Hamster Cell Mutants Research Articles

Distinct cellular phenotype linked to defective DNA interstrand crosslink repair and homologous recombination.

Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL-V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL-V8B exhibits increased sensitivity to various DNA-damaging agents, including compounds leading to DSBs formation (bleomycin and 6-thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400-fold), which is typical for HR-defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL-V8B cells. However, in contrast to other known HR mutants, CL-V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL-V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC-induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL-V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL-V8B to crosslinking agents results in S-phase arrest (as in the wild type cells), but also in larger proportion of G2/M-phase cells and apoptotic cells. CL-V8B exhibits similarities to HR- and/or FA-defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.

Crystal structure of a TAF1-TAF7 complex in human transcription factor IID reveals a promoter binding module.

The general transcription factor IID (TFIID) initiates RNA polymerase II-mediated eukaryotic transcription by nucleating pre-initiation complex formation at the core promoter of protein-encoding genes. TAF1, the largest integral subunit of TFIID, contains an evolutionarily conserved yet poorly characterized central core domain, whose specific mutation disrupts cell proliferation in the temperature-sensitive mutant hamster cell line ts13. Although the impaired TAF1 function in the ts13 mutant has been associated with defective transcriptional regulation of cell cycle genes, the mechanism by which TAF1 mediates transcription as part of TFIID remains unclear. Here, we present the crystal structure of the human TAF1 central core domain in complex with another conserved TFIID subunit, TAF7, which biochemically solubilizes TAF1. The TAF1-TAF7 complex displays an inter-digitated compact architecture, featuring an unexpected TAF1 winged helix (WH) domain mounted on top of a heterodimeric triple barrel. The single TAF1 residue altered in the ts13 mutant is buried at the junction of these two structural domains. We show that the TAF1 WH domain has intrinsic DNA-binding activity, which depends on characteristic residues that are commonly used by WH fold proteins for interacting with DNA. Importantly, mutations of these residues not only compromise DNA binding by TAF1, but also abrogate its ability to rescue the ts13 mutant phenotype. Together, our results resolve the structural organization of the TAF1-TAF7 module in TFIID and unveil a critical promoter-binding function of TAF1 in transcription regulation.

Characterization of a Chinese hamster mutant cell line with a complete defect in mitochondrial protein synthesis

Characterization of a Chinese hamster mutant cell line with a complete defect in mitochondrial protein synthesis

Abstract 3896: The interaction between BRCA2 and DSS1 is crucial for homologous recombination through controlling protein localization

Abstract The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination (HR). Human BRCA2 is huge protein and is composed of several domains including DNA/DSS1 binding domain (DBD). DSS1 has shown to be important for HR in various organisms but the precise function of DSS1 through the interaction between BRCA2 is still unclear in mammalian cells. To clarify the role of DSS1-BRCA2 interaction in the repair of DNA damage, we put several mutations into BRCA2 peptides which could restore HR in Brca2 mutant hamster cells. The mutant BRCA2 peptides with mutation which reduced DSS1 binding impaired the ability of these peptides to promote HR when compared to wild type peptides. It is of great interest that the excessive DSS1 expression partially restored HR activity of BRCA2 mutants which weakly bind DSS1. Furthermore, excessive DSS1 expression facilitate the nuclear localization of wild type BRCA2 peptide. On the other hand, excessive DSS1 expression had no effect for neither HR activity nor the subcellular localization of mutant BRCA2 peptide that could not bind to DSS1. Knockdown of DSS1 by siRNA also showed reduced HR and nuclear localization of BRCA2 protein. In conclusion, one of the essential functions of DSS1 in HR through the interaction between BRCA2 might be a regulation of BRCA2 subcellular localization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2011-3896

DDX3 Regulates Cell Growth through Translational Control of Cyclin E1

DDX3 belongs to the DEAD box family of RNA helicases, but the details of its biological function remain largely unclear. Here we show that knockdown of DDX3 expression impedes G(1)/S-phase transition of the cell cycle. To know how DDX3 may act in cell cycle control, we screened for cellular mRNA targets of DDX3. Many of the identified DDX3 targets encoded cell cycle regulators, including G(1)/S-specific cyclin E1. DDX3 depletion specifically downregulates translation of cyclin E1 mRNA. Moreover, our data suggest that DDX3 participates in translation initiation of targeted mRNAs as well as in cell growth control via its RNA helicase activity. Consistent with these findings, we show that in the temperature-sensitive DDX3 mutant hamster cell line tsET24, cyclin E1 expression is downregulated at a nonpermissive temperature that inactivates mutant DDX3. Taken together, our results indicate that DDX3 is critical for translation of cyclin E1 mRNA, which provides an alternative mechanism for regulating cyclin E1 expression during the cell cycle.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C splice heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C splice embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C splice/neo heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

