176
BRIEF
local homologies and symmetries. Nucleic
Acids
Res. 10: 247-
263. 4.
KANEHISA,
M.
I., AND
GOAD,
W.
B. (1982).
Pattern
recogni-
tion in nucleic acid sequences. II. An efficient method for finding locally stable secondary structures. Nucleic Acids Res. 10: 265-278. 5.
PROBER, J. M., TRAINOR, G. L., DAM, R. J., HOBBS, F. W., ROBERTSON, C. W., ZAGURSKY, R. J., COCUZZA, A. J., JENSEN, M. A., AND BAUMEISTER, K. A. (1987). A system for
rapid DNA sequencing with fluorescent chain-terminating clideoxynucleotides. Science 238: 336-341. 6.
SANGER, sequencing
Acad.
F., NICKLEN, S., AND with chain-terminating
Sci. USA
COULSON, A. R. (1977). inhibitors. Proc.
DNA
N&l.
74: 5463-5467.
7.
TEMPLETON, N. S., BROWN, P. D., LEVY, A. T., MARGULIES, I. M. K., LIOVA, L. A., AND STETLER-STEVENSON, W. G. (1990). Cloning and characterization of human tumor inter-
8.
WILBUR,
stitial collagenase. Cancer Res. 50: 5431-5437. W.
J., AND
LIPMAN,
D. J. (1983).
Rapid
similarity
searches of nucleic acid and protein data banks. Proc. Natl. Acad.
Sci. USA
80: 726-730.
Highly Polymorphic Sequence in lntron MAOB Gene
(CT), Repeat II of the Human
C. Konradi,* L. Ozelius,**t and X. 0. Breakefield**$ ‘Departments of Neurobiology Genera/ Hospital, Charlestown, Department and #Neuroscience Boston, Massachusetts 02 115 ReceivedJune
24, 1991;revised
and Neurology, Massachusetts Massachusetts 02129; and tGenetics Program, Harvard Medical School,
September
6, 1991
Monoamine oxidase (MAO; monoamine: 0, oxidoreductase, EC 1.4.3.4) is the primary enzyme involved in the degradation of aminergic neurotransmitters like dopamine, noradrenaline, and serotonin, and the neurotoxin precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Heikkila et al., 1985). Two isozymes, MAO-A and MAO-B, differ in their pharmacologic and biochemical properties (for review see Weyler et al., 1990). Genes for MAO-A and MAO-B are located near each other in the ~11.3 region of the human X chromosome (for review see Hsu et al., 1989). Over 50-fold variations in MAO-A and MAO-B activities have been described in control humans, as measured in cultured skin fibroblasts and platelets, respectively, and activity levels appear to be genetically determined (Hsu et al., 1989). Since variations in MAO activity can influence neurophysiology, it seems likely that individuals with different levels of activity may have differential susceptibilities to certain pathogenic processes. Biochemical measurements of MAO-B activity in peripheral tissues have not resolved the possible role of MAO-B in human diseases, presumably because a number of factors, including drugs, hormonal state, age, and diet influence levels of MAO-B activity in vivo (for review see Demisch et al., 1983). Molecular biological methods can be used to follow the inheritance of the MAOB gene in families and to evaluate rapidly the associaGENOMICS 12,176-177 (1992) 0888-7543/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
REPORTS
tion of different alleles with disease states. Here we describe a highly polymorphic (GT), repeat (Weber, 1990) within the second intron of the human MAOB gene and define primers and conditions for amplification of it using the polymerase chain reaction (PCR; Saiki et al., 1988). A human genomic DNA clone, MAOB-5, was isolated from an EMBL 3A phage library, generated from partially Sau3A-digested genomic DNA cloned into a BamHI site (Kwiatkowski et al., 1988) by screening with a partial cDNA clone, KSB2 (kindly provided by Dr. Paul Hsu, West Roxbury Veterans’ Administration, MA), corresponding to nucleotides lo-600 in the 5’ end of the published human MAO-B cDNA (Bach et al., 1988). This 30-kb genomic clone was found to contain a (GT), repeat by screening with a poly(dC-dA) . (dG-dT) oligonucleotide probe (Pharmacia, Piscataway, NJ). Following digestion of clone MAOB-5 with EcoRI and PstI and gel electrophoresis, a 1.8-kb band that hybridized to both the partial MAOB cDNA clone and the (GT), oligonucleotide