Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/ Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.bacteriophage | RNA interference | small RNA C RISPR (clustered regularly interspaced short palindromic repeats) cassettes are present in virtually every archaeon and in approximately 40% of bacteria (1-3). A CRISPR cassette consists of almost identical direct repeats that are regularly interspersed with spacers (4). In any given cassette, the length of spacers is similar, whereas their sequences vary. CRISPR cassettes are often flanked by a diverse set of CRISPR-associated (cas) genes (2,5,6).CRISPR/Cas (CRISPR-associated sequences) functions as an adaptive immunity system by excluding viruses and other mobile genetic elements that contain sequences matching CRISPR cassette spacers (6-9). Bacterial and archaeal CRISPR/Cas systems generally target DNA (10-13), although one archaeal system has been demonstrated in vitro to interfere at the level of RNA (14). Transcription of a CRISPR cassette, followed by processing with the help of dedicated endoribonucleases, creates small CRISPR RNAs (crRNAs) that guide the Cas machinery to the target, eventually resulting in target cleavage (11,(15)(16)(17)(18)(19)(20).Although a match between a single CRISPR spacer and a foreign DNA sequence called the protospacer can provide immunity to the entry of that DNA into the host, it is not sufficient. Mutations in the conserved protospacer-adjacent motif (PAM, ref. 21) abolish CRISPR-mediated immunity even in the presence of a perfect spacer-protospacer match. Likewise, some point mutations in protospacer that introduce single mismatches with the spacer abolish CRISPR/Cas function even when the PAM is intact (22). Thus, a PAM and a match between a spacer and protospacer are both required for CRISPR/Cas function.Recently, how...
Summary
Two cod stocks (western Baltic cod, WBC, and eastern Baltic cod, EBC) are managed in the Baltic Sea which is characterized by different main spawning areas and different main spawning periods. In this study we analyse the spatial and temporal occurrence of spawning individuals of both cod stocks in the main spawning grounds of the Baltic Sea based on eight microsatellite loci. Our results suggest that EBC (Gadus morhua callarias) has formed currently temporally stable, substantially homogeneous population not only in the Bornholm Sea (ICES SD: 25) but also in the Arkona Sea (ICES SD: 24). The presented analyses proved that EBC (G. m. callarias) can temporarily also spawn in the Belt Sea.
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