GENOMICS
6, 540-544
(1990)
The Gene for the RNA Component of the Mitochondrial RNAProcessing Endoribonuclease Is Located on Human Chromosome 9p and on Mouse Chromosome 4 C.-L. HSIEH,* T. A. DONLON,t B. T. DARRAS,~ D. D. CHANG,t J. N. TOPPER,7 D. A. CLAYToN,t*$ AND U. FRANCKE* *Howard
Hughes Medical Institute and Department of Genetics, tDepartment of Pathology, and *Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305; and §Department of Pediatrics, New England Medical Center, Boston, Massachusetts 02111 Received
September
18, 1989;
Academic
Prem,
Inc.
INTRODUCTION
Mitochondrial RNA-processing endoribonuclease (RNAase MRP) is a ribonucleoprotein particle (RNP) present in both the nucleus and the mitochondria of vertebrate cells. In uitro, this RNP can cleave mitochondrial RNA complementary to the light strand of the displacement loop (D-loop) region (Clayton, 1984) at a unique site (Chang and Clayton, 1987a,b). This cleavage site is one of the transition sites of primer RNA synthesis to DNA synthesis at the leading strand origin of mitochondrial DNA replication. It has been suggested that this RNP is involved in the processing of RNA primers at the origin of leading-strand DNA OSSS-7543/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
1, 1989
MATERIALS
AND
METHODS
Somatic Cell Hybrids Primary chromosomal assignment in human was carried out with a panel of 11 hybrid clones that were derived from 5 independent fusion experiments between Chinese hamster and human cell lines (for summary see Yang-Feng et al., 1986). For mapping in mouse, 10 Chinese hamster X mouse and one rat X mouse hybrid clone or subclones derived from four 540
Inc. reserved.
December
replication. An RNA component of MRP (MRP RNA) is essential for enzymatic activity of RNAase MRP. Recent isolation and sequencing of the gene for the MRP RNA revealed that this RNA is encoded in the nucleus and then imported into mitochondria. Fulllength MRP RNA is 275 nucleotides in mouse and 265 nucleotides in human (Chang and Clayton, 1989; Topper and Clayton, 1989). The coding region of this gene is highly conserved between human and mouse (84% homology), and the 715bp upstream 5’ flanking region of this gene is 70% conserved between these two species (Topper and Clayton, 1990). Little or no homology exists downstream of the gene. Members of the U series of small nuclear RNAs (snRNA) have a sequence similarity of 90-95% between human and mouse, but the 5’ flanking regions have only about 50% similarity that is limited to a few hundred base pairs upstream of transcriptional start sites (Birnstiel et al., 1988). Therefore, the gene for MRP RNA seemsto be unique among the genesfor snRNAs identified so far. We have mapped the RMRP gene to chromosome 9p21-p12 in human and chromosome 4 in mouse with human and mouse cDNA probes by using somatic cell hybrid analysis and in situ hybridization.
Mitochondrial RNA-processing endoribonuclease (RNAase MRP) has the capacity to cleave mitochondrial RNA complementary to the light strand of the displacement loop at a unique site. The enzyme is a ribonucleoprotein whose RNA component is a nuclear gene product. The 5’ flanking region of the primary transcript has control elements characteristic of RNA polymerase II transcription, and the coding region has features of RNA polymerase III transcription signals. The RNA associated with RNAase MRP is the first known RNA encoded by a single-copy gene in the nucleus and believed to be imported into mitochondria. The gene (RMRP) for this RNA component of RNAase MRP was assigned to human chromosome 9 and mouse chromosome 4 by Southern blot analyses of 11 human X rodent hybrids and 11 mouse X rodent hybrids with probe pHMl.0 and probe pSP270, respectively. In situ hybridization of probe pHSTU300 to normal human chromosomes revealed 29 of 100 cells with label on 9p and 9.6% of 302 silver grains located at 9p21-~12. 0 1990
revised
MAPPING 1
2
3
4
5
OF
RMRP
6
541
GENES
7
8
9
10
11
12
13
FIG. 1. Mapping of RMRP in the human. Hybridization of “P-labeled pHM1.0 probe to a Southern blot of BglII-digested DNA from Chinese hamster X human hybrid cell lines and controls. Lane 1, Chinese hamster cells V79/380-6; lane 2, diploid lymphoblastoid cells; lanes 3-13, Chinese hamster X human hybrid cell lines. Lanes 5 and Q-11 contain the 8.0-kb human MRP RNA sequence as well as Chinese hamster MRP RNA sequence and were scored as +. Lanes 3,4, 6-8, 12, and 13 contain only Chinese hamster MRP RNA sequences and were scored as -.
