Regional assignment of the genes for TK1, GALK, ALDC, and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer

Summary A panel of clones of mink-Chinese hamster somatic cell hybrids was analysed to obtain data for assigning the genes for thymidine kinase-1 (TK1), galactokinase (GALK), subunit C of aldolase (ALDC), and esterase D (ESD) to specific mink chromosomes. The results demonstrate that the genes for TK1, GALK, ALDC and ESD are syntenic and located on mink chromosome 8. Prometaphase analysis of transformed mouse cells obtained by transfer of mink genes by means of metaphase chromosomes demonstrated the presence of mink chromosome 8 fragments of different sizes in some of the independent transformants. Segregation analysis of these fragments and mink TK1, GALK, ALDC and ESD allowed us to assign the genes for TK1 and GALK to 8p24, ALDC to pter-8p25, and ESD to 8q24-8qter.     

Transfer of mink genes into mouse cells by means of isolated lipid-encapsulated nuclei

Summary A method for gene transfer by means of interphase nuclei encapsulated within lipid membranes was developed. The method was based on passage of interphase nuclei through a layer of organic solvents containing phospholipids. Evidence was obtained indicating that the nuclei become surrounded by a protective phospholipid membrane: measurements of bound labelled or non-labelled phospholipids; decrease in the permeability of lipid-encapsulated nuclei for high molecular compounds; visualization by direct electron microscopy. Lipid-encapsulated nuclei of mink fibroblasts were used for transformation of mutant mouse LMTK^- cells (deficient for thymidine kinase). The frequency of occurrence of HAT-resistant colonies/recipient cell was 1.9x10^-5. Biochemical analysis of 14 independent clones demonstrated that they all contained TK1 of mink origin. Analysis of 15 other biochemical markers located on 12 of the mink chromosomes revealed the activities of mink galactokinase (a syntenic marker) in 5 transformed clones, and that of mink aconitase-1 (the marker of mink chromosome 12) in 1 clone. No cytogenetically visible donor chromosomes were identified in the transformed clones. Nine transformed clones were tested for the stability of the TK⁺ phenotype; of these, the phenotype was expressed stably in 3 and unstably in 6. The method suggested is similar to the gene transfer procedure using total DNA. Its advantage is in ensuring efficient gene transfer and donor DNA integrity.     

Cotransfer of syntenic human genes into mouse cells using isolated metaphase chromosomes or cellular DNA

Summary Chromosome-mediated gene transfer (CMGT) of the human genes for hypoxanthine phosphoribosyl transferase (HPRT) and cytosol thymidine kinase (TK1) into HPRT deficient mouse A9 cells or TK deficient Swiss mouse 3T3TK^– cells was found to occur at frequencies at least one order of magnitude higher than DNA-mediated gene transfer (DMGT). The frequency of CMGT into 3T3TK^– cells was reduced by more than an order of magnitude by a posttreatment of the recipient cells with dimethyl sulphoxide (DMSO). After CMGT, expression of the non-selected genes coding for galactokinase (GALK) and acid alpha-glucosidase (GAA), both syntenic with TK1, was observed in a number of transformants. From the pattern of cotransfer, a tentative gene ordering of CENTROMERE-GALK-TK1-GAA on human chromosome 17 was deduced. Chromosome-mediated cotransfer of X-linked human phosphoglycerate kinase (PGK) with HPRT was observed in two out of 33 A9 transformants analysed. DNA-mediated cotransfer of a syntenic gene was only observed for GALK, cotransferred with TK1 in two out of 18 TK⁺ transformants of mouse LTK^– cells. Therefore, with murine cells as recipients of human donor genetic material, CMGT results in a higher frequency of transfer and a higher incidence of cotransfer of syntenic genes than DMGT using cellular DNA in the same cell system.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

Expression of human histocompatibility antigens on the surface of murine cells transformed by cosmid clones containing HLA genes

Thymidine kinase-negative C3H mouse L fibroblasts (LMTK⁻) transformed with cosmid clones containing both herpes virus-derived thymidine kinase (TK) and HLA class I genes were first selected in HAT (hypoxanthine, aminopterin, thymidine) medium and subsequently analyzed for the expression of human transplantation antigens. TK⁺-transformed cells expressing HLA class I molecules were characterized by surface radioimmunoassay, cytofluorimetric analysis and immunoperoxidase PAP technique at the light and electron microscopic levels, using a set of monoclonal antibodies. Comparisons were made with human B (Raji) and T (1 301) lymphoblastoid cell lines which respectively express high and low levels of HLA molecules on their surface. The expression of HLA class I in association with murine β2-microglobulin on the surface of transformed cells did not reduce the level of expression of H-2 molecules.     

Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Intraspecies transfer via total cellular DNA of the gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells

Summary Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.DNA has been purified from the abnormal HPRT revertant cells and incubated with mouse A9 cells (HPRT^-). After growth in selective medium one clone was isolated which expressed the electrophoretically abnormal form of HPRT. Six clones showed the normal form of HPRT due to reversion of the defective HRRT locus in A9 cells. This result indicates DNA-mediated transfer of the mouse HPRT gene at a frequency of about 0.5×10^-7. A similar frequency has been found for transfer of the variant HPRT locus via isolated metaphase chromosomes to A9 recipient cells. When placed in non-selective media the DNA-mediated transferent cells gradually lost their ability to express the HPRT transgenome at a rate of about 6% per average cell generation.     

Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine,Hoechst 33258, and visible light

The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK^– mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK⁺ cells by a factor of 10⁵ without affecting the viability of TK^– mutants. This procedure was used to isolate H4IIEC3-derived rat hepatoma cells deficient in TK activity. The properties of several TK^– hepatoma clones are discussed.The valuable technical assistance of J. M. Courvall, F. R. Parker, and M. M. Smith is acknowledged. These studies were supported by grant GM26449 from the National Institutes of Health. A.M.K. was supported by a postdoctoral fellowship from the American Cancer Society, California Division. T.G.L. is a Leukemia Society of America postdoctoral fellow. R.E.K.F. is the recipient of an American Cancer Society Faculty Research Award.     

Chinese hamster x American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase,NADP-1 and malate dehydrogenase,NAD-1

Summary Chinese hamster x American mink somatic cell hybrids were obtained and examined for chromosome content and expression of mink malate dehydrogenase, NADP (MOD-1; EC 1.1.1.40), malate dehydrogenase, NAD (MOR-1; EC 1.1.1.37), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). All the hybrid clones examined were found to segregate mink chromosomes. A clone panel containing 25 clones was set up. The possibilities and limitations of this panel for mink gene mapping are analysed. Using this panel, it is feasible to rapidly map genes located on chromosomes 1–13 and to provisionally assign genes located on chromosome 14 and the X. Based on the data obtained, the genes for MOD-1 and MOR-1 were firmly assigned to mink chromosomes 1 and 11, respectively, and the genes for G6PD and HPRT were provisionally assigned to the X.     

5-azacytidine-induced re-expression of alleles on the inactive X chromosome in a hybrid mouse cell line

In order to test the hypothesis that DNA methylation is involved in mammalian X chromosome inactivation, cells of an HPRT-deficient Mus musculus × M. caroli line, having an inactive M. caroli X, were grown in 5-azacytidine, and HPRT⁺ reactivants selected in HAT medium. Recovery of colonies depended on azacytidine concentration, and on time between treatment and selection; the highest recovery frequency was 0.3%. All colonies re-expressed the M. caroli form of HPRT, showing that the Hpt⁺ allele on the inactive M. caroli X was reactivated by azacytidine treatment. About 6% of HPRT⁺ reactivants also re-expressed M. caroli G6PD, whereas none of the 56 azacytidine-treated unselected colonies did so; thus re-expression of the Hpt and Gpd loci appears to be co-ordinated to some extent. However, no HPRT⁺ reactivants, nor unselected colonies re-expressed M. caroli PGK-A.     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Expression of SV40 Large T Antigen Stimulates Reversion of a Chromosomal Gene Duplication in Human Cells

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare “immortalization”—clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finnet al.(1989)Mol. Cell. Biol.9, 4009–4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexistingHPRT⁺cells, were confirmed asHPRT⁺by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicatedHPRTregion, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion ofHPRTmRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion toHPRT⁺was unaltered during the normalin vitrolifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; eachP< 10⁻⁵). Stimulation ofHPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10–30 doublings before replicative senescence. Since no immortal subclones arose from these clones, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.     

