- Email: [email protected]
Multiple rDNA units distributed on all chromosomes of Nosema bombycis
Journal of Invertebrate Pathology 99 (2008) 235–238
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Short Communication
Multiple rDNA units distributed on all chromosomes of Nosema bombycis Handeng Liu a, Guoqing Pan a, Shihong Song a, Jinshan Xu b, Tian Li a, Yanbo Deng c, Zeyang Zhou a,b,* a b c
The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing 400716, China Laboratory of Animal Biology Chongqing Normal University, Chongqing 400047, China School of Life Science & Technology, Beijing Institute of Technology, Beijing 100081, China
a r t i c l e
i n f o
Article history: Received 21 January 2008 Accepted 12 June 2008 Available online 1 July 2008 Keywords: Nosema bombycis rDNA Genomic survey Southern blotting Distribution
a b s t r a c t Among Microsporidia, Nosema bombycis has a novel arrangement of LSUrRNA, SSUrRNA, ITS, IGS and 5SrRNA. To determine the distribution of rDNA among the chromosomes, we performed genome-wide screening and Southern blotting with three probes (SSU, ITS and IGS). Southern blotting revealed that ribosomal RNA genes are distributed on all chromosomes of N. bombycis, which is contrary to the previous result, which concluded that the N. bombycis rRNA genes were limited to a single chromosome. This wide distribution is similar to that of the rDNA unit of Encephalitozoon cuniculi. Screening of the N. bombycis genome detected 53 LSUrRNA elements, 43 SSUrRNA elements and 36 5SrRNA elements. However, it is still difficult to determine their loci on the chromosomes as the genomic map is unfinished. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Microsporidian genomes have aroused interest since they were first karyotyped and found to be much smaller than expected for eukaryotes. Genome size in the Microsporidia ranges between 2.3 and 19.5 Mbp (Biderre et al., 1994; Méténier and Vivarès, 2001; Keeling and Fast, 2002). Current knowledge of genome organization in this group mainly derives from karyotyping studies based on pulsed field gel electrophoresis (PFGE) techniques, which revealed extensive inter-species variation in number and size of chromosomes (Munderloh et al., 1990). These findings have helped to illustrate genetic relationships among species at the subcellular level and have provided evidence for phylogenetics and species identification (Amigó et al., 2002). Nosema bombycis is an obligate intracellular parasite of the domesticated silkworm, and as the causative agent of pebrine disease, is still prevalent in sericulture (Weiss et al., 1998; Gao et al., 2001). Through PFGE, the number of chromosomes in N. bombycis was estimated at 18, ranging from approx. 380 to 1500 kbp, with a total size of approx. 15,330 kb (Kawakami et al., 1994). Nosema bombycis lacks the 5.8S rRNA gene, which is similar to other species in the group (Vossbrink and Woese, 1986). The location of the rRNA genes of several other microsporidians have been reported (Biderre et al., 1994; Peyretaillade et al., 1998). Using rDNA as a probe, the rDNA units of Encephalitozoon cuniculi were
* Corresponding author. Address: The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing 400716, China. Fax: +86 23 68251128. E-mail address: [email protected] (Z. Zhou). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.06.012
shown to be distributed on each chromosome (Brugère et al., 2000). Previous studies have reported that the small subunit rRNA gene of N. bombycis is on a single chromosome with a length of 760 kb (Kawakami et al., 1994). Later the novel arrangement of the rRNA genes in N. bombycis, i.e. LSU rRNA followed by ITS and then SSU rRNA, IGS and 5S rRNA was discovered (Wang et al., 2006; Huang et al., 2004). This arrangement is the reverse to that of Nosema ceranae and eukaryotes in general (Huang et al., 2008). Also the SSU rRNA gene was found to contain insertion sequences (Iiyama et al., 2004), which indicates that rRNA genes can be interrupted by some repeat elements. In this study, the karyotype of N. bombycis was reassessed by PFGE. 18 chromosomes were obtained, with a total size of approx. 15,330 kb, which coincides with previous findings (Kawakami et al., 1994). Using a rDNA sequence from GenBank (Huang et al., 2004) as the query sequence, we screened our genomic database for LSU rRNA, SSU rRNA and 5S rRNA genes, respectively. Southern blotting was performed to determine the distribution of the rRNA genes among the 18 chromosomes of N. bombycis. In short, we discovered that multiple ribosomal RNA genes are distributed on all chromosomes. 2. Materials and methods 2.1. Genome-wide survey Using the program RepeatMasker, and a rDNA sequence from GenBank (GenBank Accession No. AY259631, Huang et al., 2004) as the query, we screened our database for LSU rRNA, SSU rRNA and 5S rRNA elements, respectively (Xu et al., 2006). A script of perl
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programmer (drawContigStructure) was used to locate each element on contigs.
