Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1510-1514, April 1975

Localized Derepression on the Human Inactive X in Mouse-Human Cell Hybrids (X chromosome inactivation/hypoxanthine phosphoribosyltransferase reappearance/ X chromosome translocation/azaguanine-resistant fibroblasts) BRENDA KAHAN AND ROBERT DEMARS Laboratory of Genetics, University of Wisconsin, Madison, Wisc. 53706 Communicated by Boris Ephrussi, February 7, 1975

ABSTRACT Evidence for derepression of the for more vague regarding the l)roblem of how the determined hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate plhosphoribosyltransferase, EC 2.4.2.8) on states are maintained. In the explerimenits (lescribe(l below the human inactive X chromosome was obtained in hybrids we aime(l to produce new information about the mainitenance of mouse and human cells. The mouse cells lacked HPRT of rel)ression by dletectinig derepression of genies on the in- and were also deficient in adenine phosphoribosyltrans- active X (Xi). Two l)ossible processes for mainitaininhg X ferase (APIIT; AMP: pyrophosphate phosplhoribosyltrans- chromosome repressioni were consi(lere(l: rel)ression may be ferase; EC 2.4.2.7). The human female fibroblasts were HPRT-deficient as a consequence of a mutation on the cis-p)rop)agated from one or more sites on Xi or, alterniatively, active X but contained a normal HPRT gene on the in- maintenance mav require releatecl trans-active signials that active X. The two human X were further could emaniate from autosomes, the active X (Xa) or, even, distinguished by differences in morphology: the inactive Xi. These genieral alternative mechaniismis for mainteniance X was morphologically normal while the active X included of X most of the long arm of autosome no. 1 translocated to chromosome (lifferentiationi lead to importanit l)re(lictionis the distal end of the X long arm. Forty-one hybrid clones for the behavior of X chromosomes in hy-brid somatic cells. were first isolated by selection for the presence of APIRT; Derepressioni of ani Xi in hybrid cells might occur in the follow- when these clones were selected for HPlRT, six of them ing waxs: (1) by segregation of specific autosomes from Xi, yielded derivatives having human HPRT with incidences (2) by segregationi of Xa from (3) or by segregation of one of about 1 in 106 APRT-selected hybrid cells. The HPRT- Xi, positive derivatives contained a normal-appearing X lart of Xi from aniother lpart as a consequenice of breakage. chromosome indistinguishable from the inactive X of the Chromosome segreoationi anid breakage occur frequenitly in parental human fibroblasts. The active X with the trans- initersl)ecific somatic hybrid cells. Therefore, e devised a location was not found in any of the HPRT-positive exl)eriimelit in which the following questionis could be asked hybrid cells. Human plhosplhoglycerokinase (ATP: 3- in hybridized phospho-i-glycerate 1-phosphotransferase, EC 2.7.2.3) about Xi somatic cells: and glucose-6-phosplhate dehydrogenase (D-glucose 6- (1) Can (lerelpressioni of aii occur in somatic cells? phosphate: NADP l-oxidoreductase, EC 1.1.1.49), which (2) If Xi dlerel)ression occurs, (loes it involve the enitire X, are specified by X-chromosomal loci, were not detected in or is it local, and is (lerel)ression quantitatively l)artial or full' the hybrids expressing HPRT even though they contained (3) Can (lerel)ressioni be related to l)atternis of chromosome an apparently intact X chromosome. The observations are most simply explained by the infrequent, stable derepres- segregation or breakage? sion of inactive X chromosome segments that include the W~e fused mouse cells with a strain of female human cells HPRT but not the phosphoglycerokinase and glu- havinilag genietically anid morphologically (listinctive active and cose-6-phosphate dehydrogenase loci. iniactive X chromosomes, anid then selected for hybrid cells an allele l)resent oilv on the humiiian This Various theories; concernin-g X chromosome inactivation exp)ressinig Xi. dlesign eniabled us to (letect rare (lerel)ression evenits. Humani the of the choice of the X chromosomes to emphasize problem ini which Xa ande were at two loci were be activated or inactivated (1-10), but they are generally cells Xi heterozvgous create(l for this l)url)ose. Wre startedl with female fibroblasts that were already