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Internal transcribed spacer

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Internal transcribed spacer (ITS) is the spacer DNA situated between the small-subunit ribosomal RNA (rRNA) and large-subunit rRNA genes in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript.

Across life domains

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In bacteria and archaea, there is a single ITS, located between the 16S and 23S rRNA genes. Conversely, there are two ITSs in eukaryotes: ITS1 is located between 18S and 5.8S rRNA genes, while ITS2 is between 5.8S and 28S (in opisthokonts, or 25S in plants) rRNA genes. ITS1 corresponds to the ITS in bacteria and archaea, while ITS2 originated as an insertion that interrupted the ancestral 23S rRNA gene.[1][2]

Organization

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Organization of the eukaryotic nuclear ribosomal DNA tandem repeats

In bacteria and archaea, the ITS occurs in one to several copies, as do the flanking 16S and 23S genes. When there are multiple copies, these do not occur adjacent to one another. Rather, they occur in discrete locations in the circular chromosome. It is not uncommon in bacteria to carry tRNA genes in the ITS.[3][4]

In eukaryotes, genes encoding ribosomal RNA and spacers occur in tandem repeats that are thousands of copies long, each separated by regions of non-transcribed DNA termed intergenic spacer (IGS) or non-transcribed spacer (NTS).

Each eukaryotic ribosomal cluster contains the 5' external transcribed spacer (5' ETS), the 18S rRNA gene, the ITS1, the 5.8S rRNA gene, the ITS2, the 26S or 28S rRNA gene, and finally the 3' ETS.[5]

During rRNA maturation, ETS and ITS pieces are excised. As non-functional by-products of this maturation, they are rapidly degraded.[6]

Use in phylogenetic inference

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Sequence comparison of the eukaryotic ITS regions is widely used in taxonomy and molecular phylogeny because of several favorable properties:[7]

  • It is routinely amplified thanks to its small size associated to the availability of highly conserved flanking sequences.
  • It is easy to detect even from small quantities of DNA due to the high copy number of the rRNA clusters.
  • It undergoes rapid concerted evolution via unequal crossing-over and gene conversion. This promotes intra-genomic homogeneity of the repeat units, although high-throughput sequencing showed the occurrence of frequent variations within plant species.[8]
  • It has a high degree of variation even between closely related species. This can be explained by the relatively low evolutionary pressure acting on such non-coding spacer sequences.

For example, ITS markers have proven especially useful for elucidating phylogenetic relationships among the following taxa.

Taxonomic group Taxonomic level Year Authors with references
Asteraceae: Compositae Species (congeneric) 1992 Baldwin et al.[9]
Viscaceae: Arceuthobium Species (congeneric) 1994 Nickrent et al.[10]
Poaceae: Zea Species (congeneric) 1996 Buckler & Holtsford[11]
Leguminosae: Medicago Species (congeneric) 1998 Bena et al.[5]
Orchidaceae: Diseae Genera (within tribes) 1999 Douzery et al.[12]
Odonata: Calopteryx Species (congeneric) 2001 Weekers et al.[13]
Yeasts of clinical importance Genera 2001 Chen et al.[14]
Poaceae: Saccharinae Genera (within tribes) 2002 Hodkinson et al.[15]
Plantaginaceae: Plantago Species (congeneric) 2002 Rønsted et al.[16]
Jungermanniopsida: Herbertus Species (congeneric) 2004 Feldberg et al.[17]
Pinaceae: Tsuga Species (congeneric) 2008 Havill et al.[18]
Chrysomelidae: Altica Genera (congeneric) 2009 Ruhl et al.[19]
Symbiodinium Clade 2009 Stat et al.[20]
Brassicaceae Tribes (within a family) 2010 Warwick et al.[21]
Ericaceae: Erica Species (congeneric) 2011 Pirie et al.[22]
Diptera: Bactrocera Species (congeneric) 2014 Boykin et al.[23]
Scrophulariaceae: Scrophularia Species (congeneric) 2014 Scheunert & Heubl[24]
Potamogetonaceae: Potamogeton Species (congeneric) 2016 Yang et al.[25]

ITS2 is known to be more conserved than ITS1 is. All ITS2 sequences share a common core of secondary structure,[26] while ITS1 structures are only conserved in much smaller taxonomic units. Regardless of the scope of conservation, structure-assisted comparison can provide higher resolution and robustness.[27]

Mycological barcoding

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Main article: Fungal DNA barcoding

