However, we did not observe the skewing toward hydrophobic residues. frame usage are more pronounced, but also exhibit greater inter-individual variation, in IgG+ and IgA+ than in IgM+ B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during Norepinephrine hydrochloride VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is usually expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host specific DH reading frame usage bias. reason that these DRF should not be used. Their absence seems to highlight the presence of mechanistic differences in the inverted and forward rearrangements. Forward DH Gene Reading Frame Usage Distribution The absence of inverted DRF may be mediated by aspects of the rearrangement mechanism. A surprising feature of the DRF usage that cannot be explained by the rearrangement mechanism is the restricted usage of particular forward RFs. In the forward direction, the DRF usage Norepinephrine hydrochloride is usually highly skewed towards the third forward RF for most clones, whereas a minority of the clones uses first and second forward DRF (Fig. 3A) Again, as was the case for the inverted DRF, most forward DRF do not contain stop codons, which would reduce their likelihood of usage in circulating B cells. Moreover, as will be further explained, there is no a priori advantage for one DRF over the others, and no element of the rearrangement process that could readily explain this preference. This unequal distribution thus seems to hint to the presence of a clear selection mechanism for one DRF for a given DH segment. In order to check that this result is not the peculiarity of a single individual, we have compared the DRF usage among subjects and amongst antibody heavy chain isotypes (data not shown). In all cases, the DRF usage is usually skewed and the manner of skewing is very comparable in different individuals. Thus, if specific DRF are indeed selected, this selection mechanism is similar in most individuals. Note also that there is no reason for the PCR amplification to over amplify one IL23R DRF rather than the other since the region of the amplification product that contains the DRF is usually internal to and non-overlapping with the primer. As mentioned above, the frequencies at which DH genes are used in our sample vary widely among Norepinephrine hydrochloride DH genes. Thus, the average results could be the result of a small number of highly frequent DH genes. As was the case for the inverted DRF, we have repeated the analysis separating each DH. The DRF usage varies among DH genes, but it is usually again highly non-uniform for each DH gene (Fig. 3A). The non-uniform distribution is not the result of stop codons, since DRFs stop codons are practically absent. Moreover, the DRF usage pattern is usually highly reproducible among samples from different individuals (Supplemental Fig. 2) and is the result Norepinephrine hydrochloride of a large number of clones for each sample and each DH (data not shown). Supplemental Fig. 2 shows the DRF usage of clones and not of total sequences but the results for total sequences are comparable. Thus, the biased distribution cannot be the result of amplification errors or biases, since those would not affect the clone number (remember that comparable sequences comprising each clone are only counted once). For the same reason, the biased distribution is not affected.