- Email: [email protected]
LIZ shuttle vector
Mutation Research 375 Ž1997. 79–82
The recovery of a B2 insertion in the lacI gene of a rat cell line containing a lrLIZ shuttle vector Johan G. de Boer 1, Andrew Suri, David Walsh, Barry W. Glickman
)
Center for EnÕironmental Health, Department of Biology, P.O. Box 3200, UniÕersity of Victoria, Victoria BC, V8W 3N5, Canada Received 3 September 1996; revised 13 November 1996; accepted 25 November 1996
Abstract The bacterial lacI gene is in use as a reporter gene for mutation in transgenic mice, rats and in cell lines. During a mutagenesis study in which we used such a rat cell line, we recovered a mutant which features an insertion of a rat B2 repeated sequence element. This sequence element was inserted between positions 77 and 78 in the lacI gene. When the inserted sequence was compared to the published rat B2 sequences, two short deletions, possibly mediated by short repeat sequences, were revealed. This is the first demonstration of the recovery of a repeated sequence element into the lacI gene used in transgenic animals and cell lines, and confirms previous findings that such sequences indeed move around the genome. Keywords: Retroposon; Big Blue; lacI transgene; Repeated sequence
1. Introduction Repeated sequences are ubiquitous constituents of the mammalian genome. The insertion of repeated sequence elements into expressed genes has been found to be associated with a number of human diseases. For example, Wallace et al. w1x reported the insertion of an Alu sequence into an intron of the neurofibromatosis gene in an NF1 patient that resulted in the deletion of an exon during splicing. Similarly, Janinic et al. w2x identified an Alu insertion into the calcium-sensing receptor gene in a patient
) Corresponding author. Tel.: 1 250 427 4067; Fax: 1 250 472 4075; E-mail: [email protected] 1 Tel: Ž250 .-472-4067. Fax: Ž 250 . -472-4075. Email: [email protected].
with familial hypocalciuric hyperglycemia and neonatal severe hyperparathyroidism, while a patient with severe haemophilia B displayed a de novo insertion of an Alu sequence into the factor IX gene w3x. The insertion of repeated sequence elements has also been observed in animals. B2 sequence elements are short repetitive sequences present in approximately 100 000 copies in the rodent genome w4x. Their movement has been found to be associated with several mutations. For example, a B2-repeat sequence insertion changes the expression of the g-phosphorylase kinase gene in mice w5x, while B2 sequences found in the C4 gene of certain mouse strains are associated with aberrant splicing of that gene w6,7x. One event thought to have occurred in the early 1970s was the integration of a B2 element in an 18S rRNA gene in a murine cell line w8x. During a mutagenesis study in which we used a
0027-5107r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 6 . 0 0 2 5 5 - 2
80
J.G. de Boer et al.r Mutation Research 375 (1997) 79–82
rat cell line containing a stably integrated lacI mutation target gene w9x, we recovered a mutant which displayed an increased length of the PCR product that includes the lacI gene w10x. In this communication we report the first recovery of a B2 element in a target gene used for the recovery of mutations.
2. Materials and methods The rat fibroblast Rat2 cell line, transgenic for the lrLIZ vector containing a lambda chromosome and the bacterial lacI gene was obtained from Stratagene w9x. The cells were grown in Dulbecco’s Modified Eagle Medium ŽGibco BRL, Gaithersburg, MD.. The protocol has been described by Suri et al. w10x. Menadione was obtained from Sigma ŽSt. Louis, MO.. Genomic DNA was isolated and mutants were recovered following standard protocols as described by Stratagene. Briefly, cells were treated with 2.5 mgrml menadione for 30 minutes, washed, and allowed expression for 5 days. Genomic DNA was isolated from the cells, using a dialysis method ŽR. Winegar, unpublished, Stanford Research Institute, Menlo Park, CA., and individual l chromosomes were recognised and packaged with l packaging extract ŽTranspack, Stratagene.. Bacteriophage l particles were plated in the presence of X-gal and blue plaques, indicating a mutation in the lacI gene, identified and cored using a Pasteur pipette. The lacI gene was amplified from these l bacteriophage particles by PCR. The lacI gene was sequenced using
cycling sequencing and automated analysis using a Pharmacia ALF DNA sequencer. Detailed protocols have been published elsewhere w11x.