Southern Blot Hybridization Ten micrograms of DNA from each of the hybrid and parental control cell lines was digested with a fourfold excess of Bg211in high salt buffer. DNA fragments were separated by agarose gel electrophoresis and transferred to Hybond-N membranes (Amersham) by the method of Southern (1975). The probes were labeled with [32P]dCTP by oligolabeling (Feinberg and Vogelstein, 1983). The filters were prehybridized in 50% formamide (for human mapping) or 40% formamide (for mouse mapping), 5X SSC, 5X Denhardt’s, 1% SDS, and 100 pg/ml ssDNA for 4 h and then hybridized in 50% or 40% formamide, 5X SSC, 1X Denhardt’s, 1% SDS, 5% dextran sulfate, and 100 pg/ml ssDNA at 42°C for 16 h. After hybridization, filters were rinsed in 2X SSC and 0.5% SDS, washed in 2X SSC or 2X SSC and 0.5% SDS at room temperature for 15 min, and washed in 1X SSC, 1% SDS at 65°C
different series of hybrids (Francke et al., 1977; Francke and Taggart, 1979; Joyner et al., 1985) were used. Probes The probes used for Southern blot hybridization were the gel-purified inserts from clones pHM1.0 and pSP270. For in situ hybridization, pHSTU300 was used. Clone pHM1.0 is a l.O-kb human DNA fragment containing 700 bp of the 5’ flanking region, the coding region, and approximately 25 bp of 3’ flanking sequence, inserted into the (or a) PstI site of pSP65 plasmid. Clone pSP270 is a 0.3-kb mouse DNA fragment containing only the coding region, inserted into the PstI and EcoRI sites of pSP64 plasmid. Clone pHSTU300 is a 0.3-kb fragment of human DNA containing most of the coding region and a small portion of 3’ flanking sequences, inserted into the SmaI and PstI sites of pSP65 plasmid.
TABLE Correlation
of Human
RMRP
Sequences
with
Human
1
Chromosomes
in Rodent
Human 12
Hybridization/chromosome
+/+
2 112 5 7 24 2 3 31 20512414023223
-/+/-/+ Discordant Informative
hybrids hvbrids
Note. The numbers for each chromosome. excluded.
34
4 11 of hybrids Hybrids
3 11
82 11 8
that are concordant in which a particular
67
8
9
10
11
12
0 5 3
212 35 22
3 2
3 7 0
0 5 4
0 3 3
2 5 2
5 10
63 11 9
6 11
0 10
6 11
6 9
4 11
and discordant was structurally
Somatic
Cell Hybrids
chromosomes
5
(+/+ or -/-) chromosome
X Human
13
17
18
32420432231 5 3 4 5 5 110 2 4 322332352
4 0
3 11
(+/or -/+) rearranged
14
4 9
15
3 11
16
4 11
6 11
19
4 12
344552 11 11
20
21
5
3 2
11
10
22
X
22 00
10
5
with the human RMRP sequence are given or present in fewer than 10% of cells were
542
HSIEH
ET
AL.
pHSTU300
3T’ II1
13
FIG. 2. Histogram autoradiography. There
I
I
14
15
of grain distribution is a single nonrandom
16
17
In Situ Hybridization All procedures for in situ hybridization were as described previously (Donlon, 1986). The distribution of autoradiographic silver grains on chromosomes was scored in 100 metaphase cells. RESULTS
of the Human
RMRP
18
7 p1g
21
“p*pip-r
22
II
ID1 X
Y
after in situ hybridization of pHSTU300 probe to normal human metaphase chromosomes and peak at 9p21-~12 and only background label over the rest of the human chromosome complement.
for 10 min. All filters were autoradiographed at -70°C for various lengths of time with Kodak X-Omat AR film.
