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Mapping of the human ribonuclease inhibitor gene (RNH) to chromosome 11p15 by in situ hybridization
GENOMICS
8,
175-178
(1990)
SHORT COMMUNICATION Mapping of the Human Ribonuclease Inhibitor Gene (RNH) to Chromosome 11 pl5 by in Situ Hybridization SUSAN
M.
ZNEIMER,**’
DAVID
CRAWFORD,t
NANCY
R. SCHNEIDER,*
AND BRUCE BEUTLERt
*Department of Pathology and tl-loward Hughes Medical Institute and Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235 Received
February
1, 1990;
The turnover of RNA in eukaryotic cells is poorly understood, but neutral and alkaline ribonucleases are thought to play a critical role in the process. A number of these molecules have been studied extensively (Irie et al., 1988). The nucleases can be bound and held in a latent form by ribonuclease inhibitor (RNH), a 50,000-Da cytoplasmic protein (Blackburn et al, 1977). Under normal conditions, in excess of 90% of these nucleases are found in this inactive state (Roth, 1956). Thus, RNH may be essential for control of mRNA turnover. The ribonuclease/RNH interaction may be reversible in uiuo (Fominaya et aZ., 1988). Therefore, levels of free RNase may be controlled, at least in part, by the formation and dissociation of this complex. The proposal that the RNH/RNase ratio may have an imcorrespondence
should
April
23, 1990
pact on the cell’s fate is supported by observations that this ratio increases during periods of rapid cell growth and division (Quirin-Stricker et al., 1968) and decreases during periods of catabolism (Kraft et al., 1969). Although a single gene encodes RNH in humans, there are at least five distinct mRNAs that differ primarily in the 5’-untranslated regions (UTR) (Crawford et al., 1989). Recently, the cloning and sequencing of cDNAs encoding RNH from human placenta (Lee et al., 1988; Crawford et al., 1989) and HeLa cells (Schneider et al., 1988) were reported. The derived amino acid sequence shares approximately 80% homology with the porcine liver RNH sequence that was reported recently (Hofsteenge et al., 1988). The protein also has a repetitive leucine- and cysteine-rich sequence, similar to that found in a number of functionally diverse proteins, which may contribute to the conformation and function of this protein (Lee et al., 1988; Hofsteenge et al., 1988; Schneider et al., 1988). The recent cloning of RNH cDNAs has now allowed us to determine the chromosomal location of this gene by in. situ hybridization. Peripheral blood from cytogenetically normal individuals was cultured in phytohemagglutinin-supplemented medium for 72 and 96 h; metaphase cells were collected and chromosomes were fixed on slides by standard cytogenetic techniques. Slides of metaphase chromosomes were then stained with quinacrine dihydrochloride (0.005%) for 1 min, counterstained with actinomycin D (0.05%) for 15 min, and then stored overnight in the dark. Random metaphase spreads were photographed before hybridization for Q-banded chromosome identification in an unbiased sample of metaphases to be analyzed after hybridization. The probe used for hybridization consisted of a 600bp fragment of the RNH cDNA, containing about 450 bp of coding region and 150 bp of the 3’-UTR (Crawford et al., 1989). These sequences are shared by all of the
Ribonuclease inhibitor (RNH) is a protein that binds tightly to ribonucleases in cells and may be essential in the control of mRNA degradation and gene expression. The human RNH gene has been regionally localized to chromosome band 11~16 by in situ hybridization. A human placental cDNA was used to construct a 600-bp probe, which was then radiolabeled with tritium for in situ hybridization to human metaphase chromosomes. Localization of the RNH gene to IlplS, and possibly to 11~15.5, adds to a large number of genes assigned to this band, including 10 structural genes. This chromosomal region also represents an evolutionarily conserved syntenic group in the owl monkey, mouse, rat, and cow. Thus, regional assignment of RNH could facilitate the understanding of this gene and its association with ribonucleases, and perhaps extend a conserved syntenic region in mammalian genomes. 0 1990 Academic Press, Inc.
