J. Cell Sci. 25, 39-57 (1977) 30,

Printed in Great Britain

GENE TRANSFER BY MEANS OF CELL FUSION

II. THE MAPPING OF 8 LOCI ON HUMANCHROMOSOME 1.BY STATISTICAL ANALYSIS OFGENE ASSORTMENT IN SOMATIC CELL HYBRIDS

S. J. GOSS AND H. HARRISThe Sir William Dunn School of Pathology, University of Oxford,South Parks Road, Oxford OXi 3RE, England

SUMMARY

A method is described which should permit determination of the order and spacing of geneson all human chromosomes by the analysis of just one set of man-mouse hybrid cells. Thismethod is used to determine the map of 8 loci on human chromosome 1. A comparison of thestatistical maps of chromosome 1 and of the X-chromosome with the cytogenetic maps of thesechromosomes at metaphase indicates that the statistically derived distances between genes arerelated to the amount of Giemsa light-band material between the genes.

INTRODUCTION

In the preceding paper (Goss & Harris, 1977), we described how the gene map of ahuman chromosome may be derived from a consideration of the frequency withwhich pairs of syntenic genes are co-transferred by cell fusion from an irradiatedhuman cell to a rodent cell. In this paper we extend the statistical approach bymapping 8 loci on human chromosome 1. Since chromosome 1 is the largest humanchromosome, we shall be studying some of the most widely spaced syntenic humanloci. It is advantageous not to be restricted in the first instance to measuring thedistances of all loci from one selectable locus, because long distances are difficult tomeasure accurately. We have therefore adopted a method of analysis, which, unlikethat used in the preceding paper, does not depend upon a selectable locus.

Hybrid cells are made by fusing irradiated diploid human fibroblasts with cells ofthe mouse cell line, PG-19. Unlike man-hamster hybrids, man-mouse hybrids fre-quently retain human chromosomes even in the absence of any selective pressure forsuch retention. Several human chromosomes are retained in each hybrid clone, and itis found that this retention is approximately random. Genes on different human chro-mosomes should therefore assort more or less at random in the hybrids, whereas genesentering the hybrid on the same chromosome, or on the same chromosome fragment,clearly may not assort at random. By studying the randomness of assortment of pairsof different loci, it is possible to estimate the frequency with which those pairs of lociare co-transferred. A gene map may then be derived by analysing these co-transferfrequencies according to the principles described in the preceding paper.

We chose to map chromosome 1 because we could then study a large number of

40 S. J. Goss and H. Harris

loci, many of which had already been mapped by cytogenetic methods. Some of theseloci are known to be very closely linked, for example both PGM1 and UMPK havebeen assigned to band 1P32 (Burgerhout & Jongsma, 1976), while others were knownto be at the extreme tips of the chromosome arms. This situation provides a severetest for our analysis. Two of the loci studied have not yet been given any preciseregional assignment by cytogenetic studies.

The meaning of the map distances obtained by the statistical approach was investi-gated in two ways. First we measured the distances between X-linked genes in theMRC-5 human fibroblasts and compared these distances with those previously ob-tained with human lymphocytes. In this way we could examine the dependence ofmap distances on the state of condensation of the chromatin. Second, we comparedthe statistical gene map with a cytogenetic map of the metaphase chromosome. Themetaphase chromosome is, of course, differentiated along its length by the presence ofchromosome bands and a centromere, and it was reasonable to suppose that this dif-ferentiation might cause some distortion of a statistical map based on chromosomerearrangement.

MATERIALS AND METHODS

Cell culture

The mouse cell, PG-19 is a clonal derivative of a cell line derived by explanting a spontaneousmelanoma of the C57 Black mouse. PG-19 ' s resistant to 1 -5 fig/ml 6-thioguanine and exhibits agreatly reduced activity of hypoxanthine phosphoribosyl transferase (HPRT). It is killed byHAT medium (Littlefield, 1964). Details of PG-19 are given in Jonasson, Povey & Harris (1977).

The human diploid fibroblast, MRC-5, is described by Jacobs, Jones & Bailie (1970). Thecells used in the present work were all between their 17th and 25th passages from the originalexplant. Giemsa-banded chromosome spreads of MRC-5, prepared in this laboratory by DrV. G. H. Riddle, showed the presence of a pair of normal chromosomes 1 in each cell. The sizeof the variable heterochromatic region on iq was apparently equal in the two homologues.

Both cell lines were maintained as monolayer cultures in Eagle's minimal essential mediumfor PG-19 a nd Dulbecco's modification of that medium for MRC-5. A.11 media were supple-mented with 10% (v/v) foetal calf serum.

Irradiation of the MRC-5

The cells were irradiated as described in the preceding paper (Goss & Harris, 1977).

Cell fusion

3 x io6 mouse cells were fused to 1 x io6 MRC-5 cells by the method of Harris & Watkins(1965). 2000 haemagglutinating units of ultraviolet-irradiated Sendai virus were used. Beforefusion, the MRC-5 cells were left in confluent culture for 3 days without medium change. Itcould then be shown by autoradiographic studies with tritiated thymidine that over 95 % of thecells were blocked, reversibly, in the G1 phase of the cell cycle. Each human cell that subse-quently fuses with a mouse cell then contributes one diploid human genome to the heterokaryon.The fused cells were plated out, and the hybrid clones were isolated in HAT medium, asdescribed in the preceding paper.

Identification of human enzymes in the hybrids

Human enzymes were detected in the hybrids by subjecting cell lysates to Cellogel electro-phoresis. The basic procedure for electrophoresis and the method of preparing the lysates, are

Statistical mapping of human chromosome 41

given by Meera Khan (1971). The X-linked enzymes, glucose-6-phosphate dehydrogenase(G6PD), 3-phosphoglycerate kinase (PGK) and HPRT, were analysed as described in the pre-ceding paper. Table 1 lists the autosomally specified enzymes that were analysed, and Tables 2and 3 describe the electrophoretic conditions for each.

