A FAP46 Mutant Provides New Insights into the Function and Assembly of the C1d Complex of the Ciliary Central Apparatus

Virtually all motile eukaryotic cilia and flagella have a \u279+2\u27 axoneme in which nine doublet microtubules surround two singlet microtubules. Associated with the central pair of microtubules are protein complexes that form at least seven biochemically and structurally distinct central pair projections. Analysis of mutants lacking specific projections has indicated that each may play a unique role in the control of flagellar motility. One of these is the C1d projection previously shown to contain the proteins FAP54, FAP46, FAP74 and FAP221/Pcdp1, which exhibits Ca(2+)-sensitive calmodulin binding. Here we report the isolation and characterization of a Chlamydomonas reinhardtii null mutant for FAP46. This mutant, fap46-1, lacks the C1d projection and has impaired motility, confirming the importance of this projection for normal flagellar movement. Those cells that are motile have severe defects in phototaxis and the photoshock response, underscoring a role for the C1d projection in Ca(2+)-mediated flagellar behavior. The data also reveal for the first time that the C1d projection is involved in the control of interdoublet sliding velocity. Our studies further identify a novel C1d subunit that we term C1d-87, give new insight into relationships between the C1d subunits, and provide evidence for multiple sites of calmodulin interaction within the C1d projection. These results represent significant advances in our understanding of an important but little studied axonemal structure

Evidence of a Large-Scale Functional Organization of Mammalian Chromosomes

Evidence from inbred strains of mice indicates that a quarter or more of the mammalian genome consists of chromosome regions containing clusters of functionally related genes. The intense selection pressures during inbreeding favor the coinheritance of optimal sets of alleles among these genetically linked, functionally related genes, resulting in extensive domains of linkage disequilibrium (LD) among a set of 60 genetically diverse inbred strains. Recombination that disrupts the preferred combinations of alleles reduces the ability of offspring to survive further inbreeding. LD is also seen between markers on separate chromosomes, forming networks with scale-free architecture. Combining LD data with pathway and genome annotation databases, we have been able to identify the biological functions underlying several domains and networks. Given the strong conservation of gene order among mammals, the domains and networks we find in mice probably characterize all mammals, including humans

Integrating genetic and gene expression data: application to cardiovascular and metabolic traits in mice

The millions of common DNA variations that occur in the human population, or among inbred strains of mice and rats, perturb the expression (transcript levels) of a large fraction of the genes expressed in a particular tissue. The hundreds or thousands of common cis-acting variations that occur in the population may in turn affect the expression of thousands of other genes by affecting transcription factors, signaling molecules, RNA processing, and other processes that act in trans. The levels of transcripts are conveniently quantitated using expression arrays, and the cis- and trans-acting loci can be mapped using quantitative trait locus (QTL) analysis, in the same manner as loci for physiologic or clinical traits. Thousands of such expression QTL (eQTL) have been mapped in various crosses in mice, as well as other experimental organisms, and less detailed maps have been produced in studies of cells from human pedigrees. Such an integrative genetics approach (sometimes referred to as “genetical genomics”) is proving useful for identifying genes and pathways that contribute to complex clinical traits. The coincidence of clinical trait QTL and eQTL can help in the prioritization of positional candidate genes. More importantly, mathematical modeling of correlations between levels of transcripts and clinical traits in genetic crosses can allow prediction of causal interactions and the identification of “key driver” genes. An important objective of such studies will be to model biological networks in physiologic processes. When combined with high-density single nucleotide polymorphism (SNP) mapping, it should be feasible to identify genes that contribute to transcript levels using association analysis in outbred populations. In this review we discuss the basic concepts and applications of this integrative genomic approach to cardiovascular and metabolic diseases

