Download Dörte Harpke - Lehrstuhl Technische Informatik der MLU Halle

DIPLOMARBEIT
UNIVERSITÄTSZENTRUM INFORMATIK
Martin-Luther-Universität Halle-Wittenberg
Non-concerted ITS evolution and analysis of functional and
non-functional 5.8S rRNA genes in genus Mammillaria (Cactaceae)
Dörte Harpke
(2005)
 Universitätszentrum Informatik • Universität Halle
von-Seckendorff-Platz 1 • 06120 Halle (Saale)
Allgemeine Angaben
Die Diplomarbeit wurde am im Biozentrum der Universität Halle, Abteilung
Gentechnik von Dr. Angela Peterson in Zusammenarbeit mit dem Lehrstuhl für
Bioinformatik, Mustererkennung von Prof. Dr. Stefan Posch, Institut für Informatik,
Fachbereich Mathematik und Informatik der Martin-Luther-Universität angefertigt.
Zur Kontaktaufnahme benutzen Sie bitte die Email-Adresse
[email protected].
General informations:
The nuclear ribosomal RNA genes (rDNA) occur as tandem repeated arrays called nucleolus
organizer regions (NORs) on one or more loci. Their primary transcript includes small-subunit
(SSU), 5.8S and large-subunit (LSU) rRNA separated by internal transcribed spacer 1 (ITS1)
and the internal transcribed spacer 2 (ITS2). It is assumed that the 5.8S rDNA is highly conserved among angiosperms, because its secondary structure is required for a proper function
of the ribosomal complex (Suh et al. 1992, Hershkovitz & Zimmer 1996, Jobes & Thien
1997). Nuclear rDNA regions like the ITS region (ITS1, 5.8S, ITS2) are often used to estimate phylogenetic relationships (Buckler et al. 1997; Peterson et al. 2004). As a multigene
family the ITS region is subjected to concerted evolution, which means the homogenisation
among the copies through processes like gene conversion and unequal crossing over. An
incomplete concerted or non-concerted evolution caused by e.g. hybridisation, disadvantageous loci or polyploidy results in intra-individual polymorphism. Generally, intra-individual
nrDNA polymorphism has been considered to be an exception. Nevertheless, some investigations showed that individuals with polymorphic ITS copies often contain potentially nonfunctional nrDNA copies (pseudogenes) in addition to functional copies (Buckler et al., 1997;
reviewed by Bailey et al., 2003). They are expected to evolve at a high rate. Non-functional
sequences are often characterized by cytosine mutations at methylation sites, because they
are highly mutable. Thus, they become AT-rich.
Motivation:
In order to provide a closer view of phylogenetic relationships among the species of Mammillaria molecular markers were carried out. Therefore we investigated the chloroplast intergenic region trnL-trnF because this region is often used to estimate phylogenetic relationships
(e.g. Peterson et al. 2004). Our results of trnL-trnF sequence data showed only low levels of
variations for the genus Mammillaria (Harpke et al. 2005). As consequence two Mammillaria
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species were chosen to check the utility of the ITS region for phylogenetic studies. Interestingly, amplification of the ITS region revealed two bands differing in sequence length. Direct
sequencing of upper bands for the two individuals revealed an overlap of different sequences
by multiple peaks per position in the electropherogram. Based on this fact, further investigations, introduced by this diploma thesis, should reveal reasons for the observed phenomenon
of non-concerted evolution and whether it is common among the whole genus. Thus, the
idea was to study more species representing different subgenera and series of the large
subgenus Mammillaria.
Abstract:
Molecular studies of 21 species of the large Cactaceae genus Mammillaria representing a
variety of intrageneric taxonomic levels revealed a high degree of intra-individual polymorphism of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). This polymorphism
includes both spacers as well as the 5.8S rDNA. However, the high degree of intra-individual
polymorphism of up to 36 % in ITS1, up to 39 % in 5.8S and up to 41 % in ITS2 suggests a
non-concerted evolution of these loci in Mammillaria.
To identify putative pseudogenes in genomic clones of Mammillaria individuals an extensive
analysis of 5.8S rDNA sequences was done including reconstruction of the proper secondary
structure, relative rate tests, bootstrap hypothesis testing, and analysis of methylation-related
substitutions, length variation and sequence comparisons to cDNA sequences. These analyses reveal 96 out of 110 genomic clones as pseudogenes. Additionally, conserved angiosperm ITS1 and ITS2 motifs were compared between genes and pseudogenes. Some of
these motifs (e.g. ITS1 motif, ‘TGGT’ within ITS2) in combination with the determination of
GC-content, length comparisons of the spacers are helpful in the identification of pseudogene rDNA regions.
References:
Buckler IV ES, Ippolito A, Holtsford TP (1997) The Evolution of Ribosomal DNA: Divergent
Paralogues and Phylogenetic Implications. Genetics 145: 821-832.
Harpke D, Peterson A, Hoffmann MH, Röser M (2005) Phylogenetic evaluation of chloroplast
trnL-trnF DNA sequence variation in the genus Mammillaria Haworth (Cactaceae).
Schlechtendalia in press.
Hershkovitz MA & Zimmer EA (1996) Conservation patterns in angiosperm rDNA-ITS2 sequences. Nucl. Acids. Res. 24: 2857-2867.
Jobes DV & Thien LB (1997) A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is
a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences. Plant
Mol. Biol. Rep. 15: 326-334.
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Peterson A, John H, Koch E, Peterson J (2004) A molecular phylogeny of the genus Gagea
(Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences.
Plant Syst. Evol. 245: 145-162.
Suh Y, Thien LB, Zimmer EA (1992) Nucleotide sequences of the internal transcribed spacers and 5.8S rRNA gene in Canella winterana (Magnoliales; Canellaceae). Nucl. Acids. Res.
20: 6101-6102.
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