Complementation analysis of xeroderma pigmentosum variants

Experimental

Cell Research 136 (1981) 81-90

COMPLEMENTATION

ANALYSIS

PIGMENTOSUM N. G. J. JASPERS,‘,’ ‘Depur~meni

VARIANTS

G. JANSEN-VAN

of Cell Biology and Genetics, and ‘Medical Biologicul Laboratory

OF XERODERMA

Emsmus TNO.

DE KUILEN’

and D. BOOTSMA’

University Rotierdmm, 3000 DR Rotterdam. 2280 HV Rijswijk, Tl7e Netherlands

SUMMARY DNA repair after UV exposure was studied in multinucleate cells, obtained after fusion of excision-defective and variant xeroderma pigmentosum fibroblasts. Optimal fusion conditions were determined, facilitating the measurement of DNA replication in heterokaryons. In unirradiated multikaryons, entry into the S phase was depressed, when compared with unfused cells. The extent of the depression of S phase entry was dependent on the fusion conditions. In heterokaryons obtained after fusion of XP variant (6 different strains) with excision-defective XP (three cell strains from complementation groups A, C and D) both unscheduled DNA synthesis and postreplication repair after UV irradiation were restored to normal levels. In contrast, complementation was not observed after pairwise fusion of the XP variant cell strains. These results suggest that the XP variants comprise a single complementation group, different from complementation groups A, C and D.

suffering from the inherited disorder xeroderma pigmentosum (XP) can be subdivided into two categories, based on the DNA repair characteristics of their cultured fibroblasts [l, 21. Cells from patients of the first category all have a reduced level of UV-induced unscheduled DNA synthesis (UDS) [3] and a reduced capacity to remove pyrimidine dimers from DNA [4]. By somatic cell hybridization, seven different complementation groups have been identified so far, indicating extensive genetic heterogeneity [5-91. Cells from the second category of XP patients, the so-called XP variants, show normal levels of DNA repair synthesis [lo] and normal capacities to remove thymine dimers [4, 1 I]. However, these cells have a defect in DNA replication after UV irradiation Patients

[l II. In normal human cells, like in most mam-

malian cells, DNA synthesized shortly after moderate doses of UV light has a molecular weight (MW) that is smaller than that in unirradiated cells [ll, 121. Upon further incubation these newly synthesized DNA segments are assembled into long DNA molecules, a process which is called postreplication repair (PRR) [ll-161. In UVirradiated XP variant cells a stronger reduction in MW is seen than in normal cells [ 11, 15-171, whereas the rate of the increase of the MW is similar to that in normal cells [ll, 1.5, 181. So, in the XP variant cells newly synthesized DNA molecules of low MW persist for relatively long times after UV exposure. Cells from the first category of XP patients (excision-defective XP) show a similar defect in DNA replication after UV exposure, but less pronounced [9, 11, 191. Strains from all the complementation groups are intermediate

82

Jaspers, Jansen-van

Table 1. Properties

de Kuilen and Bootsma

of cell strains used

Cell strain

Residual level of UDS (%)”

Designation

Origin

Clinical symptoms

XPlRO XP4BE XPSSE XPITA XP30RO XP37RO XP25RO XP20RO XP3NE C4RO C5RO

106 90 90 92 93 102
XP Variant XP Variant XP Variant XP Variant XP Variant XP Variant Group A Group C Group D Normal Normal

Netherlands USA Japan Israel Lebanon Netherlands

Very mild Severe Moderate Severe Severe Very mild

u Determined after exposure to 9.4 Jm-’ of UV light. * By definition.

between normal and XP variant cells, with the exception of group E, which behaves normally in this respect [19]. When PRR is measured in the presence of caffeine a further reduction in the size of the nascent DNA of XP variant cells is observed and also the growth of this DNA into high MW forms after further incubation is strongly depressed [I 1, 18-201. In contrast, caffeine affects these events in normal and XP group E cell strains only weakly. The other excision-defective XP strains show an intermediate sensitivity to the drug [9, 11, 191. These effects of caffeine result in large MW differences of the nascent DNA after UV exposure of XP variant, excision-defective and normal cells, allowing an easy discrimination between the three types by the study of PRR in a pulsechase experiment [ 11, 19, 211. Utilizing these differences we have undertaken a genetic study of the XP variant cell strains. A technique has been worked out which allows the measurement of PRR in heterokaryons after cell fusion. Normal patterns of PRR in the presence of caffeine were observed in XP variant nuclei after Exp Cd

Res 136 (1981)

fusion with excision-defective XP and normal cells. Restoration of normal PRR was not found after fusion of different XP variant cell strains.

