Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.

CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.

The nonhistone, N-terminal tail of an essential, chimeric H2A variant regulates mitotic H3-S10 dephosphorylation.

H2A.Y is an essential, divergent Tetrahymena thermophila histone variant. It has a long nonhistone N terminus that contains leucine-rich repeats (LRR) and an LRR cap domain with similarity to Sds22p, a regulator of yeast protein phosphatase 1 (PP1) activity in the nucleus. In growing cells, H2A.Y is incorporated into micronuclei only during S phase, which occurs immediately after micronuclear mitosis. Depletion of H2A.Y causes prolonged retention of mitosis-associated histone H3-S10 phosphorylation and mitotic abnormalities that mimic S10E mutation. In cells where H2A.Y is depleted, an inducible chimeric gene, in which the H2A.Y N terminus is attached to H2A.X, is shown to regulate micronuclear H3-S10 phosphorylation. H2A.Y can also be specifically coimmunoprecipitated with a Tetrahymena PP1 ortholog (Ppo1p). Taken together, these results argue that the N terminus of H2A.Y functions to regulate H3-S10 dephosphorylation. This striking in vivo case of "cross-talk" between a H2A variant and a specific post-translational modification of another histone demonstrates a novel function for a histone variant.

Diversity of TRIM5α and TRIMCyp sequences in cynomolgus macaques from different geographical origins.

The TRIM5α restriction factor can protect some species of monkeys, but not humans, from HIV infection. It has also emerged that some monkeys have a cyclophilin A domain retrotransposed into the TRIM5 locus resulting in the expression of a TRIMCyp protein with anti-retroviral activity. A high degree of sequence variation in the primate TRIM5 gene has been reported that varies between populations of rhesus macaques, a widely used non-human primate model of HIV/AIDS, and recently shown to correlate with susceptibility to simian immunodeficiency viruses in this species. Cynomolgus macaques are also used widely in HIV research. A non-indigenous population on Mauritius has highly restricted genetic diversity compared with macaques from Indonesia. The relative allelic diversity of TRIM5α and TRIMCyp within these two sub-populations may impact on the susceptibility of the macaques to simian immunodeficiency virus thereby influencing the outcome of studies using these monkeys. We sought to establish the genetic diversity of these alleles in cynomolgus macaques. We identified seven TRIM5α alleles in Indonesian macaques, three of which are novel, but only three in the Mauritian-origin macaques. Strikingly, 87% of Indonesian, but none of the Mauritian macaques, possessed a retrotransposed Cyp domain. A splice acceptor site single-nucleotide polymorphism that allows formation of a TRIMCyp protein was absent for the TRIM5α alleles found in the Mauritian macaques. The level of allelic diversity reported here is greater than previously proposed for cynomolgus macaque species.

Functionally diverging molecular quasi-species evolve by crossing two enzymes.

Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.