Chromosome assignment of a human gene for argininosuccinate synthetase expression in Chinese hamster X human somatic cell hybrids

Printed in Sweden Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in my form reserved ISSN 00144827

Experimental Cell Research 106 (1977) 71-78

CHROMOSOME ASSIGNMENT OF A HUMAN GENE FOR ARGININOSUCCINATE SYNTHETASE EXPRESSION IN CHINESE HAMSTERxHUMAN SOMATIC CELL HYBRIDS B. CARRITT,

P. S. G. GOLDFARB, M. 1,. HOOPER and C. SLACK

Cancer Research Campaign

Somatic Cell Genetics Group, Institutes Glasgow GII UR, Scotland

of Genetics and Virology,

SUMMARY The Chinese hamster cell line Don lacks detectable activity of the enzyme argininosuccinate synthetase (ASS). Analysis of somatic cell hybrids formed between Don and a human fibroblast which has ASS activity suggested that ASS expression is a dominant trait which segregates out of those clones which have lost the relevant human gene(s). Forty independent primary hybrid clones were analysed with respect to the correlation of ASS with human chromosomes and linkage markers. This analysis suggested a linkage between a human ASS gene and adenylate kinase-1 on chromosome 9, and this relationship was confirmed in thirty subclones derived from four of the primary hybrid clones.

Excess amino nitrogen in mammals is converted to urea via a metabolic pathway in which the amino acids ornithine, citrulline and arginine serve as intermediates. Although the complete Krebs-Henseleit urea cycle is found only in mammalian liver, the two-step synthesis of arginine from citrulline occurs in a variety of somatic tissues [ 1, 21. Thus the arginine requirement for growth of many cultured cells, both primary and established, can be met by citrulline but not by ornithine [3,4,5-j. The synthesis of arginine from citrulline proceeds through argininosuccinic acid; argininosuccinate synthetase (ASS; EC6.3.4.5; r.-citrulline : L-aspartate ligase) condenses aspartate with citrulline yielding argininosuccinate, which is then cleaved to arginine and fumarate by argininosuccinate lyase (ASL; EC4.3.2.1; r,-argininosuccinate

arginine-lyase) . Syndromes arising from inherited deficiencies in either of these enzymes are known [6]. In one case, a patient diagnosed as citrullinemic, an ASS structural gene defect is suggested by the m-esence of an enzyme with an altered K, far citrulline [7]. This defect, together with the resulting arginine auxotrophy , is demonstrable in cells cultured from skin biopsy of the affected individual, whereas normal. skin fibroblasts are amongst those cells able to effect the conversion of citrulline to arginine [7]. The unidirectional loss of chromosomes from some interspecific somatic cell hybrids has made available a means of establishing gene-gene and gene-chromosome linkages, especially in man. Experiments of this type are based on the proposition that the non-random assortment of pairs or groups

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of human phenotypes from a large number of independent human-mouse or humanhamster hybrid clones indicates that they are specified by genes on the same chromosome (i.e. are syntenic) [8]. In the same way, a correlation between the presence of a human phenotype and a particular human chromosome in such hybrids can lead to the assignment of human genes to chromosomes. Making use of a Chinese hamster cell line which cannot convert citrulline to argininosuccinate and a diploid human fibroblast line which can, we find that the expression of ASS in somatic cell hybrids formed between them behaves as a segregating, dominant genetic trait. These hybrids were used to establish a syntenic relationship between human adenylate kinase-1 and ASS, and to confirm the expectation [9, IO] that this linkage group can be assigned to chromosome 9. MATERIALS

AND METHODS

Materials All chemicals were from Sigma with the following exceptions: Colcemid was from Ciba, one batch of aminopterin from Nutritional Biochemicals, 4-methylumbellifervl-2-acetimido-2-deoxv-B-~-alucouvranoside from Koch-Light. Enzymes were p&chased from Boehringer-Mannheim. exceot for nvntvic kinase which came from Sigma. r&arbamoyl-14C]citrulline (53-62 mCi/mmole) was obtained from the Radiochemical Centre, kmersham. Sendai virus (UV inactivated) was purchased from Searle Diagnostics Ltd., High Wycombe, Ham’s F12 medium (xl0 concentrate) from GlBCo-Biocult and plastic tissue culture ware from A/S Nunc, Coming & Linbro. Cellogel (0.5 mm thickness) was obtained through Reeve-Angel Scientific Ltd.

