- Email: [email protected]
Chromosomal distribution of ribosomal protein genes in the mouse
Cell, Vol. 24, 307-312,
May
1981,
Copyright
0 1981
by MIT
Chromosomal Distribution of Ribosomal Protein Genes in the Mouse P. D’Eustachio,* Oded Meyuhas,$$ and Robert P. Perry?
Frank
Ruddle*
*Yale University New Haven, Connecticut 06510 tlnstitute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 191 1 1
Summary The chromosomal distributions of five families of mouse r-protein genes (S16, L18, L19, L30 and L32/33) were studied by Southern blot analysis of DNA from a panel of mouse-hamster hybrid cells containing various complements of mouse chromosomes. Our results indicated that members of a particular family are often located on more than one chromosome, that extensive clustering of many rprotein gene families on a few chromosomes is unlikely, and that there is no obligatory linkage of rprotein and rRNA genes. Introduction To understand the coordinate regulation of the genes specifying a functionally related group of proteins or RNAs, it is important to know whether or not they are clustered. Such clustering is often exploited for coordinate regulation of transcription, as in the case of many operons in procaryotes or certain complex transcription units in eucaryotes. Concomitant activation of clustered genes may also be achieved in eucaryotes by zone-specific modification of chromatin structure. On the other hand, many functionally related genes are widely separated in the genome. Coordinate expression of these genes seems to require either responsiveness to a common pleotropic effector or transcriptional or posttranscriptional feedback control systems. Ribosome production in mammals requires the coordinate expression of genes encoding over 70 different proteins as well as the genes specifying 18S, 28S, 5.8s and 5s rF?NA (Perry, 1972). The synthesis of 18S, 28s and 5.8s rRNA is coordinate because the genes belong to the same complex transcription unit, but since the 5S genes are usually located on different chromosomes (Long and Dawid, 1980), another mechanism-perhaps involving feedback (Pelham and Brown, 1980)-is needed for their regulation. Until now nothing was known about the chromosomal distribution and possible clustering of mammalian rprotein genes. Recent evidence indicates that individual r proteins are encoded by multigene families with an average of about ten members (Monk et al., 1981). $- Present
address:
Jerusalem.
Israel.
Hadassah
Medical
School,
Hebrew
University.
In the studies described here we have investigated the chromosomal distribution of r-protein genes in the mouse, directing our attention primarily to their possible clustering and their location relative to rRNA genes. These genes appear to be widely dispersed throughout the genome. There is no obligatory linkage between r-protein and rRNA genes or between families of r-protein genes. Further, although some members of a particular r-protein gene family may be clustered, generally all members of the family are not located on the same chromosome.
Results These chromosomal panel of mouse x sess defined sets and the ability to protein genes in a
localization studies require a hamster hybrid cell lines that posof different mouse chromosomes, distinguish mouse from hamster rSouthern blot analysis of hybrid cell
DNAs. Such a panel has been used to determine the chromosomal locations in the mouse of immunoglobulin light and heavy chain genes (Swan et al., 1979; D’Eustachio et al., 1980), and of a-fetoprotein and albumin (D’Eustachio et al., 1981). Classical Mendelian genetic analyses of some of these genes have yielded consistent chromosomal locations (Nichols et al., 1975; Gottlieb and Durda, 1976; Meo et al., 1980), suggesting that our approach is a reliable one. Table 1 lists the mouse karyotypes of the hybrid cell lines used in the present study. Our ability to distinguish the restriction fragment patterns of mouse and hamster r-protein genes was tested in a set of preliminary Southern blots with Eco RI digests of DNA from BALB/cAn and A/HeJ mouse liver, the mouse fibroblastoid cell line A9, and the Chinese hamster fibroblastoid cell line E36, using a set of five different r-protein probes. In all cases, the mouse and hamster fragment patterns were complex but quite distinctive, whereas the patterns of the three mouse DNA samples were very similar (data not shown). The r-protein gene fragments ranged in size from about 2 td 20 kb. Mouse-specific fragments were readily detected on a hamster background, as shown in Figures la, 3a, 3c and 3e, in which digests of mouse and hamster DNA as well as 1 :l , 1:4 and 1 :16 mixtures of mouse and hamster DNA were probed for r-protein sequences. Although mouse and hamster fragments overlapped in some cases, at least five mouse-specific fragments could usually be detected when 25% or more of the total DNA was of mouse origin. We first focused our attention on the L18 family of r-protein genes, which exhibited seven distinctive mouse Eco RI fragments designated a through g (Figure 1). The genes defined by fragments c, d and g were present in the DNA of hybrids MAE28A, 7A133B3, BEM l-6 and 4A64-Al and absent in the DNA of ECm4e. Fragments c and g were also absent in the