A wild derived quantitative trait locus on mouse chromosome 2 prevents obesity

<p>Abstract</p> <p>Background</p> <p>The genetic architecture of multifactorial traits such as obesity has been poorly understood. Quantitative trait locus (QTL) analysis is widely used to localize loci affecting multifactorial traits on chromosomal regions. However, large confidence intervals and small phenotypic effects of identified QTLs and closely linked loci are impeding the identification of causative genes that underlie the QTLs. Here we developed five subcongenic mouse strains with overlapping and non-overlapping wild-derived genomic regions from an F2 intercross of a previously developed congenic strain, B6.Cg-<it>Pbwg1</it>, and its genetic background strain, C57BL/6J (B6). The subcongenic strains developed were phenotyped on low-fat standard chow and a high-fat diet to fine-map a previously identified obesity QTL. Microarray analysis was performed with Affymetrix GeneChips to search for candidate genes of the QTL.</p> <p>Results</p> <p>The obesity QTL was physically mapped to an 8.8-Mb region of mouse chromosome 2. The wild-derived allele significantly decreased white fat pad weight, body weight and serum levels of glucose and triglyceride. It was also resistant to the high-fat diet. Among 29 genes residing within the 8.8-Mb region, <it>Gpd2, Upp2, Acvr1c, March7 </it>and <it>Rbms1 </it>showed great differential expression in livers and/or gonadal fat pads between B6.Cg-<it>Pbwg1 </it>and B6 mice.</p> <p>Conclusions</p> <p>The wild-derived QTL allele prevented obesity in both mice fed a low-fat standard diet and mice fed a high-fat diet. This finding will pave the way for identification of causative genes for obesity. A further understanding of this unique QTL effect at genetic and molecular levels may lead to the discovery of new biological and pathologic pathways associated with obesity.</p

Molecular mechanism of edema formation in nephrotic syndrome: therapeutic implications

Sodium retention and edema are common features of nephrotic syndrome that are classically attributed to hypovolemia and activation of the renin–angiotensin–aldosterone system. However, numbers of clinical and experimental findings argue against this underfill theory. In this review we analyze data from the literature in both nephrotic patients and experimental models of nephrotic syndrome that converge to demonstrate that sodium retention is not related to the renin–angiotensin–aldosterone status and that fluid leakage from capillary to the interstitium does not result from an imbalance of Starling forces, but from changes of the intrinsic properties of the capillary endothelial filtration barrier. We also discuss how most recent findings on the cellular and molecular mechanisms of sodium retention has allowed the development of an efficient treatment of edema in nephrotic patients

Variable bone fragility associated with an Amish COL1A2 variant and a knock-in mouse model

Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1–4) areal bone mineral density (aBMD) Z -scores demonstrated that 73% had moderate to severe disease (less than −2), 23% had mild disease (−1 to −2), and 4% were in the unaffected range (greater than −1). A line of knock-in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F 1 lines of knock-in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI. © 2010 American Society for Bone and Mineral ResearchPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65040/1/90720_ftp.pd

The rate of change in Ca2+ concentration controls sperm chemotaxis

During chemotaxis and phototaxis, sperm, algae, marine zooplankton, and other microswimmers move on helical paths or drifting circles by rhythmically bending cell protrusions called motile cilia or flagella. Sperm of marine invertebrates navigate in a chemoattractant gradient by adjusting the flagellar waveform and, thereby, the swimming path. The waveform is periodically modulated by Ca(2+) oscillations. How Ca(2+) signals elicit steering responses and shape the path is unknown. We unveil the signal transfer between the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and path curvature (κ). We show that κ is modulated by the time derivative d[Ca(2+)](i)/dt rather than the absolute [Ca(2+)](i). Furthermore, simulation of swimming paths using various Ca(2+) waveforms reproduces the wealth of swimming paths observed for sperm of marine invertebrates. We propose a cellular mechanism for a chemical differentiator that computes a time derivative. The cytoskeleton of cilia, the axoneme, is highly conserved. Thus, motile ciliated cells in general might use a similar cellular computation to translate changes of [Ca(2+)](i) into motion

Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated