New evidence for nucleolar dominance in hybrids of Drosophila arizonae and Drosophila mulleri

Drosophila mulleri (MU) and D. arizonae (AR) are cryptic species of the mulleri complex, mulleri subgroup, repleta group. Earlier cytogenetic studies revealed that these species have different regulatory mechanisms of nucleolar organizing activity. In these species, nucleolar organizing regions are found in both the X chromosome and the microchromosome. In the salivary glands of hybrids between MU females and AR males, there is an interspecific dominance of the regulatory system of the D. arizonae nucleolar organizer involving, in males, amplification and activation of the nucleolar organizer from the microchromosome. The authors who reported these findings obtained hybrids only in that cross-direction. More recently, hybrids in the opposite direction, i.e., between MU males and AR females, have been obtained. The purpose of the present study was to evaluate, in these hybrids, the association of the nucleoli with the chromosomes inherited from parental species in order to cytogenetically confirm the dominance patterns previously described. Our results support the proposed dominance of the AR nucleolar organizer activity over that of MU, regardless of cross-direction.     

Discrimination between <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> and <Emphasis Type="Italic">H. qinghaiensis</Emphasis> based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2)

In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1–0.4%, nucleotide differences between the tested species ranged 2.1–23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

黄颡单尾虫(粘体门,双壳目)的重描述及基于28S rDNA和ITS-5.8S序列的系统地位分析

采用形态分类学方法与以28S rDNA和ITS-5.8S序列为基础的分子系统学研究方法,对采自嘉陵江重庆市磁器口江段的黄颡单尾虫Unicauda pelteobagrusMa,1998进行了形态学和分子生物学的研究。基于28S rDNA数据探讨了黄颡单尾虫以及单尾虫属与相邻种属粘孢子虫间的系统地位;基于5.8S rDNA数据比较分析了粘孢子虫的系统地位。补充了黄颡单尾虫重庆种群形态学信息和28S rDNA、ITS-5.8S rDNA序列的分子信息。     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.     

Primers<Emphasis Type="Italic">ITS1, ITS2</Emphasis> and<Emphasis Type="Italic">ITS4</Emphasis> detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi

Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.     

Study of a cloned ribosomal operon region coding for 5.8S rRNA in the loach Misgurnus fossilis L

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

Characterization of Otostrongylus circumlitus from Pacific harbor and northern elephant seals

Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.     

Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.     

毛木耳rDNA序列结构及IGS鉴别菌种初探

rDNA序列中的ITS作为DNA barcode广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。有关食用菌rDNA序列的报道较少。本研究对毛木耳Auricularia cornea单核菌株B02进行三代测序与组装,然后用二代测序数据进行校正,得到一个组装效果较好的基因组序列。比对Fibroporia vaillantiir的rDNA序列获得毛木耳rDNA重复单元的完整序列,每个重复单元包含ETS、18S rDNA、ITS1、5.8S rDNA、ITS2、28S rDNA、IGS1、5S rDNA和IGS2,长度分别为398bp、1 790bp、156bp、156bp、206bp、3 432bp、2 247bp、121bp和2 135bp,总长度10 641bp,毛木耳rDNA有310个串联重复单元,转录组和系统发育分析均支持这一结果。与其他已报道的食用菌不同,毛木耳的IGS1、IGS2序列高度保守,其中IGS1的1 400-2 200bp区域在各拷贝之间没有多态性、而在品种之间有较高频率的SNP,这一片段序列有望用于品种鉴别研究。     

Sequence Analysis of the ITS Region of Nuclear Ribosomal DNA (nrDNA) in Chinese Amaranthus and Its Systematic Utility

The sequences of the ITS region (including ITS-1, 5.8S rDNA and ITS-2) of nuclear ribosomal DNA from 15 species of the genus Amaranthus L. and outgroup Celosia cristata L. were determined. The result shows that the size of the ITS region of Amaranthus is from 629 to 632 bp, and the length variation is only found in ITS-1 (250-253 bp). On the basis of phylogenetic analysis of nucleotide sequences, the Amaranthus species in China may be devided into three sections, viz., sect. Spinosus, sect. Amaranthus and sect. Paucestamen; the cultivated species, viz., Amaranthus paniculatus L., A. cruentus L., A. caudatus L. and A. hypochondriacus L. can be treated as the subsp. of A. hybridus L.; A. taishanensis F.Z.Li and A. tenuifolius Willd are both closely related to species in the sect. Paucestamen. The study also indicated that the number of stamens has more phylogenetic information than other characters, such as the number of sepals and dehiscence/indehiscence of fruits in Amaranthus.     