’ To whom
revised
be addressed. 175
o&M-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
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COMMUNICATION
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CHROMOSOMES FIG. 1. Idiograma of the 24 human chromosomes with the distribution of grains cDNA probe. Chromosome 11 contains three times the expected number of grains.
reported RNH splice forms. The probe extends from the most downstream Sac1 restriction site in the RNH cDNA to a site approximately 50 bp upstream from the poly(A) tail. This cDNA was radiolabeled with all four tritiated nucleotides to a specific activity of 5-7 X 10’ cpm/pg DNA (Feinberg and Vogelstein, 1983, 1984). In situ hybridization was carried out according to the procedure of Harper and Saunders (1981) modified according to Zneimer and Womack (1988). Slides were then dipped in Kodak NTB-2 emulsion and stored for 2 to 3 weeks at 4°C in the dark before development. Silver grains on all chromosomes were scored from those metaphase spreads previously Q-banded and photographed. Clusters of grains, in which single grains were not clearly distinguished, were rarely seen and not scored. In three independent hybridization experiments, 48 metaphase spreads were analyzed and 37 of the 272 total grains were present on chromosome 11 (Fig. 1). Therefore, 13.7% of all grains were located on a chromosome that represents only 4.3% of the total genome length. Of the grains present on chromosome 11,68% (25 grains) were localized to band 11~15; this is 9.2% of the total number of grains scored (Fig. 2). Twentytwo of the 25 grains localized to band 11~15 were at the telomere of the chromosome, suggesting localization to subband 11~15.5 (Fig. 3). All other chromosomes contained only the expected number of grains based on their individual fractions of the total genome length. The background level of grains was fewer than 6 grains per metaphase spread.
on each chromosome
after
hybridization
with
an RNH
Regional assignment of the ribonuclease inhibitor gene to chromosome band 11~15, with data suggestive of 11~15.5, adds one more gene to this highly studied region of the human genome. Other genes mapped to
-73 15.5 15.4 15.3
15.2
:::::::$W 0.0
15.1
P
0
l4 u
23.2 23.3
t-l
a.
FIG. 2. Idiogram depicting the total grain distribution on chromosome 11. A peak of 25 grains (68%) is seen at band 11~1.5, with 22 of these grains at the telomere, suggestive of subband 11~15.5.
SHORT
COMMUNICATION
FIG. 3. Upper left: Q-banded metaphase spread with arrows indicating human chromosome spread after in situ hybridization, with the arrow indicating a hybridization event at band 11~15. different cells, showing the Q-banded pattern on the left and hybridization grains at the telomere,
11~15 include the b-globin gene cluster (HBB), insulin (INS), insulin-like growth factor 2 (IGFB), tyrosine hydroxylase (TH), parathyroid hormone (PTH), Harvey ras oncogene (HRAS), calcitonin/calcitonin gene-related peptide (CALCA), lactate dehydrogenase (LDHA), and ribonucleotide reductase (RRMl) (see Junien and McBride, 1989, for review). Most of these genes have further been given a gene order within band 11~15 based on linkage data (Holm et al., 1985; Gerhard et al., 1985). Also associated with this region are at least four cloned anonymous DNA segments and one to six red blood cell antigens recognized by monoclonal antibodies (Palmer et al, 1985; Rettig et al., 1985). Many of the genes localized to 11~15 in the human genome are spatially related to one another in other mammalian species. Of the genes localized to human 11~15, LDHA, HBB, INS, PTH, and HRAS represent a conserved syntenic group on chromosome 19 of the owl monkey. The mouse also has the conserved synteny of Ldb-1, Hbb, Ins-l, Hrasl, Calc, Th, and Rrml on chromosome 7, and the rat homologs Ldh-1 and Hrasl are syntenic on chromosome 1 (see Lalley and McKusick, 1985, for review). In contrast, genes local-
