Internal transcribed spacer

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.{{Cite journal | doi = 10.1038/35080045| pmid = 11433365| title = The function and synthesis of ribosomes| journal = Nature Reviews Molecular Cell Biology| volume = 2| issue = 7| pages = 514–520| year = 2001| last1 = Lafontaine | first1 = D. L. J. | last2 = Tollervey | first2 = D. | hdl = 1842/729| s2cid = 2637106| hdl-access = free}}{{cite book|author=Scott Orland Rogers|title=Integrated Molecular Evolution|url=https://books.google.com/books?id=xywnbQMKzXwC&pg=PA66|access-date=9 March 2015|date=27 July 2011|publisher=CRC Press|isbn=978-1-4398-1995-1|pages=65–66}}

File:Eucaryot rdna.png

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.{{cite journal |last1=Takada |first1=Hiraku |last2=Shimada |first2=Tomohiro |last3=Dey |first3=Debashish |last4=Quyyum |first4=M. Zuhaib |last5=Nakano |first5=Masahiro |last6=Ishiguro |first6=Akira |last7=Yoshida |first7=Hideji |last8=Yamamoto |first8=Kaneyoshi |last9=Sen |first9=Ranjan |last10=Ishihama |first10=Akira |title=Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli |journal=PLOS ONE |date=22 December 2016 |volume=11 |issue=12 |pages=e0163057 |doi=10.1371/journal.pone.0163057|pmid=28005933 |pmc=5179076 |bibcode=2016PLoSO..1163057T |doi-access=free }}{{cite journal |last1=Stewart |first1=Frank J. |last2=Cavanaugh |first2=Colleen M. |title=Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria |journal=Journal of Molecular Evolution |date=July 2007 |volume=65 |issue=1 |pages=44–67 |doi=10.1007/s00239-006-0235-3|pmid=17568983 |bibcode=2007JMolE..65...44S |s2cid=13536182 |citeseerx=10.1.1.456.2659 }}

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.{{Cite journal|last1=Bena|first1=Gilles|last2=Jubier|first2=Marie-France|last3=Olivieri|first3=Isabelle|last4=Lejeune|first4=Bernard|year=1998|title=Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae)|journal=Journal of Molecular Evolution|volume=46|issue=3|pages=299–306|doi=10.1007/PL00006306|pmid=9502673|issn=0022-2844|bibcode=1998JMolE..46..299B|s2cid=38838013}}

During rRNA maturation, ETS and ITS pieces are excised. As non-functional by-products of this maturation, they are rapidly degraded.{{Cite journal|last1=Michot|first1=Bernard|last2=Bachellerie|first2=Jean-Pierre|last3=Raynal|first3=Francoise|date=1983-05-25|title=Structure of mouse rRNA precursors. Complete sequence and potential folding of the spacer regions between 18S and 28S rRNA|journal=Nucleic Acids Research|volume=11|issue=10|pages=3375–3391|doi=10.1093/nar/11.10.3375|pmid=6304630|pmc=325970|issn=0305-1048}}

Sequence comparison of the eukaryotic ITS regions is widely used in taxonomy and molecular phylogeny because of several favorable properties:{{Cite journal|last1=Baldwin|first1=Bruce G.|last2=Sanderson|first2=Michael J.|last3=Porter|first3=J. Mark|last4=Wojciechowski|first4=Martin F.|last5=Campbell|first5=Christopher S.|last6=Donoghue|first6=Michael J.|date=1995-01-01|title=The ITS Region of Nuclear Ribosomal DNA: A Valuable Source of Evidence on Angiosperm Phylogeny|jstor=2399880|journal=Annals of the Missouri Botanical Garden|volume=82|issue=2|pages=247–277|doi=10.2307/2399880}}

• 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.{{Cite journal|last1=Song|first1=Jingyuan|last2=Shi|first2=Linchun|last3=Li|first3=Dezhu|last4=Sun|first4=Yongzhen|last5=Niu|first5=Yunyun|last6=Chen|first6=Zhiduan|last7=Luo|first7=Hongmei|last8=Pang|first8=Xiaohui|last9=Sun|first9=Zhiying|date=2012-08-30|title=Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA|journal=PLOS ONE|volume=7|issue=8|pages=e43971|doi=10.1371/journal.pone.0043971|issn=1932-6203|pmc=3431384|pmid=22952830|bibcode=2012PLoSO...743971S|doi-access=free}}
• 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.

