Paul D. Siebert

[20] Suppression subtractive hybridization: A versatile method for identifying differentially expressed genes

Abstract A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

SUPPRESSION SUBTRACTIVE HYBRIDIZATION

Suppression subtractive hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. In fact, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. The SSH method is based on a suppression PCR effect and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNA or genomic DNA fragments, and simplifies analysis of the subtracted library. In our hands, the SSH technique has enriched over 1000-fold for rare sequences in a single round of subtractive hybridization.

Studies on the mode of action of calciferol XIX. A 24R-hydroxyl-group can replace the 25-hydroxyl-group of 1α,25-dihydroxyvitamin D3 for optimal binding to the chick intestinal receptor☆

Abstract Various 1α-hydroxylated side chain analogs of vitamin D3 have been studied for their ability to compete with 1α,25-dihydroxy[3H]vitamin D3 for binding to the chick intestinal receptor. Of the analogs examined, 1α,24R-dihydroxyvitamin D3 was found to be nearly equivalent to 1α,25-dihydroxyvitamin D3 in its ability to compete for receptor binding. However, this near equivalence was not shared by its stereoisomer, 1α,24S-dihydroxyvitamin D3, which was only 10% as effective a competitor. It is proposed that the ability of a 24R-hydroxyl group to mimic the 25-hydroxyl group is not due to a lack of side chain specificity on the part of the receptor, but is instead due to the similar orientation of the 25-hydroxyl and the 24R-hydroxyl such that they can be accommodated equivalently by the receptor.

Cell-free translation analysis of the vitamin D-dependent calcium binding protein mRNA activity present in total RNA and polysomal extracts from chick intestine☆

Abstract To further elucidate the mechanism(s) involved in the induction of a vitamin D-dependent intestinal calcium binding protein (CaBP), by the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], we have examined by cell-free translation CaBP-mRNA activity present in total cellular RNA extracts or polysomes obtained from chick small intestine. CaBP-mRNA activity was examined by immunochemical analysis of polypeptides completed upon addition of purified intestinal RNA or polysomes to a lysate system. Intestinal polysomes isolated from vitamin D-deficient chicks treated with vitamin D3, 1,25(OH)2D3, or 1α-hydroxyvitamin D3 were found to complete the synthesis of an immunoprecipitable protein which comigrated with authentic chick intestinal CaBP on SDS polyacrylamide gels. This product was found to represent up to 0.5–1.0% of the total polypeptides directed by polysomes isolated from treated birds but could not be detected among the products directed by intestinal polysomes obtained from vitamin D-deficient birds. A detectable level of polysomal CaBP-mRNA activity was present within 3 h and a maximal activity achieved at 9 h after administration of 1,25(OH)2D3. Increases in the levels of cytoplasmic CaBP (measured by radioimmunoassay) were first detected at 6 h, which was significantly later than the polysomal CaBP-mRNA activity. CaBP-mRNA activity in total cellular RNA isolated with guanidinium thiocyanate was also found to be dependent on treatment with vitamin D3 or 1,25(OH)2D3 but represented only 0.1–0.2% of the total mRNA activity. Surprisingly, total RNA and poly(A) RNA were found to direct the synthesis and release of two additional polypeptides that were immunoprecipitated even with an affinity-purified antibody to CaBP. One, an approximately 11,000-dalton polypeptide, was clearly vitamin D dependent; the other, an approximately 38,000-dalton polypeptide, was not. The time course for appearance of CaBP-mRNA activity in total cell RNA extracts following 1,25(OH)2D3 treatment was found to be nearly identical to that observed in polysome preparations. These results indicate that induction of CaBP-biosynthesis results, at least in part, from a 1,25(OH)2D3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH)2D3.

Generation of Full-Length cDNA Libraries Enriched for Differentially Expressed Genes

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expressionready clones that can be used for further high-throughput functional screening.

Construction of cDNA Libraries from Small Quantities of Total RNA Using Template Switching Catalyzed by M-MLV Reverse Transcriptase

Generation of high quality cDNA libraries with a comprehensive representation of the original mRNA population requires relatively large amounts (5–50 μg) of poly ( A)+RNA which is difficult to obtain when the amount of biological material is limited ( e.g., rare and unstable cell lines, microdissected cancer cells, biopsy materials, pathological specimens, embryonic and neuron tissues, cells in body fluids and so on). To circumvent this problem, several PCR-based technologies for amplification of cDNA from small amounts of total RNA have been described (Froussard 1993; Bertioli et al. 1994; Korneev et al. 1994). Basically, the amplification of a total cDNA population requires that universal primer binding sites are available at both cDNA ends. An arbitrary sequence can easily be imposed at the 5′ cDNA end by priming reverse transcription from the poly (A)+RNA fraction of total RNA by oligo (dT) or anchored oligo (dT) primer. Several strategies have been developed to add a determined sequence (anchor) at the 3′ end of the first-strand cDNA. These strategies include: (1) oligo (dG) or oligo (dA) tailing by terminal deoxynu- cleotidyltransferase (Bertioli et al. 1994; Korneev et al. 1994); ( 2) the use of T4 RNA ligase to covalently attach a single-stranded (ss) anchor oligonucleotide to the 3′ end of the ss cDNA (Apte and Siebert 1993); (3) the ligation of double-stranded (ds) adaptors to both ends of the ds cDNA (Frohman et al. 1988); and (4) the removal of the 7-MeGppp cap structure followed by ligation of an anchor sequence to the 5′ end of the decapped mRNA by T4 RNA ligase (Fromont-Racine et al. 1993).