Vol.
177,
No.
June
14.
1991
2, 1991
BIOCHEMICAL
RESEARCH
COMMUNICATIONS 597-602
OF THE BAT 06-METHYLGUANINE-DNA-METHYLTRANSFERASE
Iwonna
Received
BIOPHYSICAL
Pages
cDNA CLONING
Groupe
AND
"Radiochimie
April
26,
Rahden-Staron
and Francoise
Lava1
de l'ADN", U 247 INSERM, Institut ROUSSY, 94805 Villejuif, France
Gustave
1991
A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of 06-methylguanine residues. Following three exposures to N-methyl-N*-nitro-Nnitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated . Extracts of BK2210 cells hosting the RMGMT plasmid expressed a 06-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells . The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd. The rat protein shows 68 % homology with the human 0 1991Academic Press, 1°C. transferase.
Alkylating agents form a variety of lesions in DNA ( 1 ). Among them, 06-alkylguanine is the major mutagenic and carcinogenic lesion, as it pairs with a thymine during DNA replication ( 2). 06-alkylguanine residues are also involved in the toxic effect of mono and bifunctional alkylating drugs and specially of the alkylated compounds used in cancer chemotherapy ( 3 ). These lesions are repaired by a protein , the 06-alkylguanine-DNAwhich transfers the alkyl alkyltransferase ( transferase ), group from the O6 position of guanine to one of its cysteine residue, and the alkylated protein is not regenerated ( 4 ). Two transferases exist in Escherichia coli, a 39 kD protein (Ada protein) which is inducible by treatment with alkylating 19 kD protein ( Ogt protein ) agents ( 5 ), and a noninducible activity is also present in a high ( 6 1,. A transferase proportion of mammalian cell lines ( Mer+ or Mex+ phenotype ) and the cDNA encoding the human transferase has been isolated (
7,8,9
1.
ABBREVIATIONS
MNNG: N-methy-N'-nitro-N06-meGua: 06-methylguanine; nitrosoguanidine; MNU: N-methyl-N-nitrosourea.
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
We have previously shown that the transferase actvitity was increased in different cell lines treated with DNA-damaging agents, and specially in a rat hepatoma cell line ( H4 cells ) in order to study the molecular mechanism of ( 10 1. Therefore, this enhancement and the properties of the rat protein, we have isolated a cDNA encoding the rat transferase. In this paper, we report the complete sequence of the rat transferase cDNA, named the amino acid sequence of the rat protein RMGMT, and compare with those of transferases of E. coli and human origin. MATERIALS
AND METHODS E. coli strains AB1157 and BH290 were from laboratory Cells. The BK2110 strain ( AB1157 gyrA ada ) was a gift from stocks. (11). H4 cells, epithelial cells derived from a Dr E. Seeberg medium rat hepatoma (lo), were grown at 37 "C in Dulbecco's % horse serum, supplemented with 5 % fetal calf serum g/ml). All and streptomycin (" 50 penicillin (50 units/ml) media and serums were obtained from Gibco. Plasmid pcDNA II was obtained from Invitrogen and pUC19 DNA. The [3H]MNU-treated DNA prepared was from laboratory stocks. as already described ( 10 ), had a specific activity df 1 x lo3 DNA and H4 cells DNA, isolated from c.p.m./ g . Rat liver 1~10~ cells, were prepared as already described ( 12 ). Construction of a cDNA plasmid expression library from H4 cells BH290. The cDNA library was constructed from 1x10' in E. coli cells harvested in exponential growth. 60 g of poly(A+) mRNA the were isolated and 10 g of this mRNA were used to construct from cDNA library using the Librarian II construction kit adaptors, the Invitrogen. After the addition of BstXl into the pcDNA II expression synthesized cDNA was ligated and transferrred into BH290 cells, as previously vector described ( 12 ). of the Og-methvlquanine-DNA methyltransferase Determination Bacteria and activity and polvacrylamide eel electrophoresis. H4 cells were disrupted by sonication at 0°C. After removal of the transferase assay was performed debris by centrifugation; as already described ( 10 ). For gel electrophoresis, 400 g or H4 cells proteins were incubated with 20 of bacterial [3H]MNU-treated DNA ( 2400 cpm of 06-meGua ) for 30 rniz z: then the protein separation was done in 12.5 % SDS37°C‘ polyacrylamide gels as described by Laemmli ( 13 ). The gels were fixed in methanol/acetic acid, soaked in Enhance ( New England Nuclear ) then exposed to X-ray films. In situ hybridization. Colonies resistant to MNNG were subjected to in situ hybridization as described by Maniatis ( 14 ). Briefly, colonies on nylon filters ( Hybond-N, Amersham) were lysed by alkali treatment, prehybridized then hybridized with the labeled probe. The probe was a synthetic oligonucleotide corresponding to the active site of the human transferase ( 7 ), labeled with [32 P] dATP using the 5'endlabeling Kit from Boehringer. DNA seouencino. The Xba I / Hind III fragment from the RMGMT plasmid was sequenced in Ml3 mp1S and mp19, ' chain termination with modified T7 DNA polymerase ( Sequa~~~~g( 15 ). 598
Vol.