heterozygous at the X-chromosomal locus. Deriva- Abbreviations: Xa, active X chromosome; Xi, inactive X chro- glucose-6-phosphate (lehy(lrogeniase (G61'1)) mosome; G6PI), glucose-6-phosphate dehydrogenase (D-glucose tives of theser cells that' were heterozygous at a seconid locus 6-phosphate:NAl)P 1-oxidoreductase, EC 1.1.1.49); gpd, locus were obtained by selection for azaguaniine (zG) resistance specifying G6PD); HPRT, hypoxanthine phosphoribosyltrans- after x-irradliationi. Our objective was to lproduce cells with a ferase (Ii\IP: pyrophosphate phosphoribosyltransferase, EC mutation oii Xa at the h~pt locus that caused a dleficielicv of 2.4.2.8); hpt, locus specifying HPRT; AP1RT, adenine phos- hypoxanthinie 1)hosl)hioribosyltranisferase (HI'RT) activity. phoribosyltransferase (AMIP: pyrophosphate phosphoribosyl- This created the ol)lportunitv for subsequently selectinig for transferase, EC 2.4.2.7); PGK, 3-phosphoglycerate kinase exl)ressioii of the hpt+ allele lpresenit on Xi of these cells. (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3); An iml)ortanit aspect of our experiment wvas to allow the pgk, locus specifying PGK; zG, 8-azaguanine; sG, 6-thioguanine; without iniitial selection for HAS, medium containing hypoxanthine and azaserine; HAAS, hylbrid cells to l)roliferate expres- HAS medium supplemented with adenine; D)X hybrids, in this sion of the hpt+ allele. This nionselective )eriod was included a paper, cell hybrids selected in HAS medium and apparently so that (lerel)ression exvenfts could occur over l)eriodl of time in containing an inactive X chromosome locally derepressed at the wvhich these evenits were niot requisite for hybrid survival. In hpt locus. practice we itially isolatedl hybrid cells that expressed 1510 Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Derepression on the Human Inactive X Chromosome 1511

Xc xi fr-

pgK K. pqkt ~hp+.R5 hp++ - 9pdA J gpdo

T2 X FIG. 2. Gene arrangement on the T2 active (X.) and inactive FIG. 1. Quinacrine band pattern of the X-chromosome trans- (Xi) X chromosomes of R5 human cells. hptR6 denotes an location in R5 human cells. T2 consists of an X to which most undefined change that caused HPRT deficiency, and the pat- of the long arm of a no. 1 chromosome has been translocated. terned segment denotes most of the long arm of chromosome T1 is the remainder of the no. 1 and lacks an apparent X-chro- no. 1. mosomal segment. 1 translocated to the distal end of the long arm of one X and is human adenine phosphoribosyltransferase (APRT), and relied the active X chromosome in R5. The morphologically normal on the chance retention of the human Xi in cells of some of X was identified as the Xi by autoradiography (14): in 50 these hybrids. Such APRT-selected hybrid cell populations informative metaphases of cells labeled with [3H ]thymidine for were then resubjected to selection for the presence of HPRT 30 min during the first half of S, the morphologically normal X activity. The properties of the hybrids pro(lucecl with these had incorporated little or no thymidine while incorporation procedures provide evidence that localized derepression of the had occurred along the entire length of the T2 chromosome, human Xi occurred. synchronously with the autosomes. MATERIALS AND METHODS The R5 clone produced only G6PD-A and expressed 3-phos- phoglycerate kinase (PGK). We therefore interpret the X Media. Nonselective media consisted of 85 volumes of F10 chromosomes of R5 as follows (Fig. 2): (1) T2 is the active X medium (11) minus hypoxanthine and 15 volumes of either chromosome and carries the human pgk+, hptRs, and gpdA calf serum (CSF1O medium) or fetal calf serum (FCSF1O alleles. The order of the loci is derived from gene localization medium). The following selective media were used: 8.0,AI zG exlperiments (15). We believe these loci are on the T2, and not in CSF10 (zG medium); 50 IATI thioguanine (sG) in FCSF1O the T1 chromosome, because of the properties of one hybrid, (sG medium); 0.1 mAM hypoxanthine and 1.0 /AI azaserine in 57-3-1, to be described inldetail elsewhere. Hybrid 57-3-1 FCSF10 (HAS medium); and 0.1 mM\ hypoxanthine, 0.1 mM\t ex.lressed human PGK, was HPRT-deficient and unable to adenine, and 1.0, I azaserine in FCSF10 (HAAS medium). grow in HAS, and produced G6PD-A. A T2 chromosome-was Cells. The parental mouse cell line, D7, was