Mapping
*Pq
IhI
I
Gene
The human gene was mapped with a panel of 11 Chinese hamster X human somatic cell hybrids having reduced numbers of human chromosomes. After hybridization of the 32P-labeled human cDNA probe pHM1.0 to BgZII-digested genomic DNA, a single restriction fragment of 8.0 kb was detected in human control DNA (Fig. 1, lane 2) and a 4.2-kb fragment was observed in Chinese hamster control DNA (Fig. 1, lane 1). In hybrids containing human chromosome 9, the
8.0-kb human signal as well as the Chinese hamster signal was present (Fig. 1, lanes 5, 9, 10, and 11). No human signal was detected in hybrids not containing human chromosome 9 (Fig. 1, lanes 3,4,6-8, 12, and 13). The presence or absence of the human RMRP sequence was in perfect concordance with human chromosome 9 in hybrid cell lines (Table 1). All other human chromosomes were excluded by at least 2 discordant hybrids. Silver grains were scored after in situ hybridization of 3H-labeled human cDNA probe pHSTU300 to human metaphase chromosome spreads. Twenty-nine of the 100 cells had label on 9p, and 9.6% of 302 silver grains were located at 9p21-p12 (Fig. 2). No other chromosome site was labeled above background.
Mapping
of the Murk
Rmrp Gene
The mouse gene was assigned with a panel of 11 mouse X rodent somatic cell hybrids having reduced numbers of mouse chromosomes. After hybridization of the 32P-labeledmouse cDNA probe pSP270 to BgZII-
MAPPING 0
1234667
9
10
11
543
RMRP GENES
OF
12
ster fragments was present (Fig. 3, lanes 3, 5, and 8). No mouse signal was detected in hybrids not containing mouse chromosome 4 (Fig. 3, lanes 4,6,7, and 9-11). The presence of the mouse RMRP sequence showed perfect concordance with the presence of mouse chromosome 4 in hybrid cell lines (Table 2). All other mouse chromosomes were excluded by at least 2 discordant hybrids.
42
DISCUSSION A.2
We have mapped the RMRP gene to human chromosome 9 by analysis of somatic cell hybrids. The in situ hybridization result placed this locus on the short arm of chromosome 9, at region p21-~12. Since only one fragment was observed in BglII- (this study), BamHI-, and PstI-digested (J. N. Topper, unpublished observation) human DNA on Southern blots and only one site was detected with in situ hybridization, we conclude that RMRP is a single-copy gene with no other highly homologous sequences in human. In the mouse, we have mapped this gene (locus symbol Rmrp) to chromosome 4 with somatic cell hybrids. The observation of a single fragment of 7.2 kb in BglII-digested mouse DNA also suggestedthe absenceof closely related sequences in the mouse genome. Comparative mapping of human and mouse chromosomes has revealed eight other genes,IFNA, IFNB, GGTBZ, GALT, ACOl, ALAD, ORMl, and ORM2, on mouse chromosome 4 and on human chromosome 9 (Lalley et al, 1988). The region of mouse chromosome 4 that contains these loci includes bands A3 to C6 (Lyon, 1989). In humans, the first four of these eight genes, including the interferon genes and the galactosyltransferase genes listed, have been assigned to the short arm of chromosome 9. GALT is the locus for galactose-lphosphate uridyltransferase, deficiency of which causes
12 Mch
sgl
R
II
FIG. 3. Mapping of Rmrp in the mouse. Hybridization of 32Plabeled pSP270 probe to a Southern blot of BglII-digested DNA from rodent X mouse hybrid cell lines and controls. Lane 1, mouse liver; lane 2, Chinese hamster cells V79/380-6; lane 12, rat Morris hepatoma 7777 cells; lanes 3-10, Chinese hamster X mouse hybrid cell lies; lane 11, rat X mouse hybrid cell lie. Lanes 3,S, and 8 contain mouse MRP RNA sequences as well as Chinese hamster MRP RNA sequences and were scored as +. Lanes 4,6,7, and 9-l 1 contain only Chinese hamster or rat MRP RNA sequences and were scored as -.
digested genomic DNA, a single fragment of 7.2 kb was detected in mouse control DNA (Fig. 3, lane l), two fragments of sizes 4.2 and 1.2 kb were observed in Chinese hamster control DNA (Fig. 3, lane 2), and a 2.2-kb fragment was detected in rat control DNA (Fig. 3, lane 12). In hybrids that contain mouse chromosome 4, the 7.2-kb mouse signal as well as the Chinese ham-
TABLE Correlation
2
of Mouse-Specific Rmrp Sequences with Mouse in Rodent x Mouse Somatic Cell Hybrids Mouse
Hybridization/chromosome
+/+
Note. The numbers for each chromosome. excluded.
