Functional characterization of Pura in vivo and in vitro - Pura knock-out mice and Pura DNA unwinding activity
type: CreativeWork
description: Pura is a ubiquitous DNA- and RNA-binding protein that is highly conserved in eukaryotic organisms. Pura binds preferentially to purine-rich single-stranded DNA. To date, biochemical analyses of Pura have indicated that this protein might be involved in diverse cellular functions, including DNA replication, transcription, translation and cell growth control (Gallia et al., 2000). However, the function of Pura in vivo remains unclear. In this study, Pura knock-out mice (KO) were generated using homologous recombination techniques in ES cells. The Pura gene locus was isolated from 129/SvJ Mouse Genomic Library and partially sequenced. A targeting vector using positive and negative selection was generated for homologous recombination and was finally electroporated into E14 ES cells for five times. Out of 2400 ES cell clones only one positive ES cell clone (KO 248) was identified by Southern-blot analysis. Pura KO mice generated from ES cell clone KO 248 displayed multiple phenotypes. Apparently, they were normal when born, but developed assorted abnormalities. 12-14 days after birth they developed a severe and continuous tremor phenotype similar to shiverer mice that lack myelin basic protein (MBP) (Chernoff, 1981). This tremor phenotype continued unabated until natural death occurred. Pura KO mice seemed to have a shorter life span since none of the Pura KO mice lived longer than 6 months while the wild type mice lived normally. Pura has been shown to be involved in developmental and transcriptional control of MBP (Haas et al., 1995), therefore the MBP levels in the brain stems of Pura KO mice were investigated. It could be shown that MBP levels in 16-day-old Pura KO mice were decreased by 30-40% as compared to heterozygotes and wild type. However, MBP levels of the 63-dayold Pura KO, heterozygous and wild type mice did not show significant difference. Although Pura KO mice showed a tremor phenotype comparable to shiverer mice and displayed reduced MBP levels at the b…
abstract: Pura is a ubiquitous DNA- and RNA-binding protein that is highly conserved in eukaryotic organisms. Pura binds preferentially to purine-rich single-stranded DNA. To date, biochemical analyses of Pura have indicated that this protein might be involved in diverse cellular functions, including DNA replication, transcription, translation and cell growth control (Gallia et al., 2000). However, the function of Pura in vivo remains unclear. In this study, Pura knock-out mice (KO) were generated using homologous recombination techniques in ES cells. The Pura gene locus was isolated from 129/SvJ Mouse Genomic Library and partially sequenced. A targeting vector using positive and negative selection was generated for homologous recombination and was finally electroporated into E14 ES cells for five times. Out of 2400 ES cell clones only one positive ES cell clone (KO 248) was identified by Southern-blot analysis. Pura KO mice generated from ES cell clone KO 248 displayed multiple phenotypes. Apparently, they were normal when born, but developed assorted abnormalities. 12-14 days after birth they developed a severe and continuous tremor phenotype similar to shiverer mice that lack myelin basic protein (MBP) (Chernoff, 1981). This tremor phenotype continued unabated until natural death occurred. Pura KO mice seemed to have a shorter life span since none of the Pura KO mice lived longer than 6 months while the wild type mice lived normally. Pura has been shown to be involved in developmental and transcriptional control of MBP (Haas et al., 1995), therefore the MBP levels in the brain stems of Pura KO mice were investigated. It could be shown that MBP levels in 16-day-old Pura KO mice were decreased by 30-40% as compared to heterozygotes and wild type. However, MBP levels of the 63-dayold Pura KO, heterozygous and wild type mice did not show significant difference. Although Pura KO mice showed a tremor phenotype comparable to shiverer mice and displayed reduced MBP levels at the b…
author: Ding, Haiyan
date published: 2001-12-14
date modified: 2026-07-24T02:51:24Z
provider: Ludwig Maxmilians Universität München
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Ludwig-Maximilians-Universität