probes was identified. Southern hybridization was carried out as described (Hotamisligil and Breakefield, 1991). This band was subcloned into a Bluescript SK vector (Stratagene, La Jolla, CA). Sequencing of the 1.8-kb human genomic clone 2.2 revealed a (GT),, repeat located 264 bp from the 3’ end of exon II (Grimsby et al., 1991). Double-stranded sequencing was performed by the alkaline-denaturation method (Toneguzzo et al., 1988) followed by the chain termination reaction (Sanger et al., 1977) using [35S]dATP and modified T7 DNA polymerase (United States Biochemical, Cleveland, OH). No other MAO-B exon sequence was present in clone 2.2. Primers flanking the (GT), repeat (DNA synthesizer, Cyclone, Biosearch, San Raffael, CA) had the following sequence: 5’ GAA GCA TCG AAG TTA GGA GT 3’ (CA strand) and 5’ ATT TGG CCT CAT AGA GTT AG 3’ (GT strand). PCR reactions contained 0.2 mM dATP, dCTP, dTTP, 2.5 pM dTTP, 0.08 ~1 [32P]dTTP (3000 mCi/ mmol),4 ng of each primer, 10 ng genomic DNA, and 0.05 ~1 Tuq polymerase (Perkin-Elmer Cetus) in a total volume of 10 ~1. Samples were heated to 94°C for 1.5 min, followed by 25 cycles each for 1 min at 94”C, 1 min at 55°C and 1 min at 72”C, and a final extension for 7 min at 72°C. Seven different MAOB alleles were detected by acrylamide gel electrophoresis of PCR fragments generated from 48 randomly chosen, nonrelated controls (17 males, 31 females; total 79 X chromosomes). The sizes of the fragments ranged from 172 to 184 bp. The frequency of alleles in this control population is shown in Table 1. The expected heterozygosity for
TABLE of MAOB
Frequency
alleles
1 in control
Allele
Frequency
172 174 176 178 180 182 184
0.025 0.076 0.127 0.139 0.228 0.241 0.165
population
BRIEF
this repetitive element was 0.821. Segregation of MAOB alleles in more than 10 families has confirmed Mendelian inheritance of an X-linked locus. If MAO activity levels are controlled predominantly by the genes encoding them, then variations in alleles for these genes can be used to mark activity states. A high correlation has been demonstrated between allelic status for the MAOA gene and levels of activity measured in human skin fibroblasts in culture (Hotamisligil and Breakefield, 1991). The current study describes the first reported polymorphism for the human MAOB gene, which will allow a similar comparison with activity states to be made. Further, different frequencies of MAOB alleles in control and disease populations could implicate this gene in the disease process. ACKNOWLEDGMENTS C.K. and X.O.B. express their deep gratitude to Dr. Richard Heikkila for his brilliant scientific contributions to the role of MAO in disease. We are thankful to Yun-Pung Paul Hsu, Ph.D., for providing the MAO-B cDNA clone; James Trofatter, Ph.D., for help with statistical analysis; Julie Andersen, Ph.D., for help with experiments; Ms. Donna Roman0 for synthesizing the primers; Ms. Heather McFarlane for establishing the lymphoblastoid cell lines; Ms. Deborah Schuback for technical assistance; David Kwiatkowski, M.D., Ph.D., for the gift of the phage library; and Ms. Suzanne McDavitt for skilled preparation of this manuscript. C.K. was supported by a grant from the Max Kade Foundation; X.O.B. by NIH Grant NS21921 (Senator Jacob Javits Award) and ADAMHA Grant AA08683. REFERENCES 1.
BACH, A. W. J., LAN, N. C., JOHNSON, D. L., ABELL, C. W., BEMBENEK, M. E., KWAN, S-W., SEEBURG, P. H., AND SHIH,
J. C. (1988). cDNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties. Proc. Natl. Acad. Sci. USA 85: 4934-4938. 2. DEMISCH, L., KACZMARCZYK, P., AND GJZBHART, P. (1983). Methodological problems of using platelet MAO in psychiatric research. In “Modern Problems in Pharmacopsychiatry” (H. Beckmann and P. Riederer, Eds.), Vol. 19, pp. 265-277, Karger, Basel. 3. GRIMSBY, J., CHEN, K., WANG, L-J., LAN, N. C., AND SHIH, J. C. (1991). Human monoamine oxidase A and B genes exhibit identical exon-intron organization. Proc. Natl. Acad. Sci. USAM:
3637-3641.
