No abstract

We have isolated a novel cDNA, vesl, that was induced during convulsive seizure in the rat hippocampus. The vesl gene encodes a protein of 186 amino acids that has significant homology to the EVH1 domain of the VASP/Ena family of proteins implicated in the control of microfilament dynamics. The expression of vesl mRNA was induced in the granule cell layer during persistent long-term potentiation (LTP) of the dentate gyrus in an NMDA receptor-dependent manner. Furthermore, vesl mRNA was expressed at a high level during hippocampal synaptogenesis. We suggest that the Vesl protein may be involved in the structural changes that occur at synapses during long-lasting neuronal plasticity and development.© 1997 Federation of European Biochemical Societies.Key words: Long-term potentiation; Seizure; Hippocampus; Gene regulation; PCR differential display IntroductionBehavioral and pharmacological studies have revealed that memory has at least two distinct components, short-term and long-term memory. Long-term memory differs from shortterm memory in that it requires gene expression and protein synthesis at a critical period during and/or shortly after the learning experience (for review, [3]). Various protein synthesis inhibitors block the prolonged storage of memory without affecting short-term memory, in both vertebrates and invertebrates. This suggests that gene products synthesized during learning switch the stored information from a short-lasting labile form to a long-lasting stable form [4].Activity-dependent changes in the strength of synaptic transmission have been considered to provide a plausible cellular basis for learning and memory. Long-term potentiation (LTP) is a typical form of synaptic plasticity, in which elec-*Corresponding author. Fax: (81) (427) trical stimulation causes a long-lasting increase in synaptic efficacy [5]. Importantly, LTP in the hippocampus appears to have two distinct phases. The early phase, which persists ~ 3 h, is independent of macromolecule synthesis, whereas the late phase, which lasts for several hours in vitro and even for weeks in vivo, is prevented by pharmacological inhibition of protein and RNA synthesis, without any effects on the early phase [6][7][8][9][10]. These results strongly suggest that a particular set of genes, whose expression is induced after neural activity, play crucial roles in the prolonged maintenance of plastic changes in synaptic efficacy. Recent studies have identified a number of genes that are induced following a high-frequency stimulus (HFS) that elicits the late phase of LTP [11][12][13][14][15][16][17][18][19][20]. A major class of genes identified so far encodes transcription factors such as NGFI-A (also called Zif268, Krox24 and Egrl), c-Fos, Krox20 (Egr2), and Egr3 [11,12,16,20], supporting the hypothesis that altered gene expression is a prerequisite for the prolonged maintenance of LTP. In addition to these regulatory proteins, the late phase of LTP is accompanied by the induction of effector proteins such as activin [3A and Arc [1...

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.

INTRQDUCTIQNGlycinc, the structurally simplest amino acid, is a major neurotransmittcr in the vertebrate nervous system, especially in the spinal cord and brainstem of' mammals (I], On spinal motoneurons, glycine exerts an inhibitory action due to increase in Cl" pcrmcability via activation of a specific receptor/ion channel complex [ 1,2]. Molecular cloning work with the glycinc receptor (GlyR) has been carried out using rat and human cDNA libraries, and so far 3 species of the receptor subunit cDNAs (referred to as rat and human cul, human a2 and rat P subunits) have been isolated [3-61, In the cxpression systems using Xeno,~s oocytes and embryonic human kidney cell lines, the tul or ru2 protein itself has the function of a giycine-gated Cl-channel, whereas the ability of the P protein was much less [4-tl]. Northern blot analysis shows that mRNAs hybridizing to the a1 or P subunit cDNA are present abundantly in the spinal cord of matured rats (older than 20 days), but rather less abundantly in infant animal cords [3,9]. In addition to these GlyR-mRNAs, one of the authors (I-LA.) has provided some evidence for the existence of another species of GlyR-mRNA, production of which is made preferentially in the growing spinal cord [9, lo].

We have determined the complete nucleotide sequence of the linear DNA plasmid, pSKL, isolated from Saccharomyces kluyveri. Sequence analysis showed that pSKL has a high (A + T) content of 71.7%, and that there are 10 open reading frames (ORFs) larger than 250 nucleotides. All 10 ORFs were shown to be transcribed in S. kluyveri cells by S1 nuclease mapping analysis. The localization of ORFs, direction of transcription, and the predicted amino acid sequences of each ORF were quite similar to that of pGKL2, one of the killer plasmids found in Kluyveromyces lactis. The amino acid sequences of the largest two ORFs (ORF2 and ORF6) have homology with several DNA polymerases and RNA polymerases, respectively.