Strand pairing by Rad54 and Rad51 is enhanced by chromatin

We investigated the role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, we found that the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, we observed that Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA.     

Somatic hypermutation does not require Rad54 and Rad54B-mediated homologous recombination

Secondary diversification of immunoglobulin (Ig) genes occurs through somatic hypermutation (SHM) in B cells of the germinal center (GC). The GC reaction is associated with a high frequency of DNA double-strand breaks (DSB) in the hypermutation domain of Ig genes. Homologous recombination (HR) is a prominent DSB repair pathway. Among the proteins involved in HR are the Rad-54 paralogues, Rad54 and Rad54B. To investigate whether Rad54/Rad54B-mediated HR is involved in SHM, we determined the ratio of mutated versus non-mutated Vlambda PCR products from memory (IgM-, IgD-, Vlambda1+) and GC (PNA(high), Vlambda1+) B cells, the mutation load, the mutation frequency, the base exchange pattern and the distribution of somatic mutations along the rearranged Vlambda light chain (VlambdaLC) genes. All these parameters of SHM were unaltered in memory and GC B cells lacking one or both Rad54 paralogues. Thus, our data indicate that Rad54 and Rad54B-mediated HR is not essential for SHM. In addition, the finding that the ablation of RAD51 paralogues causes an increase in SHM argues against a direct involvement of HR in promoting SHM.     

CD44 is dispensable for B lymphopoiesis

Several studies have implicated a role for the cell surface glycoprotein CD44 in lymphocyte differentiation, adhesion to the matrix, homing, activation and apoptosis. However, the requirement of CD44 in B cell maturation remains elusive. To address this point, we analyzed the generation and activation of B lymphocytes in CD44-deficient mice. Unexpectedly, both maturation and autoreconstitution of early bone marrow B cell progenitors after sublethal irradiation as well as frequencies of splenic B cell subsets are indistinguishable in wild-type and CD44-deficient mice. Furthermore, splenic CD44-deficient B lymphocytes show a normal activation pattern after stimulation with LPS, anti-IgM/IL-4 or anti-CD40/IL-4. These results show for the first time that CD44 is clearly dispensable for development, reconstitution and activation of B lymphocytes.     

Frat is dispensable for canonical Wnt signaling in mammals

Wnt-signal transduction through beta-catenin is thought to require the inhibition of GSK3 by Frat/GBP. To investigate the role of Frat in mammalian development, we have generated mice with targeted mutations in all three murine Frat homologs. We show that Frat is normally expressed at sites of active Wnt signaling. Surprisingly, Frat-deficient mice do not display gross abnormalities. Moreover, canonical Wnt signaling in primary cells is unaffected by the loss of Frat. These studies show that Frat is not an essential component of the canonical Wnt pathway in higher organisms, despite the strict requirement of Frat/GBP for maternal Wnt signaling in Xenopus.     

Gamma interferon is dispensable for neopterin production in vivo

Previous studies have indicated that neopterin is synthesized in vitro by human monocyte-derived macrophages and dendritic cells upon stimulation with gamma interferon (IFN-gamma). Neopterin production under specific conditions in vitro has also been obtained upon stimulation with IFN-alpha and/or IFN-beta. However, it is unknown if any IFN-gamma-independent neopterin synthesis is possible in vivo. In the present study we investigated the serum neopterin concentrations in patients affected by the syndrome of Mendelian susceptibility to mycobacterial disease (MSMD). Indeed, this syndrome is characterized by deeply impaired or absent IFN-gamma production or function due to severe mutations in molecules involved in IFN-gamma/interleukin-12 (IL-12)/IL-23-dependent pathway. Serum neopterin levels were measured by an enzyme-linked immunosorbent assay in 27 patients with MSMD. We found that serum neopterin levels are elevated in the complete absence of IFN-gamma activity due either to a complete deficiency of its receptor or to deleterious mutations of IL-12 or its receptor. These data clearly indicate that, as reported from in vitro studies, other stimuli are able to induce neopterin synthesis in vivo. Consequently, neopterin cannot be used as means of diagnosis of MSMD due to IFN-gamma-, IL-12-, and IL-23-dependent pathway defects.     