MATERIALS

AND

METHODS

Cell strains and culture conditions Table 1 presents a summary of the human tibroblast cell strains that were used. Post-replication repair data of the XP variants XP4BE, XPITA, XP30RO and XPSSE have been reported [9, 11, 191. XPlRO and XP37RO showed very similar MWs when tested in PRR assays described earlier [9] and were normal in UV-induced UDS. Cells were routinely cultured in 50 cm2 glass bottles with 10 ml Ham’s FlO medium, supplemented with 15% (v/v) fetal calf serum (FCS) and penicillin and streptomycin (each 100 units/ml).

Cell fusion protocols Suspension fibon. Cells from monolayer cultures kept confluent for at least 2 days were trypsinized, mixed, washed twice with glucose-free Hanks’ balanced salt solution (GFH) and then suspended in GFH containing 400 HAU of inactivated Sendai virus per ml (final cell concentration 2~10~ per ml). The cells were incubated for 10 min in ice and gently shaken for 2030 mm at 37”c, they were suspended in medium and seeded into 3.5 cm diameter Petri dishes (Costar). Monolayer fusion. Cells from monolayer cultures kept confluent for at least 2 days were trypsinized. A total of 2.5~105 cells (either one cell strain or a 1 : 1 mixture of two different strains) were seeded into 3.5 cm Petri dishes containing a round glass coverslip

Complementation

analysis of xeroderma

pigmentosum

variants

83

Estimation of the MW of DNA in heterokaryons Populations of fused cells are heterogeneous in nature. They always contain a fraction of unfused mononuclear cells and the fused cells are subdivided in heterokaryons and homokaryons. In order to estimate the MW of the DNA in the heterokaryons using alkaline sucrose gradient analysis the following theoretical considerations were made. From the definition of the weight-average molecular weight, I

2

3 NUCLEI

L PER

5

6

p6

CELL

Fig. I. The efficiency of entry into the S phase in fused cells. After monolayer fusion (closed symbols) or suspension fusion (open symbols) C4RO cells were continuously labelled with tritiated thymidine for 46 h, fixed and processed for autoradiography. Details of the fusion procedures are described in Materials and Methods. Fusion efficiencies for the monolayer and suspension fusion were 0.72 and 0.75, respectively.

MW=ZR, MJCR,, where R, and Mi are the radioactivity and the MW in the ith fraction of the gradient, one can deduce the following relations: Mm=WAA

-F)Mmon,,+FMmu,t,

(2)

F being the fusion efficiency of S phase nuclei in the fusion A XB and M being the weight-average molecular weights of the mono- and multinucleate subpopulations;

Mmulti=( 1-P)M,omo+PM,,t,,,

(3)

where P is the fraction of nuclei in multinucleate cells being present in heterokaryons. Finally, because in the fusion A XB unfused cells and homokaryons do not show complementation, we assumed that M,ix=M,,nu=Mi,,,mo.

(4)

Taking together eqs (2) to (4) results in

Mhetrro=(M/u-(1-FP)M,,,)/FP

Analysis of DNA MWs (post-replication repair) 24 hours after fusion cultures were UV-irradiated with 9.4 Jme2. incubated for 60 min, then pulse-labelled for 60 min with [3H-methyl]thymidine (Radiochemical Centre, Amersham; 40-60 Ci/mmol, 33 &i/ml) and chased for 3 h in unlabelled medium containing thymidine and deoxycytidine (10 PM). Caffeine (1.5 mM) was present in all post-irradiation incubations. Analvsis of the labelled DNA in alkaline sucrose gradients and calculation of weight-average molecula; weights (MW) were performed as described by Lehmann et al. [19]. Parallel dishes were fixed immediately after the tritium pulse and processed for autoradiography using Ilford K-2 dipping solution. After exposure for 4-6 days the percentage of nuclei in S phase in unfused mononuclear cells and in multikaryons, the fusion efficiency and the grain number over non-S phase nuclei (unscheduled DNA synthesis) were determined.