Cisplatin sensitivity is related to late DNA damage processing and checkpoint control rather than to the early DNA damage response

The present study aimed at elucidating mechanisms dictating cell death triggered by cisplatin-induced DNA damage. We show that CL-V5B hamster mutant cells, a derivative of V79B, are hypersensitive to cisplatin-induced apoptotic death. CL-V5B cells are characterized by attenuated cisplatin-induced early (2–6 h) stress response, such as phosphorylation of stress-activated protein kinases (SAPK/JNK), ATM and Rad3-related (ATR) protein kinase, histone H2AX and checkpoint kinase-1 (Chk-1). Human FANCC cells also showed a reduced phosphorylation of H2AX and SAPK/JNK at early time point after cisplatin treatment. This was not the case for BRCA2-defective VC-8 hamster cells, indicating that the FA core complex, rather than its downstream elements, is involved in early damage response. The alleviated early response of CL-V5B cells is not due to a general dysfunction in ATM/ATR-regulated signaling. It is rather due to a reduced formation of primary cisplatin-DNA adducts in the hypersensitive mutant as shown by analysis of DNA platination, DNA intra- and interstrand crosslink formation and DNA replication blockage. Despite of lower initial DNA damage and attenuated early DNA damage response (DDR), CL-V5B cells are characterized by an excessive G2/M arrest as well as an elevated frequency of DNA double-strand breaks (DSB) and chromosomal aberrations (CA) at late times (16–24 h) after cisplatin exposure. This indicates that error-prone processing of cisplatin-induced lesions, notably interstrand crosslinks (ICL), and the formation of secondary DNA lesions (i.e. DSB), results in a powerful delayed DNA damage response and massive pro-apoptotic signaling in CL-V5B cells. The data provide an example that the initial level of cisplatin-DNA adducts and the corresponding early DNA damage response do not necessarily predict the outcome of cisplatin treatment. Rather, the accuracy of DNA damage processing and late checkpoint control mechanisms determine the extent of cell death triggered by cisplatin-induced DNA lesions.

Enhanced Use of Backup Pathways of NHEJ in G2in Chinese Hamster Mutant Cells with Defects in the Classical Pathway of NHEJ

In higher eukaryotes DNA double-strand breaks (DSBs) are repaired by homologous recombination repair (HRR) or nonhomologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) using DNA ligase III and PARP1. We have reported previously that mouse embryo fibroblasts (MEFs) defective in D-NHEJ show enhanced repair of DSBs in G2 not reflecting a contribution of HRR. Here we extend these studies to Chinese hamster mutant cells with defects in the DNA-PKcs, Ku80 or XRCC4 components of D-NHEJ or in the XRCC2 and XRCC3 components of HRR. Using cell sorting to separate cells at defined times after irradiation, we measure repair of DSBs with pulsed-field gel electrophoresis in unperturbed G1- and G2-phase cells. Wild-type cells and mutants of XRCC2 and XRCC3 repair DSBs with similar efficiency in G1 and G2. Mutants of DNA-PKcs, Ku80 and XRCC4 show more pronounced repair in G2 than in G1. These and previously published results provide support for the notion that the increased efficacy of DSB repair in G2 reflects the enhanced function of B-NHEJ, which may be a general feature of rodent cells that also holds for human cells.

G2-phase chromatid break kinetics in irradiated DNA repair mutant hamster cell lines using calyculin-induced PCC and colcemid-block

We have investigated the role of the major pathways of DNA double-strand break (DSB) rejoining in the formation and kinetics of disappearance of chromatid breaks following irradiation in the G2 phase of the cell-cycle. We studied the responses of Chinese hamster cell lines xrs5, UV41 and irs1 with mutations in DNA repair genes XRCC5, ERCC4/XPF and XRCC2, involved in the non-homologous end-joining (NHEJ), single-strand annealing (SSA) and homologous recombination (HR) pathways of DSB rejoining respectively. We have used calyculin-induced PCC to study the kinetics of chromatid breaks in xrs5 and UV41 and wild-type CHOK1 cell line. xrs5 showed an elevated frequency of both spontaneous and radiation-induced chromatid breaks. However, the rate of disappearance of chromatid breaks with time was similar in xrs5 to that in its parental CHO cell line. The results with xrs5 firstly confirm our previous findings using the traditional colcemid-block technique, and secondly they demonstrate the independence of chromatid break kinetics of the radiation-induced cell-cycle checkpoint delay. The lack of correspondence between chromatid break kinetics and the deficiency in DSB rejoining in xrs5 argues strongly for an indirect involvement of DSB in the formation of chromatid breaks. The UV41 strain also showed similar chromatid break frequencies and kinetics to CHOK1 suggesting that the SSA pathway is not involved in the rejoining of DSB in the G2 phase of the cell-cycle. We found it not possible to use calyculin-induced PCC in V79-4 and irs1 cell lines. However, using colcemid block we show an elevation in both spontaneous and radiation-induced chromatid break frequency, and a similar rate of disappearance of G2 chromatid breaks with time after irradiation to its wild-type parental V79 line. Thus, deficiencies in two of the major pathways of DSB rejoining (NHEJ and HR) lead to elevated frequencies of chromatid breaks, but do not significantly influence the kinetics of their disappearance with time. We conclude from these data that chromatid breaks do not represent ‘expanded’ DSB but that they are an indirect consequence of the formation of DSB.