3. Results 3.1. Genome-wide survey of rDNA sequence in N. bombycis
2.2. Spore production and purification Nosema bombycis isolate CQ1, isolated from infected silkworms in Chongqing, China, is preserved in China Veterinary Culture Collection Center (CVCC No. 102059). The third instar molted silkworm larvae were challenged by feeding on mulberry leaves artificially contaminated by N. bombycis spores (about 2.0 105 spores for one silkworm, Wu et al., 2007). The silk gland of the infected larvae was dissected at day 4 or 5 of the fifth instar, and homogenized and centrifuged further. Spores were purified by a discontinuous Percoll gradient (25%, 50%, 75%, and 100%, v/v) and centrifuged at 30,000g for 40 min. The pellets of mature spores were rinsed and stored as pellets at 4 °C. 2.3. Preparation of probes Nosema bombycis gDNA (3 lg) was extracted by the CTAB Method (Li and Wang, 2006). Three pairs of primers corresponding to three rDNA fragments (SSU, ITS and IGS sequences) were designed and synthesized according to Huang et al. (2004). The primer sequences are as follows: SSU: ILSUF: 50 -TGGGTTTAGACCGTCGTG AG-30 , S33R: 50 -ATAGCGTCTACGTCAGGCAG-30 ; ITS: HG4F: 50 -GCGG CTTAATTTGACTCAAC-30 , 5SR: 50 -TACAGCACCCAACGTTCCCAAG-30 ; IGS: 18f: 50 -CACCAGGTTGATTCTGCC-30 , 1537r: 50 -TTATGATCCTGCT AATGGTTC-30 . Probes were obtained by PCR amplification, with the length of 1232 bp, 501 bp, 852 bp, respectively, and they are located at the end of nucleotides 2677–3908, 2209–2709, 3450– 4301 from the 50 end of rRNA gene, respectively, according to Huang et al. (2004). The composition of the PCR reactions: 2.5 ll 10 PCR reaction buffer; 1 ll 25 mmol/L MgCl2; 1 ll 10 mmol/L dNTP; 2 ll the forward and reverse primers (10 lmol/L), respectively; 1 ll template of total genome of N. bombycis; 0.5 ll DNA polymerase; add ddH2O to the total volume 25 ll. The products were amplified using the following conditions: an initial DNA denaturation step at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing (ITS at 49 °C; SSU at 56 °C; IGS at 49 °C) for 1 min, and elongation at 72 °C for 2 min. The last step was a final 10 min extension at 72 °C. Then the PCR products were purified using the DNA Fragment Quick Purification/Recover Kit (Catalog No. A014-1, DingGuo). The recovered DNA fragments were quantified by GeneQuant pro, and then labeled and detected using the DIG DNA Labeling and Detection Kit (Catalog No. 11093657910, Roche) according to the manufacturer’s protocol. 2.4. PFGE and Southern blotting The preparation of plugs for PFGE analyses was performed as follows: purified spores were inserted in 1.4% low melting point agarose gel, then the solidified agarose plugs were incubated in solution I (EDTA, 0.1 M; SDS, 5%; DTT, 0.1 M) at 50 °C for 2 h and before treatment with solution II (EDTA, 0.5 M; SLS, 0.5%; Proteinase K, 1.5 mg/mL) at 50 °C for 48 h. PFGE was run under the following conditions: 0.5 TBE buffer (45 mM Tris base, 45 mM boric acid, 1 mM EDTA), 12 °C, 5 V/cm, pulse time 60, 65, 70, 75, 80, 90 s, with a total run time of 60 h. The bands representing the segregated chromosomes were vacuum-transferred to a nylon membrane after UV pre-treatment. Finally according to the manufacturer’s instructions offered by DIG DNA Labeling and Detection Kit, Southern blotting was carried out by adding probes, which were prepared in advance (ITS, SSU, or IGS probe; 25 ng/mL), to the nylon membrane on which the chromosomes were transferred before.