Cell culture The Don cell line, an established pseudodiploid Chinese hamster lung fibroblast, was obtained from the American Type Culture Collection and maintained in Glasgow-modified Eagle’s medium+ 10% fetal calf serum. a23, a TK- derivative of Don isolated after selection in BUdR [ 1I] was very generously given to us by Dr A. Westerveld (Rotterdam) and maintained in F12+10% fetal calf serum. GM29, a HGPRT- human ,%,'2CdRes 106 (1977)

diploid fibroblast cultured from a skin biopsy of a Lesch-Nvhan natient. was purchased from the Mammalian Genetic Mu&t Cell Respository, Camden, N.J.. and grown in Glasgow-modified Eagle’s medium f 10% fetal calf serum &pplemented with non-essential amino acids, nucleosides and sodium pvruvate as __ described before [ 121. All hybrid lines were maintained in Ham’s F12 +10% fetal calf serum and containing hypoxdnthine (10m4M). aminonterin (8 x lo-‘M) and TdR (2x 10.5M) (HAT).’ ‘Mycopiasma ‘contamination seriously interferes with meaningful analyses of arginine metabolism in cultured cells [13], and so all cells were routinely screened for mycoplasma contamination by the method of Fogh & Fogh [14]. Cultures so contaminated were discarded.

Cell fusion All fusions were between a23 (Chinese hamster TK -) and GM29 (human HGPRT-) and were performed either in suspension (GMA series) or in monolayer (PF. FIV and Sx series). Essentiallv the orocedures of Giles & Ruddle [15] were followed, u&g 500-l 000 HAU UV-inactivated Sendai virus in 1 ml Hanks’ balanced salts solution lacking glucose, and a parental input ratio of 1: 1. The 415 series of hybrids were obtained using Pontecorvo’s own modification (ners. comm.) of l& polyethylene glycol method [16]. A-confluent mixed (1: 1) monolayer of a23 and GM29 were treated with a 1 : 1 (w/v) solution of polyethylene alvcol 6000 (PEG) in F12. which was immediatelv rembved. This’was replaced by a 1: 3 (w/v) PEG solution which was again immediately removed, and the procedure repeated with 1 : 7 and 1 : 15 (w/v) solutions of PEG. The monolayer was washed twice with F12 + 10% serum and incubated in this medium for 2 h at 37°C. The monolayer was then trypsinised and plated out at about 3 x lo3 cells/cm” in plastic flasks. Colonies were isolated after 2-4 weeks in F12+HAT; each primary clone considered in this study originated from a separate flask set up immediately following fusion, and is thus assumed to be the result of a unique fusion event. HAT-resistant colonies were grown directly to a population size of 5-10X IO7cells in roller culture and cell pellets, washed thrice with 50 ml phosphate-buffered isotonic saline, prepared from the same bottle for ASS assay and isozyme analysis. Parallel cultures were taken at an equivalent cumulative population doubling for the preparation of metaphase spreads and frozen stocks.

Isozyme analysis Hybrids were analysed for the Presence of the following human enzymes after electrophoresis on Cellogel: phosphoglucomutase 1 (PGM-1); isocitrate dehydrogenase, NADPdependcnt, cytoplasmic form (IDH-I); hexosaminidase A and B (Hex-A and -B); superoxide dismutase dimeric and tetrameric forms (SOD-l and 2); glutathione reductase (GR); adenylate kinase, red cell form (AK-l); glutamic-oxaloacetic transaminase, cytoplasmic form (GOT-l); lactate dehydrogenase A and B (LDH-A and B); purine nu-