2
3
24341143110 1 2 5 2 0 1 5 5 2
-/+/-/+ Discordant Informative
1
hybrids hybrids of hybrids Hybrids
7 10 that are concordant in which a particular
5 11
3 11
4
5
chromosome
7
8
9
643 3 0320132411201 013 3
5
547365
2
2
3
3 11
5 11
5 10
045 10 9
(+/+ or -/-) chromosome
6
9
3 10
and discordant was structurally
Chromosomes
10
11
12
13
14
3
2
2
0
4
12
4 11
5 11
2 10
4 11
15
16
17
18
19
4 2 333532
4
2
4
2
2 13
0
1 5
3 10
6 10
4
4
0 4
4 11
5 10
4 11
3 10
X
(+/or -/+) with the mouse Rmrp sequences are given rearranged or present in fewer than 10% of cells were
544
HSIEH
galactosemia. The AC01 gene, coding for an enzyme that catalyzes the isomerization of citrate inside of mitochondria, has been mapped to 9p22-q32. The RMRP gene appears to be a new member of this conserved syntenic group.
ET
AL. 6.
DONLON, T. A. (1986). Practical ization. Ka~ogram 12: 3-10.
7.
FEINBERG, A., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
8.
FRANCKE, U., LALLEY, P. A., Moss, W., Iw, J., AND MINNA, J. D. (1977). Gene mapping in Mus musculus by interspecies cell hybridization: Assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 12 and adenylate kinase-1 to chromosome 2. Cytogenct. Cell Genet. 19: 57-84. FRANCKE, U., AND TAGGART, R. T. (1979). Assignment of gene for cytoplasmic superoxide dismutase (Sod-I) to a region of chromosome 16 and of Hprt to a region of the X chromosome in the mouse. Proc. Natl. Acad. Sci. USA 76: 5230-5233. JOYNER, A. C., LEBO, R. V., RAN, Y. W., TJIAN, R., Cox, D. R., AND MARTIN, G. R. (1985). Comparative chromosome mapping of a conserved homeo box region in mouse and human. Nature (London) 314: 173-175.
ACKNOWLEDGMENTS The work in U. Francke’s laboratory was supported by NIH Grant GM26105 U.F. is an Investigator and C.-L.H. an Associate of the Howard Hughes Medical Institute. Isolation of the human and mouse RNAase MRP RNA genes in D. A. Clayton’s laboratory was supported by NIH Grant GM33088. D.D.C. and J.N.T. were and are, respectively, Medical Scientist Training Program Trainees supported by NIH Grant GM07365.
9.
10.
REFERENCES 1.
BIRNSTIEL, M. L., AND SCHALJFELE, F. J. (1988). Structure and function of minor snRNPs. In “Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles” (M. L. Birnsteil, Ed.), pp. 155-182, Springer-Verlag, Berlin.
2.
CHANG, D. D., AND CLAYTON, D. A. (1987a). A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J. 6: 409-417. CHANG, D. D., AND CLAYTON, D. A. (1987b). A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science 235: 1179-1184.
3.
4.
5.
CHANG, D. D., AND CLAYTON, D. A. (1989). Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate. Cell 66: 131-139. CLAYTON, tochondrial
D. A. (1984). Transcription of the mammalian genome. Annu. Reu. Biochem. 63: 573-594.
mi-
approaches
to in situ hybrid-
11.
LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIEN, S. J., RODERICK, T. H., DOOLI~LE, D. P., AND HILLYARD, A. L. (1988). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 49: 227-235.
12.
LYON, M. F. (1989). Mouse chromosome atlas. Mouse News L&t. 84: 24-25. SOUTHERN, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. TOPPER, J. N., AND CLAYTON, D. A. (1990). Characterization of human MRP/Th RNA and its nuclear gene: Full length MRP/Th RNA is an active endonuclease when assembled as an RNP. Nucleic Acids Res., in press. YANG-FENG, T. L., DEGENNARO, L. J., AND FRANME, U. (1986). Genes for synapsin I, a neuronal phosphoprotein, map to conserved regions of human and murine X chromosomes. Proc. Natl. Acad. Sci. USA 83: 8679-8683.
