Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.     

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].     

Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators.

The 5' regulatory region (-345 to +1) of the rat insulin I gene (Ins-I) was examined for binding to cellular factors with short oligodeoxynucleotide probes. Over 40 binding species were detected. The binding profiles were specific for each cell type studied. We characterized the factors binding two elements crucial for enhancer activity (the Nir and Far boxes) which bear sequence similarity to the microE1, microE2, and microE3 elements of the immunoglobulin heavy-chain enhancer. The Nir box binds three cellular factors that display preferential affinities for microE1, microE2, or microE3, and the Far box binds two factors related to microE2 or microE3. The insulin gene enhancer was mutated at the Nir box element to reflect the sequences of microE1, microE2, or microE3. Ins-microE2 was fully active, Ins-microE3 was partially active, and Ins-microE1 was inactive. Thus, factors similar or identical to nuclear factor NF-microE1, NF-microE2, or NF-microE3 may play a role in the activity of the insulin gene enhancer.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family

Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.