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No.
2, 1991
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BIOPHYSICAL
RESEARCH
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Sequence of the 3 '-end of the cDNA was done using a mixture of three 20 T oligonucleotides primers (T20G, T20C and T20A )(16). DNA sequence analysis was performed using the CITI2 system and the program HCAPLCT ( 17 ). Southern and Northern blot hvbridization. 30 g of Eco Rl, Hind III or Eco Rl/Hind III digested H4 cells or rat liver DNA were separated by agarose gel electrophoresis. 20 g of total RNA from H4 cells or rat liver were separated using agarose gel electrophoresis in the presence of formaldehyde. Nucleic acids were transferred to nitrocellulose filters by capillary action. The probe for hybridization was prepared by nick translation of the XbaI/Hind III fragment of the RMGMT plasmid. activity of the probe labeled with [32P] dATP andT~'2~~ec6%~ was 5.5~10~ cpm / The hybridization was performed as described by Maniatis gi 14 ) , then the filters were dried and exposed to X-ray films. RESULTS
AND DISCUSSION
Isolation of the rat RMGMT -* cDNA The cDNA expression library was introduced by electroporation into E.coli BK2110, which is mutated in the ada gene and is therefore more sensitive to alkylating agents than the parental strain AB1157. lo7 bacteria were incubated for 1 hour with MNNG ( 100 g/ml ) then plated on LB-amplicillin plates. After two subsequent selections with MNNG, the resistant colonies were hybridized with a labeled probe corresponding to the active site of the human transferase (7). FOUr colonies showed hybridization and contained plasmids with an insert of about 800 bp. One of these colonies contained a plasmid ( RMGMT plasmid ) which conferred resistance to MNNG to BK21.10 bacteria after retransfection. To check that the RMGMT plasmid contained the rat transferase protein coding sequence, extracts from AB1157, BK2110 containing the pUC plasmid, BK2110 containing the RMGMT plasmid and H4 cells were incubated with [3H]MNlJ-treated DNA then separated on polyacrylamide gels ( Fig. 1 ). The labeled proteins from H4 cells and from BK2110 cells harboring the RMGMT plasmid have the same molecular weight, showing that the plasmid expresses the rat transferase. 2(a) shows the Nucleotide seouence of the RMGMT cDNA. Fig. strategy used for sequencing the cDNA and the nucleotide sequence is represented in Fig. 2 (b). The cDNA is 763 bp long with an open reading frame of 209 amino acids. The protein has a calculated molecular weight of 22.2 kd, which is similar to that of the human transferase ( 21.7 kd ) ( 7,8,9 ). The rat transferase is a basic protein with a calculated p1 of 8.0 at pH 7. An alignement of the rat RMGMT protein and of the human 599
Vol.
177,
No.
BIOCHEMICAL
2, 1991 1
AND 2
BIOPHYSICAL
RESEARCH
kDa
4
3
COMMUNICATIONS
-61
-43
-30
-20
Fiq.1. Fluorography of H4 cells or E.coli extracts. Extracts were incubated for 30 min at 37°C with r3H]MNU-treated (1) H4 cells, DNA then subjected to SDS-PAGE electrophoresis. (3) BK2110 + pUC plasmid,(4) (2) BK2110 + RMGMT plasmid, AB1157.
