Proc. Nati. Acad. Sci. USA Vol. 75, No. 3, pp. 1456-1460, March 1978
Genetics
Chromosomal localization of human A3 globin gene on human chromosome 11 in somatic cell hybrids (gene mapping/cloning/cDNA/fibroblasts/molecular hybridization)
ALBERT DEISSEROTH*, ARTHUR NIENHUISt, JEANNE LAWRENCE*, RICHARD GILESt, PATRICIA TURNERt,
AND FRANK H.
RUDDLEf
Experimental Hematology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014; t Clinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014; and * Department of Biology, Yale *
University, New Haven, Connecticut 06520
Contributed by Frank H. Ruddle, December 21, 1977
ABSTRACT We have successfully used a DNA-cDNA molecular hybridization assay to directly determine the presence or absence of human 13 globin gene sequences in 20 humanmouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human .3 globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human 1, globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human 13 globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT4. 157-BNPT-4 contained the human 1 globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human y globin cDNA; two lines positive for human'3 globin gene sequences also contained human y globin gene sequences while one line was negative for both 13 and My gene sequences. On the basis of these results, the human 13 and oy globin genes have been assigned to human chromosome 11.
quisitely sensitive to detect the globin gene sequences in several human-mouse hybrid clones, each of which contains different subsets of human chromosomes (2-4). Correlation of the chromosomal content of each clone with the presence or absence of the human globin gene sequences has previously led to the assignment of the human a globin gene to chromosome 16 (2). In this report, we will present data that provide strong evidence for the assignment of the human 13 (and y) globin genes to chromosome 1 1. MATERIALS AND METHODS Cell Hybrids and Composition of Their Human Chromosomes. The parent cell used for fusion, cell culture techniques, and methods of fusion for hybrid cell lines AHA 3D, AHA 16D, AHA 16E, AIM 23 OLD, AIM 23 X-1, IL IT-5, IL II-54D, JFA 14a 5, JFA 14a 13-5, J10H7, WAV, WAV R4D, WAV R4D A19, WAIV A, WAIV A-AA, and WAIV A-DAP have been discussed in detail in our earlier study (2). Clones 157-BNPT-4 and 157-BNPT-1 were derived by fusion of the human fibroblast GM 126[46, XY, t(1,15)(p36; ql)] with the mouse fibroblagt tsCl AOH followed by isolation of the hybrid cells in Dulbecco's modified Eagle's medium (5). AIM 15a B-1 was obtained by fusion of the human fibroblast GM 17(tl5;18) with the mouse fibroblast A9 and isolation of the hybrid cells in hypoxanthine/aminopterin/thymidine as described (6). Hybrid cell WL II 24a 5C was derived by fusion of the thymidine kinase-deficient L cell (LM TI-) with the human fibroblast WI-38 and isolated in hypoxanthine/aminopterin/thymidine (7). Giemsa 11 stain was used to specifically identify mouse and human chromosomes, as outlined previously (2, 8, 9). Single metaphase spreads were stained sequentially by a combination of Giemsa-trypsin followed by Hoechst 33258 staining to identify specific human chromosomes (2, 10, 11). An average of 82 metaphase spreads (range 45-167) were analyzed for each cell line. The methods for the 22 different isozyme markers for 17 different human chromosomes were given in our earlier reports (2-12). The cells used for chromosomal and isozymal analysis were from the pool of cells from which the DNA was isolated for use in the DNA-cDNA hybridization studies (2). Preparation of Specific cDNAs. A cDNA enriched in human a globin gene sequences was obtained by one of three methods, as previously described (2). Method I used mRNA, obtained from reticulocytes.of a patient with 13 thalassemia, that was deficient in 13 globin mRNA sequences. In method II, a globin mRNA was purified from normal human reticulocyte mRNA by polyacrylamide gel electrophoresis in 98% formamide. For method III, human a globin cDNA obtained by method II was further purified by annealing the cDNA to 13 globin-enriched mRNA to a limited Cot, followed by recovery
Currently available techniques for assignment of unique genes to individual human chromosomes include linkage analysis in appropriate pedigrees, in situ hybridization of specific gene probes to metaphase chromosomes, and utilization of somatic cell hybrids in which segregation of particular chromosomes can be correlated with the presence of specific gene products (1). The first method depends upon the occurrence of informative polymorphisms of genes in individual families while the second method is limited in the sensitivity and specificity of the assay used for gene detection, which is based upon the annealing of a radioactively labeled probe with the nucleoprotein complex that makes up the metaphase chromosome. The current knowledge of the human chromosome gene map (1) has come about primarily through the use of somatic cell hybrids. The use of this method for the chromosomal localization of markers of differentiated cells has been difficult because these markers are often not expressed in hybrid cells derived by fusion of mouse and human fibroblasts. In our studies of the chromosomal localization of the human globin genes, we have used a molecular hybridization assay that is both highly specific and exThe 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. §1734 solely to indicate this fact.
1456
Genetics: Deisseroth et al.
of the a globin cDNA that remained single stranded by hydroxylapatite chromatography (2). A probe enriched in human A globin cDNA sequences was obtained with mRNA, extracted from reticulocytes of a patient with hemoglobin H disease, that was deficient in a globin mRNA sequences. A cDNA specific for human y globin genes was synthesized with mRNA from fetal erythrocytes. The a and 13 globin mRNA sequences were removed by hydroxylapatite chromatography after annealing of the mixed cDNA (a, 13, and y) to normal human reticulocyte mRNA that contains only a and 1 globin mRNA. The cycling process (annealing followed by hydroxylapatite chromatography) was repeated twice, giving a cDNA that was more than 90% y globin cDNA sequences, as shown previously (13). Mouse globin cDNA was synthesized with reticulocyte polysomal mRNA. Synthesis of cDNA was by incubation of mRNA with RNAdirected DNA polymerase (reverse transcriptase) as previously described (2, 14). [a-32P]dCTP of specific activity 150-250 Ci/mmol (supplied by Amersham/Searle or New England Nuclear) was used to give a probe the specific activity of which ranged from 150,000-250,000 cpm/ng. The cDNA probes used in annealing reactions were selected for size by alkaline sucrose gradient centrifugation (500-600 nucleotides in length) and were used within 10 days of their synthesis. Extraction of DNA and DNA-cDNA Hybridization. Cells (2-6 X 109) were used for preparation of DNA exactly as described (2). Human DNA was obtained from spleen and mouse DNA from mouse embryos. Each DNA was sonicated to a fragment size of 400-500 nucleotides. For each analysis, 1 or 2 mg of DNA was annealed in a total reaction mixture of 70 or 140,gl to 16-55 pg of cDNA (specific activity 150,000-280,000 cpm/ng) in saline/citrate that was 3 times the standard concentration and 50% formamide. Individual 12- or 20-IAl aliquots were sealed in glass capillary tubes and incubated at 520 for 10 min to 48 hr, thereby generating a Cot curve. Analysis was performed by either hydroxylapatite chromatography or SI nuclease digestion (2, 3, 14, 15). The type of human a globin cDNA (from method I, II, or III) used for analysis of DNA from the individual hybrid lines was as indicated in our previous publication (2). The four new lines reported here (157-BNPT-1, 157-BNPT-4, AIM 15a B-i, and WL II 24a 5C) were analyzed with human a globin cDNA obtained by method II. RESULTS Detection of Human , Globin Gene in Hybrid Cells by DNA*cDNA Hybridization. The species specificity of the human globin cDNA used in our studies for detection of human globin genes has been established in our previous publications (2-4, 14, 15). When DNA purified from a hybrid clone that contains human a globin gene sequences was incubated with the human 13 globin cDNA used in our studies, no significant hybridization was observed (Fig. 1). This result establishes that the human 13 globin cDNA is sufficiently free of a cDNA sequences to allow independent detection of the human 13 globin gene. Similarly, DNA from a hybrid cell line that contained human 13 globin gene sequences was annealed to the human a globin cDNA (method II); no significant hybridization occurred (Fig. 1). Thus, the human a and 13 globin cDNA probes used in our studies of globin gene chromosomal localization were specific for the human a and 13 globin genes, respectively. Significant levels of human 13 globin gene sequences were detected by hybridization assay in hybrid cell lines AHA 3D,
Proc. Nati. Acad. Sci. USA 75 (1978)
0 0
~60~50
1457
/
(3 aS
> 40-
A 30
-
20
1
2
o
3
4
1 Log Cot
2
3
a
4
FIG. 1. Specific detection of human a and flglobin genes in somatic cell hybrid lines. (A) DNA of hybrid cell line WAV-5 was annealed to mouse globin cDNA (a), human a cDNA (A) (prepared by method I as outlined in ref. 1), or human (3 cDNA (0). Analysis of hybrid formation was by batch chromatography on hydroxylapatite (3). (B) DNA from hybrid cell line AIM-iSa B-1 was annealed to the three cDNAs as outlined above. Analysis of hybrid formation in this experiment was by determination of SI nuclease resistance (2, 14). The differences in the apparent rate of annealing of the two hybrid cell DNAs to mouse cDNA (log Cotj12 in A is 2.55 and in B is 3.30) reflects the different techniques used to measure duplex formation (16).