Currently, the N. bombycis genome is being sequenced in our lab, and 200,000 shotguns reads representing the draft genome have been obtained (Xu et al., 2006). Now we are drawing the genomic fine map of N. bombycis. We surveyed the draft genome with SSUrDNA, LSUrDNA and 5SrDNA sequences using the RepeatMasker program. The copy numbers of these three elements were found to be 43, 53, 36, respectively. At this stage of assembly, there are 86 contigs which harbor rRNA genes, and only nine of them contain the complete rRNA gene. The average size of these contigs is 6.15 kb, and the longest contig is 31.59 kb. The distribution of the rRNA genes among the contigs is displayed in Fig. 1. The majority of the rRNA genes (about 72.1%) lie at the ends of the contigs (Fig. 1A–D), but rDNA sequences located in the middle of contigs were also found (Fig. 1E). Incomplete sequences of each element were also discovered, and several units were also lost, resulting in partial rDNA, such as the loss of the complete sequence of LSU (Fig. 1D) and the loss of the partial sequence of LSU (Fig. 1E). We also found fragmented SSU rDNAs, which were interrupted by insertion sequences ( Fig. 1B), a result that has been previously documented (Iiyama et al., 2004). Multiple sequence alignments for each rRNA gene were obtained using CLUSTALX (Thompson et al., 1997). Single nucleotide polymorphisms (SNPs) were found among the sequences. (The percentage similarity found between each pair of sequences in the alignment are all no less than 99%.) 3.2. Distribution of rDNA units among chromosomes Through PFGE, we found that the N. bombycis karyotype was comprised of approximately 18 chromosomes, which coincides with the previous result (Kawakami et al., 1994). After transferring chromosomes of N. bombycis to a nylon membrane, we performed Southern blotting with the SSU, IGS, ITS rDNA probe, respectively. It had been detected that the binding of these probes was specific to N. bombycis rDNA by using the total genome of the domesticated silkworm Dazao (Bombyx mori) as a control. Hybridization signals corresponding to all bands were detected, and each of the three probes gave nearly the same amount of visible signal on all chromosomes (Fig. 2). These results show that rDNA units reside on all chromosomes of N. bombycis. 4. Discussion Through this study, we obtained a PFGE pattern of 18 N. bombycis chromosomes which correlates well with the previous result (Kawakami et al., 1994). Using the RepeatMasker program, we searched our N. bombycis database for SSU rDNA, LSU rDNA and 5S rDNA sequences, respectively. Some mobile elements have been identified in the N. bombycis genome and can lead to more complicated chromosome rearrangements (Xu et al., 2006), while they probably do not exist inE. cuniculi. Those mobile elements may be the reason that some fragmented rDNA units, which contain insertion sequences, arise in N. bombycis genome. In the present genome data of N. bombycis, the contigs which contain rDNA units are still too small. The more clear localization of rDNA of N. bombycis will be given in the future. We performed Southern blotting with three probes, which were rDNA fragments, and obtained the same results, i.e. each of these three sequences lies on all chromosomes of N. bombycis. So we can conclude that the rDNA sequence of N. bombycis is distributed among all chromosomes. This result differs to the conclusion of
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Fig. 1. rDNA location on contigs of N. bombycis. Five contigs of N. bombycis display the location of rDNA and CDSs (coding sequence). The CDSs are indicated with red (forward) and blue (reverse) arrows. rDNA sequences are indicated with green arrows. (A) The complete sequences of LSU, ITS, SSU and IGS, but 5S is a partial sequence, (B) An insertion sequence (yellow) in SSU and ITS, respectively. The rDNA of A, B, C and D lies on the terminal of each contig, Contig D contains an rDNA unit missing the LSU, (E) rDNA lies in the middle of this contig and an incomplete LSU rDNA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Southern blotting results performed with IGS, ITS and SSU probes. (a) IGS probe; (b) ITS probe; (c) SSU probe. (1 and 2) The images of Southern blotting; (3 and 4) the electropherogram segregated by PFGE.I ? XVIII. The chromosomes bands from small to large segregated by PFGE of N. bombycis.
Kawakami (1994). By searching in our database with the probe sequence used by Kawakami (1994), we found that it was actually a pseudo ribosomal RNA gene sequence, only one copy of which is present in the N. bombycis genome (located on contig0213, GenBank Accession No. EU616620). This pseudogene is a noncoding sequence. We conclude that the rDNA unit exists as multiple copies and resides on each chromosome of N. bombycis. Using rDNA elements as probes, the rDNA of several Microsporidia have been located (Peyretaillade et al., 1998; Biderre et al., 1994). There are at least
two rRNA copies existing in a tandem arrangement in the microsporidian Nosema apis (Gatehouse and Malone, 1998), and a new discovery that two distinct rRNA gene copies exist in one spore of N. bombi has been reported (O’Mahony et al., 2007). So, it may be that rRNA gene exists as multiple copies in all Microsporidia (Brugère et al., 2000; Gatehouse and Malone, 1998; O’Mahony et al., 2007). In order to get a more accurate position for the rDNA loci, we need to conduct further experiments such as fluorescence in situ hybridization.
