Cytogenet. Cell Genet. 22: 281-284 (1978)
Assignment of a structural gene for type I collagen to chromosome 7 E. Solom on 1
and B. S ykes 2
'Genetics Laboratory, University of Oxford, and 2Department of Pathology, Radcliffe Infirmary, Oxford
Type I collagen, found in skin, bone, tendon, and cornea, is a hetero polymer of three polypeptide chains, two of which are identical, ai(I), and a third of which is homologous, 0^(1) (for a review, see ref. 1). We have examined the production of type I collagen in human-mouse somatic cell hybrids using species-specific anti-collagen antibodies. Human collagen production segregated in complete concordance with chromosome 7.
Supported by grants from the Lalor Foundation (E.S.) and the Nuffield Foun dation (B.S.).
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Human type I collagen was isolated and purified from acetic acid extracts of human fetal skin, and mouse type I collagen from mouse tail tendon.2 Antisera were raised in sheep and tested by double immunodiffusion against heat-denatured type I collagen. Precipitin arcs were susceptible to in situ digestion by purified bacterial collagenase. Periodate treatment of the collagen did not affect the forma tion of precipitin arcs, implying that the principal antigenic sites were not a product of post-translational glycosylation. The human and mouse antisera each crossreacted to some extent with mouse and human collagen respectively. These were therefore purified through collagen-CNBr-sepharose conjugates of allospecific collagen. These antibodies were examined by hemagglutination using sheep erythro cytes coated with heat-denatured collagens.'1 The anti-human collagen antibody had a titer of 1/2500; the anti-mouse collagen antibody had a titer of 1/640. To measure collagen production by parental and hybrid cells, we have developed an inhibition of hemagglutination assay using the purified anti-type I collagen antibodies, with human and mouse collagen-coated sheep erythrocytes. Cultured fibroblasts synthesize collagen which is secreted into the medium as the soluble precursor procollagen. With this assay we were able to use the media from confluent cultures of human and mouse cells (2 X lO11 cells in RPMI 1640 plus 10 Vo fetal calf serum) and measure directly the ability of the media to inhibit the agglutination
282
Solomon , Sykes
Somatic cell hybrids/Non-enzyme loci
of collagen-coated erythrocytes by the antibodies. The sensitivity of this assay (1/128 dilution of some culture media still gave detectable inhibition) allowed us to examine the media without concentration or any other special treatment.
We thank Ms. E. T olly for MIRL 110 and MIRL M7 and Dr. H. K oprowski for clones 10 and 21 and F4/SC/13.
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
Heat-denatured media from five human fibroblast lines (four primary and one transformed) were tested and found to inhibit hemagglutina tion by the anti-human collagen antibody and to have no effect on the anti-mouse collagen antibody. Similarly, five mouse lines (four transformed and one primary) were tested and found to inhibit the anti-mouse collagen antibody but not the human. Purified type I collagens (5 /(I) chains to inhibit agglutination was more than 10 times greater than that of aj(I) chains. Table I shows the hybrids which were examined for human and mouse collagen production, as well as two of the parental lines. Inhibition of hemagglutination is expressed as ( —logo) titer decrease compared to the end-point achieved with fresh medium. The ability to synthesize mouse collagen is retained in all of the hybrids. Four of the hybrids did not produce detectable amounts of human collagen, and therefore the following chromosomes were eliminated as candidates for the assignment: 3, 5, 8, 10, 11, 12, 13, 14, 17, 18, 20, 21, 22, X. Two hybrids, F4/SC/13 and MIRL 110, did produce human collagen, indicating that this ability is associated with chromosome 7. Two other hybrids, MIRL 7 and clone 10, have very reduced amounts of human chromosomal material but do have chromosome 7, and both produce human collagen. Finally, clone 21 has chromosome 7 as its only karyotypically visible human material (two to four copies per cell), and it also produces human collagen. Chromosome 7, therefore, appears to carry the gene for at least one of the chains of type I collagen. Because the anti-human type I collagen antibody is considerably more sensitive to «¿(I) than «i(I) chains, it is likely that we have assigned at least the a»(I) chain. Obviously, this will have to be determined by biosynthetic experiments, which are now in progress.
Table I. Production of human (H) and mouse (M) collagen and chromosome constitution of parental lines and hybrids.
