High somatic mutation frequencies in a LacZ transgene integrated on the mouse X-chromosome

To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of ⁻⁵ - 10⁻⁶. Here we report a 25–100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational ‘hot spot’ in the mammalian somatic genome in vivo.     

Paradoxical decrease in mutant frequencies and chromosomal rearrangements in a transgenic lacZ reporter gene in Ku80 null mice deficient in DNA double strand break repair

Repair of DNA double strand breaks (DSB), either by homologous recombination (HR) or nonhomologous end-joining (NHEJ), is essential to maintain genomic stability. To examine the impact of NHEJ deficiency on genomic integrity in Ku80 null (Ku-) mice, the chromosomally integrated shuttle vector pUR288, which includes a lacZ reporter gene, was used to measure mutations in vivo. Unexpectedly, a significant decrease was found in mutant frequencies of Ku- liver (5.04x10(-5)) and brain (4.55x10(-5)) compared to tissues obtained from normal (Ku+) littermates (7.92x10(-5)and 7.30x10(-5), respectively). No significant difference was found in mutant frequencies in spleen from Ku- (7.21x10(-5)) and Ku+ mice (8.16x10(-5)). The determination of the mutant spectrum in lacZ revealed the almost complete absence of chromosomal rearrangements (R) in Ku- tissues (0.5%, 3/616), a notable distinction from Ku+ controls (16.7%, 104/621). These findings suggest that accurate repair of DSB by HR and elimination of cells with unrepaired DNA damage by apoptosis are capable of maintaining genomic stability of the lacZ reporter in Ku- mice.     

Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

Ku plays a key role in multiple nuclear processes, e.g., DNA double-strand break (DSB) repair. The regulation mechanism of the localizations of Ku70 and Ku80 plays a key role in regulating the multiple functions of Ku. Although numerous biochemical studies in vitro have elucidated the DNA binding mechanism of Ku, no accumulation mechanisms of Ku70 and Ku80 at DSBs have been clarified in detail in vivo. In this study, we examined the accumulation mechanism of Ku80 at DSBs in living cells. EGFP-Ku80 accumulation at DSBs began immediately after irradiation. On the other hand, our data show that Ku70 alone, which has DNA binding activity independent of Ku80, cannot accumulate at the DSBs, whereas Ku70 bound to Ku80 can. The deletion of the C-terminal DNA-PKcs-binding domain and the mutation at the SUMOylation site of Ku80 had no effect on Ku80 accumulation. Unexpectedly, N-terminal deletion mutants of Ku80 fully lost their accumulation activity, although the mutants retained their Ku70 binding activity. Altogether, these data demonstrate that Ku80 is essential for Ku70 accumulation at DSBs. Furthermore, three domains of Ku80, i.e., the N-terminal α/β, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.     

A null mutation at the mouse Phosphoglucomutase-1 locus and a new locus,Pgm-3

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57 BL/6J. The null allele (Pgm-1 ⁿ) was shown to segregate as a Mendelian unit alternative to the Pgm-1 ^a and Pgm-1 ^b alleles. Mice expressing the Pgm-1 ⁿ allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1 ⁿ mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57 BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.Financial support for this work was provided in part by Contract #263-78-C-0393 from the National Institute of Environmental Health Sciences to the Research Triangle Institute.     

Mutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lacZ-plasmid reporter mice

Transgenic mice harboring the lacZ gene within a plasmid that can be recovered and amplified in Escherichia coli, to establish mutant frequencies and spectra, have provided crucial insights into the relationships between mutations, cancer and aging in vivo. Here, we use embryonic fibroblasts from transgenic lacZ-plasmid reporter mice to determine the relationship between cell proliferation in culture and mutations induced by ultraviolet (UV) light. A single dose of 2.5J/m2 of UVC to actively proliferating cells caused an approximately eight-fold increase in mutant frequency 24 h after irradiation. Identically treated quiescent cells showed a two-fold increase in mutant frequency. Thus, whereas proliferation facilitated the acquisition of mutations, it was not an absolute requirement. Characterization of the UV-induced mutations indicated that the lower mutant frequency in quiescent cells was due mainly to a reduction in point mutations; size-change mutations, indicative of translocations or deletions, were relatively unaffected by the growth state of the cells. To investigate long-term genomic stability after UVC-induced damage, we monitored the lacZ locus in irradiated cells passaged for many generations in culture. The results indicated the emergence of jackpot mutations of rapidly changing frequency, most likely reflecting the successive emergence and decline of dominant cell clones during long-term culture. These findings show that the lacZ-plasmid locus is a valid reporter for studying induced mutations in short-term cultures of both quiescent and proliferating fibroblasts. In long-term cultures, the locus is less suitable for studying induced mutations owing to the instability of the cell population.     

