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PD-L1 has two receptors: PD-1 and CD80. Previous reports assumed that PD-L1 and CD80 interacted in trans, but recent reports showed that only cis PD-L1/CD80 interactions existed, and prevention of cis PD-L1/CD80 interactions on antigen-presenting cells (APCs) reduced antitumor immunity via augmenting PD-L1/PD-1 and CD80/CTLA4 interactions between T and APCs. Here, using tumor-bearing mice capable of cis and trans or trans only PD-L1/CD80 interactions, we show that trans PD-L1/CD80 interactions do exist between tumor and T cells, and the effects of trans PD-L1/CD80 interactions require tumor cell expression of MHC-I and T cell expression of CD28. The blockade of PD-L1/CD80 interactions in mice with both cis and trans interactions or with only trans interactions augments antitumor immunity by expanding IFN-γ–producing CD8 + T cells and IFN-γ–dependent NOS2-expressing tumor-associated macrophages. Our studies indicate that although cis and trans PD-L1/CD80 interactions may have opposite effects on antitumor immunity, the net effect of blocking PD-L1/CD80 interactions in vivo augments CD8 + T cell-mediated antitumor immunity.

Significance Mice that have homozygous deletion of the p53 tumor suppressor protein universally die of malignancy, generally before 6 months of age. We show that hemizygous deficiency of RALBP1 (RLIP76 or Rlip) confers a degree of protection from spontaneous malignancy that has never previously been observed. This discovery introduces a paradigm for p53 function, in which Rlip plays a central role as an effector that appears necessary for the cancer susceptibility of p53 null mice. Because p53 loss has a powerful effect on genomic instability that contributes to the initiation and promotion of cancers and to drug and radiation resistance in humans, our findings provide a method for prevention and therapy of p53-deficient cancer., TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53−/−) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53−/− mice, Rlip−/− mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53−/− mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53−/− mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53−/− mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/− mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip–p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.

Significance While most mammalian genes are expressed from both chromosomal copies, many autosomal genes randomly express only one allele in a given cell, resulting in somatic cellular mosaicism. To better understand the mechanisms, developmental aspects, and evolution of autosomal monoallelic expression (MAE), we used nucleotide polymorphism differences between hybrid mice to analyze MAE of clonal neural stem cell lines as they differentiated to astrocytes. We found that genes showing MAE are highly enriched among developmental stage-specific genes. Genes showing strong skewed expression are similarly enriched. We also found evidence suggestive of balancing selection not just for genes with MAE but also, for developmental stage-specific genes., Cellular mosaicism due to monoallelic autosomal expression (MAE), with cell selection during development, is becoming increasingly recognized as prevalent in mammals, leading to interest in understanding its extent and mechanism(s). We report here use of clonal cell lines derived from the CNS of adult female F1 hybrid (C57BL/6 X JF1) mice to characterize MAE as neural stem cells (nscs) differentiate to astrocyte-like cells (asls). We found that different subsets of genes show MAE in the two populations of cells; in each case, there is strong enrichment for genes specific to the respective developmental state. Genes that exhibit MAE are 22% of nsc-specific genes and 26% of asl-specific genes. Moreover, the promoters of genes with MAE have reduced CpG dinucleotides but increased CpG differences between the two parental mouse strains. Extending the study of variability to wild populations of mice, we found evidence for balancing selection as a contributing force in evolution of those genes showing developmental specificity (i.e., expressed in either nsc or asl), not just for genes showing MAE. Furthermore, we found that genes showing skewed allelic expression (SKE) were similarly enriched among cell type-specific genes and also showed a heightened probability of balancing selection. Thus, developmental stage-specific genes and genes with MAE or SKE seem to make up overlapping classes subject to selection for increased diversity. The implications of these results for development and evolution are discussed in the context of a model with stochastic epigenetic modifications taking place only during a relatively brief developmental window.

We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9(+) (Sox9(+)) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9(+) ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9(+) ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300-450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9(+) ductal cell differentiation into β cells in adult mice.