4. HEIKKILA, R. E., MANZINO, L., CABBAT, F. S., AND DWOISIN, R. C. (1985). Studies on the oxidation of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine by monoamine oxidase B. J. Neurochem. 45: 1049-1054. 5.
HOTAMISLIGIL,
G. S., AND BREAKEFIELD,
X. 0. (1991).
HU-
man monoamine oxidase A gene determines levels of enzyme activity. Am. J. Hum. Genet. 49: 383-392. 6. Hsu, Y-P. P., POWELL, J. F., SIMS, K. B., AND BREAKEFIELD, X. 0. (1989). Molecular genetics of the monoamine oxidases. 7.
J. Neurochem. KWIATKOWSKI,
53: 12-18. D. J., MEHL,
R. M.,
AND YIN,
H. L. (1988).
Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin. J. Cell. Biol. 106: 375-384. 8.
SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH,
S. J., H. A.
177
REPORTS (1988).
Primer
a thermostable 9.
SANGER,
directed enzymatic amplification of DNA with DNA polymerase. Science 239: 487-491.
F., NICKLEN,
S., AND COULSON,
sequencing with chain terminating Acad.
Sci. USA
A. R. (1977). Proc.
inhibitors.
DNA Natl.
74: 5463-5468.
10.
TONEGUZZO, F., GLYNN, S., LEVI, E., MJOLSNESS, HAYDAY, A. (1988). Use of a chemically modified polymerase for manual and automated sequencing coiled DNA. Biotechniques 6: 460-469.
S., AND T7 DNA of super-
11.
WEBER, J. L. (1990). Informativeness dA), * (dG-dT), polymorphisms. Genomics
12.
WEYLER, W., Hsu, Y-P. P., AND BREAKEFIELD, X. 0. (1990). Biochemistry and genetics of monoamine oxidase. J. Pharmacol. Therapeut. 47: 391-417.
of human 7: 524-530.
(dC-
Assignment of the Angiogenin Gene to Mouse Chromosome 14 Using a Rapid PCR-RFLP Mapping Technique Mark E. Steinhelper and Loren J. Field Department of Medicine, Indiana Krannert Institute of Cardiology, Indianapolis, lndiana 46202 Received
June 6, 1991;revised
University School of Medicine, 1111 West 10th Street,
September
18, 1991
Gene mapping using recombinant inbred (RI) strains is one of several diverse methods available for mapping genes in the mouse (Taylor, 1978). The polymerase chain reaction (PCR, Erlich et aZ., 1988) has facilitated many biological assays and appeared to us to have several advantages over Southern hybridization for gene mapping with RI strains. Accordingly, the present report describes a strategy to map the mouse angiogenin gene to chromosome 14, near the Rib-l, Tcra, and Np-2 loci. This mapping procedure involves two steps: (a) identification of an allelic restriction fragment length polymorphism (RFLP) using radiolabeled DNA amplified from a set of progenitor mice, and (b) generation of a strain distribution pattern (SDP) for that locus in a RI set derived from these progenitors. Oligonucleotide primers (sense: GTCTCCACCCACTTAGTCTAAGTTAG; anti-sense: CCCTGACAATGAACGCTGGAACCAG) designed to amplify a portion of the mouse angiogenin sequence (Bond and Vallee, 1990) were synthesized on an Applied Biosystems DNA synthesizer. The angiogenin gene was amplified from 2 ng of template DNA in 40 ~1 of 50 mM KCl, 10 m&f Tris-HCl (pH 8.3), 2.5 mA4 MgCl*, 0.01 mg/ml gelatin, 0.25 n&f each dNTP, 1.25 pM each oligonucleotide, 1 unit Ampli-Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT). Amplification was performed over 35 cycles with 1 min of denaturation at 94”C, 2 min of annealing at 66’C, and 3 min of extension at 72°C. These conditions produced a single band of the expected size (616 bp) on agarose gel electrophoresis using C57BL/6J and DBA/2J mouse genomic DNA as template. To facilitate RFLP detection, [(w-32P]dCTP was added to a final concentration of lo-20 &i/ml. A 2-~1 portion of the radiolabeled reaction product was digested to completion in GENOMICS12,177-179(1992) osss-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF
local homologies and symmetries. Nucleic
Acids
Res. 10: 247-
263. 4.