derived from present in about 50% of the metaphases of 57-3-1. Neither a LMi(TK-) Cl. iD cells, and is deficient in APRT, HPRT, and T1 chromosome nor a morphologically normal X was present thymidine kinase (EC 2.7.1.21) activities (12). in any metaphase, whether T2 was present or not. (2) Xi is Human fibroblast Strain 129 was derived from a skin biopsy morphologically normal and carries the alleles for human of a female who was heterozygous for A - and B + alleles that pgk+, hpt+, and gpdB: G6PD-B was an indicator of clones of determine electrophoretically distinguishable forms of G6PD. strain 129 in which this X was Xa and such clones also Cells were exposed to x-rays (250 roentgens) and clone R5 was expressed PGK and HPRT. isolated by subjecting the survivors to zG selection (13). The Cell Hybridization. Hybrid cells were produced by co- HPRT specific activity of sG-isolated subclones of R5 was inoculating 106 D7 and 106 R5 cells in one P60 dish on day 0 only 2-3% of normal. The R5 subelone used in these experi- and stimulating their fusion on the following day with f3- ments was isolated as a single colony after plating R5 at 10 propiolactone-inactivated Sendai virus (12). The cells were cells per 60 mm Falcon plastic petri dish (P60) in FCSF1O suspended by trypsinization on day 2 and distributed into 50 medium. These cells were able to grow in zG and sG media, P60's containing nonselective FCSF10 medium. On day 8, but were unable to utilize exogenous hypoxanthine for selection for hybrid cells in HAAS medium was begun. Both growth in HAS selective medium. human and hybrid cells survive in HAAS selective medium, The X Chromosomes of Human Female R5 Cells. Clone R5 but hybrids can be located by their different cell and colonial was chosen for these experiments because it had by chance morphologies. acquired an x-ray-induced X chromosome translocation that Karyotype Analyses. Slides of metaphase chromosomes were made the morphology of one of the X chromosomes very prepared as previously described (12) to reveal quinacrine different from that of its normal homologue, thus providing banding patterns (16). another marker for distinguishing the two X's. The trans- located X chromosome (T2) present in R5 is shown in Fig. 1. Species Identification of . HPRT was identified as of T2 consists of almost the entire long arm of a chromosome no. human or mouse origin by means of an immunoprecipitation Downloaded by guest on September 30, 2021 1512 Genetics: Kahan and DeMars Proc. Nat. Acad. Sci. USA 72 (1975) The DX hybrids were unable to grow in medium containing azaserine but not hypoxanthine, i.e., their ability to grow in HAS did not result simply from resistance to azaserine. All DX hybrids had HPRT specific activities in the range (50-138 pmol/.ug of cell protein per hr) observed with several inde- E pendent control hybrids containing an X. chromosome with an hpt+ allele. The incidence of sG-resistant cells in popula- Cr tions constantly maintained in HAS was 4 to 8 X 10-g. This value is at least 100 times greater than that expected to result

a. from mutations causing HPRT deficiency in our iD mouse cells (unpublished results) or human fibroblasts (21), but is consistent with that expected for loss of the enzyme due to loss of the human X chromosome in mouse-human hybrid cells o 2 3 4 5 (unpublished results). (Loss of the mouse X chromosome is I1 ANTISERUM not expected to occur frequently in mouse-human hybrid cells FIG. 3. Immunoprecipitation of HPRT activity of cell and would probably be lethal to these cells.) extracts by anti-human HPRT serum. (A) mouse standard: 1D, Experiments were performed to distinguish between the (0) human standard: 434, (0) hybrid DX10-2. following three sources of HPRT activity in hybrids possessing the enzyme: (1) Reexpression of the mouse HPRT gene reaction with rabbit antibodies against purified human (reversion, in a broad sense); (2) reversion of the hptR& allele HPRT (K. Held, B. Kahan, and R. DeMars, submitted for on Xa; or (3) derepression of the hpt+ allele on Xi. no HAS- publication). HPRT specific activity was determined as We may exclude (1) since D7 mouse cells yielded described previously (17). selectable revertants in over 3 X 107 cells tested separately, The human and mouse forms of G6PD and their hybrid and mouse HPRT has not reappeared in eight independent copolymers