Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and non-random occurrence of nonnascent short chains in the DNA of Ehrlich ascites cells

The DNA of Ehrlich ascites cells was labeled with radioactive thymidine using different labeling schedules: Incubation periods between 15 s and 4 h; pulse/pulse-chase experiments with pulses in the range of a few minutes; longtime incubation followed by a longtime chase (both in the range of 1 cell generation). From the purified DNA of the labeled cells a fraction (0.3-0.4%) of short chains was separated and partially fractionated by means of a hydroxyapatite thermochromatography procedure. The evaluation of the labelling patterns of the short chains indicated that less than 5% of them can be regarded as replication intermediates ('Okazaki pieces'). The rest, termed nonnascent pieces, exhibited a slow turnover. The life span of the nonnascent pieces was estimated to be about 1 cell generation. On helical DNA, nonnascent pieces were distributed in a non-random manner. Their preferential localisation was nearby sites which caused binding of the DNA, after purification, to nitrocellulose and which occurred about every 60-80 microns on the nuclear DNA of the cells.     

The secondary structure and nitrocellulose affinity of freshly replicated DNA from Ehrlich ascites cells (author's transl)]

Hydroxyapatite chromatography and isopycnic Cs2SO4 centrifugation normally yield no indications of single-stranded DNA when that fraction of replicating DNA from Ehrlich ascites cells which can be separated by nitrocellulose chromatography is analyzed. Single-stranded DNA is detected by both methods if the DNA is fragmented by ultrasound before the nitrocellulose chromatography. The digestion of this DNA fraction by single-strand-specific nucliase leads to a loss of its binding to nitrocellulose and of the indications of single-stranded DNA. The loss for the affinity to nitrocellulose is also observed when the corresponding fraction separated from unfragmented DNA is digested by endonuclease. It is suggested that replicating DNA is bound to nitrocellulose by means of single-stranded gaps on the replication fork. These gaps are apparently too small to be detected within large, otherwise entirely double-stranded molecules by hydroxyapatite chromatography and Cs2SO4 centrifugation. In the case of nitrocellulose-binding ultrasound fragments, this relation seems to be more favorable because of the separation of most of the residual double-stranded part. It is demonstrated that sonication of helical DNA also generates a small amount of fragments with some single-stranded character. The effects observed with replicating DNA could be distinguished from these artifacts.     

Dynamic Activity of miR-125b and miR-93 during Murine Neural Stem Cell Differentiation In Vitro and in the Subventricular Zone Neurogenic Niche

Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Recent experience modulates forebrain gene-expression in response to mate-choice cues in European starlings

Mate-choice decisions can be experience dependent, but we know little about how the brain processes stimuli that release such decisions. Female European starlings (Sturnus vulgaris) prefer males with long-bout songs over males with short-bout songs, and show higher expression of the immediate early gene (IEG) ZENK in the auditory forebrain when exposed to long-bout songs than when exposed to short-bout songs. We exposed female starlings to a short-day photoperiod for one of three durations and then, on an increased photophase, exposed them to one week of long-bout or short-bout song experience. We then examined their IEG response to novel long-bout versus novel short-bout songs by quantifying ZENK protein in two song-processing areas: the caudo-medial hyperstriatum ventrale and the caudo-medial neostriatum. ZENK expression in both areas increased with tenure on short-day photoperiods, suggesting that short days sensitize females to song. The ZENK response bias toward long-bout songs was greater in females with long-bout experience than in females with short-bout experience, indicating that the forebrain response bias toward a preferred trait depends on recent experience with that category of trait. This surprising level of neuroplasticity is immediately relevant to the natural history and fitness of the organism, and may underlie a mechanism for optimizing mate-choice criteria amidst locally variable distributions of secondary sexual characteristics.     

Female European starling preference and choice for variation in conspecific male song

Data from several field studies support the hypothesis that female European starlings, Sturnus vulgaris, attend to variation among the songs of conspecific males when making mate-choice decisions. However, for a variety of methodological reasons, direct evidence for female preferences based on song in starlings has been lacking. This study presents a novel technique for assaying directly female preference and choice in European starlings by using the presentation of conspecific male song as an operant reinforcer in a controlled environment. Using an apparatus in which the playback of songs from different nestboxes is under the operant control of the subject, we demonstrate how the reinforcing properties of conspecific song can be used to measure female preference and choice. The results of the study suggest three conclusions. First, female starlings prefer naturally ordered conspecific male songs over reversed songs. Second, female starlings display robust preferences for longer compared with shorter male song bouts. Behaviour in the operant apparatus varied directly with male song bout length. Third, preferences based on song bout length are sex specific. Male starlings failed to respond differentially to the same stimuli for which females showed strong preferences. These results suggest that male-male variation in song bout length is important for mate choice among starlings. In addition, we detail the use of a novel behavioural assay for measuring female preferences that can be applied to similar behaviours in other species of songbirds. Copyright 2000 The Association for the Study of Animal Behaviour.     

Individual vocal recognition and the effect of partial lesions to HVc on discrimination, learning, and categorization of conspecific song in adult songbirds

Among songbirds, the capacity to associate particular songs with particular singers (i.e., vocal recognition) forms the cognitive basis for more complex communication behaviors such as female choice and territoriality. In the present study, we combine operant conditioning techniques and excitotoxic lesions to the forebrain nucleus HVc to examine the role of this region in the discrimination, associative learning, and categorization of conspecific song. We trained adult male and female European starlings, Sturnus vulgaris, to recognize simultaneously the songs of several conspecific males. Then, using a series of transfer procedures, we demonstrate that correct recognition does not generalize to song bouts containing novel motifs from familiar singers. This suggests that starlings do not make use of individually invariant source or filter characteristics for vocal recognition. We then lesioned a portion of HVc bilaterally with ibotenic acid, and exposed the birds to a series of manipulations testing the discrimination, associative learning, and categorization of conspecific song. The lesions attenuated song production among males, but retention of the basic recognition task (i.e., maintenance of the discrimination) was unaffected. However, when the response contingencies were reversed-as a test of associative learning independent of discrimination-the initial performance and subsequent learning rate were negatively correlated with the size of the HVc lesions. This suggests that HVc plays a role in the formation of associations between a song and some referent. The results of this study are discussed in light of earlier claims regarding the role of HVc in the perceptual processing of conspecific song.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr