A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase.

Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids. A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed. The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities. The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases. We propose that this deletion is directly responsible for the anomalous behavior of this enzyme. Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases.

A short synthetic adaptor as second-strand primer in the construction of cDNA libraries by the vector-primer method.

The construction of high quality cDNA libraries is one of the most important yet technically challenging procedures in the study of gene structure and function. The present report presents a simplification of the classical Okayama-Berg protocol for the construction of plasmid libraries. The introduction of a short synthetic oligonucleotide as a second-strand adaptor facilitates the optimization of library construction and allows for quick adaptation of almost any vector as a cDNA cloning vehicle.

A developmentally regulated trans-acting factor recognizes dissimilar G/C-rich elements controlling a class of cAMP-inducible Dictyostelium genes.

Transcriptional response elements involved in the cAMP-inducible and developmentally regulated expression of the Dictyostelium aggregate-stage gene pst-cath/CP2 have been shown to include a G/C-rich sequence element [G-box regulatory element (GBRE)]. We have recently identified a trans-acting factor, GBF (GBRE binding factor), that specifically interacts with this sequence and have shown that the binding activity of GBF to GBRE is developmentally regulated and inducible by cAMP. Here, we examine further the possible role of GBF in the regulation of pst-cath/CP2 and three other coordinately regulated, cAMP-inducible aggregate-stage genes. We show that GBF itself (or other closely related factors) recognizes dissimilar G/C-rich elements present in the 5'-flanking regions of these genes and that the ability of the individual, distinct G/C-rich elements to confer regulated expression on a promoter deletion mutant of the pst-cath/CP2 gene is correlated with the relative affinity for GBF. G/C-rich elements carrying point mutations that prevent in vitro binding of GBF to two of the G/C-rich elements fail to activate expression in vivo. An analysis of major points of contact between the GBF protein and two distinctly different binding sites suggests that binding of GBF to these sequence elements involves a considerable degree of flexibility in DNA-protein interactions. These results suggest that the regulated expression of a class of aggregate-stage cAMP-inducible genes involves the interaction of GBF or homologous factors with dissimilar G/C-rich sequence elements and that induction of GBF activity or that of homologous factors by cAMP may thus be a limiting step in the induction of this temporally coordinate set of genes during Dictyostelium development.

A trans-acting factor required for cAMP-induced gene expression in Dictyostelium is regulated developmentally and induced by cAMP.

We have identified a nuclear activity that binds specifically to a GT-rich sequence or G-box shown previously by use of deletion analysis to be required for cAMP and for developmentally induced expression of the prestalk gene pst-cathepsin (CP2). We show that the insertion of an oligonucleotide that contains the CP2 G-box restores regulated expression whereas the insertion of oligonucleotides that contain mutations in some of the G residues does not. Moreover, the mutant oligonucleotides do not compete for binding of the factor to the wild-type sequence. The activity of the G-box binding factor (GBF) is regulated developmentally with induction of activity occurring at the time of induction of pst-cathepsin expression. In a single-cell culture, GBF activity is inducible by cAMP, and its appearance is inhibited by cycloheximide, which suggests that the factor, or a protein component required for binding of the factor, is directly induced by cAMP and may be the rate-limiting factor required for cAMP induction of pst-cathepsin expression. Models for cAMP induction of prestalk genes are described.

Analysis of cis and trans elements involved in cAMP-inducible gene expression in Dictyostelium discoideum.

Expression of the Dictyostelium discoideum pst-cath (CP2) gene is transcriptionally regulated during multicellular development, and the gene is inducible in competent single cells following administration of exogenous cAMP. The 5' flanking region of pst-cath (CP2) that extends from -313 to the Cap site (+1) has previously been shown to contain sufficient cis-acting regulatory elements for proper developmental and cAMP-inducible expression of a foreign gene [Datta and Firtel, 1987, Mol Cell Biol 7:149-159]. The -283 to -201 region includes two exceptional "G-boxes" centered at -233 and -217 respectively, and this approximately 80 bp region is essential for basal as well as regulated expression of the pst-cath (CP2) gene. Here we summarize results obtained from a detailed analysis of a series of linker-scanner mutants and mutants that carry small internal deletions within the essential 80-bp region. Insertion of a synthetic oligonucleotide that includes the downstream G-box is demonstrated to rescue a low level of cAMP-inducible expression following insertion into cassette mutants. The effect of introducing a change in the relative spacing between regulatory elements has also been investigated. We have analyzed nuclear extracts for the presence of DNA-binding proteins that interact specifically with the pst-cath (CP2) regulatory region and identified two such putative trans-acting factors: 1) the AT-factor that is observed within a few hours following the onset of starvation and that binds tightly to stretches of alternating adenine-thymine residues (poly(dA-dT]; and 2) the AG-factor that is present in nuclear extracts of aggregated cells. Competition studies have demonstrated significant differences in the affinity that characterizes the binding of the two factors to G-box-containing sequences. The binding specificities of these DNA-binding proteins have been analyzed using gel mobility-shift and DNaseI footprinting assays.

Pseudomonas cepacia in 16 non-fatal cases of postoperative bacteremia derived from intrinsic contamination of the anaesthetic fentanyl. Clinical and epidemiological observations in Denmark and Holland.

From Nov. 15 to Dec. 17, 1977, Pseudomonas cepacia was isolated from the blood of 16 patients in Odense, Denmark, and Nijmegen, Holland, 2--5 days after an operation with general anaesthesia. The fever started 14--70 h after operation and lasted 2--4 days. All patients recovered. 14/15 patients examined 7--51 days later had agglutinating antibody titres of 400-3,200 against the epidemic strain. Ps. cepacia with identical biochemical characters and sensitivity pattern was isolated from unbroken vials containing the anaesthetic fentanyl, which had been given to all 16 patients. Two batches were contaminated, one heavily so (10(4)--10(5) cfu/0.1 ml). Seven other batches examined yielded no growth. The preservative added to the vials was a mixture of methyl- and propyl-p-hydroxybenzoates which not only allowed growth of the Ps. cepacia strain but could also serve as a carbon source as did citric and malonic acids. The concentration of preservative was not reduced in contaminated vials. The vials had not been sterilized after closure; too much reliance had been placed on an aseptic technique and insufficient preservatives.