Search

The timing of DNA replication in the Xq27 portion of the human X chromosome was studied in cells derived from normal and fragile X males to further characterize the replication delay on fragile X chromosomes. By examining a number of sequence-tagged sites (STSs) that span several megabases of Xq27, we found this portion of the normal active X chromosome to be composed of two large zones with different replication times in fibroblasts, lymphocytes, and lymphoblastoid cells. The centromere-proximal zone replicates very late in S, whereas the distal zone normally replicates somewhat earlier and contains FMR1, the gene responsible for fragile X syndrome when mutated. Our analysis of the region of delayed replication in fragile X cells indicates that it extends at least 400 kb 5' of FMR1 and appears to merge with the normal zone of very late replication in proximal Xq27. The distal border of delayed replication varies among different fragile X males, thereby defining three replicon-sized domains that can be affected in fragile X syndrome. The distal boundary of the largest region of delayed replication is located between 350 and 600 kb 3' of FMR1. This example of variable spreading of late replication into multiple replicons in fragile X provides a model for the spread of inactivation associated with position-effect variegation or X chromosome inactivation.

Cytosine methylation at promoter regions and late replication timing have both been implicated in the regulation of genes subject to X chromosome inactivation. Reported here are studies of X-linked gene replication in normal male and female cells as well as in cell hybrids that contain either a normal active X, a normal inactive X, or an inactive X chromosome that has been treated with the demethylating agent, 5-azacytidine (5aC). The relationship between replication timing and transcriptional activity was examined for XIST, XPCT, PGK1, HPRT, F9, FMR1, IDS, and G6PD, and earlier replication was generally found to be associated with increased transcriptional activity. The HPRT and G6PD genes in an untreated inactive X hybrid were among the few exceptions to this correlation in that they remain inactive, yet replicate earlier than their inactive X alleles present in normal human diploid cells. This condition of earlier replication timing may contribute to the high rates of 5aC-induced reactivation for HPRT and G6PD in this hybrid relative to other inactive X hybrids. Other anomalous cases include 5aC-induced advances in replication time for genes such as XIST and F9 whose transcription was unaltered by treatment. These and other data support a model for regulation of X-inactivated genes that involves at least two levels of control: (i) large chromosomal domains are placed into a transcriptionally nonpermissive state by late replication and (ii) transcription is blocked at the local level by promoter methylation. In addition, our observations of continued XIST expression in 5aC-treated hybrids with reactivated genes indicates that such expression is not sufficient for the maintenance of X inactivation.

Using a bromodeoxyuridine incorporation method to detect replicated DNA, we studied allele-specific replication of several sites within the human Prader-Willi/Angelman and IGF2/H19 imprinted regions. No obvious allele-specific differences in time of replication were detected at most loci previously reported to replicate asynchronously in the same cell types as determined by a FISH-based replication assay. Our finding of an absence of allelic replication asynchrony may be related to low levels of imprinted gene expression near these loci in the examined cells (lymphocytes, fibroblasts and lymphoblastoid cells). This view is supported by our studies of the imprinted SNRPN gene in that cells with paternal allele-specific expression (lymphocytes and lymphoblasts) replicate SNRPN alleles asynchronously, whereas cells with a low level of expression (HeLa) replicate SNRPN later and with less allelic asynchrony. In lymphoblasts, the early replicating allele of SNRPN was identified as the paternal one based on the properties of maternal allele-specific methylation and paternal allele-specific expression. Our studies suggest that FISH data implying replication asynchrony in nonexpressing cells reflect structural differences between the maternal and paternal alleles rather than differences in replication timing.

The timing of DNA replication appears to be an important epigenetic regulator of gene expression during development. Replication of active genes in expressing tissues occurs earlier than does replication of their inactive counterparts in nonexpressing tissues. This pattern is also observed for active and inactive alleles present in the same cell, as exemplified by genes subject to X chromosome inactivation in females. We find that the replication timing of the X-linked XIST gene in normal human fibroblasts provides a striking exception to this well-established pattern. Within the same cell, the expressed allele of XIST replicates late in S phase and the silent allele replicates early. This 'reverse' replication timing may have functional significance with respect to XIST or could be a passive consequence of the replication timing requirements of neighboring genes that are subject to X chromosome inactivation. Our finding of early replication for XIST in male fibroblasts contrasts with a report of late replication in such cells as determined by an in situ hybridization method [Torchia et al., (1994) Am. J. Hum. Genet. 55, 96-104]. We propose that our data and those obtained by the in situ method can be accommodated by the existence of structural features that differ between the silent and expressed alleles of XIST. Similar features may be important determinants of the replication asynchrony found by the in situ method for other genes subject to monoallelic expression.

