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L Vardimon

Publications and source records attributed to L Vardimon.

31 records · Page 2Linked to original sources
PubMed1986

Accumulation of c-src mRNA is developmentally regulated in embryonic neural retina.

Accumulation of c-src mRNA gradually increased during early development of the neural retina in chicken embryos and reached a peak by days 11 to 13 of embryonic life. Thereafter, its amount declined to a low level which persisted also in adult retina. The early increase in c-src mRNA correlated inversely with the decrease in the amount of H3.2 replication histone mRNA and with the decline in the rate of cell growth. The accumulation profile of c-src mRNA corresponded to that of pp60c-src protein, suggesting that the latter is regulated at the level of transcription.

Animals↗
PubMed1984

DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2.

The E2a gene of human adenovirus type 2 (Ad2) encodes the 72-kilodalton DNA-binding protein. We previously described perfect inverse correlations between the methylation of all 5' C-C-G-G 3' sequences in the Ad2 E2a gene in virus-transformed hamster cells containing viral DNA sequences in an integrated state and the extent to which this gene is expressed. We subsequently showed that in vitro methylation of all 14 5' C-C-G-G 3' sequences in the cloned E2a gene by prokaryotic Hpa II DNA methyltransferase leads to transcriptional inactivation after microinjection into Xenopus laevis oocytes. The unmethylated cloned E2a gene is expressed in these cells. We report here the construction of partly methylated clones of the E2a gene. In the promoter (5')-methylated construct, three 5' C-C-G-G 3' sequences at the 5' end of the subclone were methylated. One of these sites is located 215 base pairs (bp) upstream (bp 26,169 of Ad2 DNA), and two sites are located 5 and 23 bp downstream from the cap site (bp 25,931 and 25,949 of Ad2 DNA) of the E2a gene. This construct was transcriptionally inactive upon microinjection into nuclei of X. laevis oocytes. In the gene (3')-methylated construct, 11 5' C-C-G-G 3' sequences in the main part of the transcribed gene region were methylated in vitro. This construct was transcribed in X. laevis oocytes, and at least some of the Ad2-specific RNA synthesized was initiated at the same sites as in Ad2-infected human KB cells. Both mock-methylated constructs were transcribed into Ad2-specific RNA in X. laevis oocytes. These results demonstrate that DNA methylations at or close to the promoter and 5' end of the E2a gene cause transcriptional inactivation. Perhaps only one methyl group would be adequate for inactivation; in vivo methylation of more than one cytosine may be a form of safeguard or redundancy.

Adenoviruses, Human↗
PubMed1984

In Z-DNA the sequence G-C-G-C is neither methylated by Hha I methyltransferase nor cleaved by Hha I restriction endonuclease.

Plasmids carrying 24- or 32-base-pair inserts of alternating (dG-dC) residues were used to analyze the level of methylation of the G-C-G-C sites by Hha I DNA methyltransferase and their cleavage by Hha I endonuclease in the B-DNA or Z-DNA conformation. In supercoiled plasmids in which the inserts formed Z-DNA, the extent of methylation at the insert G-C-G-C sites was dramatically lower than the level of methylation at the G-C-G-C sites located outside the insert in the same plasmid. Similarly, cleavage by Hha I endonuclease was sharply lowered when the insert was in the Z-DNA form. In the relaxed plasmid, all its G-C-G-C sites were methylated to the same extent and the unmethylated sites were readily cleaved. After treatment with the methylase, the supercoiled plasmid was linearized and then digested with Hha I restriction endonuclease. This exposed unmethylated G-C-G-C sites from the insert that had been protected against cleavage in the Z conformation. A chemical reaction was used to study the distribution of the unmethylated cytosine residues. No accumulation of unmethylated cytosine residues was found anywhere along the entire 32-base-pair insert, which is consistent with a cooperative B-Z transition.

Base Sequence↗
PubMed1983

Can DNA methylation regulate gene expression?

