Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.

Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cells. The frequency distributions of mutational classes induced by UVB and by simulated sunlight in repair-deficient CHO cells were virtually identical, each showing a marked increase in tandem CC-->TT transitions relative to NER-proficient cells. A striking increase in CC-->TT events was also previously documented for mutated p53 tumor-suppressor genes from nonmelanoma tumors of NER-deficient, skin cancer-prone xeroderma pigmentosum patients, compared to normal individuals. The data therefore indicate that the aprt gene in NER-deficient cultured rodent cells irradiated with artificial solar light generates the same distinctive "fingerprint" for sunlight mutagenesis as the p53 locus in NER-deficient humans exposed to natural sunlight in vivo. Moreover, in strong contrast to the situation for repair-component CHO cells, where a significant role for UVA was previously noted, the mutagenic specificity of simulated sunlight in NER-deficient CHO cells and of natural sunlight in humans afflicted with xeroderma pigmentosum can be entirely accounted for by the UVB portion of the solar wavelength spectrum.

Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma.

The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression.

Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea.

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.

Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a single synaptic climbing fiber response, a so-called complex spike, resulted in a transient elevation of [Ca2+]i that showed distinct differences among various subcellular compartments. With conventional imaging, the Ca2+ signals were prominent in the dendrites and almost absent in the soma. Confocal recordings from the somatic region, however, revealed steep transient increases in [Ca2+]i that were confined to a submembrane shell of 2- to 3-microns thickness. In the central parts of the soma [Ca2+]i increases were much slower and had smaller amplitudes. The kinetics and amplitudes of the changes in [Ca2+]i were analyzed in more detail by using the fast, low-affinity Ca2+ indicator Calcium Green-5N (Kd approximately 17 microM). We found that brief depolarizing pulses produced [Ca2+]i increases in a narrow somatic submembrane shell that resembled those seen in the dendrites. These results provide direct experimental evidence that the surface-to-volume ratio is a critical determinant of the spatiotemporal pattern of Ca2+ signals evoked by synaptic activity in neurons.

Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer.

Contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast- versus slow-twitch myofibers. This fiber-type-specific gene regulation occurs by unknown mechanisms and does not occur within cultured myocytes. We have developed a transient, whole-animal assay using somatic gene transfer to study this phenomenon and have identified a fiber-type-specific regulatory element within the promoter region of a slow myofiber-specific gene. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow- versus fast-twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a slow troponin I promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well expressed by both muscle types when fused to a nonfiber-type-specific skeletal actin promoter. Deletion analysis of the troponin I promoter region revealed that a 157-bp enhancer conferred slow-muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow-twitch myofibers. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber-type-specific gene expression prior to the generation of transgenic mice.

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

Scrambling of the actin I gene in two Oxytricha species.

The DNA in a germ-line nucleus (a micronucleus) undergoes extensive processing when it develops into a somatic nucleus (a macronucleus) after cell mating in hypotrichous ciliates. Processing includes destruction of a large amount of spacer DNA between genes and excision of gene-sized molecules from chromosomes. Before processing, micronuclear genes are interrupted by numerous noncoding segments called internal eliminated sequences (IESs). The IESs are excised and destroyed, and the retained macro-nuclear-destined sequences (MDSs) are spliced. MDSs in some micronuclear genes are not in proper order and must be reordered during processing to create functional gene-sized molecules for the macronucleus. Here we report that the micronuclear actin I gene in Oxytricha trifallax WR consists of 10 MDSs and 9 IESs compared to the previously reported 9 MDSs and 8 IESs in the micronuclear actin I gene of Oxytricha nova. The MDSs in the actin I gene are scrambled in a similar pattern in the two species, but the positions of MDS-IES junctions are shifted by up to 14 bp for scrambled and 138 bp for the nonscrambled MDSs. The shifts in MDS-IES junctions create differences in the repeat sequences that are believed to guide MDS splicing. Also, the sizes and sequences of IESs in the micronuclear actin I genes are different in the two Oxytricha species. These observations give insight about the possible origins of IES insertion and MDS scrambling in evolution and show the extraordinary malleability of the germ-line DNA in hypotrichs.

