We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with si...We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.展开更多
Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D ...Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.展开更多
The allelic distribution of EcoRI and BamHI fragments of ras family genes between the human primary gastric cancer tissues and the corresponding adjacent normal tissues did not show any differences. Three genotypes of...The allelic distribution of EcoRI and BamHI fragments of ras family genes between the human primary gastric cancer tissues and the corresponding adjacent normal tissues did not show any differences. Three genotypes of BamHI restriction fragments length polymorphism of c-H-ras were revealed. No significant differences in the RFLPs were observed between normal individuals and gastric cancer patients. Four protooncogenes, c-H-ras, N-ras, c-myc and c-fos, were found to be transcriptionally active in the gastric cancer tissues in some cases examined. The comparison of the expression of these oncogenes between the malignant tissues and the corresponding normal tissues showed differential patterns. The expression of c-H-ras at cellular level was detected with in situ hybridization. The enhanced expression of c-H-ras in the gastric cancer cells was demonstrated, but the degree of the expession among the cancer cells was shown to be heterogeneous. In addition, the enhanced expression of c-H-ras was seen in the inflammatory cells.展开更多
文摘We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.
基金supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 5R01AR037399the UCLA Vector Core (Emmanuelle Faure and Kip Hermann) for vector and viral preparations supported by JCCC/P30 CA016042 and CURE/P30 DK41301
文摘Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.
文摘The allelic distribution of EcoRI and BamHI fragments of ras family genes between the human primary gastric cancer tissues and the corresponding adjacent normal tissues did not show any differences. Three genotypes of BamHI restriction fragments length polymorphism of c-H-ras were revealed. No significant differences in the RFLPs were observed between normal individuals and gastric cancer patients. Four protooncogenes, c-H-ras, N-ras, c-myc and c-fos, were found to be transcriptionally active in the gastric cancer tissues in some cases examined. The comparison of the expression of these oncogenes between the malignant tissues and the corresponding normal tissues showed differential patterns. The expression of c-H-ras at cellular level was detected with in situ hybridization. The enhanced expression of c-H-ras in the gastric cancer cells was demonstrated, but the degree of the expession among the cancer cells was shown to be heterogeneous. In addition, the enhanced expression of c-H-ras was seen in the inflammatory cells.