11. Upper right: The same metaphase Lower row: Chromosome 11 from seven suggestive of 11~15.5, on the right.
ized to human llp14-11~11, includingcatalase (CAT), the p subunit of follicle stimulating hormone (FSHB), and acid phosphatase (ACP2), are all syntenic on mouse chromosome 2 (Hoffman and Grieshaber, 1974; Glaser et aZ., 1985). Therefore, this separation in the mouse of genes on human 11~15 from genes on llp1411~11 records the occurrence of a translocation event in mammalian chromosomal evolution. In cattle, a syntenic group containing CAT, PTH, and HBB has been mapped to chromosome 15 (Fries et al., 1988), but LDHA has been mapped to unidentified chromosome 7 (U7) (Womack and Moll, 1986). Thus, the syntenic relationship of genes localized to chromosome 11~15 in humans has been conserved on owl monkey chromosome 19; however, at least two disruptions have occurred during speciation, such that genes syntenic on human llp have been separated onto two mouse and two cow chromosomes. Given that the human RNH gene has now been localized to human 11~15, it would be informative to determine its chromosomal location in these other mammalian species to assess whether its syntenic relationship has been preserved. Also, ribonuclease in-
178
SHORT
COMMUNICATION
hibitors are known to exist not only in many tissues of different mammalian species (Roth, 1967) but also in those of birds (Dijkstra et aZ., 1978) and insects (Aoki and Natori, 1981). Assignment of the RNH chromosomal location in humans may facilitate an understanding of this gene, of genes with which it is associated, and of its chromosomal location in other species.
11.
12. 13.
14. ACKNOWLEDGMENTS The authors thank the technologists in the Cytogenetics Lahoratory, Department of Pathology, UTSMCD, for their technical assistance in the chromosome preparations. This work was supported in part by NIH Grant ROlCA45525 and by a grant from the Tobacco Research Council. D. Crawford is supported by NIH Grant GM08014. While these studies were in progress, we were informed by Dr. B. Vallee that he and his colleagues had independently established the location of the human RNH gene using in situ hybridization techniques. Their assignment was also to chromosome 11~15.
REFERENCES 1.
AOKI, Y., AND NATORI, S. (1981). Activation of latent clease in the fat-body of fleshfly (Sarcophagaperegrina) on pupation. Biochem. J. 196: 699-703.
2. BLACKBURN, clease inhibitor 5910.
3. CRAWFORD, splice forms 5’untranslated
15.
16.
17. KRAFT, N., SHORTMAN,
18.
19.
P., WILSON, G., AND MOORE, S. (1977). Ribonufrom human placenta. J. BioL Chem. 252: 5904-
20.
D., HAGERTY, K., AND BEUTLER, B. (1989). Multiple of ribonuclease inhibitor mRNA differ only in the region. Gene 85: 527-533.
21.
4. DIJKSTRA,
22.
363-373. 5. FEINBERG,
A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
6. FEINBERG,
A. P., AND VOGELSTEIN, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity: Addendum. Anal. B&hem. 137: 266-267.
23.
25.
8. FRIES, R., HEDIGER,
R., AND STRANZINGER, G. (1988). The loci for parathyroid hormone and @-globin are closely linked and map to chromosome 15 in cattle. Genomics 3: 302-307.
9. GERHARD,
D. S., VULLIAMY, T., BRUNS, G., KRAMER, P., GuSELLA, J., KIDD, J., KIDD, K., AND HOUSMAN, D. E. (1985). Linkage relationship among nine markers on the short arm of chromosome 11. Cytogenet. Cell Gener. 40: 640.
10. GLASER, T., GERHARD,
D., JONES, C., ALBRITTON, L., LALLEY, P., AND HOUSEMAN, D. (1985). A fine structure deletion map of chromosome 11~. Cytogenet. Cell Genet. 40: 643.