were electrophoretically distinguished in acryl- HAS-selected hybrids of D7 and hpt+ human cells (unpub- of amide gel slabs at pH 9.0 in sulfate-borate buffers as described lished results). Furthermore, as Fig. 3 illustrates, the results the by Ortec, Inc. (18), except that a gel with three sections of 3.5, a specific immunoprecipitation test demonstrated that from that 6, and 8% acrylamide was used. HPRT of hybrid DX10-2 was indistinguishable The PGK enzymes of mouse and human origin were sepa- present in human fibroblasts, and in mouse-human hybrid X rated by Cellogel electrophoresis as described by Meera cells expressing a human hpt+ allele on an active (K. Held, Khan (19). B. Kahan, and R. DeMars, submitted for publication). Similar results were obtained with DX hybrids 10-4, 12, and RESULTS 36-2 (data not shown). Hybrids between two types of parental cells, each of which Alternatives (2) and (3) may be distinguished by determin- in and was HPRT-deficient and unable to use exogenous hypo- ing which human X chromosome is present the hybrids in the DX xanthine, were initially selected for their ability to grow using which other human X-linked are expressed 2 two human X chromo- adenine as the externally supplied purine source. Only hybrid hybrids. As Figs. 1 and illustrate, the of G6PD that cells that retained at least the human APRT gene on chromo- somes differ in morphology and in the forms T2 X chromo- some no. 16 formed colonies (20, 12). A total of 41 hybrid could be expressed. is the active, translocated colonies isolated from 30 dishes of the hybridization experi- some, Xa, of the human cells and expresses G6PD-A and in HAAS selective medium until three PGK. The Xi is morphologically normal and in the human ment were propagated and PGK. confluent P60 cultures (about 3-9 million cells) of each were parental cells has unexpressed alleles for G6PD-B on available. At this time, two of the cultures were stored in Alternative (2), reversion of hptRs the (T2) Xa chromosome, T2 chromosome is remaining one was subjected to selec- is unlikely for the following reasons: (i) The liquid nitrogen and the examined 400 tion in HAS medium, in which proliferation depended on the not present in any cells of each DX hybrid (over None of the six DX ability to convert hypoxanthine to purine nucleotides. metaphases from four DX hybrids). (ii) or human PGK In parallel experiments in which hypoxanthine was the sole hybrids expressed either G6PD-A (Fig. 4), of these alleles be if T2 purine source from the beginning of selection, no hybrid (Fig. 5). Expression might expected were present, or even if portions of T2 were present as a result colonies were found. However, six of the 41 hybrids initially of selected for human APRT retention yielded HAS-growing of breakage that resulted in an unrecognizable fragment of about 10-6. Presumably, this early this Xa chromosome. In control hybrids we prepared contain- colonies with incidences human period of "gratuitous" growth of the hybrid cells provided the ing Xa chromosomes from other strains of cells, were Furthermore, opportunity for the appearance of hypoxanthine-utilizing cells human G6PD and PGK always expressed. as described in Materials and Methods, hybrid 57-3-1 contain- in some hybrid clones. PGK and We believe four of the six hybrids (10-1, 10-2, 10-3, and ing the T2 chromosome also expressed human of the locus on T2 has not been 10-4) were of common origin, since they were isolated from the G6PD-A. (iii) Reversion hpt which contains the T2 chromo- same petri dish and had nearly identical chromosome comple- detected in hybrid 57-3-1, some, but not the normal X (Xi): no HAS-growing colonies ments. Although 10-1, 10-2, 10-3, 104, 12, and 36-2 represent al- only three independent hybrids, the HAS-selected populations were obtained among 1.4 X 108 cells of hybrid 57-3-1, derived from them (DX10-1, DX10-2, DX10-3, DX10-4, though 50% of them contained T2. DX12, and DX36-2) represent at least six independent events We believe that alternative (3), derepression of the hpt+ that restored the ability to utilize hypoxanthine. allele on Xi, is most likely because: (i) A morphologically Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Derepression on the Human Inactive X Chromosome 1513

1 2 3 4 5 6 a4 .. OOM6

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-A-VwAjffik.

