Internal Deletion Research Articles

Missense mutations in exon 2 of the MED12 gene are involved in IGF-2 overexpression in uterine leiomyoma.

Uterine leiomyoma (UL), the most common benign tumour found in females, is associated with many recurrent genetic aberrations, such as translocations, interstitial deletions and specific germline mutations. Among these, mutations affecting exon 2 of the mediator complex subunit 12 (MED12) gene are commonly detected in the majority of ULs. Mutational analysis of the MED12 gene, performed on 36 UL samples, revealed that 12 leiomyomas (33.4%) exhibited heterozygous missense mutations in codon 44 of exon 2 of the MED12 gene, four leiomyomas (11.1%) showed internal in-frame deletions, and two leiomyomas (5.5%) exhibited deletions involving intron 1-exon 2 junction, which caused a predicted loss of the splice acceptor. No mutations were detected in uterine myometrium (UM) and pseudocapsule (PC) samples, including those from women with a MED12 mutation in UL. These data showed that the PC is a healthy tissue that surrounds the UL to maintain UM integrity. Analysis of insulin-like growth factor 2 (IGF-2) and collagen type IV alpha 2 (COL4A2) mRNA expression levels in the same set of ULs revealed that only those with MED12 missense mutations expressed significantly higher levels of IGF-2 mRNA. In contrast, MED12 gene status does not appear to affect mRNA expression levels of the COL4A2 gene. On the basis of this finding, we suggest that the MED12 status stratifies the ULs into two mutually exclusive pathways of leiomyoma genesis, one with IGF-2 overexpression and the other with no IGF-2 activation. The occurrence of IGF-2 overexpression could be therapeutically targeted for the non-surgical treatment of leiomyomas.

Communication between binding sites is required for YqjI regulation of target promoters within the yqjH-yqjI intergenic region.

The nickel-responsive transcription factor YqjI represses its own transcription and transcription of the divergent yqjH gene, which encodes a novel ferric siderophore reductase. The intergenic region between the two promoters is complex, with multiple sequence features that may impact YqjI-dependent regulation of its two target promoters. We utilized mutagenesis and DNase I footprinting to characterize YqjI regulation of the yqjH-yqjI intergenic region. The results show that YqjI binding results in an extended footprint at the yqjI promoter (site II) compared to the yqjH promoter (site I). Mutagenesis of in vivo gene reporter constructs revealed that the two YqjI binding sites, while separated by nearly 200 bp, appear to communicate in order to provide full YqjI-dependent regulation at the two target promoters. Thus, YqjI binding at both promoters is required for full repression of either promoter, suggesting that the two YqjI binding sites cooperate to control transcription from the divergent promoters. Furthermore, internal deletions that shorten the total length of the intergenic region disrupt the ability of YqjI to regulate the yqjH promoter. Finally, mutagenesis of the repetitive extragenic palindromic (REP) elements within the yqjH-yqjI intergenic region shows that these sequences are not required for YqjI regulation. These studies provide a complex picture of novel YqjI transcriptional regulation within the yqjH-yqjI intergenic region and suggest a possible model for communication between the YqjI binding sites at each target promoter.

Identification of a SecM segment required for export-coupled release from elongation arrest.

SecM in Escherichia coli has two functionally crucial regions. The arrest motif near the C-terminus interacts with the ribosomal exit tunnel to arrest its own translational elongation. The signal sequence at the N-terminus directs the SecM nascent polypeptide to the Sec-mediated export pathway to release the arrested state of translation. Here, we addressed the importance of the central region of SecM. Characterization of internal substitution and deletion mutants revealed that a segment from residue 100 to residue 109 is required for the export-coupled release of the SecM nascent chain from the elongation-arrested state. Thus, the central region of SecM is not just a geometric linker but it participates actively in the regulation of translation arrest.

Structural elements in the Gag polyprotein of feline immunodeficiency virus involved in Gag self-association and assembly.

The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. We aimed to identify the FIV Gag domains that interact and promote Gag multimerization. To do this we generated a series of Gag subdomains and tested their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal of 37 residues from the C-terminus of FIV Gag and deletion of the N-terminal and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant protein encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Notably, the CA-NC Gag subdomain both associated with wild-type Gag and was recruited into particles in a proportion close to 50 % of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant-negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that the N-terminal and the C-terminal domains of the CA are necessary for Gag assembly. Our results demonstrate that the FIV CA-NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.

The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. Importance: Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets. Previously, we described a series of N-α-methylbenzyl-N'-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function.

