Monique Lazar

C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction

At the termination step of protein synthesis, hydrolysis of the peptidyl‐tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1‐dependent and ribosome‐dependent GTPase. The role of the eRF1⋅eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two‐hybrid assay. We show that the N‐terminal parts of eRF1 (positions 1–280) and of eRF3 (positions 1–477) are either not involved or non‐essential for binding. Two regions in each factor are critical for mutual binding: positions 478–530 and 628–637 of eRF3 and positions 281–305 and 411–415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411–415) conserved in eukaryotes and archaebacteria is critical for eRF1s ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C‐terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C‐terminally truncated eRF3 is inactive as a GTPase.

Modulation of the Distribution of Acetylcholinesterase Molecular Forms in a Murine Neuroblastoma × Sympathetic Ganglion Cell Hybrid Cell Line

Studies were carried out on the polymorphism of acetylcholinesterase (AChE, EC 3.1.1.7) in a neuroblastoma × sympathetic ganglion cell hybrid cell line (T28) and its parental clone (N18TG2). These cells contain the tetrameric (G4, 10S), dimeric (G2, 6.5S), and monomeric (GI, 4S) forms of AChE, but not the collagen‐tailed A12 (16S) form of the sympathetic ganglion. Three variants of these forms could be distinguished on the basis of their solubility properties: (i) secreted forms which do not interact with the detergent Triton X‐100; (ii) cellular forms which may be solubilized in detergent‐free buffer and which interact reversibly with Triton X‐100; (iii) cellular forms which require detergent for solubility, and aggregate in its absence. By using a nonpenetrating inhibitor, we demonstrated that, in T28 stationary cells, the cellular G4 form is associated with the plasma membrane, whereas the G1 form is intracellular. During induction of AChE activity in T28 cells, the relative proportion of the G4 form increases, suggesting, in agreement with previous observations, that GI is a metabolic precursor of G4. The evolution of AChE molecular forms released into the culture medium closely resembles that of the cellular forms. The preferential accumulation of the G4 molecules does not simply depend on the cellular level of G1. It is favoured by culture conditions which promote morphological differentiation, but does not require the actual extension of neurites. T28 cells as well as other neuroblastoma‐derived cells appear to be useful experimental materials to investigate the regulatory mechanisms underlying the maturation of AChE globular forms.

Analysis of dopamine transporter gene expression pattern − generation of DAT‐iCre transgenic mice

The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon‐improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome–dopamine transporter–iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome–dopamine transporter–iCre transgene and a construct expressing the β‐galactosidase gene after Cre‐mediated recombination. In situ studies showed that β‐galactosidase (5‐bromo‐4‐chloroindol‐3‐yl β‐d‐galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of β‐galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome–dopamine transporter–iCre transgenic line is a unique tool for targeting Cre/loxP‐mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins.

Activation of the gene encoding the glycolytic enzyme β-enolase during early myogenesis precedes an increased expression during fetal muscle development

We define the spatial and temporal patterns of expression of the gene encoding the glycolytic enzyme, beta-enolase, during mouse ontogenesis. Transcripts were detected by in situ hybridization using 35S labelled cRNA probes. The beta-enolase gene is expressed only in striated muscles. It is first detected in the embryo, in the cardiac tube and in newly formed myotomes. In the muscle masses of the limb, beta gene expression occurs at a low level in primary fibers, and subsequently greatly increases at a time which corresponds to the onset of innervation and secondary fiber formation. Later in development, it becomes undetectable in slow-twitch fibers. Our results demonstrate the multistep regulation of the beta-enolase gene. The regulation of this muscle-specific gene in somites is discussed in terms of the myogenic sequences of the MyoD family shown to be present when it is activated.

5-HT1A-iCre, a new transgenic mouse line for genetic analyses of the serotonergic pathway

The 5-HT1A receptor not only plays an important role in brain physiology but it may be also implicated in the etiology of behavioral disorders such as pathological anxiety. To further define the role of 5-HT1A receptor-expressing neurons, we generated a transgenic mouse line expressing Cre recombinase in these cells. The 5-HT1A receptor open reading frame was substituted for that of Cre recombinase in a BAC containing the 5-HT1A receptor gene. In adult transgenic brain, Cre expression perfectly matched the distribution of 5-HT1A receptor mRNA. Additionally, Cre-mediated DNA recombination was restricted to neuronal populations that express the receptor, e.g., cerebral cortex, septum, hippocampus, dorsal raphe, thalamic, hypothalamic and amygdaloid nuclei, and spinal cord. Recombination occurred as early as E13 in trigeminal nerve, spinal ganglia and spinal cord. This transgenic line will allow the generation of conditional mutant mice that lack specific gene products along the serotonergic pathways and represents a unique tool for studying 5-HT1A-mediated serotonin signaling in the developing and adult brain.

Developmental Expression of Alpha- and Gamma-Enolase Subunits and mRNA Sequences in the Mouse Brain

Nonneuronal alpha alpha- and neuron-specific alpha gamma- and gamma gamma-enolase activities were measured in the mouse brain during development. The corresponding mRNA sequences were quantified directly by hybridization with cDNA probes. The variations in alpha- and gamma-monomer levels inferred from the enzymatic activities were very similar to those of their respective mRNAs. We conclude that monomer levels are primarily controlled by the amounts of their mRNAs during mouse brain development.

Cloning, expression and mutagenesis of a subunit contact of rabbit muscle-specific (ββ) enolase

The cDNA for rabbit muscle-specific (ββ) enolase was cloned, sequenced and expressed in Escherichia coli. This ββ-enolase differs at eight positions from that sequenced by Chin (17). Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts. Recombinant wild-type and mutant enolase were purified from E. coli and compared to enolase isolated from rabbit muscle. Molecular weights were determined by mass spectrometry. All three ββ-enolases had similar kinetics, and UV and circular dichroism (CD) spectra. The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate. We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation. ΔGo for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant. In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.