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Acknowledgments The authors thank Dr. James John Becnel, the Associate Editor, and two anonymous reviewers for their helpful comments and suggestions, which have greatly improved the presentation of this manuscript. This research was funded by the grants from National Natural Science Foundation of China Key Project (No. 30771173), National Basic Research Program of China under Grant (No. 2005CB121000), State Development Fund at Risk of Callus Silk (No. M012005-000Y-00070) and Chongqing key scientific and technological Break-through Project (No. 2006AA5019). References Amigó, J.M., Gracia, M.P., Salvadó, H., Vivarés, C.P., 2002. Pulsed field gel electrophoresis of three microsporidian parasites of fish. Acta Protozool. 41, 11–16. Biderre, C., Pagès, M., Méténier, G., David, D., Bata, J., 1994. On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. C. R. Acad. Sci. III 317, 399–404. Brugère, J.F., Cornillot, E., Méténier, G., Bensimon, A., Vivarès, C.P., 2000. Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes. Nucleic Acids Res. 28 (10), 2026–2033. Gao, H., Mu, Z., Wang, Y., Zhang, S., 2001. The research progression of Nosema bombycis. North Sericult. 22 (4), 8–10. Gatehouse, H.S., Malone, L.A., 1998. The ribosomal RNA gene region of Nosema apis (Microspora): DNA sequence for small and large subunit rRNA genes and evidence of a large tandem repeat unit size. J. Invertebr. Pathol. 71, 97–105. Huang, W., Bocquet, M., Lee, K., Sung, I., Jiang, J., Chen, Y., Wang, C., 2008. The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations. J. Invertebr. Pathol. 97, 9–13. Huang, W., Tsai, S., Lo, C., Soichi, Y., Wang, C., 2004. The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis. Fungal Genet. Biol. 41, 473–481.
Iiyama, K., Chieda, Y., Yasunaga-Aoki, C., Hayasaka, S., Shimizu, S., 2004. Analyses of the ribosomal DNA region in Nosema bombycis NIS 001. J. Eukaryot. Microbiol. 51 (6), 598–604. Kawakami, Y., Inoue, T., Ito, K., Kitamizu, K., Hanawa, C., Ando, T., Iwano, H., Ishihara, R., 1994. Identification of a chromosome harboring the small subunit ribosome RNA gene of Nosema bombycis. J. Invertebr. Pathol. 64, 147–148. Keeling, P.J., Fast, N.M., 2002. Microsporidia: biology and evolution of highly reduced intracellular parasites. Annu. Rev. Microbiol. 56, 93–116. Li, F., Wang, Y., 2006. The Comparison of DNA Extraction Methods of Microsporida. Guangdong Sericult. 40 (3), 32–34. Méténier, G., Vivarès, C.P., 2001. Molecular characteristics and physiology of microsporidia. Microbes Infect. 3, 407–415. Munderloh, U.G., Kurtti, T.J., Ross, S.E., 1990. Electrophoretic characterization of chromosomal DNA from two microsporidia. J. Invertebr. Pathol. 56 (2), 243– 248. O’Mahony, E.M., Tay, W.T., Paxton, R.J., 2007. Multiple rRNA variants in a single spore of the microsporidian Nosema bombi. J. Eukaryot. Microbiol. 54 (1), 103–109. Peyretaillade, E., Biderre, C., Peyret, P., 1998. Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSUrRNA reduced to the universal core. Nucleic Acids Res. 26 (15), 3513–3520. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876– 4882. Vossbrink, C.R., Woese, C.R., 1986. Eukaryotic ribosomes that lacks a 5.8S RNA. Nature 320 (6059), 287–288. Wang, L., Chen, K., Yao, Q., Gao, G., Zhao, Y., 2006. The sequence and phylogenetic analysis of the ribosomal RNA gene and the ITS region of Nosema antheraeae. Sci. Agric. Sin. 39 (8), 1674–1679. Weiss, L.M., Vossbrinck, C.R., 1998. Microsporidiosis: molecular and diagnostic aspects. J. Adv. Parasitol. 40, 351–395. Wu, Z., Tan, X., Pan, G., Li, Y., Zhou, Z., 2007. Extraction of spore wall proteins of Nosema bombycis with improved methods. Sci. Sericult. 33 (1), 62–66. Xu, J., Pan, G., Fan, L., Li, J., Tian, X., Li, T., Zhou, Z., Xiang, Z., 2006. The varying microsporidian genome: existence of LTR retrotransposon in domesticated silkworm parasite Nosema bombycis. Int. J. Parasitol. 36, 1049–1056.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Short Communication
Multiple rDNA units distributed on all chromosomes of Nosema bombycis Handeng Liu a, Guoqing Pan a, Shihong Song a, Jinshan Xu b, Tian Li a, Yanbo Deng c, Zeyang Zhou a,b,* a b c
The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing 400716, China Laboratory of Animal Biology Chongqing Normal University, Chongqing 400047, China School of Life Science & Technology, Beijing Institute of Technology, Beijing 100081, China