Abstract 195: Characterization of DNA Modules in Regulating VWF Vascular Bed--Specific Expression

Expression of von Willebrand Factor (vWF) in the endothelium is regulated by vascular bed-specific pathways. A region of the human vWF gene between -843 and -620 was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to further delineate the responsible DNA sequences. A series of constructs containing 5’-deletions, internal deletions or point mutations of the vWF promoter coupled to LacZ were targeted to the Hprt locus of mice using homologous recombination, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that a three-base-pair fragment within vWF promoter is necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Although the sequence does not conform to established DNA binding sites, it bound to nuclear protein as determined by elecrophoretic mobility shift assay. Mass spectrometry of the DNA-protein complex revealed several potential binding transcription factors. The heart- and skeletal muscle-specific promoter region did not response to shear stress or mechanical stress. Bisulfite sequencing revealed differential methylation of the human vWF promoter in endothelial cells from expressing and non-expressing vascular beds. These differences were altered by the mutation at the short cis-element. Together, these data support a model of modular gene regulation in which distinct DNA sequences in the vWF promoter direct expression in different vascular beds.

Exploration of sequence space as the basis of viral RNA genome segmentation.

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

BuT2 is a member of the third major group of hAT transposons and is involved in horizontal transfer events in the genus Drosophila.

The hAT superfamily comprises a large and diverse array of DNA transposons found in all supergroups of eukaryotes. Here we characterized the Drosophila buzzatii BuT2 element and found that it harbors a five-exon gene encoding a 643-aa putatively functional transposase. A phylogeny built with 85 hAT transposases yielded, in addition to the two major groups already described, Ac and Buster, a third one comprising 20 sequences that includes BuT2, Tip100, hAT-4_BM, and RP-hAT1. This third group is here named Tip. In addition, we studied the phylogenetic distribution and evolution of BuT2 by in silico searches and molecular approaches. Our data revealed BuT2 was, most often, vertically transmitted during the evolution of genus Drosophila being lost independently in several species. Nevertheless, we propose the occurrence of three horizontal transfer events to explain its distribution and conservation among species. Another aspect of BuT2 evolution and life cycle is the presence of short related sequences, which contain similar 5′ and 3′ regions, including the terminal inverted repeats. These sequences that can be considered as miniature inverted repeat transposable elements probably originated by internal deletion of complete copies and show evidences of recent mobilization.

Repair of the 3' proximal and internal deletions of a satellite RNA associated with Cucumber mosaic virus is directed toward restoring structural integrity

The phenomenon of rapid turnover of 3' proximal nucleotides (nt) lost by the action of nuclease in RNA viruses is integral to replication. Here, a set of six deletions encompassing the 3' 23 nt region of a satellite RNA (satRNA) of Cucumber mosaic virus (CMV) strain Q (Q-sat), were engineered. Repair of the 3' end was not observed in the absence of CMV. However, co-expression with CMV in planta revealed that Q-sat mutants lacking the 3' 18 nt but not the 3' 23 nt are repaired and the progeny accumulation was inversely proportional to the extent of the deletion. Progeny of the 3'Δ3 mutant were repaired to wild type (wt) while those from the remaining four mutants were heterogeneous, exhibiting a wt secondary structure. Analysis of additional 3' internal deletions mutants revealed that progeny with a repaired sequence reminiscent of wt secondary structure were competent for replication and systemic spread.

Genomic landscape and evolutionary dynamics of mariner transposable elements within the Drosophila genus.

BackgroundThe mariner family of transposable elements is one of the most widespread in the Metazoa. It is subdivided into several subfamilies that do not mirror the phylogeny of these species, suggesting an ancient diversification. Previous hybridization and PCR studies allowed a partial survey of mariner diversity in the Metazoa. In this work, we used a comparative genomics approach to access the genus-wide diversity and evolution of mariner transposable elements in twenty Drosophila sequenced genomes.ResultsWe identified 36 different mariner lineages belonging to six distinct subfamilies, including a subfamily not described previously. Wide variation in lineage abundance and copy number were observed among species and among mariner lineages, suggesting continuous turn-over. Most mariner lineages are inactive and contain a high proportion of damaged copies. We showed that, in addition to substitutions that rapidly inactivate copies, internal deletion is a major mechanism contributing to element decay and the generation of non-autonomous sublineages. Hence, 23% of copies correspond to several Miniature Inverted-repeat Transposable Elements (MITE) sublineages, the first ever described in Drosophila for mariner. In the most successful MITEs, internal deletion is often associated with internal rearrangement, which sheds light on the process of MITE origin. The estimation of the transposition rates over time revealed that all lineages followed a similar progression consisting of a rapid amplification burst followed by a rapid decrease in transposition. We detected some instances of multiple or ongoing transposition bursts. Different amplification times were observed for mariner lineages shared by different species, a finding best explained by either horizontal transmission or a reactivation process. Different lineages within one species have also amplified at different times, corresponding to successive invasions. Finally, we detected a preference for insertion into short TA-rich regions, which appears to be specific to some subfamilies.ConclusionsThis analysis is the first comprehensive survey of this family of transposable elements at a genus scale. It provides precise measures of the different evolutionary processes that were hypothesized previously for this family based on PCR data analysis. mariner lineages were observed at almost all “life cycle” stages: recent amplification, subsequent decay and potential (re)-invasion or invasion of genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-727) contains supplementary material, which is available to authorized users.

