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D B Roth

Publications and source records attributed to D B Roth.

At least 55 records · Page 3Linked to original sources
PubMed1997

Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products.

Ku, a heterodimer of 70- and 86-kDa subunits, serves as the DNA binding component of the DNA-dependent protein kinase (DNA-PK). Cells deficient for the 86-kDa subunit of Ku (Ku86-deficient cells) lack Ku DNA end-binding activity and are severely defective for formation of the standard V(D)J recombination products, i.e., signal and coding joints. It has been widely hypothesized that Ku is required for protection of broken DNA ends generated during V(D)J recombination. Here we report the first analysis of V(D)J recombination intermediates in a Ku-deficient cell line. We find that full-length, ligatable signal ends are abundant in these cells. These data show that Ku86 is not required for the protection or stabilization of signal ends, suggesting that other proteins may perform this function. The presence of high levels of signal ends in Ku-deficient cells prompted us to investigate whether these ends could participate in joining reactions. We show that nonstandard V(D)J recombination products (hybrid joints), which involve joining a signal end to a coding end, form with similar efficiencies in Ku-deficient and wild-type fibroblasts. These data support the surprising conclusion that Ku is not required for some types of V(D)J joining events. We propose a novel RAG-mediated joining mechanism, analogous to disintegration reactions performed by retroviral integrases, to explain how formation of hybrid joints can bypass the requirement for Ku and DNA-PK.

3T3 Cells↗
PubMed1997

Visual results after submacular surgery for neovascularization in age-related macular degeneration.

BACKGROUND AND OBJECTIVE: Submacular membranectomy has been proposed as a treatment option for subfoveal choroidal neovascular membranes (CNVMs). The authors reviewed the visual outcomes of patients who underwent surgical removal of subfoveal CNVMs caused by age-related macular degeneration (ARMD). PATIENTS AND METHODS: Thirty-eight patients (38 eyes) were retrospectively reviewed. Selection criteria included: (1) the presence of a well-demarcated subfoveal CNVM on fluorescein angiography; (2) best-corrected Snellen visual acuity reduced to the level of 20/200 or worse; and (3) no other eye disease that could compromise visual acuity. Pars plana vitrectomy, retinotomy, and membrane removal were performed to remove the subfoveal choroidal neovascular complex with minimal disruption of the surrounding tissues. A significant change was defined as a two-line difference from best-corrected preoperative visual acuity on the Snellen chart. RESULTS: At 3 months postoperatively, 7 (18.4%) of the 38 eyes had improved, 8 (21.1%) of the eyes had worsened, and 23 (60.5%) of the eyes had remained unchanged. The final visual acuity improved in 8 (21.1%) of the eyes, worsened in 11 (28.9%) of the eyes, and remained unchanged in 19 (50%) of the eyes. The average follow-up time was 632 days. CONCLUSIONS: The authors employed a minimally invasive approach to the removal of CNVMs in ARMD. Nevertheless, the resultant visual acuity was often unsatisfactory. Therefore, the authors recommend not operating on ARMD-associated subfoveal CNVMs with the current technology.

Aged↗
PubMed1996

Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates.

Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining.

Animals↗
PubMed1996

p53 is required for both radiation-induced differentiation and rescue of V(D)J rearrangement in scid mouse thymocytes.

The murine scid mutation affects both V(D)J recombination and DNA repair. This mutation has been mapped to the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), which is activated by DNA damage in normal cells. In scid mice, antigen receptor gene rearrangements are initiated normally, but impaired joining of coding ends prevents assembly of functional receptor genes, resulting in arrest of B- and T-cell development. Others have shown that exposure of scid mice to genotoxic agents such as gamma-irradiation rescues rearrangement at the T-cell receptor (TCR) beta locus and promotes thymocyte development. Here we demonstrate that irradiation rescues rearrangements at multiple TCR loci, suggesting a general effect on the recombination mechanism. Furthermore, our data show that p53 is required for irradiation-mediated rescue of both thymocyte development and V(D)J recombination. We also find that thymocyte proliferation and differentiation in the absence of DNA damage do not require p53 and are not sufficient to rescue V(D)J recombination. These results suggest that exposure to ionizing radiation facilitates a partial bypass of the scid defect, perhaps by inducing p53-dependent DNA damage response pathways.

Animals↗
PubMed1996

Local carboplatin and radiation therapy in the treatment of murine transgenic retinoblastoma.