We have used chromosome 18-specific painting probes to analyze early stages of oogenesis in two human trisomy 18 fetuses. At leptotene, a diffuse, nonlinear chromosomal fluorescence was detected as one (27%), two (42%), or three (31%) signals in 534 cells. The variation in size of these signals implies the possibility of associations between homologs prior to zygotene. At pachytene, about 75% (339/453) of the cells had a trivalent configuration, and almost half of these cells exhibited almost complete triple synapses. Approximately 24% of the pachytene cells demonstrated a bivalent:univalent configuration, and 1% exhibited complete asynapsis. Our data imply that triple synapses may be a regular feature of meiosis involving multivalents.

Fluorescent in situ hybridization (FISH) utilizating an X chromosome whole library probe was used directly to assess the rate of aneuploidy and pairing behavior of the X chromosome in human female meiosis. Over 3000 meiotic cells obtained from fetal ovaries (gestational age 13-22 weeks) were scored for meiotic stage and evaluated for pairing abnormalities. No pairing anomalies were observed in 832 pachytenes. Twenty-two percent (88/398) of cells in zygotene were partially paired, but nonhomologous pairings could not be identified. One aneuploid preleptotene oocyte, presumably from mitotic nondisjunction was detected. To our knowledge, this is the first report of the use of FISH utilizing whole chromosome probes to evaluate the pairing behavior of chromosomes in human female meiosis. The application of this technique to study the relationship between nondisjunction and chromosome pairing behavior in maternal-age-related aneuploidy is discussed.

The fragile X syndrome is commonly associated with mutant alleles of the FMR1 gene that are hypermethylated and have large expansions of CGG repeats. We present data here on the replication timing of FMR1 that confirm predictions of delayed replication of alleles from affected males. The normal FMR1 allele replicates late in S phase, while alleles from affected males replicate later, the major peak of replication occurring in the flow cytometry fraction usually referred to as G2/M. The delayed timing of replication is not the direct result of a single replication fork stalling at the expanded CGG repeat, because delayed replication was observed for regions on both sides of the repeat. The domain of altered replication timing includes sites at least 150 kb 5' and 34 kb 3' of the repeat, indicating that genes in addition to FMR1 may be affected.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)

Many genes are known to have nuclease-sensitive sites and/or control sequences in their 3' flanking regions, but for very few genes has this region been sequenced. Previously, we mapped specific, gene activity-dependent DNAase I- and MspI-sensitive sites at the 3' end of the human X-linked housekeeping gene phosphoglycerate kinase (PGK1). Sequence information presented here shows that the 3' nuclease-sensitive site maps precisely to an Alu sequence and near a "BKM" repeat. This is the first report of an Alu sequence that has alternative chromatin configurations depending on gene activity.

It has been known since the development of nearest neighbor analysis that the frequency of the dinucleotide CpG is markedly suppressed in vertebrate DNA (i.e. less than %C x %G). This suppression appears to be heterogeneous since it was shown some years ago that three vertebrate tRNA genes did not exhibit CpG suppression. We have analyzed 13 different human tRNA genes and found that they also do not exhibit CpG suppression. Because CpG suppression has been linked, to some extent at least, to the methylation-deamination process by which a methylated CpG is mutated to TpG, we investigated whether the lack of suppression of CpG in tRNAs could originate from an absence of methylation. Three human tRNA genes were selected from Genbank (lysine, Proline, and Phenylalanine) and examined for methylation at HpaII sites by polymerase chain reaction (PCR) and Southern blot analysis. The observed patterns were consistent with the absence of methylation at the seven HpaII sites analyzed in and around the tRNA genes, and we predict that the remaining CpGs in these genes will be unmethylated. Since GC-rich promoter regions also escape CpG suppression and since they are generally unmethylated, avoidance of methylation may be a general explanation for the absence of CpG suppression in selected regions of vertebrate genomes.

