Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.

This study shows that while Cas9 expression during meiosis I promotes genotype conversion - the mechanism underlying CRISPR ’gene drive’ - in both male and female mice, timing and high levels of Cas9 protein are critical to achieve robust efficiency.     

The relationship between different types of genetically defined aggressive behavior

Aggressive behavior is not a unitary trait, and different stimuli/situations elicit different kinds of aggressive behavior. According to numerous data the genotype plays a significant role in the expression of aggressive behavior. However, it remains unclear how genetic predisposition to one kind of aggression is linked with other kinds of aggressive behavior, especially pathological aggression (infanticide). Here, we report on our investigation of the expression of defensive, offensive, predatory and asocial aggression in wild rats selectively bred for 85 generations for either a high level or a lack of aggression towards humans. We found that those rats genetically predisposed to a high level of defensive aggression showed decreased social behavior and increased pathological aggressive behavior towards juvenile males. The highly aggressive rates showed a reduced latency time of attack and an increased latency time of the first social contact. Rats genetically predisposed to defensive aggression demonstrated increased predatory aggression—latency time of muricide was shorter in highly aggressive than in tame animals. At the same time, both lines of rats did not differ significantly in intermale aggression. We conclude that the data indicate a close relation between defensive, predatory and pathological aggressive behavior that allows us to suggest that similar genetic mechanisms underlie these types of aggressive behavior.     