AHA 16D, AHA 16E, J10H7, 157-BNPT-4, and AIM 15a B-i. Human 13 globin gene sequences were not found in hybrid cell lines AIM 23 OLD, AIM 23 X-1, IL II 5, IL II 54D, JFA 14a 5, JFA 14a 13-5, WAV, WAV R4D, WAV R4D A19, WAIV A, WAIV A-AA, WAIV A-DAP, and 157-BNPT-1. Only barely detectable levels of human 13 globin gene were found in WL II 24a 5C. Identification of Human Chromosomes in Each Human-Mouse Hybrid Clone. An average, of 82 metaphase spreads (range 45-167) of each cell line were characterized by two chromosomal staining techniques to identify the human chromosomes present in each cell line. The alkaline Giemsa stain (2, 8) and a combination of Giemsa-trypsin and Hoechst 33258 centromeric staining (1, 9, 10) permitted specific identification of each human chromosome as well as the identification of any interspecific chromosomal translocations. The human chromosomal content and isozyme studies of markers for human chromosomes of hybrid lines AHA 3D, AHA 16D, AHA 16E, AIM 23 OLD, AIM 23 X-1, IL II 5, IL 11 54 D, JFA 14a 5, JFA 14a 13-5, J10H7, WAV, WAV R4D, WAV R4d A19, WAIV A, WAIV A-AA, and WAIV A-DAP were reported in table 1 of our earlier study on the human a globin gene (2). The human chromosomal content as well as the content of human 13 and y globin genes of hybrid cell lines 157-BNPT-1, 157BNPT-4, AIM 15a B-i, and WL II 24a 5C are presented in Table 1 of the present report. Analysis of these latter four lines for the presence of lactate dehydrogenase A (EC 1.1.1.27), a marker that exists on the short arm of human chromosome 11 (7), is presented in Fig. 2. Assignment of Human P Globin Gene to Human Chromosome 11. The presence of a human chromosome in greater than 10% of the metaphase spreads of a hybrid cell line that is devoid of human 13 globin genes is used as grounds for exclusion of that chromosome from candidacy. The molecular hybridization assay used to detect the globin gene was sufficiently sensitive to allow this exclusion criterion to be applied (2). All human chromosomes were excluded except for human chromosomes 6, 8, 9, 11, and 13, as shown by the data presented in Table 2. Only human chromosome 11 was present at a high
Genetics: Deisseroth et al.
1458
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Chromosomal composition and fl and y globin gene content of hybrid cell clones Hi,uman chroimosome 1 2 3 4 5 6 7
8 9 10 11
12
13 14 15
Cell line AIM 15a WL II G-157 B-1 24a 5C BNPT-1 0/57 0/77 0/59 -PEP C -PEP C -PEP C -PGM-1 -PGM-1 -PGM-1 25/57 0/77 0/57 +IDH-1 -IDH-1 -IDH-1 0/57 11/77 22/59 0/57 13/77 0/59 31/57 3/77 0/59 0/57 0/77 0/59 -MOD-1 -MOD-1 23/57 0/77 0/59 +,B GLUC -f GLUC -0 GLUC 16/57 0/77 0/59 0/57 0/77 0/59 0/57 0/77 0/59 -AD K -AD K -AD K -GOT -GOT -GOT 50/57 0/77 0/59 +LDH A -LDH A -LDH A 12/57 0/77 1/59 +LDH B -LDH B -PEP B -PEP B 12/57* 0/77 0/59 -ESD -ESD -ESD 11/57 0/77 34/59 +NP -NP +NP 7/57 0/77 0/59 +MPI -MPI -MPI +HEX A
18
0/57 -APRT 40/57 +GK 25/57
19
0/57
20
-GPI 0/57
16 17
0/77
21 22
X B Glolbin gene y Glolbin gene
G-157 BNPT-4
0/64 -PEP C -PGM-1 0/64 -IDH-1 22/64 0/64 0/64 0/64 -MOD-1 0/64 -, GLUC 0/64 0/64 0/64 -AD K -GOT 52/64 +LDH A 0/64 -LDH B
0/64 -ESD 34/64 +NP
0/64 -MPI
-HEX A
-HEX A
0/59
0/64
-APRT
-APRT
-APRT
24/77
0/59
0/64
+GK 0/77
-GK
-GK
0/59
0/64
+PEP A
0/77 -GPI 0/77
-PEP A
-PEP A
0/59
0/64
-GPI
-GPI 0/64
0/59
a.:.