The frequencies of induced mutation to ouabain (OUA) and 6-thioguanine (6TG) resistance were compared in two established mouse fibroblast lines with different doses of the active X chromosome. The activity states of the X chromosomes were determined by DNA replication studies. Mutation frequency to 6TG R, an X-linked recessive phenotype, was inversely related to dosage whereas OUA R, a codominant phenotpye, occurred with equal frequency in both lines. 6TG R clones isolated from the line containing a majority of cells with two active X chromosomes were monosomic for the active X chromosome. Enzyme activity studies of these cytologically monosomic lines yieled results that were consistent with the presence of a single active X chromosome. The findings strongly support the hypothesis that mutation frequency is dependent on gene dosage.

In this study we compared steroid sulfatase levels in XO, XX, and XY mice and carried out a clonal analysis in fibroblast cell cultures from mice heterozygous for the steroid sulfatase deficiency gene and heterozygous at the X-linked electrophoretic phosphoglycerate kinase locus. The combined results indicate that the murine steroid sulfatase locus is not dosage compensated and is not subject to X-inactivation. With respect to X-inactivation, it behaves in a somewhat different way from the closely linked sex-reversed gene and the human steroid sulfatase locus.

The human X chromosome-linked fragment, "Xrep," was sequenced because it exerts a positive effect on plasmid growth in both E. coli and Saccharomyces cerevisiae. The sequence revealed three features similar to the human BK virus replication origin: Xrep has a true palindrome, CCTCC(T)3CCTCC, which is similar to "true" palindrome-like sequences found at the replication origins of polyoma [CCTC(T/C)10CTCC], BK [CCTC(A/G)8CCTCC] and SV40 [CCTCC(A)6GCCTCC] viruses. Twenty nucleotides away from the true palindrome, Xrep has the sequence GAATCCTATTCACTTTT while BK virus, the human analogue of SV40, has GAAATCCCTATTCTTTT in exactly the same position relative to the true palindrome. These two 17-mers differ only in the positions of two nucleotides comparing Xrep and BK virus. Also similar to the replication origins of DNA viruses, Xrep appears to have a cluster of enhancers adjacent to the origin-like sequences. Potent enhancer-like activity was detected in pSV1 X CAT/Xrep constructs. Xrep may originate from an endogenous virus, or from an X chromosomal replication origin.

Mammalian X chromosome inactivation is generally considered to be a good example of stable transcriptional repression; however, there has been no satisfactory evidence for transcriptional control. We have made a test of the hypothesis of transcriptional control by Northern blot analysis of RNA from a woman heterozygous for a mutant Hpt allele which shows no detectable transcription of wild-type mRNA. Cells from this Hpt+ Hpt- woman were separated into HPRT+ and HPRT- subpopulations by selection in HAT or thioguanine. The HPRT+ population (in which the Hpt+ is on the active X) transcribed normal Hpt mRNA, while the HPRT- population (in which the Hpt+ allele is on the inactive X) did not. These results provide strong support for the hypothesis of transcriptional control.

A human XXY chromatin negative triploid culture has been found to have both the A and B isozymes of glucose-6-phosphate dehydrogenase, as well as an intermediately migrating AB (hybrid) band. This finding indicates that the cells in this culture each possess 2 active X chromosomes. Possible mechanisms producing the abnormalities seen in this disorder are presented and discussed.

Steroid sulfatase (STS) is an X-linked enzyme whose locus escapes X inactivation in human somatic cells. STS activity was determined in human fibroblasts varying in X-chromosome number from one to four. Greater STS activity was detected in 2X cell strains when compared to 1X cell strains; however, increased STS activity was not found in 3 and 4X strains as compared with the 2X strains. Greater STS activity was also observed in female hair follicles when compared with male hair follicles. These data provide evidence of a gene dosage effect at the STS locus in human tissues.

