An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.     

int-h: An int mutation of phage lambda that enhances site-specific recombination

The mutation int-h3 maps in the int gene of coliphage λ and results in the synthesis of an integrase with enhanced activity, which is manifested by an ability to support λ site-specific recombination relatively efficiently under conditions where the wild-type integrase functions inefficiently. The level of site-specific recombination seen in the presence of the int⁺ integrase in himA^? hosts is greatly reduced, as measured by lysogen formation, intramolecular site-specific integration and excision, and excision of a cryptic λ prophage. In contrast, the int-h3 integrase shows relatively high levels of activities under these conditions. Int-h3 is also more active in other host mutants (himB and hip) that reduce λ site-specific recombination. In the absence of the normal attB site, the frequency of lysogen formation (at secondary sites) by λ int⁺ is reduced 200 fold. Although λ int-h3 will integrate preferentially at the attB site if it is present, the mutant phage forms lysogens at a high frequency in attB-deleted hosts. λ int-h3 requires himA function for integration at secondary sites. The fact that the int-h3 integrase uses the same att sites as well as the same host functions as the int⁺ integrase suggests that the mutation results in a quantitative rather than a qualitative change in integrase activity; that is, the int-h3 integrase is more active. The mutant integrase supports site-specific recombination with att sites that carry the att24 mutation. We propose that the int-h3 integrase is endowed with an enhanced ability to recognize att sequences, including some that are not effectively recognized by wild-type integrase.     

An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus

Neuroligins are evolutionarily conserved postsynaptic cell-adhesion molecules that function, at least in part, by forming trans-synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino-acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin-4. We introduced the R704C mutation into mouse neuroligin-3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin-3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor-mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor-mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin-3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.     

Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1

Recent studies have demonstrated that the ATP-binding cassette transporter A1 (ABCA1) facilitates the efflux of phospholipids and cholesterol to apoprotein acceptors, leading to the synthesis of HDL. The purpose of this study was to determine the changes in the lipoprotein fractions in Abca1-deficient mice and study the mechanisms responsible for the low levels of HDL when ABCA1 is absent. Plasma phospholipid concentration was decreased by more than 75%, mostly due to a reduction of phosphatidylcholine (PC) in HDL. Abca1(-/-) HDL represents less than 2% of wild-type levels and is smaller and enriched in phospholipids (11.2-fold more than HDL from controls). Compared to wild-type littermates, Abca1(-/-) HDL had a 4-fold increase in PC, whereas lysophosphatidylcholine (LPC) (125-fold), sphingomyelin (SPH) (49-fold), and phosphatidylethanolamine (PE) (18-fold) showed even higher increases. As a consequence, the ratios of LPC/PC, SPH/PC, PE/PC, and phosphatidylinositol + phosphatidylserine (PI+PS)/PC were all much higher in HDL from Abca1(-/-), compared to wild-type HDL. Plasma phospholipid transfer protein (PLTP) and lecithin cholesterol acyltransferase (LCAT) activities were decreased by more than 80%, suggesting that the maturation of HDL is affected. To test this hypothesis, plasma from Abca1(-/-) mice was incubated with CHO cells that are known to express high levels of ABCA1 with the intent of restoring the flux of phospholipid and cholesterol onto apoAI. Compared to native plasma, no change in maturation of HDL was observed. In contrast, a 220% increase in the formation of mature HDL was observed when ABCA1 function and LCAT activities were restored. Taken together, these observations suggest that ABCA1 is necessary for the adequate lipidation of apoAI, which enables the interaction with LCAT and subsequent maturation.     

The relative contributions of recombination and point mutation to the diversification of bacterial clones

Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.     

Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase

The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F₁F_o-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc₁ complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F₁F_o-ATP synthase and NADH:ubiquinone oxidoreductase.     

Properties of Haemophilus influenzae mutants that are slightly recombination deficient and carry a mutation in the rec-1 gene region.

The highly recombination-deficient rec-1 mutants of Haemophilus influenzae are, as far as tested, equivalent to recA mutants of Escherichia coli. By selection for mutations in the rec-1 gene of H. influenzae, mutants designated ird (intermediary recombination-deficient) mutants were isolated; these mutants were much less recombination deficient (degree of transformability, 0.2 to 30% of wild-type value) than previously isolated rec-1 mutants (degree of transformability, 0.0001% of wild-type value). The ird mutants were more sensitive to ultraviolet irradiation and mytomycin C treatment than the wild type, but less sensitive than rec-1 mutants. Spontaneous production of phage HP1c1 by lysogenic MC11 cells and prophage induction by mitomycin C or ultraviolet irradiation were the same as in the wild type. In the ird mutants endogenous deoxyribonucleic acid was degraded both spontaneously and after ultraviolet irradiation to the same extent as in the wild type. Examination of one of the ird mutants revealed that recombination could be enhanced by ultraviolet irradiation, possibly because of an increased synthesis of the rec-1 gene product induced by ultraviolet irradiation.     

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.     

Genotoxicity of 2,4-D and dicamba revealed by transgenic Arabidopsis thaliana plants harboring recombination and point mutation markers

The phenoxy herbicides 2,4-D and dicamba are released daily into the environment in large amount. The mechanisms of genotoxicity and mutagenicity of these herbicides are poorly understood, and the available genotoxicity data is controversial. There is a cogent need for a novel genotoxicity monitoring system that could provide both reliable information at the molecular level, and complement existing systems.We employed the transgenic Arabidopsis thaliana 'point mutation' and 'recombination' plants to monitor the genetic effects of the herbicides 2,4-D and dicamba. We found that both herbicides had a significant effect on the frequency of homologous recombination A-->G mutation. Neither herbicides affected the T-->G mutation frequency. Interestingly, these transgenic biomonitoring plants were able to detect the presence of phenoxy herbicides at concentrations that were lower than the guideline levels for Drinking Water Quality. The results of our studies suggest that our transgenic system may be ideal for the evaluation of the genotoxicity of herbicide-contaminated water. Moreover, the unique ability of the plants to detect both double-strand breaks (homologous recombination) and point mutations provides tremendous potential in the study of molecular mechanisms of genotoxicity and mutagenicity of phenoxy herbicides.