b
1
16
31
61
46
76
AAACTCGAACTTGGCAGA ATG GCT GAG ATC TGC AAA ATG AAA TAC ACG GTG T T G GAC AGC CCT T T G GGG AAG ATA GAG CTG TCC GGC T G T blet Ala Glu Ile Cys Lys Met Lys Tyr Thr Val Leu Asp Ser Pro Leu Gly Lys Ile Glu Leu Ser Gly CyS 91
106
121
136
166
151
GAG CGA GGC CTG CAT GGG ATA CGA T T T CTC AGl GGG AAG ACC CCA AAC ACT GAC CCC ACA GAG GCl CCA CCC T G T CCT GAG GTG CTC GGT Glu Arg Gly Leu His Gly Ile Arg Phe Leu Ser Gly Lys Thr Pro Asn Thr Asp Pro Thr Glu Ala Pro Ala Cys Pro Glu Val Leu Gly 226 241 181 196 211 GGG CCA GAG GGA GTG CCA GAG CCC CTG GTG CAG TGC ACA GCC TGG CTG GA,, GCC T A T TTC CAC GM sly Pro Glu Gly Val Pro Glu Pro Leu Val Gln Cys Thr Ale Trp Leu Glu Ala Tyr Phe His Glu
256 CCT GCA GCC ACA GAG GGG CTT CCC Pro Ala Ala Thr Glu Gly Leu Pro
316 331 346 271 2% 301 T T G CCT GCT CTC CAT CAC CCT GTG TTC CAG CAA GAT TCA TTC ACC AGA CAG GTG T T A TGG AAG CTG CTG AAG G T T GTG AAA TTC CGA GAA Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu 361 376 ATG G T T TCT TAC CAG cM Met Val Ser Tyr Gin Gin
391 T T A GCA GCC CTG GCA GGC Ax Leu Ala Ala Leu ALa GLy Am
406 ccc Pro
421
436
AAA GCG GCT CGT GCA GTA CGA GGA CCC ATG AGG AGC MT CCA GTC CCC Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
466 481 496 511 526 451 ATC CTC ATC CCC TGC CAC AGG GTG A T T CGC AGT GAC GGT CCC A T T GGC AAT TAC TCT GGA GGA CGA CAG ACT GTG AAA GAG TGG CTT CTG Ile
Leu
ILe
Pro
Cys His
Arg
Vat
Ile
Arg
Ser Asp Gly
Ala
Ile
GLy Asn Tyr
Ser
GLy Gly
Gly
Gin
Thr
Vat
Lys
Glu
Trp
Leu
Leu
541 556 571 586 601 616 GCC CAT GAG GGC ATC CCA ACT CGA CAG CCG GCC TCC AA4 GGC T T G GGT CTG A T T GGG AGC TGG CTC AAG CCA TCC TTC GAG TCT TCC AGC Ala His Glu Gly ILe Pro Thr Gly Gin Pro Ala Ser Lys Gly Leu GLy Leu Ile Gly Ser Trp Leu Lys Pro Ser Phe Glu Ser Ser Ser 631 646 665 684 704 724 CCA AAG CCG TCT GGC TGA AATTGAGTMCCGTTTGAATGACACATAGATGTMTGCGGTGTTGGMGCGGATGTGTGGTGGGTACCACTATATT~GAGCTGCATGTGTCC Pro Lys Pro Ser Gly l ** 743 TGGGG-AAAAAA
763
Fig.2. a) Sequencing strategy b) Nucleotide sequence of the amino acid sequence is predicted
for the BMGMT cDNA. H4 cells transferase cDNA. from the nucleotideseguence. 600
The
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
70 10 20 30 40 50 60 MAEICK~YTVLDS~LGKIELSGCERGLHGIRFLSGKT~NTD~T~~AC~~~LGG~~G~PEPLVQCTAWL ----= = == = =======-===__--=- == -= = cc ====== = -=m====== MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSDAV~AP~V~GPE--P--~QCTAWL 60 10 20 30 40 50 110 120 130 140 80 90 100 EAYFHEPAATEGLPLPALHHPVFQQDSFTRQVLWKLLKVVS -====_= = = =-==========-==================__===============------====NAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPXAARAVGGAMRG 80 90 100 110 120 130 70 180 190 200 150 160 170 NPVPIL,IPCHRVIRSDGAIGNYSGGGQTVKEWLLAHEGIPTGQPASKGL-GLIGSWLKPSFESSSPKPCG ------= =_ = == =-=== =====c:======= ==-======-===_______== = NPVPILIPCHRWCSSGAVGNYSGG-LAVKEWLLAHEGHRLGKPGLGGSSGLAGAWLKGAGATSGSPPAG 150 160 170 180 190 200 140
Fig.3. Sequence identity between the rat and human transferase proteins. A single line indicates a conserved amino acid, a double line indicates an identical amino acid. protein ( Fig.3 ) shows about 68 % homology. Comparison of the predicted amino acid sequence of the rat transferase with the C-terminal domain of the E. coli Ada and Ogt proteins show 32 and 52 % homology, respectively ( data not shown ). Southern and Northern analysis. The Southern analysis of H4 cells and normal liver DNA are shown in Fig.4. The same patterns are obtained with the two DNA and suggest that the minimum size of the gene is about 15 kb. Northern analysis of H4 cells and rat liver RNA shows that a single transcript is produced at about 1000 bp (Fig.5). These results suggest that probably the same protein exists in transformed and normal rat liver cells. There is a high homology between the rat and the human or the E. coli transferases. This homology is not only found in the region around the active cystein residue, but also extends
1
2
I,’
(
3
I
kb
2
-23
S
-16s
Fiq.4. DNA ( digestion Rl/Hind Fiq.5. rat liver
Southern blot analysis of H4 cells( ).The DNA were probed with the Hind III with Eco Rl ( lane l), III ( lane 3 ).