LEE, F. S., Fox, E. A., HAI-MENG, Z., STRYDOM, D. J., AND VALLEE, B. L. (1988). Primary structure of human placental ribonuclease inhibitor. Biochemistry 27: 8545-8553. PALMER, D. K., MILLER, Y. E., GEYER, D., JONES, C., DANIELS, G. L., AND TIPPET, P. (1985). MERB, a new human red cell polymorphism controlled by a locus on chromosome 11. Cytogenet. Cell Genet. 40: 720. QUIRIN-STRICKER, C., GROSS, M., AND MANDEL, P. (1968). Influence dune carence en proteines sur l’activite des ribonucleases et sur celle de leur inhibiteur dans le foie de rat. B&him. Biophys. Acta 159: 75-80. RETTIG, W. J., DRACOPOLI, N. C., SPENGLER, B. A., BIEDLER, J. L., AND OLD, L. J. (1985). Somatic cell genetic analysis of human cell-surface antigens, including putative human Thy-l: Eight distinct antigenic systems controlled by chromosome 11. Cytogenet. Cell Genet. 40: 732. ROTH, J. S. (1956). Studies on the properties and distribution of ribonuclease inhibitor in the rat. Arch. Biochem. Biophys.
21:22-44. 24. ROTH, J. S. (1967).
7. FOMINAYA,
J. M., GARCIA-SEGURA, J. M., FERRERAS, M., AND GAVILANES, J. G. (1988). Theoretical treatment of tight-binding of an enzyme: Ribonuclease inhibitor as special case. Biochem. J. 253: 517-522.
K., AND JAMESON, D. (1969). The effect of X-irradiation on the balance between alkaline ribonuclease inhibitor and the alkaline ribonuclease of mammalian tissues. Radiat. Res. 39: 655-668. LALLEY, P. A., AND MCKUSICK, V. A. (1985). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 40:
536-566.
ribonularvae
J., Touw, J., HALSEMA, I., GRUBER, M., AND AB, G. (1978). Estradiol-induced synthesis of viteliogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor. B&him. Biophys. Actu 521:
HARPER, M. E., AND SAUNDERS, G. F. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. HOFFMAN, H. A., AND GRIESHABER, C. K. (1974). Genetic studies of murine catalase. J. Hered. 65: 277-279. HOFSTEENGE, J., KIEFFER, B., MA?THIES, R., HEMMINGS, B. A,, AND STONE, S. R. (1988). Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leutine-rich repeats. Biochemistry 27: 8537-8544. HOLM, T., O’CONNELL, P., LEPPERT, M., CALLAHAN, P., LALOUEL, J.-M., AND WHITE, R. (1985). Parathyroid hormone and calcitonin are tightly linked and have been placed on the genetic map of chromosome llp. Cytogenet. Cell Genet. 40: 653. IRIE, M., NITTA, R., OHGI, K., NIWATA, Y., WANATABE, H., IWAMA, M., BEINTEMA, J. J., SANDA, A., AND TAKIZAWA, Y. (1988). Primary structure of a non-secretory ribonuclease from bovine kidney. J. Biochem. 104: 289-296. JUNIEN, C., AND MCBRIDE, 0. W. (1989). Report of the committee on the genetic constitution of chromosome 11. Cytogenet. CellGenet. 51:226-258.
26.
Some observations on the assay and properties of ribonucleases in normal and tumor tissues. In “Methods of Cancer Research” (H. Busch, Ed.), Vol. 3, pp. 153-242, Academic Press, New York. SCHNEIDER, R., SCHNEIDER-SCHERZER, E., THURNHER, M., AUER, B., AND SCHWEIGER, M. (1988). The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module. EMBO J. 7: 4151-4156. WOMACK, J. E., AND MOLL, Y. D. (1986). Gene map of the COW: Conservation of linkage with mouse and man. J. Hered. 77:
2-7. 27. ZNEIMER,
S. M., AND WOMACK, J. E. (1988). Regional localization of the fibronectin and gamma crystallin genes to mouse chromosome 1 by in situ hybridization. Cytogenet. Cell Genet.
48:238-241.
8,
175-178
(1990)
SHORT COMMUNICATION Mapping of the Human Ribonuclease Inhibitor Gene (RNH) to Chromosome 11 pl5 by in Situ Hybridization SUSAN
M.
