Effects on the Underlying Water Column by Extensive Floating Treatment Wetlands.

Floating treatment wetlands (FTWs) are an emerging engineering option with promise for simultaneous water quality improvement and habitat creation. Relatively little research has been published regarding their construction or effects on the underlying water column. In this field-scale experiment, four different extensive FTW designs were constructed using minimal materials, including drainpipe, burlap, mulch, utility netting, and reused polyethylene bottles. The FTWs were then planted with spp. (cattail) and L. (common rush). Over 28 mo, the water column beneath FTWs in two test ponds was compared to that in an open water control pond. The ponds (190 ± 10 m) were fed with well water enriched with nitrate to emulate agricultural ponds. Although observed differences were relatively small, statistically significant differences were noted. With respect to the control, waters underneath FTWs had lower dissolved oxygen, sulfate, nitrate, and pH, dampened diurnal temperature fluctuations, and greater alkalinity. The FTWs created habitat and were colonized by species of insects, birds, amphibians, snails, and spiders. Results indicated that spp. is suitable for FTW creation. However, a more supportive planting matrix is suggested to encourage faster plant growth and protect against wind and wave action damage. Although plant growth was limited, results suggest that FTWs may be applied to encourage less aerobic and more organic rich and thermally insulated conditions for water quality improvement in agricultural ponds and other aquatic systems while also creating valuable habitat.

Is the hip capsule thicker in diseased hips?

OBJECTIVES: The purpose of this study was to compare the thickness of the hip capsule in patients with surgical hip disease, either with cam-femoroacetabular impingement (FAI) or non-FAI hip pathology, with that of asymptomatic control hips. METHODS: A total of 56 hips in 55 patients underwent a 3Tesla MRI of the hip. These included 40 patients with 41 hips with arthroscopically proven hip disease (16 with cam-FAI; nine men, seven women; mean age 39 years, 22 to 58) and 25 with non-FAI chondrolabral pathology (four men, 21 women; mean age 40 years, 18 to 63) as well as 15 asymptomatic volunteers, whose hips served as controls (ten men, five women; mean age 62 years, 33 to 77). The maximal capsule thickness was measured anteriorly and superiorly, and compared within and between the three groups with a gender subanalysis using student's t-test. The correlation between alpha angle and capsule thickness was determined using Pearson's correlation coefficient. RESULTS: Superiorly, the hip capsule was significantly greater in cam- (p = 0.028) and non-FAI (p = 0.048) surgical groups compared with the asymptomatic group. Within groups, the superior capsule thickness was significantly greater than the anterior in cam- (p < 0.001) and non-FAI (p < 0.001) surgical groups, but not in the control group. There was no significant correlation between the alpha angle and capsule thickness. There were no gender differences identified in the thickness of the hip capsule. CONCLUSION: The thickness of the capsule does not differ between cam- and non-FAI diseased hips, and thus may not be specific for a particular aetiology of hip disease. The capsule is, however, thicker in diseased surgical hips compared with asymptomatic control hips.Cite this article: K. S. Rakhra, A. A. Bonura, R. Nairn, M. E. Schweitzer, N. M. Kolanko, P. E. Beaule. Is the hip capsule thicker in diseased hips? Bone Joint Res 2016;5:586-593. DOI: 10.1302/2046-3758.511.2000495.

Biochemical oxygen demand and nutrient processing in a novel multi-stage raw municipal wastewater and acid mine drainage passive co-treatment system.

