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Adrenergic receptors mediate the central and peripheral actions of norepinephrine and epinephrine and are pharmacologically divided into three major types, alpha-1, alpha-2, and beta. These types are further subdivided into alpha-1A, alpha-1B, and alpha-1D; alpha-2A, alpha-2B, and alpha-2C; and beta-1, beta-2, and beta-3, respectively. Adrenergic receptor sequence information is presented in three tables with respect to species, subtype identification, GenBank accession number, source of the nucleic acid sequence, the presence of a 5' flanking region upstream of the transcription start site, and the nucleotides defined as introns, coding regions, or 3' and/or 5' untranslated but transcribed (UTR) regions. Sequences have been assigned to adrenergic subtype categories based on sequence comparison using either FASTA or denogram of Pileup from the GCG sequence analysis program rather than as described in the author definition line. Sequence information found in these tables can be important for probe development for screening libraries for isolating adrenergic receptor genes from species other than the most common species. Where commercial libraries for specific tissue or species needs are not available, we have described construction of genomic cosmid libraries or PCR-based synthesis of a cDNA library using a microgram of RNA.
difference between cdna and genomic library
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There are important differences between genomic DNA clones and cDNA clones, as illustrated in Figure 8-35. Genomic clones represent a random sample of all of the DNA sequences in an organism and, with very rare exceptions, are the same regardless of the cell type used to prepare them. By contrast, cDNA clones contain only those regions of the genome that have been transcribed into mRNA. Because the cells of different tissues produce distinct sets of mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library.
A first and elegant approach for the isolation of genomic alterations was the representational difference analysis (RDA) (3), which evolved from the subtractive hybridization technique (4). Subtractive hybridization stands for (physical) separation of tester-specific sequences from ubiquitous and/or driver-specific DNA after hybridization of tester to an excess of driver. The key idea of the RDA approach was to replace the physical separation by a PCR-aided enrichment of tester-specific fragments. In our hands, however, a successful application of RDA was hampered by the preferential amplification of repetitive sequences and by generation of artificial fusion sequences.
The aims of the key steps of Limes were (i) minimal loss of complexity during library preparation by use of TspRI-flanking PCR libraries; (ii) minimal artefact production by exclusive conversion of perfectly matched tester/tester homodimers into PCR competent sequences with a highly specific ligation reaction; and (iii) supplement prevention of artefacts by purification of such ligation products via streptavidin-coupled magnetic beads. The efficacy of this novel protocol was investigated by an experimental setting that was as close as possible to that used in the initial RDA control experiments. We have chosen the same DNA as tester (λ-DNA; size of the monitoring fragment: 519 instead of 564 bp) and mammalian genomic DNA as driver (human instead of murine DNA). The efficacy of Limes was proven by the successful isolation of two tester-specific λ sequences out of 10 enriched fragments. In addition, fusion artefacts were efficiently avoided.
The screening for genomic mutations was stopped at the point when we realized that the examined My-La subline had lost its complete Y chromosome, because large genomic deletions impeded the identification of candidate oncogenes or tumor suppressor genes. It is worth noting, however, that the majority of the analyzed genomic clones were single copy sequences indicating that the frequency of repetitive elements decreased considerably in the enriched library. 2ff7e9595c
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