a r t i c l e
i n f o
Article history: Received 21 January 2008 Accepted 12 June 2008 Available online 1 July 2008 Keywords: Nosema bombycis rDNA Genomic survey Southern blotting Distribution
a b s t r a c t Among Microsporidia, Nosema bombycis has a novel arrangement of LSUrRNA, SSUrRNA, ITS, IGS and 5SrRNA. To determine the distribution of rDNA among the chromosomes, we performed genome-wide screening and Southern blotting with three probes (SSU, ITS and IGS). Southern blotting revealed that ribosomal RNA genes are distributed on all chromosomes of N. bombycis, which is contrary to the previous result, which concluded that the N. bombycis rRNA genes were limited to a single chromosome. This wide distribution is similar to that of the rDNA unit of Encephalitozoon cuniculi. Screening of the N. bombycis genome detected 53 LSUrRNA elements, 43 SSUrRNA elements and 36 5SrRNA elements. However, it is still difficult to determine their loci on the chromosomes as the genomic map is unfinished. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Microsporidian genomes have aroused interest since they were first karyotyped and found to be much smaller than expected for eukaryotes. Genome size in the Microsporidia ranges between 2.3 and 19.5 Mbp (Biderre et al., 1994; Méténier and Vivarès, 2001; Keeling and Fast, 2002). Current knowledge of genome organization in this group mainly derives from karyotyping studies based on pulsed field gel electrophoresis (PFGE) techniques, which revealed extensive inter-species variation in number and size of chromosomes (Munderloh et al., 1990). These findings have helped to illustrate genetic relationships among species at the subcellular level and have provided evidence for phylogenetics and species identification (Amigó et al., 2002). Nosema bombycis is an obligate intracellular parasite of the domesticated silkworm, and as the causative agent of pebrine disease, is still prevalent in sericulture (Weiss et al., 1998; Gao et al., 2001). Through PFGE, the number of chromosomes in N. bombycis was estimated at 18, ranging from approx. 380 to 1500 kbp, with a total size of approx. 15,330 kb (Kawakami et al., 1994). Nosema bombycis lacks the 5.8S rRNA gene, which is similar to other species in the group (Vossbrink and Woese, 1986). The location of the rRNA genes of several other microsporidians have been reported (Biderre et al., 1994; Peyretaillade et al., 1998). Using rDNA as a probe, the rDNA units of Encephalitozoon cuniculi were
* Corresponding author. Address: The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing 400716, China. Fax: +86 23 68251128. E-mail address: [email protected] (Z. Zhou). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.06.012
shown to be distributed on each chromosome (Brugère et al., 2000). Previous studies have reported that the small subunit rRNA gene of N. bombycis is on a single chromosome with a length of 760 kb (Kawakami et al., 1994). Later the novel arrangement of the rRNA genes in N. bombycis, i.e. LSU rRNA followed by ITS and then SSU rRNA, IGS and 5S rRNA was discovered (Wang et al., 2006; Huang et al., 2004). This arrangement is the reverse to that of Nosema ceranae and eukaryotes in general (Huang et al., 2008). Also the SSU rRNA gene was found to contain insertion sequences (Iiyama et al., 2004), which indicates that rRNA genes can be interrupted by some repeat elements. In this study, the karyotype of N. bombycis was reassessed by PFGE. 18 chromosomes were obtained, with a total size of approx. 15,330 kb, which coincides with previous findings (Kawakami et al., 1994). Using a rDNA sequence from GenBank (Huang et al., 2004) as the query sequence, we screened our genomic database for LSU rRNA, SSU rRNA and 5S rRNA genes, respectively. Southern blotting was performed to determine the distribution of the rRNA genes among the 18 chromosomes of N. bombycis. In short, we discovered that multiple ribosomal RNA genes are distributed on all chromosomes. 2. Materials and methods 2.1. Genome-wide survey Using the program RepeatMasker, and a rDNA sequence from GenBank (GenBank Accession No. AY259631, Huang et al., 2004) as the query, we screened our database for LSU rRNA, SSU rRNA and 5S rRNA elements, respectively (Xu et al., 2006). A script of perl
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programmer (drawContigStructure) was used to locate each element on contigs.