13.
14.
15.
6, 540-544
(1990)
The Gene for the RNA Component of the Mitochondrial RNAProcessing Endoribonuclease Is Located on Human Chromosome 9p and on Mouse Chromosome 4 C.-L. HSIEH,* T. A. DONLON,t B. T. DARRAS,~ D. D. CHANG,t J. N. TOPPER,7 D. A. CLAYToN,t*$ AND U. FRANCKE* *Howard
Hughes Medical Institute and Department of Genetics, tDepartment of Pathology, and *Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305; and §Department of Pediatrics, New England Medical Center, Boston, Massachusetts 02111 Received
September
18, 1989;
Academic
Prem,
Inc.
INTRODUCTION
Mitochondrial RNA-processing endoribonuclease (RNAase MRP) is a ribonucleoprotein particle (RNP) present in both the nucleus and the mitochondria of vertebrate cells. In uitro, this RNP can cleave mitochondrial RNA complementary to the light strand of the displacement loop (D-loop) region (Clayton, 1984) at a unique site (Chang and Clayton, 1987a,b). This cleavage site is one of the transition sites of primer RNA synthesis to DNA synthesis at the leading strand origin of mitochondrial DNA replication. It has been suggested that this RNP is involved in the processing of RNA primers at the origin of leading-strand DNA OSSS-7543/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
1, 1989
MATERIALS
AND
METHODS
Somatic Cell Hybrids Primary chromosomal assignment in human was carried out with a panel of 11 hybrid clones that were derived from 5 independent fusion experiments between Chinese hamster and human cell lines (for summary see Yang-Feng et al., 1986). For mapping in mouse, 10 Chinese hamster X mouse and one rat X mouse hybrid clone or subclones derived from four 540
Inc. reserved.
December
replication. An RNA component of MRP (MRP RNA) is essential for enzymatic activity of RNAase MRP. Recent isolation and sequencing of the gene for the MRP RNA revealed that this RNA is encoded in the nucleus and then imported into mitochondria. Fulllength MRP RNA is 275 nucleotides in mouse and 265 nucleotides in human (Chang and Clayton, 1989; Topper and Clayton, 1989). The coding region of this gene is highly conserved between human and mouse (84% homology), and the 715bp upstream 5’ flanking region of this gene is 70% conserved between these two species (Topper and Clayton, 1990). Little or no homology exists downstream of the gene. Members of the U series of small nuclear RNAs (snRNA) have a sequence similarity of 90-95% between human and mouse, but the 5’ flanking regions have only about 50% similarity that is limited to a few hundred base pairs upstream of transcriptional start sites (Birnstiel et al., 1988). Therefore, the gene for MRP RNA seemsto be unique among the genesfor snRNAs identified so far. We have mapped the RMRP gene to chromosome 9p21-p12 in human and chromosome 4 in mouse with human and mouse cDNA probes by using somatic cell hybrid analysis and in situ hybridization.
Mitochondrial RNA-processing endoribonuclease (RNAase MRP) has the capacity to cleave mitochondrial RNA complementary to the light strand of the displacement loop at a unique site. The enzyme is a ribonucleoprotein whose RNA component is a nuclear gene product. The 5’ flanking region of the primary transcript has control elements characteristic of RNA polymerase II transcription, and the coding region has features of RNA polymerase III transcription signals. The RNA associated with RNAase MRP is the first known RNA encoded by a single-copy gene in the nucleus and believed to be imported into mitochondria. The gene (RMRP) for this RNA component of RNAase MRP was assigned to human chromosome 9 and mouse chromosome 4 by Southern blot analyses of 11 human X rodent hybrids and 11 mouse X rodent hybrids with probe pHMl.0 and probe pSP270, respectively. In situ hybridization of probe pHSTU300 to normal human chromosomes revealed 29 of 100 cells with label on 9p and 9.6% of 302 silver grains located at 9p21-~12. 0 1990
revised
MAPPING 1
2
3
4
5
OF
RMRP
6
541
GENES
7
8
9
10
11
12
13
FIG. 1. Mapping of RMRP in the human. Hybridization of “P-labeled pHM1.0 probe to a Southern blot of BglII-digested DNA from Chinese hamster X human hybrid cell lines and controls. Lane 1, Chinese hamster cells V79/380-6; lane 2, diploid lymphoblastoid cells; lanes 3-13, Chinese hamster X human hybrid cell lines. Lanes 5 and Q-11 contain the 8.0-kb human MRP RNA sequence as well as Chinese hamster MRP RNA sequence and were scored as +. Lanes 3,4, 6-8, 12, and 13 contain only Chinese hamster MRP RNA sequences and were scored as -.
