The CRE consensus sequence in the synapsin I gene promoter region confers constitutive activation but no regulation by cAMP in neuroblastoma cells.

Synapsin I is implicated in the modulation of neurotransmitter release and in synaptogenesis and is regulated by phosphorylation. The rat and human synapsin I genes both carry CRE and TRE consensus sequences in their promoter regions. This suggested that protein kinase-mediated signal pathways might also regulate synapsin I activity at the level of gene expression and thus contribute, on a slower time scale, to synaptic plasticity. We have therefore investigated, in neuroblastoma cell lines, the effects of agents that activate protein kinases on synapsin I gene expression. Unexpectedly, treatment with forskolin/IBMX was not found to enhance synapsin I mRNA levels. Rather, it causes a decrease to approximately 50% within 1 day although several CRE-dependent control genes are strongly induced. The calcium ionophore, A23187, lowers synapsin I mRNA to approximately 75%, and the phorbol ester, TPA, is without effect. Transient expression of a CAT fusion gene under the control of the synapsin I promoter region is also inhibited by forskolin/IBMX, as well as by protein kinase A (PKA) overexpression, suggesting that the decrease of synapsin I mRNA in response to forskolin/IBMX is due to the inhibition of transcription. Mutation of the CRE consensus does not affect the response to PKA, but it reduces the constitutive activity of synapsin I promoter constructs down to 30-50%. Nuclease footprinting experiments demonstrate sequence-specific binding proteins from brain, liver and NS20Y cell nuclear extracts to the CRE consensus sequence of the rat synapsin I promoter.

The 5'-flanking region of the synapsin I gene. A G+C-rich, TATA- and CAAT-less, phylogenetically conserved sequence with cell type-specific promoter function.

The 5'-terminal region of the rat gene for the neuron-specific phosphoprotein, synapsin I, was isolated and sequenced. It comprises 1472 nucleotides (nt) of 5'-flanking sequence, 507 nt of the first exon, and 242 nt of the first intron. A single transcription start site was mapped by primer extension and S1 nuclease analysis. A sequence of 340 nt upstream from the transcription start site and the first exon are G+C-rich and enriched in CpG dinucleotides, resembling a CpG island. The 5'-flanking sequence lacks TATA and CAAT consensus elements but contains a consensus motif for the cAMP-responsive element. Furthermore, we notice two potential consensus motifs which are also found in corresponding positions in the genes for the nerve growth factor receptor and the 68-kDa neurofilament protein. The 5'-terminal region of the human synapsin I gene was also cloned and sequenced. A high degree of sequence conservation between rat and human is found in the upstream 340 nt that coincides precisely with the G+C-rich domain and includes the consensus elements, and throughout the first exon including the untranslated sequence. Sequence conservation is also observed further upstream and at the beginning of the first intron. In a transient chloramphenicol acetyltransferase expression assay, 5'-flanking sequences of the rat synapsin I gene function as strong promoters in neuroblastoma cells, but not in fibroblastoid cells. 225 nt of 5'-flanking sequence and 105 nt of 5'-untranslated sequence are sufficient for cell-type specific transcription in this assay.

Molecular genetics of the extracellular lipase of Pseudomonas aeruginosa PAO1.

The structural gene (lipA) coding for the extracellular lipase of Pseudomonas aeruginosa PAO1 has been cloned on plasmid pSW118. Nucleotide sequence analysis revealed a gene of 936 bp. lipA codes for a proenzyme of 311 amino acids including a leader sequence of 26 amino acids. The mature protein was predicted to have a M(r) of 30134, an isoelectric point of 5.6, and a consensus sequence (IGHSHGG) typical of lipases. Furthermore it is highly homologous (greater than 60%) to other lipases from various pseudomonads. The lipA gene failed to hybridize detectably with genomic DNA from other Pseudomonas species except P. alcaligenes, even under relaxed stringency. Located 220 bp downstream of the lipA gene, is an open reading frame (ORF2, lipH) which encodes a hydrophilic protein (283 amino acids; M(r) 33587) that shows some homology to the limA gene product of P. cepacia. In complementation tests of lipase-defective mutants, lipH was shown to be necessary for expression of active extracellular lipase in P. aeruginosa PAO1.

The 5'-flanking region of the rat synapsin I gene directs neuron-specific and developmentally regulated reporter gene expression in transgenic mice.

The expression of the synapsin I gene is neuron-specific and developmentally regulated. As a step toward characterizing the molecular mechanisms that are responsible for its transcriptional regulation in vivo, we have generated transgenic mice that carry the chloramphenicol acetyltransferase (CAT) receptor gene under the control of approximately 4,300 nucleotides of 5'-flanking sequence of the rat synapsin I gene. In four independent transgenic mouse lines, high level CAT expression is observed specifically in the brain and other neural tissues. Two of these lines also exhibit notable CAT expression in testis. The transgene is expressed at similar levels in many different regions of the central nervous system. Immunohistochemical staining detects the CAT marker protein in various cell populations of neuronal morphology within the brain and the spinal cord. Transgene expression is developmentally regulated in a way that correlates well with the expression of the endogenous synapsin I gene. Both follow a characteristic, biphasic postnatal time course with a maximum around day 20. We conclude that the DNA region investigated contains cis-regulatory elements sufficient to drive the expression of a reporter gene in a spatial and temporal pattern that resembles the expression of the endogenous synapsin I gene.