Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids.

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.

Identification of an element crucial for the sub-synaptic expression of the acetylcholine receptor epsilon-subunit gene.

The adult neuromuscular junction displays an accumulation of both the acetylcholine receptor (AChR) protein in the subneural domain of the post-synaptic membrane and the mRNAs coding for all its subunits at the level of the subjunctional "fundamental nuclei." In the course of end plate development, the epsilon-subunit, at variance with other subunits, becomes exclusively expressed at the level of the fundamental nuclei, yet at a rather late stage (around birth). To analyze the promoter region of the epsilon-subunit gene which directs its specific expression at the synapse, we used a quantitative transient in vivo expression assay in intact muscle tissue using constructs of the epsilon-subunit promoter placed upstream of the beta-galactosidase reporter gene. One crucial element for synapse-specific expression was detected between the -11 and -6 positions. Disruption of this element, either by a scanning mutation or single base mutations, greatly diminishes, or even completely inhibits, preferential expression of the transgene at the end plate. Gel shift experiments reveal the presence of a complex in nuclear muscle extracts that bind the core sequence of this element. The identification of such a site opens the possibility to identify regulatory factors responsible for compartmentalized expression at the neuromuscular junction.

An 83-nucleotide promoter of the acetylcholine receptor epsilon-subunit gene confers preferential synaptic expression in mouse muscle.

The expression of the acetylcholine receptor epsilon-subunit gene is restricted to the endplate of adult muscle fibers. We have started to study the regulatory elements of the epsilon-subunit gene promoter that are important for its synaptic expression. We used, for this purpose, a rapid method of in vivo expression after DNA injection into the muscle tissue [Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. & Felgner, P. L. (1990) Science 247, 1465-1468]. Our results show that a construction containing 83 nucleotides upstream from the transcription start site is sufficient to obtain preferential endplate expression. Moreover, mutation of a MyoD binding site located around position-70 does not alter this synaptic expression. We also studied the expression of this promoter in vitro in muscle primary cultures and showed the presence of a positive element between positions -122 and -83. Comparison of in vivo and in vitro results reveals that the elements important for in vivo localization at the synapse and in vitro expression in cultured muscle cells may differ.

Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken.

Myogenin and CMD1, the chicken homologue of MyoD, transactivate the promoter of the alpha-subunit of the acetylcholine receptor (AChR) in chicken fibroblasts. The expression of these three genes was followed by in situ hybridization. In two-day-old embryos the CMD1 gene is expressed shortly before the AChR alpha-subunit and the myogenin genes. At day 19 extrajunctional AChR mRNA clusters have disappeared and myogenin mRNAs are no longer detected in PLD muscle. Moreover, both myogenin and CMD1 mRNA levels increase after muscle denervation in chicks. These data are compatible with a role for myogenic factors in the induction and maintenance of extra-junctional expression of the AChR genes during early muscle development. Using digoxygenin labelled RNA probes, we also show that the mRNAs for the AChR alpha-subunit display a punctated, probably perinuclear distribution, whereas mRNAs for myogenic genes accumulate in the sarcoplasm around subsets of nuclei in the muscle fiber.

Influence of innervation of myogenic factors and acetylcholine receptor alpha-subunit mRNAs.

The mRNA levels of the acetylcholine receptor alpha-subunit gene and of four members of the MyoD1 gene family, which code for candidate transcriptional activators of the former gene, were compared in the mouse during post-natal development and after denervation in the adult. During post-natal development, mRNA levels of myogenin, MyoD1, and Myf5 decrease in parallel with alpha-subunit mRNA whereas MRF4 mRNA level remains nearly constant. After denervation, increases in the levels of mRNAs coding for myogenin, MyoD1 and MRF4 accompany that of alpha-subunit mRNA, while Myf5 mRNA level varied little. A possible role of these myogenic genes in mediating nerve influence on the state of muscle differentiation and AChR alpha-subunit gene expression is discussed.

Induction of acetylcholine receptor alpha-subunit gene expression in chicken myotubes by blocking electrical activity requires ongoing protein synthesis.

The level of acetylcholine receptor alpha-subunit mRNA in primary cultures of chicken myotubes increases when the spontaneous electrical activity of the myotube is suppressed by the sodium channel blocker tetrodotoxin. This increase was prevented by two translational inhibitors: cycloheximide and anisomycin. Neither the basal level of alpha-subunit mRNA nor that of muscle-specific creatine phosphokinase mRNA was affected by these inhibitors. In contrast, cycloheximide potentiated the limited increase of alpha-subunit mRNA levels evoked by the neuropeptide calcitonin gene-related peptide. The high level of alpha-subunit mRNA elicited by tetrodotoxin treatment did not persist after subsequent addition of cycloheximide in the presence of tetrodotoxin, indicating that the continuous synthesis of protein factor(s) is necessary for this regulation. Moreover, cycloheximide decreased the high level of alpha-subunit mRNA present at early stages of in vitro maturation of muscle primary cultures without blocking the further increase of the muscle-specific creatine phosphokinase mRNA. Implications of the requirement for constant synthesis of protein factors on the induction of muscle-specific gene expression by blocking myotube electrical activity are discussed.

Acetylcholine receptor expression in primary cultures of embryonic chick myotubes--II. Comparison between the effects of spinal cord cells and calcitonin gene-related peptide.

Spinal cord cells co-cultured with primary chick myotubes caused a 1.5-3-fold increase in the number of muscle surface acetylcholine receptors assayed with [125I]alpha-bungarotoxin. This increase did not result from the metabolic stabilization of the acetylcholine receptor protein and was at least partially due to a stimulation of acetylcholine receptor biosynthesis up to the level of the accumulation of alpha-subunit mature and partially spliced precursor mRNAs. A medium conditioned by spinal cord cells also caused a rise in acetylcholine receptor number. This increase did not coincide with an augmentation of the intracellular cyclic AMP level as reported for the neuropeptide calcitonin gene-related peptide. In contrast, spinal cord cells and the medium conditioned by them potentiated the effect of calcitonin gene-related peptide on acetylcholine receptor number. Stimulation of acetylcholine receptor synthesis by the conditioned medium was blocked by the protein kinase C activator 12-O-tetradecanoyl phorbol-13-acetate and by the calcium ionophore A23187. These two compounds have already been reported to block the increase of acetylcholine receptor number produced by the voltage sensitive sodium channel antagonist tetrodotoxin which stimulates acetylcholine receptor biosynthesis by blocking spontaneous electrical activity of the cultured muscle cells. The possibility that different neural factors and second messenger systems are involved in the regulation of acetylcholine receptor biosynthesis during the development of the neuromuscular junction is discussed.