Cell 308
Table
1. Chromosome
Mouse Chromosome Number
Complements
of Hybrid
Cell Lines Tested
for Mouse
r-Protein
BEMl-6
BEMl-4
1
0.61
0.35
0.34
0.39
2
1.03
0.83
0.41
0.94
1.50
0.86
0.00
0.00
3
0.94
0.70
0.01
0.09
0.01
0.00
0.00
0.00
4
0.97
0.13
0.02
0.14
0.00
0.69t
0.00
0.00
5
0.00
0.22
0.00
0.35
0.54
0.00
0.00
0.00
6
1.97t
1.09
0.06
0.85
0.04
0.00
0.00
0.00
7
0.13
0.00
0.25
0.29
0.20
0.69
0.00
0.00
8
0.87
0.35
0.00
0.30
0.00
0.00
0.00
0.00
9
0.23
0.00
0.001
0.76
1.35
0.00
0.00
0.00
10
0.27
0.00
0.00
0.00
0.00
0.00
0.00
0.00
11
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
12
0.74
0.83
0.23t
0.88
1.77
0.11
0.00
1.03t
13
0.77
0.52
0.00
0.21
0.26
0.44
0.00
0.00
14
1.03
0.78
0.02
0.70
0.27
0.11
1 .oot
0.00
15
1.74
1 .oo
1.05
1.30
1.30
0.92
1 .oot
0.00
16
0.87
0.65
0.06
1.15
0.17
0.97
0.00
0.00
17
0.87
1.09
0.20
0.91
0.70
0.25
0.00
0.00
18
0.55
0.00
0.03
1.03
1.09
0.33
0.00
0.00
19
1.26
0.96
0.27
1.15
1.07
186
0.00
0.00
X
1.85t
0.48
0.02
0.00
0.00
0.00
0.00
1.03t
Number of cells karyotyped
31
23
101
MACH 3B9C4-1
Genes’
MACH 4A64A-1
33
MACH 7A13-383 0.00
33
* Mouse chromosomes were identified in metaphase spreads subjected to the sequential number shown is the mean number of copies of the chromosome per cell. t Includes copies of the chromosome occurring in the form of translocations.
DNA of 3B9C4-1. In contrast, the genes defined by fragments e and f were present in the DNA of 7A13383, 4A64-Al and 3B9C4-1, and absent in the DNA of MAE28A, ECm4e and BEM 1-6. These results indicate that L18 genes c, d and g are on a different chromosome than genes e and f. The data are most nearly consistent with c, d and g being on chromosome 12, and e and f being on chromosome 7. The only discordance with this assignment was the absence of genes c and g in the line 3B9C4-1, which carried chromosome 12 in a high proportion of the cells. The possibility of L18 genes c, d and g being on chromosome 12 was investigated further by a detailed comparison of hybrid lines MAE 28A, which contains a single complex mouse chromosome formed by the fusion of an entire chromosome 12 with an entire x chromosome, line MAE 4, which is similar except that the distal portion of chromosome 12 has been lost, and MAE 28A-8AgR, a subline of MAE28A that had lost the 12/X complex chromosome, derived by growth of the cell population in the presence of 8azaguanine. The Southern blot of these DNAs (Figure
MACH
4A63
0.00
36
Giemsa-Viokase-Hoechst
ECm4e 0.00
MAE28A 0.00
9
31
“33258”
technique.
The
2a) clearly shows fragments c, d and g only in MAE 28A. In the other lines nothing above the hamster background is detectable. This result further supports the assignment of these genes to chromosome 12, and suggests, moreover, that they are located on the distal portion, remote from the centromeric region where the rRNA genes are located (Henderson et al., 1974; Elsevier and Ruddle, 1975). To confirm that the rRNA genes are still present in MAE 4 we analyzed its DNA for the presence of mouse-specific Hind Ill fragments that contain rRNA sequences (Figures 2b and 2~). Using “51-labeled 28s and 18s mouse rRNAs as hybridization probes, we were able to detect a -29 kb fragment containing both 28s and 18s sequences and a -14 kb fragment containing 18s sequences (Cory and Adams, 1977) in MAE4 as well as MAE28A DNA. The low relative intensity of these bands in both hybrid lines presumably reflects the small number of mouse rRNA genes present (only those located on chromosome 12) on a background of several hundred hamster rRNA genes. Nevertheless, their presence in MAE4 DNA clearly demonstrates that the fragmentation of chromosome 12, which resulted in the loss of
~Z~mosomal
Location
of r-Protein
Genes
Figure 1. Southern Blot Genes in Eco RI-Digested Hamster and Mouse-Hamster
ab"dcI:
f-
ef-
.
(a) Calibration series of various proportions of mouse (A9 cells) and hamster (E36 cells). The mouse fragments that are distinguishable from their hamster counterparts are labeled a-g. (b) and (c) Comparison of fragment patterns in various hybrid cell lines. Abbreviations MAE28 = MAE28A; ECm4 = ECm4e; 7A13 = MACH 7A13-383; Bern = BEM l-6; 4A6 = MACH 4A64A-1; 389 = MACH 3B9C4-1. The dots (0) indicate two fragments (-2.4 and 4 kb) derived from a procaryotic contaminant which hybridizes to the pMB9 vector of the r-protein probe. These fragments were observed in the same series of blots made with other r-protein probes (see Figures 2 and 3).
e-
d-
e-
Analysis of L18 DNA from Mouse, Hybrid Cells
f.
