Microinjection of foreign DNA into fertilized mammalian eggs is a convenient means of introducing genes into the germ line. Some ofthe more important parameters that influence successful integration of foreign DNA into mouse chromosomes are described. The effects of DNA concentration, size, and form (supercoiled vs. linear with a variety of different ends) are considered as well as the site of injection (male pronucleus, female pronucleus, or cytoplasm) and buffer composition. The optimal conditions for integration entail injection of a few hundred linear molecules into the male pronucleus of fertilized one-cell eggs. Under these conditions about 25% of the mice that develop inherit one or more copies of the microinjected DNA. The overall efficiency also depends on the choice of mouse strains; for example, generating transgenic mice that express foreign growth hormone genes is about eight times easier with C57/BL6 x SJL hybrid mice than with inbred C57/BL6 mice.A number of investigators have successfully introduced foreign DNA into somatic tissues and the germ line of mice by microinjecting DNA into fertilized eggs (1)(2)(3)(4)(5)(6) (10). Our experience is that mosaic mice and mice with more than one integration site each represent 10-20% of nearly a hundred transgenic mice that we have analyzed by outbreeding. For most of the analyses described here, we do not distinguish mosaics and multiple integration sites because they are most easily detected by breeding experiments (unpublished data).In the analysis described here, we consider the influence of the number of DNA molecules injected, the form and size of the DNA, injection buffer, and the site of injection on the frequency of integration. Overall efficiency entails more than achieving a high percentage of positive fetuses. It must also include factors such as ease of obtaining fertilized eggs, ease of microinjection, survival of the eggs after microinjection, ability of the eggs to continue development after microinjection, and ability of the eggs to continue development after transfer to pseudopregnant recipients. These factors become important when considering adapting these conditions to other strains of mice or species. METHODSPreparation of DNA for Microinjection. All of the genes were propagated as plasmids or cosmids in Escherichia coli. Supercoiled molecules were isolated from bacterial cultures by standard techniques involving either lysozyme/Triton X-100 lysis or lysozyme/alkaline lysis followed by banding the supercoiled plasmids on ethidium bromine/CsCl gradients (11). Linear DNA molecules were prepared by digestion with restriction enzymes; after digestion the DNA was extracted with NaDodSO4/phenol/chloroform, precipitated with ethanol, washed thoroughly with ethanol/salt, and then dissolved in TE buffer (10 mM Tris HCl/0.25 mM EDTA, pH 7.5). When more than one fragment was generated, the fragments were generally separated by agarose gel electrophoresis and visualized by UV after staining with ethidium bromide and recovered by binding t...

Regulation of the endogenous mouse metallothionein I and II (MT-I and MT-II) genes by heavy metals and glucocorticoids was studied in cultured mouse cells. Both mRNAs were measured simultaneously by solution hybridization with [3HJMT-I cDNA and [32PJMT-II cDNA, and the absolute amount of each mRNA was calculated by using a single-stranded M13 standard that contained both mRNA sequences. Both genes responded identically to different concentrations of metals (zinc, cadmium, and copper) and dexamethasone. Furthermore, the time courses qf induction of both mRNAs were the same. However, under all conditions there was 1.2-to 1.9-fold more MT-I mRNA than MT-II mRNA. We conclude that both genes are regulated identically by receptors for glucocorticoids and metals but that the rate of transcription from the MT-I gene is slightly higher than from the MT-II gene.Metallothioneins (MTs) are cysteine-rich, heavy-metalbinding proteins found in most animal and plant species. In vertebrates, there are two major forms, designated MT-I and MT-II (10). Both forms are induced by and bind heavy metals such as Zn, Cd, and Cu (7,10,17,20). In mammals, glucocorticoids and inflammatory signals also act as inducers (3,8,11). In mice, there is one copy of each gene per haploid genome and they are closely linked on chromosome 8 (2, 24), whereas in primates there are several copies of MT-I and MT-Il genes and they reside on several chromosomes (13, 23).Because the MT isoforms appear to have similar metal binding properties, one reason for the evolution of two similar genes could be to allow djfferential regulation during development or stress (24). Indeed, the endogenous MT-Ia and MT-IIa genes in cultured human cells have been shown to be differentially regulated by heavy metals and glucocorticoids; the human MT-IIa gene is induced by Cd, Zn, and dexamethasone, whereas the human MT-Ia gene is induced principally by Cd (21). When the respective promoters were fused to the thymidine kinase gene of herpes simplex virus and transferred into rat cells, the corresponding induction phenotypes were observed (21), confirming that the effects are due to differences in the promoter regions.In contrast to the differential regulation of the human MT-Ia and MT-IIa genes, our results suggested that MT-I and MT-II genes were regulated similarly in intact mice (24). Because it is difficult to control metal and hormone levels in vivo, we proceeded to examine the regulation of these genes in cultured cells. We chose the Hepa 1A cell line to examine metal regulation because the basal level of MT gene expression in this cell line is very low in the absence of added metals. These cells do not respond well to glucocorticoids (14); therefore, mouse L cells were used to study the hormonal response (15). To facilitate quantitation of both mRNAs, we adapted the solution hybridization protocol of Durnam and Palmiter (5) by using differentially labeled cDNAs that were specific to MT-I or MT-II mRNA. In addition, we used a single-stranded M13 phage containing the mRNA st...