Molecular characterization of hard tick <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> from China by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0–2 and 0–2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1–55.2 and 37–52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.     

Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia

Species of Eimeria from chickens from Australia were characterised using a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) approach. The ribosomal DNA region spanning the second internal transcribed spacer (ITS-2) was amplified from genomic DNA by PCR, digested separately with three restriction endonucleases (CfoI, Sau3AI and TaqI) and the fragments separated by denaturing gel electrophoresis. The PCR products amplified from the six species varied from approximately 70 to 620 bp on agarose gels, with differences in size and number of bands among species, but no apparent variation within a species. The PCR-RFLP analysis of ITS-2 amplicons on denaturing gels gave characteristic profiles for individual species (except for minor variation in profiles within some species). The results indicate that ITS-2 contains useful genetic markers for the identification of six Eimeria species occurring in Australia.     

Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of <Emphasis Type="Italic">Bacillus</Emphasis> species

Restriction fragment length analysis of PCR amplified 16S rDNA with AluI revealed the presence of a 265 bp fragment in all species of Bacillus with the exception of B. cereus and B. thuringiensis, which contains two restriction sites within this fragment which results in three smaller fragments totalling to 265 bp. Some distant species of Bacillus with no evidence of this fragment could be delineated into other genera based on phenotypic and genotypic parameters. BLAST search for homologous sequences of individual species revealed that it is a highly conserved region. Multiple alignment of the fragment suggests that a region between 160 and 265 bp of the 265 bp fragment was a hypervariable region and were highly species-specific. A set of primers was designed for amplification of this hypervariable region. Partial sequencing of the hypervariable region within the 265 bp fragment seems an index for identification of Bacillus species.     

Analysis of three multigene families as useful tools in species characterization of two closely-related species, Dicentrarchus labrax, Dicentrarchus punctatus and their hybrids

By analyzing three multigene families, two closely related and commercially important species, Dicentrarchus labrax and Dicentrarchus punctatus, were characterized by cytogenetic and molecular methods. The interspecies hybrid Dicentrarchus labrax (♀) × Dicentrarchus punctatus (♂) was also analyzed. The multigene families studied were the 5S rDNA, 45S rDNA and the U2 snRNA. A microsatellite GTT motif was found within the non transcribed spacers (NTS) of the 5S rDNA from the two species. However, hexanucleotide duplication next to this microsatellite was observed in the D. labrax and hybrid clones, but not in D. punctatus. The U2 snRNA appeared to be linked to the U5 gene and showed two variant sequences, in both D. labrax and D. punctatus. They differed in one insertion/deletion of 7 nucleotides. The first internal transcribed spacer (ITS-1) region showed higher nucleotide variability in D. punctatus than in D. labrax. Nucleotide polymorphism within species and also nucleotide divergence between species were determined in the different gene regions. In a FISH analysis we obtained three chromosomal markers, because the 5S rDNA, 18S rDNA and U2 snRNA probes hybridized each in three different chromosome pairs. Hence none of them was co-localized. The 5S rDNA cluster and U2 snRNA were localized in acrocentric chromosome pairs, while the 18S rRNA gene probe hybridized in a subtelocentric pair. Finally, the usefulness of the results in developing tools for phylogenetic analysis and species identification are discussed in relation to other fish species.     

Trichosporon mycotoxinivorans sp. nov., a new yeast species useful in biological detoxification of various mycotoxins

A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon. Trichosporon mycotoxinivorans is closely related to T. loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx. 600 bp fragment of the 18S rDNA and both ITS regions. However, the two species differ at nine positions in the D1/D2 region of 26S rDNA. The IGS1 region of T. mycotoxinivorans is 401 bp long. T. mycotoxinivorans is distinguished from T. loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40 °C. The name of this newly isolated strain refers to an important characteristics of T. mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone. Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds.     

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction-restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA).

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49–56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR–RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.     

ITS-2 rDNA sequencing of Gnathostoma species (Nematoda) and elucidation of the species causing human gnathostomiasis in the Americas

From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.