A laboratory-scale, four-stage continuous flow reactor system was constructed to test the viability of high-strength acid mine drainage (AMD) and municipal wastewater (MWW) passive co-treatment. The synthetic AMD had pH 2.60 and 1860 mg/L acidity as CaCO(3) equivalent with 46, 0.25, 2, 290, 55, 1.2 and 390 mg/L of Al, As, Cd, Fe, Mn, Pb and Zn, respectively. The AMD was introduced to the system at a 1:2 ratio with raw MWW from the City of Norman, Oklahoma USA containing 265 ± 94 mg/L BOD(5), 11.5 ± 5.3 mg/L PO(4)(-3), and 20.8 ± 1.8 mg/L NH(4)(+)-N. During the 135 d experiment, PO(4)(-3) and NH(4)(+)-N were decreased to <0.75 and 7.4 ± 1.8 mg/L, respectively. BOD(5) was generally decreased to below detection limits. Nitrification increased NO(3)(-) to 4.9 ± 3.5 mg/L NO(3)(-)-N, however relatively little denitrification occurred. Results suggest that the nitrogen processing community may require an extended period to mature and reach full efficiency. Overall, results indicate that passive AMD and MWW co-treatment is a viable ecological engineering approach for the developed and developing world that can be optimized and applied to improve water quality with minimal use of fossil fuels and refined materials.

People never see us living well: an appraisal of the personal stories about mental illness in a prospective print media sample.

OBJECTIVE: Having found no discussions of self-depictions offered by psychiatric patients in the mass media we sought such items in a prospective national sample of print media and analysed how those speakers portrayed themselves. METHOD: As part of a larger study of media depictions of mental illnesses in print media all items with any mental health or illness aspect that appeared in a New Zealand publication over a four-week period were collected. The resulting collection of 600 items ranged from news briefs to full-page newspaper articles. From that set we selected and analysed items in which a person identified as having been a psychiatric patient or as having a mental disorder was either quoted by the reporter who had interviewed them, or personally described their experiences. Employing both propositional analyses and discourse analysis we explored how the speakers were positioned and identified patterns or themes in their construction of living with a mental illness. RESULTS: Only five articles (0.8%) met our criteria for a person with a mental disorder being reported directly. In those items the journalists had positioned the speakers as credible, expert sources who, in representing their lives and experiences, drew on five clusters of resources, that we titled: Ordinariness/Living Well; Vulnerability; Stigma; Crisis; and Disorder/Treatment. Ordinariness/Living Well foregrounded the role of personal strengths in living well and in overcoming adversity, particularly that associated with being stigmatized. We identified that theme as central to the ways in which these speakers depicted themselves as recognizably human and understandable. CONCLUSION: The findings are preliminary but these depictions are different from those reported by most researchers. Unlike those depictions, these speakers provided accessible and recognizably human self-portrayals. That finding intensifies our concern that most researchers appear to be unaware that these consumer voices are largely absent from mass media depictions of mental illnesses.

Nucleotide excision repair proteins and their importance for radiation-enhanced transfection.

PURPOSE: Irradiated cells transfect more efficiently than unirradiated cells because of a radiation-induced increase in plasmid integration. However, the molecular mechanism is unclear. Because of recent observations that nucleotide excision repair (NER) proteins can be involved in certain types of recombination in yeast, it was hypothesized that NER proteins might play a role in this radiation-enhanced integration. MATERIALS AND METHODS: Hamster and human cells with inactivating mutations in NER genes were irradiated at doses from 0 to 6 Gy and then immediately transfected with a linearized selectable marker plasmid. Transfection-enhancement ratios (TERs) were calculated as the ratio of the number of drug-resistant colonies in unirradiated cells to the number of transfectants in irradiated cells, corrected for cytotoxicity from radiation. RESULTS: Transfection into unirradiated rodent cells was unaffected by NER mutation status. Transfection into unirradiated human cells, however, was increased by NER mutation. The TERs were 5 and 100 for CHO and primary human fibroblasts, respectively, after exposure of the cells to 6 Gy. Mutations in ERCC1, XPA, XPB, XPC, XPF, XPG and CSB dramatically reduced TER. Mutations in ERCC1, XPC, XPF, XPG and CSB suppressed transfection so that the TER was significantly below 1. CONCLUSIONS: The mechanism of radiation-enhanced plasmid integration was distinct from that of plasmid integration in unirradiated cells, and NER gene products were critical for enhanced integration to occur.