W", "MRw

1 2 3 4 5 FIG. 5. Cellogel electrophoresis of PGK from mouse, human, tim and HAS-selected hybrid clones. (1) A 1:1 mixture of mouse a-_ UD _ -*" D7 and human 356 PGK enzymes; (2) hybrid )X1O-4; (3) mouse D7; (4) human 356; (5) hybrid 713.6, mouse-human cell hybrid with an active human X chromosome. FIG. 4. Electrophoresis of G6PD from mouse, human, and require combinations of individually improbable events. For HAS-selected hybrid clones in an acrylamide slab gel. (1) hybrid example, to explain the results by assuming the reexpression of 78.9: mouse-human cell hybrid with an active human X ex- pressing G6PD-B; (2) human 356, expressing human G6PD-B; hpt on Xa (T2) would require that the original T2 had been (3) mouse D7; (4) hybrid DX1O-2; (5) human 129.22, expressing modified so as to resemble a normal X chromosome, since no human G6PD-A; (6) hybrid 713.6: mouse-human cell hybrid T2 X chromosome was observed in any cells of the DX with an active human X expressing G6PD-A. hybrids, while a normal X was present in all DX hybrids examined; Additional changes of T2 are required to explain the normal X indistinguishable from the original Xi is present in lack of expression by the pgk+ and gpdA alleles. over 90% of the metaphases (Fig. 6) of three of four DX There are at least two other reasons for thinking it unlikely hybrids analyzed and in about 50% of the metaphases of the that the original T2 chromosome is the source of HPRT in fourth hybrid. (We presume that breakage of the normal X DX hybrids. (1) No T2 chromosomes were detected in the had already occurred in some cells of the fourth hybrid. adenine-propagated hybrids 104 or 36-2 from which two of Extensive karyotype analyses of these hybrids will be relported the 1)X hybrids were selected, while again, a normal X was in a later publication.) (ii) We can demonstrate that the hpt found in most of the metaphases. (2) Finally, the failure to locus on the normal X chromosome is the source of the detect HAS-growable derivatives from hybrid 57-3-1 indicates

HPRT, as sG selection results in the specific loss of this chro- that reversion of the hptR6 allele on T2 occurs too infrequently mosome and the simultaneous loss of HPRT activity. The to account for the reappearance of human HPRT in 1)X regularity and specificity of the loss argue against the inter- hybrids. pretation that the normal X was retained by chance in the Somatic derepression of an X chromosomal gene has not HAS-selected hybrids and that another hpt locus, possibly previously been reported, although reactivation of an X derived from *Xa, was tucked away in an unrecognized chromosome appears to occur normally during development of chromosome segment and was responsible for the presence of the germ line in females (21), and reexpression of autosomal human HPRT. genes inserted into the Xi chromosome in the mouse has been In experiments to determine the extent of derepression of described (22). Derepression of the inactive X has not been the Xi in DX hybrids, we examined DX hybrids for the presence of G6PD-B and human PGK. As already mentioned, no human PGK was expressed by any DX hybrid examined (Fig. 5); similarly, no DX hybrid expressed G6PD-B (Fig. 4), even though an apparently intact X chromosome was present in nearly every cell of three of the hybrids and in about one- half the cells of the other DX hybrid. DISCUSSION