ADAP is dispensable for NK cell development and function

NK cells are key mediators of the innate immune response and anti-tumor surveillance. Adhesion and degranulation-promoting adapter protein (ADAP, formerly known as SLAP-130 or Fyb) is a hematopoietic-specific adapter that is required for efficient TCR signaling and T cell activation. Herein, we examine a potential role for ADAP in NK development and function. ADAP is expressed in primary NK cells and in IL-2 stimulated lymphokine-activated killers. However, ADAP-deficient mice show no defects in NK development. Further, ADAP is dispensable for key NK functions, including cytotoxicity in response to engagement of activating receptors, cytokine production, conjugate formation and tumor suppression in vivo. These results indicate that, unlike events stimulated by TCR engagement, signaling events engaged by immunoreceptor tyrosine-based activation motif-associated and cytokine receptors on NK cells can occur independently of ADAP.     

Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability

Nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the two major pathways of DNA double-strand break (DSB) repair in eukaryotic cells. NHEJ repairs DSBs by ligation of cognate broken ends irrespective of homologous flanking sequences, whereas HR repairs DSBs using an undamaged homologous template. Although both NHEJ and HR have been clearly implicated in the maintenance of genome stability, how these apparently independent and mechanistically distinct pathways are coordinated remains largely unexplored. To investigate the relationship between HR and NHEJ modes of DSB repair, we generated cells doubly deficient for the NHEJ factor DNA Ligase IV (Lig4) and the HR factor Rad54. We show that Lig4 and Rad54 cooperate to support cellular proliferation, repair spontaneous DSBs, and prevent chromosome and single chromatid aberrations. These findings demonstrate a role for NHEJ in the repair of DSBs that occur spontaneously during or after DNA replication, and reveal overlapping functions for NHEJ and Rad54-dependent HR in the repair of such DSBs.     

Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome

Immortal human cells maintain telomere length by the expression of telomerase or through the alternative lengthening of telomeres (ALT). The ALT mechanism involves a recombination-like process that allows the rapid elongation of shortened telomeres. However, it is not known whether activation of the ALT pathway affects other sequences in the genome. To address this we have investigated, in ALT-expressing cell lines and tumours, the stability of tandem repeat sequences known to mutate via homologous recombination in the human germline. We have shown extraordinary somatic instability in the human minisatellite MS32 (D1S8) in ALT-expressing (ALT+) but not in normal or telomerase-expressing cell lines. The MS32 mutation frequency varied across 15 ALT+ cell lines and was on average 55-fold greater than in ALT- cell lines. The MS32 minisatellite was also highly unstable in three of eight ALT+ soft tissue sarcomas, indicating that somatic destabilization occurs in vivo. The MS32 mutation rates estimated for two ALT+ cell lines were similar to that seen in the germline. However, the internal structures of ALT and germline mutant alleles are very different, indicating differences in the underlying mutation mechanisms. Five other hypervariable minisatellites did not show elevated instability in ALT-expressing cell lines, indicating that minisatellite destabilization is not universal. The elevation of MS32 instability upon activation of the ALT pathway and telomere length maintenance suggests there is overlap between the underlying processes that may be tractable through analysis of the D1S8 locus.     

RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis

The Rho proteins are Ras-related guanosine triphosphatases (GTPases) that function in cytoskeletal reorganization, cell migration, and stress fiber and focal adhesion formation. Overexpression of RhoC enhances the ability of melanoma cells to exit the blood and colonize the lungs. However, in vivo confirmation of RhoC's role in metastasis has awaited a RhoC-deficient mouse model. Here we report the generation of RhoC-deficient mice and show that RhoC is dispensable for embryonic and post-natal development. We demonstrate that loss of RhoC does not affect tumor development but decreases tumor cell motility and metastatic cell survival leading to a drastic inhibition of metastasis.     

Sialylation of lipooligosaccharides is dispensable for the virulence of Haemophilus ducreyi in humans

Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.     

Sos2 is dispensable for NMDA-induced Erk activation and LTP induction

N-methyl-d-aspartate (NMDA) receptor-induced activation of extracellular signal-related protein kinase (Erk) plays important roles in various neuronal functions including long-term potentiation (LTP). Son of sevenless (Sos) proteins have been implicated in NMDA-induced Erk activation in neurons of young mice. However, contribution of each of the two Sos isoforms, Sos1 and Sos2, has not been clarified. In this study, Sos2 involvement in NMDA-induced Erk activation was examined. We observed no defect in Erk phosphorylation induced by NMDA treatment of cortical neuronal cultures from Sos2−/− newborn mice. Moreover, theta-burst-induced LTP induction in the hippocampus of Sos2−/− mice was also normal. Finally, Erk activation by either depolarization or BDNF treatment was also normal in cultured neurons from Sos2 knockout mice. These results imply that Sos1 is the major regulator of these well-known neuronal Sos functions and suggest that a novel function for Sos2 in neurons remains to be determined.     

Malleal processus brevis is dispensable for normal hearing in mice.