Southern Blot Hybridization Ten micrograms of DNA from each of the hybrid and parental control cell lines was digested with a fourfold excess of Bg211in high salt buffer. DNA fragments were separated by agarose gel electrophoresis and transferred to Hybond-N membranes (Amersham) by the method of Southern (1975). The probes were labeled with [32P]dCTP by oligolabeling (Feinberg and Vogelstein, 1983). The filters were prehybridized in 50% formamide (for human mapping) or 40% formamide (for mouse mapping), 5X SSC, 5X Denhardt’s, 1% SDS, and 100 pg/ml ssDNA for 4 h and then hybridized in 50% or 40% formamide, 5X SSC, 1X Denhardt’s, 1% SDS, 5% dextran sulfate, and 100 pg/ml ssDNA at 42°C for 16 h. After hybridization, filters were rinsed in 2X SSC and 0.5% SDS, washed in 2X SSC or 2X SSC and 0.5% SDS at room temperature for 15 min, and washed in 1X SSC, 1% SDS at 65°C
different series of hybrids (Francke et al., 1977; Francke and Taggart, 1979; Joyner et al., 1985) were used. Probes The probes used for Southern blot hybridization were the gel-purified inserts from clones pHM1.0 and pSP270. For in situ hybridization, pHSTU300 was used. Clone pHM1.0 is a l.O-kb human DNA fragment containing 700 bp of the 5’ flanking region, the coding region, and approximately 25 bp of 3’ flanking sequence, inserted into the (or a) PstI site of pSP65 plasmid. Clone pSP270 is a 0.3-kb mouse DNA fragment containing only the coding region, inserted into the PstI and EcoRI sites of pSP64 plasmid. Clone pHSTU300 is a 0.3-kb fragment of human DNA containing most of the coding region and a small portion of 3’ flanking sequences, inserted into the SmaI and PstI sites of pSP65 plasmid.
TABLE Correlation
of Human
RMRP
Sequences
with
Human
1
Chromosomes
in Rodent
Human 12
Hybridization/chromosome
+/+
2 112 5 7 24 2 3 31 20512414023223
-/+/-/+ Discordant Informative
hybrids hvbrids
Note. The numbers for each chromosome. excluded.
34
4 11 of hybrids Hybrids
3 11
82 11 8
that are concordant in which a particular
67
8
9
10
11
12
0 5 3
212 35 22
3 2
3 7 0
0 5 4
0 3 3
2 5 2
5 10
63 11 9
6 11
0 10
6 11
6 9
4 11
and discordant was structurally
Somatic
Cell Hybrids
chromosomes
5
(+/+ or -/-) chromosome
X Human
13
17
18
32420432231 5 3 4 5 5 110 2 4 322332352
4 0
3 11
(+/or -/+) rearranged
14
4 9
15
3 11
16
4 11
6 11
19
4 12
344552 11 11
20
21
5
3 2
11
10
22
X
22 00
10
5
with the human RMRP sequence are given or present in fewer than 10% of cells were
542
HSIEH
ET
AL.
pHSTU300
3T’ II1
13
FIG. 2. Histogram autoradiography. There
I
I
14
15
of grain distribution is a single nonrandom
16
17
In Situ Hybridization All procedures for in situ hybridization were as described previously (Donlon, 1986). The distribution of autoradiographic silver grains on chromosomes was scored in 100 metaphase cells. RESULTS
of the Human
RMRP
18
7 p1g
21
“p*pip-r
22
II
ID1 X
Y
after in situ hybridization of pHSTU300 probe to normal human metaphase chromosomes and peak at 9p21-~12 and only background label over the rest of the human chromosome complement.
for 10 min. All filters were autoradiographed at -70°C for various lengths of time with Kodak X-Omat AR film.
