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Conserved overlapping gene arrangement, restricted expression, and biochemical activities of DNA polymerase ν (POLN).

Takata K, Tomida J, Reh S, Swanhart LM, Takata M, Hukriede NA, Wood RD - J. Biol. Chem. (2015)

Bottom Line: Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development.These properties are conserved with the human enzyme.Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957, the University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, ktakata@mdanderson.org.

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Independent expression of POLN and HAUS3 and preferential expression of POLN in testis.A, RT-PCR analysis of POLN and HAUS3 in 833K, human testis, and 293T cDNA. PCR was performed with primers in exon 1 of POLN and HAUS3 (F1), ORF of HAUS3 (F3 and B1), and ORF of POLN (F2, F4, and B2). Positions of the primers are diagramed above the gel picture. β-Actin was used as a control. Expected product size (bp) are F1 + B2, 1474; F2 + B2, 1384; F4 + B2, 203; F1 + B1, 999; F3 + B1, 736; β-actin, 353. B, real time PCR analysis in human cultured cells and human testis. The y axis indicates the absolute quantity of transcripts for POLN, POLQ, and HAUS3 per 40 ng of total RNA isolated from 1618K, 833K, RKO, SUSA, TERA1, 293T, and testis. C, RT-PCR analysis of Poln and Haus3 in mouse testis and R1-ES cDNA. PCR was performed with primers in exon 1 of Poln and Haus3 (1), ORF of Haus3 (2 and 3), and ORF of Poln (4 and 5). Positions of the primers are diagramed above the gel picture. D, Rev3L and PolL were used as controls. Expected product size (bp) are 1 + 3, 1867; 2 + 3, 1713; 1 + 5, 2628; 4 + 5, 2595; Rev3L, 350 and 478; PolL, 1720. PCR products were separated on a 1% agarose gel and visualized with EZ-Vision DNA Dye (A) or ethidium bromide (C and D).
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Figure 5: Independent expression of POLN and HAUS3 and preferential expression of POLN in testis.A, RT-PCR analysis of POLN and HAUS3 in 833K, human testis, and 293T cDNA. PCR was performed with primers in exon 1 of POLN and HAUS3 (F1), ORF of HAUS3 (F3 and B1), and ORF of POLN (F2, F4, and B2). Positions of the primers are diagramed above the gel picture. β-Actin was used as a control. Expected product size (bp) are F1 + B2, 1474; F2 + B2, 1384; F4 + B2, 203; F1 + B1, 999; F3 + B1, 736; β-actin, 353. B, real time PCR analysis in human cultured cells and human testis. The y axis indicates the absolute quantity of transcripts for POLN, POLQ, and HAUS3 per 40 ng of total RNA isolated from 1618K, 833K, RKO, SUSA, TERA1, 293T, and testis. C, RT-PCR analysis of Poln and Haus3 in mouse testis and R1-ES cDNA. PCR was performed with primers in exon 1 of Poln and Haus3 (1), ORF of Haus3 (2 and 3), and ORF of Poln (4 and 5). Positions of the primers are diagramed above the gel picture. D, Rev3L and PolL were used as controls. Expected product size (bp) are 1 + 3, 1867; 2 + 3, 1713; 1 + 5, 2628; 4 + 5, 2595; Rev3L, 350 and 478; PolL, 1720. PCR products were separated on a 1% agarose gel and visualized with EZ-Vision DNA Dye (A) or ethidium bromide (C and D).

Mentions: The expression patterns of POLN and HAUS3 were also distinct in mammals. We compared expression of POLN, POLQ, and HAUS3 in mRNA from human testis and human cell lines (Fig. 5). All three genes were readily detected in testis. In cultured cells, only low levels of partial POLN transcript were detectable by RT-PCR (Fig. 5A) and by real time PCR (qPCR) (Fig. 5B). Using RT-PCR, we designed primers to test whether POLN and HAUS3 are independent mature spliced transcripts. The F1-B2 primer pair amplified a product from human testis of the size expected for POLN but not for a fusion of HAUS3 and POLN transcript (Fig. 5A). Consistently, no evidence for a fused transcript was found in the 5′-RACE experiments (Fig. 3A). Full-length POLN transcript was not detected by this primer set in the human 833K or 293T cell lines, although a transcript representing a portion of the mRNA could be detected (primer set F4 + B2). The F1 + B1 primer set yielded two major bands and one minor band. The major bands are consistent with the predicted size of the two documented transcript variants of human HAUS3 (accession numbers NM_001303143 and NM_024511). A third lower molecular weight band arising with this primer set may also be an alternatively spliced product.