The murine X-linked steroid sulfatase gene (Sts) normally escapes X inactivation. However, we have observed that most long-term murine cell cultures are deficient in STS activity even though only the L cells are known to be derived from an STS- mouse strain. To investigate this phenomenon, we developed a selective system whereby STS+ cells could be selected from STS- populations. The system is based on making cells dependent on cholesterol-sulfate as the sole source of cholesterol, allowing only STS+ cells to grow. Two STS- cell lines, after treatment with either 5-azacytidine (5AC) or ethyl methane sulfonate (EMS), yielded STS+ revertants, suggesting that their STS- phenotype was due to hypermethylation. To study the evolution of STS- cell lines, we established XO and XX primary lines from STS+ strains; the XX cell line remained STS+ after more than 200 cell doublings whereas the XO became STS- after about 100 doublings. Treatment of this STS- XO cell line with 5AC produced clones with restored STS activity. All the revertants showed a growth disadvantage compared to their STS- counterparts. It would appear that aberrant methylation is the basis for much of the STS deficiency observed in established murine lines and that its propagation is due to the growth advantage of STS- over STS+ cells.

The X-linked human Pgk gene has been cloned and partially characterized, and some preliminary results have been obtained regarding active vs. inactive gene comparisons of chromatin structure and methylation patterns. As yet we can say nothing definitive about what role, if any, these differences may play in X inactivation. The studies showing that DNA from the inactive X chromosome in mature somatic cells does not function in transformation of the Hprt gene strongly imply modification of the inactive X chromosome at the DNA level. However, methylation studies with the Hprt, Gd Pgk genes have revealed a complexity of methylation patterns including hypermethylation of parts of the active X gene. Resolution of just what difference is critical in expression, differentiating between cause and effect, and extrapolating to the spreading and initiation aspects of X inactivation are still, unfortunately, long-range goals. The Pgk system may be of special value in unraveling some of these difficult questions. A unique autosomal Pgk locus exists and should allow an informative comparison between an X-linked housekeeping gene and an autosomal, tissue-specific gene encoding proteins of identical enzymatic function. The proximity of Pgk to the X-inactivation control center may be useful in identifying the starting point of this very important event in early mammalian development.

A fluorescence in situ hybridization method using a biotinylated DNA probe specific for the centromeric region of the human X chromosome was used to differentiate the genetically active from the inactive X in interphase cells. With this technique, we were able to interpret both the relative position and the degree of condensation of the X chromosomes within the nucleus. We first established the specificity of fluorescence labelling of the hybridized probe by comparing its location and appearance (either dense or diffuse) when associated with a sex chromatin body (SCB) in early passage normal human female fibroblasts. In these cells, where the presence of inactive X chromatin was verified by identification of a 4',6-diamidino-2-phenyl indole (DAPI)-positive SCB in 85% of the cells examined, the X chromatin fluorescence was always associated with the SCB. The signal was dense in structure in 98% and peripheral in location in 80% of the nuclei. A second type of signal, diffuse in form, was observed in 85% of the nuclei and presumably represents the location of the active X chromosome. It was located peripherally or centrally with equal frequency and was not associated with any identifiable nuclear component. This diffuse signal was the major type associated with human male fibroblasts. In rodent x human hybrid cells containing a human inactive X, the fluorescent signal was associated with an SCB-like structure in only 13% of the nuclei; it was dense in 66% of the nuclei and equally peripheral or central in location. This indicates an alteration in the interphase structure of the human inactive X chromosome in hybrid cells which may explain its known instability with respect to genetic activity in such systems.

We have carried out a comparative analysis of DNase I sensitivity of the active and inactive X-linked phosphoglycerate kinase (Pgk) genes in human lymphoblast and fibroblast cultured cells. Three DNase I-sensitive regions were detected: a 5' hypersensitive site, a sensitive region in the interior of the gene and a 3' slightly sensitive site which we previously reported and have now mapped with some precision. A comparison of these sensitive sites in single and multiple X cell lines indicates that the sensitive sites are unique to the active X chromosome. A similar study of an X-linked Pgk pseudogene shows no difference in DNase I sensitivity between the pseudogenes on the active and inactive X chromosomes. These latter results imply that sex chromatin does not confer a unique level of DNase I resistance to DNA on the inactive X chromosome. The exact role of sex chromatin in differential DNase I sensitivity of genes on the inactive and active X chromosomes is discussed.