Selection of mouse x hamster hybrids using hat medium and a polyene antibiotic

Summary In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK⁻C1ID or HPRT⁻ A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-2095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Evidence for tetrameric structure of mammalian hypoxanthine phosphoribosyltransferase

A fast electrophoretic variant of hypoxanthine phosphoribosyltransferase (HPRT) has been detected in Mus musculus bactrianus, a mouse subspecies from Middle Asia (USSR). The electrophoretic HPRT pattern yielded by hybrids between the somatic cell of LMTK^– (deficient in thymidine kinase) and the splenocytes of a male of M. m. bactrianus was five-banded. The pattern obtained from the germ cells of the ovaries from 14.5-day-old embryos from laboratory CBA mice × M. m. bactrianus crosses was also composed of five bands. The hybrids between the somatic cells of human fibroblasts × LMTK^– cells gave a three-banded electrophoretic HPRT pattern because the asymmetrical heteropolymeric isozymes are probably unstable. Taken together, all the evidence is in favor of a tetrameric structure of mammalian HPRT.     

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.     

Evidence for intrachromosomal gene conversion in cultured mouse cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK^? cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK⁺ was applied to detect recombinational events between different TK^? genes. TK⁺ segregants were observed at a frequency of 10^?4–10^?5 in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK⁺ cells at frequencies of 10^?7 or less. Physical analysis of the TK⁺ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho I endonuclease digestion in each TK⁺ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.     

Reexpression of HPRT activity following cell fusion with polyethylene glycol

Polyethylene glycol-1000 (PEG-1000) induced fusion of HPRT (E.C. 2.4.2.8) deficient Chinese hamster cells with -galactosidase A (E.C. 2.3.1.22) deficient cells from a patient with Fabry's disease yielded hybrids which contained both human and hamster HPRT, G6PD (E.C. 1.1.1.49), and APRT (E.C. 2.4.2.7) and Chinese hamster -galactosidase B. Thus PEG-1000 mediated somatic cell fusion led to reexpression of Chinese hamster HPRT. It did not restore the expression of human -galactosidase. Since PEG-1000 treatment of HPRT^– Chinese hamster cells in the absence of human cells yielded no HPRT⁺ cells, it is concluded that the element responsible for the restoration of rodent HPRT was contributed by the human cells and not by the agent employed to promote fusion.This work was supported by research grants from the United States Public Health Services GM 17702, from the National Science Foundation BMS 74-21424, and from the National Foundation March of Dimes 1-377.     

Identification of two new nucleotide mutations (HPRTUtrecht and HPRTMadrid) in exon 3 of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene

Mutations in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) result in deficiencies of HPRT enzyme activity, which may cause either a severe form of gout or Lesch-Nyhan syndrome depending on the residual enzyme activity. Mutations leading to these diseases are heterogeneous and include DNA base substitutions, DNA deletions, DNA base insertions and errors in RNA splicing. Identification of mutations has been performed at the RNA and DNA level. Sequencing genomic DNA of the HPRT gene offers the possibility of direct diagnostic analysis independent on the expression of the mature HPRT mRNA. We describe a Dutch and a Spanish family, in which the Lesch-Nyhan syndrome and a severe partial HPRT-deficient phenotype, respectively, were diagnosed. Direct sequencing of the exons coding for the HPRT gene was performed in both families. Two new exon 3 mutations have been identified. At position 16676, the normally present G was substituted by an A in the Dutch kindred (HPRT_Utrecht), and led to an arginine for glycine change at residue 70. At position 16680, the G was substituted by a T in the Spanish family (HPRT_Madrid); this substitutes a valine for glycine at residue 71. These new mutations are located within one of the clusters of hotspots in exon 3 of the HPRT gene in which HPRT_Yale and HPRT_{New Haven} have previously been identified.     

Assignment of the gene for cystathionine β-synthase to human chromosome 21 in somatic cell hybrids

Summary Among several established mouse, rat, and Chinese hamster cell lines that were screened for cystathionine -synthase (CBS) activity, mouse 3T3 and Chinese hamster Don fibroblasts were found to contain no detectable activity. Somatic cell hybrids between human fibroblasts KG-7 with normal CBS activity and Don/a23TK^- cells (series XXI) were examined for CBS activity and for human chromosome content. Only chromosome 21 cosegregated with CBS activity. Because the activities measured could represent either Chinese hamster or human gene products, we have prepared a new series of hybrids between Don/a23TK^- cells and mutant human fibroblasts from a patient with homocystinuria due to deficiency of functional CBS mRNA. None of these (series XXV) hybrids contained detectable CBS activity, although collectively all human chromosomes were represented. Our results suggest that the human gene for CBS, called CBS, and thus for the most common form of homocystinuria, is located on chromosome 21.     

Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.