Mechanoregulation of h2-Calponin Gene Expression and the Role of Notch Signaling

The essential role of mechanical signals in regulating the function of living cells is universally observed. However, how mechanical signals are transduced in cells to regulate gene expression is largely unknown. We previously demonstrated that the gene encoding h2-calponin (Cnn2) is sensitively regulated by mechanical tension. In the present study, mouse genomic DNA containing the Cnn2 promoter was cloned, and a nested set of 5' truncations was studied. Transcriptional activity of the Cnn2 promoter-reporter constructs was examined in transfected NIH/3T3, HEK293, and C2C12 cells for their responses to the stiffness of culture substrate. The results showed significant transcriptional activities of the -1.00- and -1.24-kb promoter constructs, whereas the -0.61-kb construct was inactive. The -1.38-, -1.57-, and -2.12-kb constructs showed higher transcriptional activity, whereas only the -1.57- and -2.12-kb constructs exhibited repression of expression when the host cells were cultured on low stiffness substrate. Internal deletion of the segment between -1.57 and -1.38 kb in the -2.12-kb promoter construct abolished the low substrate stiffness-induced repression. Site-specific deletion or mutation of an HES-1 transcription factor binding site in this region also abolished this repression effect. The level of HES-1 increased in cells cultured under a low tension condition, corresponding to the down-regulation of h2-calponin. h2-Calponin gene expression is further affected by the treatment of cells with Notch inhibitor and activator, suggesting an upstream signaling mechanism.

Mapping the Interacting Sites Mediating Tetramerisation of Ryanodine Receptor Amino-Terminus

The cardiac ryanodine receptor (RyR2) is an intracellular calcium release channel composed of four ∼560kDa subunits. It mediates sarcoplasmic reticulum calcium release playing a critical role in cardiac excitation-contraction coupling. Recently, we presented evidence that the RyR2 N-terminus self-associates into a tetrameric form, which stabilises the closed conformation of the channel. In order to identify the interacting domains mediating RyR2 N-terminus tetramerisation, we used truncated constructs in yeast two-hybrid and chemical cross-linking assays. RyR2 residues 1-418 remained in monomeric form, whereas residues 1-530 retained self-association ability although it was much weaker compared to longer fragments. Tetramerisation was not abolished by removal of the N-terminal 160 amino acids but further truncations were detrimental. A targeted approach for putative inter-subunit contact sites was also employed testing the effect of small internal deletions within RyR2 residues 1-906. We found that two small deletions (167-178 or 335-358 amino acids) severely compromised tetramerisation ability. These findings suggest that residues 167-178 and 335-358 are the primary interacting sites, whereas additional downstream determinants further strengthen the RyR2 N-terminus self-association.

Insertion of long interspersed element-1 in theMitfgene is associated with altered neurobehavior of the black-eyed whiteMitfmi-bwmouse

Microphthalmia-associated transcription factor (Mitf) is required for the differentiation of melanoblasts of the neural crest origin. The mouse homozygous for the black-eyed white (Mitf(mi-bw) ) allele is characterized by white-coat color and deafness with black eye, due to the loss of melanoblasts during embryonic development. The Mitf(mi-bw) allele carries an insertion of long interspersed element-1 (L1) in intron 3 of the Mitf gene, which may cause the deficiency of melanocyte-specific Mitf-M. Here, we show that the L1 insertion results in the generation of alternatively spliced Mitf-M mRNA species, such as Mitf-M mRNA lacking exon 3, exon 4 or both exons 3 and 4, each of which encodes Mitf-M protein with an internal deletion. Transient expression assays showed the loss of or reduction in function of each aberrant Mitf-M protein and the dominant negative effect of Mitf-M lacking exon 4 that encodes an activation domain. Thus, the L1 insertion may decrease the expression level of functional Mitf-M. Importantly, Mitf-M mRNA is expressed in the wild-type mouse brain, with the highest expression level in the hypothalamus. Likewise, aberrant Mitf-M mRNAs are expressed in the bw mouse brain. The bw mice show the altered neurobehavior under a stressful environment, suggesting the role of Mitf-M in sensory perception.

