Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Characterization of Gilbert-like syndrome in squirrel monkeys (Saimiri sciureus)

Bolivian squirrel monkeys, unlike those of Brazilian origin, exhibit a marked fasting hyperbilirubinemia (FH) similar to that observed in Gilbert's syndrome in man. Since no delays in the hepatic clearance of sulfobromophthalein or indocyanine green are present, the Bolivian monkey appears to be similar to Gilbert's type I syndrome. FH can be significantly decreased by either phenobarbital or tin-protoporphyrin pretreatment. Nicotinic acid-induced hyperbilirubinemia and delayed tolbutamide clearance were not observed as in the human syndrome.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module

We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig. 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.