-ADA
-ADA
-ADA
-ADA
19/57 +SOD 0/57 0/57*
0/77
21/59*
0/64
-SOD
-SOD
0/77 0/77
0/59 17/59
0/64 51/64
+HPRT -PGK
-HPRT
+HPRT +PGK
+HPRT +PGK
+++ +++
A chromosome is scored as being present if it appears in greater than 10% of the metaphase spreads of that line which were studied. Isozyme abbreviations: PEP C, peptidase C (EC 3.4.11.1); PGM, phosphoglucomutase (EC 2.7.5.1); IDH, isocitrate dehydrogenase (EC 1.1.1.42); MOD, (NADP) malic enzyme cytoplasmic (EC 1.1.1.40); ,B GLUC, fl-glucuronidase (EC 3.2.1.31); AD K, adenosine kinase (EC 2.7.1.20); GOT, glutamate oxaloacetate transaminase (EC 2.6.1.1); LDH A, lactate dehydrogenase A (EC 1.1.1.27); LDH B, lactate dehydrogenase B (EC 1.1.1.27); PEP B, peptidase B (EC 3.4.11.1); ESD, esterase D (EC 3.1.1.1); NP, purine nucleoside phosphorylase (EC 2.4.2.1); MPI, mannosephosphate isomerase (EC 5.3.1.8); HEX A, hexosaminidase A (EC 3.2.1.30); APRT, adenine phosphoribosyltransferase (EC 2.4.2.7); GK, galactokinase (EC 2.7.1.6); PEP A, peptidase A (EC 3.4.11.1); GPI, glucosephosphate isomerase (EC 5.3.1.9); ADA, adenosine deaminase (EC 3.5.4.4); SOD, cytoplasmic
.:
i
..:
b
..: ::
c 0
co
:c
.s
.e
1 2
3
4
5
6
7
FIG. 2. Detection of laetate dehydrogenase A (EC 1.1.1.27), as known marker for human chromosome i1, in somatic cell hybrids. The electrophoretic method and staining technique are as previously published (11). Extracts from the following cells were analyzed: Channel 1, HeLa cells (human); 2, mouse fibroblasts (A9); 3 and 4, hybrid clone WL II 24a 5C; 5, hybrid clone AIM 15a, B-1; 6, hybrid clone 157-BNPT-1; and 7, hybrid clone 157-BNPT-4. a and b, Heteropolymers (A + B); c, mouse homopolymer (A4); d, heteropolymers (A); e, origin and heteropolymers; f, human homopolymers (A4).
frequency in all of the hybrid cell lines that contained human 13 globin genes (see Table 3). In fact, human chromosome 11 was the only human chromosome that was present in all of the hybrid clones positive for the human 13 globin gene. In contrast, human chromosomes 6, 8, 9, and 13 were present in less than 10% of the metaphase spreads of four of the six hybrid cell lines that contained the human 13 globin genes (Table 1). Hybrid cell clone WL II 24a SC, which contained a very low level of human ,B globin gene sequences, was negative for the human isozyme lactate dehydrogenase A (a known marker for human chromosome 11), as shown in Fig. 2. None of the metaphase spreads of this latter line contained examples of an intact human chromosome 11. Two translocations were found in this line. One, present in 58 of 77 metaphase spreads of this line examined, was interspecific and may have involved a portion of the long arm of human chromosome 11. The other involved a translocation of the long arm of human chromosome 17 to a mouse chromosome. The data obtained with the sister clones, 157-BNPT-1 and 157-BNPT-4, strongly support the assignment of the , globin gene to chromosome 11. The electrophoretograms presented in Fig. 2 show that 157-BNPT-1 and 157-BNPT-4 are negative and positive, respectively, for lactate dehydrogenase A. Both clones contain a high frequency of human chromosomes 3, 14, and X (Table 1). The results of the analysis of DNA purified from these two clones is presented in Fig. 3A. Clone 157BNPT-4, which is positive for lactate dehydrogenase A, was also strongly positive for the human # globin gene, while 157BNPT-1, which lacks lactate dehydrogenase A, was devoid of human 13 globin gene sequences. Chromosomal analysis of these two lines revealed that the major difference between them was the presence of chromosome 11 in 157-BNPT-4; it was not indophenol oxidase (EC 1.15.1.1); PGK, phosphoglycerate kinase (EC 2.7.2.3); HPRT, hypoxanthine phosphoribosyl transferase (EC 2.4.2.8). * Isozyme and chromosomal data disagree in 3 of the 65 instances in which both sets of data were given.
Proc. Natl. Acad. Sci. USA 75 (1978)
Genetics: Deisseroth et al.
1459
Table 2. Cell lines devoid of ,3 globin genes and containing the given chromosome in more than 10% of metaphase spreads
ChromoCell lines
some
.0 -
AIM 23 OLD (30/68), AIM 23X-1 (28/70) JFA 14a 5 (55/160) AIM 23 OLD (29/68), AIM 23X-1 (41/70) BNPT-1 (21/59) WAV (23/84), WAV R4D (27/107) AIM 23X-1 (8/70)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
X
40-
50-
240
30-
3020 -~ ~ 20
~ A
~~2200-
~ ~ ~10
/
10
IL II-5 (18/66), IL II-54D (14/70)
1
2
3
4
AIM 23 OLD (21/68), AIM 23X-1 (22/70)
AIM 23 OLD (30/68), AIM 23X-1 (20/70), IL II-5 (23/66), IL II-54D (15/70)
BNPT-1 (34/59) AIM 23X-1 (13/70), IL II-5 (11/66) WAV (54/84), WAIV A (56/95), WAIV A AA (32/46) IL II-5 (16/66), IL II-54D (42/70) JFA 14a 13-5 (106/167) IL II-5 (13/66), IL II-54D (7/70) WAV (9/84) AIM 23 OLD (34/68), AIM 23X-1 (10/70), WAV (47/59), WAV R4D (25/37), WAV R4D A19 (15/41), BNPT-1 (21/59) WAV (43/59), WAV R4D (24/37), WAV R4D A19 (15/41), WAIV A (27/95), WAIV A-DAP (24/50) AIM 23 OLD (29/68), AIM 23X-1 (37/70), BNPT-1 (17.59)
found in 157-BNPT-1. An interspecific translocation not involving chromosome 11 was also present in 157-BNPT-4. This analysis strongly confirms the assignment of human (3 globin gene to human chromosome 11. Assignment of Human y Globin Gene to Human Chromosome 11. Family studies as well as characterization of the abnormal hemoglobins "Lepore" and "Kenya," which arose from genetic crossover, have led to the conclusion that the human A and -y globin genes are closely linked (17, 18). As a formal test of the synteny of the human # and y globin structural genes and as a further test of our assignment of the human # globin gene to human chromosome I1, we determined the 'y globin sequence content of DNA from hybrid cells 157BNPT-1, 157-BNPT-4, and AIM 15a B-1. As shown in Fig. 3B, the hybrid cell lines that contain human chromosome 11 and the ft globin gene (157-BNPT-4 and AIM i5a B-1) contain 'y globin gene sequences, while DNA from 157-BNPT-1 (negative for both human chromosome 11 and the ( globin gene) lacked Table 3. Composition of human chromosomes present in hybrid cell lines positive for human jB globin genes Fraction of metaphase spreads containing human chromosome
Cell line AHA 3D AHA 16 D AHA 16 E J1OH7 AIM 15a B-1 157-BNPT-4
6
8
9
11
13
0/45 2/60 0/70 27/69 0/57 0/64
5/45 1/60 3/70 0/69 16/57 0/64
0/45 0/60 0/70 0/69 0/57 0/64
31/45 23/60 45/70 52/69 50/57 52/64
0/45 2/60 9/70 0/69 12/570/64
1
5 Log
2
3
4
5
Cot
FIG. 3. Detection of human ,B and y globin genes in somatic cell hybrids. Human fi (A) or 'y (B) globin cDNA was annealed to DNA from human spleen (0), hybrid cell 157-BNPT-4 (0), hybrid cell AIM 15a B-1 (A), or hybrid cell 157-BNPT-1 (A). Measurement of duplex formation was by Si nulcease resistance. In each instance 7000 cpm of [32P]cDNA was used in the analysis of 2 mg of DNA. The specific activity of ,B globin cDNA was 315.7 cpm/pg and that of 'y globin cDNA was 125.7 cpm/pg so that 7000 cpm represented 22.2 pg of , and 55.7 pg of y globin cDNA. This difference in cDNA input accounts for the different apparent rate and extent of hybridization between the two probes (16).
y globin gene sequences. We concluded that on the basis of these data both the human ( and y globin genes are on chromosome 11.