Table 1. Human autosomal enzymes analysed in the hybrid cells

Enzyme (trivial name) Abbreviation E.C. no.

a-L-fucosidaseAdenylate kinase iAdenylate kinase 2Fumarate hydrataseLactate dehydrogenase APeptidase CPhosphoglucomutase iPhosphopyruvate hydratase I

(enolase)Uridine-glucose pyrophosphory-

laseUridine monophosphate kinase

* McKusick (1975).f McKusick, Klinger,

aFUCA K j

AK2

FHLDH-APEPCPGMiPPHj

UGPPUMPK

Bootsma

—

2 - 7 - 4 - 32 - 7 - 4 - 34 . 2 . 1 . 21.1.1.27

3 - 4 - -2 .7 .5 .14 . 2 . 1 . 1 1

2.7.7.92.7.4.4

& Ruddle (1976).

Genecatalogue

no.*

2300010300

10302

13685150001700017190

17243

19185

I7I93

Chromo-some

assign-mentf

1

91

1

1 1

1

1

1

1

1

Table 2. Buffers used for the electrophoresis of autosomal enzymes

Enzyme Buffer

aFUCAKX & AK2FH, LDH-A

PEPC

UGPP

UMPK

5 mM citric acid, adjusted to pH 6-o with NaOHBuffer XI, of van Someren et al. (1974a)citric acid, 6 mM; NaH2PO4, 8-6 mM; adjusted to pH 7-0 withNaOH

Buffer X, of van Someren et al. (1974a)Buffer XI, of van Someren et al. (1974a)Sodium phosphate buffer, 5 mM in phosphate, +0-2 mg/1.MgCl2.6H2O. pH 7 - 5

Na4P2O7. ioH2O, 2.23 g/1., adjusted to pH 70 with ortho-phosphoric acid

Sodium phosphate buffer, 20 mM in phosphate. pH 70

Development of the gels

After electrophoresis, the gels were developed in an appropriate reaction mixture (MeeraKhan, 1971) to determine the extent of migration of the enzyme being examined. The reactionmixtures for PEPC, PGM, PPH and UGPP are described by van Someren et al. (1974a). Thatfor LDH-A is described by Meera Khan (1971), and that for UMPK by Giblett et al. (1974).The reaction mixture for FH was that of Tolley & Craig (1975), with the addition of 5 mMsodium oxamate to inhibit LDH activity. AK was developed in the same reaction mixture asUMPK, except that disodium AMP (2-5 mg/ml) was substituted for UMP, and a higherconcentration of magnesium chloride (40 mM) was used. The formula and use of the reactionmixture for aFUC are given by Turner, Beratis, Turner & Hirschhorn (1974).

42 S. J. Goss and H. Harris

Table 3. Running conditions for electrophoresis

Enzyme

aFUCAK! & AK,FHLDH-APEPCPGMj

PPHiUGPPUMPK

All electrophoresis 'thickness of Whatmar

Gap width(across whichthe voltage isapplied), cm

88

1 2

1 2

88888

was performed

Constantvoltage

(power-packoutput), V

4 0 02 0 0

2 0 0

2 5 04 0 0

47535°2 0 0

1 0 0

in a Shandon1 No. 1 grade filter paper.

Initial current(through a gel8 cm wide),

mA

4'S2-O

4'O

5°3-0

4-53-53-0

2-5

U77 tank at 4 °

Duration ofrun, h

1

2

2-5I

I

2

i -52

3

C. The wicks

Site oforigin:

distancefrom thecathodal

bridge, cm

37441

1

41

1

were a double

Electrophoretic patterns

The appearance of the stained gels is in most cases adequately described elsewhere (vanSomeren et al. 1974a; Meera Khan, 1971; Tolley & Craig, 1975; van Someren, Beijersbergenvan Henegouwen, Westerveld & Bootsma, 1974 ft). The appearance of'gels developed for AK,PGM1( UMPK, PEPC and aFUC, is illustrated in Fig. 1.

AKX and AK2 (Fig. IA)

Our identification of the human AKj and AK3 bands is based on a comparison of our gelswith the starch gels of Van Cong et al. (1974). We checked the susceptibility of AKi tooxidation by omitting the /?-mercaptoethanol from the electrophoresis buffer (Khoo & Russell,1972). The AKX band was then very much reduced in intensity, but the AK2 band was un-affected.

PGM1 (Fig. IB)

MRC-5 is heterozygous for PGM]. It was possible to identify the bands of enzyme activity onour gels by comparing them with the gels of Billardon et al. (1973), and with those of Spencer,Hopkinson & Harris (1964). Prolonged development of the gels revealed the faster movingisozymes PGM2 and PGM3 in a pattern resembling that observed by Hopkinson & Harris(1968). The PGM! alleles present in MRC-5 a r e most probably the common alleles, PGM12

and PGM,1. Each allele may give rise to two bands, the faster moving band probably being dueto a phosphorylated form of the enzyme (Spencer et al. 1964). The faster band specified by thePGM!2 allele is weak in the MRC-5 ar>d hybrid cell extracts illustrated)in Fig. 1 B. The fasterband of PGM11 co-electrophoreses in this system with the main band of mouse PGM. This isillustrated by the electrophoretic pattern of an extract of HeLa cells which are homozygousPGM11. The faster band of PGMX

2 migrates slightly further than the main band of mousePGM, and is just visible in hybrids 5 and 6 of Fig. 1B. This electrophoretic system resolvesPGMi2 satisfactorily from mouse PGM, and is thus a useful improvement on the starch geltechnique, which, in the past, has led to some ambiguities (Van Cong et al. 1974; Billardon etal. 1973).

Statistical mapping of human chromosome i 43

PG19 1

AK,

2

AK2

AK,

3

AK2

MRC-5

AK2

AK,

PG19 4PGM,

1

5PGM,2-1

6PGM,

2

MRC-5

PGM,2-1

HeLa

PGMi1

PG19 7 MRC-5

UMPK UMPK

PG19 8

PEPC

MRC-5

PEPC

PG19

Fig. i. Electrophoretograms of the enzymes (A) AK, (B) PGMb (c) UMPK, (D) PEPCand (E) IXFUC. The origin is indicated by the arrow on the right of each photograph.(Gels c and E show artifacts at the origin.) Each gel shows a mouse standard (PG19)and a human standard (MRC-5, HeLa). Hybrid cell extracts were run in the num-bered channels. The human enzymes detected in each cell type are indicated.