3. Results 3.1. Genome-wide survey of rDNA sequence in N. bombycis
2.2. Spore production and purification Nosema bombycis isolate CQ1, isolated from infected silkworms in Chongqing, China, is preserved in China Veterinary Culture Collection Center (CVCC No. 102059). The third instar molted silkworm larvae were challenged by feeding on mulberry leaves artificially contaminated by N. bombycis spores (about 2.0 105 spores for one silkworm, Wu et al., 2007). The silk gland of the infected larvae was dissected at day 4 or 5 of the fifth instar, and homogenized and centrifuged further. Spores were purified by a discontinuous Percoll gradient (25%, 50%, 75%, and 100%, v/v) and centrifuged at 30,000g for 40 min. The pellets of mature spores were rinsed and stored as pellets at 4 °C. 2.3. Preparation of probes Nosema bombycis gDNA (3 lg) was extracted by the CTAB Method (Li and Wang, 2006). Three pairs of primers corresponding to three rDNA fragments (SSU, ITS and IGS sequences) were designed and synthesized according to Huang et al. (2004). The primer sequences are as follows: SSU: ILSUF: 50 -TGGGTTTAGACCGTCGTG AG-30 , S33R: 50 -ATAGCGTCTACGTCAGGCAG-30 ; ITS: HG4F: 50 -GCGG CTTAATTTGACTCAAC-30 , 5SR: 50 -TACAGCACCCAACGTTCCCAAG-30 ; IGS: 18f: 50 -CACCAGGTTGATTCTGCC-30 , 1537r: 50 -TTATGATCCTGCT AATGGTTC-30 . Probes were obtained by PCR amplification, with the length of 1232 bp, 501 bp, 852 bp, respectively, and they are located at the end of nucleotides 2677–3908, 2209–2709, 3450– 4301 from the 50 end of rRNA gene, respectively, according to Huang et al. (2004). The composition of the PCR reactions: 2.5 ll 10 PCR reaction buffer; 1 ll 25 mmol/L MgCl2; 1 ll 10 mmol/L dNTP; 2 ll the forward and reverse primers (10 lmol/L), respectively; 1 ll template of total genome of N. bombycis; 0.5 ll DNA polymerase; add ddH2O to the total volume 25 ll. The products were amplified using the following conditions: an initial DNA denaturation step at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing (ITS at 49 °C; SSU at 56 °C; IGS at 49 °C) for 1 min, and elongation at 72 °C for 2 min. The last step was a final 10 min extension at 72 °C. Then the PCR products were purified using the DNA Fragment Quick Purification/Recover Kit (Catalog No. A014-1, DingGuo). The recovered DNA fragments were quantified by GeneQuant pro, and then labeled and detected using the DIG DNA Labeling and Detection Kit (Catalog No. 11093657910, Roche) according to the manufacturer’s protocol. 2.4. PFGE and Southern blotting The preparation of plugs for PFGE analyses was performed as follows: purified spores were inserted in 1.4% low melting point agarose gel, then the solidified agarose plugs were incubated in solution I (EDTA, 0.1 M; SDS, 5%; DTT, 0.1 M) at 50 °C for 2 h and before treatment with solution II (EDTA, 0.5 M; SLS, 0.5%; Proteinase K, 1.5 mg/mL) at 50 °C for 48 h. PFGE was run under the following conditions: 0.5 TBE buffer (45 mM Tris base, 45 mM boric acid, 1 mM EDTA), 12 °C, 5 V/cm, pulse time 60, 65, 70, 75, 80, 90 s, with a total run time of 60 h. The bands representing the segregated chromosomes were vacuum-transferred to a nylon membrane after UV pre-treatment. Finally according to the manufacturer’s instructions offered by DIG DNA Labeling and Detection Kit, Southern blotting was carried out by adding probes, which were prepared in advance (ITS, SSU, or IGS probe; 25 ng/mL), to the nylon membrane on which the chromosomes were transferred before.