DISCUSSION An analysis of 20 human-mouse hybrid cell clones by a highly specific and sensitive DNA cDNA hybridization assay has established that the human ( and y globin genes are present on human chromosome I1. The presence of human chromosomes in hybrid cell lines devoid of human , globin genes served to exclude all human chromosomes except 6, 8, 9, 11, and 13. Among these chromosomes, only human chromosome 11 was present in the hybrid cell line positive for the human # or ly globin genes. The frequency of human chromosome 11 in the subclones 157-BNPT-1 and 157-BNPT-4 correlated well with the presence of the human , and y globin genes. On the basis of the data, we have concluded that the structural genes for the human y and (3 globin chains reside on human chromosome ii. Earlier studies by others using in situ molecular hybridization suggested that the human globin genes were on human chromosomes 2, 4, or 5 (19-22). The data that we have presented in this report and in our previous study on the localization of the human a globin gene indicate that these earlier assignments based on in situ molecular hybridization were incorrect. The theoretical reasons for the disagreement of the human globin gene assignments based on in situ hybridization with our own work have been discussed previously (2, 23). The complementary DNA used for our own studies was labeled to a high specific activity, contained a full-length copy of the globin mRNA, and was shown by us to exhibit specificity for the individual human globin genes (2; Fig. 1). Such specificity was achieved by conducting the annealing reaction at a temperature above that which allowed formation of duplexes between human cDNA and the mouse globin genes. In addition, we have presented evidence in this report that the cDNA used for detection of human a and ( globin genes was of adequate purity to specifically identify the human a and ( globin genes, respectively.
1460
Genetics: Deisseroth et al.
We believe that the method we have used for chromosomal localization of the human a and globin genes may permit a rapid accumulation of information on the map positions of a number of other differentiated cell functions in human beings and other mammalian species. Specialized gene products as well as the complementary DNA species generated from them will surely permit more extensive use of the method we have developed for the chromosomal localization of markers of differentiated cells. Furthermore, application of recombinant DNA technology will yield totally pure probes for many genes which may be used to study the gene sequence content of hybrid cells. In addition, the identification of the chromosomal localization of the human a globin gene has enabled us to develop a genetic strategy of chromosomal dependent transfer of the human a globin gene into recipient mouse cells in a state that permits continued expression of this gene (24). Such methods, when applied to the human globin gene and to other differentiated cell functions, may be of use in elucidating the mechanisms governing the expression of differentiated cell functions as well as in approaching the antenatal diagnosis of mutations of these genes which are associated with disease in human beings. A.D. recognizes the generosity extended to him by Dr. Samuel Latt of the Children's Hospital Medical Center, Boston, MA, and the use of his photomicroscope and photographic facilities during a portion of this work. Elizabeth Nichols is recognized for her superb work in the isozymal analysis of the hybrid clones studied. We thank Drs. J. and D. Beard for providing RNA-directed DNA polymerase through the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute. Sylvon Von Der Pool, Ann Cunningham, and Francis Lawyer are acknowledged for the excellence of their technical skills in the support of this project. 1. McKusick, V. & Ruddle, F. (1977) Science 196,390-405. 2. Deisseroth, A., Nienhuis, A., Turner, P., Velez, R., Anderson, F. W., Ruddle, F., Lawrence, J., Creagan, R. & Kucherlapati, R. (1977) Cell 12,205-218.
Proc. Natl. Acad. Sci. USA 75 (1978) 3. Deisseroth, A., Velez, R. & Neinhuis, A. (1976) Science 191, 1962-1964. 4. Deisseroth, A. & Nienhuis, A. (1976) In Vitro 12,734-742. 5. Giles, R..(1977) Ph.D. Dissertation, (Yale University, New Haven,
CT). 6. Faber, H. E., Kucherlapati, R., Poulik, M. D., Ruddle, F. H. & Smithies, 0. (1976) Somatic CelltGenet. 2, 141-153. 7. Boone, C., Chen, T. R. & Ruddle, F. H. (1972) Proc. Nati. Acad. Sci. USA 69,510-514. 8. Bobrow, M., Madan, K. & Pearson, P. L. (1972) Nature New Biol. 238, 122-124. 9. Friend, K. K., Chen, S. & Ruddle, F. H. (1976) Somatic Cell Genet. 2, 183-188. 10. Yoshida, M. C., Ikeuchi, T. & Sadaki, M. (1975) Proc. Jpn. Acad. 51, 184-187. 11. Kozak, C. A., Lawrence, J. B. & Ruddle, F. H. (1977) Exp. Cell Res. 105, 109-117. 12. Nichols, E. A. & Ruddle, F. H. (1973) J. Histochem. Cytochem. 21, 1066-1081. 13. Neinhuis, A. W., Turner, P. & Benz, E. J., Jr. (1977) Proc. Natl. Acad. Sci. USA 74,3960-3964. 14. Benz, E. J., Geist, C. E., Steggles, A. W., Barker, J. E. & Nienhuis, A. W. (1977) J. Biol. Chem. 252, 1098-1916. 15. Deisseroth, A., Velez, R., Burke, R., Minna, J., Anderson, F. W. & Neinhuis, A. (1976) Somatic Cell Genet. 2,373-384. 16. Britten, R. J. & Davidson, E. H. (1976) Proc. Natl. Acad. Sci. USA 73,415-419. 17. Smith, D. H., Clegg, J. B., Weatherall, D. J. & Gilles, H. M. (1973) Nature New Biol. 246, 184-188. 18. Barnabas, J. & Muller, C. J. (1962) Nature 194,931-933. 19. Price, P. M. & Hirschhorn, K. (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 2227-2243. 20. Price, P. M. & Hirschhorn, K. (1975) Cytogenet. Cell Genet. 14, 255-231. 21. Price, P. M., Conover, J. H. & Hirschhorn, K. (1972) Nature 237,
340342. 22. Cheung, S. W., Tishler, P. V., Atkins, L., Sengupta, S. K., Modest, E. J. & Forget, B. (1977) Cell Biol. Int. Rep. 1, 255-262. 23. Bishop, J. 0. & Jones, K. W. (1972) Nature 240, 149-150. 24. Deisseroth, A. & Hendrick, D. (1977) Blood 50, Suppl. 1, p. 105.