ZNEIMER,**’
DAVID
CRAWFORD,t
NANCY
R. SCHNEIDER,*
AND BRUCE BEUTLERt
*Department of Pathology and tl-loward Hughes Medical Institute and Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235 Received
February
1, 1990;
The turnover of RNA in eukaryotic cells is poorly understood, but neutral and alkaline ribonucleases are thought to play a critical role in the process. A number of these molecules have been studied extensively (Irie et al., 1988). The nucleases can be bound and held in a latent form by ribonuclease inhibitor (RNH), a 50,000-Da cytoplasmic protein (Blackburn et al, 1977). Under normal conditions, in excess of 90% of these nucleases are found in this inactive state (Roth, 1956). Thus, RNH may be essential for control of mRNA turnover. The ribonuclease/RNH interaction may be reversible in uiuo (Fominaya et aZ., 1988). Therefore, levels of free RNase may be controlled, at least in part, by the formation and dissociation of this complex. The proposal that the RNH/RNase ratio may have an imcorrespondence
should
April
23, 1990
pact on the cell’s fate is supported by observations that this ratio increases during periods of rapid cell growth and division (Quirin-Stricker et al., 1968) and decreases during periods of catabolism (Kraft et al., 1969). Although a single gene encodes RNH in humans, there are at least five distinct mRNAs that differ primarily in the 5’-untranslated regions (UTR) (Crawford et al., 1989). Recently, the cloning and sequencing of cDNAs encoding RNH from human placenta (Lee et al., 1988; Crawford et al., 1989) and HeLa cells (Schneider et al., 1988) were reported. The derived amino acid sequence shares approximately 80% homology with the porcine liver RNH sequence that was reported recently (Hofsteenge et al., 1988). The protein also has a repetitive leucine- and cysteine-rich sequence, similar to that found in a number of functionally diverse proteins, which may contribute to the conformation and function of this protein (Lee et al., 1988; Hofsteenge et al., 1988; Schneider et al., 1988). The recent cloning of RNH cDNAs has now allowed us to determine the chromosomal location of this gene by in. situ hybridization. Peripheral blood from cytogenetically normal individuals was cultured in phytohemagglutinin-supplemented medium for 72 and 96 h; metaphase cells were collected and chromosomes were fixed on slides by standard cytogenetic techniques. Slides of metaphase chromosomes were then stained with quinacrine dihydrochloride (0.005%) for 1 min, counterstained with actinomycin D (0.05%) for 15 min, and then stored overnight in the dark. Random metaphase spreads were photographed before hybridization for Q-banded chromosome identification in an unbiased sample of metaphases to be analyzed after hybridization. The probe used for hybridization consisted of a 600bp fragment of the RNH cDNA, containing about 450 bp of coding region and 150 bp of the 3’-UTR (Crawford et al., 1989). These sequences are shared by all of the
Ribonuclease inhibitor (RNH) is a protein that binds tightly to ribonucleases in cells and may be essential in the control of mRNA degradation and gene expression. The human RNH gene has been regionally localized to chromosome band 11~16 by in situ hybridization. A human placental cDNA was used to construct a 600-bp probe, which was then radiolabeled with tritium for in situ hybridization to human metaphase chromosomes. Localization of the RNH gene to IlplS, and possibly to 11~15.5, adds to a large number of genes assigned to this band, including 10 structural genes. This chromosomal region also represents an evolutionarily conserved syntenic group in the owl monkey, mouse, rat, and cow. Thus, regional assignment of RNH could facilitate the understanding of this gene and its association with ribonucleases, and perhaps extend a conserved syntenic region in mammalian genomes. 0 1990 Academic Press, Inc.