b
Northern blot analysis RNA ( lane b ).
of
601
H4 cells
a ) and rat liver RMGMT cDNA after ( lane 2 ) or Eco RNA ( lane
a )
or
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BIOCHEMICAL
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RESEARCH
COMMUNICATIONS
to the N- and C-terminal of the human protein and to the CA high degree of homology terminal of the bacterial proteins. also exists between the rat ( 12 ), the human ( 18 ) and the E. suggesting that a high coli 3-methyladenine-DNA-glycosylases, degree of conservation might exist in DNA repair proteins . The level of the rat transferase can be increased in cells treated with DNA-damaging agents ( 10 ) and this cDNA has been used to show an enhanced transcription of the gene in the treated cells ( 19 1. Acknowledgments Lescot for the synthesis of The authors thank Dr. E. O'Connor for helpful discussions. oligonucleotides and Dr.T.R. The excellent technical assistance of Mrs. Martine Letourneur was a recipient of a was greatly appeciated. I. Rahden-Staron fellowship from the Institut National de la Sante et de la Recherche Medicale (INSERM) while on leave from Institute of Poland. This work was Biopharmacy, Warsaw Medical School, supported by Grants from INSERM and Association pour la Recherche sur le Cancer ( Villejuif ). REFERENCES
1. Singer,B. and Grunberger,D. ( 1983 ) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York. 2. Loechler,E.L., Green,C.L., and Essigman,J.M. ( 1984 ) Proc. Natl.Acad.Sci.USA 81, 6271-6275 3. Ludlum,D.B. ( 1990 ) Mut. Res. 233, 117-126. 4. Demple,B., Jacobson,A., Olsson,M., Robins,P. and Lindahl,T. ( 1982 ) J. Biol. Chem. 257, 13776-13780 5. Lindahl,T., Sedgwick,B., Sekiguchi,M. and Nakabeppu,Y. ( 1988 ) Ann. Rev. Biochem. 57, 133-157 6. Potter,P.M., Wilkinson,M.C., Fitton,J., Carr,F.J., Brennand,J., Cooper,D.P. and Margison,G.P. ( 1987 ) Nucleic Acids Res. 15, 9177-9193 7. Rydberg,B., Spurr,N. and Karran,P. ( 1990 ) Jal. Biol. Chem. 265, 9563-9569 8. Tano,K., Shiota,S., Collier,J., Foote,R.S. and Mitra,S. ( 1990 ) Proc. Natl. Acad. Sci. USA, 87, 686-690 9. Koike,G., Maki,H., Takeya,H., Hayakawa,H. and Sekiguchi,M. ( 1990 ) Jal. Biol. Chem. 265, 14754-14762 lO.Lefebvre,P. and Laval,F. ( 1986 ) Cancer Res. 46,5701-5705 ll.Evensen,G. and Seeberg,E. ( 1982 ) Nature 296, 773-775 12.0'Connor,T.R. and Laval,F. ( 1990 ) EMBO J. 9, 3337-3342 13.Laemmli,U.K. ( 1970 ) Nature 227, 680-685 14.Maniatis,T., Fritsch,E.F. and Sambroock,J. ( 1989 ) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. 15,Tabor,S. and Richardson,C.C. ( 1978 ) Proc. Natl. Acad. Sci. USA. 84, 4767-4771 16.Thweatt,R., Goldstein,S. and Shmookler Reis,R.J. ( 1990 ) Anal. Biochem. 190, 314-316. 17.Gaboriaud,C. Bissery,V., Benchetrit,T. and Mornon,J.P.(1987) FEBS Lett. 224, 149-155 18.0'Connor,T.R. and Laval,J. ( 1991 ) Biochem. Biophys. Res. Commun. in press. 19. Laval,F., ( 1991 ) Biochem. Biophys. Res. Commun. in press. 602
177,
No.