Déficience dans la réparation par excision de nucléotides en phase S induite par la séquestration du facteur de réplication RPA

Introduction Les lésions induites par les rayons UV peuvent causer des blocages dans la réplication de l'ADN. Ces dommages sont éliminés par le processus moléculaire très conservé de réparation par excision de nucléotides (NER). Nous avons précédemment démontré que la protéine ATR, une kinase majeure impliquée dans le stress réplicatif, est requise pour une NER efficace, et ce exclusivement durant la phase S. Des résultats subséquents ont suggéré que ce prérequis n’était pas lié à la réponse induite par ATR, mais plutôt d’une conséquence globale causée par la présence de stress réplicatif. En ce sens, nous mettons l’emphase qu’après irradiation UV, le complexe RPA joue un rôle crucial dans l'activation des mécanismes de NER ainsi que dans le redémarrage des fourches de réplication bloquées. Hypothèses: En général, les mutations qui confèrent une augmentation du stress réplicatif engendrent une séquestration excessive du facteur RPA aux fourches de réplication bloquées ce qui réduit son accessibilité pour le NER. Méthodes et résultats: Le modèle de la levure a été choisi pour vérifier cette hypothèse. Nous avons développé un essai de NER spécifique à chacune des phases du cycle cellulaire pour démontrer que les cellules déficientes en Mec1, l’homologue d’ATR, sont défectives dans la réparation par excision de nucléotides spécifiquement en phase S. De plus, plusieurs autres mutants de levure, caractérisés par un niveau de dommages spontanés élevé, ont aussi exhibé un défaut similaire. Ces mutants ont démontré une fréquence et une intensité de formation de foyers de RPA plus élevée. Finalement, une diminution partielle de RPA dans les levures a induit un défaut significatif dans le NER spécifiquement durant la phase S. Conclusion: Nos résultats supportent la notion que la séquestration de RPA aux fourches de réplication endommagées durant la phase S prévient son utilisation pour la réparation par excision de nucléotides ce qui inhibe fortement l'efficacité de réparation. Cette étude chez la levure facilite l’élucidation du phénomène analogue chez l’humain et, ultimement, comprend des implications majeures dans la compréhension du mécanisme de développement des cancers UV-dépendants.

Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: Null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells

Human pyruvate dehydrogenase complex (PDC) catalyzes a key step in the generation of cellular energy and is composed by three catalytic elements (E1, E2, E3), one structural subunit (E3-binding protein), and specific regulatory elements, phosphatases and kinases (PDKs, PDPs). The E1α subunit exists as two isoforms encoded by different genes: PDHA1 located on Xp22.1 and expressed in somatic tissues, and the intronless PDHA2 located on chromosome 4 and only detected in human spermatocytes and spermatids. We report on a young adult female patient who has PDC deficiency associated with a compound heterozygosity in PDHX encoding the E3-binding protein. Additionally, in the patient and in all members of her immediate family, a full-length testis-specific PDHA2 mRNA and a 5′UTR-truncated PDHA1 mRNA were detected in circulating lymphocytes and cultured fibroblasts, being bothmRNAs translated into full-length PDHA2 and PDHA1 proteins, resulting in the co-existence of both PDHA isoforms in somatic cells.Moreover, we observed that DNA hypomethylation of a CpG island in the coding region of PDHA2 gene is associatedwith the somatic activation of this gene transcription in these individuals. This study represents the first natural model of the de-repression of the testis-specific PDHA2 gene in human somatic cells, and raises some questions related to the somatic activation of this gene as a potential therapeutic approach for most forms of PDC deficiency.

Late somatic effects of radiation.

Reprinted from Fundamental nuclear energy research - 1966.