Chromosome assignment ofASS Table 1. Argininosuccinate synthetase (ASS) uctivity in Chinese hamster liver, humun and Chinese hamster cell lines ASS activity (unitslmg protein) Chinese hamster liver” Human fibroblast (CO~)~ yGL%; human fibroblast Don TK- Don (a23)

18.4k1.2 (5) 16.7k1.3 (3) 15.8+0.5 (8) co. 1 (2) CO.1 (6)

The table shows enzyme specific activity -IS.E.M.; figures in parentheses give the number of separate determinations. a Chinese hamster livers were perfused with phosphate-buffered isotonic saline, excised and homogenised in 0.05 M Tris-HCl (pH 8.3). The supematant after centrifugation at 90000 g for 1 h served as a source of enzyme. a Co1 isolated in this laboratory from a skin biopsy of a normal female donor.

cleoside phosphorylase (NP); pyruvic kinase3 (PK-3); peptidase A (PEP-A); glucose phosphate isomerase (GPI); adenosine deaminase (ADA); glucose&phosphate dehydrogenase (G6PD). Conditions of electrophoresis and“ staining were either those described by Meera Khan and co-workers [17, 181 or, for GOT-l, NP, PK-3, PEP-A, GPI and ADA in some early experiments, were adapted for Cellogel from Nichols & Ruddle [19].

Karyotype analysis Metaphase spreads were prepared from logarithmic cultures using 0.04 pglml colcemid for 45 min as dcscribed before [12]. Trypsin-Giemsa banding was performed either as described [12] or using 0.05% w/v trypsin (Difco) in phosphate-buffered isotonic saline at pH 7 for 2-3 min at room temperature. For the determination of the human chromosome complement in a hybrid clone, a minimum of twentyfive banded metaphases were examined, and a particular chromosome considered to be present when detected in 35 % or more of cells.

Argininosuccinate assay

synthetase

ASS was assayed using a modification of Schimke’s method I201. 1-5X 1Or cells were son&ted briefly in 200-300 -pl ‘0.05 M Tris-HCl (pH 8.3) and the homogenate clarified by low-speed centrifugation. 0.2-l mg protein as 100 ~1 extract-were added to a reaction mixture at 0°C containing, in 0.4 ml, aspartic acid 1 pmole, ATP 1.2 &mole, MgC& 1.2 pmolc, KC1 10 ymole, phospho(enol)pyruvate 5 kmole, pyruvic kinase 40 pg, r.~carbamoyl-14C]citrulline 0.5 pmole (2.5 &i), Tris-

43

HCl (pH 8.3) 50 pmole. A 100 ~1 aliquot was removed to 50 ~1 ice-cold 30% trichloroacetic acid (TCA) and the reaction mixture incubated at 37°C. At 5, IO and 20 min 100 ~1 aliquots were removed to 50 ~1 30% TCA, and, after centrifugation, the supernatants were mixed thoroughly with 5 vol diethyl ether. Forty pal samples of the aqueous phase were mixed with 10 ~1 of arginine, argininosuccinate and citrulline solution (10 mg/ml each) and applied to silica gel thin layers (Merck). The plates were subjected to ascending chromatography overnight in 75 % aqueous phenol @DH) containing 0.2 r&ml sodium cyanide @DA). Ninhydrin-positive areas corresponding to argininosuccinic acid, arginine and citrulline were scraped into toluene-based scintillation fluid and counted. Counts in argininosuccinic acid at 0 min were subtracted from those at other times; the rate of formation of argininosuccinic acid was usually constant for at least 10 min. The further metabolism of argininosuccinic acid to arginine was negligible. Enzyme activity was calculated for the linear portion of the time course, and one unit of ASS activity defined as that which converts one nmole of citrulline to argininosuccinate per hour at 37°C. RESULTS

