Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA.

A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.

The role of PTF1-P48 in pancreatic acinar gene expression.

The 100-base pair ELA1 transcriptional enhancer drives high level transcription to pancreatic acinar cells of transgenic mice and in transfected pancreatic acinar cells in culture. The A element within the enhancer is the sole positively acting element for acinar specificity. We show that the acinar cell-specific bHLH protein PTF1-P48 and the common bHLH cofactor HEB are part of the PTF1 complex that binds the A element and mediates its activity. Acinar-like activity of the enhancer can be reconstituted in HeLa cells by the introduction of P48, HEB, and the PDX1-containing trimeric homeodomain complex that binds the second pancreatic element of the enhancer. The 5' region of the mouse Ptf1-p48 gene from -12.5 to +0.2 kilobase pairs contains the regulatory information to direct expression in transgenic mice to the pancreas and other organs of the gut that express the endogenous Ptf1-p48 gene. The 5'-flanking sequence contains two activating regions, one of which is specific for acinar cells, and a repressing domain active in non-pancreatic cells. Comparison of the 5'-gene flanking regions of the mouse, rat, and human genes identified conserved sequence blocks containing binding sites for known gut transcription factors within the acinar cell-specific control region.

Inactivation of the G alpha i2 and G alpha o genes by homologous recombination.

G proteins couple receptors to effectors and thus regulate multiple biological processes. Here we report on the phenotypes of G alpha i2-deficient and G alpha o-deficient mice. G alpha i2-deficient mice display a blunted inhibitory regulation of adenylyl cyclase, alterations in T cell maturation and function, a growth retardation and also develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. G alpha o-deficient mice are also viable, but significantly smaller than wild-type controls.