Afffinity tag excision strategies for Car9-tagged proteins

Thesis (Master's)--University of Washington, 2016-06

Green turtle somatic growth dynamics in a spatially disjunct Great Barrier Reef metapopulation

The growth dynamics of green sea turtles resident in four separate foraging grounds of the southern Great Barrier Reef genetic stock were assessed using a nonparametric regression modeling approach. Juveniles recruit to these grounds at the same size, but grow at foraging-ground-dependent rates that result in significant differences in expected size- or age-at-maturity. Mean age-at-maturity was estimated to vary from 25-50 years depending on the ground. This stock comprises mainly the same mtDNA haplotype, so geographic variability might be due to local environmental conditions rather than genetic factors, although the variability was not a function of latitudinal variation in environmental conditions or whether the food stock was seagrass or algae. Temporal variability in growth rates was evident in response to local environmental stochasticity, so geographic variability might be due to local food stock dynamics. Despite such variability, the expected size-specific growth rate function at all grounds displayed a similar nonmonotonic growth pattern with a juvenile growth spurt at 60-70 cm curved carapace length, (CCL) or 15-20 years of age. Sex-specific growth differences were also evident with females tending to grow faster than similar-sized males after the Juvenile growth spurt. It is clear that slow sex-specific growth displaying both spatial and temporal variability and a juvenile growth spurt are distinct growth behaviors of green turtles from this stock.

Spatial and temporal variability in somatic growth of green sea turtles (Chelonia mydas) resident in the Hawaiian Archipelago

The somatic growth dynamics of green turtles ( Chelonia mydas) resident in five separate foraging grounds within the Hawaiian Archipelago were assessed using a robust non-parametric regression modelling approach. The foraging grounds range from coral reef habitats at the north-western end of the archipelago, to coastal habitats around the main islands at the southeastern end of the archipelago. Pelagic juveniles recruit to these neritic foraging grounds from ca. 35 cm SCL or 5 kg ( similar to 6 years of age), but grow at foraging-ground-specific rates, which results in quite different size- and age-specific growth rate functions. Growth rates were estimated for the five populations as change in straight carapace length ( cm SCL year) 1) and, for two of the populations, also as change in body mass ( kg year) 1). Expected growth rates varied from ca. 0 - 2.5 cm SCL year) 1, depending on the foraging-ground population, which is indicative of slow growth and decades to sexual maturity, since expected size of first-time nesters is greater than or equal to 80 cm SCL. The expected size- specific growth rate functions for four populations sampled in the southeastern archipelago displayed a non-monotonic function, with an immature growth spurt at ca. 50 - 53 cm SCL ( similar to 18 - 23 kg) or ca. 13 - 19 years of age. The growth spurt for the Midway atoll population in the northwestern archipelago occurs at a much larger size ( ca. 65 cm SCL or 36 kg), because of slower immature growth rates that might be due to a limited food stock and cooler sea surface temperature. Expected age-at-maturity was estimated to be ca. 35 - 40 years for the four populations sampled at the south-eastern end of the archipelago, but it might well be > 50 years for the Midway population. The Hawaiian stock comprises mainly the same mtDNA haplotype, with no differences in mtDNA stock composition between foraging-ground populations, so that the geographic variability in somatic growth rates within the archipelago is more likely due to local environmental factors rather than genetic factors. Significant temporal variability was also evident, with expected growth rates declining over the last 10 - 20 years, while green turtle abundance within the archipelago has increased significantly since the mid-1970s. This inverse relationship between somatic growth rates and population abundance suggests a density-dependent effect on somatic growth dynamics that has also been reported recently for a Caribbean green turtle stock. The Hawaiian green turtle stock is characterised by slow growth rates displaying significant spatial and temporal variation and an immature growth spurt. This is consistent with similar findings for a Great Barrier Reef green turtle stock that also comprises many foraging-ground populations spanning a wide geographic range.