. cl(a)
(b)
Li8 in the same way, as shown in Figure 3 and summarized in Table 2. Members of these families showed no general tendency to cluster on one or a few chromosomes. For example, besides the L18 genes, MAE28A contained some L30 genes (b, c, d), but lacked most or all of the genes for S16, L19 and L32/ 33. Many of the r-protein genes absent from MAE28A were clearly detected in the hybrid lines 7A13-3B3 or BEM 1-6, which together cover almost the entire complement of mouse chromosomes. On the basis of this survey, one or more genes could be assigned to each of chromosomes 3,5 and 6, and genes could be tentatively assigned as well to chromosomes 2, 11, 16 and X. None of the r-protein genes tested was present in line ECm4e, which contains mouse chromosome 15, a chromosome that has been reported to bear rRNA genes (Henderson et al., 1974, 1976). Discussion
Li8 Figure 2. Locatlon of L18 Genes c. d and g on the Distal Chromosome 12: Separation from rRNA Genes
Arm of
(a) Comparison of L18 Eco RI fragment patterns m MAE28A, MAE4 and MAE28A-8AgR (abbreviated MAE Ag8). (b) and (c) Hind Illdlgested DNA probed with 28s and 18s rRNA, respectively.
L18 genes, did not This result provides clustering between Four other families
result in the loss of rRNA genes. direct evidence for a lack of tight rRNA and r-protein genes. of r-protein genes were analyzed
In order to study the chromosomal locations of rprotein gene families in the mouse, we have used cDNA probes corresponding to members of five of these families to examine DNA from somatic cell hybrids carrying restricted sets of mouse chromosomes on a constant background of Chinese hamster chromosomes. Each family examined in this way consisted of 7-20 distinct DNA fragments (Monk et al., 19811, of which at least five could be detected against the background of hamster crossreactive material. Within this limit, and the additional limit imposed by the
Cell
310
r: p: g--=A=== O\AL
E E
(0, r. Q) f E
E Ee
&
b #,
C
E Iwrcr
s
Figure 3. Southern Blot Analysis of 5516, S19, L32/33 and L30 Genes in Eco RI-Digested DNA from Mouse. Hamster and Mouse-Hamster Hybrid Cells (a) (c) and (e) Calibration series as in Figure l a(b)(d)(f) and (g) Comparison of hybrid lines as in Figure 1 b.
a-
a--Iy,
Q) 2 cu*,z o’$E;g
c-
.
.
cl-
g-
(a)
S16
lb)
Cder: g.
hi-
((4) L30 (e)
L 32/33
(f)
particular distribution of mouse chromosome in our hybrid cell panel, nine fragments were assigned to individual mouse chromosomes and 11 were tentatively assigned to individual chromosomes or a small group of chromosomes (Table 2). None of the r-protein gene families was localized entirely on a single mouse chromosome. Further, we found no obvious correlation between the distributions of r-protein genes and of r-RNA genes. We have characterized only a small fraction of the hundreds of r-protein genes. Nevertheless, to the extent that our sample is typical, extensive clustering by family in the mouse genome appears unlikely. Some clustering, however, remains possible. Whereas most of our data suggest that the L18 c and g DNA fragments reside on mouse chromosome 12, we failed to detect these fragments in hybrid 3B9C4-1, even though this cell population contained mouse chromosome 12 at a high frequency. The probable explanation of this discrepancy is that the chromosome 12
present in 3B9C4-1 suffered a small deletion, not detectable by conventional karyotyping procedures. If fragments c and g represent two separate genes and not parts of a single L18 gene, this explanation would imply that these genes are located close together on the chromosome. At least one other fragment (L30 c) shows a similar pattern of probable assignment to chromosome 12 and absence from hybrid 3B9C4-1. If there is any functionally significant clustering, it must involve only certain subsets of rprotein genes, as appears to be the case in Escherichia coli (Nomura et al., 1977). The distribution of rprotein genes on different linkage groups has also been noted in yeast (Woolford et al., 1979). Without any convincing evidence to the contrary, it seems reasonable to consider the mammalian r-protein genes as belonging to highly dispersed multigene families, and to anticipate regulatory mechanisms that would be appropriate for this type of gene arrangement.
Chromosomal 311
Table
2.
Location
Distribution
of
r-Protein
of Mouse
Genes
r-Protein
Presence
Gene
in Hybrid
Segments
among
Mouse-Hamster
Hybrid
Cell Lines
Cell Line MACH
r-Protein Fragment
Genomic
S16
BEM a
-
b e
-e?
L19
l-4
MACH 3B9C4-1
7A13383
MACH 4A63
ECm4e
MAE28A
-
+
n.d.