Significance Mixed chimerism has shown good potential to cure some autoimmune diseases and prevent tissue rejection. It is known that MHC-mismatched but not -matched mixed chimerism effectively tolerizes autoreactive T cells, even those noncross-reactive T cells that do not directly recognize donor-type antigen presenting cells [i.e., dendritic cells (DCs)]. How this is accomplished remains unknown. These studies have shown that tolerizing peripheral residual host-type noncross-reactive autoreactive T cells requires engraftment of donor-type DCs and involves a host-type DC-mediated increase in donor-type Treg cells, which associates with restoration of tolerogenic features of host-type plasmacytoid DCs and expansion of host-type Treg cells. This study suggests a previously unrecognized tolerance network among donor- and host-type DCs and Treg cells in MHC-mismatched mixed chimeras., Autoimmune type 1 diabetes (T1D) and other autoimmune diseases are associated with particular MHC haplotypes and expansion of autoreactive T cells. Induction of MHC-mismatched but not -matched mixed chimerism by hematopoietic cell transplantation effectively reverses autoimmunity in diabetic nonobese diabetic (NOD) mice, even those with established diabetes. As expected, MHC-mismatched mixed chimerism mediates deletion in the thymus of host-type autoreactive T cells that have T-cell receptor (TCR) recognizing (cross-reacting with) donor-type antigen presenting cells (APCs), which have come to reside in the thymus. However, how MHC-mismatched mixed chimerism tolerizes host autoreactive T cells that recognize only self-MHC–peptide complexes remains unknown. Here, using NOD.Rag1−/−.BDC2.5 or NOD.Rag1−/−.BDC12-4.1 mice that have only noncross-reactive transgenic autoreactive T cells, we show that induction of MHC-mismatched but not -matched mixed chimerism restores immune tolerance of peripheral noncross-reactive autoreactive T cells. MHC-mismatched mixed chimerism results in increased percentages of both donor- and host-type Foxp3+ Treg cells and up-regulated expression of programmed death-ligand 1 (PD-L1) by host-type plasmacytoid dendritic cells (pDCs). Furthermore, adoptive transfer experiments showed that engraftment of donor-type dendritic cells (DCs) and expansion of donor-type Treg cells are required for tolerizing the noncross-reactive autoreactive T cells in the periphery, which are in association with up-regulation of host-type DC expression of PD-L1 and increased percentage of host-type Treg cells. Thus, induction of MHC-mismatched mixed chimerism may establish a peripheral tolerogenic DC and Treg network that actively tolerizes autoreactive T cells, even those with no TCR recognition of the donor APCs.

Significance We report here that a recently developed Bayesian network (BN) methodology and software platform yield useful information when applied to the analysis of intrachromosomal interaction datasets combined with Encyclopedia of DNA Elements publicly available datasets for the B-lymphocyte cell line GM12878. Of 106 variables analyzed, interaction strength between DNA segments was found to be directly dependent on only four types of variables: distance, Rad21 or SMC3 (cohesin components), transcription at transcription start sites, and the number of CCCTC-binding factor (CTCF)–cohesin complexes between interacting DNA segments. The importance of directionally oriented ctcf motifs was confirmed not only for loops but also for enhancer–promoter interactions. Purely data-driven BN analyses also identified known critical, lineage-determining transcription factors (TFs) as well as some potentially new dependencies between TFs., Long-range intrachromosomal interactions play an important role in 3D chromosome structure and function, but our understanding of how various factors contribute to the strength of these interactions remains poor. In this study we used a recently developed analysis framework for Bayesian network (BN) modeling to analyze publicly available datasets for intrachromosomal interactions. We investigated how 106 variables affect the pairwise interactions of over 10 million 5-kb DNA segments in the B-lymphocyte cell line GB12878. Strictly data-driven BN modeling indicates that the strength of intrachromosomal interactions (hic_strength) is directly influenced by only four types of factors: distance between segments, Rad21 or SMC3 (cohesin components),transcription at transcription start sites (TSS), and the number of CCCTC-binding factor (CTCF)–cohesin complexes between the interacting DNA segments. Subsequent studies confirmed that most high-intensity interactions have a CTCF–cohesin complex in at least one of the interacting segments. However, 46% have CTCF on only one side, and 32% are without CTCF. As expected, high-intensity interactions are strongly dependent on the orientation of the ctcf motif, and, moreover, we find that the interaction between enhancers and promoters is similarly dependent on ctcf motif orientation. Dependency relationships between transcription factors were also revealed, including known lineage-determining B-cell transcription factors (e.g., Ebf1) as well as potential novel relationships. Thus, BN analysis of large intrachromosomal interaction datasets is a useful tool for gaining insight into DNA–DNA, protein–DNA, and protein–protein interactions.