Purified fetal mouse germ cell preparations have been analyzed for the presence of a heterochromatic X chromosome at cell division. A single large heterochromatic chromosome is found in oogonia stage germ cells, but such a chromosome is not present in 16-day fetal oocytes. These results support the idea that mitotic stage germ cells are subject to X-chromosome inactivation and that they go through a reactivation step at meiosis.

In order to map human PGK sequences, DNA was prepared from 55 human-mouse somatic cell lines. The DNA was digested to completion with HindIII and Southern filters prepared. These filters were hybridized at high stringency conditions to a human PGK cDNA. Mouse and human X-linked and autosomal bands were distinguished and, in addition to known X-linked sequences, two autosomal PGK sequences were mapped: a 1-kb band to chromosome 19 and a 5-kb band to chromosome 6. The PGK cDNA probe was also hybridized to flow-sorted chromosomes confirming the presence of PGK sequences on the X chromosome and chromosomes 6 and 19.

Nuclei from a variety of human cell lines and tissues were digested with gradually increasing levels of DNase I. The DNA was then purified, treated with restriction enzymes and subjected to Southern blot hybridization using a cloned cDNA probe to 3-phosphoglycerate kinase (PGK) a housekeeping enzyme. At relatively high levels of DNase I, a specific, slightly sensitive site in chromatin sequences encoding PGK was observed in all of the cell types examined. This slightly sensitive site resides on the active X-chromosome since cell lines with increased numbers of inactive X-chromosomes do not show an increase in the region of chromatin which is sensitive. Except for this restricted region of enhanced sensitivity on the active X-chromosome, the data suggest that, for PGK encoding sequences, chromatin configurations on the active and inactive X-chromosomes are similar.

X-Chromosome inactivation leads to the formation of mosaic cell populations in the somatic cells of mammalian females. Cells have either a maternal or paternal X-chromosome active. If an individual is heterozygous for a cell autonomous X-linked trait, then a set of built-in cellular markers is provided for the investigation of various developmental phenomena, including selection. In the absence of somatic cell selection the tissues of an X-linked heterozygote should all be mosaic and should have a mosaic composition approaching 1:1. If somatic cell selection is occurring, it should be detectable by a significance shift from the random expected 1:1 mosaicism. The system is effective at detecting selection acting on X-linked loci and on newly arisen somatic autosomal variants, and several examples of somatic cell selection are described. However, it is concluded that somatic cell selection, as described above, is not a normal aspect of ontogeny.

The steroid sulfatase (STS) levels in mature oocytes of XX and XO mice were assayed along with lactate dehydrogenase (LDH), an autosomal marker, and glucose-6-phosphate dehydrogenase (G6PD), a known X-linked gene. LDH levels in XX and XO oocytes were equal, whereas STS and G6PD levels were approximately twice as high in XX oocytes as in XO oocytes. These results indicate that the STS gene is X-linked in the mouse just as it is in humans. Assays of STS in kidney tissue of XX and XO mice indicated dosage compensation for the gene, which is different from that observed in humans.

Five embryonic mouse cultures and one human fibroblast culture were transformed with SV40. The cultures were studied cytologically to see if the normal pattern of sex chromosome replication was maintained in SV40 transformed cells. Characteristic late replication patterns were observed for both the X and Y chromosomes, and there was no evidence for loss of the inactive X chromosome, even in cells with 4 or more X chromosomes. The human line was heterozygous at two X-linked loci and a clonal analysis showed that the expression of X-linked genes was not affected by SV40 transformation.