Currently, the N. bombycis genome is being sequenced in our lab, and 200,000 shotguns reads representing the draft genome have been obtained (Xu et al., 2006). Now we are drawing the genomic fine map of N. bombycis. We surveyed the draft genome with SSUrDNA, LSUrDNA and 5SrDNA sequences using the RepeatMasker program. The copy numbers of these three elements were found to be 43, 53, 36, respectively. At this stage of assembly, there are 86 contigs which harbor rRNA genes, and only nine of them contain the complete rRNA gene. The average size of these contigs is 6.15 kb, and the longest contig is 31.59 kb. The distribution of the rRNA genes among the contigs is displayed in Fig. 1. The majority of the rRNA genes (about 72.1%) lie at the ends of the contigs (Fig. 1A–D), but rDNA sequences located in the middle of contigs were also found (Fig. 1E). Incomplete sequences of each element were also discovered, and several units were also lost, resulting in partial rDNA, such as the loss of the complete sequence of LSU (Fig. 1D) and the loss of the partial sequence of LSU (Fig. 1E). We also found fragmented SSU rDNAs, which were interrupted by insertion sequences ( Fig. 1B), a result that has been previously documented (Iiyama et al., 2004). Multiple sequence alignments for each rRNA gene were obtained using CLUSTALX (Thompson et al., 1997). Single nucleotide polymorphisms (SNPs) were found among the sequences. (The percentage similarity found between each pair of sequences in the alignment are all no less than 99%.) 3.2. Distribution of rDNA units among chromosomes Through PFGE, we found that the N. bombycis karyotype was comprised of approximately 18 chromosomes, which coincides with the previous result (Kawakami et al., 1994). After transferring chromosomes of N. bombycis to a nylon membrane, we performed Southern blotting with the SSU, IGS, ITS rDNA probe, respectively. It had been detected that the binding of these probes was specific to N. bombycis rDNA by using the total genome of the domesticated silkworm Dazao (Bombyx mori) as a control. Hybridization signals corresponding to all bands were detected, and each of the three probes gave nearly the same amount of visible signal on all chromosomes (Fig. 2). These results show that rDNA units reside on all chromosomes of N. bombycis. 4. Discussion Through this study, we obtained a PFGE pattern of 18 N. bombycis chromosomes which correlates well with the previous result (Kawakami et al., 1994). Using the RepeatMasker program, we searched our N. bombycis database for SSU rDNA, LSU rDNA and 5S rDNA sequences, respectively. Some mobile elements have been identified in the N. bombycis genome and can lead to more complicated chromosome rearrangements (Xu et al., 2006), while they probably do not exist inE. cuniculi. Those mobile elements may be the reason that some fragmented rDNA units, which contain insertion sequences, arise in N. bombycis genome. In the present genome data of N. bombycis, the contigs which contain rDNA units are still too small. The more clear localization of rDNA of N. bombycis will be given in the future. We performed Southern blotting with three probes, which were rDNA fragments, and obtained the same results, i.e. each of these three sequences lies on all chromosomes of N. bombycis. So we can conclude that the rDNA sequence of N. bombycis is distributed among all chromosomes. This result differs to the conclusion of
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Fig. 1. rDNA location on contigs of N. bombycis. Five contigs of N. bombycis display the location of rDNA and CDSs (coding sequence). The CDSs are indicated with red (forward) and blue (reverse) arrows. rDNA sequences are indicated with green arrows. (A) The complete sequences of LSU, ITS, SSU and IGS, but 5S is a partial sequence, (B) An insertion sequence (yellow) in SSU and ITS, respectively. The rDNA of A, B, C and D lies on the terminal of each contig, Contig D contains an rDNA unit missing the LSU, (E) rDNA lies in the middle of this contig and an incomplete LSU rDNA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Southern blotting results performed with IGS, ITS and SSU probes. (a) IGS probe; (b) ITS probe; (c) SSU probe. (1 and 2) The images of Southern blotting; (3 and 4) the electropherogram segregated by PFGE.I ? XVIII. The chromosomes bands from small to large segregated by PFGE of N. bombycis.
Kawakami (1994). By searching in our database with the probe sequence used by Kawakami (1994), we found that it was actually a pseudo ribosomal RNA gene sequence, only one copy of which is present in the N. bombycis genome (located on contig0213, GenBank Accession No. EU616620). This pseudogene is a noncoding sequence. We conclude that the rDNA unit exists as multiple copies and resides on each chromosome of N. bombycis. Using rDNA elements as probes, the rDNA of several Microsporidia have been located (Peyretaillade et al., 1998; Biderre et al., 1994). There are at least
two rRNA copies existing in a tandem arrangement in the microsporidian Nosema apis (Gatehouse and Malone, 1998), and a new discovery that two distinct rRNA gene copies exist in one spore of N. bombi has been reported (O’Mahony et al., 2007). So, it may be that rRNA gene exists as multiple copies in all Microsporidia (Brugère et al., 2000; Gatehouse and Malone, 1998; O’Mahony et al., 2007). In order to get a more accurate position for the rDNA loci, we need to conduct further experiments such as fluorescence in situ hybridization.