ASS nctivity in Don and human fibroblasts The Chinese hamster cell line Don has a growth requirement for arginine which can be satisifed by argininosuceinate but not by citrulline (our unpublished results). This suggests a specific deficiency in ASS, the first of the two enzymes catalysing the conversion of citrulline to arginine, Attempts to detect ASS in Don are shown in table 1. It appears that, whereas Chinese hamster liver and human fibroblasts from two sources had comparable levels of ASS activity, Don and its TK- derivative a23 bad no detectable activity. Results not shown here suggest that Don has a level of ASL, the second step enzyme, activity similar to that found in HeLa [20]. Attempts to raise ASS activity in Don by maintaining cells for 72 h in medium containing 0.02 M citrulhne in place of arginine [2Q] were unsuccessful. ASS activity in human-Chinese hamster hybrids ASS activities were determined in a total of forty independently isolated primary hybrid Exp Ceil Kr.s IN6 (!977)

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Table 2. ASS activities in human-Chinese hamster hybrid primary clones Clone GMA All Al5 A21 A25 A41 A.51 PF 2A5 2B2 4Al 4A2 4Bl 4B3 Twenty-eight others of GMA, PF, FIV, 415 and SX series

ASS activity (U/mg protein) 7.8 8.0 8.5 8.0 ii:: 8.0 17.0 6.4 5.0 16.0 4.0
ASS activities are the average of at least two separate determinations.

reflect a defect in the structural gene for ASS or an inactive structural gene capable of re-activation by the introduction of a human positive control element. ASS activities in the primary clones were rarely equal to that of the human parent (16 Ulmg) and were commonly one half of this or less (table 2). This is probably due, at least in part, to clonal heterogeneity, a conclusion suggested both by inspection of karyotypes and by the fact that some subclones of the low activity ASS’ primary clones had ASS activities greater than those of their parent clones (see later). Whether there are, in addition, gene dosage effects superimposed on this heterogeneity is not clear from our data.

clones obtained from five separate fusions Linkage relations of ASS in between the Don TK- line a23 and a human Primary clones HGPRT- diploid fibroblast. Table 2 shows Initial attempts to correlate the ASS phenoASS activities in the twelve primary hybrid type with a particular human chromosome clones which had specific activities of 4 were made by isozyme and karyotype U/mg or greater; these clones are con- analyses of the twelve ASS primary clones sidered to have the ASS’ phenotype. The shown in table 2 and thirteen ASS- primary remaining twenty-eight primary hybrid clones from the GMA and PF series of hyclones had ASS specific activities of 0.5 U/ brids. Table 3 shows the results of these mg or less, and were considered to be analyses. The presence of human chromoASS-. somes 3, 4, 7, 13, 16, 17 and 22 was tested Since the loss of human chromosomes for by inspection of Giemsa-banded metafrom human-Chinese hamster hybrids is phase spreads alone. The presence of the well established [ll], these data suggest remainder of the human complement was that the expression of ASS in these hybrids initially inferred from the presence of the is a genetically dominant trait which segre- human linkage markers shown in table 3, gates out of those clones which have lost and subsequently confirmed in the majority the relevant human gene(s). We cannot at of clones by karyotype inspection. The present, however, assert that the segre- presence of human chromosome 17 in all gating human gene is a structural gene for hybrids is suggested since this chromosome ASS, since we have been unable to dis- carries the human TK gene [21] which is tinguish human from Chinese hamster ASS, required for survival in HAT medium. An and are unable, therefore, to identify the intact chromosome 17 was observed in species type of ASS in the ASS+ hybrids. metaphases of twenty-three of the twentyMoreover, the basis of the ASS phenotype five clones considered here. of Don is not known, and could equally Correlations statistically significant at the Exp Cell Res 106 (1977)

Chromosome

Table 3. Correlation primary

of ASS phenotype

with human chromosomes

assignment ofASS

75

and linkage markers in

hybrid clones ASS/marker or chromosome (no. of clones)

Chromosome

Marker PGM-1 IDH-l/MDH HexB SOD-B

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

GR AK-1 GOT LDH-A LDH-B NP PK-3/HexA PEPA GPI ADA SOD-A G6PD

+I+ 7 5 2 3 6 10 7 11 9 9 5 3 9 10 I 11 11 6 8 4 9 8 4

+I-

-I+

-I10

7 7 ; 6 2 5 1 5 : 2 ; 4

12 3 6 8 4

1 7 4 6 12 6 3 8 6 4 10

: 5 9 10 5 13 11 8 9

x2 0.96 1.10 0.02 0.85 0.64 0.03 4.89a 0.03 14.89b 6.17” 1.85 0.07 0.07 3.38” 0.21 0.01 2.49 0.32 0.11 0.03 1.99 0.10 0.25

