24 research outputs found
Structural basis for spumavirus GAG tethering to chromatin
The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A–H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards
Exploiting Highly Ordered Subnanoliter Volume Microcapillaries as Microtools for the Analysis of Antibody Producing Cells
The interrogation of highly diverse
repertoires of heterogeneous
cell populations on a single cell basis increases the likelihood that
a cell with unique characteristics will be identified. We have developed
a new single cell analysis system comprising millions of bundled subnanoliter
volume bioincubation chambers for the identification and recovery
of target specific antibody secreting cells (ASCs). This platform
integrates dual surface screening with dedicated user driven data
analysis and automated cell recovery enabling multiple biophysical
parameters to be tracked for millions of antibody leads in parallel.
This direct clone analysis and selection technology is a clear deviation
from current microfabricated well-based approaches and offers drastically
enhanced screening throughput, simultaneous dual surface analysis,
and rapid automated single cell recovery. The technology is also applicable
to screening both bacterial and mammalian antibody secreting cells.
We demonstrate the implementation and feasibility of this platform
in identifying target specific antibodies from bacterial, hybridoma,
and B cell libraries
A regulatory role for the cohesin loader NIPBL in nonhomologous end joining during immunoglobulin class switch recombination.
DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR
Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole
Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium
Plant reproduction in the alpine landscape : reproductive ecology, genetic diversity and gene flow of the rare monocarpic "Campanula thyrsoides" in the Swiss Alps
Aims & Objectives
The work presented in this thesis forms part of a larger project “How patchy
habitat and isolation affect alpine plant life: genetic diversity, gene flow and mating
systems”, which includes the PhD studies of Patrick Kuss and the author under the
supervision of Professor Jürg Stöcklin.
This doctoral thesis investigates the consequences of the natural fragmentation
and patchiness of alpine landscapes on the life of alpine plant populations. The central
focus of the thesis is on the mating system, the role of inbreeding and/or outbreeding
depression, genetic diversity and geographic structure within and among populations
of the rare Alpine monocarpic perennial Campanula thyrsoides. The main objectives
and research questions addressed are:
• Is Campanula thyrsoides self-compatible (SI) and if not, does the SI system
break down with flower age? Do inbred C. thyrsoides offspring in the common
garden suffer from inbreeding depression?
• Do we find a distance related inbreeding depression (poorer reproducive
output) or outbreeding depression (increased reproductive output) in field
populations of C. thyrsoides following crosses of different crossing distances
(selfing, 1m, 10m, 100m and among distant populations)?
• How much genetic diversity exists within populations of C. thyrsoides and
how does it relate to population size and altitude? Has the natural habitat
fragmentation let to strong genetic differentiation and restricted gene flow
among populations of C. thyrsoides resulting in a pronounced geographic
structure?
Study species
In order to seek answers to our research questions, we choose to study a
yellow bellflower; Campanula thyrsoides. The choice was based on the information
that C. thyrsoides is a rare plant species, which is only found on calcarious soils
within the European Alps and adjacent mountain ranges (Aeschimann et al. 2005).
The plants selectiveness for carbonate bearing soils together with the fact that its
seeds are not adapted to long-distance dispersal (Tackenberg 2003) are the main
reasons for the isolation and small sizes of many of its populations. These population
characteristics, therefore, made C. thyrsoides a suitable study species. Another
important characteristic of C. thyrsoides, and one of the main reasons for its inclusion
in the study is because it is a monocarpic perennial which flowers once and
subsequently dies (Jäger 2000). Monocarpic plants species, which are more
commonly found in subtropical and tropical mountain systems (e.g. the giant rosettes
of Puya spp, Espeletia spp., Echium spp. etc., Smith & Young 1987; Young &
Augspurger 1991) are rare amidst the temperate alpine flora (for the Alps, see
Aeschimann et al. 2005). Monocarpy can promote genetic differentiation between
populations by reducing the effective population size due to a shorter generation time
and lower density of populations (Loveless & Hamrick 1984; Vitalis et al. 2004).
When studying the effects of population isolation and habitat fragmentation on
plant reproduction (e.g. mating system and inbreeding depression), it is, moreover,
ideal to study a Campanula species. Although most Campanula species are selfincompatible
and allogamous (Nyman 1993), both a break-down in the SI system with
flower age (Vogler et al. 1998) and an evolution towards complete self-compatibility
(Ægisdóttir & Thórhallsdóttir 2006) have been recorded.
Design
We studied the reproductive ecology and genetic diversity of Campanula
thyrsoides by firstly setting up pollination experiments in the common garden and in
the field and secondly by sampling leaf material in 32 field populations in
Switzerland. In the common garden study, we set up a pollination experiment in order
to study the breeding system of C. thyrsoides, including the consequences of selfing,
half-sibling crossings and outcrossing on reproductive output and seedling
performance. Moreover, field experiments in four populations were set up in the
Swiss Alps in order to study the effect of different crossing distances on reproduction
in C. thyrsoides and to see if evidence would be found of hidden inbreeding
depression or outbreeding depression following large-distance crossings compared to
within-population crossings. In addition, we studied the genetic diversity, gene flow
and geographical structure within and among 32 field populations of C. thyrsoides in
Switzerland, covering both large geographical and altitudinal ranges. The genetic
study was conducted using 5 co-dominant microsatellite markers. In addition, we
studied the genetic diversity in C. thyrsoides and two other alpine plants using random
amplified polymorphic DNA (RAPD) marker as well as studing the evolutionary
demography of C. thyrsoides