UMPK {Fig. it)The method for separating human from rodent UMPK was kindly communicated to us by

W. G. Burgerhout. The human enzyme when present in man-mouse hybrids generally givesrise to a much fainter band than the mouse enzyme.

PEPC (Fig. ID)

The peptidase illustrated in Fig. 1 D showed no activity with the substrates valyl-leucine,leucyl-glycyl-glycine and leucyl-proline. The gel was developed with lysyl-leucine as substrate.

44 S. J. Goss and H. Harris

This pattern of substrate specificity defines PEPC (Lewis & Harris, 1967). The most anodalhuman peptidase, a very faint band, is probably PEPE. Both human and mouse PEPC havegiven rise to a double band on the gel shown in Fig. 1 D. The relative intensity of the bands ineach doublet varied with the age of the cell lysate. This probably reflects different redox statesof the enzyme (Lewis & Harris, 1967).

aFUC (Fig. IE)

aFUC is a glycoprotein and occurs in many different forms which can only be completelyresolved by isoelectric focussing. Even after prolonged treatment with neuraminidase, thisenzyme still exhibits three or more electrophoretically different species (Turner, Beratis,Turner & Hirschorn, 1975; Turner et al. 1976). We found that electrophoresis of cell lysatesat pH 6-o enabled usto distinguish two types of man-mouse hybrid cell. All hybrid cells showedaFUC activity that migrated to the cathode, like the enzyme in PG-19; but some hybrids showedadditional activity that migrated to the anode. Since MRC-5 has an aFUC that migrates to theanode, any hybrid showing enzyme activity migrating to the anode was assumed to be expressingthe human form of the enzyme. Our subsequent analysis showed that this trait could be mappedto a single locus on chromosome 1; so it appears that this electrophoretic system does providea resolution adequate for our purposes. Fig. 1 E shows a hybrid expressing the human enzyme(channel 9) and a hybrid lacking the enzyme (channel 10).

Presentation of data

Space does not permit a full presentation of all our 'raw' data. We have instead presented theresults of our calculations on those data. A complete copy of the results of the enzyme assays isavailable on application to S.J.G.

RESULTS

The retention of human chromosomes in PG19 x MRC-5 hybrids

The level of HPRT in PG-19 is so low that the cells cannot survive in HATmedium. It follows that clones isolated in HAT from a fusion between PG-19 andMRC-5 must be either hybrid cells, containing human HPRT, or revertants of PG-19to the wild type HPRT phenotype. Most of the clones isolated in HAT were found toexpress several human genes and were therefore clearly hybrid cells; but a smallnumber expressed none of the 12 human enzymes for which we regularly tested. Thehybrid origin of these clones was confirmed by checking that their HPRT co-electro-phoresed with HPRT from MRC-5 cells. No revertant of PG-19 w a s found in theseexperiments, in agreement with the general finding in this laboratory that PG-19 hasa remarkably stable phenotype in this respect. We therefore assume that all hybridsretain the human gene for HPRT, and we would then expect that the hybrids wouldalso retain those X-linked markers that are co-transferred with HPRT. In addition,it is found that unselected human chromosomes are frequently retained in the hybrids.It is these that constitute the main object of discussion in this paper. This unselectedretention of human genes is illustrated in Table 4, which lists the frequencies withwhich various autosomally specified human enzymes were detected in the hybrids.There are clearly enough clones expressing any given human enzyme for us to beable conveniently to study the randomness of assortment of the genes specifying theseenzymes.

Statistical mapping of human chromosome I 45

Mathematical models for use in estimating the co-transfer of loci, from the degreeof randomness of gene assortment in the hybrids

We shall define the frequency of co-transfer of two loci in exactly the same way as inthe preceding paper. We can then derive a gene map from a set of co-transfer frequen-cies, using the principles found to be applicable to the X-linked loci. The co-transferfrequency, Po, for two syntenic loci, is the frequency with which the linkage betweenthose two loci is not disrupted by segregational events. This definition refers to aunique pair of loci on a single chromosome. In the case of a pair of autosomal loci, thefrequency with which the loci remain linked on both homologues is simply P0

2.Since PG-19 x MRC-5 hybrids frequently retain unselected human chromosomal

material, two human loci may often be simultaneously retained in a hybrid althoughthose loci are not linked. The co-expression of two genes is thus not directly a measureof the co-transfer of those genes to the hybrid. To derive the relationship betweengene retention in hybrids and gene co-transfer, we shall assume that the retention ofhuman chromosome fragments is such that the retention of any one fragment isindependent of the retention of all other fragments. The validity of this assumptionis tested below. To simplify the derivation, we shall further assume that all fragmentshave a similar chance of retention, regardless of their informational content. Thissimplification is suggested in Table 4, which shows that different genes are retainedwith very similar frequencies. Thus, when considering a given pair of loci, we shallwork with the mean retention frequency for that pair of loci. It is possible by the useof more complex models to avoid this assumption, but the modification that thisproduces in the final results is trivial, so that the extra sophistication is not necessary.

The case for the unique chromosome

We shall consider, in the first case, the pattern of retention of a pair of loci, A andB, normally situated on a unique chromosome. At fusion, A and B are co-transferredto a fraction, Po, of the hybrids. The remaining fraction of the hybrids, (1 — Po),receive A on one chromosome fragment, and B on another. The frequency with whichany chromosome fragment is lost from the hybrids will be denoted by /. and thefrequency of retention of any given fragment will then be (1 — /). The value of I isestimated directly from the results, by the relation:

/ = o-5(frequency of clones not expressing A + frequency of those not expressing

B).i.e. / = o-5(freq. A~ + freq. B~).

