Desensitization contributes to the synaptic response of gain-of-function mutants of the muscle nicotinic receptor

Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e., gain-of-function mutations) increase the extent to which AChRs desensitize upon ACh removal. In this paper, we confirm this prediction by applying high-frequency trains of brief ( approximately 1 ms) ACh pulses to outside-out membrane patches expressing either lab-engineered or naturally occurring (disease-causing) gain-of-function mutants. Entry into desensitization was evident in our experiments as a frequency-dependent depression in the peak value of succesive macroscopic current responses, in a manner that is remarkably consistent with the theoretical expectation. We conclude that the comparatively small depression of the macroscopic currents observed upon repetitive stimulation of the wild-type AChR is due, not to desensitization being exceedingly slow but, rather, to the particular balance between gating, entry into desensitization, and ACh dissociation rate constants. Disruption of this fine balance by, for example, mutations can lead to enhanced desensitization even if the kinetics of entry into, and recovery from, desensitization themselves are not affected. It follows that accounting for the (usually overlooked) desensitization phenomenon is essential for the correct interpretation of mutagenesis-driven structure-function relationships and for the understanding of pathological synaptic transmission at the vertebrate neuromuscular junction.     

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2^−/− (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro-computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.     

Identification of the gene whose mutation leads to hypocotyl tropism in Arabidopsis thaliana

Genetic and molecular analysis of a mutant of Arabidopsis thaliana with bended hypocotyl from a previously obtained collection of insertion mutants is presented. The examined mutation was shown to be recessive and based on a single insertion of pLD3 vector T-region into the A. thaliana genome. Computer-aided analysis of a DNA region adjacent to the left border of the insertion revealed a putative site of T-DNA insertion, the At1g15760 gene from 609-bp chromosome 1 represented by a single exon.     

Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages

whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although bldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for bldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. bldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.     

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.     

Molecular cloning and overexpression of the tyrosine aminotransferase (TAT) gene leads to increased rosmarinic acid yield in Perilla frutescens

Perilla frutescens is a medicinal plant that produces various bioactive compounds, including unsaturated fatty acids and phenolic compounds such as rosmarinic acid (α-O-caffeoyl-3, 4 -dihydroxyphenylacetic acid, RA). Tyrosine aminotransferase (TAT) catalyzes the first step in the tyrosine-derived branch of RA biosynthesis, and TAT is presumed to have a role in RA accumulation. Here, we report the isolation of full-length TAT cDNA (designated PfTAT) from P. frutescens. Sequence analysis revealed that PfTAT contained 1,535 bp long and an open reading frame of 1,233 bp encoding 411 amino acid residues. Analysis of PfTAT genomic DNA revealed 7 exons and 5 introns. The 5′ flanking sequence of PfTAT was also cloned, and a group of putative cis-acting elements such TATA box, CAAT box, TC-rich repeats and G box were identified. Quantitative real-time PCR analysis indicated that constitutive expression of PfTAT in leaves was much higher than in roots and stems. A vector was constructed containing the PfTAT gene derived by the cauliflower mosaic virus 35S promoter. Transgenic P. frutescens overexpressing PfTAT was obtained with an Agrobacterium tumefaciens-mediated transformation system and overexpression was confirmed by PCR and Southern blot. PfTAT mRNA expression in transgenic plant lines measured by real-time quantitative PCR was 2–3 times greater than PfTAT expression in the untransformed plant line. Also, enhanced gene expression corresponded to significantly increased RA in PfTAT-transgenic lines, as quantified by HPLC. Our data emphasize the importance of PfTAT in the production of RA in P. frutescens.     

Lack of the murine homeobox gene Hesx1 leads to a posterior transformation of the anterior forebrain

The homeobox gene Hesx1 is an essential repressor that is required within the anterior neural plate for normal forebrain development in mouse and humans. Combining genetic cell labelling and marker analyses, we demonstrate that the absence of Hesx1 leads to a posterior transformation of the anterior forebrain (AFB) during mouse development. Our data suggest that the mechanism underlying this transformation is the ectopic activation of Wnt/beta-catenin signalling within the Hesx1 expression domain in the AFB. When ectopically expressed in the developing mouse embryo, Hesx1 alone cannot alter the normal fate of posterior neural tissue. However, conditional expression of Hesx1 within the AFB can rescue the forebrain defects observed in the Hesx1 mutants. The results presented here provide new insights into the function of Hesx1 in forebrain formation.     

Identifying targets of the rough homeobox gene of Drosophila: evidence that rhomboid functions in eye development.