The ITS region is the most widely sequenced DNA region in molecular ecology of fungi[28] and has been recommended as the universal fungal barcode sequence.[29] It has typically been most useful for molecular systematics at the species to genus level, and even within species (e.g., to identify geographic races). Because of its higher degree of variation than other genic regions of rDNA (for example, small- and large-subunit rRNA), variation among individual rDNA repeats can sometimes be observed within both the ITS and IGS regions. In addition to the universal ITS1+ITS4 primers[30][31] used by many labs, several taxon-specific primers have been described that allow selective amplification of fungal sequences (e.g., see Gardes & Bruns 1993 paper describing amplification of basidiomycete ITS sequences from mycorrhiza samples).[32] Despite shotgun sequencing methods becoming increasingly utilized in microbial sequencing, the low biomass of fungi in clinical samples make the ITS region amplification an area of ongoing research.[33][34]

References

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  1. ^ Lafontaine, D. L. J.; Tollervey, D. (2001). "The function and synthesis of ribosomes". Nature Reviews Molecular Cell Biology. 2 (7): 514–520. doi:10.1038/35080045. hdl:1842/729. PMID 11433365. S2CID 2637106.
  2. ^ Scott Orland Rogers (27 July 2011). Integrated Molecular Evolution. CRC Press. pp. 65–66. ISBN 978-1-4398-1995-1. Retrieved 9 March 2015.
  3. ^ Takada, Hiraku; Shimada, Tomohiro; Dey, Debashish; Quyyum, M. Zuhaib; Nakano, Masahiro; Ishiguro, Akira; Yoshida, Hideji; Yamamoto, Kaneyoshi; Sen, Ranjan; Ishihama, Akira (22 December 2016). "Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli". PLOS ONE. 11 (12): e0163057. Bibcode:2016PLoSO..1163057T. doi:10.1371/journal.pone.0163057. PMC 5179076. PMID 28005933.
  4. ^ Stewart, Frank J.; Cavanaugh, Colleen M. (July 2007). "Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria". Journal of Molecular Evolution. 65 (1): 44–67. Bibcode:2007JMolE..65...44S. CiteSeerX 10.1.1.456.2659. doi:10.1007/s00239-006-0235-3. PMID 17568983. S2CID 13536182.
  5. ^ a b Bena, Gilles; Jubier, Marie-France; Olivieri, Isabelle; Lejeune, Bernard (1998). "Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae)". Journal of Molecular Evolution. 46 (3): 299–306. Bibcode:1998JMolE..46..299B. doi:10.1007/PL00006306. ISSN 0022-2844. PMID 9502673. S2CID 38838013.
  6. ^ Michot, Bernard; Bachellerie, Jean-Pierre; Raynal, Francoise (1983-05-25). "Structure of mouse rRNA precursors. Complete sequence and potential folding of the spacer regions between 18S and 28S rRNA". Nucleic Acids Research. 11 (10): 3375–3391. doi:10.1093/nar/11.10.3375. ISSN 0305-1048. PMC 325970. PMID 6304630.
  7. ^ Baldwin, Bruce G.; Sanderson, Michael J.; Porter, J. Mark; Wojciechowski, Martin F.; Campbell, Christopher S.; Donoghue, Michael J. (1995-01-01). "The ITS Region of Nuclear Ribosomal DNA: A Valuable Source of Evidence on Angiosperm Phylogeny". Annals of the Missouri Botanical Garden. 82 (2): 247–277. doi:10.2307/2399880. JSTOR 2399880.
  8. ^ Song, Jingyuan; Shi, Linchun; Li, Dezhu; Sun, Yongzhen; Niu, Yunyun; Chen, Zhiduan; Luo, Hongmei; Pang, Xiaohui; Sun, Zhiying (2012-08-30). "Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA". PLOS ONE. 7 (8): e43971. Bibcode:2012PLoSO...743971S. doi:10.1371/journal.pone.0043971. ISSN 1932-6203. PMC 3431384. PMID 22952830.
  9. ^ Baldwin, B.G. (1992). "Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae". Molecular Phylogenetics and Evolution. 1 (1): 3–16. doi:10.1016/1055-7903(92)90030-K. PMID 1342921.
