Promoter sequences in the RI beta subunit gene of cAMP-dependent protein kinase required for transgene expression in mouse brain.

Neural-specific expression of the mouse regulatory type-I beta (RI beta) subunit gene of cAMP-dependent protein kinase is controlled by a fragment of genomic DNA comprised of a TATA-less promoter flanked by 1.5 kilobases of 5'-upstream sequence and a 1.8-kilobase intron. This DNA contains a complex arrangement of transcription factor binding motifs, and previous experiments have shown that many of these are recognized by proteins found in brain nuclear extract. To identify sequences critical for RI beta expression in functional neurons, we performed a deletion analysis in transgenic mice. Evidence is presented that the GC-rich proximal promoter is responsible for cell type-specific expression in vivo because RI beta DNA containing as little as 17 base pairs (bp) of 5'-upstream sequence was functional in mouse brain. One likely regulatory element coincides with the start of transcription and includes an EGR-1 motif and 3 consecutive SP1 sites within a 21-bp interval. Maximal RI beta promoter activity required the adjacent 663 bp of 5'-upstream DNA where most, but not all, of the regulatory activity was localized between position -663 and -333. A 37-bp direct repeat lies within this region that contains 2 basic helix-loop-helix binding sites, each of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence. Intron I sequences were also tested, and deletion of a 388-bp region containing numerous Sp1-like sequences lowered transgene activity significantly. These results have identified specific regions of the RI beta promoter that are required for the expression of this signal transduction protein in mouse neurons.

Promoter for the regulatory type I beta subunit of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase directs transgene expression in the central nervous system.

Cyclic AMP-dependent protein kinase (cAPK) modulates synaptic transmission and influences memory and learning. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian cAPK, only the regulatory type I beta (RI beta) subunit is unique to nervous tissue. The requirement for RI beta in neurons is presently unknown. Previous studies demonstrate that holoenzyme containing RI beta activates at lower concentrations of cAMP compared to other forms of cAPK. Thus, neurons that induce RI beta expression may become more sensitive to subsequent hormonal signals and maintain more long-term phosphorylation events. To further elucidate the function of this novel protein, we have begun to investigate its gene. Here we report the isolation of the mouse RI beta promoter as determined by S1 nuclease analysis and transgenic mouse expression. A beta-galactosidase fusion gene containing 1.5 kilobases of 5'-nontranscribed RI beta DNA and 2 kilobases of intron 1 was expressed preferentially in the cortex and hippocampus of the brain and within the spinal cord. In addition to mimicking the location of endogenous RI beta expression, the transgene was activated at a similar time (embryonic day 11.5) during mouse fetal development. Isolation of the RI beta promoter will help identify the elements that direct transcription in a subset of neurons and illuminate the physiological conditions that may regulate RI beta expression. This promoter can also be used to target the expression of wild type and mutant cAPK subunit genes in order to investigate synaptic plasticity in animals.

Experimental phenocopy of a minute maternal-effect mutation alters blastoderm determination in embryos of Drosophila melanogaster.

Maternal haploinsufficiency for a third chromosome Minute, M(3)i55, lowers rates of protein synthesis by approximately 30% during the syncytial nuclear cycles of early embryogenesis. The maternal effect of Mi55 also produces segmentation defects (denticle belt fusions) in the posterior abdomen of larvae. Furthermore, embryos from Minute mothers show abnormal expression patterns of the segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage of embryogenesis. We developed a computer-aided analysis to describe the deviations in ftz expression which demonstrates that abnormally narrow ftz stripes occur in segment primordia that become fused in the larva. Unexpectedly, an abnormally wide ftz stripe occurs in segment primordia which do not develop abnormally. In addition, Mi55 produces a general narrowing of all ftz- interstripes. We phenocopied the Minute mutation by injecting wild-type embryos with cycloheximide concentrations which decreased protein synthesis rates to levels comparable with those of Minute embryos. Thus, a general decrease in protein synthesis during early embryogenesis leads to abnormal determination of posterior abdominal segment primordia.