Evolution by gene duplication is generally accepted as one of the crucial driving forces for the gain of new complexity and functions, but the formation of pseudogenes remains a problem for this mechanism. Here we expand on earlier ideas that epigenetic modifications can drive neo- and subfunctionalization in evolution by gene duplication. We explore the effects of stochastic epigenetic modifications on the evolution (and thus development) of complex organisms in a constant environment. Modeling is done both using a modified genetic drift analytical treatment and computer simulations, which were found to agree. A transposon silencing model is also explored. Some key assumptions made include (i) stochastic, incomplete removal (or addition) of repressive epigenetic marks takes place during a window(s) of opportunity in the zygote and early embryo; (ii) there is no statistical variation of the marks after the window closes; and (iii) the genes affected are sensitive to dosage. Our genetic drift treatment takes into account that after gene duplication the prevailing case upon which selection operates is a duplicate/singlet heterozygote; to the best of our knowledge, this has not been considered in previous treatments. We conclude from our modeling that stochastic epigenetic modifications, with rates consistent with experimental observation, can both increase the rate of gene fixation and decrease pseudogenization, thus dramatically improving the efficacy of evolution by gene duplication. We also find that a transposon silencing model is advantageous for fixation of recessive genes in diploid organisms, especially with large effective population sizes.

Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report that (i) TET1/2/3 and TDG can be direct targets of the microRNA miR-26a, (ii) murine TETs, especially TET2 and TDG, are down-regulated in islets during postnatal differentiation, whereas miR-26a is up-regulated, (iii) changes in 5-hydroxymethylcytosine accompany changes in TET mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcytosine are also seen in an in vitro differentiation system initiated with FACS-sorted adult ductal progenitor-like cells, and (v) overexpression of miR-26a in mice increases postnatal islet cell number in vivo and endocrine/acinar colonies in vitro. These results establish a previously unknown link between miRNAs and TET expression levels, and suggest a potential role for miR-26a and TET family proteins in pancreatic cell differentiation.

Dysregulated inflammation in adipose tissue, marked by increased proinflammatory T-cell accumulation and reduced regulatory T cells (Tregs), contributes to obesity-associated insulin resistance. The molecular mechanisms underlying T-cell-mediated inflammation in adipose tissue remain largely unknown, however. Here we show a crucial role for signal transducer and activator of transcription 3 (Stat3) in T cells in skewing adaptive immunity in visceral adipose tissue (VAT), thereby contributing to diet-induced obesity (DIO) and insulin resistance. Stat3 activity is elevated in obese VAT and in VAT-resident T cells. Functional ablation of Stat3 in T cells reduces DIO, improves insulin sensitivity and glucose tolerance, and suppresses VAT inflammation. Importantly, Stat3 ablation reverses the high Th1/Treg ratio in VAT of DIO mice that is likely secondary to elevated IL-6 production, leading in turn to suppression of Tregs. In addition, Stat3 in T cells in DIO mice affects adipose tissue macrophage accumulation and M2 phenotype. Our study identifies Stat3 in VAT-resident T cells as an important mediator and direct target for regulating adipose tissue inflammation, DIO, and its associated metabolic dysfunctions.