The mechanism of X-chromosome inactivation has been investigated recently using DNA-mediated transformation of the X-linked hypoxanthine phosphoribosyl transferase (hprt) locus. Several experiments indicate that inactive X-chromosomal DNA does not function in HPRT transformation. Liskay and Evans used DNA from hamster or mouse cells which had an hprt- allele on the active X chromosome and an hprt+ allele on the inactive X chromosome. We and others used rodent-human hybrid cell lines which had an hprt+ allele on the inactive human X chromosome alone. DNA from all of these cells failed to transform HPRT- recipients. Recently, Chapman et al. have shown that inactive X-chromosome DNA from several tissues of adult female mice is strikingly inefficient in genetic transformation for the hprt gene. On the other hand, de Jonge et al., using simian virus 40 (SV40)-transformed fibroblasts from a human heterozygous for an HPRT deficiency, observed HPRT transformation regardless of whether the hprt+ allele was on the active or the inactive X chromosome of the donor cells. We have done an experiment similar to that of deJonge et al., and report here results which clearly indicate that DNA from the inactive X chromosome functions very poorly in HPRT transformation, thus supporting the original interpretation of Liskay and Evans that inactive X-chromosomal DNA is structurally modified.

In a population at equilibrium for a sex-linked lethal, one-third of the genes for that lethal must arise anew each generation. Therefore, one-third of all cases of Lesch-Nyhan disease, a severe X-linked recessive lethal disorder, should be new mutants. To test this hypothesis, we have collected 47 families, 20 with a single proband and 27 with multiple affected males in which the patients' mothers and other female relatives had been studied for heterozygosity. Available carrier detection tests identify heterozygous for HPRT deficiency in hair roots and skin fibroblasts. Only four mothers were found not to be carriers. This result deviates significantly from expected (P less than .001). Statistical tests for ascertainment effects indicated absence of bias for multiple proband families but strong bias in favor of families with many heterozygous females. When the analysis was limited to single proband families, the deviation from expected was still significant (P less than .01). The incidence of new mutants among the heterozygous mothers, as determined by the ratio of +/+ to +/- maternal grandmothers, should be one-half (see Appendix). Of all 20 maternal grandmothers studied, five were +/+ and 15 were +/- (P less than .05). Considering only the single proband families, the ratio of 5 +/+ to 8 +/- was not significantly different from expected. In four of the five cases in which the heterozygous mother of an affected individual was a new mutation, the age of her parents was considerably higher than the mean parental age in the population. This raises the possibility of a paternal age effect on X-linked mutations. There appears to be a true deficiency of new mutatnts among males but not among females. Data on additional Lesch-Nyhan families are needed before conclusions regarding a possible higher mutation rate in males can be drawn.

Nick translation assays of fixed interphase female fibroblasts with tritiated nucleotides demonstrated a characteristic absence of label over sex chromatin. The chromatin bodies were nearly always peripheral in location and a ribbon of nick translatable DNA was detected between the sex chromatin and the nuclear envelope. High voltage electron microscopy indicated the possibility of a special nuclear envelope attachment region. The apparent resistance of sex chromatin to nick translation did not appear to be due to resistance to DNase I attack.

Inactivation of the X chromosome during mammalian spermatogenesis has been postulated to occur by the same mechanism that controls female somatic X chromosome inactivation. We have used DNA-mediated transformation of HPRT- cells to test this idea, because it has been shown previously that inactive X chromosome DNA from somatic cells will not transform HPRT- cells. Isolated DNA from the mature sperm of five mammals (human, mouse, horse, bull, rabbit) were all capable of HPRT transformation, and transformants were confirmed electrophoretically. Measures were taken to ensure that the transformation frequencies observed could not be due to somatic contamination. The positive HPRT transformation result indicates that mature sperm X chromosomal DNA is not modified in the same manner as that of female inactive X chromosomal DNA. Since there is evidence for methylation of the somatic inactive X chromosome, it is possible that methylation, at least for the genes studied, is not involved in sperm X chromosome inactivation.

Three genes on the human inactive X chromosome retained in the Chinese hamster X human hybrid cell line X8/6T2 have been reactivated using the demethylating agent, 5-azacytidine (5-aza-CR). Pulse-labeling and histochemical methods permitted detection and measurement of reactivation rates of the hypoxanthine phosphoribosyltransferase (Hpt) and glucose-6-phosphate dehydrogenase (G6pd) genes within 48 h of treatment. About 50% of the cells became active for these genes, which represents a reactivation rate some 30-fold greater than previously reported in similar systems. The phosphoglycerate kinase (Pgk) gene was not reactivated as frequently as the Hpt or G6pd genes. Segregation analysis of progeny of treated cells showed that enzyme-positive and enzyme-negative cells were produced in proportions supporting the notion that 5-aza-CR causes demethylation by replicative loss and that demethylation leads to reactivation.