-
+ + -
+ + -
+ +
n.d. n.d. n.d.
-
-
+
n.d. n.d. nd. n.d. n.d.
-
-
-
-
-
-
+ + -
-
-
? ?
? ?
(2. 17. 19)
-
+ + + -
X? 12?
-
27 -
+ -
c
+
n.d.
+
-
d e f
+ -
9
+
n.d. n.d. n.d. nd.
+ + + +
+ + + -
+ + + +
a c d
+
+
-
+ + + +
+ + + + *? + ? + + f?
+
+
+ 2?? f? + -
? + -
f? ? + +
n.d. n.d.
n.d. n.d.
n.d. n.d.
L30
L32/33
BEM + + f? -
+ -
9 L18
l-6
c. d f-j
n.d. n.d.
Possible Chromosome Assignmerits
MACH 4A64A-1
+ + -
-
+ i? -
? + ? ?
+
n.d. n.d.
-
5
(2, 17.19) (2, 17. 19)
-
(11,
+ + -
-
+
6 6? 13? 3. x
12 (2, 17.19) 3 16?
unlinked
Data from experiments shown in Figures 1-3 plus additional experiments not shown. A + or - means unequivocal presence fragment, respectively; a %? indicates that there is some discrepancy among different experiments; a ? means autoradiogram means not done. Groups of chromosomes in parentheses could not be resolved by our hybrid panel.
Experimental
D‘Eustachio.
Procedures
P.. Ingram,
Som. Cell Genet., The mouse-hamster cell lines used in these studies have been described previously (D’Eustachro et al., 1980). They all contain a near-tetraploid number of Chinese hamster chromosomes derived from cell line E36 derived from primary
and various numbers of mouse fibroblasts from BALB/c embryos
chromosomes (BEM serves).
peritoneal macrophage from A/He J mice (MACH series) or selected cell lines (ECm4e and MAE serves). Methods for preparahon of Southern blots (Scangos et al., 1979) and hybridization with r-protein probes (Monk et al., 1981) were as described elsewhere. Acknowledgments This research was supported by grants from the NSF and NIH, a fellowship to P.D’E. from the Leukemia Society of America, Inc., and an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
December
22, 1980:
revised
February
9. 1981
Ft., Tilghman.
J. M. (1977). the 18S, 285
A very large repeating and 5.8s rRNA genes.
D’Eustachio, P.. Pravtcheva. D., Marcu, K. and Ruddle. Location of the structural gene cluster encoding murine ulin heavy chains. J. Exp. Med. 751, 1545-l 550.
unit of Cell 1 I,
F. H. (1980). immunoglob-
S. and Ruddle.
S. M. and Ruddle,
F. H. (1975).
Locahon
for 18s and 28s nbosomal Chromosoma 52, 219-228.
RNA wrthin the genome
Gottlieb.
P. J. (1976).
P. D. and
or absence not clear;
of a n.d.
F. H. (1981).
in press.
Durda,
of genes
coding
of Mus musculus.
The I.-peptide
marker
the Ly3-surface alloantigen: structural studies of a V,-region morphism and a T-cell marker determined by linked genes. Spring Harbor Symp. Quant. Biol. 47, 805-815.
and polyCold
Henderson, A. S.. Either, E. M.. Yu. M. T. and Atwood, K. C. (1974). The chromosomal location of ribosomal DNA in the mouse. Chromosoma 49, 155-l 60. Henderson, A. S.. Either. E. M.. Yu. M. T. and Atwood, K. C. (1976). Variation in ribosomal RNA gene number in mouse chromosomes. Cytogenet. Cell Genet. 7 7, 307-316. Long, E. 0. and Dawid, I. B. (1980). Ann. Rev. Biochem. 49, 727-764.
Repeated
genes
in eukaryotes’
*.
Meo, T., Johnson, J., Beechey, C. V.. Andrew% S. J., Peters, J and Searle, A. D. (1980). Linkage analyses of murine immunoglobulin heavy chain and serum prealbumin genes establish their location on chromosome 12 proximal to theT(5:12)31 H breakpoint m band 12Fl. Proc. Nat. Acad. Sci. USA 77, 550-553. Monk, R., Meyuhas. 0. and Perry, multiple genes for individual ribosomal
References Gory. S. and Adams, mouse DNA containing 795-805.
Elsevier.
101
12 12 7 7 12
R. P. (1981). Mammals have proteins. Cell 24, 301-306.
Nichols, E. A., Ruddle, F. H. and Petras, M. L. (1975). Linkage of the locus for serum albumin in the house mouse, Mus musculus. Biochem. Genet. 73, 551-555. Nomura. bacterial
M., Morgan, E. A. and Jaskunas, S. P. (1977). ribosomes. Ann. Rev. Genet. 7 7, 297-347.
Pelham.
H. R. B. and
Brown,
D. D. (1980).
A specific
Genetics transcnption
of
Cell 312
factor Acad.
that can bind either the 55 RNA gene or 55 RNA. Proc. Sci. USA 77, 4170-4174.