Genetics
Chromosomal localization of human A3 globin gene on human chromosome 11 in somatic cell hybrids (gene mapping/cloning/cDNA/fibroblasts/molecular hybridization)
ALBERT DEISSEROTH*, ARTHUR NIENHUISt, JEANNE LAWRENCE*, RICHARD GILESt, PATRICIA TURNERt,
AND FRANK H.
RUDDLEf
Experimental Hematology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014; t Clinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014; and * Department of Biology, Yale *
University, New Haven, Connecticut 06520
Contributed by Frank H. Ruddle, December 21, 1977
ABSTRACT We have successfully used a DNA-cDNA molecular hybridization assay to directly determine the presence or absence of human 13 globin gene sequences in 20 humanmouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human .3 globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human 1, globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human 13 globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT4. 157-BNPT-4 contained the human 1 globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human y globin cDNA; two lines positive for human'3 globin gene sequences also contained human y globin gene sequences while one line was negative for both 13 and My gene sequences. On the basis of these results, the human 13 and oy globin genes have been assigned to human chromosome 11.
quisitely sensitive to detect the globin gene sequences in several human-mouse hybrid clones, each of which contains different subsets of human chromosomes (2-4). Correlation of the chromosomal content of each clone with the presence or absence of the human globin gene sequences has previously led to the assignment of the human a globin gene to chromosome 16 (2). In this report, we will present data that provide strong evidence for the assignment of the human 13 (and y) globin genes to chromosome 1 1. MATERIALS AND METHODS Cell Hybrids and Composition of Their Human Chromosomes. The parent cell used for fusion, cell culture techniques, and methods of fusion for hybrid cell lines AHA 3D, AHA 16D, AHA 16E, AIM 23 OLD, AIM 23 X-1, IL IT-5, IL II-54D, JFA 14a 5, JFA 14a 13-5, J10H7, WAV, WAV R4D, WAV R4D A19, WAIV A, WAIV A-AA, and WAIV A-DAP have been discussed in detail in our earlier study (2). Clones 157-BNPT-4 and 157-BNPT-1 were derived by fusion of the human fibroblast GM 126[46, XY, t(1,15)(p36; ql)] with the mouse fibroblagt tsCl AOH followed by isolation of the hybrid cells in Dulbecco's modified Eagle's medium (5). AIM 15a B-1 was obtained by fusion of the human fibroblast GM 17(tl5;18) with the mouse fibroblast A9 and isolation of the hybrid cells in hypoxanthine/aminopterin/thymidine as described (6). Hybrid cell WL II 24a 5C was derived by fusion of the thymidine kinase-deficient L cell (LM TI-) with the human fibroblast WI-38 and isolated in hypoxanthine/aminopterin/thymidine (7). Giemsa 11 stain was used to specifically identify mouse and human chromosomes, as outlined previously (2, 8, 9). Single metaphase spreads were stained sequentially by a combination of Giemsa-trypsin followed by Hoechst 33258 staining to identify specific human chromosomes (2, 10, 11). An average of 82 metaphase spreads (range 45-167) were analyzed for each cell line. The methods for the 22 different isozyme markers for 17 different human chromosomes were given in our earlier reports (2-12). The cells used for chromosomal and isozymal analysis were from the pool of cells from which the DNA was isolated for use in the DNA-cDNA hybridization studies (2). Preparation of Specific cDNAs. A cDNA enriched in human a globin gene sequences was obtained by one of three methods, as previously described (2). Method I used mRNA, obtained from reticulocytes.of a patient with 13 thalassemia, that was deficient in 13 globin mRNA sequences. In method II, a globin mRNA was purified from normal human reticulocyte mRNA by polyacrylamide gel electrophoresis in 98% formamide. For method III, human a globin cDNA obtained by method II was further purified by annealing the cDNA to 13 globin-enriched mRNA to a limited Cot, followed by recovery
Currently available techniques for assignment of unique genes to individual human chromosomes include linkage analysis in appropriate pedigrees, in situ hybridization of specific gene probes to metaphase chromosomes, and utilization of somatic cell hybrids in which segregation of particular chromosomes can be correlated with the presence of specific gene products (1). The first method depends upon the occurrence of informative polymorphisms of genes in individual families while the second method is limited in the sensitivity and specificity of the assay used for gene detection, which is based upon the annealing of a radioactively labeled probe with the nucleoprotein complex that makes up the metaphase chromosome. The current knowledge of the human chromosome gene map (1) has come about primarily through the use of somatic cell hybrids. The use of this method for the chromosomal localization of markers of differentiated cells has been difficult because these markers are often not expressed in hybrid cells derived by fusion of mouse and human fibroblasts. In our studies of the chromosomal localization of the human globin genes, we have used a molecular hybridization assay that is both highly specific and exThe 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. §1734 solely to indicate this fact.
1456
Genetics: Deisseroth et al.
of the a globin cDNA that remained single stranded by hydroxylapatite chromatography (2). A probe enriched in human A globin cDNA sequences was obtained with mRNA, extracted from reticulocytes of a patient with hemoglobin H disease, that was deficient in a globin mRNA sequences. A cDNA specific for human y globin genes was synthesized with mRNA from fetal erythrocytes. The a and 13 globin mRNA sequences were removed by hydroxylapatite chromatography after annealing of the mixed cDNA (a, 13, and y) to normal human reticulocyte mRNA that contains only a and 1 globin mRNA. The cycling process (annealing followed by hydroxylapatite chromatography) was repeated twice, giving a cDNA that was more than 90% y globin cDNA sequences, as shown previously (13). Mouse globin cDNA was synthesized with reticulocyte polysomal mRNA. Synthesis of cDNA was by incubation of mRNA with RNAdirected DNA polymerase (reverse transcriptase) as previously described (2, 14). [a-32P]dCTP of specific activity 150-250 Ci/mmol (supplied by Amersham/Searle or New England Nuclear) was used to give a probe the specific activity of which ranged from 150,000-250,000 cpm/ng. The cDNA probes used in annealing reactions were selected for size by alkaline sucrose gradient centrifugation (500-600 nucleotides in length) and were used within 10 days of their synthesis. Extraction of DNA and DNA-cDNA Hybridization. Cells (2-6 X 109) were used for preparation of DNA exactly as described (2). Human DNA was obtained from spleen and mouse DNA from mouse embryos. Each DNA was sonicated to a fragment size of 400-500 nucleotides. For each analysis, 1 or 2 mg of DNA was annealed in a total reaction mixture of 70 or 140,gl to 16-55 pg of cDNA (specific activity 150,000-280,000 cpm/ng) in saline/citrate that was 3 times the standard concentration and 50% formamide. Individual 12- or 20-IAl aliquots were sealed in glass capillary tubes and incubated at 520 for 10 min to 48 hr, thereby generating a Cot curve. Analysis was performed by either hydroxylapatite chromatography or SI nuclease digestion (2, 3, 14, 15). The type of human a globin cDNA (from method I, II, or III) used for analysis of DNA from the individual hybrid lines was as indicated in our previous publication (2). The four new lines reported here (157-BNPT-1, 157-BNPT-4, AIM 15a B-i, and WL II 24a 5C) were analyzed with human a globin cDNA obtained by method II. RESULTS Detection of Human , Globin Gene in Hybrid Cells by DNA*cDNA Hybridization. The species specificity of the human globin cDNA used in our studies for detection of human globin genes has been established in our previous publications (2-4, 14, 15). When DNA purified from a hybrid clone that contains human a globin gene sequences was incubated with the human 13 globin cDNA used in our studies, no significant hybridization was observed (Fig. 1). This result establishes that the human 13 globin cDNA is sufficiently free of a cDNA sequences to allow independent detection of the human 13 globin gene. Similarly, DNA from a hybrid cell line that contained human 13 globin gene sequences was annealed to the human a globin cDNA (method II); no significant hybridization occurred (Fig. 1). Thus, the human a and 13 globin cDNA probes used in our studies of globin gene chromosomal localization were specific for the human a and 13 globin genes, respectively. Significant levels of human 13 globin gene sequences were detected by hybridization assay in hybrid cell lines AHA 3D,
Proc. Nati. Acad. Sci. USA 75 (1978)
0 0
~60~50
1457
/
(3 aS
> 40-
A 30
-
20
1
2
o
3
4
1 Log Cot
2
3
a
4
FIG. 1. Specific detection of human a and flglobin genes in somatic cell hybrid lines. (A) DNA of hybrid cell line WAV-5 was annealed to mouse globin cDNA (a), human a cDNA (A) (prepared by method I as outlined in ref. 1), or human (3 cDNA (0). Analysis of hybrid formation was by batch chromatography on hydroxylapatite (3). (B) DNA from hybrid cell line AIM-iSa B-1 was annealed to the three cDNAs as outlined above. Analysis of hybrid formation in this experiment was by determination of SI nuclease resistance (2, 14). The differences in the apparent rate of annealing of the two hybrid cell DNAs to mouse cDNA (log Cotj12 in A is 2.55 and in B is 3.30) reflects the different techniques used to measure duplex formation (16).
