Internal transcribed spacer explained

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

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

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

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

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

Taxonomic groupTaxonomic levelYearAuthors with references
Asteraceae

Compositae

Species (congeneric)1992Baldwin et al.[9]
Viscaceae

Arceuthobium

Species (congeneric)1994Nickrent et al.[10]
Poaceae

Zea

Species (congeneric)1996Buckler & Holtsford[11]
Leguminosae

Medicago

Species (congeneric)1998Bena et al.
Orchidaceae

Diseae

Genera (within tribes)1999Douzery et al.[12]
Odonata

Calopteryx

Species (congeneric)2001Weekers et al.[13]
Yeasts of clinical importanceGenera2001Chen et al.[14]
Poaceae

Saccharinae

Genera (within tribes)2002Hodkinson et al.[15]
Plantaginaceae

Plantago

Species (congeneric)2002Rønsted et al.[16]
Jungermanniopsida

Herbertus

Species (congeneric)2004Feldberg et al.[17]
Pinaceae

Tsuga

Species (congeneric)2008Havill et al.[18]
Genera (congeneric)2009Ruhl et al.[19]
SymbiodiniumClade2009Stat et al.[20]
BrassicaceaeTribes (within a family)2010Warwick et al.[21]
Ericaceae

Erica

Species (congeneric)2011Pirie et al.[22]
Diptera

Bactrocera

Species (congeneric)2014Boykin et al.[23]
Scrophulariaceae

Scrophularia

Species (congeneric)2014Scheunert & Heubl[24]
Potamogetonaceae

Potamogeton

Species (congeneric)2016Yang 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

See 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]

External links

Notes and References

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  2. Book: Scott Orland Rogers. Integrated Molecular Evolution. 9 March 2015. 27 July 2011. CRC Press. 978-1-4398-1995-1. 65–66.
  3. Takada . Hiraku . Shimada . Tomohiro . Dey . Debashish . Quyyum . M. Zuhaib . Nakano . Masahiro . Ishiguro . Akira . Yoshida . Hideji . Yamamoto . Kaneyoshi . Sen . Ranjan . Ishihama . Akira . Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli . PLOS ONE . 22 December 2016 . 11 . 12 . e0163057 . 10.1371/journal.pone.0163057. 28005933 . 5179076 . 2016PLoSO..1163057T . free .
  4. Stewart . Frank J. . Cavanaugh . Colleen M. . Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria . Journal of Molecular Evolution . July 2007 . 65 . 1 . 44–67 . 10.1007/s00239-006-0235-3. 17568983 . 2007JMolE..65...44S . 13536182 . 10.1.1.456.2659 .
  5. 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. 10.1007/PL00006306. 9502673. 0022-2844. 1998JMolE..46..299B. 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. 10.1093/nar/11.10.3375. 6304630. 325970. 0305-1048.
  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. 2399880. Annals of the Missouri Botanical Garden. 82. 2. 247–277. 10.2307/2399880.
  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. 10.1371/journal.pone.0043971. 1932-6203. 3431384. 22952830. 2012PLoSO...743971S. free.
  9. Baldwin, B.G.. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae. Molecular Phylogenetics and Evolution. 1992 . 3–16. 1 . 10.1016/1055-7903(92)90030-K . 1342921 . 1.
  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. 2445477. American Journal of Botany. 81. 9. 1149–1160. 10.2307/2445477.
  11. Buckler. E. S.. Holtsford. T. P.. 1996-04-01. Zea systematics: ribosomal ITS evidence.. Molecular Biology and Evolution. 13. 4. 612–622. 0737-4038. 8882504. 10.1093/oxfordjournals.molbev.a025621.
  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. 0002-9122. 10371730. 10.2307/2656709. 2656709.
  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. 10.1006/mpev.2001.0947. 11421650.
  14. Chen, Y-C, J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. Lafe, A. P. Limaye, and B. T. Cookson. Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts. J. Clin. Microbiol.. 2001 . 10.1128/JCM.39.11.4042-4051.2001. 4042–4051. 39. 11682528 . 11 . 88485.
  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. 10.1007/s10265-002-0049-3. 12579363. 2002JPlR..115..381H . 22971617. 0918-9440.
  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. 10.1046/j.1095-8339.2002.00070.x. 1095-8339. free.
  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. 10.1007/s00606-004-0221-4. 2004PSyEv.249..247F . 21538862. 0378-2697.
  18. Havill. Nathan P.. Nathan Havill . 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. 10.1600/036364408785679770. 26668467.
  19. Ruhl. Michael W.. Wolf. Matthias. Jenkins. Tracie M.. 2010. Compensatory base changes illuminate morphologically difficult taxonomy. Molecular Phylogenetics and Evolution. en. 54. 2. 664–669. 10.1016/j.ympev.2009.07.036. 19660561.
  20. Stat. Michael. Pochon. Xavier. 2008-07-02. Specificity in communities of Symbiodinium in corals from Johnston Atoll. Marine Ecology Progress Series. 386. 83–96. 10.3354/meps08080. free.
  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. 10.1007/s00606-010-0271-8. 2010PSyEv.285..209W . 28199415. 0378-2697.
  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. 10.1016/j.ympev.2011.06.007. 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. 10.1111/jen.12047. 82003038. 1439-0418.
  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. 10.1016/j.ympev.2013.09.023. 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. 10.1371/journal.pone.0166177. 1932-6203. 5113904. 27855191. 2016PLoSO..1166177Y. free.
  26. Schultz . J . Maisel . S . Gerlach . D . Müller . T . Wolf . M . A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. . RNA . April 2005 . 11 . 4 . 361–4 . 10.1261/rna.7204505 . 15769870 . 1370725.
  27. Koetschan . C . Kittelmann . S . Lu . J . Al-Halbouni . D . Jarvis . GN . Müller . T . Wolf . M . Janssen . PH . Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota). . PLOS ONE . 2014 . 9 . 3 . e91928 . 10.1371/journal.pone.0091928 . 24663345 . 3963862 . 2014PLoSO...991928K . free.
  28. Peay K.G. . Kennedy P.G. . Bruns T.D. . Fungal community ecology: a hybrid beast with a molecular master. BioScience. 2008 . 799–810. 58. 9 . 10.1641/b580907. 18363490 . free .
  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. . Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi . PNAS. 2012 . 10.1073/pnas.1117018109. 6241–6246. 109 . 16. etal . 22454494 . 3341068. free .
  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.. ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhiza and rusts. Molecular Ecology. 1993 . 10.1111/j.1365-294X.1993.tb00005.x. 113–118. 2. 8180733 . 2. 24316407.
  33. Usyk. Mykhaylo. Zolnik. Christine P.. Patel. Hitesh. Levi. Michael H.. Burk. Robert D.. 2017-12-13. Mitchell. Aaron P.. Novel ITS1 Fungal Primers for Characterization of the Mycobiome. mSphere. 2. 6. e00488–17, /msphere/2/6/mSphere0488–17.atom. 10.1128/mSphere.00488-17. 2379-5042. 5729218. 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. 10.1038/s41579-018-0116-y. 30442909. 53438777. 1740-1534.

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