(1)

where M,i, represents the MW of a 1: I mixture of the fused populations A XA and B XB and R and M their contributing radioactivities and weight-average molecular weights; M,w=(l

(3 cm e) and cultured for 14-16 h at 37°C. This culture period allowed the cells to attach and spread out to ensure optimal contact at the time of fusion. After two rinses with ice-cold GFH 0.3-0.4 ml ice-cold virus suspension (750 HAU per ml) was added onto the coverslip of each dish. After 10 min at 4°C 1 ml of GFH of37”C was added and dishes were left at 37°C for an additional 20-30 min. Then GFH was removed and warm medium was added for further culture. For assessment of the fusion efficiency the cells were fixed 24 h after fusion and stained with MayGriinwald-Giemsa. Fusion efticiencv was defined as the fraction of a total of I 000-2006 nuclei that was present in the fused cells.

MA~+R~~MBR)I(R~~+R~6)

(3

which is the formula that was used to correct for the presence of mononuclear cells and homokaryons in the measurement of PRR after fusion.

RESULTS DNA synthesis in multinucleate cells Analysis of post-replication repair requires a relatively high percentage of cells in S phase at the time of UV exposure. In order to make complementation studies feasible, this requirement should also be met in multinucleate cells obtained after cell fusion. In this respect, we have tested two Exp CdRes

136 (IY81)

Jaspers,

84

10

Jansen-van

20 TIME

30 AFTER

de Kuilen and Bootsma

10 FUSION

20

30

(HRS)

Fig. 2. The kinetics of entry into the S phase in fused cells. C4RO cells were pulse-labelled with tritiated thymidine for 30 min at different times after monolayer fusion, then fixed and processed for autoradiography. N, number of nuclei/cell. For each point 600-I 000 nuclei were counted.

different fusion procedures, both using inactivated Sendai virus as the fusing agent. The cells were either trypsinized, mixed (1 : 1) and fused in suspension, or Sendai virus was added onto monolayer cultures that had been mixed before. Coverslip cultures of fused C4RO cells were cultivated for 46 h in the presence or [“H]TdR (2 Cilmmol, 2 &i/ml), fixed and processed for autoradiography. The percentage of labelled nuclei was determined in the mononuclear and multinucleate cells. Fig. 1 shows that in the unfused cells the fraction of labelled nuclei was similar after the two fusion protocols. However, the entry into S phase was less efficient in the multinucleate cells and dependent on the number of nuclei per cell. After suspension fusion S phase entry in multinucleate cells was more strongly inhibited than after fusion of the cells in monolayer. This difference between the two fusion protocols was qualitatively reproducible and observed after fusing other cell strains (C5R0, XP7TA) as well. We concluded Exp

Cell

Rrs

136 (I%+/)

GRAINS

PER

NUCLEUS

Fig. 3. Unscheduled DNA synthesis after exposure to 9.4 JmmL of UV light in different binuclear cells. Abscissu: grain number above one nucleus of a binuclear cell; ordinate: grain number above the other nucleus of the same binuclear cell. 0, XP20ROx XP20RO (25 cells counted); +, XP30ROxXP30RO (25 cells counted); X, XPZOROxXP30RO (50 cells counted).

that the monolayer fusion protocol was the best for analysis of PRR in fused cells. For a study of the kinetics of the entry into the S phase, fused C4RO ceils were pulse-labelled with [3H]TdR at different times after monolayer fusion and fixed. Autoradiography was performed and the percentage of S phase nuclei in the different categories of fused cells was determined. As is illustrated by fig. 2, the nuclei started entering S phase around 10 h after fusion. At about 15 h an S phase peak was observed in mononuclear cells. With an increase in the number of nuclei per cell this peak tended to shift to later times. The appearance of a peak in the graphs of fig. 2 was related to the use of confluent cultures of cells as starting material for fusion. When log phase cultures were used, a gradual decrease from about 25 % S phase after 5 h

Complementation

Table 2. Unscheduled Cell strains

DNA

Variant

Excision-deficient

Binuclear cells counted

XP7TA XPIRO XP4BE XPSSE XP30RO XP37RO XP7TA XP30RO

XP25RO XP25RO XP25RO XP25RO XP25RO XP25RO XPZORO XPZORO

50 40 40 40 50 50 50 50

n Valhes

fused

synthesis

in parentheses:

(A) (A) (A) (A) (A) (A) (C) (C) percentages

analysis afterfusion

pigmentosum

of different

I

II

III

12 (24)a 9 (23) IO (25) 11 (27) 12 (24) 12 (24) I1 (22) 14 (28)