Perry, R. P. (1972). Regulation Biochem. Sot. Symp. 37, 105-l
of ribosome 16.
content
Nat.
in eukaryotes.
Scangos. G. A., Huttner, K. M., Silverstein, S. and Ruddle, F. H. (1979). Molecular analysis of chromosome-mediated gene transfer. Proc. Nat. Acad. Sci. USA 76, 3987-3990. Swan, D., D’Eustachio, P., Leinwand, L.. Seidman, J., Keithley. D. and Ruddle. F. H. (1979). Chromosomal assignment of the mouse IC light chain genes. Proc. Nat. Acad. Sci. USA 76, 2735-2739. Woolford, J. L. Jr., Hereford, L. M. and Rosbash, M. (1979). Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae. Cell 18. 1247-l 259.
May
1981,
Copyright
0 1981
by MIT
Chromosomal Distribution of Ribosomal Protein Genes in the Mouse P. D’Eustachio,* Oded Meyuhas,$$ and Robert P. Perry?
Frank
Ruddle*
*Yale University New Haven, Connecticut 06510 tlnstitute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 191 1 1
Summary The chromosomal distributions of five families of mouse r-protein genes (S16, L18, L19, L30 and L32/33) were studied by Southern blot analysis of DNA from a panel of mouse-hamster hybrid cells containing various complements of mouse chromosomes. Our results indicated that members of a particular family are often located on more than one chromosome, that extensive clustering of many rprotein gene families on a few chromosomes is unlikely, and that there is no obligatory linkage of rprotein and rRNA genes. Introduction To understand the coordinate regulation of the genes specifying a functionally related group of proteins or RNAs, it is important to know whether or not they are clustered. Such clustering is often exploited for coordinate regulation of transcription, as in the case of many operons in procaryotes or certain complex transcription units in eucaryotes. Concomitant activation of clustered genes may also be achieved in eucaryotes by zone-specific modification of chromatin structure. On the other hand, many functionally related genes are widely separated in the genome. Coordinate expression of these genes seems to require either responsiveness to a common pleotropic effector or transcriptional or posttranscriptional feedback control systems. Ribosome production in mammals requires the coordinate expression of genes encoding over 70 different proteins as well as the genes specifying 18S, 28S, 5.8s and 5s rF?NA (Perry, 1972). The synthesis of 18S, 28s and 5.8s rRNA is coordinate because the genes belong to the same complex transcription unit, but since the 5S genes are usually located on different chromosomes (Long and Dawid, 1980), another mechanism-perhaps involving feedback (Pelham and Brown, 1980)-is needed for their regulation. Until now nothing was known about the chromosomal distribution and possible clustering of mammalian rprotein genes. Recent evidence indicates that individual r proteins are encoded by multigene families with an average of about ten members (Monk et al., 1981). $- Present
address:
Jerusalem.
Israel.
Hadassah
Medical
School,
Hebrew
University.
In the studies described here we have investigated the chromosomal distribution of r-protein genes in the mouse, directing our attention primarily to their possible clustering and their location relative to rRNA genes. These genes appear to be widely dispersed throughout the genome. There is no obligatory linkage between r-protein and rRNA genes or between families of r-protein genes. Further, although some members of a particular r-protein gene family may be clustered, generally all members of the family are not located on the same chromosome.
Results These chromosomal panel of mouse x sess defined sets and the ability to protein genes in a
localization studies require a hamster hybrid cell lines that posof different mouse chromosomes, distinguish mouse from hamster rSouthern blot analysis of hybrid cell
DNAs. Such a panel has been used to determine the chromosomal locations in the mouse of immunoglobulin light and heavy chain genes (Swan et al., 1979; D’Eustachio et al., 1980), and of a-fetoprotein and albumin (D’Eustachio et al., 1981). Classical Mendelian genetic analyses of some of these genes have yielded consistent chromosomal locations (Nichols et al., 1975; Gottlieb and Durda, 1976; Meo et al., 1980), suggesting that our approach is a reliable one. Table 1 lists the mouse karyotypes of the hybrid cell lines used in the present study. Our ability to distinguish the restriction fragment patterns of mouse and hamster r-protein genes was tested in a set of preliminary Southern blots with Eco RI digests of DNA from BALB/cAn and A/HeJ mouse liver, the mouse fibroblastoid cell line A9, and the Chinese hamster fibroblastoid cell line E36, using a set of five different r-protein probes. In all cases, the mouse and hamster fragment patterns