KANEHISA,
M.
I., AND
GOAD,
W.
B. (1982).
Pattern
recogni-
tion in nucleic acid sequences. II. An efficient method for finding locally stable secondary structures. Nucleic Acids Res. 10: 265-278. 5.
PROBER, J. M., TRAINOR, G. L., DAM, R. J., HOBBS, F. W., ROBERTSON, C. W., ZAGURSKY, R. J., COCUZZA, A. J., JENSEN, M. A., AND BAUMEISTER, K. A. (1987). A system for
rapid DNA sequencing with fluorescent chain-terminating clideoxynucleotides. Science 238: 336-341. 6.
SANGER, sequencing
Acad.
F., NICKLEN, S., AND with chain-terminating
Sci. USA
COULSON, A. R. (1977). inhibitors. Proc.
DNA
N&l.
74: 5463-5467.
7.
TEMPLETON, N. S., BROWN, P. D., LEVY, A. T., MARGULIES, I. M. K., LIOVA, L. A., AND STETLER-STEVENSON, W. G. (1990). Cloning and characterization of human tumor inter-
8.
WILBUR,
stitial collagenase. Cancer Res. 50: 5431-5437. W.
J., AND
LIPMAN,
D. J. (1983).
Rapid
similarity
searches of nucleic acid and protein data banks. Proc. Natl. Acad.
Sci. USA
80: 726-730.
Highly Polymorphic Sequence in lntron MAOB Gene
(CT), Repeat II of the Human
C. Konradi,* L. Ozelius,**t and X. 0. Breakefield**$ ‘Departments of Neurobiology Genera/ Hospital, Charlestown, Department and #Neuroscience Boston, Massachusetts 02 115 ReceivedJune
24, 1991;revised
and Neurology, Massachusetts Massachusetts 02129; and tGenetics Program, Harvard Medical School,
September
6, 1991
Monoamine oxidase (MAO; monoamine: 0, oxidoreductase, EC 1.4.3.4) is the primary enzyme involved in the degradation of aminergic neurotransmitters like dopamine, noradrenaline, and serotonin, and the neurotoxin precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Heikkila et al., 1985). Two isozymes, MAO-A and MAO-B, differ in their pharmacologic and biochemical properties (for review see Weyler et al., 1990). Genes for MAO-A and MAO-B are located near each other in the ~11.3 region of the human X chromosome (for review see Hsu et al., 1989). Over 50-fold variations in MAO-A and MAO-B activities have been described in control humans, as measured in cultured skin fibroblasts and platelets, respectively, and activity levels appear to be genetically determined (Hsu et al., 1989). Since variations in MAO activity can influence neurophysiology, it seems likely that individuals with different levels of activity may have differential susceptibilities to certain pathogenic processes. Biochemical measurements of MAO-B activity in peripheral tissues have not resolved the possible role of MAO-B in human diseases, presumably because a number of factors, including drugs, hormonal state, age, and diet influence levels of MAO-B activity in vivo (for review see Demisch et al., 1983). Molecular biological methods can be used to follow the inheritance of the MAOB gene in families and to evaluate rapidly the associaGENOMICS 12,176-177 (1992) 0888-7543/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
REPORTS
tion of different alleles with disease states. Here we describe a highly polymorphic (GT), repeat (Weber, 1990) within the second intron of the human MAOB gene and define primers and conditions for amplification of it using the polymerase chain reaction (PCR; Saiki et al., 1988). A human genomic DNA clone, MAOB-5, was isolated from an EMBL 3A phage library, generated from partially Sau3A-digested genomic DNA cloned into a BamHI site (Kwiatkowski et al., 1988) by screening with a partial cDNA clone, KSB2 (kindly provided by Dr. Paul Hsu, West Roxbury Veterans’ Administration, MA), corresponding to nucleotides lo-600 in the 5’ end of the published human MAO-B cDNA (Bach et al., 1988). This 30-kb genomic clone was found to contain a (GT), repeat by screening with a poly(dC-dA) . (dG-dT) oligonucleotide probe (Pharmacia, Piscataway, NJ). Following digestion of clone MAOB-5 with EcoRI and PstI and gel electrophoresis, a 1.8-kb band that hybridized to both the partial MAOB cDNA clone and the (GT), oligonucleotide probes was identified. Southern hybridization