The high rate of endoreduplication in the repair deficient CHO mutant EM9 parallels a reduced level of methylated deoxycytidine in DNA

It has been recently proposed that hypomethylation of DNA induced by 5-azacytidine (5-azaC) leads to reduced chromatid decatenation that ends up in endoreduplication, most likely due to a failure in topo II function [S. Mateos, I. Domínguez, N. Pastor, G. Cantero, F. Cortés, The DNA demethylating 5-azaC induces endoreduplication in cultured Chinese hamster cells, Mutat. Res. 578 (2005) 33–42]. The Chinese hamster mutant cell line EM9 has a high spontaneous frequency of endoreduplication as compared to its parental line AA8. In order to see if this is related to the degree of DNA methylation, we have investigated the basal levels of both endpoints in AA8 and EM9, as well as the effect of extensive 5-azaC-induced demethylation on the production of endoreduplication. Based on the correlation between the levels of DNA methylation and indices of endoreduplication we propose that genomic DNA hypomethylation in EM9 cell line is probably an important factor that bears significance in relation to the high basal level of endoreduplication observed in this cell line.

Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells [H. Nagasawa, Y. Peng, P.F. Wilson, Y.C. Lio, D.J. Chen, J.S. Bedford, J.B. Little, Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations, Radiat. Res. 164 (2005) 141–147]. In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23 to 0.33 SCE/chromosome, 0–30% lower than wild-type cells. SCEs were induced significantly 20–50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after α-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

Investigations of the potential effects of phosphorylation of the MWFE and ESSS subunits on complex I activity and assembly

There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant.

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.

Human DDX3Y, the Y-encoded isoform of RNA helicase DDX3, rescues a hamster temperature-sensitive ET24 mutant cell line with a DDX3X mutation

We investigated the function of DDX3Y, the Y chromosome AZFa region encoding a putative DEAD-box RNA helicase protein, the loss of which results in oligozoospermia or azoospermia in humans. The human DDX3Y amino acid sequence is similar to that of the X chromosome gene DDX3X (91.7% homology). Here we report that human Y- and X-encoded DEAD box RNA helicase proteins DDX3Y and DDX3X are interchangeable and have an essential function: both proteins rescued a temperature-sensitive mutant hamster cell line (tsET24) that was otherwise incapable of growth at a nonpermissive temperature. Mouse homologues Ddx3y and D1Pas1–PL10 also rescued the mutant cell line at a nonpermissive temperature. In situ hybridization revealed that Ddx3x mRNA was expressed in almost every cell in mouse testis, suggesting that Ddx3x is involved in spermatogenesis. A comparative study of DDX3X and DDX3Y was performed to determine the significance of DDX3Y for cell growth and spermatogenesis. Both DDX3X and DDX3Y promoter DNAs produced a similar degree of transcription in vivo, whereas deletion studies of the promoter DNAs indicated that these genes are differentially regulated. DDX3Y, similar to DDX3X, shuttles between the nucleus and cytoplasm in a crm1-dependent manner.

Impairment of homologous recombination control in a Fanconi anemia-like Chinese Hamster Cell mutant

DNA interstrand cross-links (ICL)-inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL-inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL-inducing agents, we examined spontaneous and ICL-induced HR in rodent FA-like V-H4 cells. In this report, we show that MMC-hypersensitive V-H4 cells exhibit an increased spontaneous homology-directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51-dependent recombination, without affecting non-conservative single-strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA-like V-H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA-like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V-H4 cells and emphasize the involvement of the FA pathway in HR-mediated repair.

Cellular response of X-ray sensitive hamster mutant cell lines to gemcitabine, cisplatin and 5-fluorouracil.