3. Results and discussion During a study designed to measure the mutagenic effect of menadione and magnetic fields in a rat cell line containing a stably integrated lacI mutation target gene w10x, we recovered mutants after exposure to 2.5 mgrml menadione. The mutant frequency after this exposure was 15 = 10y5 . After PCR amplification of a 1280 base pair fragment containing the lacI gene of these mutants, one mutant was observed to yield a fragment of approximately 1415 base pairs, indicating an insertion. Subsequent sequencing of this DNA fragment revealed an insertion of 135 base pairs between nucleotide positions 77 and 78 ŽFig. 1A., which was not a duplication of a sequence found elsewhere in the wild-type lacI gene. No other alterations were detected. A sequence homology search of GenBank revealed a close homology with the rat B2 repeated sequence ŽEMBL entry X14119., w12x. Fig. 1 shows an alignment between the inserted sequence and the Žinverted. B2 sequence. The inserted sequence differs from the published rat B2 sequence w12x. An eleven base pair sequence, flanked by a 5X-CA-3X dinucleotide direct repeat has been deleted from the B2 sequence. In addition, a 12-basepair sequence
X X Fig. 1. ŽA. Site of integration in the lacI sequence Žbetween position 77 and 78.. ŽB. Sequence 5 to 3 as found in the lacI gene Žreverse compared to the B2 sequence.. Top line: the inserted sequence; Bottom line: the rat B2 sequence ŽEMBL-X14119.. Lower case letters indicate a mismatch. Underlined letters indicate a repeated sequence.
J.G. de Boer et al.r Mutation Research 375 (1997) 79–82
X Fig. 2. The top line is the 5 -end of the B2 sequence ŽEMBLX X14119.. In the insertion it is shown as the 3 -end in Fig. 1. The underlined nucleotides indicate the end of the sequence that is inserted into the lacI gene. The bottom line is the sequence from position 66 to 88 in the lacI gene. The space indicates the site of insertion. Lower case letters indicate a mismatch.
was deleted, which resulted in the generation of a repeat of the sequence 5X-TGGTTG-3X which was found at the 5X side of this deletion. At the 5X endpoint of the insertion a stretch of nine thymidine residues is found. This end of the insertion corresponds to the 3X end of the B2 sequence, and is indicative of the residual polyŽA. tail sequence. In addition, the inserted sequence contains at least 3 Žoverlapping. polyadenylation signals ŽAATAAA, or TTTATT in the reported sequence.. The sequence flanking the 3X-end of the inserted part of the B2 element Ž5X-GTGCTCTTAAC-3X , which is actually the 5X-end of the B2 sequence. shows a 9 out of 11 nucleotide homology to the sequence just 5X of the insertion site in the lacI gene. The alignment is shown in Fig. 2. This homology suggests a sequence directed invasion into the gene. The orientation of the inserted sequence Ž3X ™ 5X . is the same as the sequence homology. The endpoints of inserted sequence elements frequently feature short direct repeat sequences. The insertion of a B2 element in an 18S rDNA gene in the murine cell line PYS-2r12 w8x has a perfect 15 b a se p a ir fla n k in g d ire c t re p e a t Ž 5 X AAAAAGCTCGTAGTT-3X .. The insertion into the lacI gene features only a 2-basepair flanking repeat Ž5X-AT-3X .. However, the complement of the sequence 5X of the insertion site is 5X-AAGAGACACC3X . This sequence is similar to 5X-CAGAGGAGGACAG-3X , which is found as the short repeated flanking sequences of the B2 insertion in intron 13 of the mouse C4 gene w7x. The flanking repeats are however not always the same. A B2 insertion in the mouse g-phosphorylase kinase gene is flanked by 5XAAAGATTTATG-3X repeats w5x. As far as we are aware, this is the first reported case of the recovery of a retroposon jump into a mutation target gene. The mutant was recovered after treatment with menadione, a compound that gener-
81
ates superoxide radicals w13x and induces single- and double-strand breaks w14x. We have no way of knowing whether this treatment triggered the B2 insertion event, however, this insertion is the first to be found in over 8000 mutants we have sequenced from lacI transgenic animals and cell lines. B2 retroposon sequences are specific to rodents, including mice, rats, and Chinese hamsters, and occur in large numbers w4x. These sequences are very active in transcription, with most transcripts found in hnRNA, and some in mRNA w4x. Such active transcription may facilitate insertion in other sites in the genome. Heng et al. w15x reported that the 1.8 Mb insert of 40 copies of lrLIZ DNA into the genome of the Big Blue w mice is not contained in the chromatin domain of chromosome 4, and does not interact with the chromosome core. Nevertheless, our finding indicates that retroposon jumps can occur into heterologous DNA, and extends the finding by Oberbaumer w8x that these sequences can move throughout a mammalian genome.