AHA 16D, AHA 16E, J10H7, 157-BNPT-4, and AIM 15a B-i. Human 13 globin gene sequences were not found in hybrid cell lines AIM 23 OLD, AIM 23 X-1, IL II 5, IL II 54D, JFA 14a 5, JFA 14a 13-5, WAV, WAV R4D, WAV R4D A19, WAIV A, WAIV A-AA, WAIV A-DAP, and 157-BNPT-1. Only barely detectable levels of human 13 globin gene were found in WL II 24a 5C. Identification of Human Chromosomes in Each Human-Mouse Hybrid Clone. An average, of 82 metaphase spreads (range 45-167) of each cell line were characterized by two chromosomal staining techniques to identify the human chromosomes present in each cell line. The alkaline Giemsa stain (2, 8) and a combination of Giemsa-trypsin and Hoechst 33258 centromeric staining (1, 9, 10) permitted specific identification of each human chromosome as well as the identification of any interspecific chromosomal translocations. The human chromosomal content and isozyme studies of markers for human chromosomes of hybrid lines AHA 3D, AHA 16D, AHA 16E, AIM 23 OLD, AIM 23 X-1, IL II 5, IL 11 54 D, JFA 14a 5, JFA 14a 13-5, J10H7, WAV, WAV R4D, WAV R4d A19, WAIV A, WAIV A-AA, and WAIV A-DAP were reported in table 1 of our earlier study on the human a globin gene (2). The human chromosomal content as well as the content of human 13 and y globin genes of hybrid cell lines 157-BNPT-1, 157BNPT-4, AIM 15a B-i, and WL II 24a 5C are presented in Table 1 of the present report. Analysis of these latter four lines for the presence of lactate dehydrogenase A (EC 1.1.1.27), a marker that exists on the short arm of human chromosome 11 (7), is presented in Fig. 2. Assignment of Human P Globin Gene to Human Chromosome 11. The presence of a human chromosome in greater than 10% of the metaphase spreads of a hybrid cell line that is devoid of human 13 globin genes is used as grounds for exclusion of that chromosome from candidacy. The molecular hybridization assay used to detect the globin gene was sufficiently sensitive to allow this exclusion criterion to be applied (2). All human chromosomes were excluded except for human chromosomes 6, 8, 9, 11, and 13, as shown by the data presented in Table 2. Only human chromosome 11 was present at a high
Genetics: Deisseroth et al.
1458
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Chromosomal composition and fl and y globin gene content of hybrid cell clones Hi,uman chroimosome 1 2 3 4 5 6 7
8 9 10 11
12
13 14 15
Cell line AIM 15a WL II G-157 B-1 24a 5C BNPT-1 0/57 0/77 0/59 -PEP C -PEP C -PEP C -PGM-1 -PGM-1 -PGM-1 25/57 0/77 0/57 +IDH-1 -IDH-1 -IDH-1 0/57 11/77 22/59 0/57 13/77 0/59 31/57 3/77 0/59 0/57 0/77 0/59 -MOD-1 -MOD-1 23/57 0/77 0/59 +,B GLUC -f GLUC -0 GLUC 16/57 0/77 0/59 0/57 0/77 0/59 0/57 0/77 0/59 -AD K -AD K -AD K -GOT -GOT -GOT 50/57 0/77 0/59 +LDH A -LDH A -LDH A 12/57 0/77 1/59 +LDH B -LDH B -PEP B -PEP B 12/57* 0/77 0/59 -ESD -ESD -ESD 11/57 0/77 34/59 +NP -NP +NP 7/57 0/77 0/59 +MPI -MPI -MPI +HEX A
18
0/57 -APRT 40/57 +GK 25/57
19
0/57
20
-GPI 0/57
16 17
0/77
21 22
X B Glolbin gene y Glolbin gene
G-157 BNPT-4
0/64 -PEP C -PGM-1 0/64 -IDH-1 22/64 0/64 0/64 0/64 -MOD-1 0/64 -, GLUC 0/64 0/64 0/64 -AD K -GOT 52/64 +LDH A 0/64 -LDH B
0/64 -ESD 34/64 +NP
0/64 -MPI
-HEX A
-HEX A
0/59
0/64
-APRT
-APRT
-APRT
24/77
0/59
0/64
+GK 0/77
-GK
-GK
0/59
0/64
+PEP A
0/77 -GPI 0/77
-PEP A
-PEP A
0/59
0/64
-GPI
-GPI 0/64
0/59
a.:.
