The rise and fall of CRISPRs - dynamics of spacer acquisition and loss
Westra, E.R. ; Brouns, S.J.J. - \ 2012
Molecular Microbiology 85 (2012)6. - ISSN 0950-382X - p. 1021 - 1025.
immune-system - streptococcus-thermophilus - acquired-resistance - antiviral defense - escherichia-coli - seed sequence - cas systems - rna - dna - repeats
Bacteria and Archaea are continuously exposed to mobile genetic elements (MGE), such as viruses and plasmids. MGEs may provide a selective advantage, may be neutral or may cause cell damage. To protect against invading DNA, prokaryotes utilize a number of defence systems, including the CRISPR/Cas system. CRISPR/Cas systems rely on integration of invader sequences (spacers) into CRISPR loci that act as a genetic memory of past invasions. Processed CRISPR transcripts are utilized as guides by Cas proteins to cleave complementary invader nucleic acids. In this issue, two groups report on spacer acquisition and turnover dynamics of CRISPR loci in a thermoacidophilic archeon and a pathogenic bacterium. Erdmann and Garrett demonstrate that three of the six CRISPR loci of Sulfolobus solfataricus rapidly acquire new spacer sequences from a conjugative plasmid present in a virus mixture. Intriguingly, two distinct mechanisms of spacer integration are utilized: leader adjacent and internal CRISPR spacer acquisition. Lopez-Sanchez and co-workers studied the type II system of Streptococcus agalactiae and observe heterogeneity in the bacterial population. A fraction of the population lost one or more anti-mobilome spacer sequences during its cultivation, allowing the transfer of a MGE in this subpopulation and a rapid response to altering selection pressures
Structures of the RNA-guided surveillance complex from a bacterial immune system
Wiedenheft, B. ; Jore, M.M. ; Brouns, S.J.J. ; Oost, J. van der - \ 2011
Nature 477 (2011). - ISSN 0028-0836 - p. 486 - 489.
crispr rna - electron-microscopy - target recognition - seed sequence - prokaryotes - interference - repeats - dna - endoribonuclease - maturation
Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors1, 2, 3, 4, 5, 6. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger7, 8, 9, 10, 11, 12. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages13, 14. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5' end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences
Evolution and classification of the CRISPR-Cas systems
Makarova, K.S. ; Brouns, S.J.J. ; Oost, J. van der - \ 2011
Nature Reviews Microbiology 9 (2011)6. - ISSN 1740-1526 - p. 467 - 477.
provides acquired-resistance - dna-repair system - immune-system - small rna - repeats - prokaryotes - bacteria - defense - archaea - protein
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci
Structural variation in the chicken genome identified by paired-end next-generation DNA sequencing of reduced representation libraries
Kerstens, H.H.D. ; Crooijmans, R.P.M.A. ; Dibbits, B.W. ; Vereijken, A. ; Okimoto, R. ; Groenen, M.A.M. - \ 2011
BMC Genomics 12 (2011). - ISSN 1471-2164 - 16 p.
copy-number variation - deletion polymorphism - rearrangements - nucleotide - expression - repeats - disease - impact
Background Variation within individual genomes ranges from single nucleotide polymorphisms (SNPs) to kilobase, and even megabase, sized structural variants (SVs), such as deletions, insertions, inversions, and more complex rearrangements. Although much is known about the extent of SVs in humans and mice, species in which they exert significant effects on phenotypes, very little is known about the extent of SVs in the 2.5-times smaller and less repetitive genome of the chicken. Results We identified hundreds of shared and divergent SVs in four commercial chicken lines relative to the reference chicken genome. The majority of SVs were found in intronic and intergenic regions, and we also found SVs in the coding regions. To identify the SVs, we combined high-throughput short read paired-end sequencing of genomic reduced representation libraries (RRLs) of pooled samples from 25 individuals and computational mapping of DNA sequences from a reference genome. Conclusion We provide a first glimpse of the high abundance of small structural genomic variations in the chicken. Extrapolating our results, we estimate that there are thousands of rearrangements in the chicken genome, the majority of which are located in non-coding regions. We observed that structural variation contributes to genetic differentiation among current domesticated chicken breeds and the Red Jungle Fowl. We expect that, because of their high abundance, SVs might explain phenotypic differences and play a role in the evolution of the chicken genome. Finally, our study exemplifies an efficient and cost-effective approach for identifying structural variation in sequenced genomes.
