Repair defect mutants of Proteus mirabilis. II. Excision of pyrimidine dimers from the DNA of ultraviolet-irradiated P. mirabilis wildtype and UV-sensitive mutants

Summary Two photoproducts, thymine-thymine and cytosine-thymine-dimers were identified after UV-irradiation of Proteus mirabilis. It was found that 1 erg/mm² at 253 nm produced approximately 2.9×10⁶ pyrimidine dimers/thymine residues or about 8 dimers per 10⁷ nucleotides. Both photoproducts were excised at the same rate from the DNA of ultraviolet-resistant wildtype cells (PG 273, PG 758), but remained in acid precipitable DNA in ultraviolet-sensitive HC R-mutants (PG 678, PG 686).The excised dimers appeared both in the TCA-soluble cell fraction and in the medium outside the cells. EXR-mutants (PG 693, PG 699) also demonstrated excision capability. The excision ability of the REC-mutant (PG 672) could not be unambiguously demonstrated, because of high DNA-degradation. The number of excised dimers depended on the UV-dose. In contrast to HC R-mutants of Escherichia coli, HC R-mutants of P. mirabilis showed DNA-degradation at about the same rate as the wildtype strain during repair after UV-irradiation.     

Studies on the conformation of protonated DNA

Experiments were made to demonstrate the predominant protonation effects and structural changes of the ordered double helical DNA structure and denatured state of DNA. Spectrophotometric titrations performed at different wavelengths indicate that cytosine can be protonated in the DNA double helical molecule to a high extent without breakdown of the secondary structure. With DNA heat-denatured under severe conditions the protonation of cytosine can be measured at 280, 295, and 300 mμ: the apparent pK value obtained was ～4.6. The protonated double helical conformation of the DNA molecule differs from the unprotonated state, which follows from the decrease of the thermal stability and from changes in the ORD curves. The ORD of a GC-rich DNA indicates a novel Cotton effect with positive rotations at ～260 mμ in 0.02M KCl below pH 4.0 to pH 3.3. The occurrence of the new peak parallels the extent of protonated cytosine measured by the spectrophotometric titrations. It is concluded that the protonated cytosine in the double helical structure is responsible for the difference between the protonated DNA conformation and the native state at neutral pH.     

Wachstum von Hefeprotoplasten und ihre Reversion zu intakten Hefezellen

Zusammenfassung Die durch enzymatische Lyse der Zellwand erhaltenen Protoplasten verschiedener Hefen waren lebensfähig. Sie zeigten in Nährmedien, die zur Stabilisation der Protoplasten KCl enthielten, Wachstumserscheinungen.In flüssigen Medien war eine Regeneration zur intakten Hefezelle und damit eine Neusynthese der Zellwand nicht möglich. Die Protoplasten blieben osmotisch labil und lysierten in Aqua dest. Die nach mehrstündiger Kultur gebildeten Membranen waren nicht in der Lage, die Funktionen der Zellwand zu übernehmen.Durch Kultivieren in 80% iger Bierwürzegelatine konnten die Protoplasten jedoch zu Hefezellen mit intakter Zellwand regenerieren. Die entstandenen Hefezellen besaßen bezüglich ihrer Gärungseigenschaften die gleichen Merkmale wie die Ausgangshefen. Nur Protoplasten mit einem Kern waren zur Regeneration fähig.Während des Wachstums der Protoplasten ging der Vermehrung der Cytoplasmabestandteile eine Anreicherung des DNA-haltigen Materials parallel. Diese drückte sich in einer Vervielfachung der Kernzahl aus. Eine Bestimmung des Nucleinsäuregehaltes wachsender Protoplasten ergab, daß sich nach 12 stündiger Kultur der Gehalt an DNA auf etwa das Fünffache erhöht hatte, während der RNA-Gehalt nur um etwa das 2,5fache gestiegen war. Das Verhältnis RNA/DNA verschob sich demgemäß im Verlaufe des Wachstums immer mehr zugunsten der DNA.     

Altered cleavage and localization of PINK1 to aggresomes in the presence of proteasomal stress

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.     

Rifampicin exposure reveals within-host Mycobacterium tuberculosis diversity in patients with delayed culture conversion

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management.     

Addressing heterogeneity of individual blood cancers: the need for single cell analysis

Cancer heterogeneity is a significant factor in response to treatment and escape leading to relapse. Within an individual cancer, especially blood cancers, there exists multiple subclones as well as distinct clonal expansions unrelated to the clinically detected, dominant clone. Over time, multiple subclones and clones undergo emergence, expansion, and extinction. Although sometimes this intra-clonal and inter-clonal heterogeneity can be detected and/or quantified in tests that measure aggregate populations of cells, frequently, such heterogeneity can only be detected using single cell analysis to determine its frequency and to detect minor clones that may subsequently emerge to become drug resistant and dominant. Most genetic/genomic tests look at the pooled tumor population as a whole rather than at its individual cellular components. Yet, minor clones and cancer stem cells are unlikely to be detected against the background of expanded major clones. Because selective pressures are likely to govern much of what is seen clinically, single cell analysis allows identification of otherwise cryptic compartments of the malignancy that may ultimately mediate progression and relapse. Single cell analysis can track intra- or inter-clonal heterogeneity and provide useful clinical information, often before changes in the disease are detectable in the clinic. To a very limited extent, single cell analysis has already found roles in clinical care. Because inter- and intra-clonal heterogeneity likely occurs more frequently than can be currently appreciated on a clinical level, future use of single cell analysis is likely to have profound clinical utility.