The frequency of clones showing discordant expression of A and B, that is thefrequency of clones expressing only one of the genes A or B, is then given by:

freq. A+B- + freq. A-B+ = 2./(1 - / ) (1 -Po).

If A and B were not syntenic, then Po = o, and the frequency of clones showingdiscordant expression of A and B becomes:

freq. discordant clones = 2.l.(i—l).4 C E L 25

46 S. J. Goss and H. Harris

It follows thatfreq. discordant clones observed

0 freq. discordant clones expected if A and B are not linked'

freq. A+B- +freq. A-B+ie- P ° = I ~ a / : ( l / ) •

The case of two homologous chromosomes

We assume that the two homologous chromosomes behave identically, but inde-pendently, in the hybrids. The co-transfer frequency of A and B is then the same forboth chromosomes, and the frequency with which the genes derived from eitherchromosome are retained in the hybrids is the same.

If /A denotes the chance that a specified locus of the homologous pair of loci, A, islost from the hybrids, it follows that the frequency, LA, of clones having lost bothhomologues, is /A

2. Likewise, the frequency, LB, of clones having lost both loci, is/2iS. It is convenient to estimate /, of equation (1), as follows:

1 = jr.,where L = 0-5 (LA + LB).

There will be a fraction of clones, X, that express neither A nor B. These clonesmust have lost the loci A and the loci B derived from both chromosomes. The proba-bility of losing from the hybrids both the locus A and the locus B derived from onespecified chromosome of the homologous pair must then be ^IX. The frequency withwhich the locus A from one of the homologues is lost from a hybrid that retains locusB from the same homologue is then:

where the subscripts ' 1' indicate that A and B are derived from the same chromosomeof the homologous pair. Likewise,

freq. A / B f = lB-JX.But,

freq. A ^ B ^ = freq. A / B j -

so we may conveniently write:

freq. A ^ B ^ + freq. Ax+Br = 2 (l-JX) = 2 (yJL-JX).This is the frequency of discordant segregation of the loci A and B derived from a

single chromosome of the homologous pair and may thus be introduced into equation(1) in order to evaluate the co-transfer frequency, Po, of A and B.

This is our estimate of the co-transfer of autosomal genes. Both equations (1) and

Statistical mapping of human chromosome i 47

(2) are based on the assumption that each hybrid initially contains a single humangenome. Some hybrids may have received two human genomes at fusion. For thisset of hybrids, equation (2) becomes:

Po =

For simplicity, we have assumed that the majority of hybrids are derived from singlehuman cells, and thus that equation (2) gives an adequate approximation to Po.Gene co-transfer frequencies calculated using equation (2) are found to give anacceptable map of chromosome 1.

Estimation of the co-transfer of X-linked genes

It is of interest to study the co-transfer of X-linked genes in these clones. Becauseunselected genes are retained in the present clones with a frequency (1 — /), thefrequency of co-expression of an X-linked gene and HPRT will be an overestimate ofthe co-transfer of that gene with HPRT. The appropriate correction is:

/

where F is the frequency of co-expression of HPRT and the X-linked gene, I isestimated as ^L', where L' is the mean of the frequencies, L, of clones lacking speci-fied autosomally determined enzymes.

Tests of the randomness of assortment of asyntenic genes

It is possible to test that unlinked genes assort at random in the hybrids by checkingthat the Po values for known asyntenic genes are not significantly different from zero.Table 5 shows a test for the independent segregation of a pair of homologous chromo-somes. As MRC-5 is heterozygous for PGA^ (see Materials and methods), it followsthat the two PGMj alleles are segregating in the hybrids. Po values have been cal-culated for the co-transfer of the two alleles, PGM1

1 and PGMX2. It is clear that these

values do not differ significantly from zero. The retention in the hybrids of eitherallele is therefore independent of the retention of its homologue.

Whilst we cannot detect any 'spurious linkage' between two different asyntenicalleles, we do find some spurious linkage between homologous pairs of asyntenicautosomal loci. It is, of course, more likely in the latter case that a small degree ofinterdependence in the retention of unlinked fragments will have a significant effecton the value of Po; since there are two homologous copies of each autosomal genethere is a greater opportunity for the occurrence of interdependent segregation. Table6 presents the frequencies with which each of 8 loci on chromosome 1 is spuriouslylinked either with AKX (a locus on chromosome 9) or with LDH-A (a locus on chromo-some 11). In subsequent analysis of the co-transfer of syntenic loci, we shall have totake account of this background of spurious linkage for autosomal loci.

4-2

48 S. jf. Goss and H. Harris

The gene order of human chromosome i

Table 7 presents the co-transfer frequencies for loci on chromosome 1 in hybridsbetween PG-19 and MRC-5 cells exposed to 10 J kg"1 y-rays before fusion. Most ofthese values are well in excess of the mean value for spurious linkage, i.e. 27-6 ± 2%.The order of the genes along the chromosome can be derived on the assumption thatthe most closely situated genes have the highest co-transfer frequencies. Inspection ofTable 7 reveals that the most likely gene order is:

FH-PEPC-UGPP-PGMi-UMPK-AK^aFUC-PPH!

This is consistent with the gene order derived by cytogenetic mapping (Hamerton,1976). Burgerhout, Leupe-de-Smit & Jongsma (1977) have recently revised the orderof PEPC and FH, and now place PEPC between FH and UGPP. The present experi-ment confirms this revision. A detailed comparison of the cytogenetic and statisticalmaps is shown in Fig. 2 (pp. 50-51).

Table 8 presents the co-transfer frequencies for the same loci on chromosome 1measured in hybrids produced with MRC-5 c e u s giy e n 20 J kg""1 of radiation. Theco-transfer frequencies are reduced at this higher radiation dose, but this reduction issmaller than one might expect on the assumption that the logarithm of the co-transferfrequencies is proportional to (dose)1'7, the relationship found to be applicable to theco-transfer of X-linked loci (see preceding paper). This is an indication that spontan-eous breakage is occurring in these clones, a problem that is considered further below.