HAS-selected mouse-human cell hybrids express human HPRT and contain a normal-appearing human X chromo- some identified as the Xi in the parental human cells. The human gpd and pgk genes, flanking the hpt locus, are not expressed even though the X chromosome is apparently in- tact. These observations are most simply explained by local FIG. 6. A portion of a fluorescent metaphase of HAS-selected derepression events involving a segment of Xi containing the hybrid D)X10-4. The morphologically normal human X chromo- hpt locus but not the gpd or pgk loci. Alternative explanations some is identified by an arrow. Downloaded by guest on September 30, 2021 1514 Genetics: Kahan and DeMars Proc. Nat. Acad. Sci. USA 72 (1975) detected inl previous experiments waith human cells (23'26) or We thank I)r. Oliver Smithies and Dr. Oliver Nelson for sug- with human-human or mouse-human hybrid cells (27-29, 26). gesting improvements in our manuscript. We are grateful to Our observations indicate that reactivation events on the in- Ms. Beth Trend for performing the PGK analyses and for other assistance. This work was supported by grants from the National active X are both rare and local. Only 10% (3/30) of our Institutes of Health (GiM-06983, G.M-15422, and NIEHS initial independent hybrids gave positive results, and in only 722095). Support for B.K. near the end of this study was gra- about one cell per million. We believe there are several reasons ciously provided by Dr. Robert Auerbach from Grant CA-13548. why we detected these rare events where others have failed: This is Paper no. 1827 from the Laboratory of Genetics, Uni- (a) The hybrids were initially isolated by selection for APRT, versity of Wisconsin, Madison. permitting them to form large populations before selection for 1. Lyon, -M. F. (1972) Biol. Rev. 47, 1-35. HPRT was applied. (b) 'Many independent hybrid clones were 2. Russell, L. B. (1963) Science 140, 976-978. 3. Grumbach, 1\I. M.,Morishima, A. & Taylor, J. H. (1963) examined. (c) We used human parental cells having a mutant Proc. Nat. Acad. Sci. USA 49, 581-589. hpt allele on the active X with no positive selective value for 4. Delvfars, R. (1967) Nat. Cancer Inst. Monogr. 26, 327-351. the hybrid cells, permitting strong selection for reexpression of 5. Cattanach, B. MI., Pollard, C. E. & Perez, J. N. (1969) hpt+ on Xi. (d) Human cells that wA-ere heterozygous for gpd Genet. Res. 14, 223-235. alleles and for a striking X chromosome translocation were 6. Eicher, E. AI. (1970) Advan. Genet. 15, 175-259. 7. Cooper, 1). W. (1971) Nature 230, 292-294. used, providing multiple criteria for detecting derepression. 8. Ohno, S. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, If we had simply observed the absence of G6PD-B in our 155-164. HAS-selected hybrids we might have concluded that derepres- 9. Brown, S. W. & Chandra, H. S. (1973) Proc. Nat. Acad. sion had not occurred. We cannot account for the negative Sci. USA 70, 195-199. 10. Cook, P. R. (1974) Biol. Rev. 49, 51-84. results we and others have had with human cells alone but can 11. Ham, 1t. C. (1963) Exp. Cell Res. 29, 515-526. only suggest that (lisrul)tion of the processes for maintaining 12. Kahan, B., Held, K. R. & De.Iars, R. (1974) Genetics 78, X chromosome repression may be enhanced in interspecies in press. hybrid cells. D)erepression of other, non-X chromosomal 13. Albertini, R. J. & D)eMars, R. (1973) M11utat. Res. 18, 199- 224. genes that are developmentally restricted in certain cell types 14. Brody, S. (1969) Ph.). Dissertation, University of Wis- has been observed in interspecies hybrid cells (30-34). consin, Madison. The genetically relevant information available for our study 15. Pearson, P. L., Sanger, R. & Brown, J. A. (1974) Rotterdam makes possible a relatively detailed interlpretation of our Conference (1974): Second International