was carried out as described (Hotamisligil and Breakefield, 1991). This band was subcloned into a Bluescript SK vector (Stratagene, La Jolla, CA). Sequencing of the 1.8-kb human genomic clone 2.2 revealed a (GT),, repeat located 264 bp from the 3’ end of exon II (Grimsby et al., 1991). Double-stranded sequencing was performed by the alkaline-denaturation method (Toneguzzo et al., 1988) followed by the chain termination reaction (Sanger et al., 1977) using [35S]dATP and modified T7 DNA polymerase (United States Biochemical, Cleveland, OH). No other MAO-B exon sequence was present in clone 2.2. Primers flanking the (GT), repeat (DNA synthesizer, Cyclone, Biosearch, San Raffael, CA) had the following sequence: 5’ GAA GCA TCG AAG TTA GGA GT 3’ (CA strand) and 5’ ATT TGG CCT CAT AGA GTT AG 3’ (GT strand). PCR reactions contained 0.2 mM dATP, dCTP, dTTP, 2.5 pM dTTP, 0.08 ~1 [32P]dTTP (3000 mCi/ mmol),4 ng of each primer, 10 ng genomic DNA, and 0.05 ~1 Tuq polymerase (Perkin-Elmer Cetus) in a total volume of 10 ~1. Samples were heated to 94°C for 1.5 min, followed by 25 cycles each for 1 min at 94”C, 1 min at 55°C and 1 min at 72”C, and a final extension for 7 min at 72°C. Seven different MAOB alleles were detected by acrylamide gel electrophoresis of PCR fragments generated from 48 randomly chosen, nonrelated controls (17 males, 31 females; total 79 X chromosomes). The sizes of the fragments ranged from 172 to 184 bp. The frequency of alleles in this control population is shown in Table 1. The expected heterozygosity for
TABLE of MAOB
Frequency
alleles
1 in control
Allele
Frequency
172 174 176 178 180 182 184
0.025 0.076 0.127 0.139 0.228 0.241 0.165
population
BRIEF
this repetitive element was 0.821. Segregation of MAOB alleles in more than 10 families has confirmed Mendelian inheritance of an X-linked locus. If MAO activity levels are controlled predominantly by the genes encoding them, then variations in alleles for these genes can be used to mark activity states. A high correlation has been demonstrated between allelic status for the MAOA gene and levels of activity measured in human skin fibroblasts in culture (Hotamisligil and Breakefield, 1991). The current study describes the first reported polymorphism for the human MAOB gene, which will allow a similar comparison with activity states to be made. Further, different frequencies of MAOB alleles in control and disease populations could implicate this gene in the disease process. ACKNOWLEDGMENTS C.K. and X.O.B. express their deep gratitude to Dr. Richard Heikkila for his brilliant scientific contributions to the role of MAO in disease. We are thankful to Yun-Pung Paul Hsu, Ph.D., for providing the MAO-B cDNA clone; James Trofatter, Ph.D., for help with statistical analysis; Julie Andersen, Ph.D., for help with experiments; Ms. Donna Roman0 for synthesizing the primers; Ms. Heather McFarlane for establishing the lymphoblastoid cell lines; Ms. Deborah Schuback for technical assistance; David Kwiatkowski, M.D., Ph.D., for the gift of the phage library; and Ms. Suzanne McDavitt for skilled preparation of this manuscript. C.K. was supported by a grant from the Max Kade Foundation; X.O.B. by NIH Grant NS21921 (Senator Jacob Javits Award) and ADAMHA Grant AA08683. REFERENCES 1.
BACH, A. W. J., LAN, N. C., JOHNSON, D. L., ABELL, C. W., BEMBENEK, M. E., KWAN, S-W., SEEBURG, P. H., AND SHIH,
J. C. (1988). cDNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties. Proc. Natl. Acad. Sci. USA 85: 4934-4938. 2. DEMISCH, L., KACZMARCZYK, P., AND GJZBHART, P. (1983). Methodological problems of using platelet MAO in psychiatric research. In “Modern Problems in Pharmacopsychiatry” (H. Beckmann and P. Riederer, Eds.), Vol. 19, pp. 265-277, Karger, Basel. 3. GRIMSBY, J., CHEN, K., WANG, L-J., LAN, N. C., AND SHIH, J. C. (1991). Human monoamine oxidase A and B genes exhibit identical exon-intron organization. Proc. Natl. Acad. Sci. USAM:
3637-3641.