Mapping
*Pq
IhI
I
Gene
The human gene was mapped with a panel of 11 Chinese hamster X human somatic cell hybrids having reduced numbers of human chromosomes. After hybridization of the 32P-labeled human cDNA probe pHM1.0 to BgZII-digested genomic DNA, a single restriction fragment of 8.0 kb was detected in human control DNA (Fig. 1, lane 2) and a 4.2-kb fragment was observed in Chinese hamster control DNA (Fig. 1, lane 1). In hybrids containing human chromosome 9, the
8.0-kb human signal as well as the Chinese hamster signal was present (Fig. 1, lanes 5, 9, 10, and 11). No human signal was detected in hybrids not containing human chromosome 9 (Fig. 1, lanes 3,4,6-8, 12, and 13). The presence or absence of the human RMRP sequence was in perfect concordance with human chromosome 9 in hybrid cell lines (Table 1). All other human chromosomes were excluded by at least 2 discordant hybrids. Silver grains were scored after in situ hybridization of 3H-labeled human cDNA probe pHSTU300 to human metaphase chromosome spreads. Twenty-nine of the 100 cells had label on 9p, and 9.6% of 302 silver grains were located at 9p21-p12 (Fig. 2). No other chromosome site was labeled above background.
Mapping
of the Murk
Rmrp Gene
The mouse gene was assigned with a panel of 11 mouse X rodent somatic cell hybrids having reduced numbers of mouse chromosomes. After hybridization of the 32P-labeledmouse cDNA probe pSP270 to BgZII-
MAPPING 0
1234667
9
10
11
543
RMRP GENES
OF
12
ster fragments was present (Fig. 3, lanes 3, 5, and 8). No mouse signal was detected in hybrids not containing mouse chromosome 4 (Fig. 3, lanes 4,6,7, and 9-11). The presence of the mouse RMRP sequence showed perfect concordance with the presence of mouse chromosome 4 in hybrid cell lines (Table 2). All other mouse chromosomes were excluded by at least 2 discordant hybrids.
42
DISCUSSION A.2
We have mapped the RMRP gene to human chromosome 9 by analysis of somatic cell hybrids. The in situ hybridization result placed this locus on the short arm of chromosome 9, at region p21-~12. Since only one fragment was observed in BglII- (this study), BamHI-, and PstI-digested (J. N. Topper, unpublished observation) human DNA on Southern blots and only one site was detected with in situ hybridization, we conclude that RMRP is a single-copy gene with no other highly homologous sequences in human. In the mouse, we have mapped this gene (locus symbol Rmrp) to chromosome 4 with somatic cell hybrids. The observation of a single fragment of 7.2 kb in BglII-digested mouse DNA also suggestedthe absenceof closely related sequences in the mouse genome. Comparative mapping of human and mouse chromosomes has revealed eight other genes,IFNA, IFNB, GGTBZ, GALT, ACOl, ALAD, ORMl, and ORM2, on mouse chromosome 4 and on human chromosome 9 (Lalley et al, 1988). The region of mouse chromosome 4 that contains these loci includes bands A3 to C6 (Lyon, 1989). In humans, the first four of these eight genes, including the interferon genes and the galactosyltransferase genes listed, have been assigned to the short arm of chromosome 9. GALT is the locus for galactose-lphosphate uridyltransferase, deficiency of which causes
12 Mch
sgl
R
II
FIG. 3. Mapping of Rmrp in the mouse. Hybridization of 32Plabeled pSP270 probe to a Southern blot of BglII-digested DNA from rodent X mouse hybrid cell lines and controls. Lane 1, mouse liver; lane 2, Chinese hamster cells V79/380-6; lane 12, rat Morris hepatoma 7777 cells; lanes 3-10, Chinese hamster X mouse hybrid cell lies; lane 11, rat X mouse hybrid cell lie. Lanes 3,S, and 8 contain mouse MRP RNA sequences as well as Chinese hamster MRP RNA sequences and were scored as +. Lanes 4,6,7, and 9-l 1 contain only Chinese hamster or rat MRP RNA sequences and were scored as -.
digested genomic DNA, a single fragment of 7.2 kb was detected in mouse control DNA (Fig. 3, lane l), two fragments of sizes 4.2 and 1.2 kb were observed in Chinese hamster control DNA (Fig. 3, lane 2), and a 2.2-kb fragment was detected in rat control DNA (Fig. 3, lane 12). In hybrids that contain mouse chromosome 4, the 7.2-kb mouse signal as well as the Chinese ham-
TABLE Correlation
2
of Mouse-Specific Rmrp Sequences with Mouse in Rodent x Mouse Somatic Cell Hybrids Mouse
Hybridization/chromosome
+/+
Note. The numbers for each chromosome. excluded.
