The mammalian EGLN family contains three paralagous genes (EGLN1, EGLN2, and EGLN3) encoding prolyl hydroxylase isoforms that mediate the oxygen-dependent targeting of the transcription factor hypoxia inducible factor alpha to the proteosome. The rat orthologue of EGLN3 (SM-20) exhibits tissue-restricted expression, is induced by growth factors in cultured vascular smooth muscle, and is up-regulated during myogenesis. To determine if all three EGLN genes are coordinately regulated, we examined their mRNA expression in murine tissues and in cultured cells. We now report that the three murine EGLN mRNAs have unique but overlapping patterns of tissue expression. The most striking differences were in the heart, where EGLN3 had its highest levels of expression, and the testis, where EGLN2 was the only isoform expressed. In cultured vascular smooth muscle cells, serum treatment led to up-regulation of EGLN1 and EGLN3, but not EGLN2, and only EGLN3 was superinduced by cyclohexamide. In cultured C2C12 myocytes, EGLN3 was up-regulated during differentiation, whereas EGLN1 and EGLN2 were constitutively expressed. The abundance of EGLN3 mRNA in the heart, its induction by growth factors in vascular smooth muscle, and its regulation during C2C12 differentiation suggest a unique role for EGLN3 and might justify the development of isoformspecific inhibitors.

The vast majority of past and present efforts in the molecular cloning of expressed sequences involve isolation of clones from cDNA libraries constructed in bacteriophage lambda (1,2). As discussed in Chapter 6 , screening these cDNA libraries using labeled probes remains the most straightforward method to isolate full length cDNAs for which some partial sequence information is known. Although the availability of high quality reagents and kits over the past decade has made the process of library construction increasingly straightforward, generation of high-quality libraries is a task that still requires a fair amount of dedicated effort. Because alternative PCR-based cloning strategies have become increasingly popular alternatives to cDNA library screening, it is useful to consider the advantages and disadvantages of each strategy before embarking on a project to construct a cDNA library (Table 1). In our opinion, it is worthwhile to construct a cDNA library when the transcript of interest is not exceedingly rare (i.e., can readily be detected by Northern blot analysis of total RNA), when multiple cDNAs will need to be cloned over a period of time, and in situations where occasional mutations can not be tolerated (for example, if the cDNA is to be expressed in mammalian cells to examine function). In situations where the transcript of interest is expressed at exceedingly low levels, or when only a single cDNA needs to be cloned, a PCR-based strategy should be considered. When the tissue source is precious (such as a unique clinical specimen), successful construction of a phage library provides a resource that can be amplified and used for multiple cloning projects over many years, but runs the risk of consuming the available RNA if the library construction fails. Table 1 Comparison of Relative Advantages of cDNA Cloning from Lambda Phage Libraries by Plaque Hybridization Compared to Newer PCR- Based Strategies Lambda phage cDNA library PCR-based strategy Freedom from error ++ +/- Able to detect very rare transcripts - ++ Reusable ++ - Useful for rare/precious tissue samples - ++