0

38 29 27 29 36 38 35 35

of cells in category

and

Autoradiographic analysis (excision repair). The experimental protocol for PRR

analysis involves labelling of newly synthesized DNA after UV exposure. With this procedure unscheduled DNA synthesis (UDS) will occur in the non-S phase nuclei. Therefore, after fusing different XP variant cells with excision-deficient cells from different complementation groups both autoradiographic and biochemical analysis of the labelled cells was performed. Fig. 3 shows the results from grain counts carried out after UV exposure and radiolabelling of fused XP20RO (excision-deficient complementation group C) and fused

s 0 2 0 4 1

variants

85

types of XP cells

Categories

to about 5% after 35 h was observed in multinucleate cells (data not shown). Apparently, seeding the cells from plateau phase cultures allows some degree of synchronization. After fusion of other cell strains (C5R0, XP7TA) the kinetics of entry into the S phase were similar to those shown in fig. 2. For analysis of PRR optimal levels of S phase were reached between 15 and 30 h after fusion. We have chosen 24 h as a standard time point in all further fusions. Fusion between XP variants excision-deficient XP

of xeroderma

I.

XP30RO (variant) cells. Nuclei in binucleate XP20RO cells showed a reduced level of UDS compared with those in binucleate XP30RO cells. In fig. 3 lines were drawn between these two grain distributions resulting in a subdivision into three categories: I, both nuclei low UDS; III, both nuclei high (normal) UDS and II, mixed-type binucleate cells. When grains above nuclei in binucleate cells obtained after fusion of XP20RO with XP30RO were counted, about one-quarter of these randomly selected cells were found to be in category I and three-quarters in category III. This pattern is expected when the UDS of XP20RO nuclei is restored to normal levels by the presence of a variant nucleus in the heterokaryon and agrees with the statistical expectation in a random 1 : 1 fusion. As shown by table 2, similar results were obtained after fusion of other combinations of XP variant and excision-deficient strains. All the variants tested restored UDS to normal levels in the excision-deficient cells. The population of fused cells with nuclei showing a low UDS was assumed to represent homokaryons, made up from excision-deficient XP cells. When the entire population of fused cells was included, E.rp

Cell

Rus

136 (1981)

86

Jaspers,

Jansen-van

de Kuilen and Bootsma Biochemical analysis (post-replication repair)

M7.25=122x106 k <0

10

Mmfx :98x106

6

FRACTION

NUMBER

Fig. 4. Radioactivity profiles in alkaline sucrose gradients obtained after analysis of different fused cell populations. Cells were treated as described in Materials and Methods. Sedimentation is from right to left. O-O, XP7TAxXWTA; X-X, XP25ROx XP25RO; O-O, XWTAxXP25RO; . . ., calculated profile of a 1 : 1 mixture of XWTAxXP7TA and XP25ROxXP25RO cell populations (M,,J.

doubling of the fraction of nuclei present in cells with a low UDS gives the total fraction of nuclei present in the two types of homokaryons. The heterokaryon fraction (P) was defined as one minus the homokaryon fraction. From the same autoradiograms also the frequency of nuclei in S phase in fused and unfused cells was determined. F was defined as the fraction of all S phase nuclei that was present in fused cells. The frequency of nuclei in S phase was also determined in preparations from the homogeneous fusions of the type “A xA” and ‘B xB”. Within all the fusions there were no gross differences in proliferative activity between the two cell types. The fraction of nuclei in S phase ranged from 0.28 to 0.41 in the various experiments. Exp Cdl

RPS 136 (1981)

In order to test whether excision-deficient and variant XP strains were able to complement each other with respect to their defects in PRR, fused cells were analysed on alkaline sucrose gradients. In such an experiment the weight-average DNA molecular weight (MW) of the mixed fusion was compared with the MWs of both fused parental strains, measured in the same experiment. Typical results are shown in fig. 4. XP variant fusion XP7TAxXP7TA yielded a lower MW than the excisiondeficient XP25ROx XP25R0, and the latter was again lower than that found in fused normal cells (C4RO and C5R0, mean MW = 135-t5 million D), tested in six separate experiments. The MW of XP7TAxXP25RO (M7.25) was slightly higher than that obtained for XP25ROxXP25RO and did not reach the normal level. A correction for the presence in the population of unfused cells and homokaryons was made as described in Materials and Methods. The values Mi,25 and Mmix were derived from the centrifugation data and F and P were determined in the autoradiograms as described above. Substitution of M ,,,,=122~ l@, M,i,=98X 106, F=0.751 and P=O.828 into eq. (5) yields Mhetero =137x lo6 D, a number that agrees well with the MW in normal cells. This indicates that XP7TA and XP25RO cells complement each other for their defects in PRR. A similar conclusion could be drawn after testing other fused combinations, involving six XP variants and three excision-deficient XP cell strains from different complementation groups (table 3). In all cases the MW was significantly higher than the Mmix value, that was obtained from both parental fused cell strains. Also, the estimated M hetei-o always reached a value that com-

