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Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I.

Xu L, Yang L, Hashimoto K, Anderson M, Kohlhagen G, Pommier Y, D'Arpa P - BMC Genomics (2002)

Bottom Line: BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.Epitope-tagged BTBD2 localized to cytoplasmic bodies.The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA. lixu@usuhs.mil

ABSTRACT

Background: Two-hybrid screening for proteins that interact with the core domain of human topoisomerase I identified two novel proteins, BTBD1 and BTBD2, which share 80% amino acid identities.

Results: The interactions were confirmed by co-precipitation assays demonstrating the physical interaction of BTBD1 and BTBD2 with 100 kDa topoisomerase I from HeLa cells. Deletion mapping using two-hybrid and GST-pulldown assays demonstrated that less than the C-terminal half of BTBD1 is sufficient for binding topoisomerase I. The topoisomerase I sequences sufficient to bind BTBD2 were mapped to residues 215 to 329. BTBD2 with an epitope tag localized to cytoplasmic bodies. Using truncated versions that direct BTBD2 and TOP1 to the same cellular compartment, either the nucleus or the cytoplasm, co-localization was demonstrated in co-transfected Hela cells. The supercoil relaxation and DNA cleavage activities of topoisomerase I in vitro were affected little or none by co-incubation with BTBD2. Northern analysis revealed only a single sized mRNA for each BTBD1 and BTBD2 in all human tissues tested. Characterization of BTBD2 mRNA revealed a 255 nucleotide 90% GC-rich region predicted to encode the N-terminus. BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.

Conclusions: BTBD1 and BTBD2 belong to a small family of uncharacterized proteins that appear to be specific to animals. Epitope-tagged BTBD2 localized to cytoplasmic bodies. The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

No MeSH data available.


RT-PCR identifies a highly GC-rich sequence predicted to encode the ammo terminal region of BTBD2. A. RT-PCR strategy and the sequence of the amplified region are shown: BTBD2 coding region (open arrow), 3'-UTR (narrow open bar) and exons (wide open bar, 1 – 9), sense primers (UO and UI), antisense primers (LI and LO), GC-rich region (solid bar and sequence). B. Products of the RT-PCR are shown on an ethidium bromide gel. Reverse transcription was performed with antisense primers complementary to exon 2. The schematic shows the relevant portion of the mRNA template, the RT primers (LI and LO), the sense primers (UO and UI), and the amplified products (460 and 306 bp) that correspond to the bands on the agarose gel stained with ethidium bromide (lanes 1 & 2, DNA size standards; lane 3, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UO and LI; lane 4, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UI and LO.
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Figure 1: RT-PCR identifies a highly GC-rich sequence predicted to encode the ammo terminal region of BTBD2. A. RT-PCR strategy and the sequence of the amplified region are shown: BTBD2 coding region (open arrow), 3'-UTR (narrow open bar) and exons (wide open bar, 1 – 9), sense primers (UO and UI), antisense primers (LI and LO), GC-rich region (solid bar and sequence). B. Products of the RT-PCR are shown on an ethidium bromide gel. Reverse transcription was performed with antisense primers complementary to exon 2. The schematic shows the relevant portion of the mRNA template, the RT primers (LI and LO), the sense primers (UO and UI), and the amplified products (460 and 306 bp) that correspond to the bands on the agarose gel stained with ethidium bromide (lanes 1 & 2, DNA size standards; lane 3, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UO and LI; lane 4, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UI and LO.

Mentions: The longest BTBD2 cDNA obtained from two-hybrid screening (2413 bp) contained an ORF starting at the 5' end. To obtain the missing 5' cDNA, a nested PCR strategy was used to amplify the sequence from four independent cDNA libraries, but no extended sequences were obtained. 5'-RACE (Life Technologies) also failed to produce longer clones. This difficulty to obtain longer cDNA clones was seen by others [21]. The GC richness at the 5' end (85 nucleotides are 79% GC) may form secondary structures that block reverse transcriptase. Analysis of the BTBD2 gene immediately upstream of the 5' end of our cDNA predicts an ORF that begins with an ATG and encodes 85 amino acids in-frame with the ORF of our longest BTBD2 clone; however, the 255 nucleotides encoding these in-frame amino acids sequence are 89% GC rich. To determine if this sequence might be part of the BTBD2 mRNA, RT-PCR followed by nested PCR was used. Two antisense primers complementary to exon 2 were used to prime reverse transcription. Sense primers for PCR amplification were located within predicted exon 1 (Fig. 1A). Amplified products of 460 and 306 bp that are predicted from the mRNA template resulted (Fig. 1B). If genomic DNA had been the template, products greater than 18 kb would have been obtained. Furthermore, sequencing of the 460 bp-amplification product confirmed it to be the predicted mRNA (Fig. 1A). The cDNA starting at the predicted start codon was deposited in GenBank (AF355797).


Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I.