Cell Cycle Regulation of Microtubule Interactomes: Multi-layered Regulation Is Critical for the Interphase/Mitosis Transition

Microtubules dramatically change their dynamics and organization at the entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we used an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that the suppression of the microtubule affinity of this mitotic MAP in combination with nuclear localization is essential for microtubule organization in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.

Highly improved gene targeting by germline-specific Cas9 expression in Drosophila.

We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.

Birth of Three Stowaway-like MITE Families via Microhomology-Mediated Miniaturization of a Tc1/Mariner Element in the Yellow Fever Mosquito

Eukaryotic genomes contain numerous DNA transposons that move by a cut-and-paste mechanism. The majority of these elements are self-insufficient and dependent on their autonomous relatives to transpose. Miniature inverted repeat transposable elements (MITEs) are often the most numerous nonautonomous DNA elements in a higher eukaryotic genome. Little is known about the origin of these MITE families as few of them are accompanied by their direct ancestral elements in a genome. Analyses of MITEs in the yellow fever mosquito identified its youngest MITE family, designated as Gnome, that contains at least 116 identical copies. Genome-wide search for direct ancestral autonomous elements of Gnome revealed an elusive single copy Tc1/Mariner-like element, named as Ozma, that encodes a transposase with a DD37E triad motif. Strikingly, Ozma also gave rise to two additional MITE families, designated as Elf and Goblin. These three MITE families were derived at different times during evolution and bear internal sequences originated from different regions of Ozma. Upon close inspection of the sequence junctions, the internal deletions during the formation of these three MITE families always occurred between two microhomologous sites (6–8 bp). These results suggest that multiple MITE families may originate from a single ancestral autonomous element, and formation of MITEs can be mediated by sequence microhomology. Ozma and its related MITEs are exceptional candidates for the long sought-after endogenous active transposon tool in genetic control of mosquitoes.

Internal deletions of transposable elements: the case of Lemi elements

Mobile elements using a "cut and paste" mechanism of transposition (Class II) are frequently prone to internal deletions and the question of the origin of these copies remains elusive. In this study, we looked for copies belonging to the Lemi Family (Tc1-mariner-IS630 SuperFamily) in the plant genomes, and copies within internal deletions were analyzed in detail. Lemi elements are found exclusively in Eudicots, and more than half of the copies have been deleted. All deletions occur between microhomologies (direct repeats from 2 to 13 bp). Copies less than 500 bp long, similar to MITEs, are frequent. These copies seem to result from large deletions occurring between microhomologies present within a region of 300 bp at both extremities of the element. These regions are particularly A/T rich, compared to the internal part of the element, which increases the probability of observing short direct repeats. Most of the molecular mechanisms responsible for double strand break repair are able to induce deletions between microhomologies during the repair process. This could be a quick way to reduce the population of active copies within a genome and, more generally, to reduce the overall activity of the element after it has entered a naive genome.

P.13.10 Unexpected findings in array analysis with possible implications for dystrophinopathy

Since the introduction of array analysis in molecular diagnostics for patients suffering from mental retardation and developmental disorders as for prenatal diagnostics many unexpected findings were made. So far, more than 20 different cases with possible implications for dystrophinopathy have been reported to our centre. About an equal amount of gross deletions and duplications within the DMD gene were observed. In nearly all cases array results could be confirmed and gross deletions/duplications were accurately mapped by means of Multiplex Ligation Probe Amplification of the DMD gene. In about half of the families the mutations found may have implications for the patients and/or their families. In the majority of cases with duplications, no effect was found by means of segregation studies in families. Also some FISH experiments showed that duplications were located in other parts of the genome. A number of duplications extended towards both the 5′ or 3′ end of the DMD gene and even further. Most deletions were internal gene deletions except for one and in the majority of cases novel DMD carriers were identified. In a few cases the mutations found yielded new information on their pathogenicity. For instance, deletions which could not be found in DMD databases possibly indicating that these mutations might be harmless deletions occurring in the general population. In one case prenatal DNA analysis identified a male fetus with an out-of-frame deletion indicating a possible novel DMD patient.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

FixL-like sensor FlbS of Brucella abortus binds haem and is necessary for survival within eukaryotic cells

Replication of Brucella inside eukaryotic cells is essential for pathogenesis, and successful infection requires rapid adaptation to the intracellular milieu. Close relatives of Brucella use the two-component system FixLJ to survive inside the host. We aimed to identify a homologous sensor in Brucella abortus. A predicted protein with transmembrane and conserved histidine kinase domains was identified as the Fix-like Brucella sensor, FlbS. Although it lacks the PAS domain, recombinant FlbS binds haem in vitro. An internal in-frame deletion in flbS severely decreased B. abortus survival inside professional and non-professional phagocytes. This phenotype was reverted by genetic complementation. These results indicate the critical role of this haemoprotein in the intracellular lifestyle of Brucella.