Attention is drawn to the possibility of half chromatid and early somatic mutations and to several implications of these mosaic-yielding events. There is suggestive evidence that spontaneous mutations can result in mosaics. A world-wide cooperative study of Lesch-Nyhan families could determine the extent of half chromatid mutation transmission and early somatic mutation in humans.

Dosage studies and the inheritance pattern of the gene for steroid sulfatase (Sts) in the mouse have previously provided indirect evidence for a functional Y-linked allele which recombines obligatorily with its X-linked allele in male meiosis. In this study, we have investigated the linkage relationship of Sts and the sex-reversed mutation (Sxr), a gene which is known to reside in the pairing region of the Y chromosome. The results clearly demonstrate that Sxr and Sts are linked in a region of obligatory recombination and Sts maps proximal to Sxr with most recombinants occurring proximal to the two genes.

Three types of cell mosaics have been used in mammalian studies: hemopoietic shimeras, mosaics formed by aggregation of preimplatation embryos, and mosaics resulting from X-chromosome inactivation. The problems investigated with these cell mosaics have included normal tissue orgaization, cell selection, primordial cell pool sizes, and tumor cell kinetics. The emphasis in this review is on the application of X-chromosome inactivation mosaics to the analysis of tumor cell proliferation. The first application of mosaicism to tumor ontogeny involved leiomyomas and demonstrated single cell and independent origin of the tumors. Other tumor studies are reviewed including those of presumed multiple cell origin, especially those of hereditary origin and viral etiology. The concept of target size is invoked to explain these multiple cell origin tumors. The recent reports on the clonal nature of atherosclerotic plaques is also discussed. Emphasis is placed on resolving the relationship between the multiclonal underlying fatty streak and the clonal plaque in order to understand the implications of the clonal plaques.

Two-dimensional electrophoresis has been used to examine the pattern of proteins synthesized in germ cells at the beginning of oogenesis in the mouse. Three stage-specific changes in proteins were detected during the three observational periods studied (12, 14, and 17 days of gestation). One protein is present in 12 day female germ cells and nongerminal gonadal preparations, but disappears by day 14. Two of the proteins appear for the first time in fourteen day female germ cell preparations, and are not detectable in nongerminal gonadal tissue. No stage-specific changes in proteins were observed in preparations of male germ cells of the same age. A preliminary DNA-binding protein analysis suggests that none of the stage-specific proteins mentioned above are DNA-binding proteins.

Mammalian X-chromosome inactivation involves a coordinate shutting down of physically linked genes. Several proposed models require the presence of specific sequences near genes to permit the spread of inactivation into these regions. If such models are correct, one might predict that heterologous genes transferred onto the X chromosome might lack the appropriate signal sequences and therefore escape inactivation. To determine whether a foreign gene inserted into the X chromosome is subject to inactivation, transgenic mice harboring 11 copies of the complete, 17-kilobase chicken transferrin gene on the X chromosome were used. Male mice hemizygous for this insert were bred with females bearing Searle's translocation, an X-chromosome rearrangement that is always active in heterozygous females (the unrearranged X chromosome is inactive). Female offspring bearing the Searle's translocation and the chicken transferrin gene had the same amount of chicken transferrin messenger RNA in liver as did transgenic male mice or transgenic female mice lacking the Searle's chromosome. This result shows that the inserted gene is not subject to X-chromosome inactivation and suggests that the inactivation process cannot spread over 187 kilobases of DNA in the absence of specific signal sequences required for inactivation.

The available data on chemical carcinogenesis with methylcholanthrene in mosaic mice and studies of atherosclerotic plaques in human and experimental animal G6PD mosaics have been reviewed. The MC studies in two different kinds of mosaic mice demonstrate the importance of understanding normal tissue architecture before making inferences regarding the cellular kinetics of tumor origin. The analysis of the MC studies also indicates a large cellular target size for this carcinogen. The current data on atheromas in mosaic humans and animals do not support the classical idea of an injury-repair mechanism of plaque origin, and leave the possibility open that the monoclonal tendency of atheromas could indicate a tumorigenic type of mechanism in their development.