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
178 A
HinPl
Mbol
Mspl
B
Neil
BDBDBDBD
REPORTS TABLE
Mspl BBDFl256
Loci
Mapped
by the Rapid
2 PCR-RFLP
Technique
i
. .:
Locus Aw Rb-1 Ren Pnd Pndr Tip53 Ttr
FIG. 1. Rapid PCR-RFLP mapping of the mouse angiogenin gene. (A) Partial survey of restriction enzymes to distinguish C57BL/6J (B) and DBA/LJ (D) angiogenin alleles. (B) MspI RFLP in C57BL/6J (B), DBA/2J (D), BXD Fl (F), and select BXD RI strains (1,2,5,6).
20 ~1 with various restriction enzymes according to manufacturers’ recommendations. A portion of each digestion (5-10 ~1) was separated on 8% polyacrylamide gels as described (Sambrook et al., 1989). The gels were dried and exposed for 4 h to Kodak X-AR film. As shown in Fig. lA, this method readily identified MspI and NciI RFLPs between the two progenitors at the angiogenin locus. The strain distribution pattern (SDP) for the angiogenin MspI RFLP was determined for the BXD RI set (Jackson Laboratory, Bar Harbor, ME). Representative digestion patterns from RI strains (1,2,5, and 6) as well as progenitor and Fl mice are shown in Fig. 1B. As expected, each RI sample contained only one progenitor allele, whereas Fl mice contained both alleles. The SDP pattern for the angiogenin MspI RFLP (Table 1) was identical to the SDP of Rib-l, Tcra, and Np-2 (Dembic et al., 1985; Elliot et al., that the 1986; Kranz et al., 1985; Taylor, 1981), indicating angiogenin gene is located on mouse chromosome 14. This result is supported by the observation that the human angiogenin gene is located on a homologous region of chromosome 14qll (Weremowicz et aZ., 1990). The general applicability of this protocol has been evaluated for six additional loci as summarized in Table 2. We have used oligonucleotide primers approximately 20-30 nucleotides in length with about 50% G+C content to yield PCR products of the expected size under our standard assay
TABLE
1
Segregation of Angiogenin Recombinant Inbred
in BXD
BXD strain with allele
Allele B D
Alleles Strains
1, 5, 13, 20, 21, 22, 23, 24, 25, 27, 28, 30 9, 11, 12, 14, 15, 16, 18, 19, 29, 31, 32
2, 6, 8,
Oligonucleotide
set
Product
RFLP
1 1 1 1 1 1
+ + + + + +
+ + + + +
2
+
+
1
+
-
conditions (Steinhelper et al., 1990), without optimizing for magnesium concentration. While the expected amplification product was obtained for all primer sets, in some instances (i.e., angiogenin) it was necessary to increase the annealing temperature to 66°C to generate a single amplification product. Furthermore, RFLPs were detected for five of the seven loci investigated upon screening of the initial target sequence. The absence of a detectable RFLP for Trp53 in the initial screening was remedied by the synthesis of a second primer set; a second primer set for the Ttr locus has not yet been synthesized. That a RFLP was obtained for six of the seven loci investigated using this protocol attests to its general applicability. Finally, although the SDP for a given RI set may not adequately represent the chromosomal region that harbors the gene in question, the use of multiple RI strains can circumvent this limitation. Broader application of this technique should facilitate the development of the mouse linkage map. ACKNOWLEDGMENTS We thank Joyce Dwulet for oligonucleotide synthesis and Phyllis Weinberg for technical assistance. This work was supported by the NHLBI (HL-07992, M.E.S.; HL-38605 and HL-45453, L.J.F.) and a grant in aid from the American Heart Association Indiana Affiliate, Inc., to M.E.S. REFERENCES 1. BOND, M. D., AND VALLEE, B. L. (1990). Isolation and sequencing of mouse angiogenin DNA. &o&n. Biophys. Res. Commun. 171: 988-995. 2. DEMBIC, Z., BANNWARTH, W., TAYLOR, B. A., AND STEINMETZ, M. (1985). The gene encoding the T-cell receptor 01chain maps close to the Np-2 locus on mouse chromosome 14. Nature 314: 271-273. 3. ELLIOT, R. W., SAMUELSON, L. C., LAMBERT, M. S., AND MEISLER, M. H. (1986). Assignment of pancreatic ribonuclease gene to mouse chromosome 14. Cytogenet. Cell Genet. 42: 110-112. 4. ERLICH, H. A., GELFAND, D. H., AND SAIKI, R. H. (1988). Specific DNA amplification. Nature 331: 461-462. 5. KRANZ, D. M., SAITO, H., DISTECHE, C. M., SWISSHELM, K., PFUVTCHEVA, D., RUDDLE, F. H., AND EISEN, H. N. (1985). Chromosomal location of the murine T-cell receptor a-chain gene and the T-cell T gene. Science 227: 941-945. 6. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989).