We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.

Autoreactive pathogenic T cells (Tpaths) and regulatory T cells (Tregs) express a distinct gene profiles; however, the genes and associated genetic/signaling pathways responsible for the functional determination of Tpaths vs. Tregs remain unknown. Here we show that Skp2, an E3 ubiquitin ligase that affects cell cycle control and death, plays a critical role in the function of diabetogenic Tpaths and Tregs. Down-regulation of Skp2 in diabetogenic Tpaths converts them into Foxp3-expressing Tregs. The suppressive function of the Tpath-converted Tregs is dependent on increased production of TGF-β/IL-10, and these Tregs are able to inhibit spontaneous diabetes in NOD mice. Like naturally arising Foxp3 + nTregs, the converted Tregs are anergic cells with decreased proliferation and activation-induced cell death. Skp2 down-regulation leads to Tpath–Treg conversion due at least in part to up-regulation of several genes involved in cell cycle control and genes in the Foxo family. Down-regulation of the cyclin-dependent kinase inhibitor p27 alone significantly attenuates the effect of Skp2 on Tpaths and reduces the suppressive function of converted Tregs; its effect is further improved with concomitant down-regulation of p21, Foxo1, and Foxo3. In comparison, Skp2 overexpression does not change Tpath function, but significantly decreases Foxp3 expression and abrogates the suppressive function of nTregs. These findings support the critical role of Skp2 in functional specification of Tpaths and Tregs, and demonstrate an important molecular mechanism mediating Skp2 function in balancing immune tolerance during autoimmune disease development.

Drosha-processed microRNAs (miRNAs) have been shown to be exported from the nucleus to the cytoplasm by Exportin 5, where they are processed a second time to generate mature miRNAs. In this work we show that miRNAs also use CRM1 for nuclear-cytoplasmic shuttling. Inhibition of CRM1 by Leptomycin B results in nuclear accumulation of miRNA guide sequences. Nuclear to cytoplasmic transport can be actively competed by synthetic small interfering RNAs, indicating that this pathway is shared by different classes of processed small RNAs. We also find that CRM1 coimmunoprecipitates with Ago-1, Ago-2, Topo2α, EzH2, and Mta, consistent with a role of Argonautes and small RNAs in chromatin remodeling.

X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0–100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, ≈12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.

De novo methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four HOX gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as SIX , LHX , PAX , DLX , and Engrailed , were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous HOX genes showed that not all paralogues undergo cancer-associated methylation simultaneously. The HOXA cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7 - and HOXA9 -associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.

The UV components of sunlight (UVA and UVB) are implicated in the etiology of human skin cancer. The underlying mechanism of action for UVB carcinogenicity is well defined; however, the mechanistic involvement of UVA in carcinogenesis is not fully delineated. We investigated the genotoxicity of UVA1 versus UVB in the overall genome and in the p53 tumor suppressor gene in normal human skin fibroblasts. Immuno-dot blot analysis identified the cis - syn cyclobutane pyrimidine-dimer (CPD) as a distinctive UVB-induced lesion and confirmed its formation in the genomic DNA of UVA1-irradiated cells dependent on radiation dose. HPLC/tandem MS analysis showed an induction of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the genomic DNA of UVA1-irradiated cells only. Mapping of DNA damages by terminal transferase-dependent PCR revealed preferential, but not identical, formation of polymerase-blocking lesions and/or strand breaks along exons 5-8 of the p53 gene in UVB- and UVA1-irradiated cells. The UVB-induced lesions detected by terminal transferase-PCR were almost exclusively mapped to pyrimidine-rich sequences; however, the UVA1-induced lesions were mapped to purine- and pyrimidine-containing sequences along the p53 gene. Cleavage assays with lesion-specific DNA repair enzymes coupled to ligation-mediated PCR showed preferential, but not identical, formation of CPDs along the p53 gene in UVB- and UVA1-irradiated cells. Additionally, dose-dependent formation of oxidized and ring-opened purines and abasic sites was established in the p53 gene in only UVA1-irradiated cells. We conclude that UVA1 induces promutagenic CPDs and oxidative DNA damage at both the genomic and nucleotide resolution level in normal human skin fibroblasts.

Genes subject to monoallelic expression are expressed from only one of the two alleles either selected at random (random monoallelic genes) or in a parent-of-origin specific manner (imprinted genes). Because high densities of long interspersed nuclear element (LINE)-1 transposon sequence have been implicated in X-inactivation, we asked whether monoallelically expressed autosomal genes are also flanked by high densities of LINE-1 sequence. A statistical analysis of repeat content in the regions surrounding monoallelically and biallelically expressed genes revealed that random monoallelic genes were flanked by significantly higher densities of LINE-1 sequence, evolutionarily more recent and less truncated LINE-1 elements, fewer CpG islands, and fewer base-pairs of short interspersed nuclear elements (SINEs) sequence than biallelically expressed genes. Random monoallelic and imprinted genes were pooled and subjected to a clustering analysis algorithm, which found two clusters on the basis of aforementioned sequence characteristics. Interestingly, these clusters did not follow the random monoallelic vs. imprinted classifications. We infer that chromosomal sequence context plays a role in monoallelic gene expression and may involve the recognition of long repeats or other features. The sequence characteristics that distinguished the high-LINE-1 category were used to identify more than 1,000 additional genes from the human and mouse genomes as candidate genes for monoallelic expression.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

The preferential inactivation of the paternal X chromosome in extraembryonic cells during early mouse development is an example of parental imprinting, but it has not been studied at the transcriptional level because standard methods of measuring RNA levels do not allow detection of allele-specific RNAs in individual early embryos. We sought to determine whether the paternal allele of the X chromosome-linked gene for 3-phosphoglycerate kinase 1 (Pgk-1), which is located very near the center of X chromosome inactivation, is transcribed prior to differentiation of extraembryonic lineages. Previous reports indicated that in heterozygous embryos there is a delay in the appearance of the phosphoglycerate kinase 1 allozyme encoded by the paternal X chromosome until 2 days after the appearance of the corresponding maternal allozyme. We report results obtained by use of a reverse transcription/PCR-based method which allows the quantitative measurement of allele-specific RNA. The assay is sensitive enough for the quantitative analysis in single embryos of allele-specific transcripts differing by only one nucleotide. We have used this assay to analyze mouse embryos heterozygous at the Pgk-1 and Hprt [hypoxanthine (guanine) phosphoribosyltransferase] loci, and we find that individual 8-cell and blastocyst embryos express both Hprt and Pgk-1 paternal transcripts, as do pooled 2- to 4-cell embryos. These results are discussed in view of the apparent temporal delay in paternal expression of the Pgk-1 gene at the enzyme level.

Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

To investigate the evolution of the mammalian sex chromosomes, we have compared the gene content of the X chromosomes in the mammalian groups most distantly related to man (marsupials and monotremes). Previous work established that genes on the long arm of the human X chromosome are conserved on the X chromosomes in all mammals, revealing that this region was part of an ancient mammalian X chromosome. However, we now report that several genes located on the short arm of the human X chromosome are absent from the platypus X chromosome, as well as from the marsupial X chromosome. Because monotremes and marsupials diverged independently from eutherian mammals, this finding implies that the whole human X short arm region is a relatively recent addition to the X chromosome in eutherian mammals.