Keeping 53BP1 out of focus in mitosis

A recent study published in Science reveals the mechanism and biological importance of DNA damage response abrogation in mitotic cells.For many years, much research has focused on understanding how cells maintain genome integrity despite DNA being constantly challenged by factors of both endogenous and exogenous nature. DNA double-strand breaks (DSBs) are the most deleterious DNA lesions, and if left unrepaired or repaired incorrectly, a single DSB can trigger genome instability or even cell death¹. Therefore, any DSB has to be recognized and repaired by processes encompassed within the DNA damage response (DDR). Notably, while the ends of mammalian linear chromosomes naturally resemble DSBs, their structure and association with the so-called “Shelterin” complex normally makes them invisible to the DDR².As soon as a DSB is formed, it is sensed and directly bound by the Ku70-Ku80 and/or MRE11-NBS1-RAD50 protein complexes, which recruit and activate the DDR kinases DNA-PKcs and ATM, respectively. The first steps in the DDR to DSBs are followed by cascades of events involving protein post-translational modifications (PTMs) and formation of large protein assemblies at DSB sites known as ionizing radiation-induced foci (IRIF)³. Protein phosphorylation and ubiquitylation are at the heart of these signaling processes³. For example, following recruitment of the DDR mediator protein MDC1 to the phospho-epitope created by ATM and DNA-PKcs on variant histone H2AX, MDC1 is itself phosphorylated by ATM on multiple serines and threonines⁴. MDC1 phosphorylation on a group of threonines near its N-terminus and conforming to the consensus TQXF generates binding sites for the FHA domain of E3-ubiquitin ligase RNF8^5,6. Together with the E2-conjugating enzyme UBC13, RNF8, and another E3 ligase, RNF168, trigger formation of mainly lysine 63-linked ubiquitin adducts in DSB-proximal chromatin, promoting recruitment of downstream factors necessary for DNA repair, such as the RAP80-Abraxas-BRCA1 complex and 53BP1³.Significantly, the full DDR happens only in interphase cells, whereas if mitotic cells sustain DSBs, the process appears to be blocked at the stage of RNF8 recruitment, resulting in IRIF devoid of detectable ubiquitin conjugates⁷. Consequently, 53BP1 and BRCA1 are not recruited to IRIF during mitosis. Even more strikingly, although RNF8 and RNF168 are associated with mitotic IRIF in anaphase, hyperphosphorylated 53BP1 remains excluded from chromatin until cells progress into G1 phase⁷. Based on these findings, it was hypothesized that mitosis-specific PTMs on RNF8 and 53BP1 might preclude formation of repair-competent IRIF⁷. However, the precise mechanistic explanation of the “interrupted” DDR in mitosis remained to be unravelled.A recent study published in Science by the group of Daniel Durocher addressed the question of how full IRIF assembly and DSB repair are prevented in mitotic cells⁸. First, Orthwein et al. focused on the mechanism that abrogates RNF8 recruitment to DSBs during mitosis. They demonstrated that CDK1-dependent mitosis-specific phosphorylation of RNF8 on T198 abolished interaction between RNF8 and its target phospho-TQXF motifs in MDC1. This important finding was somewhat surprising, given that MDC1 binding is mediated by the RNF8 FHA domain^5,6 and T198 is located some distance away from this domain. It will thus be interesting to see how T198 phosphorylation abrogates MDC1 binding, for example via T198 being juxtaposed to the FHA domain in the RNF8 3D structure, through phosphorylated T198 docking with the phospho-binding region of the FHA domain, or via another mechanism. In this regard, we note that T198 is part of an STP motif, which upon modification by CDK1 could constitute a priming site for PLK1 kinase⁹. Thus, T198 phosphorylation might be followed by PLK1-mediated RNF8 phosphorylation. Interestingly, certain sites in RNF8 conform to the PLK1 consensus motif, with those at T39 and T316 being evolutionarily conserved in vertebrates. Moreover, T39 is located in the FHA domain, close to R42, mutation of which abolishes RNF8 interaction with MDC1^5,6. It would therefore be worthwhile mutating these potential PLK1 sites and establishing whether this affects mitotic control of RNF8 binding to MDC1.After identifying T198 as critical for preventing RNF8 recruitment to DSBs during mitosis, Orthwein et al. observed that, while mutating this residue to alanine restored recruitment of RNF8 (and BRCA1) to mitotic IRIF, 53BP1 still remained excluded from DSB sites. This prompted the authors to look for mitosis-specific PTMs of 53BP1 by mass spectrometry, leading to the discovery of two novel phosphosites mapped to the recently described ubiquitin-dependent recruitment (UDR) motif, which mediates binding to ubiquitylated H2A and is required for 53BP1 IRIF formation¹⁰. Notably, the same residues, T1609 and S1618, were also identified by Chowdhury and colleagues¹¹ as target sites for the PP4C/R3β phosphatase. This group showed that T1609 and S1618 must be dephosphorylated for 53BP1 to form IRIF. In accord with these findings, Orthwein et al. established that when T1609 and S1618 were mutated to alanines, the ensuing “53BP1-TASA” protein was recruited to sites of DNA damage during mitosis in cells expressing RNF8-T198A. Moreover, unlike normal cells, cells co-expressing RNF8-T198A and 53BP1-TASA carried out DSB joining reactions during mitosis and were extremely hypersensitive to ionizing radiation (IR). The authors also found that, following irradiation in mitosis, cells carrying these mutant RNF8 and 53BP1 proteins displayed increased rates of kinetochore-positive micronucleus formation, suggesting mis-segregation of full chromosomes. In addition, chromosomes in these cells were prone to sister telomere fusions, thereby helping to explain their elevated levels of aneuploidy and IR hypersensitivity.The research described above has not only revealed how DSB repair is suppressed in mitosis but has also established that this suppression is biologically important. Orthwein et al. propose that, as mitotic telomeres become “underprotected” when mitosis is prolonged upon stress¹², this could lead to telomere fusion if DNA end-joining pathway is active. The suppression of DSB signaling and repair mediated by RNF8 and 53BP1 mitotic phosphorylation therefore probably evolved as a mechanism to mitigate this threat to genome stability. A key question that still remains is why mitotic telomeres become underprotected in the first place? Also, what features in telomere structure or replication and segregation processes make it more beneficial for the cells to keep chromosome ends less protected at the cost of inhibiting the DDR during mitosis? Finally, given that cancers often harbor cell cycle and/or DDR defects¹, it will be of interest to see whether defective mitotic control of DSB repair might play a role in tumor evolution, or could provide opportunities for developing better anti-cancer therapies.     

Action of surface-active substances on biological membranes IV. Hemolytic and membrane-perturbing action of homologous series of β-d-glucopyranosyl-1-alkylphosphates

The hemolytic action of a homologous series of β-d-glucopyranosyl-1-alkylphosphates on human erythrocytes has been examined. The agent's affinity for the red cell membrane and the mean number of the agent's molecules which, upon interaction with an erythrocyte, make it undergo hemolysis have been measured. The contribution of the head group and that of a CH₂ group of the surfactants to the free energy of the agents' binding to the cell membrane have been estimated. The effect of the surfactants on the red cell volume and the lytic concentrations of the agents have been measured. The contribution of a CH₂ group to the free energy of the interaction of the amphiphiles embedded in the membrane bilayer with their environment has been evaluated and is proposed to be used as a measure of the membrane matrix stability.     