From source material to news story in New Zealand print media: a prospective study of the stigmatizing processes in depicting mental illness.

OBJECTIVE: The aim of this study was to analyse how newspaper articles that depict mental illnesses are generated from source materials. METHOD: From a prospectively collected national sample of print materials involving mental illness, 50 published items that related to the Privacy Commissioner's opinion about disclosure of a psychiatric patient's health information were identified. A copy of the Privacy Commissioner's original Case Note and three news stories about the Case Note distributed by the New Zealand Press Association constituted the database. These materials were subjected to discourse analysis. We identified themes and their transformation from the Case Note through the news stories and examined the impact of these transformations on the stigmatization of mental illness. RESULTS: Four themes were identified: human rights, vulnerability, risk of dangerousness and threat, and mental illness/psychiatric patient. The only potentially positive theme, human rights, was limited both by being fragmented in the source material, and by being utilized, in the published news stories to undermine the legitimacy of the patient's right to privacy. Use of the other themes was consistent with stereotypes about mental illness. CONCLUSIONS: Although there were no inaccuracies in the content of the news stories they were substantially more negative than the source material in their depiction of the identified patient. A potentially positive discourse (human rights) was not by itself sufficient to ensure a positive portrayal of mental illness. An understanding of the transformations is important for efforts to effectively combat the stigmatization of those with mental illness.

MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

A laboratory investigation of the role of guide planes in the retention of cast cobalt-chromium alloy partial denture frameworks.

OBJECTIVES: This in vitro study examined the resistance to displacement offered by guide planes and the displacement mechanics of a bilateral bounded saddle cast cobalt chrome alloy removable partial denture framework under conditions simulating an average initial fit. METHODS: A specially designed model mouth jig fitted with strain gauges to monitor the forces across the guiding plane/guiding surface interfaces was used. Guiding planes of enamel, silver tin amalgam and dental composite with seven combinations of guide planes and three angles of withdrawal were examined. Guiding plane size was also examined for dental enamel. RESULTS: A statistically significant difference in retention was found for all factors examined. Retention increased with increasing angle of withdrawal. Size effects were found but considered unreliable. CONCLUSIONS: It was concluded that with a tight fit guiding plane retention may be readily predicted for any given material, combination of mesial, distal and lingual guiding planes and angulation conditions by the application of mechanical principles providing the basic parameters are known. Attention is drawn to the reasons for further work to fully understand the clinical situation.

Characterization of the CDKN2A and ARF genes in UV-induced melanocytic hyperplasias and melanomas of an opossum (Monodelphis domestica).

We examined the involvement of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in the pathogenesis of ultraviolet (UV) radiation-induced melanomas in an opossum (Monodelphis domestica) melanoma model in which suckling young were exposed to UVB to produce melanocytic lesions. Monodelphis CDKN2A and alternated reading frame (ARF) cDNAs were cloned and sequenced, and the expression patterns of these genes were determined by reverse transcription-polymerase chain reaction in normal tissues, 39 primary melanocytic skin lesions, and two tumor-derived cell lines, one nonmetastatic and one metastatic. Primary melanocytic lesions, including hyperplasias, benign melanomas, melanomas metastatic to lymph nodes, and melanomas metastatic to nodes and additional visceral organs, were categorized accordingly as types I-IV. Levels of CDKN2A transcripts were most abundant in type III tumor samples and the metastatic cell line but absent in the nonmetastatic cell line. ARF transcripts were expressed in all tumors and cell lines. A UV-signature mutation was detected with the wild-type allele at the CDKN2A locus in type II and III primary tumor samples and in the nonmetastatic cell line. Interestingly, in the metastatic cell line, only the mutant allele was present and expressed. These data suggest dynamic changes in the expression and/or structure of the CDKN2A and ARF genes represent one molecular defect associated with the etiology of melanoma formation and progression in the Monodelphis model system.

Self-efficacy for coping with cancer: revision of the Cancer Behavior Inventory (version 2.0).