’ To whom
revised
be addressed. 175
o&M-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
SHORT
III
v
I
COMMUNICATION
I III
lllllllll
mnpUI~II~mIInq
Illll
q 1
Ill1 I
P
I II I
III
pC1mmpmCLBIulj
q
2
IIIII
I p.IIfm~:Cm.q
P
3
4
q
III
I
pm
I
I III
P 5
5
0 F lllll
II
Ill1
17-FTY9F 13
14
III
Ill1
-7
154
IIIIII
FyFg1 16
llllII
yfy.3 17
III
yP~9 16
II Ill
I
II
Ill
III
FyFy TFq’ E-9 fy;i 19
26
21
III
II
f’-Jyl-y~T
22
X
y=$ Y
CHROMOSOMES FIG. 1. Idiograma of the 24 human chromosomes with the distribution of grains cDNA probe. Chromosome 11 contains three times the expected number of grains.
reported RNH splice forms. The probe extends from the most downstream Sac1 restriction site in the RNH cDNA to a site approximately 50 bp upstream from the poly(A) tail. This cDNA was radiolabeled with all four tritiated nucleotides to a specific activity of 5-7 X 10’ cpm/pg DNA (Feinberg and Vogelstein, 1983, 1984). In situ hybridization was carried out according to the procedure of Harper and Saunders (1981) modified according to Zneimer and Womack (1988). Slides were then dipped in Kodak NTB-2 emulsion and stored for 2 to 3 weeks at 4°C in the dark before development. Silver grains on all chromosomes were scored from those metaphase spreads previously Q-banded and photographed. Clusters of grains, in which single grains were not clearly distinguished, were rarely seen and not scored. In three independent hybridization experiments, 48 metaphase spreads were analyzed and 37 of the 272 total grains were present on chromosome 11 (Fig. 1). Therefore, 13.7% of all grains were located on a chromosome that represents only 4.3% of the total genome length. Of the grains present on chromosome 11,68% (25 grains) were localized to band 11~15; this is 9.2% of the total number of grains scored (Fig. 2). Twentytwo of the 25 grains localized to band 11~15 were at the telomere of the chromosome, suggesting localization to subband 11~15.5 (Fig. 3). All other chromosomes contained only the expected number of grains based on their individual fractions of the total genome length. The background level of grains was fewer than 6 grains per metaphase spread.
on each chromosome
after
hybridization
with
an RNH
Regional assignment of the ribonuclease inhibitor gene to chromosome band 11~15, with data suggestive of 11~15.5, adds one more gene to this highly studied region of the human genome. Other genes mapped to
-73 15.5 15.4 15.3
15.2
:::::::$W 0.0
15.1
P
0
l4 u
23.2 23.3
t-l
a.
FIG. 2. Idiogram depicting the total grain distribution on chromosome 11. A peak of 25 grains (68%) is seen at band 11~1.5, with 22 of these grains at the telomere, suggestive of subband 11~15.5.
SHORT
COMMUNICATION
FIG. 3. Upper left: Q-banded metaphase spread with arrows indicating human chromosome spread after in situ hybridization, with the arrow indicating a hybridization event at band 11~15. different cells, showing the Q-banded pattern on the left and hybridization grains at the telomere,
11~15 include the b-globin gene cluster (HBB), insulin (INS), insulin-like growth factor 2 (IGFB), tyrosine hydroxylase (TH), parathyroid hormone (PTH), Harvey ras oncogene (HRAS), calcitonin/calcitonin gene-related peptide (CALCA), lactate dehydrogenase (LDHA), and ribonucleotide reductase (RRMl) (see Junien and McBride, 1989, for review). Most of these genes have further been given a gene order within band 11~15 based on linkage data (Holm et al., 1985; Gerhard et al., 1985). Also associated with this region are at least four cloned anonymous DNA segments and one to six red blood cell antigens recognized by monoclonal antibodies (Palmer et al, 1985; Rettig et al., 1985). Many of the genes localized to 11~15 in the human genome are spatially related to one another in other mammalian species. Of the genes localized to human 11~15, LDHA, HBB, INS, PTH, and HRAS represent a conserved syntenic group on chromosome 19 of the owl monkey. The mouse also has the conserved synteny of Ldb-1, Hbb, Ins-l, Hrasl, Calc, Th, and Rrml on chromosome 7, and the rat homologs Ldh-1 and Hrasl are syntenic on chromosome 1 (see Lalley and McKusick, 1985, for review). In contrast, genes local-