The data shown in Table 8 are generally consistent with the gene order derivedfrom Table 9. There are some anomalies in the co-transfer frequencies for the mostwidely separated markers, but this is of little significance because at 20 J kg"1 the Po

values are all very low (approximately 30%), and are therefore liable to be relativelyinaccurate. For this reason, only the data from the 10 J kg"1 fusions are subjected tothe full analysis used to derive the distances between loci.

The co-transfer of X-linked loci

By the application of equation (3), we have calculated the co-transfer frequencies ofPGK and HPRT, and of G6PD and HPRT, at 10 and 20 J kg"1. The results aresummarized in Table 9 and compared with the analogous co-transfer frequenciesdetermined from gene retention studies in hybrids between hamster cells and humanlymphocytes (see preceding paper).

It is apparent from Table 9 that the co-transfer frequencies are lower in the PG-19 x MRC-5 hybrids, than in the Wg3-h x lymphocyte hybrids. However, the incre-ment in log Po, on raising the dose of y-rays, is very similar in both sets of hybrids.This indicates that the y-ray sensitivity of the X-chromosome is very similar in thetwo hybrid cell systems. The target for the segregation of two loci is thus not signifi-cantly modified by the different degrees of chromatin condensation in the fibroblastand the lymphocyte nuclei. The lower co-transfer frequency in the man-mousesystem must then be ascribed to spontaneous chromosome breakage. A small set ofhybrids was made between PG-19 and unirradiated MRC-5 cells. In the absence ofspontaneous breakage, all these hybrids should retain human PGK and G6PD.

Statistical mapping of human chromosome I 49

However, out of 14 clones, only 8 retained PGK, and only 10 retained G6PD,confirming the occurrence of a significant level of spontaneous chromosome breakagein PG-19 x MRC-5 hybrids.

The ratio of the target G6PD/HPRT to the target PGK/HPRT, estimated by themethod described in the preceding paper is o-6i + 0-13 in the man-mouse clones. Thisresult is not significantly different from this ratio, 0-55 +0-08, derived from the man-hamster clones. Since a large part of the chromosome rearrangement in the man-mouseclones is spontaneous, we can conclude that the distribution on the chromosome ofspontaneous rearrangements is similar to that produced by radiation, a conclusion sup-ported by the cytogenetic observations of Aula & von Koskall (1976). Thus, althoughthe chromosome rearrangements in the man-mouse hybrids are produced in a some-what different way from those in the man-hamster hybrids, we can still justifiablycompare the relative map distances obtained from the two systems. The general applic-ability of equation (2) to the man-mouse hybrids (see Tables 7, 8) indicates that mostof the spontaneous rearrangement must occur at an early stage of the growth of thehybrids, before much loss of human material has occurred.

The relative map distances between 8 loci on chromosome 1, and between 4 X-linkedloci: a comparison of the statistical and cytogenetic maps

We have calculated the map distances between the loci on chromosome 1 from theco-transfer frequencies of these loci from cells given 10 J kg"1 of radiation. Thecalculations involved the application of the model developed in the preceding paper inwhich segregational events are described in terms of the exchange hypothesis ofchromosome aberration. The co-transfer frequencies were first adjusted to take ac-count of spurious linkage by applying the equation:

where c = 0-276 (the mean frequency of spurious linkage for asyntenic loci). Tofacilitate the comparison of map distances on chromosome 1 with those measured inthe preceding paper on the X-chromosome, all map distances are expressed as fractionsof the map distance PGK-HPRT. The results of these transformations are tabulatedin Table 10. Fig. 2 shows a scale drawing of the map distances presented in Table 10.The purpose of these transformations is to convert the table of co-transfer frequenciesinto a diagram which can be understood at a glance as a gene map. (All the informationin the final diagram is also in the original table of co-transfer frequencies, so that thevalidity of the conclusions drawn from the final diagram is not dependent on the pre-cise suitability of the transformations used to derive the pictorial presentation shownin Fig. 2.)

Fig. 2 also shows diagrams of chromosomes 1 and X at metaphase. Regional geneassignments based on cytogenetic studies are marked on them. These diagrams weresimply traced from the standard karyotype presented at the Paris Conference (Hamer-ton, Jacobs & Klinger, 1972). It was found that if the Giemsa dark bands were omittedfrom the metaphase diagrams, then the cytogenetic and statistical maps came into

AK,

FH PEPC

c4-4 4-2 3-2

UGPP

2-5 2-3 2-1 1-3 2-2

a i

3-2 3-4

PPH,

3-6

FH

PEPC

UGPP

FH PEPC UGPP

FH

FH

FH

UGPP

PEPC

PEPC

PEPC

UGPP

UGPP

UGPP

UGPP

PEPC

FH

FH

PGM

PGM UMPK

UMPK AK

PGM, UMPK ^ 'PGM AK

UMPK

AK

PGM

UMPK

AK

PGM

UMPK

AK

PPH

PPH

PPH

PPH

aFUC

aFUC

PPH

aFUC

PPH

aFUCI

PPH

a

55

Fig. 2A. For legend see facing page

Statistical mapping of human chromosome i

B PGK

G6PD HPRT aGAL

( 2 8

G6PD

G6PD

2-6

HPRT

HPRT

2-4 2-2

Xq

aGAL

1•3N_ 1

PGK

PGK

PGK

G6PD aGAL

Fig. 2. The map distances given in Table 10 are drawn out to scale. The statisticalgene maps (A) of chromosome 1 and (B) of the X-chromosome are compared withcytogenetic maps of the metaphase chromosomes. Only the Giemsa light-bands of themetaphase chromosomes are included in the diagram.

register both for chromosome 1 and for the X-chromosome. A considerable discrep-ancy is produced if the dark bands are included, because the long arm of chromosome1, iq, is then considerably expanded, but the upper part of the short arm, ip, whichis relatively free of dark band material, is only slightly lengthened. As the statisticalmap is based on the occurrence of chromosome rearrangements, this result stronglyimplies that chromosome rearrangement occurs predominantly in the Giemsa-lightbands. This is in excellent agreement with the cytogenetic studies of Holmberg &Jonasson (1973), and of Seabright (1973).