H
M
DUV* (primary human fibroblast) lR j (mouse L cell)
5 0
0 2.5
Hybrids
F4/SC/13 MIRL 110 M1RL M7 CLONE 10 CLONE 21
Human chromosomes
H
1 2 3 4
M
1° Fib X 1R 1° Fib X 1R 1° Fib X Cl ID 1° Fib X Cl ID transformed Fib X RAG 1° Fib X 1R 1° Fib X 1R LNSV X MACf LNSV X MAC'
0 0 0 0
4.5 3 2 2
2 1 3 4 3
3 3 4.5 4.5 5
+ +
5
6 7
8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 X
+
+ + + + + a + + + +
+
li + +« +d +o +
+ +
+ + + + + +
+ + + + + + + +
+ +
283
■'Contains an X/15 translocation: 46,X,t(X;15)(pll;qll). >' Contains a 3p deletion. «Contains an X/7 and 7/X, 46,Y,t(X;7)(q22—>24;q22), and an X/7 with a 7p deletion, a 7p3—>-7pl5, in addition to an intact 7. 'I Contains an X/7 with 7p deletion as above, in addition to an intact 7. « Contains an unidentified human-mouse translocation, in addition to an intact 7. f Peritoneal macrophages1'; these hybrids lack normal macrophage markers (S. G ordon , personal communication), and medium from cultured mouse macrophages does not contain measurable mouse collagen by this test.
Somatic cell hybrids/Non-enzyme loci
DUR 4 DT1-2R P2A P7A
Parents
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
Collagen
Solomon, Sykes
Parental lines
284
Solomon , Sykes
Somatic cell hybrids/Non-cnzyme loci
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
1 P iez , K.A.: Primary structure. In G.N. R amachandran and A.H. R eddi, eds.: Biochemistry of collagen, pp. 1-44 (Plenum Press, New York 1976). 2 P eiz , K.A.; E igner, E.Z., and L ewis , M.S.: The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2: 58-66 (1963). 3 Biel , W.; T im pl , R., and F urthmayr: Conformation dependence of antigenic determinants on the collagen molecule. Immunology 24: 13-24 (1973). 4 Solomon, E.; Bobrow, M.; G oodfellow, P.N.; Bodmer, W.F.; Swallow, D.M.; Povey, S., and N oel, B.: Human gene mapping using an X/autosome transloca tion. Somat. Cell Genet. 2: 125-140 (1976). 5 N abholz, M.; M iggiano, V., and Bodmer , W.F.: Genetics analysis with humanmouse somatic cell hybrids. Nature, Lond. 223: 358-363 (1963). 6 C roce, C.M. and K oprow ski , H.: Positive control of the transformed phenotype by the human chromosome 7 carrying the SV40 genome. J. exp. Med. 140: 1221-1229 (1974).
Assignment of a structural gene for type I collagen to chromosome 7 E. Solom on 1
and B. S ykes 2
'Genetics Laboratory, University of Oxford, and 2Department of Pathology, Radcliffe Infirmary, Oxford
Type I collagen, found in skin, bone, tendon, and cornea, is a hetero polymer of three polypeptide chains, two of which are identical, ai(I), and a third of which is homologous, 0^(1) (for a review, see ref. 1). We have examined the production of type I collagen in human-mouse somatic cell hybrids using species-specific anti-collagen antibodies. Human collagen production segregated in complete concordance with chromosome 7.
Supported by grants from the Lalor Foundation (E.S.) and the Nuffield Foun dation (B.S.).
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
Human type I collagen was isolated and purified from acetic acid extracts of human fetal skin, and mouse type I collagen from mouse tail tendon.2 Antisera were raised in sheep and tested by double immunodiffusion against heat-denatured type I collagen. Precipitin arcs were susceptible to in situ digestion by purified bacterial collagenase. Periodate treatment of the collagen did not affect the forma tion of precipitin arcs, implying that the principal antigenic sites were not a product of post-translational glycosylation. The human and mouse antisera each crossreacted to some extent with mouse and human collagen respectively. These were therefore purified through collagen-CNBr-sepharose conjugates of allospecific collagen. These antibodies were examined by hemagglutination using sheep erythro cytes coated with heat-denatured collagens.'1 The anti-human collagen antibody had a titer of 1/2500; the anti-mouse collagen antibody had a titer of 1/640. To measure collagen production by parental and hybrid cells, we have developed an inhibition of hemagglutination assay using the purified anti-type I collagen antibodies, with human and mouse collagen-coated sheep erythrocytes. Cultured fibroblasts synthesize collagen which is secreted into the medium as the soluble precursor procollagen. With this assay we were able to use the media from confluent cultures of human and mouse cells (2 X lO11 cells in RPMI 1640 plus 10 Vo fetal calf serum) and measure directly the ability of the media to inhibit the agglutination
282
Solomon , Sykes
Somatic cell hybrids/Non-enzyme loci
of collagen-coated erythrocytes by the antibodies. The sensitivity of this assay (1/128 dilution of some culture media still gave detectable inhibition) allowed us to examine the media without concentration or any other special treatment.