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells – spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.     

Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.     

Cleavage at the carboxyl-terminus of Ku80 during apoptosis in human Jurkat T cells

We have previously reported that the amount of Apg-2, an Hsp110 family protein, decreases during apoptosis in Jurkat T cells. Since we hypothesized that Apg-2 would be cleaved by caspase-3 during apoptosis, a cleavage-site-directed antibody was raised against the carboxyl-terminus of the Apg-2 fragment that appears after the cleavage by caspase-3. Although this antibody could not detect the Apg-2 fragment in apoptotic cells, three additional fragments were unexpectedly detected. Based on the results of microsequencing, one of these fragments was identified as Ku80. Ku80 is a nuclear protein and a component of DNA-dependent protein kinase (DNA-PK). In this study, we observed that Ku80 is cleaved at Asp-730 residue during apoptosis, and this cleavage occurs in the nucleus in the early apoptotic phase. Furthermore, Ku80 is distributed in the cytoplasm of nuclear fragmented apoptotic cells, although the cleaved fragment contains the nuclear-localization signal (NLS). Our study clearly shows that Ku80 is cleaved in the nucleus, and distributes in the cytoplasm during apoptosis.     

Selection and mutation at a diallelic X-linked locus

Equilibria and convergence of gene frequencies are studied in the case of a diallelic X-linked locus under the influence of selection and mutation. The model used is that of an infinite diploid population with nonoverlapping discrete generations and random mating. It is proved that if the mutation rates and fitnesses are constant and the mutation rates are less than one-third, then global convergence of gene frequencies to equilibria occurs. The phase portraits of the dynamical system describing the change of allelic frequencies from one generation to the next are determined. Convergence of gene frequencies is monotone from a certain generation on if every other generation is skipped. In the case without mutation, our proof of this monotone convergence simplifies G. Palm's original proof [37].     

人牙本质涎磷蛋白启动子驱动LacZ报告基因在人牙胚间充质细胞中表达

牙本质涎磷蛋白(DSPP)的表达是细胞向成牙本质细胞分化的标志。试图分析人DSPP启动子及构建人DSPP启动子驱动的Lac Z基因表达的报告体系,从而方便快捷检测细胞是否向成牙本质细胞分化。为了建立能表达DSPP的细胞体系,分离了人牙胚间充质细胞,并用地塞米松诱导培养液进行诱导,结果显示,该诱导培养液能有效地诱导人牙胚间充质细胞DSPP基因的表达。利用双荧光素酶报告系统对4段人DSPP基因5′上游区域(-4 000-+54、-2 500-+54、-1 447-+54和-1 027-+54)进行分析,结果显示-2 500-+54区域的启动子活性最高。5′上游区从?2 500 bp延长到?4 000 bp时,启动子活性下降;5′上游区从-2 500 bp缩短至-1 447 bp时,启动子活性下降;再次将-1 447 bp缩短至-1 027 bp时,启动子活性进一步下降。结果暗示在-4 000 bp至-2 500 bp区域存在转录抑制元件,-2 500 bp至-1 027 bp区域存在转录激活元件。用-2 500-+54启动子区域和Lac Z基因构建ph DSPP-Lac Z慢病毒报告载体,并分别在人牙胚间充质细胞和永生化人牙胚间充质细胞系ih EDMC4上检测ph DSPP-Lac Z报告载体的功能,通过X-Gal染色,结果显示在2种细胞牙向分化过程中均可检测到Lac Z基因的表达。研究构建的ph DSPP-Lac Z慢病毒报告载体可为诱导人源细胞牙向分化、牙齿发育、牙齿再生工程等研究中DSPP的表达检测提供一种更加便捷的手段。