A final point of interest is the apparent lack of any significant influence of thecentromere on the statistical gene map. It will be seen from Fig. 2 that distancesmeasured across the centromere of chromosome 1 are perhaps slightly shortenedrelative to the cytogenetic map, but this effect is small. One might have expected theaction of the centromere to distort the statistical gene map, because one might presumethat a centric chromosome fragment would more often be stably maintained in ahybrid cell than an acentric fragment. However, the absence of any net effect of thecentromere on gene segregation in these hybrids was in fact indicated from the outsetof these experiments by the lack of any significant difference between the retentionfrequencies of centromere-proximal and distal loci (see Table 4).

DISCUSSION

By analysing the deviations from randomness in the assortment of human genes inhybrids between PG-19 and MRC-5 cells, it is possible to derive a map of chromosome1 showing the order of the genes and the approximate distances between them. Twoloci that have been assigned to other autosomes were included in the present studymainly as controls to detect spurious linkage; but it is clear that these asyntenic lociare retained in the hybrids with much the same frequency as the loci on chromosome1. We could therefore probably have mapped any human chromosome with the presentset of hybrids. This means that with our approach to gene mapping, one set of hybrid

52 S. J. Goss and H. Harris

clones should suffice to map the entire human chromosome complement. The genemap presented in Fig. 2 was based on data collected from only 100 clones. The power ofthe statistical approach could be further increased by the development of a systemof multipoint mapping similar to those used in classical genetical studies (Meyers et al.

1975)-Our study of chromosome 1 has confirmed the gene order derived by cytogenetic

studies for the loci FH, PEPC, UGPP, PGM1( UMPK and PPH^ The locus AK2 hasbeen assigned to ip, but there is some disagreement over whether AK2 is proximal ordistal to PGM! (Grzeschik, 1975; Hamerton, 1976). Our results confirm those ofGrzeschik in placing AK2 distal to PCM^ and we would further localize AK2 to asite between UMPK and PPHX. The gene aFUC has been assigned to chromosome 1by Turner et al. (1976). The present work confirms this assignment and indicatesfurther that the gene aFUC is located between AK2 and PPH.

We have estimated the distances between the genes on two different human chromo-somes and, in the case of the X-linked genes, have made our measurements on hybridsproduced from both lymphocytes and fibroblasts. The sensitivity of the X-chromosometo y-rays, and the relative distances between the X-linked genes, were not detectablymodified by the different degrees of chromatin condensation in the two cell types. Acomparison of the cytogenetic maps of the two metaphase chromosomes with thestatistical gene maps indicated that a map distance measured by the statistical approachis probably a measurement of the amount of Giemsa-light band material between thegenes.

REFERENCES

AULA, P. & VON KOSKALL, H. (1976). Distribution of spontaneous chromosome breaks in humanchromosomes. Humangenetik 32, 143-148.

BILLARDON, C , VAN CONG, N., PICARD, J.-Y., L E BORGNE DE KAOUEL, C , REBOUCET, R.,

WEIL, D., FEINGOLD, J. & FREZAL, J. (1973). Linkage studies of enzyme markers in man-mouse somatic cell hybrids. Ann. hum. Genet. 36, 273-284.

BURCERHOUT, W. G. & JONGSMA, A. P. M. (1976). The regional map of chromosome i of man.Human Gene Mapping 3. Birth Defects: Original article series, XII, no. 7, 101-104.

BURCERHOUT, W., LEUPE-DE-SMIT, S. & JONGSMA, A. (1977). Regional assignment of sevengenes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. II .Results obtained after induction of breaks in chromosome 1 by X-irradiation. Cytogenet.Cell Genet, (in Press).

GIBLETT, E. R., ANDERSON, J. E., CHEN, S.-H., TENG, Y.-S. & COHEN, F. (1974). Uridinemonophosphate kinase: a new genetic polymorphism with possible clinical implications.Am. J. hum. Genet. 26, 627-635.

Goss, S. J. & HARRIS, H. (1977). Gene transfer by means of cell fusion. I. Statistical mapping ofthe human X-chromosome by analysis of radiation-induced gene segregation. J. Cell Sci. 25,17-37-

GRZESCHIK, K.-H. (1975). Regional mapping of human chromosomes 1 and 11. Human GeneMapping 2. Birth Defects: Original article series, XI, no. 3, 172-175.

HAMERTON, J. L. (1976). Report of the committee on the genetic constitution of chromosomes1 and 2. Human Gene Mapping 3. Birth Defects: Original article series, XII, no. 7, 7-23.

HAMERTON, J. L., JACOBS, P. A. & KLINGER, H. P. (1972). Standardization in human cytogenet-ics (Paris Conference 1971). Birth Defects: Original article series, VIII, no. 7.

HARRIS, H. & WATKINS, J. F. (1965). Hybrid cells derived from mouse and man: artificialheterokaryons of mammalian cells derived from different species. Nature, Lond. 205, 640-646.

Statistical mapping of human chromosome i 53

HOLMBERG, M. & JONASSON, J. (1973). Preferential location of X-ray induced chromosomebreakage in the R-bands of human chromosomes. Hereditas 74, 57-68.

HOPKINSON, D. A. & HARRIS, H. (1968). A third phosphoglucomutase locus in man. Ann. hum.Genet. 31, 359-367.

JACOBS, J. P., JONES, C. M. & BAILLE, J. P. (1970). Characteristics of a human diploid celldesignated MRC-5. Nature, Lond. 227, 168-170.

JONASSON, J., POVEY, S. & HARRIS, H. (1977). The analysis of malignancy by cell fusion. VII.Cytogenetic analysis of hybrids between malignant and diploid cells and of tumours derivedfrom them. J. Cell Sci. 24, 217-254.

KHOO, J. C. & RUSSELL, P. J. (1972). Isozymes of adenylate kinase in human tissue. Bioclum.biophys. Acta 268, 98-101.