ITS2 is known to be more conserved than ITS1 is. All ITS2 sequences share a common core of secondary structure,{{cite journal |last1=Schultz |first1=J |last2=Maisel |first2=S |last3=Gerlach |first3=D |last4=Müller |first4=T |last5=Wolf |first5=M |title=A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. |journal=RNA |date=April 2005 |volume=11 |issue=4 |pages=361–4 |doi=10.1261/rna.7204505 |pmid=15769870 |pmc=1370725}} 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.{{cite journal |last1=Koetschan |first1=C |last2=Kittelmann |first2=S |last3=Lu |first3=J |last4=Al-Halbouni |first4=D |last5=Jarvis |first5=GN |last6=Müller |first6=T |last7=Wolf |first7=M |last8=Janssen |first8=PH |title=Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota). |journal=PLOS ONE |date=2014 |volume=9 |issue=3 |pages=e91928 |doi=10.1371/journal.pone.0091928 |pmid=24663345 |pmc=3963862 |bibcode=2014PLoSO...991928K |doi-access=free}}

{{Main|Fungal DNA barcoding}}

The ITS region is the most widely sequenced DNA region in molecular ecology of fungi{{cite journal |author1=Peay K.G. |author2=Kennedy P.G. |author3=Bruns T.D. | title=Fungal community ecology: a hybrid beast with a molecular master| journal=BioScience| year=2008 |pages=799–810|volume=58|issue=9 | doi=10.1641/b580907|s2cid=18363490 |doi-access=free }} and has been recommended as the universal fungal barcode sequence.{{cite journal | author=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. | title=Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi | journal=PNAS| year=2012 |doi= 10.1073/pnas.1117018109| pages=6241–6246|volume=109 | issue=16|display-authors=etal | pmid=22454494 | pmc=3341068| doi-access=free }} 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 primersWhite, 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.The ITS1 primer covers ITS1-5.8S-ITS2 from the 5', and ITS4 covers the same area from the 3'. 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).{{cite journal |author1=Gardes, M. |author2=Bruns, T.D.| title=ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhiza and rusts| journal=Molecular Ecology| year=1993 |doi= 10.1111/j.1365-294X.1993.tb00005.x| pages=113–118|volume=2|pmid=8180733 | issue=2|s2cid=24316407}} 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.{{Cite journal|last1=Usyk|first1=Mykhaylo|last2=Zolnik|first2=Christine P.|last3=Patel|first3=Hitesh|last4=Levi|first4=Michael H.|last5=Burk|first5=Robert D.|date=2017-12-13|editor-last=Mitchell|editor-first=Aaron P.|title=Novel ITS1 Fungal Primers for Characterization of the Mycobiome|journal=mSphere|volume=2|issue=6|pages=e00488–17, /msphere/2/6/mSphere0488–17.atom|doi=10.1128/mSphere.00488-17|issn=2379-5042|pmc=5729218|pmid=29242834}}{{Cite journal|last1=Nilsson|first1=R. Henrik|last2=Anslan|first2=Sten|last3=Bahram|first3=Mohammad|last4=Wurzbacher|first4=Christian|last5=Baldrian|first5=Petr|last6=Tedersoo|first6=Leho|date=February 2019|title=Mycobiome diversity: high-throughput sequencing and identification of fungi|journal=Nature Reviews Microbiology|volume=17|issue=2|pages=95–109|doi=10.1038/s41579-018-0116-y|pmid=30442909|s2cid=53438777|issn=1740-1534}}

{{reflist|30em}}

• [http://depts.washington.edu/molmicdx/mdx/tests/yeast.shtml University of Washington Laboratory Medicine: Molecular Diagnosis | Yeast Sequencing]
• [http://itsonedb.cloud.ba.infn.it/ ITSone DB]
• [https://bio.tools/its2 ITS2 database] (Schultz et al.)

{{DEFAULTSORT:Internal Transcribed Spacer}}

Category:DNA

Category:Phylogenetics