Intronless Homing and Marker Exclusion

The first clue that free-standing endonucleases of phage genomes are mobile genetic elements came from 30-year-old studies on the inheritance of genetic markers in crosses of the closely related phages T2 and T4 (Pees and de Groot 1970; Russell and Huskey 1974). In measuring the frequency of T2 genes in the progeny of mixed infection with phage T4, Russell and Huskey found that the corresponding T4 genetic markers predominated in the progeny (Fig. 1). This result was unexpected as T4 and T2 are closely related T-even phage, and the expectation was that T2 markers would be inherited in 50% of progeny. Russell and Huskey called this phenomenon partial marker exclusion, whereby T2 markers were, on average, present in only 20% of progeny. Interestingly, two specific regions of the T2 genome, centered on genes 32 and 56, were

Fig. 1. Exclusion of T2 markers from the progeny of mixed infection with T4 (adapted from Russell and Huskey 1974). Shown is a linear representation of the T4 genetic map with the position of amber markers in essential genes indicated mainly by numbers, as well as the positions of T4-encoded endonucleases (underscored). For each genetic cross, the frequency of progeny carrying the T2 markers is indicated by a dot at the appropriate map position

Fig. 1. Exclusion of T2 markers from the progeny of mixed infection with T4 (adapted from Russell and Huskey 1974). Shown is a linear representation of the T4 genetic map with the position of amber markers in essential genes indicated mainly by numbers, as well as the positions of T4-encoded endonucleases (underscored). For each genetic cross, the frequency of progeny carrying the T2 markers is indicated by a dot at the appropriate map position almost completely excluded, present in <1% of progeny (Fig. 1). In the absence of DNA sequence data or the knowledge of site-specific DNA endonucleases, Russell and Huskey hypothesized that localized marker exclusion was likely the result of a T4 gene that specified a protein with excluding activity, and that would be capable of recognizing specific DNA sequences.

We now know that the regions of localized marker exclusion in genes 32 and 56 correspond to locations in the T4 genome that are near the insertion sites of the GIY-YIG free-standing endonucleases, segG and segF, respectively (Miller et al. 2003). Indeed, SegG and SegF make DSBs in T2 genes 32 and 56, respectively (Belle et al. 2002; Liu et al. 2003). In the case of SegG-induced DSBs, analysis of the co-conversion tracts in progeny phage reveals that co-conversion is quite extensive 3' to the SegG cleavage site, which is correlated with the very low level of inheritance of T2 markers (Liu et al. 2003). Mutation of segG or segF activity reduces the dominance of T4 over T2 markers in the gene 32 or 56 regions, consistent with a role for SegG and SegF in the marker exclusion phenomenon. Because of the similarity to endonuclease-medi-

Protein Splicing Images

Fig. 2. Endonuclease-mediated homing pathways. Comparison of intron and intronless homing. Homing is a DNA-dependent pathway, initiated by the intron-encoded homing endonuclease (ENDO, red rectangle) encoded within a group I intron (indicated by black rectangles). The endonuclease (grey dumbbell) initiates the homing pathway by binding to and cleaving a sequence in recipient or intronless alleles (grey rectangles). For intronless homing, the endonuclease gene (red rectangle) is not intron- or intein-encoded but is instead free standing, inserted between genes (open and dark grey rectangles) conserved between different phage genomes. The endonuclease (grey dumbbell) binds and cleaves in a gene adjacent to the endonuclease insertion site

Fig. 2. Endonuclease-mediated homing pathways. Comparison of intron and intronless homing. Homing is a DNA-dependent pathway, initiated by the intron-encoded homing endonuclease (ENDO, red rectangle) encoded within a group I intron (indicated by black rectangles). The endonuclease (grey dumbbell) initiates the homing pathway by binding to and cleaving a sequence in recipient or intronless alleles (grey rectangles). For intronless homing, the endonuclease gene (red rectangle) is not intron- or intein-encoded but is instead free standing, inserted between genes (open and dark grey rectangles) conserved between different phage genomes. The endonuclease (grey dumbbell) binds and cleaves in a gene adjacent to the endonuclease insertion site ated homing of group I introns, the spread of free-standing endonucleases to genomes that lacked the endonuclease gene was named intronless homing (Belle et al. 2002; Fig. 2).

Of the remaining free-standing endonucleases in T4, only segE, adjacent to uvsW (Miller et al. 2003), has also been shown to exclude genetic markers in crosses between T4 and RB30, a related T-even phage (Kadyrov et al. 1997). Interestingly, Russell and Huskey's data shows that the uvsW region of the T4 genome does not completely dominate over that of T2 in progeny phage, as the frequency of T2 markers in this region is -20%, rather than <1% expected from localized marker exclusion (Russell and Huskey 1974). This may reflect the paucity of essential genes in this region of the T4 and T2 genomes for which Russell and Huskey could obtain amber mutants, and thus measure exclusion. The same is true of three other seg genes (segB, segC and segD) en coded in the same genomic region as segE (Miller et al. 2003), where none of the surrounding genetic markers dominate over the corresponding T2 markers in progeny phage (Russell and Huskey 1974), but none of these seg genes have been shown to encode active endonucleases.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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