The E2a region of the Ad2 genome encodes the Ad2-specific DBP. An inverse correlation between the level of DNA methylation at the 5'-CC*GG-3' sites of the E2a region and the extent of expression of DBP has been demonstrated in Ad2-transformed hamster cell lines (Vardimon et al. 1980). Four different leaders are used in the transcription of the E2a region in cells productively infected with Ad2. The leader located at coordinate 75 on the viral genome is used early after infection and the other three leaders are used late after infection (Chow et al. 1979). The analysis of the integration patterns of the viral DNA in the Ad2-transformed cell lines has revealed that the early leader is deleted in the cell lines which do not express the DBP (Vardimon and Doerfler 1981). The late leader located at coordinate 72 on the viral genome is present. The region encoding that late leader has been subcloned, and the cytoplasmic RNA from the cell line which expresses the DBP has been analyzed. It has been shown that the late leader is used in transformed cells. Hence the absence of the early leader cannot be the immediate reason for the lack of expression of the DBP. Correlations between DNA methylation and the absence of gene expression may indicate that methylation regulates gene expression or that methylation is the consequence of lacking gene expression. In order to decide between these alternatives an in vitro system has been employed. The HindIII A fragment of the Ad2 DNA which encodes the DBP has been methylated in vitro by the HpaII DNA methyltransferase. Methylated or unmethylated HindIII A fragment has been microinjected into the nuclei of Xenopus laevis oocytes. Unmethylated HindIII A fragment has been found to be expressed as specific viral RNA, whereas no viral RNA can be found in oocytes microinjected with methylated HindIII A fragment. The possibility of a nonspecific inhibitory factor in the methylated DNA preparation has been ruled out by the simultaneous microinjection of sea urchin histone gene DNA together with the methylated HindIII A fragment. Histone genes are expressed, while the expression of the methylated viral gene is blocked. By using the single-strand-specific endonuclease S1 we have shown that in Xenopus laevis oocytes initiation of transcription of the E2a region starts exactly at the same site as in Ad2 productively infected cells. These results provide direct evidence for the notion that DNA methylation at specific sites is involved in the regulation of gene expression.

5-Methylcytosine↗
PubMed1982

Patch homologies and the integration of adenovirus DNA in mammalian cells.

The hamster cell line HE5 has been derived from primary hamster embryo cells by transformation with human adenovirus type 2 (Ad2). Each cell contains 2-3 copies of Ad2 DNA inserted into host DNA at apparently identical sites. The site of the junction between the right terminus of Ad2 DNA and hamster cell DNA was cloned and sequenced. The eight [corrected] right terminal nucleotides of Ad2 DNA were deleted. The unoccupied cellular DNA sequence in cell line HE5 , corresponding to the site of the junction between Ad2 and hamster cell DNA, was also cloned; 120-130 nucleotides in the cellular DNA were found to be identical to the cellular DNA sequence in the cloned junction DNA fragment, up to the site of the junction. The unoccupied and the occupied cellular DNAs and the adjacent viral DNA exhibited a few short nucleotide homologies. Patch homologies ranging in length from dodeca - to octanucleotides were detected by computer analyses at locations more remote from the junction site. When the right terminal nucleotide sequence of Ad2 DNA was matched to randomly selected sequences of 401 nucleotides from vertebrate or prokaryotic DNA, similar homologies were observed. It is likely that foreign (viral) DNA can be inserted via short sequence homologies at many different sites of cellular DNA.

Adenoviruses, Human↗
PubMed1982

Expression of a cloned adenovirus gene is inhibited by in vitro methylation.

In many viral and nonviral eukaryotic systems, an inverse correlation has been observed between the extent of DNA methylation at 5'-C-C-G-G-3' sites and the extent of expression of specific genes as mRNA. The E2a region of adenovirus serotype 2 (Ad2) DNA encodes the Ad2-specific DNA binding protein required for viral DNA replication. In three lines of Ad2 transformed hamster cells (HE1, HE2, and HE3), multiple copies of the major part of the Ad2 genome persist in an integrated state. Cell lines HE2 and HE3 do not express the DNA-binding protein whereas line HE1 does so. It has been shown that, in cell line HE1, all 5'-C-C-G-G-3' (Hpa II/MspI) sites in the E2a region remain unmethylated. Conversely, in lines HE2 and HE3 lacking expression of the E2a region all Hpa II sites are methylated. The cloned E2a region of Ad2 DNA, the HindIII A fragment in pBR322, was methylated in vitro by using Hpa II DNA methyltransferase (5'-C-C*G-G-3') or was left unmethylated. In vitro methylation did not break or nick supercoiled circular DNA. Methylated or unmethylated DNA was then microinjected into the nuclei of Xenopus laevis oocytes, and the subsequent synthesis of Ad2-specific RNA was monitored. In vitro-methylated DNA remained in the methylated state for 24 hr on microinjection into nuclei of xenopus oocytes; unmethylated DNA remained unmethylated. When the injected DNA had been methylated by using Hpa H DNA methyltransferase, Ad2-specific RNA was not synthesized as late as 24 hr after microinjection. Unmethylated DNA was readily expressed into Ad2-specific RNA. As an internal control, unmethylated histone genes (h22 DNA) from sea urchin were microinjected together with methylated E2a DNA from Ad2. Ad2-specific RNA was not found; h22 DNA-specific RNA was readily detected. This finding ruled out nonspecific inhibitory effects in the methylated DNA preparation. Ir was also shown that transcription of the unmethylated HindIII A fragment of Ad2 DNA in Xenopus oocytes was initiated on the late promoter of the E2a region. The same promoter was used in productively infected KB cells. Methylation by BsuRI methylse (5'-G'G-C*C-3') did not inactivate the HindIII A fragment. These results provide evidence for the notion that methylated sequences at highly specific sites are involved in the regulation of gene expression. The actual nature of the regulatory signal is not yet understood.

Adenoviruses, Human↗
PubMed1982

In vitro methylation of the BsuRI (5'-GGCC-3') sites in the E2a region of adenovirus type 2 DNA does not affect expression in Xenopus laevis oocytes.

The early region 2a (E2a) of adenovirus type 2 (Ad2) DNA codes for a 72,000-dalton DNA-binding protein and is expressed in the Ad2-transformed hamster cell line HE1 but not in cell lines HE2 and HE3 (H. Esche, J. Virol. 41:1076-1082, 1982; K. Johansson et al., J. Virol. 27:628-639, 1978). An inverse correlation between DNA methylation at the 5'-CCGG-3' sites of the E2a region and of gene expression in these cell lines has been observed (L. Vardimon et al., Nucleic Acids Res. 8:2461-2473, 1980). When the cloned E2a region of Ad2 DNA is methylated in vitro at the 5'-CCGG-3' sites, the gene is not transcribed after being injected into the nuclei of Xenopus laevis oocytes, whereas unmethylated DNA is expressed (L. Vardimon et al., Eur. J. Cell Biol. 25:13-15, 1981; L. Vardimon et al., Proc. Natl. Acad. Sci. U.S.A. 79:1073-1077, 1982). These data demonstrate that DNA methylation is directly involved in the shut-off of transcription. In the present communication we investigated in detail the control region of the gene for the DNA-binding protein in Ad2-transformed cell lines and showed that the first late control region (map coordinate 72 on the viral DNA) of the E2a region is present in its entirety in cell lines HE1, HE2, and HE3. The HaeIII sites (5'-GGCC-3') in the E2a region in all three cell lines were not methylated. When the DNA methyltransferase BsuRI was used, all 5'-GGCC-3' sites in the cloned E2a region of Ad2 DNA were methylated in vitro. It was shown that methylation of these sites did not inhibit the expression of this viral gene in X. laevis oocytes. Thus, for methylation to affect gene expression in the E2a region it has to occur at specific sites (e.g., 5'-CCGG-3') which may be different for other genes.

Adenoviridae↗
PubMed1981

Methylation of adenovirus genes in transformed cells and in vitro: influence on the regulation of gene expression?

An inverse correlation has been described between the levels of DNA methylation in specific segments of adenovirus DNA integrated into the genomes of transformed and tumor cells and the extent to which these segments are expressed as messenger RNA. In the adenovirus type 2 (Ad2)-transformed hamster cell lines HE2 and HE3, the virus-specific DNA binding protein (DBP) is not expressed, and the DNA in the DBP gene is completely methylated in all 5'-CCGG-3' sites. At least part of the late promoter/leader sequence of the DBP gene is present in cell lines HE2 and HE3. In line HE1, on the other hand, the DBP is expressed, and the DNA in the DBP gene is unmethylated at the 5'-CCGG-3' (HpaII) sites. The late promotor/leader sequence of the DBP gene is expressed in cytoplasmic RNA isolated from line HE1. The effect of DNA methylation has also been tested in vitro in a microinjection system using Xenopus laevis oocytes. Unmethylated DNA fragments of Ad2 (E2a region) have been found to serve as active templates. When the same fragments are methylated at the 5'-CCGG-3' sites by the HpaII DNA-methyltransferase, viral RNA synthesis is inhibited upon microinjection into oocyte nuclei. These results provide direct evidence for the notion that DNA methylated at highly specific sites is somehow involved in the regulation of gene expression.

5-Methylcytosine↗
PubMed1980

DNA methylation and viral gene expression in adenovirus-transformed and -infected cells.

The level of DNA methylation in adenovirus type 2 (Ad2) and type 12 (Ad12) DNA was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. As previously reported virion DNA of Ad2 and Ad12 is not methylated. Parental or newly synthesized Ad2 DNA in productively infected human KB or HEK cells is not methylated either, nor is the integrated form of Ad2 DNA in productively infected cells. Hamster cells and Muntiacus muntjak cells are abortively infected by Ad12. We have not detected methylation of Ad12 DNA in hamster or Muntiacus muntjak cells. An inverse correlation between the level of methylation and the extent of expression of viral DNA in Ad12-transformed hamster cells has been described earlier. A similar relation has been found for the EcoRI fragment B of Ad2 DNA which is not methylated but is expressed as the Ad2 DNA-binding (72K) protein in the Ad2-transformed hamster line HE1. Conversely, the same segment is completely methylated in lines HE2 and HE3, and there is apparently no evidence for the expression of the 72K protein in these cell lines.

Adenoviruses, Human↗