June
14.
1991
2, 1991
BIOCHEMICAL
RESEARCH
COMMUNICATIONS 597-602
OF THE BAT 06-METHYLGUANINE-DNA-METHYLTRANSFERASE
Iwonna
Received
BIOPHYSICAL
Pages
cDNA CLONING
Groupe
AND
"Radiochimie
April
26,
Rahden-Staron
and Francoise
Lava1
de l'ADN", U 247 INSERM, Institut ROUSSY, 94805 Villejuif, France
Gustave
1991
A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of 06-methylguanine residues. Following three exposures to N-methyl-N*-nitro-Nnitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated . Extracts of BK2210 cells hosting the RMGMT plasmid expressed a 06-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells . The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd. The rat protein shows 68 % homology with the human 0 1991Academic Press, 1°C. transferase.
Alkylating agents form a variety of lesions in DNA ( 1 ). Among them, 06-alkylguanine is the major mutagenic and carcinogenic lesion, as it pairs with a thymine during DNA replication ( 2). 06-alkylguanine residues are also involved in the toxic effect of mono and bifunctional alkylating drugs and specially of the alkylated compounds used in cancer chemotherapy ( 3 ). These lesions are repaired by a protein , the 06-alkylguanine-DNAwhich transfers the alkyl alkyltransferase ( transferase ), group from the O6 position of guanine to one of its cysteine residue, and the alkylated protein is not regenerated ( 4 ). Two transferases exist in Escherichia coli, a 39 kD protein (Ada protein) which is inducible by treatment with alkylating 19 kD protein ( Ogt protein ) agents ( 5 ), and a noninducible activity is also present in a high ( 6 1,. A transferase proportion of mammalian cell lines ( Mer+ or Mex+ phenotype ) and the cDNA encoding the human transferase has been isolated (
7,8,9
1.
ABBREVIATIONS
MNNG: N-methy-N'-nitro-N06-meGua: 06-methylguanine; nitrosoguanidine; MNU: N-methyl-N-nitrosourea.
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
We have previously shown that the transferase actvitity was increased in different cell lines treated with DNA-damaging agents, and specially in a rat hepatoma cell line ( H4 cells ) in order to study the molecular mechanism of ( 10 1. Therefore, this enhancement and the properties of the rat protein, we have isolated a cDNA encoding the rat transferase. In this paper, we report the complete sequence of the rat transferase cDNA, named the amino acid sequence of the rat protein RMGMT, and compare with those of transferases of E. coli and human origin. MATERIALS
AND METHODS E. coli strains AB1157 and BH290 were from laboratory Cells. The BK2110 strain ( AB1157 gyrA ada ) was a gift from stocks. (11). H4 cells, epithelial cells derived from a Dr E. Seeberg medium rat hepatoma (lo), were grown at 37 "C in Dulbecco's % horse serum, supplemented with 5 % fetal calf serum g/ml). All and streptomycin (" 50 penicillin (50 units/ml) media and serums were obtained from Gibco. Plasmid pcDNA II was obtained from Invitrogen and pUC19 DNA. The [3H]MNU-treated DNA prepared was from laboratory stocks. as already described ( 10 ), had a specific activity df 1 x lo3 DNA and H4 cells DNA, isolated from c.p.m./ g . Rat liver 1~10~ cells, were prepared as already described ( 12 ). Construction of a cDNA plasmid expression library from H4 cells BH290. The cDNA library was constructed from 1x10' in E. coli cells harvested in exponential growth. 60 g of poly(A+) mRNA the were isolated and 10 g of this mRNA were used to construct from cDNA library using the Librarian II construction kit adaptors, the Invitrogen. After the addition of BstXl into the pcDNA II expression synthesized cDNA was ligated and transferrred into BH290 cells, as previously vector described ( 12 ). of the Og-methvlquanine-DNA methyltransferase Determination Bacteria and activity and polvacrylamide eel electrophoresis. H4 cells were disrupted by sonication at 0°C. After removal of the transferase assay was performed debris by centrifugation; as already described ( 10 ). For gel electrophoresis, 400 g or H4 cells proteins were incubated with 20 of bacterial [3H]MNU-treated DNA ( 2400 cpm of 06-meGua ) for 30 rniz z: then the protein separation was done in 12.5 % SDS37°C‘ polyacrylamide gels as described by Laemmli ( 13 ). The gels were fixed in methanol/acetic acid, soaked in Enhance ( New England Nuclear ) then exposed to X-ray films. In situ hybridization. Colonies resistant to MNNG were subjected to in situ hybridization as described by Maniatis ( 14 ). Briefly, colonies on nylon filters ( Hybond-N, Amersham) were lysed by alkali treatment, prehybridized then hybridized with the labeled probe. The probe was a synthetic oligonucleotide corresponding to the active site of the human transferase ( 7 ), labeled with [32 P] dATP using the 5'endlabeling Kit from Boehringer. DNA seouencino. The Xba I / Hind III fragment from the RMGMT plasmid was sequenced in Ml3 mp1S and mp19, ' chain termination with modified T7 DNA polymerase ( Sequa~~~~g( 15 ). 598
Vol.