BRIEF “Molecular Cloning: A Laboratory Spring Harbor Laboratory Press, 7.
8.
Manual,” Cold Spring
2nd ed., Cold Harbor, NY.
STEINHELPER, M. E., COCHRANJZ, K. L., AND FIELD, L. J. (1990). Hypotension in transgenic mice expressing atria1 natriuretic factor fusion genes. Hypertension 16: 301-30'7. TAYLOR, B. A. (1978). Recombinant inbred strains: Use in gene mapping. In “Origins of Inbred Strains” (H. C. Morse III, Ed.), pp. 423-438, Academic Press, New York.
9. 10.
TAYLOR,
B. A. (1981).
Linkage
28. WEREMOWICZ, S.,Fox,
of Np-2.
Mouse
News Lett.
E. A.,MoRToN,C.C.,
61:
AND VALLEE,
B. L. (1990). Localization of the human angiogenin gene to chromosome band 14ql1, proximal to the T cell receptor a/6 locus. Am. J. Hum. Genet. 47: 973-981.
Assignment of the Interleukin-2 Receptor #I Chain Gene (Il-2rb) Band E on Mouse Chromosome
to 15
Structure and Function, Research School of The Australian National University, Canberra; tDepartment of Genetics, The Queen Elizabeth Hospital, Woodville, South Australia; $/nstitute for Molecular and Cellular Biology, Osaka University, l-3, Yamadaoka, Suita-shi, Osaka 565, Japan; and SDivision of Biochemistry and Molecular Biology, John Curtin School of Medical Research, The Australian National University, Canberra 1. 1991;
revised
August
30, 1991
Interleukin-2 (IL-2) is a cytokine that stimulates the antigen-specific clonal expansion of T lymphocytes via inter56r
is a member
of a new
gene
superfamily
that
receptors for other cytokines, as well as for growth hormone and prolactin (Bazan, 1990). The human IL-2 receptor N chain gene maps to chromosome lOp14-~15 (Waldmann, 1989) and the murine CYchain gene to bands 2A2 or 2A3 on chromosome 2 (Webb et al., 1990). We now report the mapping of the murine IL-2 receptor /? chain gene (IL-2rb) to chromosome 15. The Zl-2rb probe was a 0.5-kb PuuII fragment from XmIL-2R/3-26 (Kono et al., 1990), labeled by nick translation with tritiated dATP, dCTP, and TTP to a specific activity of 7.2 X lo7 cpm/pg. Preparation of BALB/c mitotic chromosomes, in situ hybridization, and staining were as previously described (Webb et al., 1989). Exposure to L4 emulsion was for 17 days. Silver grains over the chromoor within idiograms
one chromatid of the mouse
width of them were karyotype (Nesbitt
scored and
I5 were
redrawn
to suit
the
G-bands
we ob-
served after incorporation of 5-BrdU during early S-phase. A total of 229 grains over - 100 cells was scored for the whole karyotype; 32.5% of the grains were over chromosome 15 with 92% of these grains over the distal half of 15 (Fig. 1). The tallest peak of 56 (24% of all grains) was over band 15E with the second tallest peak of 30 grains over 15F and the third of 16 grains over subband 15D3. Background grains on the rest of the karyotype were evenly distributed with maximal peaks of 5 grains over the distal regions of chromosomes 12 and 18. Grains over the detailed idiogram of chromosome 15 were scored from -90 high quality cells, -60% of which were independent of the karyotypic scor-
I
I FIG. 1. In situ hybridization grain distribution in approximately
gene
includes
chromosome
for Molecular
April
chain
Francke, 1973), or onto a more detailed idiogram of chromosome 15 (Sawyer and Hozier, 1986); the bandwidths for
Biological Sciences,
Received
action with the high-affinity IL-2 receptor (Waldmann, 1989). Genes for the (Y and fl chains that constitute the receptor have been cloned from human and mouse (Shibuya et al., 1990; Kono et al., 1990). The IL-2 receptor fi
somes onto
Hugh D. Campbell,* Graham C. Webb,t Takeshi Kono,S Tadatsugu Taniguchi,$ Judith H. Ford,t and Ian G. Young§ ‘Centre
179
REPORTS
16
17
of mouse IL-2 receptor 100 cells is shown,
18 (3 chain cDNA with the arrow
19
X
Y
probe to mouse mitotic metaphase chromosomes. indicating a peak of grains over band 15E.
A histogram
of the
GENOMICS l&179-lSO(l992) 0888.7543/92
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.