H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO
Westra, E.R. ; Pul, Ü. ; Heidrich, N. ; Jore, M.M. ; Lundgren, N.M.J. ; Stratmann, T. ; Wurm, R. ; Raine, A. ; Mescher, M. ; Heereveld, L. van; Mastop, M. ; Wagner, E.G.H. ; Schnetz, K. ; Oost, J. van der; Wagner, R. ; Brouns, S.J.J. - \ 2010
Molecular Microbiology 77 (2010)6. - ISSN 0950-382X - p. 1380 - 1393.
coli k-12 - salmonella-typhimurium - gene-expression - regulator leuo - foreign dna - in-vivo - protein - bacteria - repeats - rna
The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity
Small CRISPR RNAs guide antiviral defense in prokaryotes
Brouns, S.J.J. ; Jore, M.M. ; Lundgren, M. ; Westra, E.R. ; Slijkhuis, R.J. ; Snijders, A.P. ; Dickman, M.J. ; Makarova, K.S. ; Koonin, E.V. ; Oost, J. van der - \ 2008
Science 321 (2008)5891. - ISSN 0036-8075 - p. 960 - 964.
provides acquired-resistance - streptococcus-thermophilus - repeats - identification - elements - dna - evolutionary - sequence - viruses - system
Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense
Variation in effective pollination rates in relation to the spatial and temporal distribution of pollen release in rejuvenated perennial ryegrass
Treuren, R. van; Goossens, P.J. ; Sevcikova, M. - \ 2006
Euphytica 147 (2006)3. - ISSN 0014-2336 - p. 367 - 382.
genetically-modified grasses - effective population-size - lolium-perenne - ssr markers - l. - diversity - cultivars - dna - dispersal - repeats
Genebank accessions stored as seed populations require periodic rejuvenation in order to maintain sufficient numbers of viable seeds. During rejuvenation the genetic composition of accessions may be altered for a variety of reasons, of which variation in pollination rates between plants is the least understood. In the present study, a paternity exclusion analysis was performed on a rejuvenated accession of perennial ryegrass. In addition, flowering data of the 49 parental plants were collected during the flowering season. The aim of the study was to determine how accurate variation in pollination rates between plants can be predicted from data on the spatial and temporal distribution of pollen release. The parental population and a total of 551 offspring from 12 progeny arrays were genotyped by means of molecular analysis. Using 25 microsatellites, paternity was identified for 81.9% of the offspring, while remaining ambiguities were resolved by AFLP analysis, except in four cases. Within the total sample 9 cases of contamination were observed. Mating within the study population was clearly non-random, as 61.9% of the identified pollen donors were located within I m distance from the mother plant. Observed pollination rates were very well described by an inverse quadratic function of inter-plant distance between potential mating pairs. Incorporation of the recorded flowering data in the calculation of expected pollination rates improved the goodness of fit with observed values by only 0.77%. Suggestions to reduce the variance in paternal contributions were presented. However, contamination was considered more threatening to the genetic integrity of perennial ryegrass germplasm than variation in pollination rates between plants, and indicated the need for improved measures to avoid gene flow from other germplasm.
Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p
Boteva, R. ; Koek, A. ; Visser, N.V. ; Visser, A.J.W.G. ; Krieger, E. ; Zlateva, T. ; Veenhuis, M. ; Klei, I. van der - \ 2003
European Journal of Biochemistry 270 (2003). - ISSN 0014-2956 - p. 4332 - 4338.
protein-protein interactions - import - matrix - repeats - pex14
Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We studied Hansenula polymorpha Pex5p (HpPex5p) using fluorescence spectroscopy. The intensity of Trp fluorescence of purified HpPex5p increased by 25% upon shifting the pH from pH 6.0 to pH 7.2. Together with the results of fluorescence quenching by acrylamide, these data suggest that the conformation of HpPex5p differs at these two pH values. Fluorescence anisotropy decay measurements revealed that the pH affected the oligomeric state of HpPex5p, possibly from monomers/dimers at pH 6.0 to larger oligomeric forms at pH 7.2. Addition of dansylated peptides containing a PTS1, caused some shortening of the average fluorescence lifetime of the Trp residues, which was most pronounced at pH 7.2. Our data are discussed in relation to a molecular model of HpPex5p based on the three-dimensional structure of human Pex5p.
Identification of cut-rose (Rosa hybrida) and rootstock varieties using robust Sequence Tagged Microsatellite markers
Esselink, D. ; Smulders, M.J.M. ; Vosman, B. - \ 2003
Theoretical and Applied Genetics 106 (2003). - ISSN 0040-5752 - p. 277 - 286.
dna fingerprint analysis - persica l. batsch - populus-nigra l. - cultivar identification - nucleotide addition - length-polymorphism - molecular markers - repeats - abundance - loci
In this study a DNA fingerprinting protocol was developed for the identification of rose varieties based on the variability of microsatellites. Microsatellites were isolated from Rosa hybrida L. using enriched small insert libraries. In total 24 polymorphic sequenced tagged microsatellite site (STMS) markers with easily scorable allele profiles, from six different linkage groups, were used to characterize 46 Hybrid Tea varieties and 30 rootstock varieties belonging to different species (Rosa canina L., Rosa indica Thory., Rosa chinensis Jacq., Rosa rubiginosa L., and Rosa rubrifolia glauca Pour.). Clones and known flower color mutants were identified as being identical, all other varieties were differentiated by a unique pattern with as few as three STMS markers. The high discriminating power of the loci suggests that a selection of the most-robust STMS markers may be able to differentiate any two varieties within rootstocks or Hybrid Teas except for mutants. The selected STMS markers will be useful as a tool for reference collection management, for assessing essential derivation of varieties and illegal propagation