with the coding region of rDNA. Each intervening sequence is flanked by a short direct repeat of variable length, being only once present in the uninterrupted rDNA. The intervening sequences of A. lumbricoides show striking similarity to the organization of type I insertion family in dipteran flies, even though they are inserted at different positions in the 26S coding region. Additional rDNA intervening sequences may be present outside of the rDNA cluster, but in not more than 15-20 homologous copies per haploid genome.     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

Identification and chromosomal location of tandemly repeated DNA sequences in Allium cepa

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between Cheterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

Construction of a high-resolution linkage map of Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility conferred by DCGMS cytoplasm in radish (Raphanus sativus L.) using synteny between radish and Arabidopsis genomes

Cytoplasmic male sterility caused by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome-3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group-2 co-segregated with male fertility in an F(2) population. Using radish ESTs positioned in linkage group-2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome-3. Synteny between the radish and Arabidopsis genomes in the 985-kb interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome-3.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket,Acheta domesticus (Orthoptera:Gryllidae)

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

Characterization of the aac(6'')-Ik gene of Acinetobacter sp. 6

Abstract Chromosomal DNA of different species of mycobacteria, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium and Mycobacterium smegmatis , has been submitted to polymerase chain reaction using two oligonucleotide primers highly homologous to DNA sequences flanking the quinolone resistance-determining region in the gyrA gene of Escherichia coli and Staphylococcus aureus . For each of these mycobacterial species, a 150-bp DNA fragment hybridizing with an intragenic probe of the gyrA gene of E. coli K12 was obtained. The nucleotide sequences of the 108-bp fragments amplified from M. tuberculosis and M. avium were determined. The two sequences were 87% homologous. Except for one residue, their deduced amino acid sequences were identical and shared 67% homology with the quinolone resistance-determining region of the gyrase A subunits of E. coli and S. aureus . Sequencing of the 108-bp fragment amplified from an in vitro mutant of M. avium , highly resistant to fluoroquinolones, showed a point mutation leading to the substitution of Ala for Val at a position corresponding to residues involved in quinolone resistance in E. coli and S. aureus , i.e. Ser 83 for E. coli and Ser 84 for S. aureus .     

Chromosomal localization and polymorphisms of ribosomal DNA in oat (Avena spp.).

The 17S/5.8S/26S ribosomal DNA (rDNA) sequences were mapped to the three satellited (SAT) chromosomes in the common hexaploid cultivated oat Avena sativa (2n = 6x = 42, AACCDD genomes). In situ hybridization and Southern hybridization of maize and (or) wheat rDNA probes to DNA from nullisomics derived from the cultivar 'Sun II' allowed the placement of rDNA sequences to the physical chromosomes. A restriction map was produced for the rDNA sequences of 'Sun II' using a maize probe from the transcribed region of the 17S/26S rDNA repeat. The set of rDNA repeats on SAT 2 of 'Sun II' possesses a 10.5-kb EcoRI fragment not found in the rDNA repeats of SAT 1 and SAT 8. This 10.5-kb fragment results from the absence of an EcoRI site in the intergenic spacer (IGS) of SAT 2 repeats. Extensive polymorphisms were demonstrated for three hexaploid Avena species, namely, the Mediterranean-type cultivated oat A. byzantina and the wild species A. sterilis and A. fatua. However, geographically diverse A. sativa cultivars displayed little rDNA variation. In contrast with all of the A. sativa cultivars examined, the A. sterilis accessions generally lacked the 10.5-kb EcoRI fragment. The results support the hypothesis that A. sativa accessions descend from a limited ancestral cultivated population. The rDNA polymorphisms are attributed to differences in lengths and restriction sites of the IGS.     

An AluI fragment isolated from lake trout (Salvelinus namaycush), maps to the intergenic spacer region of the rDNA cistron

The relationship between a 217-bp AluI fragment (SnAluI-33c) from lake trout (Salvelinus namaycush) which hybridizes to the nucleolar organizer regions (NORs) and the ribosomal RNA genes was examined by Southern analysis and comparative hybridization. Restriction enzymes with recognition sites mapped in the lake trout rDNA cistron were used to digest genomic DNA into fragments of predetermined size. Comparison of the hybridization pattern of SnAluI-33c with those of two rDNA-specific probes placed this fragment within the intergenic spacer region of the rDNA cistron, approximately 3 kb upstream (5′) of the 18S gene. This finding is consistent with in situ hybridization experiments showing hybridization of this fragment to sites of rDNA [Reed, K.M. and Phillips, R.B., Cytogenet. Cell Genet. 70 (1995) 104–107]. Based on cross hybridization and sequence comparisons, homologous sequences are present in other salmonid species.