LEWIS, W. H. P. & HARRIS, H. (1967). Human red cell peptidases. Nature, Lond. 215, 351-355.LITTLEFIELD, J. W. (1964). Selection of hybrids from matings of fibroblasts in vitro and their

presumed recombinants. Science, N.Y. 145, 709-710MCKUSICK, V. A. (1975). Mendelian Inheritance in Man, 4th edn. Baltimore: Johns Hopkins

University Press.MCKUSICK, V. A., KLINGER, H. P., BOOTSMA, D. & RUDDLE, F. H. (1976). Human Gene Map-

ping 3. Birth Defects: Original article series, XII, no. 7.MEERA KHAN, P. (1971). Enzyme electrophoresis on cellulose acetate gel: zymogram patterns

in man-mouse and man-Chinese hamster somatic cell hybrids. Arc/is Biochem. Biophys. 145,470-483.

MEYERS, D. A., CONNEALLY, P. M., HECHT, F., LOVRIEN, E. W., MAGENSIS, E., MERRIT A. D.,

PALMER, C. G., RIVAS, M. L. & WANG, L. (1975). Linkage group 1: multipoint mapping.Human Gene Mapping 2. Birth Defects: Original article series, XI, no. 3, 211-219.

SEABRIGHT, M. (1973). High resolution studies on the pattern of induced exchanges in thehuman karyotype. Chromosoma 40, 333-346.

SPENCER, N., HOPKINSON, D. A. & HARRIS, H. (1964). Phosphoglucomutase polymorphism inman. Nature, Lond. 204, 742-745.

TOLLEY, E. & CRAIG, I. (1975). Presence of two forms of fumarase in mammalian cells: immu-nological characterisation and genetic analysis in somatic cell hybrids. Confirmation of theassignment of a gene necessary for the enzyme expression to human chromosome 1. Biochem.Genet. 13, 867-883.

TURNER, B. M., BERATIS, N. G., TURNER, V. S. & HIRSCHHORN, K. (1974). Isozymes of humana-L-fucosidase detectable by starch gel electrophoresis. Clin. chim. Acta 57, 29-35.

TURNER, B. M., BERATIS, N. G., TURNER, V. S. & HIRSCHHORN, K. (1975). Silent allele asgenetic basis of fucosidosis. Nature, Lond. 257, 391-392.

TURNER, V. S., TURNER, B. M., KUCHERLAPATI, R., RUDDLE, F. H. & HIRSCHHORN, K. (1976).

Assignment of the human a-L-fucosidase gene locus to chromosome 1 by use of a 'clonepanel'. Human Gene Mapping 3. Birth Defects: Original article series, XII, no. 7, 238-240.

VAN CONG, N. G., REBOURCET, R., WEIL, D., COUILLIN, P., HORS, M . - C , JAMI, J. & FREZAL,

J. (1974). Assignment of the second locus of adenylate kinase to chromosome ip : preliminarydata. Cytogenet. Cell Genet. 13, 173-178.

VAN SOMEREN, H., BEIJERSBERGEN VAN HENEGOUWEN, H., LOS, W., WURZER-FIGURELLI, E.,

DOPPERT, B., VERVLOET, M. & MEERA KHAN, P. (1974a). Enzyme electrophoresis oncellulose acetate gel II . Zymogram patterns in man-Chinese hamster somatic cell hybrids.Humangenetik 25, 189-201.

VAN SOMEREN, H., BEIJERSBERGEN VAN HENEGOUWEN, H., WESTERVELD, A. & BOOTSMA, D.

(19746). Synteny of the human loci for fumarate hydratase and UDPG pyrophosphorylasewith chromosome 1 markers in somatic cell hybrids. Cytogenet. Cell Genet. 13, 551-557.

[Received 29 November 1976)

54 S. jf. Goss and H. Harris

Note added in proof

Corney et al. have recently demonstrated by family studies that aFUC is closelylinked to the Rhesus blood group locus. Since the Rhesus locus is known to lie be-tween UMPK and PPHX (Hamerton, 1976), the results from family studies confirmthe regional assignment of aFUC given in the present paper. The location of AK2 distalto PGMj, originally reported by Grzeschik(io.75) and supported by the present work,has also been deduced by Bruns & Gerald (1976).

BRUNS, G. A. P. & GERALD, P. S. (1976). Expression of the human adenylate kinase isozymes,phosphopyruvate hydratase, and phosphoglucomutase-i in man-rodent somatic cell hybrids.Biochem. Genet. 14, 1-17.

CORNEY, G., FISHER, R. A., COOK, P. J. L., NOADES, J. & ROBSON, E. B. (1977). Linkage be-tween a-fucosidase and the Rhesus blood group. Ann. Hum. Genet, (in Press).

We thank Dr F. H. C. Marriot for advice on the statistical analysis of our resultsand Mrs Jean Kerr-Harnott for technical assistance. The work was supported by theCancer Research Campaign.

Table 4. The presence of human autosomal enzymes in PG-19 x MRC-5 hybrids

Enzyme

AK2

FHaFUCLDH-APEPCPGMj

PPH1UGPPUMPK

Percentage of hybrids containing thespecified human enzyme:

A

(i)in 64 hybrids made afterexposure of the MRC-5cells to 10 J kg""1 y-rays

55675673756372

756970

(ii)in 58 hybrids made afterexposure of the MRC-5cells to 20 J kg"1 y-rays

52

555959645962626060

N.B. A small number of clones showed the presence of trace amounts of human enzyme afterprolonged development of the gels. This indicates that the hybrids are in some cases stillsegregating human material at the time of assay. Such hybrids were scored as lacking theenzyme. Most hybrids gave either clear positive or negative results in the electrophoretic enzymetests.