Acknowledgements The authors thank Andrew McArthur for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Strategic grant to BWG.
References w1x Wallace, M.R., Andersen, L.B., Saulino, A.M., Gregory, P.E., Glover, T.W. and Collins, F.S. Ž1991. A de novo Alu insertion results in neurofibromatosis type 1. Nature, 353, 864–866. w2x Janicic, N., Pausova, Z., Cole, D.E. and Hendy, G.N. Ž1995. Insertion of an Alu sequence in the CaŽ2q.-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Am. J. Hum. Genet., 56, 880– 886. w3x Vidaud, D., Vidaud, M., Bahnak, B.R., Siguret, V., Gispert Sanchez, S., Laurian, Y., Meyer, D., Goossens, M. and Lavergne, J.M. Ž1993. Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene. Eur. J. Hum. Genet., 1, 30–36. w4x Rogers, J.H. Ž1985. The origin and evolution of retroposons. Inter. Rev. Cytol., 93, 187–279.
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J.G. de Boer et al.r Mutation Research 375 (1997) 79–82
w5x Maichele, A., Farwell, N.J. and Chamberlain, J.S. Ž1993. A B2 repeat insertion generates alternate structures of the mouse muscle gamma-phosphorylase kinase gene. Genomics, 16, 139–149. w6x Zheng, J.H., Natsuume-Sakai, S., Takahashi, M. and Nonaka, M. Ž1992. Insertion of the B2 sequence into intron 13 is the only defect of the H-2k C4 gene which causes low C4 production. Nucleic Acids Res., 20, 4975–4979. w7x Pattanakitsakul, S., Zheng, J.H., Natsuume-Sakai, S., Takahashi, M. and Nonaka, M. Ž1992. Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice. J. Biol. Chem., 267, 7814–7820. w8x Oberbaumer, I. Ž1992. Retroposons do jump: a B2 element recently integrated in an 18S rDNA gene. Nucleic Acids Res., 20, 671–677. w9x Wyborski, D., Malkhosyan, S., Moores, J., Perucho, M. and Short, J. Ž1995. Development of a rat cell line containing stably integrated copies of a lambdar lacI shuttle vector. Mutation Res., 334, 161–166. w10x Suri, A., De Boer, J.G., Kusser, W.C. and Glickman, B.W. Ž1996. A 3 milliTesla 60 Hz magnetic field is neither muta-
w11x
w12x
w13x
w14x
w15x
genic nor co-mutagenic in the presence of menadione and MNU in a transgenic rat cell line. Mutation Res., 372, 23–31. Erfle, H., Walsh, D., Holcroft, J., Hague, N., De Boer, J.G. and Glickman, B.W. Ž1996. An efficient laboratory protocol for the sequencing of large numbers of lacI mutants recovered from Big Blue transgenic animals. Environ. Mol. Mutagen., 28, 393–396. Den Dunnen, J.T. and Schoenmakers, J.G.G. Ž1987. Consensus sequences of the Rattus norvegicus B1- and B2 repeats. Nucleic Acids Res., 15, 2772. Sawada, M., Sofuni, T. and Ishidate, Jr. Ž1991. Isolation of a menadione-resistant subclone from Chinese hamster lung ŽCHL. cells in culture. Mutation Res., 249, 7–17. Nutter, L.M., Ngo, E.O., Fisher, G.R. and Gutierrez, P.L. Ž1992. DNA strand scission and free radical production in menadione treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase. J. Biol. Chem., 267, 2474–2478. Heng, H.H.Q., Tsui, L.-C. and Moens, P.B. Ž1994. Organization of heterologous DNA inserts on the mouse meiotic chromosome core. Chromosoma, 103, 401–407.