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No.
2, 1991
BIOCHEMICAL
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Sequence of the 3 '-end of the cDNA was done using a mixture of three 20 T oligonucleotides primers (T20G, T20C and T20A )(16). DNA sequence analysis was performed using the CITI2 system and the program HCAPLCT ( 17 ). Southern and Northern blot hvbridization. 30 g of Eco Rl, Hind III or Eco Rl/Hind III digested H4 cells or rat liver DNA were separated by agarose gel electrophoresis. 20 g of total RNA from H4 cells or rat liver were separated using agarose gel electrophoresis in the presence of formaldehyde. Nucleic acids were transferred to nitrocellulose filters by capillary action. The probe for hybridization was prepared by nick translation of the XbaI/Hind III fragment of the RMGMT plasmid. activity of the probe labeled with [32P] dATP andT~'2~~ec6%~ was 5.5~10~ cpm / The hybridization was performed as described by Maniatis gi 14 ) , then the filters were dried and exposed to X-ray films. RESULTS
AND DISCUSSION
Isolation of the rat RMGMT -* cDNA The cDNA expression library was introduced by electroporation into E.coli BK2110, which is mutated in the ada gene and is therefore more sensitive to alkylating agents than the parental strain AB1157. lo7 bacteria were incubated for 1 hour with MNNG ( 100 g/ml ) then plated on LB-amplicillin plates. After two subsequent selections with MNNG, the resistant colonies were hybridized with a labeled probe corresponding to the active site of the human transferase (7). FOUr colonies showed hybridization and contained plasmids with an insert of about 800 bp. One of these colonies contained a plasmid ( RMGMT plasmid ) which conferred resistance to MNNG to BK21.10 bacteria after retransfection. To check that the RMGMT plasmid contained the rat transferase protein coding sequence, extracts from AB1157, BK2110 containing the pUC plasmid, BK2110 containing the RMGMT plasmid and H4 cells were incubated with [3H]MNlJ-treated DNA then separated on polyacrylamide gels ( Fig. 1 ). The labeled proteins from H4 cells and from BK2110 cells harboring the RMGMT plasmid have the same molecular weight, showing that the plasmid expresses the rat transferase. 2(a) shows the Nucleotide seouence of the RMGMT cDNA. Fig. strategy used for sequencing the cDNA and the nucleotide sequence is represented in Fig. 2 (b). The cDNA is 763 bp long with an open reading frame of 209 amino acids. The protein has a calculated molecular weight of 22.2 kd, which is similar to that of the human transferase ( 21.7 kd ) ( 7,8,9 ). The rat transferase is a basic protein with a calculated p1 of 8.0 at pH 7. An alignement of the rat RMGMT protein and of the human 599
Vol.
177,
No.
BIOCHEMICAL
2, 1991 1
AND 2
BIOPHYSICAL
RESEARCH
kDa
4
3
COMMUNICATIONS
-61
-43
-30
-20
Fiq.1. Fluorography of H4 cells or E.coli extracts. Extracts were incubated for 30 min at 37°C with r3H]MNU-treated (1) H4 cells, DNA then subjected to SDS-PAGE electrophoresis. (3) BK2110 + pUC plasmid,(4) (2) BK2110 + RMGMT plasmid, AB1157.
