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. 2000 Jun 15;28(12):2277-85.
doi: 10.1093/nar/28.12.2277.

Analysis of uracil-DNA glycosylases from the murine Ung gene reveals differential expression in tissues and in embryonic development and a subcellular sorting pattern that differs from the human homologues

H Nilsen  1 , K S Steinsbekk, M Otterlei, G Slupphaug, P A Aas, H E Krokan
Affiliations

Analysis of uracil-DNA glycosylases from the murine Ung gene reveals differential expression in tissues and in embryonic development and a subcellular sorting pattern that differs from the human homologues

H Nilsen et al. Nucleic Acids Res. .

Abstract

The murine UNG: gene encodes both mitochondrial (Ung1) and nuclear (Ung2) forms of uracil-DNA glyco-sylase. The gene contains seven exons organised like the human counterpart. While the putative Ung1 promoter (P(B)) and the human P(B) contain essentially the same, although differently organised, transcription factor binding elements, the Ung2 promoter (P(A)) shows limited homology to the human counterpart. Transient transfection of chimaeric promoter-luciferase constructs demonstrated that both promoters are functional and that P(B) drives transcription more efficiently than P(A). mRNAs for Ung1 and Ung2 are found in all adult tissues analysed, but they are differentially expressed. Furthermore, transcription of both mRNA forms, particularly Ung2, is induced in mid-gestation embryos. Except for a strong conservation of the 26 N-terminal residues in Ung2, the subcellular targeting sequences in the encoded proteins have limited homology. Ung2 is transported exclusively to the nucleus in NIH 3T3 cells as expected. In contrast, Ung1 was sorted both to nuclei and mitochondria. These results demonstrate that although the catalytic domain of uracil-DNA glycosylase is highly conserved in mouse and man, regulatory elements in the gene and subcellular sorting sequences in the proteins differ both structurally and functionally, resulting in altered contribution of the isoforms to total uracil-DNA glycosylase activity.

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Figures

Figure 1
Figure 1
Structure of the murine uracil-DNA glycosylase (Ung) gene. Exons are depicted as boxes with their start and stop positions indicated above. Arrows indicate the positions of the promoter regions. Blocks of mouse repetitive sequences are schematically illustrated as grey boxes under the line. Details can be found in GenBank under the accession number AF174485.
Figure 2
Figure 2
Activity of promoter–luciferase constructs. The chimaeric promoter constructs are illustrated schematically with the luciferase coding regions depicted as hatched boxes and the exons 1A as grey boxes. Putative transcription factor binding elements are shown as triangles and the positions in the Ung gene are indicated. The pGL2-Basic vector is omitted for simplicity. Activities of promoter constructs after transient transfection in NIH 3T3 cells were measured as the ratio of firefly and renilla luciferase activities and are given as percent of the activity expressed from ProA+B (the pGL2-Basic negative control had only 0.45% activity). The data shown are the mean ± SD of three independent experiments each performed in triplicate. Due to the presence of stop codons in intron 1a/PB, translation from exon 1A would not give a functional luciferase protein. Thus, luciferase activity resulting from transfection of ProA+B and ProA+B5′ are due to transcription from PB.
Figure 3
Figure 3
Schematic presentation of mouse promoter A. Exon 1A is shown as a black box with an arrow indicating translation start. Putative binding sites for transcription factors are identified by search for highly correlated sequence fragments versus the TFMATRIX database (35). The nucleotide sequence of a highly conserved region (1270–1314) is shown as well as the sequence and localisation of some putative cis-acting elements for binding of transcription factors.
Figure 4
Figure 4
Expression of Ung1, Ung2 and β-actin mRNA in different tissues of adult mice (A) and from different stages of embryonic development (B). The blots were successively hybridised with Ung1, Ung2, Ung1 + Ung2 and β-actin probes. The blots were stripped prior to each new hybridisation. The images were quantified on a Molecular Dynamics PhosphorImager.
Figure 5
Figure 5
Distribution of uracil-DNA glycosylase activity between different cell compartments in mouse cells. Pulse labelling with [3H]thymidine of density inhibited (triangles) and freely cycling (diamonds) NIH 3T3 cells (A). The data are means ± SEM for three independent experiments. Freely cycling and density inhibited NIH 3T3 cells were fractionated and uracil-DNA glycosylase activity was measured in nuclei (black bar) and mitochondria (white bar) (B).
Figure 6
Figure 6
The N-terminal sequences of the murine Ung1 and human UNG1 proteins may form amphiphilic helices. Alignment of the N-terminal sequences of human UNG1 and mouse Ung1 (A). Potential amphiphilic helix-forming residues are underlined. When amino acids 11–28 of UNG1 (B) and 4–21 of Ung1 (C) are plotted in an α-helical wheel with 3.6 amino acids/turn their intrinsic potential to form amphiphilic helices emerges. Charged and polar residues are shaded and positively charged residues are indicated by +.
Figure 7
Figure 7
Subcellular localisation of murine and human mitochondrial forms in fusion with EGFP in NIH 3T3 and HeLa cells. Cells were transient transfected with constructs expressing Ung11–20EGFP (A), Ung1EGFP (B), UNG11–28EGFP (C) and UNG1EGFP (D) in NIH 3T3. Ung11–20EGFP (E) and Ung1EGFP (F), UNG11–28EGFP (G) and UNG1EGFP (H) in NIH 3T3.
Figure 8
Figure 8
Subcellular localisation of murine and human nuclear forms in fusion with EGFP in NIH 3T3 and HeLa cells. Ung2EGFP in NIH 3T3 (A) and HeLa (B) and Ung21–48EGFP in NIH 3T3 (C) and HeLa (D). EGFP control is shown in (E).
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