4. HEIKKILA, R. E., MANZINO, L., CABBAT, F. S., AND DWOISIN, R. C. (1985). Studies on the oxidation of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine by monoamine oxidase B. J. Neurochem. 45: 1049-1054. 5.
HOTAMISLIGIL,
G. S., AND BREAKEFIELD,
X. 0. (1991).
HU-
man monoamine oxidase A gene determines levels of enzyme activity. Am. J. Hum. Genet. 49: 383-392. 6. Hsu, Y-P. P., POWELL, J. F., SIMS, K. B., AND BREAKEFIELD, X. 0. (1989). Molecular genetics of the monoamine oxidases. 7.
J. Neurochem. KWIATKOWSKI,
53: 12-18. D. J., MEHL,
R. M.,
AND YIN,
H. L. (1988).
Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin. J. Cell. Biol. 106: 375-384. 8.
SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH,
S. J., H. A.
177
REPORTS (1988).
Primer
a thermostable 9.
SANGER,
directed enzymatic amplification of DNA with DNA polymerase. Science 239: 487-491.
F., NICKLEN,
S., AND COULSON,
sequencing with chain terminating Acad.
Sci. USA
A. R. (1977). Proc.
inhibitors.
DNA Natl.
74: 5463-5468.
10.
TONEGUZZO, F., GLYNN, S., LEVI, E., MJOLSNESS, HAYDAY, A. (1988). Use of a chemically modified polymerase for manual and automated sequencing coiled DNA. Biotechniques 6: 460-469.
S., AND T7 DNA of super-
11.
WEBER, J. L. (1990). Informativeness dA), * (dG-dT), polymorphisms. Genomics
12.
WEYLER, W., Hsu, Y-P. P., AND BREAKEFIELD, X. 0. (1990). Biochemistry and genetics of monoamine oxidase. J. Pharmacol. Therapeut. 47: 391-417.
of human 7: 524-530.
(dC-
Assignment of the Angiogenin Gene to Mouse Chromosome 14 Using a Rapid PCR-RFLP Mapping Technique Mark E. Steinhelper and Loren J. Field Department of Medicine, Indiana Krannert Institute of Cardiology, Indianapolis, lndiana 46202 Received
June 6, 1991;revised
University School of Medicine, 1111 West 10th Street,
September
18, 1991
Gene mapping using recombinant inbred (RI) strains is one of several diverse methods available for mapping genes in the mouse (Taylor, 1978). The polymerase chain reaction (PCR, Erlich et aZ., 1988) has facilitated many biological assays and appeared to us to have several advantages over Southern hybridization for gene mapping with RI strains. Accordingly, the present report describes a strategy to map the mouse angiogenin gene to chromosome 14, near the Rib-l, Tcra, and Np-2 loci. This mapping procedure involves two steps: (a) identification of an allelic restriction fragment length polymorphism (RFLP) using radiolabeled DNA amplified from a set of progenitor mice, and (b) generation of a strain distribution pattern (SDP) for that locus in a RI set derived from these progenitors. Oligonucleotide primers (sense: GTCTCCACCCACTTAGTCTAAGTTAG; anti-sense: CCCTGACAATGAACGCTGGAACCAG) designed to amplify a portion of the mouse angiogenin sequence (Bond and Vallee, 1990) were synthesized on an Applied Biosystems DNA synthesizer. The angiogenin gene was amplified from 2 ng of template DNA in 40 ~1 of 50 mM KCl, 10 m&f Tris-HCl (pH 8.3), 2.5 mA4 MgCl*, 0.01 mg/ml gelatin, 0.25 n&f each dNTP, 1.25 pM each oligonucleotide, 1 unit Ampli-Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT). Amplification was performed over 35 cycles with 1 min of denaturation at 94”C, 2 min of annealing at 66’C, and 3 min of extension at 72°C. These conditions produced a single band of the expected size (616 bp) on agarose gel electrophoresis using C57BL/6J and DBA/2J mouse genomic DNA as template. To facilitate RFLP detection, [(w-32P]dCTP was added to a final concentration of lo-20 &i/ml. A 2-~1 portion of the radiolabeled reaction product was digested to completion in GENOMICS12,177-179(1992) osss-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