The recovery of a B2 insertion in the lacI gene of a rat cell line containing a lrLIZ shuttle vector Johan G. de Boer 1, Andrew Suri, David Walsh, Barry W. Glickman
)
Center for EnÕironmental Health, Department of Biology, P.O. Box 3200, UniÕersity of Victoria, Victoria BC, V8W 3N5, Canada Received 3 September 1996; revised 13 November 1996; accepted 25 November 1996
Abstract The bacterial lacI gene is in use as a reporter gene for mutation in transgenic mice, rats and in cell lines. During a mutagenesis study in which we used such a rat cell line, we recovered a mutant which features an insertion of a rat B2 repeated sequence element. This sequence element was inserted between positions 77 and 78 in the lacI gene. When the inserted sequence was compared to the published rat B2 sequences, two short deletions, possibly mediated by short repeat sequences, were revealed. This is the first demonstration of the recovery of a repeated sequence element into the lacI gene used in transgenic animals and cell lines, and confirms previous findings that such sequences indeed move around the genome. Keywords: Retroposon; Big Blue; lacI transgene; Repeated sequence
1. Introduction Repeated sequences are ubiquitous constituents of the mammalian genome. The insertion of repeated sequence elements into expressed genes has been found to be associated with a number of human diseases. For example, Wallace et al. w1x reported the insertion of an Alu sequence into an intron of the neurofibromatosis gene in an NF1 patient that resulted in the deletion of an exon during splicing. Similarly, Janinic et al. w2x identified an Alu insertion into the calcium-sensing receptor gene in a patient
) Corresponding author. Tel.: 1 250 427 4067; Fax: 1 250 472 4075; E-mail: [email protected] 1 Tel: Ž250 .-472-4067. Fax: Ž 250 . -472-4075. Email: [email protected].
with familial hypocalciuric hyperglycemia and neonatal severe hyperparathyroidism, while a patient with severe haemophilia B displayed a de novo insertion of an Alu sequence into the factor IX gene w3x. The insertion of repeated sequence elements has also been observed in animals. B2 sequence elements are short repetitive sequences present in approximately 100 000 copies in the rodent genome w4x. Their movement has been found to be associated with several mutations. For example, a B2-repeat sequence insertion changes the expression of the g-phosphorylase kinase gene in mice w5x, while B2 sequences found in the C4 gene of certain mouse strains are associated with aberrant splicing of that gene w6,7x. One event thought to have occurred in the early 1970s was the integration of a B2 element in an 18S rRNA gene in a murine cell line w8x. During a mutagenesis study in which we used a
0027-5107r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 6 . 0 0 2 5 5 - 2
80
J.G. de Boer et al.r Mutation Research 375 (1997) 79–82
rat cell line containing a stably integrated lacI mutation target gene w9x, we recovered a mutant which displayed an increased length of the PCR product that includes the lacI gene w10x. In this communication we report the first recovery of a B2 element in a target gene used for the recovery of mutations.
2. Materials and methods The rat fibroblast Rat2 cell line, transgenic for the lrLIZ vector containing a lambda chromosome and the bacterial lacI gene was obtained from Stratagene w9x. The cells were grown in Dulbecco’s Modified Eagle Medium ŽGibco BRL, Gaithersburg, MD.. The protocol has been described by Suri et al. w10x. Menadione was obtained from Sigma ŽSt. Louis, MO.. Genomic DNA was isolated and mutants were recovered following standard protocols as described by Stratagene. Briefly, cells were treated with 2.5 mgrml menadione for 30 minutes, washed, and allowed expression for 5 days. Genomic DNA was isolated from the cells, using a dialysis method ŽR. Winegar, unpublished, Stanford Research Institute, Menlo Park, CA., and individual l chromosomes were recognised and packaged with l packaging extract ŽTranspack, Stratagene.. Bacteriophage l particles were plated in the presence of X-gal and blue plaques, indicating a mutation in the lacI gene, identified and cored using a Pasteur pipette. The lacI gene was amplified from these l bacteriophage particles by PCR. The lacI gene was sequenced using
cycling sequencing and automated analysis using a Pharmacia ALF DNA sequencer. Detailed protocols have been published elsewhere w11x.
