Search

An efficient cDNA amplification procedure is described for determining of the 5′ and 3′ ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5′- or 3′-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5′and 3′rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of “vectorette PCR” and “suppression PCR” technologies that significantly reduce background during amplification. The application of “long and accurate PCR” (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5′- and 3′-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3–1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

A rapid and reliable method is described for preparing competitive DNA fragments for quantitative PCR. Synthetic DNAs complementary to previously established PCR primers are ligated together with the primers to both ends of a generic DNA fragment whose length differs from the natural target gene PCR product. After a short ligation step, the properly constructed ligation products (i.e., those that have the correct primer templates on opposite sides of the generic DNA fragment) are preferentially amplified by PCR. The generation of competitive PCR fragments, MIMICS, can be completed in a single day. To perform quantitative PCR, known quantities of PCR MIMICS are spiked into PCR amplification reactions containing the experimental cDNA samples. A visual or radioactive comparison of the PCR products can then be used to determine the initial quantity of target gene. We show that competitive PCR MIMICS can be used to accurately measure small changes in mRNA levels.

We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.

The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) was found to induce the biosynthesis and deposition of two cytoskeletal components in the human erythroleukemic cell line K562 with apparent Mr = 55,000 and 46,000. A time course showed that maximal changes in these cytoskeletal components occurred as early as 24 h after treatment and were maintained at least as long as 72 h. By immunoprecipitation, one of the induced cytoskeletal components was identified as vimentin. There was approximately a 10-fold induction of vimentin biosynthesis following TPA treatment. The other TPA-induced protein was found to co-migrate with purified actin in sodium dodecyl sulfate-polyacrylamide gels. Neither of these proteins was induced in the closely related human erythroleukemic cell line, HEL. By in vitro protein synthesis directed by total cellular RNA isolated from K562 cells, induction of vimentin biosynthesis was found to correlate with increases in the level of vimentin mRNA activity. A time course showed that vimentin mRNA activity was detectably elevated as early as 3 h after TPA treatment and reached a maximum by 12 h then the level decreased. By RNA dot blot and Northern gel hybridization using a cloned human actin cDNA, we found that there was also an induction of actin mRNA. A time course showed that there was an elevation of actin mRNA as early as 1 h after TPA treatment. The level then increased to a maximum at 6 h, after which the level decreased. These results are consistent with the induction of vimentin and actin gene transcription by TPA in K562 cells.

In culture the human teratocarcinoma cell line HT-H generates both adherent monolayer and free-floating aggregates. Some populations of aggregated cells develop further to form cystic bodies. A previous study showed the morphological resemblance of the cystic bodies to cells of blastocyst of preimplantation embryo. In this study, HT-H adherent cells were further separated into two subpopulations, fast adhering and slow adhering cells. Fast adhering cells produce fibronectin, spread well onto substratum, and do not proliferate. In contrast, slow adhering cells do not produce fibronectin. Trophoblastic markers were examined in each morphological stage of HT-H cells and the following results were obtained. Only fast adhering cells produce progesterone. Human chorionic gonadotropin was secreted preferentially by fast adhering cells, about six times less by slow adhering cells, and was not secreted by aggregates or cystic bodies. All stages of HT-H cells express c-fos but only fast adhering cells express c-fms oncogene. Cytokeratin 18 was expressed in all stages of HT-H cells. The level of cytokeratin 18 is modestly decreased from adherent to aggregates further into cystic bodies. These results indicate that HT-H cells share properties with cells in trophoblast, placenta, and extraembryonic endoderm. Spontaneous differentiation of HT-H cultures results in the appearance of fast adhering cells which exhibit biochemical properties expected for syncytiotrophoblast.

Human glycophorin A belongs to a family of structurally related cell surface glycoproteins which are expressed in erythroid cells. We have recently isolated several human glycophorin A-specific cDNA clones and have determined their nucleotide sequence (Siebert, P. D., and Fukuda, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1665-1669). In this report by using cDNA and exact sequence synthetic oligodeoxyribonucleotides as hybridization probes, we provide evidence that human glycophorin A and B are encoded by separate and distinct genes present as single copies in the human genome. Furthermore, we show that the expression of the glycophorin A and B genes are coordinately regulated by tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate.