Non-transcribed Spacer Research Articles

PCR and PCR–RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum

Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the Lamiaceae family; its active ingredient, the neoclerodane diterpene salvinorin A, is a psychotropic molecule that produces hallucinations. A comparative evaluation of S. divinorum fresh and dried leaves, S. officinalis fresh leaves, and dried powdered leaves claimed to be S. divinorum was done. HPLC–MS data confirmed the presence of salvinorin A in both S. divinorun leaf extracts and the powdered leaves, whereas no salvinorin A was found in S. officinalis. The non-transcribed spacer (NTS) in the 5S-rRNA gene of all leaf samples and the dried powdered leaves was amplified by PCR using a pair of primers located at the 3′ and 5′ ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 500 bp for S. divinorum and 300 bp for S. officinalis) were gel purified, subcloned into pGEM ®-T Easy vector and sequenced. By aligning the isolated nucleotide sequences, great diversities were found in the spacer region of the two species. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR–restriction fragment length polymorphism (PCR–RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in S. divinorum NTS region at 428–433 bp. For TaqI, multiple sites (161–164, 170–173, and 217–220 bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235–238 bp). The results of this work show that the combined use of analytical chemical (HPLC–MS) and molecular (DNA fingerprinting) methods lead to the precise and unequivocal identification of S. divinorum.

The Tof1p–Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae

Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase ("sweepase"). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.

Species Identification of Crassostrea and Ostrea Oysters by Polymerase Chain Reaction Amplification of the 5S rRNA Gene

A specific multiplex polymerase chain reaction (PCR) was developed for the identification of Crassostrea angulata, C. gigas, Ostrea edulis, and O. stentina oyster species. Universal primers were used for the amplification of complete repetition units of 5S rDNA in each of the 4 species. The alignment of the obtained sequences was the basis for the specific design of species-specific primers (ED1, ED2, ST1, ST2, CR1, and CR2) located in the nontranscribed spacer regions. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed identification of Crassostrea and Ostrea species. A multiplex PCR with a set of the 6 designed primers showed that they did not interfere with each other and bound specifically to the DNA target. This genetic marker can be very useful for traceability of the species, application in the management of oyster cultures, and conservation of the genetic resources of the species.

Molecular characterization and chromosomal mapping of the 5S rRNA gene in Solea senegalensis: a new linkage to the U1, U2, and U5 small nuclear RNA genes

Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227- and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.

Nucleotide sequence, genomic organization and chromosome localization of 5S rDNA in two species of Curimatidae (Teleostei, Characiformes)

The 5S ribosomal DNA (5S rDNA) of higher eukaryotes is organized in repeat units of tandem arrays composed of a 5S rDNA coding region, conserved even among non-related taxa, and a variable non-transcribed spacer sequence (NTS). To contribute to knowledge on the organization and evolution of vertebrate 5S rDNA we used PCR, nucleotide sequencing, Southern blot hybridization and chromosome fluorescence in situ hybridization (FISH) to investigate 5S rDNA tandem repeats in the South American Curimatidae fish Steindachnerina insculpta and Cyphocharax modesta. 5S rDNA repeats of 180 base pairs (bp) from both species were PCR-generated and sequenced evidencing the shortest 5S rDNA monomer so far described in eukaryote species. Southern blotting revealed that both species contained two tandem 5S rDNA classes, the PCR amplified fragment composed of 180 bp monomers and a class of 1600 bp monomers not detected by PCR. Chromosome mapping of the 5S rDNA repeats identified a major locus in both species and a second minor locus only in C. modesta. The Southern blot and chromosome mapping data indicate the presence of different types of 5S rDNA tandem repeats in the Curimatidae genome.

Length polymorphism in the ribosomal DNA intergenic spacers of Lens species

Analysis of the ribosomal DNA (rDNA) intergenic spacers (IGS) (i.e., the IGS of 18S, 5.8S and 24S rDNA, and the NTS or non-transcribed spacer of 5S rDNA) of lentil cultivars and landraces (L. culinaris spp. culinaris) showed to have very low intraspecific length variability. PCR products of approximately 3200 bp were obtained from all cultivated materials; no significant electrophoretic differences were found between them. However, observable intraspecific polymorphism increased with the digestion of PCR-amplified IGSs by the restriction endonucleases BspHI, StuI and BstBI. This was also the case when specific PCR primers designed to amplify a particularly variable segment of the IGS were used. This segment includes two families of short, tandemly arranged, repeated sequences. Both methods revealed differences between some of the cultivated materials, yielding genetic markers that might be useful in further genetic mapping and breeding studies. Several NTS amplification products were observed in each plant, ranging from approximately 300 to 900 bp in the cultivated lentil. Differences between L. culinaris spp. culinaris and its related wild species (including the wild ancestor of the cultigen, L. culinaris spp. orientalis) were observed in the total length of their IGSs, the restriction patterns of the latter, and in their NTS variants.

5S rDNA and U2 snRNA are linked in the genome of Crassostrea angulata and Crassostrea gigas oysters: does the (CT)n·(GA)n microsatellite stabilize this novel linkage of large tandem arrays?

The 5S rRNA genes from 2 species of the Ostreidae family, Crassostrea angulata and Crassostrea gigas, were molecularly characterized. The genes were amplified, cloned, and sequenced. The results revealed a 5S rDNA tandem array with a nucleotide sequence in an inverted position within the nontranscribed spacer region that corresponded to the U2 small nuclear RNA (snRNA) gene. The sequence analysis indicated that both genes could be functionally active. The presence of the microsatellite (CT)n x (GA)n at the 3' end of both genes and the possible involvement of concerted evolution are discussed.

THE H3/H4 HISTONE GENE CLUSTER OF LAND SNAILS (GASTROPODA: STYLOMMATOPHORA): TS/TV RATIO, GC3 DRIVE AND SIGNALS IN STYLOMMATOPHORAN PHYLOGENY

Histone gene primers were developed for land snails (Stylommatophora). The partial H3/H4 histone gene cluster was cloned and sequenced for 18 species. Transcription of the H3 and H4 genes was divergent (each gene is transcribed in the opposite direction) as has been found for other protostome and diploblast animals, with the exception of Mytilus. In the bivalve Mytilus transcription of both genes occurs in the same direction, i.e. land snails and bivalves seem to differ in their histone gene organization. The non-transcribed H3/H4 spacer varied in length between 279 and 691 basepairs. Nucleotide polymorphisms in this non-transcribed spacer might be of significance to study phylogenetics and systematics of closely related species and genera. As expected, the coding regions exhibited no amino acid substitution among land snail species. However, one amino acid substitution was found in comparison between land snails and Drosophila. The transition/transversion (TS/TV) ratio of H3 and H4 was predominately shaped by the third codon position and ranged in most cases from 1.0 to 2.0, indicating low nucleotide saturation. GC content was calculated for the third codon position (GC3 index at the ‘wobble’ base position). The histone GC3 values were far lower in land snails than values currently available for other genomes (i.e. mammals). This indicates that H3/H4 histone wobble bases of land snails evolve without strong GC drive. Phylogenetic trees were reconstructed from the coding regions. We used Succinea putris (Elasmognatha) as outgroup. Trichia villosa (Helicoidea) showed six apomorphic nucleotide signals. Moreover, the nucleotide signals give evidence that the Cochlicopidae, Vertiginidae and Valloniidae are paraphyletic family categories. The paraphyletic status of cochlicopid, vertiginid and valloniid gastropods is also supported by our unpublished ribosomal DNA trees.

5S rDNA genome regions of Lens species

The length variability of the nontranscribed spacer (NTS) of the 5S rDNA repeats was analyzed in species of the genus Lens by means of PCR amplification. The NTS ranged from approximately 227 to approximately 952 bp. The polymorphism detected was higher than previous NTS polymorphisms described in this genus. Three NTS length variants from Lens culinaris subsp. culinaris and 2 from Lens culinaris subsp. orientalis were sequenced. The culinaris NTS fragment lengths were 239, 371, and 838 bp, whereas the orientalis ones were 472 bp and 506 bp, respectively. As a result of sequence similarities, 2 families of sequences were distinguished, 1 including the sequences of 838 and 506 bp, and others with the sequences of 239, 371, and 472 bp. The 1st family was characterized by the presence of a repeated sequence designated A, whereas the 2nd family showed a single A sequence and other repeated sequences designated B, C, and D. The presence of an (AT)n microsatellite was also observed in the 2nd family of sequences. The fragments, which included the 239-bp and 838-bp NTS sequences, as well as the intergenic spacer (IGS) of the 18S-5.8S-26S ribosomal DNA also from L. culinaris subsp. culinaris, were used to localize the nucleolar organizer region (NOR) and the 5S rDNA loci in the chromosomes of several species of the genus Lens by means of fluorescence in situ hybridization (FISH). The selective hybridization of the 2 NTS probes allowed us to distinguish between different 5S rDNA chromosomal loci.

The Mating Type Switch-Activating Protein Sap1 Is Required for Replication Fork Arrest at the rRNA Genes of Fission Yeast

Schizosaccharomyces pombe rRNA genes contain three replication fork barriers (RFB1-3) located in the nontranscribed spacer. RFB2 and RFB3 require binding of the transcription terminator factor Reb1p to two identical recognition sequences that colocalize with these barriers. RFB1, which is the strongest of the three barriers, functions in a Reb1p-independent manner, and cognate DNA-binding proteins for this barrier have not been identified yet. Here we functionally define RFB1 within a 78-bp sequence located near the 3' end of the rRNA coding region. A protein that specifically binds to this sequence was purified by affinity chromatography and identified as Sap1p by mass spectrometry. Specific binding to RFB1 was confirmed by using Sap1p expressed in Escherichia coli. Sap1p is essential for viability and is required for efficient mating-type switching. Mutations in RFB1 that precluded formation of the Sap1p-RFB1 complex systematically abolished replication barrier function, indicating that Sap1p is required for replication fork blockage at RFB1.

Phylogeny of <I>Brassaiopsis</I> (Araliaceae) in Asia Based on Nuclear ITS and 5S-NTS DNA Sequences

Abstract The phylogeny of Brassaiopsis (Araliaceae), a morphologically diverse and widely distributed genus in Asia, was estimated based on sequences of the internal transcribed spacer regions (ITS) and 5S nontranscribed spacer (5S-NTS) of the nuclear ribosomal DNA. Two main paralogous copies of 5S-NTS were found for Brassaiopsis but both were not recovered for all species. All but one species of Brassaiopsis was represented by 5S-NTS (copy 1). Separate parsimony analyses of ITS and 5S-NTS (copy 1) generated largely congruent topologies, but several paralogous 5S-NTS sequences precluded data combination. A sister relationship of Brassaiopsis with Trevesia was strongly inferred. The divergence of Brassaiopsis from Trevesia was estimated to have occurred ca. 20 million years ago (MYA) during a period of mountain building and climatic change in SE Asia. Our analysis suggests a rapid evolutionary radiation of Brassaiopsis in the Sino-Himalayan region and Indochina at ca. 8 MYA. Several clades within the genus...

Ｔｒｉｃｈｏｐｈｙｔｏｎ ｔｏｎｓｕｒａｎｓ感染症の現状と対策

At the 48th Annual Meeting of The Society for Japanese Medical Mycology, held in October, 2004, we reported our findings from a survey on Trichophyton tonsurans infections in the Hokuriku and Kinki regions of Japan. The survey revealed that a few epidemics had occurred across these regions. In this article, we introduce our subsequent studies relating to 1) molecular epidemiology of isolates taken from people in many parts of Japan and 2) factors affecting adequate sampling of the scalp with hairbrushes, essential for surveying and monitoring the infection. In total, 198 isolates of Trichophyton tonsurans were analyzed using restriction fragment length polymorphisms of the non-transcribed spacer regions of ribosomal RNA genes. The restriction enzyme Mva I indicated two molecular types of strains, implying that the causative agents of the epidemic had different origins. None of the isolates obtained from the epidemic showed the same restriction profile as that of isolates from aged and sporadic cases. The published hairbrush method suitable for obtaining samples from the scalp of Judo trainees was reevaluated by changing several factors. We found that sampling should not be done soon after the students' physical training because other fungal elements may give a false positive, samples should not be obtained from students who have recently applied topical antimycotics, and samples should be taken under the guidance of qualified instructors familiar with the sampling method.

Limitations on the use of polymerase chain reaction – restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the speciesSaccharomyces cerevisiae

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.

Human ribosomal RNA gene arrays display a broad range of palindromic structures

The standard model of eukaryotic ribosomal RNA (rRNA) genes involves tandem arrays with hundreds of units in clusters, the nucleolus organizer regions (NORs). A first genomic overview for human cells is reported here for these regions, which have never been sequenced in their totality, by using molecular combing. The rRNA-coding regions are examined by fluorescence on single molecules of DNA with two specific probes that cover their entire length. The standard organization assumed for rDNA units is a transcribed region followed by a nontranscribed spacer. While we confirmed this arrangement in many cases, unorthodox patterns were also observed in normal individuals, with one-third of the rDNA units rearranged to form apparently palindromic structures (noncanonical units) independent of the age of the donors. In cells from individuals with a deficiency in the WRN RecQ helicase (Werner syndrome), the proportion of palindromes increased to one-half. These findings, supported by Southern blot analyses, show that rRNA genes are a mosaic of canonical and (presumably nonfunctional) palindromic units that may be altered by factors associated with genomic instability and pathology.

Rapid PCR-RFLP method for discrimination of imported and domestic mackerel

With the ever-decreasing domestic fishery catch of Japanese mackerel Scomber japonicus, alternative Atlantic mackerel Scomber scombrus has been increasingly imported and currently accounts for approximately 34% of mackerel consumption in Japan. As there is no morphologic difference between the species after removal of their skin, not only fresh and frozen fillets but also processed seafood of S. scombrus are frequently marketed with mislabeling as S. japonicus. In this study, a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed to discriminate imported mackerel S. scombrus and domestic mackerel S. japonicus. PCR amplification for the nuclear 5S ribosomal DNA nontranscribed spacer was performed using Scomber-specific primers. Direct digestions of the PCR products using either PvuII or HaeIII restriction enzymes generated species-specific profiles, indicating that both enzymes enable the accurate identification of S. scombrus and S. japonicus. This robust and reproducible method can serve as molecular-based routine food inspection program to enforce labeling regulations.

RNA Polymerase I Transcription Factors in Active Yeast rRNA Gene Promoters Enhance UV Damage Formation and Inhibit Repair

UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.

Identification of an EcoRI restriction site for a rapid and precise determination of β-asarone-free Acorus calamus cytotypes

Calamus ( Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of β-asarone [( Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid β-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography–mass spectrometry (GC–MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of β-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-β-ocimene (3.28%), camphor (1.54%), calarene (1.42%), α-selinene (5.02%) and τ-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), β-sesquiphellandrene (3.28%), pre iso calamendiol (22.81%) and acorone (26.33%), and completely lacked of β-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3′ and 5′ ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM®-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid β-asarone-free A. calamus was also discussed.

Genomic organization and evolution of the 5S ribosomal DNA in the ancient fish sturgeon

Ribosomal DNA in sturgeon is informative when analyzed at the molecular level because it bears unique characteristics that are, to a certain extent, ancestral within vertebrates. In this paper, we examine the structure and the molecular evolution of the 5S ribosomal DNA (rDNA) region in 13 sturgeon species, comparing both the 5S ribosomal RNA (rRNA) genes and the non-transcribed spacer (NTS) sequences between the coding regions. We have found that different NTS and 5S gene variants are intermixed in the 5S rDNA arrays of the different sturgeon species and that all variants are ancestral, having been maintained over many millions of years. Using predictive models, we have found similar levels of sequence diversity in the coding regions, as well as in the non-coding region, but fixed interspecific differences are underrepresented for 5S genes. However, contrary to the expectations, we have not found fixed differences between NTS sequences when comparing many pairs of species. Specifically, when they belong to the same phylogeographic clade of the four into which the sturgeon is divided, but fixation of mutations and divergence is found between species belonging to different phylogeographic clades. Our results suggest that the evolution of the two parts of the 5S rDNA region cannot be explained exclusively as the outcome of a balance between mutational, homogenizing (i.e., gene conversion as a predominant force in sturgeon), and selective forces. Rather, they suggest that other factors (i.e., hybridization) might be superimposed over those forces and thus could to some extent be masking their effects.

Two classes of 5S rDNA unit arrays of the silver fir, Abies alba Mill.: structure, localization and evolution

The structure and organization of the 5S ribosomal DNA units of the silver fir, Abies alba Mill., as well as their position in the chromosome complement were investigated. PCR amplification of the gene and nontranscribed spacer region, sequence analysis and Southern hybridization, using a homologous probe, detected DNA sequences of approximately 550 bp and 700 bp. Sequence analysis of the spacers revealed that the difference in length between the sequences occurred in the middle spacer region as a result of the amplification of a 75-bp sequence of the short unit class, which is organized in four 54- to 68-bp tandem repeats in the long spacer unit. The 5S rDNA transcribed region is 120 bp long and shows high sequence similarity with other gymnosperm species. The comparative analysis of 5' and 3' flanking sequences of 5S rRNA genes of silver fir and other gymnosperms indicates that A. alba spacer units have the same rate of evolution and are more closely related to Larix and Pseudotsuga than to Pinus and Picea. Southern hybridization and fluorescence in situ hybridization of metaphase chromosomes of A. alba suggest that the short and long spacer units are organized as separate tandem arrays at two chromosomal loci on chromosomes V and XI.

PCR-RFLP Analysis of Nuclear Nontranscribed Spacer for Mackerel Species Identification

Scomber mackerel have been marketed in fresh and frozen forms and as processed seafood worldwide, and three species of Japanese mackerel S. japonicus, Pacific mackerel S. australasicus, and Atlantic mackerel S. scombrus have constituted a significant part of absolute Scombrid consumption in Japan. The present study was undertaken to develop a rapid and reliable method not only for differentiation of Scomber mackerel from related Scombrid fish by PCR amplification using Scomber genus-specific primers but also for identification of three Scomber mackerel species by PCR-RFLP analysis. Alignment of nucleotide sequences of the nuclear 5S ribosomal RNA gene (5S rDNA) among Scombrid fish allowed the selection of oligonucleotide primers specific for the Scomber genus. These primers enabled amplification of the nontranscribed spacer (NTS) of the 5S rDNA from S. japonicus, S. australasicus, and S. scombrus, whereas no amplification was demonstrated from other Scombrid fish. RFLP analysis of the PCR products with ScaI endonuclease generated unique restriction patterns for each Scomber species. This simple, robust, and reproducible PCR-RFLP technique using Scomber genus-specific primers can serve as a routine food inspection program to enforce labeling regulations of marketed Scombrid fish.