Five mutant Chinese hamster cell lines deficient in DNA repair with the corresponding parental cell lines were used to determine their sensitivity to cisplatin, 5-fluorouracil and gemcitabine. The mutations in the cell lines led to defective single strand break repair (EM-C11), defective recombination mediated repair (irs1SF), defective double strand break repair (XR-V15B, a Ku-80 mutant and CR-C1, a DNA-PKcs mutant) and an AT-like mutation (VC-4). All mutant cell lines had an impaired doubling time during exponential growth and an increased sensitivity to X-irradiation. We may conclude that for cisplatin-induced cytotoxicity the homologous recombination-associated DNA repair plays an important role in the repair of the cisplatin induced lesions, confirming previous results. In 5-FU and gemcitabine induced toxicity to cells, repair processes involved with radiation-induced damage were not implicated. This is in striking contrast to the role of cisplatin in radiosensitization where inhibition of the NHEJ pathway is implicated, and to the role of gemcitabine in sensitization where specific interference with the HR pathway is implicated.

Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells

The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C ( RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant. We thus, provide direct evidence for a role of mammalian Rad51C in HR processes. The reduced HR events described here help to explain the deficient phenotypes observed in Rad51C mutants and support an accessory role of Rad51C in Rad51-mediated recombination.

Assignment of GRP78 gene to swine chromosome 1q2.12-->q2.13 by fluorescent in situ hybridization.

HereditasVolume 121, Issue 2 p. 203-205 Open Access Assignment of GRP78 Gene to Swine Chromosome lq2.12→q2.13 by Fluorescent in Situ Hybridization Hiroshi Yasue, Corresponding Author Hiroshi Yasue Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan*To whom all correspondence should he addressed.Search for more papers by this authorTakashi Harumi, Takashi Harumi Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, JapanSearch for more papers by this authorTakashi Awata, Takashi Awata Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan Laboratory of Animal Breeding, Tohoku National Agricultural Experiment Station, Morioka 020–01, JapanSearch for more papers by this author Hiroshi Yasue, Corresponding Author Hiroshi Yasue Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan*To whom all correspondence should he addressed.Search for more papers by this authorTakashi Harumi, Takashi Harumi Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, JapanSearch for more papers by this authorTakashi Awata, Takashi Awata Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan Laboratory of Animal Breeding, Tohoku National Agricultural Experiment Station, Morioka 020–01, JapanSearch for more papers by this author First published: November 1994 https://doi.org/10.1111/j.1601-5223.1994.t01-1-00203.xCitations: 1 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References Awata, T., Yamakuchi, H., Kijmagai, M. and Yasue, H. 1994. Assignment of the tenascin gene (HXB) to swine chromosome lq21.1→q21.3 by fluorescence in situ hybridization. Cytogenet. Cell Genet. In press Day, A. R. and Lee, A. S. 1989. Transcriptional regulation of the gene encoding the 78–kd glucose–regulated protein GRP78 in mouse Sertoli cells binding of specific factors to the GRP78 promoter. DNA 8: 301– 310 Kozutsumi, Y., Segal, M., Normington, K., Gethinig, M–J. and Sambrook, J. 1988. The presence of malfolded proteins in the endoplasmatic reticulum signals the induction of glucoseregulated proteins. Nature 332: 462– 464 Lee, A. S. 1987. Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. Trends Biochem. Sci. 12: 20– 23 Lee, A. S., Wells, S., Kim, K. S. and Scheffler, I. E. 1986. Enhanced synthesis of the glucose/calcium regulated proteins in a hamster cell mutant deficient in transfer of oligosaccharide core to polypeptides. J. Cell Physiol. 129: 277– 282 Mellink, C. H. M., Lahbib-Mansais, Y., Yrlb, E. M. and Gellin, J. 1993. Localization of four new markers to pig chromosomes 1, 6 and 14 by radioactive in situ hybridization. Cytogener. Cell Genet. 64: 256– 260 Pelham, H. R. B. 1986. Speculations on the functions of the major heat shock and glucose–regulated proteins. Cell 46: 959– 961 Resendez, E. Jr., Attenello, J. W., Grafsky, A., Chang, S. S. and Lee, A. S. 1985. Calcium ionophore A23187 induces expression of glucose–regulated genes and their heterologous fusion genes. Mol. Cell Biol. 5: 1212– 1219 Rønne, M., Siefanoval, E., Di Berardino, D. and Poulsen, B. S. 1987. The R–banded karyotype of the domestic pig (Sus scrofa domestica L.). Hereditas 106: 219– 231 Shiu, R. P. C., Pouyssegur, J. and Pastan, I. 1977. Glucose depletion accounts for the induction of two transformationsensitive membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74: 3840– 3844 Ziegler, A., Hagmann, J., Kiefer, B. and Nagamine, Y. 1990. Ca2+ potentiates CAMP–dependent expression of urokinase–type plasminogen activator gene through a almodulinand protein kinase C–independent mechanism. J. Biol. Chem. 265: 21194– 21201 Citing Literature Volume121, Issue2November 1994Pages 203-205 ReferencesRelatedInformation

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl- l-leucyl- l-leucyl- l-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.