We thank Ms. E. T olly for MIRL 110 and MIRL M7 and Dr. H. K oprowski for clones 10 and 21 and F4/SC/13.
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
Heat-denatured media from five human fibroblast lines (four primary and one transformed) were tested and found to inhibit hemagglutina tion by the anti-human collagen antibody and to have no effect on the anti-mouse collagen antibody. Similarly, five mouse lines (four transformed and one primary) were tested and found to inhibit the anti-mouse collagen antibody but not the human. Purified type I collagens (5 /(I) chains to inhibit agglutination was more than 10 times greater than that of aj(I) chains. Table I shows the hybrids which were examined for human and mouse collagen production, as well as two of the parental lines. Inhibition of hemagglutination is expressed as ( —logo) titer decrease compared to the end-point achieved with fresh medium. The ability to synthesize mouse collagen is retained in all of the hybrids. Four of the hybrids did not produce detectable amounts of human collagen, and therefore the following chromosomes were eliminated as candidates for the assignment: 3, 5, 8, 10, 11, 12, 13, 14, 17, 18, 20, 21, 22, X. Two hybrids, F4/SC/13 and MIRL 110, did produce human collagen, indicating that this ability is associated with chromosome 7. Two other hybrids, MIRL 7 and clone 10, have very reduced amounts of human chromosomal material but do have chromosome 7, and both produce human collagen. Finally, clone 21 has chromosome 7 as its only karyotypically visible human material (two to four copies per cell), and it also produces human collagen. Chromosome 7, therefore, appears to carry the gene for at least one of the chains of type I collagen. Because the anti-human type I collagen antibody is considerably more sensitive to «¿(I) than «i(I) chains, it is likely that we have assigned at least the a»(I) chain. Obviously, this will have to be determined by biosynthetic experiments, which are now in progress.
Table I. Production of human (H) and mouse (M) collagen and chromosome constitution of parental lines and hybrids.
H
M
DUV* (primary human fibroblast) lR j (mouse L cell)
5 0
0 2.5
Hybrids
F4/SC/13 MIRL 110 M1RL M7 CLONE 10 CLONE 21
Human chromosomes
H
1 2 3 4
M
1° Fib X 1R 1° Fib X 1R 1° Fib X Cl ID 1° Fib X Cl ID transformed Fib X RAG 1° Fib X 1R 1° Fib X 1R LNSV X MACf LNSV X MAC'
0 0 0 0
4.5 3 2 2
2 1 3 4 3
3 3 4.5 4.5 5
+ +
5
6 7
8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 X
+
+ + + + + a + + + +
+
li + +« +d +o +
+ +
+ + + + + +
+ + + + + + + +
+ +
283
■'Contains an X/15 translocation: 46,X,t(X;15)(pll;qll). >' Contains a 3p deletion. «Contains an X/7 and 7/X, 46,Y,t(X;7)(q22—>24;q22), and an X/7 with a 7p deletion, a 7p3—>-7pl5, in addition to an intact 7. 'I Contains an X/7 with 7p deletion as above, in addition to an intact 7. « Contains an unidentified human-mouse translocation, in addition to an intact 7. f Peritoneal macrophages1'; these hybrids lack normal macrophage markers (S. G ordon , personal communication), and medium from cultured mouse macrophages does not contain measurable mouse collagen by this test.
Somatic cell hybrids/Non-enzyme loci
DUR 4 DT1-2R P2A P7A
Parents
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
Collagen
Solomon, Sykes
Parental lines
284
Solomon , Sykes
Somatic cell hybrids/Non-cnzyme loci
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:29:15 PM
1 P iez , K.A.: Primary structure. In G.N. R amachandran and A.H. R eddi, eds.: Biochemistry of collagen, pp. 1-44 (Plenum Press, New York 1976). 2 P eiz , K.A.; E igner, E.Z., and L ewis , M.S.: The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2: 58-66 (1963). 3 Biel , W.; T im pl , R., and F urthmayr: Conformation dependence of antigenic determinants on the collagen molecule. Immunology 24: 13-24 (1973). 4 Solomon, E.; Bobrow, M.; G oodfellow, P.N.; Bodmer, W.F.; Swallow, D.M.; Povey, S., and N oel, B.: Human gene mapping using an X/autosome transloca tion. Somat. Cell Genet. 2: 125-140 (1976). 5 N abholz, M.; M iggiano, V., and Bodmer , W.F.: Genetics analysis with humanmouse somatic cell hybrids. Nature, Lond. 223: 358-363 (1963). 6 C roce, C.M. and K oprow ski , H.: Positive control of the transformed phenotype by the human chromosome 7 carrying the SV40 genome. J. exp. Med. 140: 1221-1229 (1974).
