Large-scale identification of differentially expressed genes during neurogenesis.

We report here a modified mRNA differential display method and its application for the analysis of differential gene expression in NGF-treated PC12 cells and in embryonic rat spinal cord. The optimized protocol is based on low fidelity priming of multiple cDNAs followed by high fidelity amplification. In PC12 cells induction by nerve growth factor (NGF) altered the expression of 4% of the 466 transcripts evaluated. During neurogenesis of the spinal cord we found that 30% of the 288 examined products changed. The differential expression of the characterized genes was confirmed by independent quantitative PCR. We conclude this method is suitable for the identification of increases and decreases of mRNA levels and allows the discovery of differentially expressed unknown transcripts.

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

Novel differentially expressed genes induced by kainic acid in hippocampus: putative molecular effectors of plasticity and injury.

Systemic kainic acid administration in rats induces acute limbic status epilepticus and subsequent neuronal degeneration and development of chronic hyperexcitability with similarities to human temporal lobe epilepsy. The mechanisms mediating the responses to kainic acid likely involve transcriptional changes in genes of importance for cellular injury, protection, and plasticity. We have used an arbitrarily primed PCR technique to identify such changes in the rat dentate gyrus. Three previously uncharacterized transcripts were found to be upregulated in the dentate gyrus 4 h following systemic kainic acid. In situ hybridization using riboprobes transcribed from the cloned PCR fragments were used to confirm differential expression specifically in dentate granule neurons following seizure. Basal expression for all three transcripts is widespread throughout the rat brain, with the highest levels seen in the hippocampal pyramidal and granule cell layers. The novel sequences do not match any known full-length cDNAs and may belong to novel gene families. However, they all showed high homology to human partial cDNA sequences (ESTs) that are expressed in brain as well as several other tissues. Two additional transcripts identified in this study corroborate earlier findings on differential expression of heat-shock proteins after seizure. The novel transcripts found in this study may be involved in epileptogenesis and neuronal responses to damage following seizure.

Synthesis of a gene for human serum albumin and its expression in Saccharomyces cerevisiae.

A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.