The mammalian middle ear cavity contains a chain of three ossicles (the malleus, incus, and stapes), which develop from the mesenchyme of the first two branchial arches. Mice deficient in the Msx1 homeobox gene exhibit craniofacial abnormalities, including the absence of the malleal processus brevis that is normally attached to the upper part of the tympanic membrane. Here, we show that the expression of Msx1 and Msx2 overlaps in the malleal primordium during early embryonic development. A functional redundancy of Msx1 and Msx2 in the development of the middle ear is suggested by the stronger hypomorphism in the malleus of Msx1(-/-)/Msx2(-/-) embryos, including the absence of the malleal manubrium and the malleal processus brevis. The expression of Bmp4, a known downstream target of Msx1 in several developing craniofacial organs, was down-regulated in the malleal primordium, particularly in the region of the developing malleal manubrium, of Msx1 and Msx1(-/-)/Msx2(-/-) embryos. Msx genes, thus, appear to act in a cell autonomous manner, possibly by regulating Bmp4 expression, in the formation of the malleus. Transgenic rescue of the cleft palate of Msx1(-/-) mice overcame the neonatal lethality and allowed Msx1(-/-) mice to grow into adulthood but retain the phenotype of the absence of the malleal processus brevis. The availability of this animal model for the first time allowed us to measure auditory evoked potentials to assess the functional significance of the malleal processus brevis. The results demonstrated unimpaired auditory function in Msx1(-/-) mice. In addition, mutant mice appeared normal in balance behavior and in the vestibular evoked potential screening test. These results indicate that the malleal processus brevis is not necessary for sound transmission and seems dispensable for normal hearing and balance in mammals.     

Protein kinase Cepsilon is dispensable for TCR/CD3-signaling

PKCepsilon has been strongly linked to cell activation and proliferation in many cell types, including leukemic T-cell lines. In particularly, an essential role of PKCepsilon has been established in the IKK-beta/I-kappaB/NF-kappaB transactivation cascade. To study the physiological function of PKCepsilon in primary T-cells, we used our newly established PKCepsilon null mice. Unexpectedly, however, we did not reveal any defect in the development and function of CD3+ T-cells. Proliferative responses as well as IL-2 cytokine secretion of PKCepsilon-deficient T-cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca2+ ionophore were comparable with wild-type controls. Consistently, after CD3/CD28 engagement, deficiency of PKCepsilon did not impair NF-kappaB transactivation as well as CD25, CD44 and CD69 induction. Thus, PKCepsilon-deficient T-cells had similar physiological thresholds for activation in vitro. This finding suggests that PKCepsilon plays a redundant role in TCR-induced regulation of T-cell proliferation.     

Borrelia burgdorferi linear plasmid 38 is dispensable for completion of the mouse-tick infectious cycle

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a complex enzootic cycle, transiting between its vector, Ixodes ticks, and a diverse range of vertebrate hosts. B. burgdorferi linear plasmid 38 (lp38) contains several genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that these plasmid-borne genes may encode proteins important for the B. burgdorferi infectious cycle. Some of these genes encode potential virulence factors, including hypothetical lipoproteins as well as a putative membrane transport system. To characterize the role of lp38 in the B. burgdorferi infectious cycle, we constructed a shuttle vector to selectively displace lp38 from the B. burgdorferi genome and analyzed the resulting clones to confirm the loss of lp38. We found that, in vitro, clones lacking lp38 were similar to isogenic wild-type bacteria, both in growth rate and in antigenic protein production. We analyzed these strains in an experimental mouse-tick infectious cycle, and our results demonstrate that clones lacking lp38 are fully infectious in a mouse, can efficiently colonize the tick vector, and are readily transmitted to a naive host.     

Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B

Cyclin B3 has been conserved during higher eukaryote evolution as evidenced by its identification in chicken, nematodes, and insects. We demonstrate that Cyclin B3 is present in addition to Cyclins A and B in mitotically proliferating cells and not detectable in endoreduplicating tissues of Drosophila embryos. Cyclin B3 is coimmunoprecipitated with Cdk1(Cdc2) but not with Cdk2(Cdc2c). It is degraded abruptly during mitosis like Cyclins A and B. In contrast to these latter cyclins, which accumulate predominantly in the cytoplasm during interphase, Cyclin B3 is a nuclear protein. Genetic analyses indicate functional redundancies. Double and triple mutant analyses demonstrate that Cyclins A, B, and B3 cooperate to regulate mitosis, but surprisingly single mutants reveal that neither Cyclin B3 nor Cyclin B is required for mitosis. However, both are required for female fertility and Cyclin B also for male fertility.