Mutant Hamster Research Articles

Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation

Murine cells homozygous for the severe combined immune deficiency mutation ( scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PK cs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PK cs, gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PK cs, is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

Isolation of a Human cDNA That Complements a Mutant Hamster Cell Defective in Methotrexate Uptake

A clone has been isolated from a human lymphoblastic cDNA expression library that complements a mutant Chinese hamster cell defective in the uptake of the folate analogue methotrexate. When transfected with this clone the mutant cells regain the ability to transport the drug and, as a consequence, become sensitive to its cytotoxic action. The clone is 2863 base pairs long and has an open reading frame of 1770 base pairs that codes for a putative protein of 64 kDa. The putative protein has 51 and 50% identity at the amino acid level with the mouse and hamster functions, respectively, involved in the transport of reduced folates. Together these three proteins share 47% identity and have similar predicted structural features. The data are consistent with this human clone encoding either the reduced folate transporter or an auxiliary function that interacts with this transporter.

Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells.

The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation. XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating that Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced approximately 4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group. Decreased levels of polypeptide thus account for most of the approximately 6-fold reduced DNA ligase III activity observed previously in EM9. Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (> 10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.

Neuroendocrine regulation of growth hormone secretion

Growth hormone (GH) secretion is controlled by many factors, including stage of development, age, gonadal steroids, body composition, nutritional state, time of day and whether the subject is asleep or awake. Understanding regulation of GH secretion is important since this hormone regulates not only growth, but also the partitioning of nutrients and body composition. There is increasing evidence that there is a basic ultradian rhythm of GH secretion. The NSF Center studies will be facilitated by 3 major efforts: (a) improvement of sensitivity of GH assays to permit accurate description of GH pulses; (b) use of biomathematical models to objectively determine GH pulse characteristics, as well as calculation of secretion rates to facilitate the study of the relationship between neural controls and GH secretion; and (c) use of the tau mutant hamster and the new mouse mutant animal models. By manipulation of the endogenous circadian clock in these animal models it will be possible to study the relationship between endogenous circadian systems and ultradian GH rhythms.

Construction of human chromosome-3-specific radiation hybrids and characterization by Alu-PCR

Using a modification of the procedure developed by Cox et al. [Genomics 4 (1989) 397–407], we isolated and characterized 60 radiation hybrids (RH) prepared by fusing an X-ray-irradiated Chinese hamster-human chromosome 3 (Chr 3) cell line (Q314-2) with a UrdA Chinese hamster mutant cell line. The RH were screened for human DNA content by PCR amplification using primers directed to the human Alu repeat sequences. Over 80% (5060) were scored as positive for the retention of human DNA. Of them, 18 were characterized with Chr-3-specific single-copy DNA probes of known map location. These experiments demonstrated that the RH analyzed contained distinct subrogions of human Chr 3. The RH that we have produced constitute a bank of cellular clones containing small segments of Chr 3. In the accompanying paper [Atchison et al., Gene 151 (1994) 325–328], we present the construction of rare-restrictionsite linking libraries and the sequence tagged site characterization of in situ localized clones.

The novel antiepileptic drug, lamotrigine, exerts prodystonic effects in a mutant hamster model of generalized dystonia.

Recent findings of antidystonic effects of NMDA and non-NMDA receptor antagonists in an inbred line of Syrian hamsters with primary generalized dystonia prompted us to investigate the effects of lamotrigine, an inhibitor of veratrine-induced glutamate release, on the severity of dystonia in mutant hamsters. In mutant dystonic hamsters the dystonic attacks which can be induced by mild environmental stimuli or handling are age-dependent with maximum severity between days 30 and 40 of life (maximum period). Thereafter the severity of dystonia slowly declines (post-maximum period) until the susceptibility to induction of dystonia disappears completely at an age of about 70 days. Lamotrigine (5.0, 10.0 or 30.0 mg/kg i.p.) dose dependently decreased the latency to onset of dystonic attacks. Furthermore, at a dose of 30 mg/kg the dystonic attacks were aggravated when lamotrigine was administered during the max and post-max period. Even in mutant hamsters older than 70 days, i.e. after spontaneous remission of dystonia, and in an inbred line of non-dystonic Syrian hamsters with genetic origin similar to the mutant hamsters, lamotrigine (10.0 or 30.0 mg/kg i.p. and 30.0 mg/kg p.o.) provoked dystonic disturbances. In a genetically different outbred line of Syrian hamsters, lamotrigine did not cause dystonic movements. The unexpected finding that lamotrigine exerts prodystonic effects in genetically susceptible hamsters may be due to the lack of selectivity of lamotrigine to block glutamate release. Tentatively, simultaneous inhibition of GABA (gamma-aminobutyric acid) release might be critically involved in the prodystonic activity of lamotrigine.

