XB-ART-24271Proc Natl Acad Sci U S A December 15, 1991; 88 (24): 11505-9.
A gene expression screen.
A gene expression screen identifies mRNAs that differ in abundance between two mRNA mixtures by a subtractive hybridization method. The two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for polymerase chain reaction (PCR) amplification. The multiple cDNA fragments isolated from any given gene can be treated as alleles in a genetic screen. Probability analysis of the frequency with which multiple alleles are found provides an estimation of the total number of up- and down-regulated genes. We have applied this method to genes that are differentially expressed in amphibian tadpole tail tissue in the first 24 hr after thyroid hormone treatment, which ultimately induces tail resorption. We estimate that there are about 30 up-regulated genes; 16 have been isolated.
PubMed ID: 1722336
PMC ID: PMC53164
Article link: Proc Natl Acad Sci U S A
Species referenced: Xenopus
Genes referenced: dio3 fap fosl2 hhipl2 itga11 klf9 mmp11 mmp13l paaf1 rem1 thdl17 thdl18 thdl20 thibz thra thrb
Article Images: [+] show captions
|FIG. 1. Flow diagram for isolation of up-regulated genes. A plus sign (+) refers to the mRNA isolated from tadpole tails treated with thyroid hormone (3,3',5-triiodo-L-thyronine, T3) for 24 hr, as well as the cDNAs derived from this + mRNA; - refers to mRNA and cDNAs from untreated tadpoles. LH, long hybridization; SH, short hybridization; BD, biotinylated driver DNA. The exact opposite protocol is carried out simultaneously to obtain the down-regulated genes.|
|FIG. 2. Enrichment of up-regulated genes (A), using + cDNA as tracer and - cDNA as driver; and enrichment of down-regulated genes (B), using - cDNA as tracer and + cDNA as driver. The enrichment was assayed by PCR Southern analysis. The flow chart (Fig. 1) and text detail each enrichment step. *, Up-regulated gene 6 (thyroid hormone receptorB); o, up-regulated gene 10; o, actin (22); A, thyroid hormone receptor a; A, down-regulated gene 17. The cDNA abundance is the number of molecules of each fragment for every 500,000 cDNA molecules at each step of enrichment. Upper arrow, minimum abundance of a specific DNA fragment required (in probes) to generate hybridization signal for colonies that contain the fragment in this screen; lower arrow, detection limit ofPCR Southern blot analysis using a cloned probe.|
|Fig. 3. A comparison of differentially expressed genes in control (-) and thyroid hormone-treated (+)-tadpole tail by Northern blot and PCR Southern blot. About 10 pg of total RNA (left two lanes) or 1.5 ,ug of - or + cDNA (right two lanes) was loaded in each lane. (A) Up-regulated gene 6 (thyroid hormone receptor P). (B) Downregulated gene 17. (C) Xenopus actin (22).|
|FiG. 4. Poisson distribution analysis of the up-r mRNAs identified by this gene expression screen. Th line shows the theoretil Poisson distribution curve that-best fitsthe data points, P'(n) P(n)N =JeAR-fnfN, where A = 0.8 and is theanumber of non-cross-hybridizing f sbiated for any cDNA. By analogy to a genetic screen isi the number of alles ond for each gene. P is the probability in Poisson distribution, P(n) is a conversion ofP by a constant N, the total number of esmated up-related ges, such that P(n) equals the number of deret cDNAs that hive n isolated non-ross-hybing . (pen circles represent the number of isolated cDNAs in the gen expression screen with n isolated "on-cross-hybridizing fragments. Where th t i Poisson distribution curve meets the vertical axis anem of the number of unidentified up-regulated cONAs genes) still present in the library.|
References [+] :
Alt, Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. 1978, Pubmed