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Acknowledgments The authors thank Dr. James John Becnel, the Associate Editor, and two anonymous reviewers for their helpful comments and suggestions, which have greatly improved the presentation of this manuscript. This research was funded by the grants from National Natural Science Foundation of China Key Project (No. 30771173), National Basic Research Program of China under Grant (No. 2005CB121000), State Development Fund at Risk of Callus Silk (No. M012005-000Y-00070) and Chongqing key scientific and technological Break-through Project (No. 2006AA5019). References Amigó, J.M., Gracia, M.P., Salvadó, H., Vivarés, C.P., 2002. Pulsed field gel electrophoresis of three microsporidian parasites of fish. Acta Protozool. 41, 11–16. Biderre, C., Pagès, M., Méténier, G., David, D., Bata, J., 1994. On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. C. R. Acad. Sci. III 317, 399–404. Brugère, J.F., Cornillot, E., Méténier, G., Bensimon, A., Vivarès, C.P., 2000. Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes. Nucleic Acids Res. 28 (10), 2026–2033. Gao, H., Mu, Z., Wang, Y., Zhang, S., 2001. The research progression of Nosema bombycis. North Sericult. 22 (4), 8–10. Gatehouse, H.S., Malone, L.A., 1998. The ribosomal RNA gene region of Nosema apis (Microspora): DNA sequence for small and large subunit rRNA genes and evidence of a large tandem repeat unit size. J. Invertebr. Pathol. 71, 97–105. Huang, W., Bocquet, M., Lee, K., Sung, I., Jiang, J., Chen, Y., Wang, C., 2008. The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations. J. Invertebr. Pathol. 97, 9–13. Huang, W., Tsai, S., Lo, C., Soichi, Y., Wang, C., 2004. The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis. Fungal Genet. Biol. 41, 473–481.
Iiyama, K., Chieda, Y., Yasunaga-Aoki, C., Hayasaka, S., Shimizu, S., 2004. Analyses of the ribosomal DNA region in Nosema bombycis NIS 001. J. Eukaryot. Microbiol. 51 (6), 598–604. Kawakami, Y., Inoue, T., Ito, K., Kitamizu, K., Hanawa, C., Ando, T., Iwano, H., Ishihara, R., 1994. Identification of a chromosome harboring the small subunit ribosome RNA gene of Nosema bombycis. J. Invertebr. Pathol. 64, 147–148. Keeling, P.J., Fast, N.M., 2002. Microsporidia: biology and evolution of highly reduced intracellular parasites. Annu. Rev. Microbiol. 56, 93–116. Li, F., Wang, Y., 2006. The Comparison of DNA Extraction Methods of Microsporida. Guangdong Sericult. 40 (3), 32–34. Méténier, G., Vivarès, C.P., 2001. Molecular characteristics and physiology of microsporidia. Microbes Infect. 3, 407–415. Munderloh, U.G., Kurtti, T.J., Ross, S.E., 1990. Electrophoretic characterization of chromosomal DNA from two microsporidia. J. Invertebr. Pathol. 56 (2), 243– 248. O’Mahony, E.M., Tay, W.T., Paxton, R.J., 2007. Multiple rRNA variants in a single spore of the microsporidian Nosema bombi. J. Eukaryot. Microbiol. 54 (1), 103–109. Peyretaillade, E., Biderre, C., Peyret, P., 1998. Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSUrRNA reduced to the universal core. Nucleic Acids Res. 26 (15), 3513–3520. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876– 4882. Vossbrink, C.R., Woese, C.R., 1986. Eukaryotic ribosomes that lacks a 5.8S RNA. Nature 320 (6059), 287–288. Wang, L., Chen, K., Yao, Q., Gao, G., Zhao, Y., 2006. The sequence and phylogenetic analysis of the ribosomal RNA gene and the ITS region of Nosema antheraeae. Sci. Agric. Sin. 39 (8), 1674–1679. Weiss, L.M., Vossbrinck, C.R., 1998. Microsporidiosis: molecular and diagnostic aspects. J. Adv. Parasitol. 40, 351–395. Wu, Z., Tan, X., Pan, G., Li, Y., Zhou, Z., 2007. Extraction of spore wall proteins of Nosema bombycis with improved methods. Sci. Sericult. 33 (1), 62–66. Xu, J., Pan, G., Fan, L., Li, J., Tian, X., Li, T., Zhou, Z., Xiang, Z., 2006. The varying microsporidian genome: existence of LTR retrotransposon in domesticated silkworm parasite Nosema bombycis. Int. J. Parasitol. 36, 1049–1056.