Novel side reaction accompanying cyclization of Glu(R1)‐Glu(R2) dipeptides via lactamization of the Glu(R1) residue

A series of linear peptides with the general formula H‐Glu(R1)‐Glu(R2)‐OH was subjected to cyclization under standard conditions. Formation of respective 2,5‐diketopiperazines was accompanied by transformation of the N‐terminal Glu(R1) to pyroglutamic acid residue. Even in the case R1 is an amino acid residue attached to the N‐terminal γ‐carboxyl group, lactamization leads to its elimination. The observed reaction has not been reported so far in the literature. Correspondingly, an alternative route to Glu(R1)‐Glu(R2)‐containing 2,5‐diketopiperazines was applied to improve the overall yields.     

Identification of hydrogen bonds to the Rieske cluster through the weakly coupled nitrogens detected by electron spin echo envelope modulation spectroscopy

The interaction of the reduced[2Fe-2S] cluster of isolated Rieske fragment from the bc1 complex of Rhodobacter sphaeroides with nitrogens (14N and 15N) from the local protein environment has been studied by X- and S-band pulsed EPR spectroscopy. The two-dimensional electron spin echo envelope modulation spectra of uniformly 15N-labeled protein show two well resolved cross-peaks with weak couplings of approximately 0.3-0.4 and 1.1 MHz in addition to couplings in the range of 6-8 MHz from two coordinating Ndelta of histidine ligands. The quadrupole coupling constants for weakly coupled nitrogens determined from S-band electron spin echo envelope modulation spectra identify them as Nepsilon of histidine ligands and peptide nitrogen (Np), respectively. Analysis of the line intensities in orientation-selected S-band spectra indicated that Np is the backbone N-atom of Leu-132 residue. The hyperfine couplings from Nepsilon and Np demonstrate the predominantly isotropic character resulting from the transfer of unpaired spin density onto the 2s orbitals of the nitrogens. Spectra also show that other peptide nitrogens in the protein environment must carry a 5-10 times smaller amount of spin density than the Np of Leu-132 residue. The appearance of the excess unpaired spin density on the Np of Leu-132 residue indicates its involvement in hydrogen bond formation with the bridging sulfur of the Rieske cluster. The configuration of the hydrogen bond therefore provides a preferred path for spin density transfer. Observation of similar splittings in the 15N spectra of other Rieske-type proteins and [2Fe-2S] ferredoxins suggests that a hydrogen bond between the bridging sulfur and peptide nitrogen is a common structural feature of [2Fe-2S] clusters.     

Taxonomic note on some paleozoic marine algae (Uzbekistan, Central Asia)

The paleozoic marine algae of Central Asia were recently investigated and attributed to warm-water and Tethyan microflora. If the majority of the taxa (114 out of 117) were already described from other basins, a few elements merit attention. Two chlorophyte species are new (Atractyliopsis nuratauensis nov. sp., Amarellina moscoviensis nov. sp.) and a new Hoeegonites sp. is left in open nomenclature. The flora also contains representatives of the puzzling Moniliporelleae, a rhodophyte family only reported from Kazakhstan with the genus Furcatoporella. An unusual in situ cluster of calcispheres is also illustrated.     

Effect of ultrafine-grained titanium surfaces on adhesion of bacteria

The influence of the ultrafine crystallinity of commercial purity grade 2 (as-received) titanium and titanium modified by equal channel angular pressing (modified titanium) on bacterial attachment was studied. A topographic profile analysis of the surface of the modified titanium revealed a complex morphology of the surface. Its prominent micro- and nano-scale features were 100–200-nm-scale undulations with 10–15 μm spacing. The undulating surfaces were nano-smooth, with height variations not exceeding 5–10 nm. These surface topography characteristics were distinctly different from those of the as-received samples, where broad valleys (up to 40–60 μm) were detected, whose inner surfaces exhibited asperities approximately 100 nm in height spaced at 1–2 μm. It was found that each of the three bacteria strains used in this study as adsorbates, viz. Staphylococcus aureus CIP 68.5, Pseudomonas aeruginosa ATCC 9025 and Escherichia coli K12, responded differently to the two types of titanium surfaces. Extreme grain refinement by ECAP resulted in substantially increased numbers of cells attached to the surface compared to as-received titanium. This enhanced degree of attachment was accompanied with an increased level of extracellular polymeric substances (EPS) production by the bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two-dimensional pulsed electron spin resonance characterization of N-labeled archaeal Rieske-type ferredoxin

Two-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly ¹⁵N-labeled archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems.