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P Lavia

Publications and source records attributed to P Lavia.

30 records · Page 2Linked to original sources
PubMed1991

Transcription factors binding to the mouse HTF9 housekeeping promoter differ between cell types.

The mouse CpG island HTF9 harbours a bidirectional promoter shared by two housekeeping genes that are arranged head-to-head. We have previously identified several protein binding-elements across the CpG island, yet a short region around the initiation region was found to be capable of bidirectional transcription in transient expression assays, suggesting that the multiple elements of the HTF9 promoter are functionally redundant. We have now compared the binding activities in nuclear extracts from different cell types. Two protein-binding elements of HTF9 interact with widely distributed factors. A potentially strong Sp1 binding site was also identified, however Sp1 appeared to bind efficiently to its target sequence with extracts prepared from proliferating cultured cells, but not from adult organs. On the other hand, the CCAAT box upstream of one gene (HTF9-A) interacted with a liver-enriched factor, whereas no binding was detected with cultured fibroblasts extracts. Consistently, deletion of the CCAAT box affected transient expression from the HTF9-A promoter in hepatocyte, but not in fibroblast, cultures. Our results suggest that ubiquitous expression of housekeeping promoters results from the activation of alternative elements in different cell types.

Animals↗
PubMed1991

Characterization of the opposite-strand genes from the mouse bidirectionally transcribed HTF9 locus.

The mouse HTF9 locus contains two genes that are bidirectionally transcribed with opposite polarity from a shared CpG-rich island. Both genes were previously shown to be expressed in a housekeeping fashion in mouse. We have now determined the molecular organization of the genes over 12 kb surrounding the island. In addition, we show that the HTF9 locus resides in the proximal region of mouse chromosome 16. We have sequenced the cDNAs corresponding to both divergent transcripts. Both genes appear to code for novel proteins that are structurally unrelated to each other. Finally, we show that both genes are highly conserved and efficiently expressed in human cells.

Amino Acid Sequence↗
PubMed1991

The housekeeping promoter from the mouse CpG island HTF9 contains multiple protein-binding elements that are functionally redundant.

The mouse CpG-rich island HTF9 harbours the divergent RNA initiation sites shared by two genes that are both expressed in a housekeeping fashion. In this work we have analyzed the architecture of the HTF9 promoter. Gel shift assays were first employed to locate nuclear factor-binding sites within HTF9. Multiple protein-binding sites were identified across a 500 bp-long region, two of which appear to interact with novel factors. Deletion analysis was used to determine the requirements for the different sites in transient expression of a CAT reporter gene. Although multiple elements contributed to the overall promoter strength in each orientation, extensive deletions failed to affect the basal level of transcription from HTF9 in either direction. Thus, only a subset of elements is necessary to activate transcription from HTF9. Functional redundancy may be a general feature of housekeeping CpG-rich promoters.

Animals↗
PubMed1989

In vitro methylation of CpG-rich islands.

CpG islands are distinguishable from the bulk of vertebrate DNA for being unmethylated and CpG-rich. Since CpG doublets are the specific target of eukaryotic DNA methyltransferases, CpG-rich sequences might be expected to be good methyl-accepting substrates in vitro, despite their unmethylated in vivo condition. This was tested using a partially purified DNA-methyltransferase from human placenta and several cloned CpG-rich or CpG-depleted sequences. The efficiency of methylation was found to be proportional to the CpG content for CpG-depleted regions, which are representative of the bulk genome. However, methylation was much less efficient for CpG frequencies higher than 1 in 12 nucleotides, reaching only 60% of the expected level. That suggests that the close CpG spacing typical of CpG-islands somehow inhibits mammalian DNA methyltransferase. The implications of these findings on the in vivo pattern of DNA methylation are discussed.

Base Sequence↗
PubMed1987

Coincident start sites for divergent transcripts at a randomly selected CpG-rich island of mouse.

We determined the nucleotide sequence of two HTF islands that were selected at random from mouse chromosomal DNA. Both were non-methylated, G + C rich, and contained CpG at close to the expected frequency. When used as probes, the two islands detected multiple transcripts in RNA from several mouse tissues. Cloned cDNAs for the major transcripts of one island (HTF9) were isolated and used to construct a transcriptional map. We found that HTF9 contains the origin of a pair of divergent transcripts that are probably messenger RNAs. The bidirectional promoter is different from those previously observed as the major transcription start sites for each orientation are coincident on opposite strands of the DNA. The results support the view that HTF islands often mark genes, and they suggest that bidirectional transcription may be a common feature of island promoters.

Amino Acid Sequence↗
PubMed1985

Differential gene activity visualized on sister chromatids after replication in the presence of 5-azacytidine.

Mitotic chromosomes with sister chromatids bearing differentially active ribosomal gene clusters were recovered from human lymphocytes exposed to 5-azacytidine. The hypothesis was that the differential activity was determined by the hypomethylation of one of the two sister chromatids. The verification was carried out by "labeling" the 5-azacytidine-substituted chromatid with BUdR, and then checking the location of active clusters by specific staining techniques. Data obtained confirmed that the chromatid bearing the active cluster was indeed the 5-azacytidine-substituted one.

Azacitidine↗
PubMed1985

Effect of 5-azacytidine (5-azaC) on the induction of chromatid aberrations (CA) and sister-chromatid exchanges (SCE).

Experiments were carried out using human lymphocytes from a male donor in order to test the action of 5-azaC treatment on the induction of SCE and chromatid aberrations. The 5-azaC was found to increase the frequency of both baseline and MMC-induced SCEs. Using the same 5-azaC treatment conditions it was found that the frequency of X-ray-induced CA did not increase.

Azacitidine↗
PubMed1983

Activation of human ribosomal genes by 5-azacytidine.

Cultured human fibroblasts were exposed to 5-azacytidine which inhibits methylation of newly synthesized DNA. A significant increase in the mean number of Ag-stained active nucleolus organizers has been observed in treated cells. This suggests that DNA methylation is involved in modulation of human rRNA synthesis.

Azacitidine↗
PubMed1982

Silver staining of the nucleolus organizer regions (NOR) requires clusters of sulfhydryl groups.

Silver stainability of nucleolus organizer regions (NORs) appears to be correlated with the presence of grouped sulfhydryl (SH) side chains of proteins. In fact, heavy metals with high affinity for SH groups, such as Hg and Cu, do prevent the silver staining reaction. Ferricyanide, which is known to oxidize SH to disulfides, also prevents any further reaction with silver. On the other hand, alkali and reducing agents (mercaptoethanol, cyanide) do not affect silver stainability of the NORs. These results show that the silver staining reaction is not related to disulfide or persulfide groups and that alkali-soluble, acidic nuclear proteins per se do not play a major role in this process.

Cell Nucleolus↗
PubMed1981

Clonal inheritance of rRNA gene activity: cytological evidence in human cells.

Clone-specific silver staining patterns of the nucleolus organizers (NOs) were observed in clones deriving from a human diploid fibroblast culture. Each specific staining pattern corresponds to one of the nine patterns observed in the general population. Since silver stainability of the NOs is related to rRNA gene function, these data demonstrate the clonal inheritance of rRNA gene activity.

Cells, Cultured↗