Repair of 6-4 photoproducts and cyclobutane pyrimidine dimers in rad mutants of Saccharomyces cerevisiae

Repair rates of both pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane pyrimidine dimers have been measured in the UV-sensitive mutants of Saccharomyces cerevisiae: rad1 to rad12 and rad14 to rad24. A dot blot immunoassay for UV photoproducts was used which measures lesions in the genome as a whole and which distinguishes 6-4 photoproducts from cyclobutane dimers. The principal findings are: (1) Wild-type yeast cells, like normal mammalian cells, repair 6-4 photoproducts more rapidly than cyclobutane domers. (2) All mutants that are defective in repair are defective in repair of both lesions. (3) The most sensitive alleles of rad1, rad2, rad3, rad4 and rad10 show no repair of either lesion. (4) Leaky alleles of rad1, rad3 and rad4 show a very marked difference in repair rates of the two lesions, rather like the human XPA revertant cell line XP129 and the Chinese hamster mutants UV61 and V-H1. (5) No mutant repairs cyclobutane dimers more rapidly than 6-4 photoproducts.

Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks.

The Ku autoantigen is a well-characterized heterodimer of 70 and 86 kDa that binds to DNA ends, but its cellular function has been obscure. An electrophoretic mobility-shift assay and Ku antisera were used to show that Ku or a closely related protein was deficient in three mutant hamster cell lines from x-ray-sensitive complementation group 5, which is characterized by defects in DNA double-strand break repair and V(D)J recombination. Furthermore, Ku protein expression was restored when the cells reverted to x-ray resistance. The Ku p86 gene maps to human chromosome 2q33-35, and group 5 cells are rescued by almost precisely the same region, 2q34-36. Thus, biochemical and genetic evidence suggests that Ku is involved in pathways for DNA recombination and repair. By its association with a DNA-dependent protein kinase activated by DNA ends, Ku may also initiate a signaling pathway induced by DNA damage, perhaps for cell cycle arrest.

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.

Isolation and Characterization of Mouse Xrcc-1, a DNA Repair Gene Affecting Ligation

Human DNA repair gene XRCC1 complements the strand-break rejoining defect in Chinese hamster mutant EM9 and encodes a protein that is apparently required for optimal activity of DNA ligase III. Toward the goal of producing transgenic mice that carry a mutation in the Xrcc-1 locus, the murine homolog of XRCC1 was cloned from both cosmid genomic and cDNA libraries. Upon transfection into EM9 cells, cosmids containing the functional mouse gene efficiently corrected (94-100%) the high sister-chromatid-exchange defect. Mouse Xrcc-1 is 26 kb in length, contains 17 exons, and maps by metaphase in situ hybridization to the 7A3-7B2 region of mouse chromosome 7. Isolated cDNA clones were highly truncated and were extended by anchored polymerase chain reactions. The 1893-bp open reading frame of mouse Xrcc-1 encodes 631 amino acids, compared with 633 for the human homolog. The predicted mouse Xrcc-1 protein of 69.1 kDa and pI of 5.95 is 86% identical and 93% similar to human XRCC1.

Mitochondrial metabolism of 12- and 15-hydroxyeicosatetraenoic acids.