  10. ^ Nickrent, Daniel L.; Schuette, Kevin P.; Starr, Ellen M. (1994-01-01). "A Molecular Phylogeny of Arceuthobium (Viscaceae) Based on Nuclear Ribosomal DNA Internal Transcribed Spacer Sequences". American Journal of Botany. 81 (9): 1149–1160. doi:10.2307/2445477. JSTOR 2445477.
  11. ^ Buckler, E. S.; Holtsford, T. P. (1996-04-01). "Zea systematics: ribosomal ITS evidence". Molecular Biology and Evolution. 13 (4): 612–622. doi:10.1093/oxfordjournals.molbev.a025621. ISSN 0737-4038. PMID 8882504.
  12. ^ Douzery, Emmanuel J. P.; Pridgeon, Alec M.; Kores, Paul; Linder, H. P.; Kurzweil, Hubert; Chase, Mark W. (1999-06-01). "Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences". American Journal of Botany. 86 (6): 887–899. doi:10.2307/2656709. ISSN 0002-9122. JSTOR 2656709. PMID 10371730. (subscription required)
  13. ^ Weekers, Peter H. H.; De Jonckheere, Johan F.; Dumont, Henri J. (2001-07-01). "Phylogenetic Relationships Inferred from Ribosomal ITS Sequences and Biogeographic Patterns in Representatives of the Genus Calopteryx (Insecta: Odonata) of the West Mediterranean and Adjacent West European Zone". Molecular Phylogenetics and Evolution. 20 (1): 89–99. doi:10.1006/mpev.2001.0947. PMID 11421650.
  14. ^ Chen, Y-C, J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. Lafe, A. P. Limaye, and B. T. Cookson (2001). "Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts". J. Clin. Microbiol. 39 (11): 4042–4051. doi:10.1128/JCM.39.11.4042-4051.2001. PMC 88485. PMID 11682528.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ^ Hodkinson, Trevor R.; Chase, Mark W.; Lledó, Dolores M.; Salamin, Nicolas; Renvoize, Stephen A. (2002). "Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers". Journal of Plant Research. 115 (5): 381–392. Bibcode:2002JPlR..115..381H. doi:10.1007/s10265-002-0049-3. ISSN 0918-9440. PMID 12579363. S2CID 22971617.
  16. ^ Rønsted, Nina; Chase, Mark W.; Albach, Dirk C.; Bello, Maria Angelica (2002-08-01). "Phylogenetic relationships within Plantago (Plantaginaceae): evidence from nuclear ribosomal ITS and plastid trnL-F sequence data". Botanical Journal of the Linnean Society. 139 (4): 323–338. doi:10.1046/j.1095-8339.2002.00070.x. ISSN 1095-8339.
  17. ^ Feldberg, K.; Groth, H.; Wilson, R.; Schäfer-Verwimp, A.; Heinrichs, J. (2004-11-04). "Cryptic speciation in Herbertus (Herbertaceae, Jungermanniopsida): Range and morphology of Herbertus sendtneri inferred from nrITS sequences". Plant Systematics and Evolution. 249 (3–4): 247–261. Bibcode:2004PSyEv.249..247F. doi:10.1007/s00606-004-0221-4. ISSN 0378-2697. S2CID 21538862.
  18. ^ Havill, Nathan P.; Campbell, Christopher S.; Vining, Thomas F.; LePage, Ben; Bayer, Randall J.; Donoghue, Michael J. (2008-07-01). "Phylogeny and Biogeography of Tsuga (Pinaceae) Inferred from Nuclear Ribosomal ITS and Chloroplast DNA Sequence Data". Systematic Botany. 33 (3): 478–489. doi:10.1600/036364408785679770. S2CID 26668467.
  19. ^ Ruhl, Michael W.; Wolf, Matthias; Jenkins, Tracie M. (2010). "Compensatory base changes illuminate morphologically difficult taxonomy". Molecular Phylogenetics and Evolution. 54 (2): 664–669. doi:10.1016/j.ympev.2009.07.036. PMID 19660561.
  20. ^ Stat, Michael; Pochon, Xavier (2008-07-02). "Specificity in communities of Symbiodinium in corals from Johnston Atoll" (PDF). Marine Ecology Progress Series. 386: 83–96. doi:10.3354/meps08080.
  21. ^ Warwick, Suzanne I.; Mummenhoff, Klaus; Sauder, Connie A.; Koch, Marcus A.; Al-Shehbaz, Ihsan A. (2010-04-13). "Closing the gaps: phylogenetic relationships in the Brassicaceae based on DNA sequence data of nuclear ribosomal ITS region". Plant Systematics and Evolution. 285 (3–4): 209–232. Bibcode:2010PSyEv.285..209W. doi:10.1007/s00606-010-0271-8. ISSN 0378-2697. S2CID 28199415.