were complex but quite distinctive, whereas the patterns of the three mouse DNA samples were very similar (data not shown). The r-protein gene fragments ranged in size from about 2 td 20 kb. Mouse-specific fragments were readily detected on a hamster background, as shown in Figures la, 3a, 3c and 3e, in which digests of mouse and hamster DNA as well as 1 :l , 1:4 and 1 :16 mixtures of mouse and hamster DNA were probed for r-protein sequences. Although mouse and hamster fragments overlapped in some cases, at least five mouse-specific fragments could usually be detected when 25% or more of the total DNA was of mouse origin. We first focused our attention on the L18 family of r-protein genes, which exhibited seven distinctive mouse Eco RI fragments designated a through g (Figure 1). The genes defined by fragments c, d and g were present in the DNA of hybrids MAE28A, 7A133B3, BEM l-6 and 4A64-Al and absent in the DNA of ECm4e. Fragments c and g were also absent in the
Cell 308
Table
1. Chromosome
Mouse Chromosome Number
Complements
of Hybrid
Cell Lines Tested
for Mouse
r-Protein
BEMl-6
BEMl-4
1
0.61
0.35
0.34
0.39
2
1.03
0.83
0.41
0.94
1.50
0.86
0.00
0.00
3
0.94
0.70
0.01
0.09
0.01
0.00
0.00
0.00
4
0.97
0.13
0.02
0.14
0.00
0.69t
0.00
0.00
5
0.00
0.22
0.00
0.35
0.54
0.00
0.00
0.00
6
1.97t
1.09
0.06
0.85
0.04
0.00
0.00
0.00
7
0.13
0.00
0.25
0.29
0.20
0.69
0.00
0.00
8
0.87
0.35
0.00
0.30
0.00
0.00
0.00
0.00
9
0.23
0.00
0.001
0.76
1.35
0.00
0.00
0.00
10
0.27
0.00
0.00
0.00
0.00
0.00
0.00
0.00
11
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
12
0.74
0.83
0.23t
0.88
1.77
0.11
0.00
1.03t
13
0.77
0.52
0.00
0.21
0.26
0.44
0.00
0.00
14
1.03
0.78
0.02
0.70
0.27
0.11
1 .oot
0.00
15
1.74
1 .oo
1.05
1.30
1.30
0.92
1 .oot
0.00
16
0.87
0.65
0.06
1.15
0.17
0.97
0.00
0.00
17
0.87
1.09
0.20
0.91
0.70
0.25
0.00
0.00
18
0.55
0.00
0.03
1.03
1.09
0.33
0.00
0.00
19
1.26
0.96
0.27
1.15
1.07
186
0.00
0.00
X
1.85t
0.48
0.02
0.00
0.00
0.00
0.00
1.03t
Number of cells karyotyped
31
23
101
MACH 3B9C4-1
Genes’
MACH 4A64A-1
33
MACH 7A13-383 0.00
33
* Mouse chromosomes were identified in metaphase spreads subjected to the sequential number shown is the mean number of copies of the chromosome per cell. t Includes copies of the chromosome occurring in the form of translocations.
DNA of 3B9C4-1. In contrast, the genes defined by fragments e and f were present in the DNA of 7A13383, 4A64-Al and 3B9C4-1, and absent in the DNA of MAE28A, ECm4e and BEM 1-6. These results indicate that L18 genes c, d and g are on a different chromosome than genes e and f. The data are most nearly consistent with c, d and g being on chromosome 12, and e and f being on chromosome 7. The only discordance with this assignment was the absence of genes c and g in the line 3B9C4-1, which carried chromosome 12 in a high proportion of the cells. The possibility of L18 genes c, d and g being on chromosome 12 was investigated further by a detailed comparison of hybrid lines MAE 28A, which contains a single complex mouse chromosome formed by the fusion of an entire chromosome 12 with an entire x chromosome, line MAE 4, which is similar except that the distal portion of chromosome 12 has been lost, and MAE 28A-8AgR, a subline of MAE28A that had lost the 12/X complex chromosome, derived by growth of the cell population in the presence of 8azaguanine. The Southern blot of these DNAs (Figure
MACH
4A63
0.00
36
Giemsa-Viokase-Hoechst
ECm4e 0.00
MAE28A 0.00
9
31
“33258”
technique.
The
2a) clearly shows fragments c, d and g only in MAE 28A. In the other lines nothing above the hamster background is detectable. This result further supports the assignment of these genes to chromosome 12, and suggests, moreover, that they are located on the distal portion, remote from the centromeric region where the rRNA genes are located (Henderson et al., 1974; Elsevier and Ruddle, 1975). To confirm that the rRNA genes are still present in MAE 4 we analyzed its DNA for the presence of mouse-specific Hind Ill fragments that contain rRNA sequences (Figures 2b and 2~). Using “51-labeled 28s and 18s mouse rRNAs as hybridization probes, we were able to detect a -29 kb fragment containing both 28s and 18s sequences and a -14 kb fragment containing 18s sequences (Cory and Adams, 1977) in MAE4 as well as MAE28A DNA. The low relative intensity of these bands in both hybrid lines presumably reflects the small number of mouse rRNA genes present (only those located on chromosome 12) on a background of several hundred hamster rRNA genes. Nevertheless, their presence in MAE4 DNA clearly demonstrates that the fragmentation of chromosome 12, which resulted in the loss of
~Z~mosomal
Location
of r-Protein
Genes
Figure 1. Southern Blot Genes in Eco RI-Digested Hamster and Mouse-Hamster
ab"dcI:
f-
ef-
.