We have previously demonstrated that peroxisomal-deficient human skin fibroblasts and mutant Chinese hamster ovary cells do not convert 12- and 15-hydroxyeicosatetraenoic acids (HETEs) to chain-shortened, polar metabolites, suggesting that peroxisomes are the intracellular location for beta-oxidation of these compounds. This implies that mitochondria do not beta-oxidize HETEs. To test this hypothesis we incubated highly purified rat liver mitochondria with [3H]12-(S)- and [3H]15-(S)-HETE in the presence of carnitine and an acylcoenzyme A-generating system. Extracts obtained from these incubations were analyzed for radiolabeled polar metabolites. Both HETEs were converted to apparent products of beta-oxidation, although the 12-HETE compound was a markedly better substrate. The presence of 50 microM 2-tetradecyloxirane carboxylate, a potent inhibitor of carnitine palmitoyl transferase, completely blocked 12- and 15-HETE conversion to these metabolites as did omission of carnitine from the medium. These data demonstrate carnitine-dependent beta-oxidation of HETEs in isolated mitochondria and suggest that mitochondria are competent to carry out this metabolic process in eukaryotic cells. Prevailing metabolic conditions in subcellular compartments may have precluded observation of mitochondrial activity in our earlier work with cultured cells. Alternatively, transport mechanisms may exist in the cell types studied that distribute 12-(S)- and 15-(S)-HETEs specifically to peroxisomes.

The novel selective and silent 5-HT 1A receptor antagonist (+)-WAY-100135 aggravates dystonic movements in a mutant hamster model

Several clinical and experimental findings suggest that abnormal serotonin (5-HT) function may be involved in movement disorders such as dystonia, and it was proposed that selective 5-HT 1A receptor antagonists may be of benefit in treating such disorders. In the present study, the novel, highly selective and silent 5-HT 1A receptor antagonist (+)-WAY-100135 ( N- tert-butyl-3(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylpropionamide) was tested in an inbred line of Syrian hamsters with generalized dystonia, i.e. a frequent movement disorder in humans. In order to demonstrate that WAY-100135 acts as a 5-HT 1A receptor antagonist in the hamster, the drug was shown to antagonize the behavioral syndrome induced by 8-hydroxy-2-(di- n-propylamino)tetralin. When administered at 5-HT 1A receptor antagonistic doses in dystonic hamsters, (+)-WAY-100135 dramatically aggravated the dystonic attacks. The data thus suggest that, in contrast to previous theoretical proposals, 5-HT 1A receptor antagonists provide no movel therapeutic approach to involuntary movement disorders such as dystonia.

Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5.

Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. The rate of DNase I digestion of unirradiated CHO K1 and xrs-5 was not significantly different, but the extent of digestion was greater in unirradiated CHO K1 cells that in xrs-5 cells suggesting that less xrs-5 cell chromatin at DNA attachment points is accessible to the enzyme DNase I. The extents of digestion in irradiated (1 Gy) CHO K1 and xrs-5 cell nuclei also differed but the relationship was reversed. The irradiated xrs-5 cell samples were digested to a greater extent compared with CHO K1 cells. These chromatin digestion data suggest that the matrix attachment regions in xrs-5 cells are different from CHO K1 cells. The different DNA attachment organization in the xrs-5 cells may play a role in modulating radiation sensitivity.

The atypical neuroleptic, clozapine, exerts antidystonic activity in a mutant hamster model. Comparison with haloperidol

The effects of the atypical neuroleptic clozapine were studied in an inbred line of Syrian golden hamsters with generalized dystonia, i.e. a frequent movement disorder in humans. The effects of clozapine were compared with those of the classical neuroleptic, haloperidol. Clozapine, 7.5–20 mg/kg i.p., potently reduced the severity of dystonic attacks in the mutant hamster model, but induced marked sedation at these doses. Lower doses were ineffective. Haloperidol, 0.5 mg/kg i.p., significantly reduced the severity of dystonia without marked sedation. The finding that clozapine possesses antidystonic potency similar to that of haloperidol in a genetic model of dystonia might suggest that this atypical neuroleptic is an effective alternative in the treatment of dystonic patients who respond to neuroleptics, particularly because of the clinical evidence that clozapine is almost devoid of extrapyramidal adverse effects.