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.

Polymerase stalling results in uncoupling of DNA polymerase and the replicative helicase, which generates single-stranded DNA (ssDNA). After stalling, RAD51 accumulates at stalled replication forks to stabilize the fork and to repair by homologous recombination (HR) double-strand breaks (DSBs) that accumulate there. We showed recently that SUMO modification of the BLM helicase is required in order for RAD51 to accumulate at stalled forks. In order to investigate how BLM SUMOylation controls RAD51 accumulation, we characterized the function of HR proteins and ssDNA-binding protein RPA in cells that stably expressed either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM). In HU-treated SM-BLM cells, mediators BRCA2 and RAD52, which normally substitute RAD51 for RPA on ssDNA, failed to accumulate normally at stalled forks; instead, excess RPA accumulated. SM-BLM cells also exhibited higher levels of HU-induced chromatin-bound RPA than BLM+ cells did. The excess RPA did not result from excessive intrinsic BLM helicase activity, because in vitro SUMOylated BLM unwound similar amounts of replication-fork substrate as unSUMOylated BLM. Nor did BLM SUMOylation inhibit binding of RPA to BLM in vitro; however, in immunoprecipitation experiments, more BLM-RPA complex formed in HU-treated SM-BLM cells, indicating that BLM SUMOylation controls the amount of BLM-RPA complex normally formed at stalled forks. Together, these results showed that BLM SUMOylation regulates the amount of ssDNA that accumulates during polymerase stalling. We conclude that BLM SUMOylation functions as a licensing mechanism that permits and regulates HR at damaged replication forks.

The von Recklinghausen neurofibromatosis 1 (NFI) locus has been previously assigned to the proximal long arm of chromosome 17, and two NF1 patients have been identified who have constitutional balanced translocations involving 17q11.2. We have constructed a cosmid library from a chromosome-mediated gene transfectant, KLTS, that contains approximately 10% of chromosome 17, including 17q11.2. Cosmids isolated from this library have been mapped across a panel of somatic cell hybrids, including the hybrids from the two patients, and have been localized to seven small regions of proximal 17q. We have 5 cosmids that map directly above the two NFI translocations, and 11 cosmids that map directly below. Of these, 2 cosmids in each region are linked to the disease locus and 3 of these cosmids show no recombination.One distal cosmid, 2B/B35, detects the two NFI translocations by pulsed-field gel analysis and has been used to produce a long-range restriction map that covers the translocations.Von Recklinghausen neurofibromatosis 1 (NF1) is one of the most common dominantly inherited disorders in man with an estimated frequency of 1/3000 and a high proportion of new mutations, estimated between 30 and 50%o (1). Its expression is extremely variable, even within affected families, and ranges from minor skin manifestations (e.g., cafe-au-lait spots) to severely disabling and sometimes lethal neurological tumors. Linkage of this disorder to markers on proximal 17q has been demonstrated (2, 3) and linked flanking markers have been defined (4, 5). As with several other dominantly inherited tumor-forming syndromes (e.g., retinoblastoma or adenomatous polyposis coli), it seemed likely that NF1 might be the result of mutations in a tumor-suppressor gene. However, there are as yet no reports of loss of heterozygosity in NF1 for markers close to the NFI locus.The finding of two individuals with NF1 carrying different constitutional balanced reciprocal translocations with breaks at 17q11.2 (6, 7) strongly suggested that the region of these translocations would contain the NFI gene. Probes that map close to the translocations have been used to make long-range maps of this region (8,9). By using a chromosome-mediated gene transfectant (CMGT), KLT8, containing about 10% of chromosome 17, including the NFl region (10), we have isolated cosmids flanking the NFI translocations. Restriction fragment length polymorphisms (RFLPs) in these cosmids have allowed us to map them by genetic linkage. Our closest probe lies distal to the translocations and has no recombinants with NFI. It contains a CpG-rich island and has been used by analysis of pulsed-field gel electrophoresis (PFGE) results to define a long-range restriction map covering the translocations that differ from the published maps (27). In this paper we present the genetic linkage data on our flanking probes and our physical map of the translocation region. MATERIALS AND METHODSCell Lines. KLT8, PLT6B, and PLT8 are CMGTs containing fragments of chromosome 17 (10). PCTBA1.8 (11) i...

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

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BLM is sumoylated in response to replication stress. We have studied the role of BLM sumoylation in physiologically normal and replication-stressed conditions by expressing in BLM-deficient cells a BLM with SUMO acceptor-site mutations, which we refer to as SUMO-mutant BLM cells. SUMO-mutant BLM cells exhibited multiple defects in both stressed and unstressed DNA replication conditions, including, in hydroxyurea-treated cells, reduced fork restart and increased fork collapse and, in untreated cells, slower fork velocity and increased fork instability as assayed by track-length asymmetry. We further showed by fluorescence recovery after photobleaching that SUMO-mutant BLM protein was less dynamic than normal BLM and comprised a higher immobile fraction at collapsed replication forks. BLM sumoylation has previously been linked to the recruitment of RAD51 to stressed forks in hydroxyurea-treated cells. An important unresolved question is whether the failure to efficiently recruit RAD51 is the explanation for replication stress in untreated SUMO-mutant BLM cells.