Structure and expression of the Zea mays mutS-homologs Mus1 and Mus2

Horwath, M.; Kramer, Wilfried; Kunze, Reinhard

doi:10.1007/s00122-002-0955-8

Cited by 17 publications

(10 citation statements)

References 49 publications

(35 reference statements)

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“…Thus, both RT-PCR experiments and Genevestigator data show the same biologically relevant trend in AtMSH2 gene expression in rapidly dividing tissues. This is consistent with previous reports indicating higher levels of MSH2 activity in actively dividing cells from A. thaliana [39], Zea mays [40], and Solanum lycopersicum [45] as compared to cells in mature tissues.…”

Section: Analysis Of Atmsh2 Transcript Levelssupporting

confidence: 94%

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High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit

2010

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Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His(6)-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.

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Section: Analysis Of Atmsh2 Transcript Levelssupporting

confidence: 94%

“…Previous reports indicate that MSH2 genes are expressed at very low levels in A. thaliana and maize as investigated by Northern blot [39,40]. Here, the starting copy number of the AtMSH2 gene was quantified in calli and seedlings using standard curves generated with the pET32b-MSH2 plasmid as template.…”

Section: Analysis Of Atmsh2 Transcript Levelsmentioning

confidence: 99%

High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit

2010

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“…Instead, only by replacing the plant tissues with mitotically dividing Arabidopsis cell suspensions did they manage to identify mRNAs for MSH2, 3 and 6-2, with high levels of MSH6-2 transcripts in the early exponential growth phase of the cell culture. Similarly, in maize, it was reported that MUS1 (MSH2) and MUS2 (MSH6-like) RNA expressions were only successfully detected in young maize seedlings (at low levels) using RNA gel-blot analyses (Horwath et al 2002 ). The tissues of young leaves and floral buds used in our study would contain a source of more actively dividing cells, when compared to mature leaves or other parts of the plant.…”

Section: Discussionmentioning

confidence: 99%

Characterization and comparative sequence analysis of the DNA mismatch repair MSH2 and MSH7 genes from tomato

et al. 2009

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DNA mismatch repair proteins play an essential role in maintaining genomic integrity during replication and genetic recombination. We successfully isolated a full length MSH2 and partial MSH7 cDNAs from tomato, based on sequence similarity between MutS and plant MSH homologues. Semi-quantitative RT-PCR reveals higher levels of mRNA expression of both genes in young leaves and floral buds. Genetic mapping placed MSH2 and MSH7 on chromosomes 6 and 7, respectively, and indicates that these genes exist as single copies in the tomato genome. Analysis of protein sequences and phylogeny of the plant MSH gene family show that these proteins are evolutionarily conserved, and follow the classical model of asymmetric protein evolution. Genetic manipulation of the expression of these MSH genes in tomato will provide a potentially useful tool for modifying genetic recombination and hybrid fertility between wide crosses.Electronic supplementary materialThe online version of this article (doi:10.1007/s10709-009-9398-3) contains supplementary material, which is available to authorized users.

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“…In that regard, it may be noteworthy that post‐replication MMR, a genetic‐fidelity function normally coupled to DNA replication during cell division, appears to be weak at best in Arabidopsis leaves. Analyses of RNA in whole Arabidopsis or maize seedlings have revealed very low mRNA levels expressed by MLH1 , MSH2 , MSH7 , and, apparently, other MSH genes (Ade et al ., 1999; Horwath et al ., 2002; Jean et al ., 1999). Consistent with this evidence for reduced MMR in Arabidopsis leaves is the surprisingly small effect of MMR deficiency on frameshift reversion in GUS reporter transgenes, scored mostly in leaves, most likely because of slip mispairing at nucleotide‐repeat sequences – a prime MMR substrate.…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis thaliana AtPOLK encodes a DinB‐like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues

et al. 2004

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SummaryCell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolk, is a homolog of Escherichia coli pol IV and human pol k, and thus belongs to the DinB subfamily. We puri®ed AtPolk and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer±terminal mispairs. The activity and processivity of AtPolk are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of b-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.

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Structure and expression of the Zea mays mutS-homologs Mus1 and Mus2

Cited by 17 publications

References 49 publications

High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit

High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit

Characterization and comparative sequence analysis of the DNA mismatch repair MSH2 and MSH7 genes from tomato

Arabidopsis thaliana AtPOLK encodes a DinB‐like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues

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