A total of 25 independent primary clones were used in this experiment; clones for which isozyme data were ambiguous or in which chromosome identification was uncertain are not included. Chromosome assignments for the markers were taken from Ruddle & Creagen [8] and were confirmed by karyotype analysis in samples comprising at least 50% of clones. x2 was calculated using Yates’s correction for correlation in a low-frequency 2x2 contingency table with 1 degree of freedom. a 0.01
5 % level are seen for ASS with the markers AK-1 (chromosome 9), GOT-l (chromosome lo), PK-3/Hex-A (chromosome 15) and with chromosome 7, although the correlation with AK-l is clearly the strongest. The single ASS clone in which AK-1 was absent (clone PF4Al) had no recognisable human chromosomes and many bizarre rearrangement products. Such rearrangement might be expected to dissociate many genuine genetic linkages. In many of our preparations, there was some ambiguity over the identification of human chromosome 13. Although we cannot therefore finally ex-

elude this chromosome as a candidate for involvement in ASS expression, the available data do not support the idea. A further fifteen independent primary Don-human hybrid clones were then analysed with respect to the AK-I/ASS linkage and for the presence of a limited number of human chromosomes and markers. All fifteen clones were ASS-; none had human AK-l, or chromosomes 9 or 7. Two clones had human PK-3, one had GOT-l 1 4 had GR, 2 had LDH-A and B, four had GGPD and one had PGM-1. The data on these additional primary clones therefore argue

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Table 4. Correlation of ASS phenotype with AK-I and chromosome 9 in subclones of four ASS+ hybrid clones ASS/AK-l

(no. of subclones)

Primary clone

+I+

+I-

-I+

-I-

PF 2A5 PF 2B2 PF 4A2 PF 4Bl Total

8 5 1 5 19

0 0 0 0 0

0 0 0 1 1

3 0 6 1 10

Subclones were also tested for the presence of human chromosome 9 by karyotype inspection. All subclones with ASS and AK-l had chromosome 9 in greater than 35% of cells examined, whereas in those with neither ASS nor AK-l chromosome 9 was absent. Karyotypes were not available for the single discordant clone, which had low AK-l activity and no ASS (see text).

tivity was detected, and yet a weak band of AK-1 was observed. Since reconstruction experiments revealed that the assay for AK-l was almost three times more sensitive than that for ASS, this difference in sensitivity could explain the discrepancy. These data therefore strongly support the assignment of a human gene responsible for ASS expression in Don-human hybrids to chromosome 9.

DISCUSSION The demonstration that, in a large number of independent human-rodent hybrid clones, two or more human phenotypes are invariably lost or retained together provides strong evidence for their being coded for by genes on the same chromosome. Modern against the involvement of chromosomes 10 techniques of chromosome identification and 15 in ASS expression and support a make it possible to assign these genes to inchromosome 9/AK-l/ASS linkage. How- dividual human chromosomes in the same ever, the correlation of ASS with chromo- way; a rapidly expanding list of gene-gene some 7 was not weakened by the additional and gene-chromosome linkages in man is clones. available as a result of such studies [8]. In experiments using human-mouse and ASSIAK-I linkage in subclones human-Chinese hamster somatic cell hyFour ASS+ primary clones from the PF brids [9, lo], adenylate kinase-1 has reseries of a23-human hybrids were recloned cently been assigned to human chromoand thirty subclones tested for ASS ac- some 9. Our results suggest a linkage of a tivity. The proportion of ASS subclones human ASS gene to AK-1 and confirm the recovered from a primary clone was assignment of this linkage group to chroroughly proportional to the ASS specific ac- mosome 9. AK-3 and cis-aconitase have tivity in that primary clone, suggesting that also been assigned to chromosome 9 [lo], that activity is a reflection of the number of although the data for the latter assignment ASS cells in the original heterogeneous are inconsistent with those of Westerveld primary clone. Furthermore, ASS activities et al. [9]. Since family studies have indiin the ASS subclones were usually higher cated a linkage between the ABO blood than that of their parent clone. group, Nail-Patella syndrome and AK-l, All subclones were tested for the pres- these may also be assigned to chromosome ence of human chromosome 7 and 9 and for 9 [91. human AK-I. Only chromosome 9 and We have not been able to identify the AK-I segregated concordantly with ASS in species type of ASS in our hybrids and thus the subclones (table 4) with a single excep- we cannot confirm that it is the human tion. In this subclone (PF4B1.6) no ASS ac- structural gene for ASS that we have asExp Cell Res 106 (1977)