3. Results and discussion During a study designed to measure the mutagenic effect of menadione and magnetic fields in a rat cell line containing a stably integrated lacI mutation target gene w10x, we recovered mutants after exposure to 2.5 mgrml menadione. The mutant frequency after this exposure was 15 = 10y5 . After PCR amplification of a 1280 base pair fragment containing the lacI gene of these mutants, one mutant was observed to yield a fragment of approximately 1415 base pairs, indicating an insertion. Subsequent sequencing of this DNA fragment revealed an insertion of 135 base pairs between nucleotide positions 77 and 78 ŽFig. 1A., which was not a duplication of a sequence found elsewhere in the wild-type lacI gene. No other alterations were detected. A sequence homology search of GenBank revealed a close homology with the rat B2 repeated sequence ŽEMBL entry X14119., w12x. Fig. 1 shows an alignment between the inserted sequence and the Žinverted. B2 sequence. The inserted sequence differs from the published rat B2 sequence w12x. An eleven base pair sequence, flanked by a 5X-CA-3X dinucleotide direct repeat has been deleted from the B2 sequence. In addition, a 12-basepair sequence
X X Fig. 1. ŽA. Site of integration in the lacI sequence Žbetween position 77 and 78.. ŽB. Sequence 5 to 3 as found in the lacI gene Žreverse compared to the B2 sequence.. Top line: the inserted sequence; Bottom line: the rat B2 sequence ŽEMBL-X14119.. Lower case letters indicate a mismatch. Underlined letters indicate a repeated sequence.
J.G. de Boer et al.r Mutation Research 375 (1997) 79–82
X Fig. 2. The top line is the 5 -end of the B2 sequence ŽEMBLX X14119.. In the insertion it is shown as the 3 -end in Fig. 1. The underlined nucleotides indicate the end of the sequence that is inserted into the lacI gene. The bottom line is the sequence from position 66 to 88 in the lacI gene. The space indicates the site of insertion. Lower case letters indicate a mismatch.
was deleted, which resulted in the generation of a repeat of the sequence 5X-TGGTTG-3X which was found at the 5X side of this deletion. At the 5X endpoint of the insertion a stretch of nine thymidine residues is found. This end of the insertion corresponds to the 3X end of the B2 sequence, and is indicative of the residual polyŽA. tail sequence. In addition, the inserted sequence contains at least 3 Žoverlapping. polyadenylation signals ŽAATAAA, or TTTATT in the reported sequence.. The sequence flanking the 3X-end of the inserted part of the B2 element Ž5X-GTGCTCTTAAC-3X , which is actually the 5X-end of the B2 sequence. shows a 9 out of 11 nucleotide homology to the sequence just 5X of the insertion site in the lacI gene. The alignment is shown in Fig. 2. This homology suggests a sequence directed invasion into the gene. The orientation of the inserted sequence Ž3X ™ 5X . is the same as the sequence homology. The endpoints of inserted sequence elements frequently feature short direct repeat sequences. The insertion of a B2 element in an 18S rDNA gene in the murine cell line PYS-2r12 w8x has a perfect 15 b a se p a ir fla n k in g d ire c t re p e a t Ž 5 X AAAAAGCTCGTAGTT-3X .. The insertion into the lacI gene features only a 2-basepair flanking repeat Ž5X-AT-3X .. However, the complement of the sequence 5X of the insertion site is 5X-AAGAGACACC3X . This sequence is similar to 5X-CAGAGGAGGACAG-3X , which is found as the short repeated flanking sequences of the B2 insertion in intron 13 of the mouse C4 gene w7x. The flanking repeats are however not always the same. A B2 insertion in the mouse g-phosphorylase kinase gene is flanked by 5XAAAGATTTATG-3X repeats w5x. As far as we are aware, this is the first reported case of the recovery of a retroposon jump into a mutation target gene. The mutant was recovered after treatment with menadione, a compound that gener-
81
ates superoxide radicals w13x and induces single- and double-strand breaks w14x. We have no way of knowing whether this treatment triggered the B2 insertion event, however, this insertion is the first to be found in over 8000 mutants we have sequenced from lacI transgenic animals and cell lines. B2 retroposon sequences are specific to rodents, including mice, rats, and Chinese hamsters, and occur in large numbers w4x. These sequences are very active in transcription, with most transcripts found in hnRNA, and some in mRNA w4x. Such active transcription may facilitate insertion in other sites in the genome. Heng et al. w15x reported that the 1.8 Mb insert of 40 copies of lrLIZ DNA into the genome of the Big Blue w mice is not contained in the chromatin domain of chromosome 4, and does not interact with the chromosome core. Nevertheless, our finding indicates that retroposon jumps can occur into heterologous DNA, and extends the finding by Oberbaumer w8x that these sequences can move throughout a mammalian genome.
