Limits...
The role of AtMUS81 in interference-insensitive crossovers in A. thaliana.

Berchowitz LE, Francis KE, Bey AL, Copenhaver GP - PLoS Genet. (2007)

Bottom Line: The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment.Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination.These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad-based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.

Show MeSH

Related in: MedlinePlus

The AtMUS81 Gene Structure, T-DNA Insertion Mutant, and Expression(A) An illustration of the AtMUS81 (At4g30870) locus showing the exon/intron organization of AtMUS81. Solid boxes represent transcribed regions including protein coding (black) and untranslated regions (gray). The T-DNA insertion site for the mutant used in this study is shown. Conserved domains are shown above. Below are products for the following primers that were used for genotyping (M81_F/M81_R), RT-PCR (M81S1_F/M81S1R and M81S2_F/M81S2_R), and real-time qPCR (M81RTRT3_F/M81RTRT_R).(B) Whole seedling (10-d) RT-PCR of wild type and the Atmus81 mutant. Primers (S1 and S2) downstream of the T-DNA insertion site were used in the RT-PCR reaction with and without reverse transcriptase (RT) using RNA from wild-type (WT) and mutant (mus) plants. The APT1 transcript was used as a control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1941751&req=5

pgen-0030132-g001: The AtMUS81 Gene Structure, T-DNA Insertion Mutant, and Expression(A) An illustration of the AtMUS81 (At4g30870) locus showing the exon/intron organization of AtMUS81. Solid boxes represent transcribed regions including protein coding (black) and untranslated regions (gray). The T-DNA insertion site for the mutant used in this study is shown. Conserved domains are shown above. Below are products for the following primers that were used for genotyping (M81_F/M81_R), RT-PCR (M81S1_F/M81S1R and M81S2_F/M81S2_R), and real-time qPCR (M81RTRT3_F/M81RTRT_R).(B) Whole seedling (10-d) RT-PCR of wild type and the Atmus81 mutant. Primers (S1 and S2) downstream of the T-DNA insertion site were used in the RT-PCR reaction with and without reverse transcriptase (RT) using RNA from wild-type (WT) and mutant (mus) plants. The APT1 transcript was used as a control.

Mentions: The SALK laboratories SIGnAL database of transfer DNA (T-DNA) insertions contains two intronic T-DNA insertions within the open reading frame of AtMUS81 (http://signal.salk.edu/cgi-bin/tdnaexpress) SALK_107515, and SALK_113F11 (Figure 1A). We used the former, as we were able to show that this is a bona fide insertion using PCR with primers spanning the insert (Figure 1A). Hartung et al. used the SALK_107515 allele (AtMUS81–1) and a second allele (AtMUS81–2) from an independent T-DNA collection (GABI, http://www.gabi-kat.de/) and showed that both produced identical mitotic phenotypes [27]. We used PCR to genotype individuals as homozygous wild type, heterozygous, or homozygous for the insert. Sequence analysis of PCR products from the homozygous insertion line confirmed the T-DNA insertion junction reported in the SALK database. RT-PCR using primers spanning the insertion in lines homozygous for this insertion showed no product (unpublished data). RT-PCR using primers downstream of the insertion showed that transcript levels were greatly reduced in lines homozygous for the insertion (Figure 1B).


The role of AtMUS81 in interference-insensitive crossovers in A. thaliana.

Berchowitz LE, Francis KE, Bey AL, Copenhaver GP - PLoS Genet. (2007)

The AtMUS81 Gene Structure, T-DNA Insertion Mutant, and Expression(A) An illustration of the AtMUS81 (At4g30870) locus showing the exon/intron organization of AtMUS81. Solid boxes represent transcribed regions including protein coding (black) and untranslated regions (gray). The T-DNA insertion site for the mutant used in this study is shown. Conserved domains are shown above. Below are products for the following primers that were used for genotyping (M81_F/M81_R), RT-PCR (M81S1_F/M81S1R and M81S2_F/M81S2_R), and real-time qPCR (M81RTRT3_F/M81RTRT_R).(B) Whole seedling (10-d) RT-PCR of wild type and the Atmus81 mutant. Primers (S1 and S2) downstream of the T-DNA insertion site were used in the RT-PCR reaction with and without reverse transcriptase (RT) using RNA from wild-type (WT) and mutant (mus) plants. The APT1 transcript was used as a control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1941751&req=5

pgen-0030132-g001: The AtMUS81 Gene Structure, T-DNA Insertion Mutant, and Expression(A) An illustration of the AtMUS81 (At4g30870) locus showing the exon/intron organization of AtMUS81. Solid boxes represent transcribed regions including protein coding (black) and untranslated regions (gray). The T-DNA insertion site for the mutant used in this study is shown. Conserved domains are shown above. Below are products for the following primers that were used for genotyping (M81_F/M81_R), RT-PCR (M81S1_F/M81S1R and M81S2_F/M81S2_R), and real-time qPCR (M81RTRT3_F/M81RTRT_R).(B) Whole seedling (10-d) RT-PCR of wild type and the Atmus81 mutant. Primers (S1 and S2) downstream of the T-DNA insertion site were used in the RT-PCR reaction with and without reverse transcriptase (RT) using RNA from wild-type (WT) and mutant (mus) plants. The APT1 transcript was used as a control.
Mentions: The SALK laboratories SIGnAL database of transfer DNA (T-DNA) insertions contains two intronic T-DNA insertions within the open reading frame of AtMUS81 (http://signal.salk.edu/cgi-bin/tdnaexpress) SALK_107515, and SALK_113F11 (Figure 1A). We used the former, as we were able to show that this is a bona fide insertion using PCR with primers spanning the insert (Figure 1A). Hartung et al. used the SALK_107515 allele (AtMUS81–1) and a second allele (AtMUS81–2) from an independent T-DNA collection (GABI, http://www.gabi-kat.de/) and showed that both produced identical mitotic phenotypes [27]. We used PCR to genotype individuals as homozygous wild type, heterozygous, or homozygous for the insert. Sequence analysis of PCR products from the homozygous insertion line confirmed the T-DNA insertion junction reported in the SALK database. RT-PCR using primers spanning the insertion in lines homozygous for this insertion showed no product (unpublished data). RT-PCR using primers downstream of the insertion showed that transcript levels were greatly reduced in lines homozygous for the insertion (Figure 1B).

Bottom Line: The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment.Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination.These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad-based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.

Show MeSH
Related in: MedlinePlus