Cellular characteristics of Chinese hamster cell mutants resembling ataxia telangiectasia cells

The radiosensitive Chinese hamster V79 cell mutants (V-C4, V-E5 and V-G8), isolated previously in our laboratory, have been shown to resemble human ataxia telangiectasia (A-T) cells. These hamster cell mutants were further characterized with respect to cross-sensitivity to different radiomimetic agents and to mutation induction by X-rays. The data on cell survival (D10 Values) show that they are hypersensitive to adriamycin (2–3-fold increase), etoposide (3-fold for V-G8 and 6-fold for V-E5 and V-C4), calicheamicin γ1I (4-fold) and streptonigrin (3-fold for V-G8 and V-C4, and 12-fold for V-E5). The frequency of X-ray-induced hprt mutations is slightly enhanced in the hamster mutant cells treated with the same dose. However, the mutants show similar mutability as parental V79 cells when considering the same survival level. The overall conclusion from these studies is that these hamsters cell mutants mimic the phenotypic characteristics observed in cultured cells from A-T patients and therefore, may be defective in the same repair pathway as their human counterparts.

Loss of Transcriptional Activation of Three Sterol-Regulated Genes in Mutant Hamster Cells

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

The glycine/NMDA receptor ligand (+)-HA-966 but not D-cycloserine has potent antidystonic efficacy in a genetic animal model of dystonia

The effects of R-(+)-HA-966 ((+)-3-amino-1-hydroxypyrrolid-2-one), a low-efficacy partial agonist of the glycine modulatory site of the NMDA receptor complex, were studied in an inbred line of Syrian golden hamsters with generalized dystonia, a frequent movement disorder in humans. The effects of R-(+)-HA-966 were compared with those of D-cycloserine, a glycine/NMDA receptor ligand with higher intrinsic activity. R-(+)-HA-966, 30–60 mg/kg i.p., potently reduced the severity of dystonic attacks in the mutant hamster model of dystonia without inducing any behavioural adverse effects. D-Cycloserine did not exert antidystonic activity at i.p. doses of 10–40 mg/kg, which might be due to its much higher intrinsic activity at the glycine site. The data indicate that the antidystonic effect of (+)-HA-966 is related to antagonism of NMDA receptor-mediated excitatory neurotransmission.

Functional equivalence of human X– and Y–encoded isoforms of ribosomal protein S4 consistent with a role in Turner syndrome

Several genes are found on both the human X and Y chromosomes in regions that do not recombine during male meiosis. In each case, nucleotide sequence analysis suggests that these X-Y gene pairs encode similar but nonidentical proteins. Here we show that the human Y- and X-encoded ribosomal proteins, RPS4Y and RPS4X, are interchangeable and provide an essential function: either protein rescued a mutant hamster cell line that was otherwise incapable of growth at modestly elevated temperatures. These findings are consistent with the hypothesis that RPS4 deficiency has a role in Turner syndrome, a complex human phenotype associated with monosomy X.

The κ-opioid receptor agonist, U50,488H, exerts antidystonic activity in a mutant hamster model of generalized dystonia

The κ-opioid receptor agonist, U50,488H (trans-(±)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide), has recently been reported to induce dystonia after s.c. administration of 5–10 mg/kg in guinea pigs. The dystonic movements observed in response to U50,488H resembled those previously reported to occur spontaneously or in response to mild environmental stimuli in a mutant hamster model of paroxysmal generalized dystonia. This prompted us to study the effects of opioid receptor antagonists and of U50,488H in the mutant hamster model. Naloxene and naltrexone, 1 and 10 mg/kg i.p., were either ineffective or tended to induce prodystonic effects in the mutant hamsters. In contrast, U50,488H markedly reduced the severity of dystonic movements at doses of 1–10 mg/kg s.c. In non-dystonic hamsters, U50,488H reduced locomotor activity but did not produce dystonic-like symptoms. The data from mutant hamsters demonstrate that stimulation of κ-opioid receptors is a powerful means of attenuating dystonic movements in a genetic animal model of idiopathic dystonia. The antidystonic effects of U50,488H might relate to interactions between κ-opioid receptors and dopaminergic and glutamatergic neurotransmission.