In order to identify potential target genes of the rough homeodomain protein, which is known to specify some aspects of the R2/R5 photoreceptor subtype in the Drosophila eye, we have carried out a search for enhancer trap lines whose expression is rough-dependent. We crossed 101 enhancer traps that are expressed in the developing eye into a rough mutant background, and have identified seven lines that have altered expression patterns. One of these putative rough target genes is rhomboid, a gene known to be required for dorsoventral patterning and development of some of the nervous system in the embryo. We have examined the role of rhomboid in eye development and find that, while mutant clones have only a subtle phenotype, ectopic expression of the gene causes the non-neuronal mystery cells to be transformed into photoreceptors. We propose that rhomboid is a part of a partially redundant network of genes that specify photoreceptor cell fate.     

Identification of the gene whose mutation leads to the morphological changes in the hypocotyl of Arabidopsis thaliana

The results of genetic and molecular genetic analysis of line 176 of Arabidopsis thaliana with reduced hypocotyls obtained from a previously obtained collection of insertion mutants, are presented. The examined mutation proved to be recessive and based on a single insertion of the T-DNA vector pLD3 into the A. thaliana genome. Computer-aided analysis of the DNA region adjacent to the left border of the insertion revealed a putative site of T-DNA insertion, the 2.5-kb At2g09920 gene located in the long arm of chromosome 2, near the centromere.     

Identification of the gene whose mutation leads to the appearance of recessive lethal germlings in Arabidopsis thaliana

The data are presented on genetic and molecular-genetic analysis of a mutant from the collection of morphological insertion mutants of Arabidopsis thaliana we obtained earlier, which belongs to the phenotypic class of recessive lethal germlings. A nucleotide DNA sequence, 147 bp in size, was identified, which adheres to the left border area of T-DNA insertion. The site of localization of the insertion was determined using computer analysis.     

The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation

Muscular dysgenesis (mdg) is a mutation in mice which causes the failure of excitation-contraction coupling in skeletal muscle. Although the sarcolemma, the sarcoplasmic reticulum, and the contractile apparatus all maintain nearly normal function, sarcolemmal depolarization fails to cause calcium release from the sarcoplasmic reticulum. Recently, the primary genetic defect in this mutation was shown to be located in the structural gene for the dihydropyridine receptor. We have examined the developmental expression from Fetal Day 15 onward, in normal and mutant muscle, of several unidentified genes as well as genes which are known markers of muscle differentiation. We find that the majority of mRNA sequences are found at similar concentrations in normal and dysgenic muscles at birth. Many differentiation-related genes also are expressed at normal levels early during myogenesis in mutant mice. However, as late fetal development progresses in dysgenic muscle, the mRNA concentrations for these genes fail to undergo the rapid rise which is characteristic of normal muscle. Several additional, unidentified genes, which normally would be down-regulated during development, remain expressed at a high level in dysgenic muscle. Thus, the primary absence of a functional dihydropyridine receptor appears to prevent the changes in gene expression which are necessary for maturation of skeletal muscle.     

Evidence for the assignment of GUK 1 gene locus to 1q32 leads to q43 segment from gene dosage effect

A male infant with dup (1) (q32 leads to q43) constitution is reported. He had mental and physical retardation and a constellation of dysmorphisms, which are considered characteristic of trisomics for the distal one-third of the long arm of chromosome 1. The assay for guanylate kinase 1 (GUK 1) activity showed a gene dosage effect and confirmed the regional assignment of this marker in the chromosomal region indicated by data derived from somatic hybrids.     

Expression of the organizer specific homeobox gene goosecoid (gsc) in porcine embryos

The homeobox gene goosecoid is one of the first genes expressed in the organizer region of vertebrates and specifies future dorsal regions along the anterior/posterior axis of the embryo. Goosecoid (gsc) expression marks the posterior end of the anterior/posterior axis and might be a good marker to visualise early events in embryonic axis formation and differentiation processes in the epiblast at the onset of gastrulation. The aim of the present study was to evaluate gsc expression in porcine embryos. For this the homeobox containing region of the porcine gsc was isolated using RT-PCR. The sequence of the PCR product appeared to be highly homologous to the sequence in the mouse, human, and chicken. We concluded that the isolated region represents part of the porcine gsc messenger. Relative levels of gsc expression were estimated in porcine embryos from day 9 to day 12 of pregnancy. Gsc was expressed in embryos of all ages and localisation on one side of the embryoblast was demonstrated with in situ hybridisation on whole- mount embryos at day 10 of pregnancy. In embryos collected at day 13 of pregnancy gsc expression was localised anterior to the primitive streak. The correlation between embryo size and level of gsc expression was low. Levels and pattern of expression varied within and between litters collected at similar days of pregnancy. It is concluded that gsc expression can be used as an early marker of differentiation and to describe embryo diversity in the pig.