An upstream repressor element plays a role in Igf2 imprinting.

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans- acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo.

DNA demethylation in vitro: involvement of RNA.

An in vitro system for studying DNA demethylation has been established using extracts from tissue culture cells. This reaction, which is unusually resistant to proteinase K, takes place through the removal of a 5-methylcytosine nucleotide unit from the DNA substrate and its conversion to an RNase-sensitive form. It is likely that this represents the in vivo mechanism, as well, since extracts from L8 myoblasts specifically demethylate an alpha-actin gene, while extracts from F9 teratocarcinoma cells specifically demodify the Aprt CpG island. After pretreatment with proteinase K, these extracts demethylate both genes equally, suggesting that gene specificity may be controlled by protein factors.

Sp1 elements protect a CpG island from de novo methylation.

Animal somatic cell DNA is characterized by a bimodal pattern of methylation: tissue-specific genes are methylated in most cell types whereas housekeeping genes have 5' CpG islands which are constitutively unmethylated. Because methyl moieties derived from the gametes are erased in the morula and early blastula, this profile must be re-established in every generation; this is apparently accomplished by a wave of non-CpG island de novo methylation that occurs at implantation. Using transfection into embryonic stem cells and transgenic mice as a model system, we now show that Sp1 elements play a key role in protecting a CpG island in the adenine phosphoribosyltransferase (APRT) gene from de novo methylation. This recognition mechanism represents a critical step in embryogenesis, as it is responsible for setting up the correct genome methylation pattern which, in turn, is involved in regulating basal gene expression in the organism.

Replication structure of the human beta-globin gene domain.

The animal cell genome is organized into a series of replicons with an average size of 50-300 kilobases; each of these units is characterized by its own origin of replication which serves as the point of initiation for DNA synthesis. In animal viruses, origin usage can be regulated by cis-acting elements, and in some cases, replication may be cell-type specific. Little is known, however, about the organization and control of endogenous tissue-specific gene replication. To understand this process, we have used a replication direction assay to examine DNA fragments covering more than 200 kilobases of the human beta-like globin domain, and have identified a single bidirectional origin located upstream of the beta-globin itself. This locus is used to initiate DNA synthesis in expressing cells, where the globin domain replicates early, and in non-expressing cells, which are characterized by late replication of the same region. Deletion of this origin sequence, as occurs in the haemoglobin Lepore syndrome, cancels bidirectional DNA synthesis at this site and leads to a striking reversal of replication direction upstream to the locus. This represents the first genetic proof of the existence of specific, discrete origins of replication in animal cells.

Allele-specific replication timing of imprinted gene regions.

Several lines of evidence suggest that the paternal and maternal genomes may have different expression patterns in the developing organism and this has been confirmed by the identification of endogenous genes that are parentally imprinted in the mouse. Little is known about the precise mechanisms involved in the process, but structural differences between the two alleles must somehow provide cis-acting signals for directing parental-specific transcription. Cell-cycle replication time is one parameter that has been shown to be associated with both tissue-specific gene expression and the allele-specific transcription patterns of the X chromosomes in female cells. For this reason we have examined the replication timing patterns for the chromosomal regions containing the imprinted genes Igf2, Igf2r, H19 and Snrpn in the mouse. At all of these sites, and their corresponding positions in the human genome, the two homologous alleles replicate asynchronously and it is always the paternal allele that is early-replicating. Thus imprinted genes appear to be embedded in large DNA domains with differential replication patterns, which may provide a structural imprint for parental identity.

Demethylation of CpG islands in embryonic cells.

DNA in differentiated somatic cells has a fixed pattern of methylation, which is faithfully copied after replication. By contrast, the methylation patterns of many tissue-specific and some housekeeping genes are altered during normal development. This modification of DNA methylation in the embryo has also been observed in transgenic mice and in transfection experiments. Here we report the fate in mice of an in vitro-methylated adenine phosphoribosyltransferase transgene. The entire 5' CpG island region became demethylated, whereas the 3' end of the gene remained modified and was even methylated de novo at additional sites. Transfection experiments in vitro show that the demethylation is rapid, is specific for embryonic cell-types and affects a variety of different CpG island sequences. This suggests that gene sequences can be recognized in the early embryo and imprinted with the correct methylation pattern through a combination of demethylation and de novo methylation.

Dynamics of demethylation and activation of the alpha-actin gene in myoblasts.

Transient transfection into L8 myoblasts has been used to study the rat alpha-actin gene promoter. Demodification of specific sites occurs in two stages, with a hemimethylated intermediate formed within a few hours after entry of the alpha-actin gene construct into the cell. The removal of the methyl moiety from the complementary strand takes place after a delay of at least 48 hr, and both events are actively carried out in the absence of DNA replication. By assaying gene activity during the course of the transfection, it was possible to demonstrate that demethylation of both strands at the critical CpG loci is essential to activate transcription. Genetic analysis revealed the existence of cis-acting elements required for demethylation. The recognition of these sites early in the differentiation process probably leads to the demodification events required to make the gene accessible to its transcription factors.

The effect of insulin on progesterone production and cellular growth in long-term cultures of human granulosa lutein cells.

The direct action of insulin on human granulosa lutein cells (GLCs) in long-term cultures obtained from in vitro fertilization (IVF) cycles was investigated. Progesterone (P) secretion by GLC increased progressively in both basal and human chorionic gonadotropin (hCG; 100 mIU/ml) stimulated conditions up to 4 days in culture, and plateaued thereafter. Insulin (0.0025 mU/ml to 2500 mU/ml) had no effect on either basal or hCG stimulation during the culture period. GLC in culture formed a monolayer and multiplied at a rate of approximately once every 3 days. Neither morphology nor cell division was affected by insulin in supraphysiologic levels (25 mU/ml). These results suggest that GLC obtained from preovulatory follicles in an IVF program are already stimulated maximally by in vivo exposure to high doses of human menopausal gonadotropin (hMG)/hCG administered to the women. Contrary to its stimulatory effect on early preovulatory granulosa cells, insulin dose not affect P production, cellular morphology, or growth rate of luteinized granulosa cells.

DNA methylation affects the formation of active chromatin.

To study the mechanism of gene repression by DNA methylation, M13 gene constructs were methylated to completion and inserted into mouse L cells by DNA-mediated gene transfer. All unmethylated sequences, regardless of their source, integrated into the DNA in a potentially active DNAase I-sensitive conformation. Total CpG methylation prevented the formation of this structure and rendered these sequences DNAase I-insensitive over the entire methylated domain. Whereas unmethylated DNA demonstrated additional conformational features of active genes, such as DNAase I hypersensitivity and restriction endonuclease-sensitive segments, these markers were not present when methylated DNA was used for transfection. The use of micrococcal nuclease to probe for active or inactive supranucleosome particles also showed that DNA methylation directs DNA into an inactive type of structure. The results suggest that DNA methylation may exert its effect on gene transcription by altering both specific and nonspecific interactions between DNA and nuclear proteins.

Effect of regional DNA methylation on gene expression.

The effect of DNA methylation on the transcriptional activity of the hamster adenine phosphoribosyltransferase (aprt) and the herpes thymidine kinase (tk) genes has been investigated. By using M13 constructs containing these gene sequences, specific segments of each gene were methylated in vitro by restriction fragment primer-directed second-strand synthesis using the substrate 2'-deoxy-5-methyl-cytidine triphosphate (dmCTP). These hybrid-methylated molecules were inserted into mouse Ltk- cells by DNA-mediated cotransfer. In all cases, the integrated sequences retained the in vitro-directed methylation pattern. The aprt gene was inhibited by CpG methylation in the 5' region but was unaffected by methylation at the 3' end or in adjacent M13 sequences. In contrast to this, DNA methylation in both the 5' promoter region and the 3' structural region of the tk gene had a strong inhibitory effect. This suggests that this modification may affect transcription by mechanisms that do not involve the direct alteration of recognition sequences for RNA polymerase.

Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.

Structure of simian virus 40-phiX174 recombinant genomes isolated from single cells.

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.

Indiscriminate recombination in simian virus 40-infected monkey cells.

DNA transfection of African green monkey BSC-1 cells with simian virus 40 (SV40) DNA and bacterial virus phi X174 replicative form DNA ("cotransfection") yielded stocks containing SV40/phi X174 recombinant virus, which was detected by an infectious-center in situ plaque hybridization procedure and which was sensitive to anti-SV40 antiserum. The recombinant virus replicated during serial passage. Restriction endonuclease cleavage of the SV40/phi X174 DNA indicated that several different types of recombinant DNA structures had arisen. Similar SV40 DNA cotransfection experiments with polyoma virus DNA, bacterial plasmid (pBR322) DNA, and a plasmid-cloned segment of the mouse genome (coding for intracisternal type A particles) yielded stocks that generated recombinant plaques as judged by in situ plaque hybridization with the appropriate labeled probe. It appears, therefore, that an active indiscriminate recombination process, incapable of distinguishing between diverse DNAs of prokaryotic and eukaryotic origin, occurs in SV40-infected monkey cells.

Association of mouse major histocompatibility and Rauscher murine leukaemia virus envelope glycoprotein antigens on leukaemia cells and their recognition by syngeneic virus-immune-cytotoxic T-lymphocytes.

Physical association was measured between MLV gp70, the envelope glycoprotein of Rauscher murine leukaemia virus (R-MLV), and serologically defined H-2 antigens on the surface of R-MLV transformed C57BL/6 (H-2DbKb) mouse leukaemia cells (RBL-5A). Capping and patching with antisera against H-2Db caused specific co-capping and co-patching of R-MLV gp70 antigens as seen by fluorescence microscopy. Despite the physical proximity of R-MLV gp70 and H-2Db antigens, high titre alphaR-MLV gp70 sera had no effect in blocking syngeneic T-lymphocyte mediated cytolysis of RBL-2A cells whereas alphaH-2Db sera were effective.