(a) Calibration series of various proportions of mouse (A9 cells) and hamster (E36 cells). The mouse fragments that are distinguishable from their hamster counterparts are labeled a-g. (b) and (c) Comparison of fragment patterns in various hybrid cell lines. Abbreviations MAE28 = MAE28A; ECm4 = ECm4e; 7A13 = MACH 7A13-383; Bern = BEM l-6; 4A6 = MACH 4A64A-1; 389 = MACH 3B9C4-1. The dots (0) indicate two fragments (-2.4 and 4 kb) derived from a procaryotic contaminant which hybridizes to the pMB9 vector of the r-protein probe. These fragments were observed in the same series of blots made with other r-protein probes (see Figures 2 and 3).
e-
d-
e-
Analysis of L18 DNA from Mouse, Hybrid Cells
f.
. cl(a)
(b)
Li8 in the same way, as shown in Figure 3 and summarized in Table 2. Members of these families showed no general tendency to cluster on one or a few chromosomes. For example, besides the L18 genes, MAE28A contained some L30 genes (b, c, d), but lacked most or all of the genes for S16, L19 and L32/ 33. Many of the r-protein genes absent from MAE28A were clearly detected in the hybrid lines 7A13-3B3 or BEM 1-6, which together cover almost the entire complement of mouse chromosomes. On the basis of this survey, one or more genes could be assigned to each of chromosomes 3,5 and 6, and genes could be tentatively assigned as well to chromosomes 2, 11, 16 and X. None of the r-protein genes tested was present in line ECm4e, which contains mouse chromosome 15, a chromosome that has been reported to bear rRNA genes (Henderson et al., 1974, 1976). Discussion
Li8 Figure 2. Locatlon of L18 Genes c. d and g on the Distal Chromosome 12: Separation from rRNA Genes
Arm of
(a) Comparison of L18 Eco RI fragment patterns m MAE28A, MAE4 and MAE28A-8AgR (abbreviated MAE Ag8). (b) and (c) Hind Illdlgested DNA probed with 28s and 18s rRNA, respectively.
L18 genes, did not This result provides clustering between Four other families
result in the loss of rRNA genes. direct evidence for a lack of tight rRNA and r-protein genes. of r-protein genes were analyzed
In order to study the chromosomal locations of rprotein gene families in the mouse, we have used cDNA probes corresponding to members of five of these families to examine DNA from somatic cell hybrids carrying restricted sets of mouse chromosomes on a constant background of Chinese hamster chromosomes. Each family examined in this way consisted of 7-20 distinct DNA fragments (Monk et al., 19811, of which at least five could be detected against the background of hamster crossreactive material. Within this limit, and the additional limit imposed by the
Cell
310
r: p: g--=A=== O\AL
E E
(0, r. Q) f E
E Ee
&
b #,
C
E Iwrcr
s
Figure 3. Southern Blot Analysis of 5516, S19, L32/33 and L30 Genes in Eco RI-Digested DNA from Mouse. Hamster and Mouse-Hamster Hybrid Cells (a) (c) and (e) Calibration series as in Figure l a(b)(d)(f) and (g) Comparison of hybrid lines as in Figure 1 b.
a-
a--Iy,
Q) 2 cu*,z o’$E;g
c-
.
.
cl-
g-
(a)
S16
lb)
Cder: g.
hi-
((4) L30 (e)
L 32/33
(f)
particular distribution of mouse chromosome in our hybrid cell panel, nine fragments were assigned to individual mouse chromosomes and 11 were tentatively assigned to individual chromosomes or a small group of chromosomes (Table 2). None of the r-protein gene families was localized entirely on a single mouse chromosome. Further, we found no obvious correlation between the distributions of r-protein genes and of r-RNA genes. We have characterized only a small fraction of the hundreds of r-protein genes. Nevertheless, to the extent that our sample is typical, extensive clustering by family in the mouse genome appears unlikely. Some clustering, however, remains possible. Whereas most of our data suggest that the L18 c and g DNA fragments reside on mouse chromosome 12, we failed to detect these fragments in hybrid 3B9C4-1, even though this cell population contained mouse chromosome 12 at a high frequency. The probable explanation of this discrepancy is that the chromosome 12
present in 3B9C4-1 suffered a small deletion, not detectable by conventional karyotyping procedures. If fragments c and g represent two separate genes and not parts of a single L18 gene, this explanation would imply that these genes are located close together on the chromosome. At least one other fragment (L30 c) shows a similar pattern of probable assignment to chromosome 12 and absence from hybrid 3B9C4-1. If there is any functionally significant clustering, it must involve only certain subsets of rprotein genes, as appears to be the case in Escherichia coli (Nomura et al., 1977). The distribution of rprotein genes on different linkage groups has also been noted in yeast (Woolford et al., 1979). Without any convincing evidence to the contrary, it seems reasonable to consider the mammalian r-protein genes as belonging to highly dispersed multigene families, and to anticipate regulatory mechanisms that would be appropriate for this type of gene arrangement.