Xu L, Yang L, Hashimoto K, Anderson M, Kohlhagen G, Pommier Y, D'Arpa P - BMC Genomics (2002)

RT-PCR identifies a highly GC-rich sequence predicted to encode the ammo terminal region of BTBD2. A. RT-PCR strategy and the sequence of the amplified region are shown: BTBD2 coding region (open arrow), 3'-UTR (narrow open bar) and exons (wide open bar, 1 – 9), sense primers (UO and UI), antisense primers (LI and LO), GC-rich region (solid bar and sequence). B. Products of the RT-PCR are shown on an ethidium bromide gel. Reverse transcription was performed with antisense primers complementary to exon 2. The schematic shows the relevant portion of the mRNA template, the RT primers (LI and LO), the sense primers (UO and UI), and the amplified products (460 and 306 bp) that correspond to the bands on the agarose gel stained with ethidium bromide (lanes 1 & 2, DNA size standards; lane 3, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UO and LI; lane 4, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UI and LO.
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Related In: Results  -  Collection

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Figure 1: RT-PCR identifies a highly GC-rich sequence predicted to encode the ammo terminal region of BTBD2. A. RT-PCR strategy and the sequence of the amplified region are shown: BTBD2 coding region (open arrow), 3'-UTR (narrow open bar) and exons (wide open bar, 1 – 9), sense primers (UO and UI), antisense primers (LI and LO), GC-rich region (solid bar and sequence). B. Products of the RT-PCR are shown on an ethidium bromide gel. Reverse transcription was performed with antisense primers complementary to exon 2. The schematic shows the relevant portion of the mRNA template, the RT primers (LI and LO), the sense primers (UO and UI), and the amplified products (460 and 306 bp) that correspond to the bands on the agarose gel stained with ethidium bromide (lanes 1 & 2, DNA size standards; lane 3, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UO and LI; lane 4, product of reverse transcription with LO, 1st round PCR with UO and LO, and 2nd round PCR with UI and LO.
Mentions: The longest BTBD2 cDNA obtained from two-hybrid screening (2413 bp) contained an ORF starting at the 5' end. To obtain the missing 5' cDNA, a nested PCR strategy was used to amplify the sequence from four independent cDNA libraries, but no extended sequences were obtained. 5'-RACE (Life Technologies) also failed to produce longer clones. This difficulty to obtain longer cDNA clones was seen by others [21]. The GC richness at the 5' end (85 nucleotides are 79% GC) may form secondary structures that block reverse transcriptase. Analysis of the BTBD2 gene immediately upstream of the 5' end of our cDNA predicts an ORF that begins with an ATG and encodes 85 amino acids in-frame with the ORF of our longest BTBD2 clone; however, the 255 nucleotides encoding these in-frame amino acids sequence are 89% GC rich. To determine if this sequence might be part of the BTBD2 mRNA, RT-PCR followed by nested PCR was used. Two antisense primers complementary to exon 2 were used to prime reverse transcription. Sense primers for PCR amplification were located within predicted exon 1 (Fig. 1A). Amplified products of 460 and 306 bp that are predicted from the mRNA template resulted (Fig. 1B). If genomic DNA had been the template, products greater than 18 kb would have been obtained. Furthermore, sequencing of the 460 bp-amplification product confirmed it to be the predicted mRNA (Fig. 1A). The cDNA starting at the predicted start codon was deposited in GenBank (AF355797).

Bottom Line: BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.Epitope-tagged BTBD2 localized to cytoplasmic bodies.The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA. lixu@usuhs.mil

ABSTRACT

Background: Two-hybrid screening for proteins that interact with the core domain of human topoisomerase I identified two novel proteins, BTBD1 and BTBD2, which share 80% amino acid identities.

Results: The interactions were confirmed by co-precipitation assays demonstrating the physical interaction of BTBD1 and BTBD2 with 100 kDa topoisomerase I from HeLa cells. Deletion mapping using two-hybrid and GST-pulldown assays demonstrated that less than the C-terminal half of BTBD1 is sufficient for binding topoisomerase I. The topoisomerase I sequences sufficient to bind BTBD2 were mapped to residues 215 to 329. BTBD2 with an epitope tag localized to cytoplasmic bodies. Using truncated versions that direct BTBD2 and TOP1 to the same cellular compartment, either the nucleus or the cytoplasm, co-localization was demonstrated in co-transfected Hela cells. The supercoil relaxation and DNA cleavage activities of topoisomerase I in vitro were affected little or none by co-incubation with BTBD2. Northern analysis revealed only a single sized mRNA for each BTBD1 and BTBD2 in all human tissues tested. Characterization of BTBD2 mRNA revealed a 255 nucleotide 90% GC-rich region predicted to encode the N-terminus. BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.

Conclusions: BTBD1 and BTBD2 belong to a small family of uncharacterized proteins that appear to be specific to animals. Epitope-tagged BTBD2 localized to cytoplasmic bodies. The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

No MeSH data available.