The team was inspired by the success of its initial collaborative studies, which were among the first applications of what would later become known as genomics to neurobiology. Bloom, Milner and John Morrison moved from Salk to Scripps, and together with JGS initiated a program project aimed at expanding the effort. The Sutcliffe laboratory's role in the program was that of discovery of novel brain-specific proteins.
Which of the thousands of brain-specific mRNAs to characterize? The early studies demonstrated that many neuronal mRNAs exhibited differential distributions within the CNS; however, their expression was generally not restricted to a few discrete loci that could be attributed to specialized functions, but rather was variegated across the CNS. The studies therefore evolved to focus upon the identification of mRNAs that show a high degree of regional enrichment within the CNS. The logic that motivated this focus was that mRNA molecules with restricted expression were likely to encode proteins that are singularly associated with the unique functions of the cells that contain them and, perhaps, might be preferentially associated with particular physiological or behavioral processes compared with molecules with more general patterns of expression. Hence, their functional roles might be more transparent to investigation. Furthermore, such molecules might, in the future, provide highly specific targets for pharmaceuticals that would act only at the restricted site of target expression.
In order to enrich our libraries for such mRNAs, and the team turned to subtractive hybridization. Subtractive hybridization refers to a series of methodologies that compare cDNA sequences from one RNA sample, the target, with those from a second sample, the driver. Nowadays, the two complementary, antiparallel strands of cDNA can be produced in either of their single-stranded orientations, sense or antisense, using modern cloning and enzymological procedures. When sense strand from the driver is supplied in great excess over antisense strand from the target and these reagents are coincubated under conditions that favor the formation of double-strand hybrids, most of the mass of those sequences that the target and driver have in common becomes double stranded, whereas sequences from the target population that are absent from the driver population remain single stranded. The single-stranded material is isolated and used to create cDNA libraries enriched for target-specific mRNAs or enriched, radioactive probes for screening libraries. This methodology, originally developed by Timberlake7 for studies on gene expression in fungi, has been progressively improved in the ensuing decades to a degree that it has allowed identification of mRNAs selectively expressed within complex mammalian nervous tissue.
Gabe Travis joined the group and introduced mixed-phase methods for increasing the apparent concentrations of the target and driver nucleic acids, thus vastly increasing the extent of their hybridization and hence enhancing enrichment for target-specific sequences.8 He and Miles Brennan applied the improved subtraction method to isolate mouse retinal photoreceptor-specific mRNAs, including that corresponding to the product of the retinal degeneration slow gene, whose human homologue accounts for a considerable portion of heritable late-onset blindness.9 Joe Watson had success in the identification of forebrain-enriched mRNAs, including those RC3/neurogranin10 and G protein y7.n Nevertheless, the method, although occasionally effective, was cumbersome and inconsistent.
These shortcomings were overcome with the advent of PCR. Hiroshi Usui, with design input from Mark Erlander, developed a method (simplified here) in which the target cDNA is cloned into a vector that introduces PCR primer binding sites on both sides of the cDNA insert. After hybridization with the driver, the single-stranded target is PCR amplified and cloned. The refined method, called directional tag PCR subtractive hybridization,12 was used to prepare cDNA libraries enriched for clones of mRNAs specific to the striatum. Screening the large number of clones produced required high-throughput in situ hybridization. LdL developed a free-floating section method and brought anatomical expertise to the group.
As a result of implementing the refined subtraction method and high throughput in situ hybridization, we identified cortistatin, a neuropeptide of the somatostatin family expressed in the neocortex and hippocampus. 13
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