Complementation Table 3. Post-replication

analysis of xeroderma

pigmentosum

variants

87

repair after fusion of different types of XP cells

Cell strains fused

Molecular weights (lo6 D)

Variant

Excision-deficient

Measured”

Mm,,*

M,,,,,"

Mhetemb

P*

XP7TA XP7TA XP7TA XP30RO XP30RO XP30RO XPIRO XP4BE XPSSE XP37RO

XP25RO (A) XPZORO (C) XP3NE (D) XP25RO (A) XP20RO (C) XP3NE (D) XP25RO (A) XP25RO (A) XP25RO (A) XP25RO (A)

122 115 115

95 88 86 60 76 90 80

133 127 12.5 100 107 125 120 110 110 115

137 140

0.83 0.75 -0 0.60 0.63 -c 0.74 0.68 0.64 0.77

E 116 103 95 95 98

ii 68

130 126 132 129 130 131

0 Mean of two independent experiments. b Calculated from one experiment. ’ Determination ofP not possible by analysis of UDS.

pared well to the MW found in normal fused cells. For the D-group cells this estimation was difficult because of a considerable overlap in UDS levels from the fusion partners, due to the relatively high residual excision repair activity in the D-group cell strain. However, by determining the relative frequencies of the different types of multinucleate cells with respect to their number of nuclei per cell, an estimation of the heterokaryon fraction could also be obtained. Assuming a random 1 : 1 fusion, a multinucleate cell with N nuclei will have a probability of 1-2”-N) to be a heterokaryon. Thus we obtained for the fusions XP7TAxXP3NE and XP30ROxXP3NE

Table 4. Post-replication Molecular Al

B-+

XPlRO XP4BE XPSSE XP7TA XP30RO

the heterokaryon fractions 0.80 and 0.79 values of 136 and 133 million and Mhetero D respectively. The same way of estimating the heterokaryon fraction P was also applied to a fusion of XP7TA and C4RO cells. Here too, the obtained Mhetero was equal to the MW found in the fusion C4RO x C4RO (data not shown). Fusion of different XP variants The experimental procedure used for the analysis of fused populations of variant and excision-deficient XP cells was also applied to complementation analysis of XP variant cells. Table 4 summarizes the re-

repair after fusion of different XP variant cells weights (millions of D)U

XP37RO

XP30RO

XP7TA

XPSSE

XP4BE

40 (45)b

77 (78) 44 (40)

70 (68) -

75 (72)

64 (65) 57 (57) 72 (73)

60 (61) 39 (42) 66 (64)

80 (79)

a Mean of two independent experiments. * Values in parentheses: calculated M,,. E.rp Cd

Rcs

136 (1981)

88

Jaspers, Jansen-van

de Kuilen and Bootsma

sults from these experiments. In contrast to the fusions with excision-deficient cells, in all cases tested, there was no significant difference between the MWs in the “A XB” fusion and the corresponding Mmix values. However, the F-values were at least 0.69 and similar to those obtained after fusing XP variant with excision-deficient cells. We conclude, that under these conditions there is no detectable complementation between the different XP variant cell strains. DISCUSSION In experiments performed to optimize the conditions for the analysis of PRR in fused cells it was found that the efficiency of entry into S phase by fibroblasts decreased, when the number of nuclei per fused cell increased. The extent of this inhibition of S phase entry was influenced by the conditions of fusion. The two procedures tested in this respect both used confluent cultures as starting material, but they differed in the order of cell fusion and the release from the contact-inhibited state. When the contact-inhibited cells were fused before reseeding (suspension fusion), the S phase entry in the multikaryons was more strongly depressed than when release of contact-inhibition preceded the cell fusion. It appears that cells sufficiently advanced into Gl phase show a tendency of commitment to enter S phase. The kinetic studies presented here are not detailed enough to provide support for the existence of a control point somewhere in the Gl phase which has been postulated by some investigators [22-241. Autoradiographic analysis of cells after fusion of excision-defective XP and variant XP tibroblasts showed normal levels of UV-induced UDS in the heterokaryons. This result is in line with the expectation Exp Cell