Statistical mapping of human chromosome 55

Table 5. To show that the alleles of human PGM± may segregateindependently of one another

Dose of y-raysadministered to

MRC-5 cellsbefore fusionwith PG-19cells, J kg"1

1 0

1 0

2 0

2 0

Fusion no.*

iiiiiiIV

Co-transfer

p 0/ro> /o

1 5 6— 1

2 0

I O

frequencyallelesf

of the

I S.E. P o

1 2 3

1 6 513

17

No. of clonesstudied

JV

64365834

* The fusions analysed in this paper were done in two batches separated by an interval of2 months. Because of the difficulty in reproducing the cloning conditions exactly, the retentionfrequency of unselected human genes was different on the two occasions. Each batch of cloneswas therefore analysed separately. For a specified pair of loci, the values, Po, obtained from dif-ferent batches at any one dose, do not differ significantly. In subsequent tables a mean value,Po, is therefore given for each dose.

f Po is obtained by equation (1) because the alleles of PGM! are unique genes. If the allelessegregate independently, Po = o (see text). It can be shown that the variance of Po, calculatedfrom eq. (i), is:

Jf QZpy t*(l " *> -** (* -*)* + o-5 Q + x-zl*) **],

where JV = no. of clones studied, as = frequency of clones having lost both alleles, and z =

Table 6. The apparent co-transfer ('spurious linkage') of asyntenicautosomal loci

Dose of y-raysadministered to

MRC-5 cells, J kg-1

Apparent co-transfer frequency, Po %, of locus A with each of thenamed loci (figures in brackets = i S.E. Po.

Locus A PGM, AK2 UMPK aFUC UGPP FH PEPC

10

10

20

20

AK, 33 (10)LDH-A 25 (12)AK, - 5 ( 1 3 )LDH-A 13 (13)

35346

3°

(11)

(11)

(13)(11)

241818

24

(11)

(12)

(12)

(11)

2533

- 715

(11)(11)

(14)(13)

192 2

IO

28

(II )(12)(12)(II)

3329

62 1

(10)(11)(13)(12)

4 1

191 0

35

(9)(11)(13)(11)

432 3

2723

(9)(11)

(11)

(11)

The values, Po, were calculated for each batch of fusions by equation (2). Each value Poshown in the figure is the weighted mean of the Po values for two batches. (A weight of theinverse of the variance of Po was applied.) It can be shown that the variance of Po> calcula-ted by eq. (2) is:

-Trt IT T,A^X)4N (y/ll — l,)'

Where N = no. of clones studied, and Z =

56 S. J. Goss and H. Harris

Table 7. The co-transfer frequencies of loci on human chromosome 1. TheMRC-5 cells were exposed to 10 J kg-1 y-rays before fusion

PPHi AK2 UMPK aFUC UGPP FH

PPHjAKa

UMPKaFUCUGPPFHPEPC

6170

8567695°51

(8)(7)(6)(8)(8)(9)(9)

636480

513444

So"

(9)(6)(10)

(10)

(10)

8870

484146

(5)(8)(9)(10)

(10)

67594647

(8)(9)(10)

(10)

4842

37

(9)(10)

(10)

60(8)75(7) 75(6)

The entry at each intersection of a row and a column is the co-transfer frequency, Po %, forthe pair of loci specified in the row and column. The figure in brackets is 1 S.E. of Po.

Table 8. The co-transfer frequencies, Po%, of loci on human chromosome 1(the M-RC-5 cells were exposed to 20 J kg*1 y-rays before fusion)

PPHiAK2UMPKaFUCUGPPFHPEPC

P(

51647662

353446

5 M j

(9)(8)(7)(8)(11)(11)(10)

PPH!

53(9)55(9)65(8)43 (10)39 (10)38(n)

See

AK2

58(9)72(7)38 (10)27(11)41 (10)

explanation

UMPK

69(7)33(n)3 3 d i )35 (11)

of Table 7.

aFUC

3V (11)18 (12)

25 (11)

UGPP

30 (11)51 (10)

FH

55(9)

Table 9. Comparison of the co-transfer of X-linked loci in PG-19 x irradiatedMRC-5 hybrids, with that in Wgy-h x irradiated lymphocyte hybrids

Co-transfer, P c %, at — log Po at each doseeach dose , A >

1 * . (a) (b) SlogP0

Hybrid Linkage 10 J kg"1 20 1kg-1 10 J kg-1 20 J kg-1 (b — a)

PG-19 x PGK-HPRT 0-49 034 0-31 047 016MRC-5 G6PD-HPRT 061 054 021 026 005

\Vg3-hx PGK-HPRT 082 054 009 027 018Lym G6PD-HPRT 088 075 006 012 006

The values Po are calculated in PG-19 x MRC-5 hybrids by equation 3 (see text), and in thecase of Wg3-h x lymphocyte hybrids they are taken from the preceding paper.

Statistical mapping of human chromosome l 57

Table 10. Relative map distances between loci on chromosome i andbetween loci on the X-chromosome

PPHLAK2

UMPKaFUCUGPPFHPEPC

aGALHPRTG6PD

PGM,

i i S0 5 80-18°'750642-172 0 8

PGK0 5 21

1-38

PPHj

I-O2

0 9 4O-282-08

4772-88

aGAL

o-47o-8i

AK2

0 1 4

0-582-4

3'32 6 2

HPRT

0-37

UMPK

0 7 5i-342-622 5 1

G6PD

aFUC

2-4

3 1 34-01

UGPP

1 2 30 4 0

FH

0 4 0

The distances between the loci on chromosome 1 are calculated from the values, Po, given inTable 7. A correction for spurious linkage is made as described in the text. The distances, d,are computed to satisfy the equation:

T = dx'-di'(i-o-$e-dl").

This equation was derived in the preceding paper, which also gives the method for its solu-tion (Goss & Harris, 1977).

If T is lnP0(oori.), the appropriate values of x' and i' are:

x' = 1 '409, i' = 1-127.

The value d for PGK-HPRT is found to be i-8, derived from the value Po, for this pair ofloci, 049 (see Table 9). These values of x', i' and d are in the same ratios as the values of x, i and{poK-HiMtT) which were found to be most suitable for relating d to T in the preceding paper.

To facilitate the comparison of map distances, all distances are expressed relative to thedistance PGK-HPRT. The map distances between X-linked loci are those determined in thepreceding paper, also expressed relative to the distance PGK-HPRT.