BACKGROUND: Combined modality therapy in the treatment of retinoblastoma may decrease treatment-related morbidity and second tumor-associated mortality, while maintaining excellent tumor control rates. OBJECTIVE: To evaluate tumor control and potential synergy between intravitreally delivered carboplatin and external beam radiation therapy (EBRT), using a transgenic murine model of spontaneous heritable retinoblastoma. METHODS: Sixty-six mouse eyes from 4-week-old transgenic mice positive for the simian virus 40 large T antigen were evaluated. Thirty-three mice were treated with 5 intravitreal injections of carboplatin (ranging from 0.1-4.0 micrograms) combined with concurrent bilateral EBRT (ranging from 10-30 Gy) delivered in twice daily 5-Gy fractions. All eyes were followed up for treatment complications. Twelve weeks following final treatment, all eyes were enucleated, serial histologic sections obtained, and the eyes examined for the presence of retinoblastoma. RESULTS: No eye treated with 0.1 microgram of carboplatin and EBRT exhibited tumor control. Three (75%) of 4 mice receiving 1.0 microgram of carboplatin combined with 10-Gy EBRT had complete tumor control. Four (100%) of 4 mice receiving 1.0 microgram of carboplatin combined with 30-Gy EBRT had complete tumor control. Nine (100%) of 9 mice receiving 4.0 micrograms of carboplatin in combination with EBRT had complete tumor control. The chemotherapeutic enhancement ratio ranged from 1.07 to 3.24. CONCLUSIONS: Combined administration of intravitreal carboplatin and EBRT enhances local tumor control in murine retinoblastoma. Combining these treatment modalities may allow tumor control in selected patients with retinoblastoma while decreasing treatment-related morbidity and the mutagenic risks associated with radiation and systemic chemotherapy.

Animals↗
PubMed1996

Thymocyte differentiation in gamma-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor alpha locus.

Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) beta, gamma and delta loci, but not at the TCR alpha locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR alpha locus. We found that J alpha-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4+ CD8+ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR alpha is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of gamma-irradiation after abundant CD4+ CD8+ thymocytes and hairpin TCR alpha coding ends had accumulated. This treatment failed to stimulate rescue of TCR alpha coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice.

Animals↗
PubMed1996

Mechanism of V(D)J recombination.

V(D)J recombination can be separated into two basic operations: DNA cleavage and joining of broken ends. Our understanding of both reactions has increased substantially in the past year. Major advances include the development of a cell-free system capable of cleavage and the identification of several proteins involved in both V(D)J recombination and double-strand break repair.

Animals↗
PubMed1996

The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo.

BACKGROUND: V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both. RESULTS: We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo. CONCLUSIONS: These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo.

Animals↗
PubMed1996

High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome.

To examine the mechanisms of recombination governing the illegitimate integration of transfected DNA into a mammalian genome, we developed a cell system that selects for integration events in defined genomic regions. Cell lines with chromosomal copies of the 3' portion of the adenine phosphoribosyltransferase (APRT) gene (targets) were established. The 5' portion of the APRT gene, which has no homology to the integrated 3' portion, was then electroporated into the target cell lines, and selection for APRT gene function was applied. The reconstruction of the APRT gene was detected at frequencies ranging from less than 10(-7) to 10(-6) per electroporated cell. Twenty-seven junction sequences between the integrated 5' APRT and its chromosomal target were analyzed. They were found to be randomly distributed in a 2-kb region immediately in front of the 3' portion of the APRT gene. The junctions fell into two main classes: those with short homologies (microhomologies) and those with inserted DNA of uncertain origin. Three long inserts were shown to preexist elsewhere in the genome. Reconstructed cell lines were analyzed for rearrangements at the target site by Southern blotting; a variety of simple and complex rearrangements were detected. Similar analysis of individual clones of the parental cell lines revealed analogous types of rearrangement, indicating that the target sites are unstable. Given the high frequency of integration events at these sites, we speculate that transfected DNA may preferentially integrate at unstable mammalian loci. The results are discussed in relation to possible mechanisms of DNA integration.

Adenine Phosphoribosyltransferase↗
PubMed1996

Mechanism of V(D)J recombination.

THe V(D)J recombination reaction can be separated into two fundamental operations: site specific cleavage and joining of broken ends. Much has been learned about the mechanisms of these steps in the past few years. Recent experiments have shown that cleavage is catalyzed by the RAG1 and RAG2 proteins and generates an asymmetric set of broken ends: hairpin coding ends and blunt signal ends. A cell free system capable of performing cleavages has been established, and detailed biochemical information about the reaction should accumulate rapidly. In vivo studies have provided insights into the regulation of cleavage. Recent experiments using artificial recombination substrates in cultured cells have shown that efficient cleavage requires a pair of RSS, one with a 12 nucleotide spacer and one with a 23 nucleotide spacer. Our understanding of the joining mechanism has also increased substantially, as several proteins involved in coding joint and signal joint formation (as well as in DSB repair) were recently identified.