RES 136 (198/j

that defects in different factors are responsible for the excision-defective and variant XP phenotypes. It implies that 24 h after fusion the excision of thymine dimers in the heterokaryons proceeds at the same rate as in UV-irradiated normal cells. In these heterokaryons also the molecular weight of the DNA synthesized shortly after UV exposure was comparable to that in UV-irradiated normal fibroblasts. This indicates that in both types of nuclei in the heterokaryons PRR was normal. The fact that the two XP-types complement each other with respect to both UDS and PRR is consistent with the idea, that in excisiondefective XP cells the defect in PRR is a secondary consequence of the inability to excise thymine dimers. A factor that functions normally in the excision-defective cells must be impaired in the XP variant. The results obtained after fusion of excision-defective and variant XP cells indicate that under our experimental conditions there is sufficient interaction of gene products in the heterokaryons to accomplish a normal PRR phenotype in the XP variant nucleus. The fraction of nuclei in the multikaryons that was in S phase at the moment of labelling was generally around 0.3 (see fig. 2). These S phase nuclei were distributed over the different multikaryons according to Poisson statistics (data not shown). This means that in a large group of heterokaryons only one type of nucleus was in S phase. Apparently, even if only a variant nucleus synthesized DNA in the heterokaryon, this DNA still had a normal molecular weight. This suggests that the presence of the complementing factor for PRR in the excision-defective cell is not confined to the S phase. For complementation analysis within the group of XP variants cell strains were used that originate from all over the world. The

Complementation

analysis of xeroderma

strains are also from donors that differ in the clinical manifestations of the disease. This choice was made to minimize the probability of familial relationships. In none of the fused combinations complementation could be observed. This result can be explained in two different ways: either the six XP variants tested are all affected in the same gene, or at least some variants are mutated in different genes. The latter possibility arises when in the variant heterokaryons there is insufficient interaction of the gene products involved in the restoration of normal PRR. This is not very probable in view of the results obtained with fusions of excision-defective and variant XP cells. It is also possible, that in the variant fusions de novo protein synthesis is necessary for full expression of normal PRR activity. The culture period of 24 h after fusion might not be sufficiently long then to accumulate enough active proteins. If this is so, then the complementation between excision-defective and variant XP cells would rely on active diffusable factors, which are already present in the excisiondefective cells at the moment of cell fusion. In our experimental conditions we cannot test this possibility, because after much longer incubation times (e.g. 48 h or more) no S phases are present in the multinucleate cells (unpublished data). This question might be answered by studying proliferating hybrids obtained from different pairs of variant cells. Various reports have shown, that such an approach should be feasible [25-271. A complicating factor in interpreting these complementation data is the fact that always caffeine was used. Several experiments were carried out in the absence of the drug as well, but in fusions of excisiondefective and variant XP cells the observed differences between MAB and Mmix were too

pigmentosum

variants

89

small to clearly demonstrate complementation in the heterokaryons. In fusions between different variants no difference between MAB and Mm,, was seen also in the absence of caffeine (results not shown). Fujiwara [20] has investigated twenty XP variants, most of them originating from Japan. He reported heterogeneity with respect’to the effect of caffeine on PRR and he was able to subdivide these patients into three subgroups on the basis of the sensitivity to this drug. All our variants tested showed a clearly caffeine-sensitive PRR, which would place them into Fujiwara’s subgroups I and Il. Therefore, variants from subgroup Ill (weakly caffeine-sensitive) seem to be of interest for complementation analysis. However, the possibility exists, that the heterogeneity reported by Fujiwara is not based on genetic differences, but rather a reflection of variation in phenotypic expression. This assumption is supported by his observation, that two XP variant sisters fall into separate subgroups I and Ill [20]. In conclusion, the data in this report suggest, that the XP variants tested comprise a single complementation group, different from group A, C and D. We propose to assign these strains to complementation group V. Apparently, the XP variant phenotype is far less genetically heterogeneous than the category of excision-defective XP patients. This work was supported by a Eurdtom grant, contract no. 196-76 BION and EUR 200-76 BION and by the Netherlands Organization for the Advancement of Pure Research (ZWO).

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