b
1
16
31
61
46
76
AAACTCGAACTTGGCAGA ATG GCT GAG ATC TGC AAA ATG AAA TAC ACG GTG T T G GAC AGC CCT T T G GGG AAG ATA GAG CTG TCC GGC T G T blet Ala Glu Ile Cys Lys Met Lys Tyr Thr Val Leu Asp Ser Pro Leu Gly Lys Ile Glu Leu Ser Gly CyS 91
106
121
136
166
151
GAG CGA GGC CTG CAT GGG ATA CGA T T T CTC AGl GGG AAG ACC CCA AAC ACT GAC CCC ACA GAG GCl CCA CCC T G T CCT GAG GTG CTC GGT Glu Arg Gly Leu His Gly Ile Arg Phe Leu Ser Gly Lys Thr Pro Asn Thr Asp Pro Thr Glu Ala Pro Ala Cys Pro Glu Val Leu Gly 226 241 181 196 211 GGG CCA GAG GGA GTG CCA GAG CCC CTG GTG CAG TGC ACA GCC TGG CTG GA,, GCC T A T TTC CAC GM sly Pro Glu Gly Val Pro Glu Pro Leu Val Gln Cys Thr Ale Trp Leu Glu Ala Tyr Phe His Glu
256 CCT GCA GCC ACA GAG GGG CTT CCC Pro Ala Ala Thr Glu Gly Leu Pro
316 331 346 271 2% 301 T T G CCT GCT CTC CAT CAC CCT GTG TTC CAG CAA GAT TCA TTC ACC AGA CAG GTG T T A TGG AAG CTG CTG AAG G T T GTG AAA TTC CGA GAA Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu 361 376 ATG G T T TCT TAC CAG cM Met Val Ser Tyr Gin Gin
391 T T A GCA GCC CTG GCA GGC Ax Leu Ala Ala Leu ALa GLy Am
406 ccc Pro
421
436
AAA GCG GCT CGT GCA GTA CGA GGA CCC ATG AGG AGC MT CCA GTC CCC Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
466 481 496 511 526 451 ATC CTC ATC CCC TGC CAC AGG GTG A T T CGC AGT GAC GGT CCC A T T GGC AAT TAC TCT GGA GGA CGA CAG ACT GTG AAA GAG TGG CTT CTG Ile
Leu
ILe
Pro
Cys His
Arg
Vat
Ile
Arg
Ser Asp Gly
Ala
Ile
GLy Asn Tyr
Ser
GLy Gly
Gly
Gin
Thr
Vat
Lys
Glu
Trp
Leu
Leu
541 556 571 586 601 616 GCC CAT GAG GGC ATC CCA ACT CGA CAG CCG GCC TCC AA4 GGC T T G GGT CTG A T T GGG AGC TGG CTC AAG CCA TCC TTC GAG TCT TCC AGC Ala His Glu Gly ILe Pro Thr Gly Gin Pro Ala Ser Lys Gly Leu GLy Leu Ile Gly Ser Trp Leu Lys Pro Ser Phe Glu Ser Ser Ser 631 646 665 684 704 724 CCA AAG CCG TCT GGC TGA AATTGAGTMCCGTTTGAATGACACATAGATGTMTGCGGTGTTGGMGCGGATGTGTGGTGGGTACCACTATATT~GAGCTGCATGTGTCC Pro Lys Pro Ser Gly l ** 743 TGGGG-AAAAAA
763
Fig.2. a) Sequencing strategy b) Nucleotide sequence of the amino acid sequence is predicted
for the BMGMT cDNA. H4 cells transferase cDNA. from the nucleotideseguence. 600
The
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
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70 10 20 30 40 50 60 MAEICK~YTVLDS~LGKIELSGCERGLHGIRFLSGKT~NTD~T~~AC~~~LGG~~G~PEPLVQCTAWL ----= = == = =======-===__--=- == -= = cc ====== = -=m====== MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSDAV~AP~V~GPE--P--~QCTAWL 60 10 20 30 40 50 110 120 130 140 80 90 100 EAYFHEPAATEGLPLPALHHPVFQQDSFTRQVLWKLLKVVS -====_= = = =-==========-==================__===============------====NAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPXAARAVGGAMRG 80 90 100 110 120 130 70 180 190 200 150 160 170 NPVPIL,IPCHRVIRSDGAIGNYSGGGQTVKEWLLAHEGIPTGQPASKGL-GLIGSWLKPSFESSSPKPCG ------= =_ = == =-=== =====c:======= ==-======-===_______== = NPVPILIPCHRWCSSGAVGNYSGG-LAVKEWLLAHEGHRLGKPGLGGSSGLAGAWLKGAGATSGSPPAG 150 160 170 180 190 200 140
Fig.3. Sequence identity between the rat and human transferase proteins. A single line indicates a conserved amino acid, a double line indicates an identical amino acid. protein ( Fig.3 ) shows about 68 % homology. Comparison of the predicted amino acid sequence of the rat transferase with the C-terminal domain of the E. coli Ada and Ogt proteins show 32 and 52 % homology, respectively ( data not shown ). Southern and Northern analysis. The Southern analysis of H4 cells and normal liver DNA are shown in Fig.4. The same patterns are obtained with the two DNA and suggest that the minimum size of the gene is about 15 kb. Northern analysis of H4 cells and rat liver RNA shows that a single transcript is produced at about 1000 bp (Fig.5). These results suggest that probably the same protein exists in transformed and normal rat liver cells. There is a high homology between the rat and the human or the E. coli transferases. This homology is not only found in the region around the active cystein residue, but also extends
1
2
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(
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2
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Fiq.4. DNA ( digestion Rl/Hind Fiq.5. rat liver
Southern blot analysis of H4 cells( ).The DNA were probed with the Hind III with Eco Rl ( lane l), III ( lane 3 ).