Chromosomal 311
Table
2.
Location
Distribution
of
r-Protein
of Mouse
Genes
r-Protein
Presence
Gene
in Hybrid
Segments
among
Mouse-Hamster
Hybrid
Cell Lines
Cell Line MACH
r-Protein Fragment
Genomic
S16
BEM a
-
b e
-e?
L19
l-4
MACH 3B9C4-1
7A13383
MACH 4A63
ECm4e
MAE28A
-
+
n.d.
-
+ + -
+ + -
+ +
n.d. n.d. n.d.
-
-
+
n.d. n.d. nd. n.d. n.d.
-
-
-
-
-
-
+ + -
-
-
? ?
? ?
(2. 17. 19)
-
+ + + -
X? 12?
-
27 -
+ -
c
+
n.d.
+
-
d e f
+ -
9
+
n.d. n.d. n.d. nd.
+ + + +
+ + + -
+ + + +
a c d
+
+
-
+ + + +
+ + + + *? + ? + + f?
+
+
+ 2?? f? + -
? + -
f? ? + +
n.d. n.d.
n.d. n.d.
n.d. n.d.
L30
L32/33
BEM + + f? -
+ -
9 L18
l-6
c. d f-j
n.d. n.d.
Possible Chromosome Assignmerits
MACH 4A64A-1
+ + -
-
+ i? -
? + ? ?
+
n.d. n.d.
-
5
(2, 17.19) (2, 17. 19)
-
(11,
+ + -
-
+
6 6? 13? 3. x
12 (2, 17.19) 3 16?
unlinked
Data from experiments shown in Figures 1-3 plus additional experiments not shown. A + or - means unequivocal presence fragment, respectively; a %? indicates that there is some discrepancy among different experiments; a ? means autoradiogram means not done. Groups of chromosomes in parentheses could not be resolved by our hybrid panel.
Experimental
D‘Eustachio.
Procedures
P.. Ingram,
Som. Cell Genet., The mouse-hamster cell lines used in these studies have been described previously (D’Eustachro et al., 1980). They all contain a near-tetraploid number of Chinese hamster chromosomes derived from cell line E36 derived from primary
and various numbers of mouse fibroblasts from BALB/c embryos
chromosomes (BEM serves).
peritoneal macrophage from A/He J mice (MACH series) or selected cell lines (ECm4e and MAE serves). Methods for preparahon of Southern blots (Scangos et al., 1979) and hybridization with r-protein probes (Monk et al., 1981) were as described elsewhere. Acknowledgments This research was supported by grants from the NSF and NIH, a fellowship to P.D’E. from the Leukemia Society of America, Inc., and an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
December
22, 1980:
revised
February
9. 1981
Ft., Tilghman.
J. M. (1977). the 18S, 285
A very large repeating and 5.8s rRNA genes.
D’Eustachio, P.. Pravtcheva. D., Marcu, K. and Ruddle. Location of the structural gene cluster encoding murine ulin heavy chains. J. Exp. Med. 751, 1545-l 550.
unit of Cell 1 I,
F. H. (1980). immunoglob-
S. and Ruddle.
S. M. and Ruddle,
F. H. (1975).
Locahon
for 18s and 28s nbosomal Chromosoma 52, 219-228.
RNA wrthin the genome
Gottlieb.
P. J. (1976).
P. D. and
or absence not clear;
of a n.d.
F. H. (1981).
in press.
Durda,
of genes
coding
of Mus musculus.
The I.-peptide
marker
the Ly3-surface alloantigen: structural studies of a V,-region morphism and a T-cell marker determined by linked genes. Spring Harbor Symp. Quant. Biol. 47, 805-815.
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Henderson, A. S.. Either, E. M.. Yu. M. T. and Atwood, K. C. (1974). The chromosomal location of ribosomal DNA in the mouse. Chromosoma 49, 155-l 60. Henderson, A. S.. Either. E. M.. Yu. M. T. and Atwood, K. C. (1976). Variation in ribosomal RNA gene number in mouse chromosomes. Cytogenet. Cell Genet. 7 7, 307-316. Long, E. 0. and Dawid, I. B. (1980). Ann. Rev. Biochem. 49, 727-764.
Repeated
genes
in eukaryotes’
*.
Meo, T., Johnson, J., Beechey, C. V.. Andrew% S. J., Peters, J and Searle, A. D. (1980). Linkage analyses of murine immunoglobulin heavy chain and serum prealbumin genes establish their location on chromosome 12 proximal to theT(5:12)31 H breakpoint m band 12Fl. Proc. Nat. Acad. Sci. USA 77, 550-553. Monk, R., Meyuhas. 0. and Perry, multiple genes for individual ribosomal
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