Animals↗
PubMed1995

Hairpin opening by single-strand-specific nucleases.

DNA molecules with covalently sealed (hairpin) ends are probable intermediates in V(D)J recombination. According to current models hairpin ends are opened to produce short single-stranded extensions that are thought to be precursors of a particular type of extra nucleotides, termed P nucleotides, which are frequently present at recombination junctions. Nothing is known about the activities responsible for hairpin opening. We have used two single-strand-specific nucleases to explore the effects of loop sequence on the hairpin opening reaction. Here we show that a variety of hairpin ends are opened by P1 nuclease and mung bean nuclease (MBN) to leave short, 1-2 nt single-stranded extensions. Analysis of 22 different hairpin sequences demonstrates that the terminal 4 nt of the hairpin loop strongly influence the sites of cleavage. Correlation of the nuclease digestion patterns with structural (NMR) data for some of the hairpin loops studied here provides new insights into the structural features recognized by these enzymes.

Base Sequence↗
PubMed1995

Repair and recombination. How to make ends meet.

The repair of double-stranded breaks in DNA and the recombination of antibody gene V(D)J segments share a common pathway involving the Ku protein, which binds DNA ends, and its associated protein kinase.

Animals↗
PubMed1995

Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V(D)J recombination.

We previously identified possible intermediates in V(D)J recombination at the TCR delta locus and characterized molecules with signal ends and with covalently sealed (hairpin) coding ends in thymocytes of scid mice by Southern blotting. Here, we use a sensitive ligation-mediated PCR assay to demonstrate that all coding ends detected in scid thymocytes are covalently sealed. Neither coding nor signal ends exhibit loss or addition of nucleotides. These data imply that hairpin formation is coupled to the initial cleavage at the signal/coding border, and that the cleavage step in V(D)J recombination is conservative. In scid/+ or wild-type thymocytes, hairpin coding ends are at least 1000-fold less abundant than signal ends. These results provide insight into the mechanism of V(D)J recombination.

Animals↗
PubMed1995

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage.

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.

Animals↗
PubMed1995

Paediatric post-traumatic endophthalmitis.

AIMS: A retrospective analysis of children with post-traumatic endophthalmitis was performed to determine if microbiological differences exist between this disease in the paediatric population compared with this disease in adults. METHOD: Twelve cases of post-traumatic endophthalmitis in children were analysed to determine characteristics of this disease in youth. Patient ages varied from 18 months to 13 years; the mean age was 8 years. Gram positive organisms were isolated in eight eyes, Gram negative organisms from four eyes, fungus from one eye, and negative cultures in three eyes. The most common isolates were streptococcal species (56.6%) and staphylococcal species (22.2%). Vitrectomy was performed on eight (66.7%) eyes. RESULTS: Visual acuity of 20/200 or better was obtained in eight eyes (66.7%). Three eyes had vision less than 5/200. One eye developed phthisis bulbi. Nine (75%) patients were younger than 10 years of age, and six (66.7%) of these nine obtained a final visual acuity of 20/200 or better. CONCLUSION: Useful vision can be obtained in children with post-traumatic endophthalmitis with early, aggressive treatment. The microbiology of paediatric post-traumatic endophthalmitis differs from adult disease with streptococcal species as the most common infecting organisms.

Adolescent↗
PubMed1993

Characterization of broken DNA molecules associated with V(D)J recombination.

We previously demonstrated that DNA molecules with double-strand breaks at variable-(diversity)-joining [V(D)J] recombination signal sequences are relatively abundant in mouse thymocytes. This abundance strongly suggests that the mechanism of V(D)J recombination involves double-strand cleavage at recombination signals. As a first step toward understanding the mechanism of cleavage, we used a sensitive PCR assay to characterize the structure of one class of cleavage products, the signal ends, in detail. Here we demonstrate that most of these ends are blunt and terminate in 5' phosphoryl groups. Virtually all of the flush signal ends are full length. A minor subpopulation of broken ends terminates in short single-strand extensions. We have found no evidence for covalent DNA-protein linkages involving the signal ends. These data allow further refinement of the double-strand cleavage model for V(D)J recombination.

Animals↗