Conserved overlapping gene arrangement, restricted expression, and biochemical activities of DNA polymerase ν (POLN).

Takata K, Tomida J, Reh S, Swanhart LM, Takata M, Hukriede NA, Wood RD - J. Biol. Chem. (2015)

Independent expression of POLN and HAUS3 and preferential expression of POLN in testis.A, RT-PCR analysis of POLN and HAUS3 in 833K, human testis, and 293T cDNA. PCR was performed with primers in exon 1 of POLN and HAUS3 (F1), ORF of HAUS3 (F3 and B1), and ORF of POLN (F2, F4, and B2). Positions of the primers are diagramed above the gel picture. β-Actin was used as a control. Expected product size (bp) are F1 + B2, 1474; F2 + B2, 1384; F4 + B2, 203; F1 + B1, 999; F3 + B1, 736; β-actin, 353. B, real time PCR analysis in human cultured cells and human testis. The y axis indicates the absolute quantity of transcripts for POLN, POLQ, and HAUS3 per 40 ng of total RNA isolated from 1618K, 833K, RKO, SUSA, TERA1, 293T, and testis. C, RT-PCR analysis of Poln and Haus3 in mouse testis and R1-ES cDNA. PCR was performed with primers in exon 1 of Poln and Haus3 (1), ORF of Haus3 (2 and 3), and ORF of Poln (4 and 5). Positions of the primers are diagramed above the gel picture. D, Rev3L and PolL were used as controls. Expected product size (bp) are 1 + 3, 1867; 2 + 3, 1713; 1 + 5, 2628; 4 + 5, 2595; Rev3L, 350 and 478; PolL, 1720. PCR products were separated on a 1% agarose gel and visualized with EZ-Vision DNA Dye (A) or ethidium bromide (C and D).
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Figure 5: Independent expression of POLN and HAUS3 and preferential expression of POLN in testis.A, RT-PCR analysis of POLN and HAUS3 in 833K, human testis, and 293T cDNA. PCR was performed with primers in exon 1 of POLN and HAUS3 (F1), ORF of HAUS3 (F3 and B1), and ORF of POLN (F2, F4, and B2). Positions of the primers are diagramed above the gel picture. β-Actin was used as a control. Expected product size (bp) are F1 + B2, 1474; F2 + B2, 1384; F4 + B2, 203; F1 + B1, 999; F3 + B1, 736; β-actin, 353. B, real time PCR analysis in human cultured cells and human testis. The y axis indicates the absolute quantity of transcripts for POLN, POLQ, and HAUS3 per 40 ng of total RNA isolated from 1618K, 833K, RKO, SUSA, TERA1, 293T, and testis. C, RT-PCR analysis of Poln and Haus3 in mouse testis and R1-ES cDNA. PCR was performed with primers in exon 1 of Poln and Haus3 (1), ORF of Haus3 (2 and 3), and ORF of Poln (4 and 5). Positions of the primers are diagramed above the gel picture. D, Rev3L and PolL were used as controls. Expected product size (bp) are 1 + 3, 1867; 2 + 3, 1713; 1 + 5, 2628; 4 + 5, 2595; Rev3L, 350 and 478; PolL, 1720. PCR products were separated on a 1% agarose gel and visualized with EZ-Vision DNA Dye (A) or ethidium bromide (C and D).
Mentions: The expression patterns of POLN and HAUS3 were also distinct in mammals. We compared expression of POLN, POLQ, and HAUS3 in mRNA from human testis and human cell lines (Fig. 5). All three genes were readily detected in testis. In cultured cells, only low levels of partial POLN transcript were detectable by RT-PCR (Fig. 5A) and by real time PCR (qPCR) (Fig. 5B). Using RT-PCR, we designed primers to test whether POLN and HAUS3 are independent mature spliced transcripts. The F1-B2 primer pair amplified a product from human testis of the size expected for POLN but not for a fusion of HAUS3 and POLN transcript (Fig. 5A). Consistently, no evidence for a fused transcript was found in the 5′-RACE experiments (Fig. 3A). Full-length POLN transcript was not detected by this primer set in the human 833K or 293T cell lines, although a transcript representing a portion of the mRNA could be detected (primer set F4 + B2). The F1 + B1 primer set yielded two major bands and one minor band. The major bands are consistent with the predicted size of the two documented transcript variants of human HAUS3 (accession numbers NM_001303143 and NM_024511). A third lower molecular weight band arising with this primer set may also be an alternatively spliced product.

Bottom Line: Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development.These properties are conserved with the human enzyme.Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957, the University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, ktakata@mdanderson.org.

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Related in: MedlinePlus