2
3
24341143110 1 2 5 2 0 1 5 5 2
-/+/-/+ Discordant Informative
1
hybrids hybrids of hybrids Hybrids
7 10 that are concordant in which a particular
5 11
3 11
4
5
chromosome
7
8
9
643 3 0320132411201 013 3
5
547365
2
2
3
3 11
5 11
5 10
045 10 9
(+/+ or -/-) chromosome
6
9
3 10
and discordant was structurally
Chromosomes
10
11
12
13
14
3
2
2
0
4
12
4 11
5 11
2 10
4 11
15
16
17
18
19
4 2 333532
4
2
4
2
2 13
0
1 5
3 10
6 10
4
4
0 4
4 11
5 10
4 11
3 10
X
(+/or -/+) with the mouse Rmrp sequences are given rearranged or present in fewer than 10% of cells were
544
HSIEH
galactosemia. The AC01 gene, coding for an enzyme that catalyzes the isomerization of citrate inside of mitochondria, has been mapped to 9p22-q32. The RMRP gene appears to be a new member of this conserved syntenic group.
ET
AL. 6.
DONLON, T. A. (1986). Practical ization. Ka~ogram 12: 3-10.
7.
FEINBERG, A., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
8.
FRANCKE, U., LALLEY, P. A., Moss, W., Iw, J., AND MINNA, J. D. (1977). Gene mapping in Mus musculus by interspecies cell hybridization: Assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 12 and adenylate kinase-1 to chromosome 2. Cytogenct. Cell Genet. 19: 57-84. FRANCKE, U., AND TAGGART, R. T. (1979). Assignment of gene for cytoplasmic superoxide dismutase (Sod-I) to a region of chromosome 16 and of Hprt to a region of the X chromosome in the mouse. Proc. Natl. Acad. Sci. USA 76: 5230-5233. JOYNER, A. C., LEBO, R. V., RAN, Y. W., TJIAN, R., Cox, D. R., AND MARTIN, G. R. (1985). Comparative chromosome mapping of a conserved homeo box region in mouse and human. Nature (London) 314: 173-175.
ACKNOWLEDGMENTS The work in U. Francke’s laboratory was supported by NIH Grant GM26105 U.F. is an Investigator and C.-L.H. an Associate of the Howard Hughes Medical Institute. Isolation of the human and mouse RNAase MRP RNA genes in D. A. Clayton’s laboratory was supported by NIH Grant GM33088. D.D.C. and J.N.T. were and are, respectively, Medical Scientist Training Program Trainees supported by NIH Grant GM07365.
9.
10.
REFERENCES 1.
BIRNSTIEL, M. L., AND SCHALJFELE, F. J. (1988). Structure and function of minor snRNPs. In “Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles” (M. L. Birnsteil, Ed.), pp. 155-182, Springer-Verlag, Berlin.
2.
CHANG, D. D., AND CLAYTON, D. A. (1987a). A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J. 6: 409-417. CHANG, D. D., AND CLAYTON, D. A. (1987b). A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science 235: 1179-1184.
3.
4.
5.
CHANG, D. D., AND CLAYTON, D. A. (1989). Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate. Cell 66: 131-139. CLAYTON, tochondrial
D. A. (1984). Transcription of the mammalian genome. Annu. Reu. Biochem. 63: 573-594.
mi-
approaches
to in situ hybrid-
11.
LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIEN, S. J., RODERICK, T. H., DOOLI~LE, D. P., AND HILLYARD, A. L. (1988). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 49: 227-235.
12.
LYON, M. F. (1989). Mouse chromosome atlas. Mouse News L&t. 84: 24-25. SOUTHERN, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. TOPPER, J. N., AND CLAYTON, D. A. (1990). Characterization of human MRP/Th RNA and its nuclear gene: Full length MRP/Th RNA is an active endonuclease when assembled as an RNP. Nucleic Acids Res., in press. YANG-FENG, T. L., DEGENNARO, L. J., AND FRANME, U. (1986). Genes for synapsin I, a neuronal phosphoprotein, map to conserved regions of human and murine X chromosomes. Proc. Natl. Acad. Sci. USA 83: 8679-8683.
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