Workshop on results: derepression involved at least one gene in a larger Human Gene Mapping, Cytogenet. Cell Genet. in press, 1975; or Birth Defects: Original Articles Series, in press, block of genes that were coordinately repressed in the cis 1975; The National Foundation, New York. arrangement on the inactive X. These local derepressions are 16. Caspersson, T., Zech, L., Johansson, C. & Modest, E. J. consequences of genetic or epigenetic events, which remain to (1970) Chromosonia 30, 215-227. be defined. In this context, our experiments suggest: (1) 17. DeZlars, R. & Held, K. (1972) Humangenetik 16, 87-110. 18. Ortec Inc. (1973) Application note AN32A, Oak Ridge, Derepression of hpt in DX hybrid 10-4 is p)robably not due to Tenn. the loss of any specific human chromosomes, since the spec- 19. _Meera Khan, P. (1971) Arch. Biochein. Biophys. 145, 470- trum of human chromosomes present before and after hpt 483. reactivation is virtually the same, as determined by compar- 20. Tischfield, J. A. & Ruddle, F. H. (1974) Proc. Nat. Acad. ing HAAS and HAS-selected populations. (2) D)erepression of Sci. USA 71, 45-49. hpt does not require of the since in- 21. Gartler, S. M., Liskay, R. A., Campbell, B. K., Sparkes, breakage Xi, apparently R. & Grant, N. (1972) Cell Differentiation 1, 215-218. tact normal X chromosomes were observed in four DX 22. Cattanach, B. -iM. (1974) Genet. Res. 23, 291-306. hybrids examined, although about half the cells of one hybrid 23. Comings, D. E. (1966) Lancet ii, 1137-1138. probably contained a broken X. (3) Repression of other 24. Felix, J. S. (1971) Ph.). Dissertation, University of Wis- genes syntenic to hpt on Xi (gpd and pgk) does not require consin, Madison. since 25. Sato, K., Slesinski, R. S. & Littlefield, J. W. (1972) Proc. the presence of any specific human chromosomes, active Nat. Acad. Sci. USA 69, 1244-1248. X's were absent from all l)X hybrids, and almost all human 26. AMigeon, B. R. (1972) Nature 239, 87-89. autosomes were missing from some DX hybrids. The in- 27. Siniscalo, M., Klinger, H. P., Eagle, H., Koprowski, H., cidences of human autosomes that were present varied among Fujimato, W. Y. & Seegmiller, J. E. (1969) Proc. Nat. Acad. the DX hybrids. (4) An important new inference that can be Sci. USA 62, 793-799. drawn from our observations is that the human Xi consists of 28. Silagi, S., 1)arlington, G. & Bruce, S. A. (1969) Proc. Nat. at least three Acad. Sci. USA 62, 1085-1092. separately repressible units, containing pgk, 29. .Migeon, B. R., Norum, R. A. & Corsaro, C. -M. (1974) hpt, and gpd, respectively. Proc. Nat. Acad. Sci. USA 71, 937-941. Our experiments appear to provide a new means for experi- 30. Peterson, J. A. & Weiss, 'M. C. (1972) Proc. Nat. Acad. Sci. mentally studying the mechanism of X chromosome repres- USA 69, 571-575. sion. Hybrid clones that by chance retain the inactive X in 31. Darlington, G. J., Bernhard, H. P. & Ruddle, F. H. (1974) many cells are subjects for systematic attempts to induce Science 185, 859-862. derepression of hpt+. Propagation of the DX hybrids in HAS 32. Colten, H. R. & Parkman, R. (1972) Science 176, 1029- of loci 1031. results in the long-term retention repressed (e.g., pgk 33. Levy, N. L., Snyderman, R., Ladda, R. L. & Lieberman, and gpd) that are syntenic to the hpt locus, permitting at- R. (1973) Proc. Nat. Acad. Sci. USA 70, 3125-3129. tempts to detect their derepression. One can now attempt to 34. Carlsson, S. A., Ringertz, N. R. & Savage, R. (1971) Exp. determine the origin and nature of the repressive signals. Cell Res. 67, 243-244. Downloaded by guest on September 30, 2021