Background: Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgeneTM Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis. Methods: We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 °C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR). Results: Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors. Conclusions: Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.

A second regulatory locus (blaRi) required for the induction of I-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced. The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the j8-lactamase (blaP) and repressor (blal) genes. Bacillus subtilis BD224 carrying these three genes synthesized P-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme. An open reading frame of 1,803 bases was identified as the blaRI gene by subcloning and DNA sequencing. The gene started 2 bases downstream of the termination codon of blal and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases. The deduced blaRi product (601 amino acids) had a molecular weight of 68,425. Five transmembrane regions were predicted from the hydrophobicity profile. The region around Phe-Ala-Pro-AlaSer-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the ,-lactamases. The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E. coli. The blaR1 gene encodes a potential penicillin receptor which is required for the induction of I8-lactamase in B. licheniformis 749.The ,-lactamase of Bacillus licheniformis is a secreted protein whose synthesis is induced by ,-lactam antibiotics. On induction, the enzyme is synthesized very slowly. The rate of synthesis reaches a maximum after 1 to 1.5 h of induction and then decreases slowly over the next hour. The rate remains severalfold higher than that of the uninduced culture for 2 or 3 h longer (11). This prolonged induction is not the result of unusual mRNA stability, because the half-life of the ,-lactamase mRNA is only 2 to 3 min (32). Sherratt and Collins reported three possible regulatory loci for the synthesis of 1-lactamase (35). The blaI gene, which is 90% linked to the 3-lactamase gene (blaP), encodes the repressor to blaP. The blaRi locus is 50% linked to blaP, and mutations (R14 or R27) thought to lie in this region cause low induction of the enzyme. Mutation blaR31 in the unlinked blaR2 locus results in a low basal level of the enzyme and a defect in induction. Penicillins irreversibly bind to the cell surface of B. licheniformis (13). Consequently, there must be a system of signal transduction through the membrane, and the stimulus may reach the repressor protein through a novel process. The blaRi and blaR2 loci are possibly involved in this transduction of the stimulus.The blaP gene has been cloned and sequenced (5,18,22,24,30 expected from the DNA sequence (M. J. Grossman and J. 0. Lampen, unpublished results). The mechanism of induction is still obscure, because Escherichia coli and Bacillus subtilis carrying the 4.2-kbp EcoRI DNA fragment do not show detectable induction of the ,B-lactamase (22). The absence of the blaRl and blaR2 genes may be responsible ...

The repressor gene, hlul, for the /I-lactamase of Bacillus lichenijormis 749 was functional when cloned in Escherichia coli, but addition of a D-lactam did not lead to induction. One plasmid contained fragments from the inducible strain (source of repressor), the other carried fragments from the blal-mutant 749/C (target). hlal lies just 5' to the promoter for the structural gene, blaP, and the target is the promoter region between the two genes. Interaction with both promoters seemed necessary for full repression. Blal is a hydrophilic protein (Mr I5 036) with the some structural similarities to repressors from Gram-negative bacteria./I-Lactamase; Repressor; Regulation; (Bacillus licheniformis, Escherlchia co/i)