Acknowledgements The authors thank Andrew McArthur for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Strategic grant to BWG.
References w1x Wallace, M.R., Andersen, L.B., Saulino, A.M., Gregory, P.E., Glover, T.W. and Collins, F.S. Ž1991. A de novo Alu insertion results in neurofibromatosis type 1. Nature, 353, 864–866. w2x Janicic, N., Pausova, Z., Cole, D.E. and Hendy, G.N. Ž1995. Insertion of an Alu sequence in the CaŽ2q.-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Am. J. Hum. Genet., 56, 880– 886. w3x Vidaud, D., Vidaud, M., Bahnak, B.R., Siguret, V., Gispert Sanchez, S., Laurian, Y., Meyer, D., Goossens, M. and Lavergne, J.M. Ž1993. Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene. Eur. J. Hum. Genet., 1, 30–36. w4x Rogers, J.H. Ž1985. The origin and evolution of retroposons. Inter. Rev. Cytol., 93, 187–279.
82
J.G. de Boer et al.r Mutation Research 375 (1997) 79–82
w5x Maichele, A., Farwell, N.J. and Chamberlain, J.S. Ž1993. A B2 repeat insertion generates alternate structures of the mouse muscle gamma-phosphorylase kinase gene. Genomics, 16, 139–149. w6x Zheng, J.H., Natsuume-Sakai, S., Takahashi, M. and Nonaka, M. Ž1992. Insertion of the B2 sequence into intron 13 is the only defect of the H-2k C4 gene which causes low C4 production. Nucleic Acids Res., 20, 4975–4979. w7x Pattanakitsakul, S., Zheng, J.H., Natsuume-Sakai, S., Takahashi, M. and Nonaka, M. Ž1992. Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice. J. Biol. Chem., 267, 7814–7820. w8x Oberbaumer, I. Ž1992. Retroposons do jump: a B2 element recently integrated in an 18S rDNA gene. Nucleic Acids Res., 20, 671–677. w9x Wyborski, D., Malkhosyan, S., Moores, J., Perucho, M. and Short, J. Ž1995. Development of a rat cell line containing stably integrated copies of a lambdar lacI shuttle vector. Mutation Res., 334, 161–166. w10x Suri, A., De Boer, J.G., Kusser, W.C. and Glickman, B.W. Ž1996. A 3 milliTesla 60 Hz magnetic field is neither muta-
w11x
w12x
w13x
w14x
w15x
genic nor co-mutagenic in the presence of menadione and MNU in a transgenic rat cell line. Mutation Res., 372, 23–31. Erfle, H., Walsh, D., Holcroft, J., Hague, N., De Boer, J.G. and Glickman, B.W. Ž1996. An efficient laboratory protocol for the sequencing of large numbers of lacI mutants recovered from Big Blue transgenic animals. Environ. Mol. Mutagen., 28, 393–396. Den Dunnen, J.T. and Schoenmakers, J.G.G. Ž1987. Consensus sequences of the Rattus norvegicus B1- and B2 repeats. Nucleic Acids Res., 15, 2772. Sawada, M., Sofuni, T. and Ishidate, Jr. Ž1991. Isolation of a menadione-resistant subclone from Chinese hamster lung ŽCHL. cells in culture. Mutation Res., 249, 7–17. Nutter, L.M., Ngo, E.O., Fisher, G.R. and Gutierrez, P.L. Ž1992. DNA strand scission and free radical production in menadione treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase. J. Biol. Chem., 267, 2474–2478. Heng, H.H.Q., Tsui, L.-C. and Moens, P.B. Ž1994. Organization of heterologous DNA inserts on the mouse meiotic chromosome core. Chromosoma, 103, 401–407.