The Cancer Behavior Inventory (CBI), a measure of self-efficacy for coping with cancer, was revised by adding a new stress management scale and reducing its length from 43 to 33 items. The 33-item CBI was administered to 280 cancer patients. A principal factors analysis with varimax rotation yielded the hypothesized seven factors (alphas in parentheses): (1) maintenance of activity and independence (alpha=0.86), (2) seeking and understanding medical information (alpha=0.88), (3) stress management (alpha=0.86), (4) coping with treatment-related side-effects (alpha=0.82), (5) accepting cancer/maintaining positive attitude (alpha=0.86), (6) affective regulation (alpha=0.81), and (7) seeking support (alpha=0.80). The alpha for the entire CBI was 0.94, the test-retest (1 week) reliability coefficient was 0.74, and correlations with measures of quality of life and coping supported its validity. The CBI may be useful to researchers and clinicians and can be integrated into a self-regulation model of coping.

Nutrient conversions by photosynthetic bacteria in a concentrated animal feeding operation lagoon system.

A diurnal examination was conducted to determine the effect of photosynthetic bacteria on nutrient conversions in a two-stage concentrated animal feeding operation (CAFO) lagoon system in west-central Oklahoma. Changes in nutrients, microbial populations, and physical parameters were examined at three depths (0, 1.5, and 3.0 m) every 3 h over a 36-h period. The south lagoon (SL) was anaerobic (dissolved oxygen [DO] = 0.09 +/- 0.12 mg/L) while the north lagoon (NL) was facultative (DO ranged from 4.0-0.1 mg/L over 36-h period). Negative sulfide-sulfate (-0.85) and bacteriochlorophyll a (bchl a)-sulfate (-0.83) correlations, as well as positive bchl a-sulfide (0.87) and light intensity (I)-bchl a (0.89) correlations revealed that the SL was dominated by sulfur conversions driven by the photosynthetic purple sulfur bacteria (PSB). The correlation data was supported by diurnal trends for sulfate, sulfide, and bchl a. Both nitrogen and sulfur conversions played a role in the NL; however, nitrogen conversions appeared to dominate this system because of the activity of cyanobacteria. This was shown by positive chlorophyll a (chl a)-I (0.91) and chl a-nitrate (0.98) correlations and the negative correlation between ammonium and nitrite (-0.88). Correlation data was further supported by diurnal trends observed for chl a, DO, and ammonium. For both lagoons, the dominant photosynthetic microbial species determined which nutrient conversion processes were most important.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC(1)) hybrids produced from backcrossing F(1) hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC(1) hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC(1) hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC(1) hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianus CDKN2X alleles in BC(1) hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC(1) hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

Genetic analysis of neoplasia induced by N-nitroso-N-methylurea in Xiphophorus hybrid fish.

Interspecific crosses within the genus Xiphophorus have historically been used to study the genetic aspects of melanoma formation. Melanomas typically occur as a result of deregulation of polymorphic, naturally occurring macromelanophore pigment patterns. Hybrid crosses also have been used to study the inducibility of melanoma by physical sources (such as UV light) and chemicals (such as N-methyl-N-nitrosourea, MNU). We previously defined a genomic region that is implicated in fish melanomagenesis and identified a candidate tumor suppressor gene (CDKN2X) within this genomic area. Highly significant associations between BC(1)-hybrid CDKN2X genotypes and UV-induced melanoma formation exist in a backcross produced from 2 inbred parental lines. However, when BC(1) hybrids are exposed to MNU as the tumor induction agent, a significant association between inheritance of CDKN2X alleles and tumor development is not observed. These data suggest there is mechanistic and genetic heterogeneity in melanomas derived from different etiologies within BC(1) hybrid fish.

A proposed classification scheme for Xiphophorus melanomas based on histopathologic analyses.

We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous-macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.

Xiphophorus genetic linkage map: beginnings of comparative gene mapping in fishes.

