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The pathway to detangle a scrambled gene.

Möllenbeck M, Zhou Y, Cavalcanti AR, Jönsson F, Higgins BP, Chang WJ, Juranek S, Doak TG, Rozenberg G, Lipps HJ, Landweber LF - PLoS ONE (2008)

Bottom Line: The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones.The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University Witten/Herdecke, Witten, Germany.

ABSTRACT

Background: Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20-30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development.

Methodology/principal findings: To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.

Conclusions/significance: This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

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Related in: MedlinePlus

Schematic representation of the micronuclear precursor and resulting macronuclear gene structures (to scale) for (A) Stylonychia lemnae (D strain) actin I, (B) Oxytricha trifallax actin I, and (C) Oxytricha trifallax TEBPα.MDSs represented in blue and labeled m (upside down if inverted); IESs (labeled i) and flanking DNA in yellow; pointer sequences, when shown, in maroon. Triangles mark the positions of start (pointing down) and stop codons (pointing up). Black boxes on both ends of the macronuclear structure represent telomeres.
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pone-0002330-g001: Schematic representation of the micronuclear precursor and resulting macronuclear gene structures (to scale) for (A) Stylonychia lemnae (D strain) actin I, (B) Oxytricha trifallax actin I, and (C) Oxytricha trifallax TEBPα.MDSs represented in blue and labeled m (upside down if inverted); IESs (labeled i) and flanking DNA in yellow; pointer sequences, when shown, in maroon. Triangles mark the positions of start (pointing down) and stop codons (pointing up). Black boxes on both ends of the macronuclear structure represent telomeres.

Mentions: The structures of the micronuclear genes for actin I in Stylonychia lemnae and its ortholog in Oxytricha trifallax, as well as O. trifallax TEBPα are shown in Figure 1. The S. lemnae actin I gene contains ten MDSs in the scrambled germline order 3 4 5 6 7 8*10*-2-1*9, with segments 1 and 2 inverted. Of the nine IESs, six are conventionally spliced and three, indicated by asterisks, define scrambled junctions. Assembly of actin I in S. lemnae therefore requires at least six events of conventional DNA deletion (joining segments 3–8 and 1–2), plus insertion of segment 9 between segments 8 and 10 and inversion of segments 1 and 2, as well as their translocation to the 5′ end of the molecule (or inversion of segments 3–10) (see Results section on DNA Permutation for more details). Note that the joining of segments 2 to 3, 8 to 9, and 9 to 10 all require permutations. The repeats (pointers) at the boundaries between MDS and IES in these scrambled genes range from 3–19 bp (Table 1–3). In [37] we examined the evolution of the actin I gene structure in two geographically isolated strains of S. lemnae, which differ significantly in non-coding regions. This study uses only the German strain.


The pathway to detangle a scrambled gene.

Möllenbeck M, Zhou Y, Cavalcanti AR, Jönsson F, Higgins BP, Chang WJ, Juranek S, Doak TG, Rozenberg G, Lipps HJ, Landweber LF - PLoS ONE (2008)

Schematic representation of the micronuclear precursor and resulting macronuclear gene structures (to scale) for (A) Stylonychia lemnae (D strain) actin I, (B) Oxytricha trifallax actin I, and (C) Oxytricha trifallax TEBPα.MDSs represented in blue and labeled m (upside down if inverted); IESs (labeled i) and flanking DNA in yellow; pointer sequences, when shown, in maroon. Triangles mark the positions of start (pointing down) and stop codons (pointing up). Black boxes on both ends of the macronuclear structure represent telomeres.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2394655&req=5

pone-0002330-g001: Schematic representation of the micronuclear precursor and resulting macronuclear gene structures (to scale) for (A) Stylonychia lemnae (D strain) actin I, (B) Oxytricha trifallax actin I, and (C) Oxytricha trifallax TEBPα.MDSs represented in blue and labeled m (upside down if inverted); IESs (labeled i) and flanking DNA in yellow; pointer sequences, when shown, in maroon. Triangles mark the positions of start (pointing down) and stop codons (pointing up). Black boxes on both ends of the macronuclear structure represent telomeres.
Mentions: The structures of the micronuclear genes for actin I in Stylonychia lemnae and its ortholog in Oxytricha trifallax, as well as O. trifallax TEBPα are shown in Figure 1. The S. lemnae actin I gene contains ten MDSs in the scrambled germline order 3 4 5 6 7 8*10*-2-1*9, with segments 1 and 2 inverted. Of the nine IESs, six are conventionally spliced and three, indicated by asterisks, define scrambled junctions. Assembly of actin I in S. lemnae therefore requires at least six events of conventional DNA deletion (joining segments 3–8 and 1–2), plus insertion of segment 9 between segments 8 and 10 and inversion of segments 1 and 2, as well as their translocation to the 5′ end of the molecule (or inversion of segments 3–10) (see Results section on DNA Permutation for more details). Note that the joining of segments 2 to 3, 8 to 9, and 9 to 10 all require permutations. The repeats (pointers) at the boundaries between MDS and IES in these scrambled genes range from 3–19 bp (Table 1–3). In [37] we examined the evolution of the actin I gene structure in two geographically isolated strains of S. lemnae, which differ significantly in non-coding regions. This study uses only the German strain.

Bottom Line: The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones.The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University Witten/Herdecke, Witten, Germany.

ABSTRACT

Background: Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20-30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development.

Methodology/principal findings: To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.

Conclusions/significance: This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

Show MeSH
Related in: MedlinePlus