In E. coli , the methyl directed MMR system consists of the three proteins MutH, MutL and MutS. The MutS-protein recognizes and binds to mispaired nucleotides, small insertions and deletions, and subsequently recruits MutL and MutH. MutS-homologous (MSH) and MutL-homologous proteins were found in many prokaryotes, yeast, insects, and vertebrates. In eukaryotes the organization of the MMR system is more complex than in E. coli. In yeast, for example, six msh genes have been identified.
To date, the genes and proteins of the plant MMR system are not known. As a first step towards the investigation of the plant MMR system, we have isolated a mutS-homologous gene from maize. As in all known MutS-homologous proteins certain amino acid sequence motifs and the distance between them are highly conserved, we used degenerate oligonucleotides (Fig. 1) as primers for RT-PCR on maize seedling mRNA. The RT-PCR products were gel-fractionated and a band of the expected size was eluted and ligated into an M13 vector. Among 16 sequenced inserts we found two putative msh sequences, that hybridized to maize single copy sequences on a Southern blot. One of these also hybridized to a weakly expressed transcript on a Northern blot.
With the latter probe we isolated a 3 kb cDNA clone from a maize seedling cDNA library (kindly supplied by Monika Frey, Technical University of Munich). Sequencing of this cDNA clone confirmed it as a putative maize mutS-homolog; we termed the gene mus1. The cDNA clone included a poly(A)-sequence at the 3'-end, and the 5'-end of the transcript was determined by 5'-RACE. The mus1 transcript has the coding capacity for a 942 amino acid protein that is most similar and colinear with the MSH2 protein from S. cerevisiae. Figure 1 shows the alignment of the most highly conserved segments of MutS-homologous proteins from various organisms with the MUS1 protein. The positions of the degenerate primers used for the initial RT-PCR are highlighted by bold letters.
In collaboration with Monika Frey (Technical University of Munich) we RFLP-mapped the mus1 gene by using a recombinant inbred population (Burr ;et al., Genetics 118:519-526, 1988 Burr and Burr, TIG 7:55-60, 1991). mus1 maps on chromosome 7L, by coincidence with the b32B locus [rip1] that encodes a ribosome inactivation protein.
EcoMutS RMLIITGPNMGGKSTYMRQTALIALMAYIGSYVPAQKVEIGPIDRIFTRVGAADDLASGR StyMutS RMLIITGPNMGGKSTYMRQTALIALLAYIGSYVPAQNVEIGPIDRIFTRVGAADDLASGR SpneuHexA SIQLVTGPNMSGKSTYMRQLAMTAVMAQLGSYVPAESAHLPIFDAIFTRIGAADDLVSGQ BsubMutS QMLLITGPNMSGKSTYMRQIALISIMAQIGCFVPAKKAVLPIFDQIFTRIGAADDLISGQ AviMutS RMLIITGPNMGGKSTYMRQTALIVLLAHIGSFVPAQSCELSLVDRIFTRIGSSDDLAGGR HinfMutS HLLVITGPNMGGKSTYMRQTALITLLAYIGSFVPADSARIGPIDRIFTRIGASDDLASGR TaquaMutS ELVLITGPNMAGKSTFLRQTALIALLAQVGSFVPAEEAHLPLFDGIYTRIGASDDLAGGK Swi4 RCLLITGPNMGGKSSFVKQLALSAIMAQSGCFVPAKSALLPIFDSILIRMGSSDNLSVNM Spel1 NMFIITGPNMGGKSTYIRSVGTAVLMAHIGAFVPCSLATISMVDSILGRVGASDNIIKGL mRep3 RVMIITGPNMGGKSSYIKQVALVTIMAQIGSYVPAEEATIGIVDGIFTRMGAADNIYKGR mMSH2 MFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILARVGAGDSQLKGV RnorvMSH2 MFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILARVGAGDSQLKGV hMSH2 MFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILARVGAGDSQLKGV yMSH2 DFLIITGPNMGGKSTYIRQVGVISLMAQIGCFVPCEEAEIAIVDAILCRVGAGDSQLKGV zmmus1 WFQIITGPNMGGKSTFIRQVGVNVLMAQVGSFVPCDQASISVRDCIFARVGAGDCQLHGV
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EcoMutS STFMVEMTETANILHNATEYSLVLMDEIGRGTSTYDGLSLAWACAENLANKIKALTLFAT StyMutS STFMVEMTETANILHNATENSLVLMDEIGRGTSTYDGLSLAWACAENLANKIKALTLFAT SpneuHexA STFMVEMMEANNAISHATKNSLILFDELGRGTATYDGMALAQSIIEYIHEHIGAKTLFAT BsubMutS STFMVEMLEAKNAIVNATKNSLILFDEIGRGTSTYDGMALAQAIIEYVHDHIGAKTLFST AviMutS STFMVEMSETANILHNASERSLVLMDEVGRGTSTFDGLSLAWAAAEHLAG-LRAWTLFAT HinfMutS STFMVEMTEMANILHQATAQSLVLIDEIGRGTSTYDGLSLAWACAEWLSKKIRSLTLFAT TaquaMutS STFMVEMEEVALILKEATENSLVLLDEVGRGTSSLDGVAIATAVAEALHE-RRAYTLFAT Swi4 STFMVEMLETKEVLSKATEKSMVIIDELGRGTSTIDGEAISYAVLHYLNQYIKSYLLFVT Spel1 STFMVEMIETSGIIRTATDKSLVIIDELGRGTSTYEGCGIAWSIAEHLAKETKCFTLFAT mRep3 STFMEQLTDTAEIIRRASPQSLVILDELGRGTSTHDGIAIAYATLEYFIRDVKSLTLFVT mMSH2 STFMAEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAISDYIATKIGAFCMFAT RnorvMSH2 STFMAEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAISEYIATNIGAFCMFAT hMSH2 STFMAEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAISEYIATKIGAFCMFAT yMSH2 STFMVEILETASILKNASKNSLIIVDELGRGTSTYDGFGLAWAIAEHIASKIGCFALFAT zmmus1 STFMQEMLETASILKGASDKSLIIIDELGRGTSTYDGFGLAWAICEHLMEVTRAPTLFAT
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Figure 1. Alignment of the highly conserved C-terminal region of various MutS homologous proteins. The putative maize homolog, MUS1, is underlined.
EcoMutS - E. coli, StyMutS - Salmonella typhimurium, SpneuHexA - Streptococcus pneumoniae, BsubMutS - Bacillus subtilis, AviMutS - Acetobacter vinelandii, HinfMutS - Haemophilus influenzae, TaquaMutS - Thermus aquaticus, yMSH2 - Saccharomyces cerevisiae, Swi4 - Schizosaccharomyces pombe, Spel1 - Drosophila melanogaster, mMSH2 and mRep3 - Mus musculus, RnorvMSH2 - Rattus norvegicus, hMSH2 - Homo sapiens, zmmus1 - Zea mays
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