-ADA
-ADA
-ADA
-ADA
19/57 +SOD 0/57 0/57*
0/77
21/59*
0/64
-SOD
-SOD
0/77 0/77
0/59 17/59
0/64 51/64
+HPRT -PGK
-HPRT
+HPRT +PGK
+HPRT +PGK
+++ +++
A chromosome is scored as being present if it appears in greater than 10% of the metaphase spreads of that line which were studied. Isozyme abbreviations: PEP C, peptidase C (EC 3.4.11.1); PGM, phosphoglucomutase (EC 2.7.5.1); IDH, isocitrate dehydrogenase (EC 1.1.1.42); MOD, (NADP) malic enzyme cytoplasmic (EC 1.1.1.40); ,B GLUC, fl-glucuronidase (EC 3.2.1.31); AD K, adenosine kinase (EC 2.7.1.20); GOT, glutamate oxaloacetate transaminase (EC 2.6.1.1); LDH A, lactate dehydrogenase A (EC 1.1.1.27); LDH B, lactate dehydrogenase B (EC 1.1.1.27); PEP B, peptidase B (EC 3.4.11.1); ESD, esterase D (EC 3.1.1.1); NP, purine nucleoside phosphorylase (EC 2.4.2.1); MPI, mannosephosphate isomerase (EC 5.3.1.8); HEX A, hexosaminidase A (EC 3.2.1.30); APRT, adenine phosphoribosyltransferase (EC 2.4.2.7); GK, galactokinase (EC 2.7.1.6); PEP A, peptidase A (EC 3.4.11.1); GPI, glucosephosphate isomerase (EC 5.3.1.9); ADA, adenosine deaminase (EC 3.5.4.4); SOD, cytoplasmic
.:
i
..:
b
..: ::
c 0
co
:c
.s
.e
1 2
3
4
5
6
7
FIG. 2. Detection of laetate dehydrogenase A (EC 1.1.1.27), as known marker for human chromosome i1, in somatic cell hybrids. The electrophoretic method and staining technique are as previously published (11). Extracts from the following cells were analyzed: Channel 1, HeLa cells (human); 2, mouse fibroblasts (A9); 3 and 4, hybrid clone WL II 24a 5C; 5, hybrid clone AIM 15a, B-1; 6, hybrid clone 157-BNPT-1; and 7, hybrid clone 157-BNPT-4. a and b, Heteropolymers (A + B); c, mouse homopolymer (A4); d, heteropolymers (A); e, origin and heteropolymers; f, human homopolymers (A4).
frequency in all of the hybrid cell lines that contained human 13 globin genes (see Table 3). In fact, human chromosome 11 was the only human chromosome that was present in all of the hybrid clones positive for the human 13 globin gene. In contrast, human chromosomes 6, 8, 9, and 13 were present in less than 10% of the metaphase spreads of four of the six hybrid cell lines that contained the human 13 globin genes (Table 1). Hybrid cell clone WL II 24a SC, which contained a very low level of human ,B globin gene sequences, was negative for the human isozyme lactate dehydrogenase A (a known marker for human chromosome 11), as shown in Fig. 2. None of the metaphase spreads of this latter line contained examples of an intact human chromosome 11. Two translocations were found in this line. One, present in 58 of 77 metaphase spreads of this line examined, was interspecific and may have involved a portion of the long arm of human chromosome 11. The other involved a translocation of the long arm of human chromosome 17 to a mouse chromosome. The data obtained with the sister clones, 157-BNPT-1 and 157-BNPT-4, strongly support the assignment of the , globin gene to chromosome 11. The electrophoretograms presented in Fig. 2 show that 157-BNPT-1 and 157-BNPT-4 are negative and positive, respectively, for lactate dehydrogenase A. Both clones contain a high frequency of human chromosomes 3, 14, and X (Table 1). The results of the analysis of DNA purified from these two clones is presented in Fig. 3A. Clone 157BNPT-4, which is positive for lactate dehydrogenase A, was also strongly positive for the human # globin gene, while 157BNPT-1, which lacks lactate dehydrogenase A, was devoid of human 13 globin gene sequences. Chromosomal analysis of these two lines revealed that the major difference between them was the presence of chromosome 11 in 157-BNPT-4; it was not indophenol oxidase (EC 1.15.1.1); PGK, phosphoglycerate kinase (EC 2.7.2.3); HPRT, hypoxanthine phosphoribosyl transferase (EC 2.4.2.8). * Isozyme and chromosomal data disagree in 3 of the 65 instances in which both sets of data were given.
Proc. Natl. Acad. Sci. USA 75 (1978)
Genetics: Deisseroth et al.
1459
Table 2. Cell lines devoid of ,3 globin genes and containing the given chromosome in more than 10% of metaphase spreads
ChromoCell lines
some
.0 -
AIM 23 OLD (30/68), AIM 23X-1 (28/70) JFA 14a 5 (55/160) AIM 23 OLD (29/68), AIM 23X-1 (41/70) BNPT-1 (21/59) WAV (23/84), WAV R4D (27/107) AIM 23X-1 (8/70)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
X
40-
50-
240
30-
3020 -~ ~ 20
~ A
~~2200-
~ ~ ~10
/
10
IL II-5 (18/66), IL II-54D (14/70)
1
2
3
4
AIM 23 OLD (21/68), AIM 23X-1 (22/70)
AIM 23 OLD (30/68), AIM 23X-1 (20/70), IL II-5 (23/66), IL II-54D (15/70)
BNPT-1 (34/59) AIM 23X-1 (13/70), IL II-5 (11/66) WAV (54/84), WAIV A (56/95), WAIV A AA (32/46) IL II-5 (16/66), IL II-54D (42/70) JFA 14a 13-5 (106/167) IL II-5 (13/66), IL II-54D (7/70) WAV (9/84) AIM 23 OLD (34/68), AIM 23X-1 (10/70), WAV (47/59), WAV R4D (25/37), WAV R4D A19 (15/41), BNPT-1 (21/59) WAV (43/59), WAV R4D (24/37), WAV R4D A19 (15/41), WAIV A (27/95), WAIV A-DAP (24/50) AIM 23 OLD (29/68), AIM 23X-1 (37/70), BNPT-1 (17.59)
found in 157-BNPT-1. An interspecific translocation not involving chromosome 11 was also present in 157-BNPT-4. This analysis strongly confirms the assignment of human (3 globin gene to human chromosome 11. Assignment of Human y Globin Gene to Human Chromosome 11. Family studies as well as characterization of the abnormal hemoglobins "Lepore" and "Kenya," which arose from genetic crossover, have led to the conclusion that the human A and -y globin genes are closely linked (17, 18). As a formal test of the synteny of the human # and y globin structural genes and as a further test of our assignment of the human # globin gene to human chromosome I1, we determined the 'y globin sequence content of DNA from hybrid cells 157BNPT-1, 157-BNPT-4, and AIM 15a B-1. As shown in Fig. 3B, the hybrid cell lines that contain human chromosome 11 and the ft globin gene (157-BNPT-4 and AIM i5a B-1) contain 'y globin gene sequences, while DNA from 157-BNPT-1 (negative for both human chromosome 11 and the ( globin gene) lacked Table 3. Composition of human chromosomes present in hybrid cell lines positive for human jB globin genes Fraction of metaphase spreads containing human chromosome
Cell line AHA 3D AHA 16 D AHA 16 E J1OH7 AIM 15a B-1 157-BNPT-4
6
8
9
11
13
0/45 2/60 0/70 27/69 0/57 0/64
5/45 1/60 3/70 0/69 16/57 0/64
0/45 0/60 0/70 0/69 0/57 0/64
31/45 23/60 45/70 52/69 50/57 52/64
0/45 2/60 9/70 0/69 12/570/64
1
5 Log
2
3
4
5
Cot
FIG. 3. Detection of human ,B and y globin genes in somatic cell hybrids. Human fi (A) or 'y (B) globin cDNA was annealed to DNA from human spleen (0), hybrid cell 157-BNPT-4 (0), hybrid cell AIM 15a B-1 (A), or hybrid cell 157-BNPT-1 (A). Measurement of duplex formation was by Si nulcease resistance. In each instance 7000 cpm of [32P]cDNA was used in the analysis of 2 mg of DNA. The specific activity of ,B globin cDNA was 315.7 cpm/pg and that of 'y globin cDNA was 125.7 cpm/pg so that 7000 cpm represented 22.2 pg of , and 55.7 pg of y globin cDNA. This difference in cDNA input accounts for the different apparent rate and extent of hybridization between the two probes (16).
y globin gene sequences. We concluded that on the basis of these data both the human ( and y globin genes are on chromosome 11.