b
Northern blot analysis RNA ( lane b ).
of
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to the N- and C-terminal of the human protein and to the CA high degree of homology terminal of the bacterial proteins. also exists between the rat ( 12 ), the human ( 18 ) and the E. suggesting that a high coli 3-methyladenine-DNA-glycosylases, degree of conservation might exist in DNA repair proteins . The level of the rat transferase can be increased in cells treated with DNA-damaging agents ( 10 ) and this cDNA has been used to show an enhanced transcription of the gene in the treated cells ( 19 1. Acknowledgments Lescot for the synthesis of The authors thank Dr. E. O'Connor for helpful discussions. oligonucleotides and Dr.T.R. The excellent technical assistance of Mrs. Martine Letourneur was a recipient of a was greatly appeciated. I. Rahden-Staron fellowship from the Institut National de la Sante et de la Recherche Medicale (INSERM) while on leave from Institute of Poland. This work was Biopharmacy, Warsaw Medical School, supported by Grants from INSERM and Association pour la Recherche sur le Cancer ( Villejuif ). REFERENCES
1. Singer,B. and Grunberger,D. ( 1983 ) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York. 2. Loechler,E.L., Green,C.L., and Essigman,J.M. ( 1984 ) Proc. Natl.Acad.Sci.USA 81, 6271-6275 3. Ludlum,D.B. ( 1990 ) Mut. Res. 233, 117-126. 4. Demple,B., Jacobson,A., Olsson,M., Robins,P. and Lindahl,T. ( 1982 ) J. Biol. Chem. 257, 13776-13780 5. Lindahl,T., Sedgwick,B., Sekiguchi,M. and Nakabeppu,Y. ( 1988 ) Ann. Rev. Biochem. 57, 133-157 6. Potter,P.M., Wilkinson,M.C., Fitton,J., Carr,F.J., Brennand,J., Cooper,D.P. and Margison,G.P. ( 1987 ) Nucleic Acids Res. 15, 9177-9193 7. Rydberg,B., Spurr,N. and Karran,P. ( 1990 ) Jal. Biol. Chem. 265, 9563-9569 8. Tano,K., Shiota,S., Collier,J., Foote,R.S. and Mitra,S. ( 1990 ) Proc. Natl. Acad. Sci. USA, 87, 686-690 9. Koike,G., Maki,H., Takeya,H., Hayakawa,H. and Sekiguchi,M. ( 1990 ) Jal. Biol. Chem. 265, 14754-14762 lO.Lefebvre,P. and Laval,F. ( 1986 ) Cancer Res. 46,5701-5705 ll.Evensen,G. and Seeberg,E. ( 1982 ) Nature 296, 773-775 12.0'Connor,T.R. and Laval,F. ( 1990 ) EMBO J. 9, 3337-3342 13.Laemmli,U.K. ( 1970 ) Nature 227, 680-685 14.Maniatis,T., Fritsch,E.F. and Sambroock,J. ( 1989 ) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. 15,Tabor,S. and Richardson,C.C. ( 1978 ) Proc. Natl. Acad. Sci. USA. 84, 4767-4771 16.Thweatt,R., Goldstein,S. and Shmookler Reis,R.J. ( 1990 ) Anal. Biochem. 190, 314-316. 17.Gaboriaud,C. Bissery,V., Benchetrit,T. and Mornon,J.P.(1987) FEBS Lett. 224, 149-155 18.0'Connor,T.R. and Laval,J. ( 1991 ) Biochem. Biophys. Res. Commun. in press. 19. Laval,F., ( 1991 ) Biochem. Biophys. Res. Commun. in press. 602
