Limits...
Expression and sub-cellular localization of human ABH family molecules.

Tsujikawa K, Koike K, Kitae K, Shinkawa A, Arima H, Suzuki T, Tsuchiya M, Makino Y, Furukawa T, Konishi N, Yamamoto H - J. Cell. Mol. Med. (2007 Sep-Oct)

Bottom Line: Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern.In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm.These observations provide important information for the future annotation of the hABH family of molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. tujikawa@phs.osaka-u.ac.jp

ABSTRACT
AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1-methyladenine and 3-methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2-oxoglutarate- and Fe(II)-dependent (2OG-Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG-Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4-hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG-Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG-Fe(II) oxygenase domain but both an RNA-binding motif and a methyl-transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub-cellular localization of EmGFP-hABH8 was restricted to the cytoplasm. EmGFP-hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern. In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules.

Show MeSH

Related in: MedlinePlus

Real-time PCR analysis of the hABH family molecules in normal human tissues. The tissue-specific expression of hABH1–8 mRNAs was determined by a SYBR Green I-based quantitative PCR assay in normalized cDNA samples derived from 16 different human tissues. The expression levels of the hABH transcripts in the human tissues were normalized for the expression levels of the glyceraldehyde-3-phophate dehydrogenase (GAPDH) transcript in the same cDNAs. 1: brain, 2: thy-mus, 3:heart, 4:lung, 5: liver, 6: spleen, 7: pancreas, 8: small intestine, 9: colon, 10: kidney, 11: prostate, 12: testis, 13: ovary, 14: skeletal muscle, 15: peripheral blood leukocytes and 16: placenta. The results shown are the averages of three independent measurements per tissue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4401260&req=5

fig04: Real-time PCR analysis of the hABH family molecules in normal human tissues. The tissue-specific expression of hABH1–8 mRNAs was determined by a SYBR Green I-based quantitative PCR assay in normalized cDNA samples derived from 16 different human tissues. The expression levels of the hABH transcripts in the human tissues were normalized for the expression levels of the glyceraldehyde-3-phophate dehydrogenase (GAPDH) transcript in the same cDNAs. 1: brain, 2: thy-mus, 3:heart, 4:lung, 5: liver, 6: spleen, 7: pancreas, 8: small intestine, 9: colon, 10: kidney, 11: prostate, 12: testis, 13: ovary, 14: skeletal muscle, 15: peripheral blood leukocytes and 16: placenta. The results shown are the averages of three independent measurements per tissue.

Mentions: The expression levels of hABH1 to hABH8 mRNAs in normal human tissues were determined in a normalized panel of 16 human tissues by quantitative realtime PCR. Table 2 lists the specific primer pairs for each hABH amplification and the number of nucleotide in the amplified fragments within the hABH ORFs. The hABH2 primers were purchased from Qiagen (Cat. No. QT00219093) and their nucleotide sequence are not disclosed. A 117-bp fragment within GAPDH was amplified as a housekeeping gene and the results were expressed as a relative fold against GAPDH. As shown in Figure 4, relatively high expression levels of hABH1 to hABH8 transcripts were detected in the pancreas and testis, although their expression was ubiquitously observed in all the human tissues examined.


Expression and sub-cellular localization of human ABH family molecules.

Tsujikawa K, Koike K, Kitae K, Shinkawa A, Arima H, Suzuki T, Tsuchiya M, Makino Y, Furukawa T, Konishi N, Yamamoto H - J. Cell. Mol. Med. (2007 Sep-Oct)

Real-time PCR analysis of the hABH family molecules in normal human tissues. The tissue-specific expression of hABH1–8 mRNAs was determined by a SYBR Green I-based quantitative PCR assay in normalized cDNA samples derived from 16 different human tissues. The expression levels of the hABH transcripts in the human tissues were normalized for the expression levels of the glyceraldehyde-3-phophate dehydrogenase (GAPDH) transcript in the same cDNAs. 1: brain, 2: thy-mus, 3:heart, 4:lung, 5: liver, 6: spleen, 7: pancreas, 8: small intestine, 9: colon, 10: kidney, 11: prostate, 12: testis, 13: ovary, 14: skeletal muscle, 15: peripheral blood leukocytes and 16: placenta. The results shown are the averages of three independent measurements per tissue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401260&req=5

fig04: Real-time PCR analysis of the hABH family molecules in normal human tissues. The tissue-specific expression of hABH1–8 mRNAs was determined by a SYBR Green I-based quantitative PCR assay in normalized cDNA samples derived from 16 different human tissues. The expression levels of the hABH transcripts in the human tissues were normalized for the expression levels of the glyceraldehyde-3-phophate dehydrogenase (GAPDH) transcript in the same cDNAs. 1: brain, 2: thy-mus, 3:heart, 4:lung, 5: liver, 6: spleen, 7: pancreas, 8: small intestine, 9: colon, 10: kidney, 11: prostate, 12: testis, 13: ovary, 14: skeletal muscle, 15: peripheral blood leukocytes and 16: placenta. The results shown are the averages of three independent measurements per tissue.
Mentions: The expression levels of hABH1 to hABH8 mRNAs in normal human tissues were determined in a normalized panel of 16 human tissues by quantitative realtime PCR. Table 2 lists the specific primer pairs for each hABH amplification and the number of nucleotide in the amplified fragments within the hABH ORFs. The hABH2 primers were purchased from Qiagen (Cat. No. QT00219093) and their nucleotide sequence are not disclosed. A 117-bp fragment within GAPDH was amplified as a housekeeping gene and the results were expressed as a relative fold against GAPDH. As shown in Figure 4, relatively high expression levels of hABH1 to hABH8 transcripts were detected in the pancreas and testis, although their expression was ubiquitously observed in all the human tissues examined.

Bottom Line: Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern.In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm.These observations provide important information for the future annotation of the hABH family of molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. tujikawa@phs.osaka-u.ac.jp

ABSTRACT
AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1-methyladenine and 3-methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2-oxoglutarate- and Fe(II)-dependent (2OG-Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG-Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4-hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG-Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG-Fe(II) oxygenase domain but both an RNA-binding motif and a methyl-transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub-cellular localization of EmGFP-hABH8 was restricted to the cytoplasm. EmGFP-hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern. In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules.

Show MeSH
Related in: MedlinePlus