Chromosome assignment ofASS signed. Neither do we know why Don cells fail to express Chinese hamster ASS. It might be, for example, that we are here seeing the segregation of a human positive control element on chromosome 9 which is of sufficiently broad specificity to activate an inactive ASS structural gene in Don. If this were the case, it must be assumed that the random presence of an asyntenic human structural gene for ASS would merely impose quantitative variations in the level of total activity in hybrids possessing chromosome 9. Although our results do not exclude an interpretation of this sort, a requirement for the co-presence of two asyntenic human genes for the manifestation of a single phenotype in human-rodent hybrids has been demonstrated only in the case of human interferon [22]. Since the loss of human chromosomes continues at a slow but finite rate throughout the life of a human-Chinese hamster hybrid [ll], heterogeneity with respect to human phenotypes is to be expected in primary hybrid clones. The fact that ASS subclones frequently had ASS activities exceeding that of their parent clone suggests that such heterogeneity in primary clones can fully account for all cases where ASS activities in primary clones were less than that of the human fibroblast parent. However, it is conceivable that there are, in addition gene dosage effects operating both in homogeneous and heterogeneous clones. If, for example, ASS activities equal to that of the human parent (16 U/mg) represent the activity of two copies of the human ASS gene in a homogeneous primary clone, then ASS activities less than this may reflect clonal heterogeneity both with respect to the presence or absence of this gene and to the number of copies present. Our data are insufficient to allow a rigorous test of this concept.

‘74

In this study we have encountered three situations well known as hazards in linkage analyses of this type, namely the difference in sensitivity of assay for two linked markers, extensive chromosome rearrangement and the non-random retention of human chromosomes in primary clones. The first two situations, although not sufficiently serious in our experiments to invalidate our main conclusion, can I the apparent or real disruption of 1 groups. The use of pre-existing chromosome rearrangement in human tissue biopsy [23] or its induction by X-rays [24] have been exploited as ways of deter order and map distance within age groups. In order to test for the rdndomness of human chromosome loss from our hybrids, we have constructed the full matrix of correlations for every chromosome with every other in twenty-five primary clones, and find statistically significant correlations at the 5 % level for eight pairs-of chromosomes (not shown). We believe that this accounts for the correlation between ASS and chromosome 7, 10 and 15 found in our primary clones. We also find that chromosomes 11, 14,115and 18 tend to be present in a clone when any of the chromosomes I-10 are present, a finding for human-Chinese hamster hybrids also suggested by the data of Douglas et al. [25]. In preliminary experiments not shown here, we have found it possible to distinguish between ASS” and ASS- hybrid clones on the basis of their ability to grow in medium containing citrulline in place of arginine . After passage of ASS+ primary clones in such medium, the spread about the mode for total numbers of chromosomes decreases, and the frequency of occurrence of chromosome 9 in the population increases. It thus appears that, for this particular parental combination at least, a

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Carritt et al.

positive selection for human chromosome 9 in hybrids now exists. We wish to thank Pat Hoover, Ronnie McKail and Rosemary Morgan for skilful technical assistance, and Professors J. A. Pateman and J. H. Subak-Sharpe for encouragement. This work was supported by a grant from the Cancer Research Campaign to Professors Pateman and SubakSharpe.

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Exp Cell Res 106 (1977)

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