DNA adducts in unique sequences along the mammalian genome are mapped in vivo at single-nucleotide resolution. Pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts] represent one of the two major adduct classes found after UV irradiation of DNA and were shown to play an important role in UV-induced mutagenesis. After UV light treatment of cells, DNA is prepared and chemically cleaved at (6-4) photoproducts with piperidine. Gene-specific fragments are then amplified from total genomic DNA by use of a ligation-mediated polymerase chain reaction. Analysis of the human chromosome X-linked phosphoglycerate kinase (PGK1) gene's promoter has shown that the frequency of (6-4) photoproducts expressed as piperidine-labile sites is (i) high at TpC and CpC dinucleotides, (ii) dependent on the nearest-neighbor bases, (iii) inhibited by the binding of a transcription factor, and (iv) different for DNA derived from the active and inactive X chromosome. This latter difference is mainly a consequence of the presence of 5-methylcytosine (m5C) in CpG dinucleotides on the inactive X chromosome. 5-Methylcytosine in the sequences Tm5CG and Cm5CG inhibits the formation of (6-4) photoproducts. Thus, in addition to in vivo mapping of a DNA adduct at nucleotide resolution, we also report another method for methylation analysis and photofootprinting.

Eight genes, located on the long arm of the human X chromosome and present on the marsupial X chromosome, were mapped by in situ hybridization to the chromosomes of the platypus Ornithorhynchus anatinus, one of the three species of monotreme mammals. All were located on the X chromosome. We conclude that the long arm of the human X chromosome represents a highly conserved region that formed part of the X chromosome in a mammalian ancestor at least 150 million years ago. Since three of these genes are located on the long arm of the platypus X chromosome, which is G-band homologous to the Y chromosome and apparently exempt from X chromosome inactivation, the conservation of this region has evidently not depended on isolation by X-Y chromosome differentiation and X chromosome inactivation.

The transducing phage λh80 dlac carries the lac operator, whereas wild-type λh80 does not. We find that in high salt (0.18 M KCl), ultraviolet radiation causes the formation of a very stable complex between repressor and 5-bromodeoxyuridine (BrdU)-substituted λh80 dlac but not to BrdU-λh80 DNA. Studies with inducers of the lac operon confirm the specificity of attachment. In low slat (0.01 M KCl), ultraviolet radiation will also attach repressor nonspecifically to BrdU-λh80 DNA. The stability of the complex suggests that covalent bonds are formed. We also report that another regulatory protein, the catabolite gene activator protein, can be attached similarly to DNA.

We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

A protein required for the activation of the lac operon has been extensively purified and partly characterized. This protein, called CGA protein (catabolite gene activator protein, sometimes named CAP), is a dimer with subunits of 22,000 daltons. Purified CGA protein has a substantial affinity for DNA; this affinity is greatly strengthened by cAMP and strongly inhibited by cGMP. Other studies have shown that these cyclic nucleotides compete for a binding site on CGA protein. The opposing effects of the two cyclic compounds in DNA-CGA protein binding show a parallel behavior to their effects on the expression of the lac operon. Thus cAMP, in addition to CGA protein, is required for expression of the lac operon, whereas cGMP inhibits the expression. The obvious inference is that CGA protein activates the lac operon by binding to the DNA under the influence of cAMP. Thus, CGA protein seems to be a new type of regulatory protein: a DNA-binding activator.

Binding of lac repressor to 20 synthetic DNAs of high molecular weight with defined repeating sequences was investigated by competition experiments. Although none of these DNAs binds repressor as tightly as does lac operator, most do bind to a measurable extent. Their affinity for repressor varies greatly and is a function of both nucleotide composition and sequence. Poly(dC-dC)·poly(dG-dC) competes for repressor 200-times less well than either poly(dA-dT)·poly(dA-dT) or poly(dT-dT-dG)·poly(dC-dA-dA). The other DNAs show a broad spectrum of affinities for repressor between these extremes. These results show that the lac repressor has affinity for, and can distinguish between, sequences distantly related to its operator.

As measured by a decreased rate of dissociation, lac repressor binds 10-times tighter to 5-bromodeoxyuridine-substituted lac operator than it does to normal lac operator. This result is obtained both in the absence and in the presence of isopropylthiogalactoside, an inducing ligand. These data are significant with regard to the mechanism of sequence-specific protein-DNA interaction, and also suggest a possible explanation for the effects of bromodeoxyuridine on the expression of differentiated functions in eukaryotic cells.