  22. ^ Pirie, Michael D.; Oliver, E. G. H.; Bellstedt, Dirk U. (2011-11-01). "A densely sampled ITS phylogeny of the Cape flagship genus Erica L. suggests numerous shifts in floral macro-morphology". Molecular Phylogenetics and Evolution. 61 (2): 593–601. doi:10.1016/j.ympev.2011.06.007. PMID 21722743.
  23. ^ Boykin, L. M.; Schutze, M. K.; Krosch, M. N.; Chomič, A.; Chapman, T. A.; Englezou, A.; Armstrong, K. F.; Clarke, A. R.; Hailstones, D. (2014-05-01). "Multi-gene phylogenetic analysis of south-east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy". Journal of Applied Entomology. 138 (4): 235–253. doi:10.1111/jen.12047. ISSN 1439-0418. S2CID 82003038.
  24. ^ Scheunert, Agnes; Heubl, Günther (2014-01-01). "Diversification of Scrophularia (Scrophulariaceae) in the Western Mediterranean and Macaronesia – Phylogenetic relationships, reticulate evolution and biogeographic patterns". Molecular Phylogenetics and Evolution. 70: 296–313. doi:10.1016/j.ympev.2013.09.023. PMID 24096055.
  25. ^ Yang, Tao; Zhang, Tian-lei; Guo, You-hao; Liu, Xing (2016-11-17). "Identification of Hybrids in Potamogeton: Incongruence between Plastid and ITS Regions Solved by a Novel Barcoding Marker PHYB". PLOS ONE. 11 (11): e0166177. Bibcode:2016PLoSO..1166177Y. doi:10.1371/journal.pone.0166177. ISSN 1932-6203. PMC 5113904. PMID 27855191.
  26. ^ Schultz, J; Maisel, S; Gerlach, D; Müller, T; Wolf, M (April 2005). "A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota". RNA. 11 (4): 361–4. doi:10.1261/rna.7204505. PMC 1370725. PMID 15769870.
  27. ^ Koetschan, C; Kittelmann, S; Lu, J; Al-Halbouni, D; Jarvis, GN; Müller, T; Wolf, M; Janssen, PH (2014). "Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota)". PLOS ONE. 9 (3): e91928. Bibcode:2014PLoSO...991928K. doi:10.1371/journal.pone.0091928. PMC 3963862. PMID 24663345.
  28. ^ Peay K.G.; Kennedy P.G.; Bruns T.D. (2008). "Fungal community ecology: a hybrid beast with a molecular master". BioScience. 58 (9): 799–810. doi:10.1641/b580907. S2CID 18363490.
  29. ^ Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W., Bolchacova, E., Voigt, K., Crous, P.W.; et al. (2012). "Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi". PNAS. 109 (16): 6241–6246. doi:10.1073/pnas.1117018109. PMC 3341068. PMID 22454494.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  30. ^ White, T.J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a Guide to Methods and Applications 18, 315–322.
  31. ^ The ITS1 primer covers ITS1-5.8S-ITS2 from the 5', and ITS4 covers the same area from the 3'.
  32. ^ Gardes, M.; Bruns, T.D. (1993). "ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhiza and rusts". Molecular Ecology. 2 (2): 113–118. doi:10.1111/j.1365-294X.1993.tb00005.x. PMID 8180733. S2CID 24316407.
  33. ^ Usyk, Mykhaylo; Zolnik, Christine P.; Patel, Hitesh; Levi, Michael H.; Burk, Robert D. (2017-12-13). Mitchell, Aaron P. (ed.). "Novel ITS1 Fungal Primers for Characterization of the Mycobiome". mSphere. 2 (6): e00488–17, /msphere/2/6/mSphere0488–17.atom. doi:10.1128/mSphere.00488-17. ISSN 2379-5042. PMC 5729218. PMID 29242834.
  34. ^ Nilsson, R. Henrik; Anslan, Sten; Bahram, Mohammad; Wurzbacher, Christian; Baldrian, Petr; Tedersoo, Leho (February 2019). "Mycobiome diversity: high-throughput sequencing and identification of fungi". Nature Reviews Microbiology. 17 (2): 95–109. doi:10.1038/s41579-018-0116-y. ISSN 1740-1534. PMID 30442909. S2CID 53438777.
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