A simple and efficient method for the isolation of differentially expressed genes.

A simple and reproducible general approach for the isolation of differentially expressed genes is described. Digestion of cDNAs with a class IIs restriction endonuclease produces fragments with every combination of possible bases in the cohesive ends. Under stringent conditions, the specific ligation of adaptors with perfectly complementary overhangs partitions the cDNA fragments into non-overlapping subpopulations. Internal cDNA restriction fragments are exponentially amplified by adaptor primer PCR and visualised by non-denaturing polyacrylamide gel electrophoresis. The power of the technology was demonstrated using a rat model of pressure-induced left-ventricular hypertrophy (LVH). A set of 29 fragments, derived from a sample (6 %) of the possible adaptor pool combinations, displayed apparent differential expression. The differential expression of 19 (66 %) were confirmed by Northern blot analysis. Sequence analysis identified both genes known to be upregulated in LVH, and novel genes. The fidelity of adaptor ligation was demonstrated by the isolation of known gene fragments by appropriate adaptor combinations. The spiking of mRNA populations with known amounts of a synthetic mRNA demonstrated a current sensitivity equivalent to the detection of transcripts expressed at the level of as little as 1 in 10,000 molecules.

Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation.

Calreticulin is now considered to be a multifunctional Ca(2+)-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.