11. Upper right: The same metaphase Lower row: Chromosome 11 from seven suggestive of 11~15.5, on the right.
ized to human llp14-11~11, includingcatalase (CAT), the p subunit of follicle stimulating hormone (FSHB), and acid phosphatase (ACP2), are all syntenic on mouse chromosome 2 (Hoffman and Grieshaber, 1974; Glaser et aZ., 1985). Therefore, this separation in the mouse of genes on human 11~15 from genes on llp1411~11 records the occurrence of a translocation event in mammalian chromosomal evolution. In cattle, a syntenic group containing CAT, PTH, and HBB has been mapped to chromosome 15 (Fries et al., 1988), but LDHA has been mapped to unidentified chromosome 7 (U7) (Womack and Moll, 1986). Thus, the syntenic relationship of genes localized to chromosome 11~15 in humans has been conserved on owl monkey chromosome 19; however, at least two disruptions have occurred during speciation, such that genes syntenic on human llp have been separated onto two mouse and two cow chromosomes. Given that the human RNH gene has now been localized to human 11~15, it would be informative to determine its chromosomal location in these other mammalian species to assess whether its syntenic relationship has been preserved. Also, ribonuclease in-
178
SHORT
COMMUNICATION
hibitors are known to exist not only in many tissues of different mammalian species (Roth, 1967) but also in those of birds (Dijkstra et aZ., 1978) and insects (Aoki and Natori, 1981). Assignment of the RNH chromosomal location in humans may facilitate an understanding of this gene, of genes with which it is associated, and of its chromosomal location in other species.
11.
12. 13.
14. ACKNOWLEDGMENTS The authors thank the technologists in the Cytogenetics Lahoratory, Department of Pathology, UTSMCD, for their technical assistance in the chromosome preparations. This work was supported in part by NIH Grant ROlCA45525 and by a grant from the Tobacco Research Council. D. Crawford is supported by NIH Grant GM08014. While these studies were in progress, we were informed by Dr. B. Vallee that he and his colleagues had independently established the location of the human RNH gene using in situ hybridization techniques. Their assignment was also to chromosome 11~15.
REFERENCES 1.
AOKI, Y., AND NATORI, S. (1981). Activation of latent clease in the fat-body of fleshfly (Sarcophagaperegrina) on pupation. Biochem. J. 196: 699-703.
2. BLACKBURN, clease inhibitor 5910.
3. CRAWFORD, splice forms 5’untranslated
15.
16.
17. KRAFT, N., SHORTMAN,
18.
19.
P., WILSON, G., AND MOORE, S. (1977). Ribonufrom human placenta. J. BioL Chem. 252: 5904-
20.
D., HAGERTY, K., AND BEUTLER, B. (1989). Multiple of ribonuclease inhibitor mRNA differ only in the region. Gene 85: 527-533.
21.
4. DIJKSTRA,
22.
363-373. 5. FEINBERG,
A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
6. FEINBERG,
A. P., AND VOGELSTEIN, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity: Addendum. Anal. B&hem. 137: 266-267.
23.
25.
8. FRIES, R., HEDIGER,
R., AND STRANZINGER, G. (1988). The loci for parathyroid hormone and @-globin are closely linked and map to chromosome 15 in cattle. Genomics 3: 302-307.
9. GERHARD,
D. S., VULLIAMY, T., BRUNS, G., KRAMER, P., GuSELLA, J., KIDD, J., KIDD, K., AND HOUSMAN, D. E. (1985). Linkage relationship among nine markers on the short arm of chromosome 11. Cytogenet. Cell Gener. 40: 640.
10. GLASER, T., GERHARD,
D., JONES, C., ALBRITTON, L., LALLEY, P., AND HOUSEMAN, D. (1985). A fine structure deletion map of chromosome 11~. Cytogenet. Cell Genet. 40: 643.
