Knockout Mutations (knockout + mutation)

Distribution by Scientific Domains

Selected Abstracts

Mutation spectrum in UVB-exposed skin epidermis of Xpa -knockout mice: Frequent recovery of triplet mutations

Hironobu Ikehata
Abstract Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the ,-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa,/,) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa,/, and wild-type mice. Neither photolesion was removed in the Xpa,/, epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m2 UVB increased the lacZ mutant frequency in the epidermis of Xpa,/, mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa,/, mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa,/, and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C , T transitions at dipyrimidine sites; however, Xpa,/, mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC , TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds. Environ. Mol. Mutagen., 2007. © 2006 Wiley-Liss, Inc. [source]

Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis

Gyöngyi Székely
Summary ,-1-pyrroline-5-carboxylate synthetase enzymes, which catalyse the rate-limiting step of proline biosynthesis, are encoded by two closely related P5CS genes in Arabidopsis. Transcription of the P5CS genes is differentially regulated by drought, salinity and abscisic acid, suggesting that these genes play specific roles in the control of proline biosynthesis. Here we describe the genetic characterization of p5cs insertion mutants, which indicates that P5CS1 is required for proline accumulation under osmotic stress. Knockout mutations of P5CS1 result in the reduction of stress-induced proline synthesis, hypersensitivity to salt stress, and accumulation of reactive oxygen species. By contrast, p5cs2 mutations cause embryo abortion during late stages of seed development. The desiccation sensitivity of p5cs2 embryos does not reflect differential control of transcription, as both P5CS mRNAs are detectable throughout embryonic development. Cellular localization studies with P5CS,GFP gene fusions indicate that P5CS1 is sequestered into subcellular bodies in embryonic cells, where P5CS2 is dominantly cytoplasmic. Although proline feeding rescues the viability of mutant embryos, p5cs2 seedlings undergo aberrant development and fail to produce fertile plants even when grown on proline. In seedlings, specific expression of P5CS2,GFP is seen in leaf primordia where P5CS1,GFP levels are very low, and P5CS2,GFP also shows a distinct cell-type-specific and subcellular localization pattern compared to P5CS1,GFP in root tips, leaves and flower organs. These data demonstrate that the Arabidopsis P5CS enzymes perform non-redundant functions, and that P5CS1 is insufficient for compensation of developmental defects caused by inactivation of P5CS2. [source]

Double Oestrogen Receptor , and , Knockout Mice Reveal Differences in Neural Oestrogen-Mediated Progestin Receptor Induction and Female Sexual Behaviour

A. E. Kudwa
Abstract To test the hypothesis that oestrogen receptor , (ER,) and ER, act together to mediate the actions of oestrogen in the ventromedial hypothalamus (VMH), we used mice with single or double knockout mutations of the ER, and ER, genes. Ovariectomized mice were implanted with 17,-oestradiol and killed 5 days later. Oestradiol treatment promoted progestin receptor (PR)-immunoreactivity (-ir) in the VMH of all genotypes, but was maximal in brains of wild-type and ER,KO females. Analysis of specific VMH subregions revealed that PR-ir induction was limited to the caudal VMH in ER,KO and ER,,KO mice. In the rostral VMH, oestradiol only induced PR-ir in wild-type and ER,KO mice, and the number of PR-ir neurones in this region was greater in ER,KO than wild-type females. Next, we tested the ability of a dopamine agonist and progesterone to facilitate sexual behaviour in females lacking functional ER,, ER,, or both receptors. Ovariectomized mice were implanted with oestradiol, and tested for sexual behaviour three times after administration of the dopamine agonist, apomorphine, followed by two tests concurrent with progesterone treatment and a final test with just apomorphine treatment. ER,KO and ER,,KO females failed to display lordosis under any testing conditions, while ER,KO females exhibited lordosis behaviour equal to that of wild-type females. Our data show that a subpopulation of PR-ir neurones is induced by oestradiol in the caudal VMH of female mice lacking both ER, and ER, genes. We hypothesize that this action of oestradiol is either mediated by a novel ER or by the mutant portion of the AF2 subregion of the ER, gene present in ER,KO brain. However, despite the presence of PR in VMH, females lacking a functional ER, gene do not display sexual behaviour, via either ligand-dependent or -independent activation. [source]

White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C4 -type pyruvate orthophosphate dikinase gene (OsPPDKB)

Hong-Gyu Kang
Summary We have isolated a floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion. Scanning electron microscopic analysis revealed that this abnormal endosperm consisted of loosely packed starch granules. The mutant phenotype was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophosphate dikinase (PPDK). Plants containing flo4-1 produced no OsPPDKB transcript or the OsPPDKB protein in their developing kernels and leaves. We obtained two additional alleles, flo4-2 and flo4-3, that also showed the same white-core endosperm phenotype. The flo4 kernels weighed about 6% less than wild-type ones. Starch contents in both kernel types were similar, but the total protein content was slightly higher in the mutant kernels. Moreover, lipid contents were significantly increased in the flo4 kernels. Expression analyses demonstrated that the cytosolic mRNA of OsPPDKB was induced in the reproductive organs after pollination, and greatly increased until about 10 days after fertilization. This mRNA was localized mainly in the endosperm, aleurone, and scutellum of the developing kernel. Our results suggest that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling. [source]