The explosive expansion of gene maps of mouse and man has provided strong support for hypotheses first advanced from comparing fish and mammalian genomes that the vertebrate genome was derived from multiple ancestral tetraploidizations with subsequent preferential translocations among paralogous chromosomes. At least two genome duplication events have become widely accepted in lineages leading to vertebrates, and a third has been proposed either before, or after, divergence of fishes and tetrapods. Cytogenetic and comparative gene mapping studies suggest that teleost gene maps have diverged more slowly from gene arrangements in the vertebrate ancestor than have those of mammals. The recent assembly of extensive maps of >100 genes in three fish species, medaka (Beloniformes), Xiphophorus swordtails and platyfishes (Cyprinodontiformes), and zebrafish (Cypriniformes) and the development of less extensive maps in several other fish orders provides the first salient opportunity to assess homology of most or all chromosomes among fishes.

Aquaria fish models of human disease: reports and recommendations from the working groups.

On September 21-24, 2000, the National Cancer Institute, Southwest Texas State University, and the Roy and Joan Mitte Foundation sponsored an international conference entitled "Aquaria Fish Models of Human Disease" at Southwest Texas State University (SWT), San Marcos, Texas, USA. Over 100 scientists, representing various fish model systems, participated in four roundtable working groups. We considered the first step in promoting the exciting research with fish models was to unify the efforts within this scientific community towards accomplishing specific goals. With this objective in mind, the following four working groups were convened: (1) fish cancer models: sustenance and enhancement; (2) fish genomics and transgenics: resources and technology; (3) fish pathology: standards for tumor pathology classification; and, (4) resources underpinning aquaria fish research. Each working group was charged with preparing a report of their discussions with recommendations on how researchers and funding agencies might best direct and strengthen research support to ensure a healthy future for such work. Included are the final reports from these working groups, together with a brief summary of the discussions held during the sessions and the consensus recommendations from each group.

Condylar repositioning in mandibular retrusion.

STATEMENT OF PROBLEM: There has been debate about which anteroposterior maxillomandibular relationship should be used in fixed and removable prosthodontics for patients without a definite maximal intercuspation. The choice has important implications for complete denture fabrication in which any guidance to the previous dentulous maximal intercuspal position is missing. PURPOSE: This study was designed to estimate the posterior displacement that takes place at the mandibular condyles and occlusal surfaces when the mandible is moved from maximal intercuspal position to the most retruded mandibular position. MATERIAL AND METHODS: Articulated occlusal models of 18 subjects with a natural dentition and well-defined maximal intercuspation were studied. Models were rearticulated in retruded contact position, and the original vertical dimension of occlusion was restored by eliminating all interfering occlusal contacts on the retruded path of closure. Measurement of change in condylar position was estimated using a SAM2 articulator with condylar position indicator. Posterior displacement at the occlusal surfaces was measured with a traveling microscope. RESULTS: Condylar analog retrusion in the horizontal plane at the initial retruded contact position varied from 0.6 to 2.4 mm (mean 1.0 +/- 0.4 mm); however, after occlusal adjustment to restore the original vertical dimension of occlusion, retrusion was reduced to a range of 0 to 0.4 mm (mean 0.2 +/- 0.1 mm). Retrusion at the occlusal surfaces was found to vary from 0.4 to 1.5 mm (mean 0.7 +/- 0.3 mm) at retruded contact position; however, after occlusal adjustment, retrusion was reduced to a range of 0 to 0.5 mm (mean 0.2 +/- 0.1 mm). CONCLUSION: Posterior displacement at both the occlusal surfaces and the condyles was small when interfering occlusal contacts on the retruded path of closure were removed.

Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination.

The XpF/Ercc1 structure-specific endonuclease performs the 5' incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1(+) and ERCC1(-) cell lines with insertion vectors having long or short terminal non-homologies flanking each side of a double-strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT(+) recombinants recovered from ERCC1(-) cells using targeting vectors with long terminal non-homologies-almost complete ablation of gap repair and single-reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1(+) cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non-homologous tails from invading homologous strands.

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells.

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.