LEE, F. S., Fox, E. A., HAI-MENG, Z., STRYDOM, D. J., AND VALLEE, B. L. (1988). Primary structure of human placental ribonuclease inhibitor. Biochemistry 27: 8545-8553. PALMER, D. K., MILLER, Y. E., GEYER, D., JONES, C., DANIELS, G. L., AND TIPPET, P. (1985). MERB, a new human red cell polymorphism controlled by a locus on chromosome 11. Cytogenet. Cell Genet. 40: 720. QUIRIN-STRICKER, C., GROSS, M., AND MANDEL, P. (1968). Influence dune carence en proteines sur l’activite des ribonucleases et sur celle de leur inhibiteur dans le foie de rat. B&him. Biophys. Acta 159: 75-80. RETTIG, W. J., DRACOPOLI, N. C., SPENGLER, B. A., BIEDLER, J. L., AND OLD, L. J. (1985). Somatic cell genetic analysis of human cell-surface antigens, including putative human Thy-l: Eight distinct antigenic systems controlled by chromosome 11. Cytogenet. Cell Genet. 40: 732. ROTH, J. S. (1956). Studies on the properties and distribution of ribonuclease inhibitor in the rat. Arch. Biochem. Biophys.
21:22-44. 24. ROTH, J. S. (1967).
7. FOMINAYA,
J. M., GARCIA-SEGURA, J. M., FERRERAS, M., AND GAVILANES, J. G. (1988). Theoretical treatment of tight-binding of an enzyme: Ribonuclease inhibitor as special case. Biochem. J. 253: 517-522.
K., AND JAMESON, D. (1969). The effect of X-irradiation on the balance between alkaline ribonuclease inhibitor and the alkaline ribonuclease of mammalian tissues. Radiat. Res. 39: 655-668. LALLEY, P. A., AND MCKUSICK, V. A. (1985). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 40:
536-566.
ribonularvae
J., Touw, J., HALSEMA, I., GRUBER, M., AND AB, G. (1978). Estradiol-induced synthesis of viteliogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor. B&him. Biophys. Actu 521:
HARPER, M. E., AND SAUNDERS, G. F. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. HOFFMAN, H. A., AND GRIESHABER, C. K. (1974). Genetic studies of murine catalase. J. Hered. 65: 277-279. HOFSTEENGE, J., KIEFFER, B., MA?THIES, R., HEMMINGS, B. A,, AND STONE, S. R. (1988). Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leutine-rich repeats. Biochemistry 27: 8537-8544. HOLM, T., O’CONNELL, P., LEPPERT, M., CALLAHAN, P., LALOUEL, J.-M., AND WHITE, R. (1985). Parathyroid hormone and calcitonin are tightly linked and have been placed on the genetic map of chromosome llp. Cytogenet. Cell Genet. 40: 653. IRIE, M., NITTA, R., OHGI, K., NIWATA, Y., WANATABE, H., IWAMA, M., BEINTEMA, J. J., SANDA, A., AND TAKIZAWA, Y. (1988). Primary structure of a non-secretory ribonuclease from bovine kidney. J. Biochem. 104: 289-296. JUNIEN, C., AND MCBRIDE, 0. W. (1989). Report of the committee on the genetic constitution of chromosome 11. Cytogenet. CellGenet. 51:226-258.
26.
Some observations on the assay and properties of ribonucleases in normal and tumor tissues. In “Methods of Cancer Research” (H. Busch, Ed.), Vol. 3, pp. 153-242, Academic Press, New York. SCHNEIDER, R., SCHNEIDER-SCHERZER, E., THURNHER, M., AUER, B., AND SCHWEIGER, M. (1988). The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module. EMBO J. 7: 4151-4156. WOMACK, J. E., AND MOLL, Y. D. (1986). Gene map of the COW: Conservation of linkage with mouse and man. J. Hered. 77:
2-7. 27. ZNEIMER,
S. M., AND WOMACK, J. E. (1988). Regional localization of the fibronectin and gamma crystallin genes to mouse chromosome 1 by in situ hybridization. Cytogenet. Cell Genet.
48:238-241.