DISCUSSION An analysis of 20 human-mouse hybrid cell clones by a highly specific and sensitive DNA cDNA hybridization assay has established that the human ( and y globin genes are present on human chromosome I1. The presence of human chromosomes in hybrid cell lines devoid of human , globin genes served to exclude all human chromosomes except 6, 8, 9, 11, and 13. Among these chromosomes, only human chromosome 11 was present in the hybrid cell line positive for the human # or ly globin genes. The frequency of human chromosome 11 in the subclones 157-BNPT-1 and 157-BNPT-4 correlated well with the presence of the human , and y globin genes. On the basis of the data, we have concluded that the structural genes for the human y and (3 globin chains reside on human chromosome ii. Earlier studies by others using in situ molecular hybridization suggested that the human globin genes were on human chromosomes 2, 4, or 5 (19-22). The data that we have presented in this report and in our previous study on the localization of the human a globin gene indicate that these earlier assignments based on in situ molecular hybridization were incorrect. The theoretical reasons for the disagreement of the human globin gene assignments based on in situ hybridization with our own work have been discussed previously (2, 23). The complementary DNA used for our own studies was labeled to a high specific activity, contained a full-length copy of the globin mRNA, and was shown by us to exhibit specificity for the individual human globin genes (2; Fig. 1). Such specificity was achieved by conducting the annealing reaction at a temperature above that which allowed formation of duplexes between human cDNA and the mouse globin genes. In addition, we have presented evidence in this report that the cDNA used for detection of human a and ( globin genes was of adequate purity to specifically identify the human a and ( globin genes, respectively.
1460
Genetics: Deisseroth et al.
We believe that the method we have used for chromosomal localization of the human a and globin genes may permit a rapid accumulation of information on the map positions of a number of other differentiated cell functions in human beings and other mammalian species. Specialized gene products as well as the complementary DNA species generated from them will surely permit more extensive use of the method we have developed for the chromosomal localization of markers of differentiated cells. Furthermore, application of recombinant DNA technology will yield totally pure probes for many genes which may be used to study the gene sequence content of hybrid cells. In addition, the identification of the chromosomal localization of the human a globin gene has enabled us to develop a genetic strategy of chromosomal dependent transfer of the human a globin gene into recipient mouse cells in a state that permits continued expression of this gene (24). Such methods, when applied to the human globin gene and to other differentiated cell functions, may be of use in elucidating the mechanisms governing the expression of differentiated cell functions as well as in approaching the antenatal diagnosis of mutations of these genes which are associated with disease in human beings. A.D. recognizes the generosity extended to him by Dr. Samuel Latt of the Children's Hospital Medical Center, Boston, MA, and the use of his photomicroscope and photographic facilities during a portion of this work. Elizabeth Nichols is recognized for her superb work in the isozymal analysis of the hybrid clones studied. We thank Drs. J. and D. Beard for providing RNA-directed DNA polymerase through the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute. Sylvon Von Der Pool, Ann Cunningham, and Francis Lawyer are acknowledged for the excellence of their technical skills in the support of this project. 1. McKusick, V. & Ruddle, F. (1977) Science 196,390-405. 2. Deisseroth, A., Nienhuis, A., Turner, P., Velez, R., Anderson, F. W., Ruddle, F., Lawrence, J., Creagan, R. & Kucherlapati, R. (1977) Cell 12,205-218.
Proc. Natl. Acad. Sci. USA 75 (1978) 3. Deisseroth, A., Velez, R. & Neinhuis, A. (1976) Science 191, 1962-1964. 4. Deisseroth, A. & Nienhuis, A. (1976) In Vitro 12,734-742. 5. Giles, R..(1977) Ph.D. Dissertation, (Yale University, New Haven,
CT). 6. Faber, H. E., Kucherlapati, R., Poulik, M. D., Ruddle, F. H. & Smithies, 0. (1976) Somatic CelltGenet. 2, 141-153. 7. Boone, C., Chen, T. R. & Ruddle, F. H. (1972) Proc. Nati. Acad. Sci. USA 69,510-514. 8. Bobrow, M., Madan, K. & Pearson, P. L. (1972) Nature New Biol. 238, 122-124. 9. Friend, K. K., Chen, S. & Ruddle, F. H. (1976) Somatic Cell Genet. 2, 183-188. 10. Yoshida, M. C., Ikeuchi, T. & Sadaki, M. (1975) Proc. Jpn. Acad. 51, 184-187. 11. Kozak, C. A., Lawrence, J. B. & Ruddle, F. H. (1977) Exp. Cell Res. 105, 109-117. 12. Nichols, E. A. & Ruddle, F. H. (1973) J. Histochem. Cytochem. 21, 1066-1081. 13. Neinhuis, A. W., Turner, P. & Benz, E. J., Jr. (1977) Proc. Natl. Acad. Sci. USA 74,3960-3964. 14. Benz, E. J., Geist, C. E., Steggles, A. W., Barker, J. E. & Nienhuis, A. W. (1977) J. Biol. Chem. 252, 1098-1916. 15. Deisseroth, A., Velez, R., Burke, R., Minna, J., Anderson, F. W. & Neinhuis, A. (1976) Somatic Cell Genet. 2,373-384. 16. Britten, R. J. & Davidson, E. H. (1976) Proc. Natl. Acad. Sci. USA 73,415-419. 17. Smith, D. H., Clegg, J. B., Weatherall, D. J. & Gilles, H. M. (1973) Nature New Biol. 246, 184-188. 18. Barnabas, J. & Muller, C. J. (1962) Nature 194,931-933. 19. Price, P. M. & Hirschhorn, K. (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 2227-2243. 20. Price, P. M. & Hirschhorn, K. (1975) Cytogenet. Cell Genet. 14, 255-231. 21. Price, P. M., Conover, J. H. & Hirschhorn, K. (1972) Nature 237,
340342. 22. Cheung, S. W., Tishler, P. V., Atkins, L., Sengupta, S. K., Modest, E. J. & Forget, B. (1977) Cell Biol. Int. Rep. 1, 255-262. 23. Bishop, J. 0. & Jones, K. W. (1972) Nature 240, 149-150. 24. Deisseroth, A. & Hendrick, D. (1977) Blood 50, Suppl. 1, p. 105.
