Biomedical subjects

R K Pearson

Publications and source records attributed to R K Pearson.

11 recordsLinked to original sources

PubMed1992

The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors.

The sloppy paired locus is involved in the establishment of the metameric body plan of the Drosophila embryo. We have cloned the sloppy paired locus by P-element-mediated enhancer detection. The locus is composed of two genes, slp1 and slp2, that are structurally and functionally related. They belong to a novel class of putative transcription factors containing a fork head domain that has also been found in mammalian hepatocyte transcription factors. The spatial expression patterns of the two transcripts are very similar, suggesting common regulation of the two genes. We recovered additional sloppy paired alleles by remobilization of an enhancer detector transposon. Genetic analysis suggests that both genes contribute to the segmentation phenotype that has characteristics of both, pair-rule and segment polarity genes, and that they interact functionally. The two genes appear to share an enhancer element situated upstream of slp1 that acts on both the proximal slp1 promoter and the distal slp2 promoter.

Amino Acid Sequence↗

PubMed1992

Expression of the rat carboxypeptidase-E gene in neuroendocrine and nonneuroendocrine cell lines.

To identify cis-acting elements involved with the expression of the rat carboxypeptidase-E (CPE) gene, constructs containing various regions of the 5'-flanking region of the CPE gene attached to the luciferase reporter gene were transiently expressed in cell lines derived from pituitary (AtT-20 and GH4C1), liver (SK-HEP-1), and kidney (HEK293 and COS1). Regions of the CPE gene spanning the major transcription initiation site (-12 to 47) are sufficient for low levels of transcription. Activity is enhanced 3- to 15-fold by sequences present between -12 and -395 in all cell lines examined. Sequences between -395 and -3081 influenced transcription activity up to 5-fold in some, but not all, cell lines. There was no correlation between the transcription activities of the various constructs and the level of endogenous CPE mRNA in the cell lines, indicating that the tissue-specific elements responsible for the large variations in endogenous CPE mRNA levels are not present within -3081 to 47. The region between -395 and 45 was examined in greater detail using transient expression assays and DNase-I protection analysis. Transcription activity is enhanced in GH4C1 and HEK293 cells by sequence present between -12 and -84; this region contains a potential GC box, which binds factors present in GH4C1 nuclear extracts. Other regions between -340 and 80 that bind proteins in the GH4C1 nuclear extracts include the major transcription initiation site, which has homology to the initiator sequence; the pituitary-specific transcription initiation sites (-101 and -105); and sequences with homology to NF-1, Pan-1, simian virus-40 enhancer core, and AP-2-binding sites. Taken together, these results suggest that basal expression of the CPE gene from its major transcription initiation site, which does not contain an up-stream TATA box, is primarily under the control of an initiator-like element together with an upstream GC box.

Animals↗

PubMed1991

Structural characterization of the rat carboxypeptidase-E gene.

Several genomic clones encoding carboxypeptidase-E (CPE) have been isolated and partially sequenced. Southern blot analysis indicates that a single copy of this gene is present in the rat genome. The entire gene spans approximately 50 kilobases and consists of nine exons, each of which contains protein-coding regions. Only one of the exon/intron junctions of the rat CPE gene is present in a comparable position within the genes for carboxypeptidase-A and -B, both of which are only 17-21% homologous to CPE at the amino acid level. Nuclease protection analysis shows that alternative splicing of exons 7, 8, and 9 does not occur, indicating that the heterogeneity of the C-terminal region of CPE is due to posttranslational processing. Primer extension and nuclease protection analyses have identified the 5' end of CPE mRNA to be 105 nucleotides up-stream from the ATG used for protein translation. The 5' flanking region does not contain TATA and/or CCAAT boxes in the near vicinity of the transcription initiation site. The 5' flanking region is GC rich, containing 70% GC residues over nucleotides -1 to -150 (relative to the transcription initiation site). Putative consensus sites for the enhancer elements SP-1, NF-1, Pan-1, and AP-2 are present in the region from -60 to -330. Since this report describes the first neuropeptide-processing enzyme gene to be partially sequenced, it is not possible to compare the sequence with those of other processing enzymes that show similar tissue-specific expression. However, comparison of the CPE sequence with 5' flanking regions of other neuroendocrine genes has revealed a short region (12-18 nucleotides) that is highly conserved among CPE, neuropeptide-Y, oxytocin, insulin, and tyrosine hydroxylase genes.

Amino Acid Sequence↗

PubMed1990

P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila.

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.

Animals↗

PubMed1989

P-element-mediated enhancer detection: a versatile method to study development in Drosophila.

We generated and characterized greater than 500 Drosophila strains that carry single copies of a novel P-element enhancer detector. In the majority of the strains, the beta-galactosidase reporter gene in the P-transposon responds to nearby transcriptional regulatory sequences in the genome. A remarkable diversity of spatially and temporally regulated staining patterns is observed in embryos carrying different insertions. We selected numerous strains as markers for different embryonic organs, tissues, and cells. Many of these strains should allow the study of complex developmental processes, such as nervous system development, which have not been convenient to analyze previously. Also, we present genetic evidence that some of the detected regulatory elements control nearby Drosophila genes. In light of our results, we discuss the diversity and complexity of cis-acting regulatory elements in the genome and the general applications of the enhancer detector method for the study of Drosophila development.

Animals↗

PubMed1989

P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila.

We describe a new approach for identifying and studying genes involved in Drosophila development. Single copies of an enhancer detector transposon, P[1ArB], have been introduced into flies at many different genomic locations. The beta-galactosidase reporter gene in this construct is influenced by a wide range of genomic transcriptional regulatory elements in its vicinity. Our results suggest that a significant proportion of these regulatory sequences are control elements of nearby Drosophila genes. These genes need not be disrupted for their regulatory elements to be identified by P[1ArB]. The P[1ArB] transposon has been designed to facilitate both rapid cloning and deletion analysis of genomic sequences into which it inserts. Therefore, the enhancer detection system is an efficient method of screening for genes primarily on the basis of their expression pattern and then rapidly analyzing those of particular interest at the molecular and genetic levels.

Animals↗

PubMed1989

Structural analysis of a distinct subtype of CCK receptor on human gastric smooth muscle tumors.

Human gastric smooth muscle tumors (leiomyosarcomas) have been shown to express cholecystokinin (CCK) binding sites that are functionally similar to physiologically important receptors present on their cells of origin. In this work, we have applied affinity-labeling techniques using 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) to attempt to define the ligand-binding subunit of this receptor, and we have used the receptor antagonist L364,718 and deglycosylating enzymes to compare this molecule with well-defined CCK receptors on the classical peripheral targets (pancreas and gallbladder) of this hormone. To validate the use of 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) for this tissue, we demonstrated that it bound to leiomyosarcoma membranes in a rapid, reversible, saturable, specific, and high-affinity manner (Kd = 0.8 nM). Although previous affinity labeling of this tissue with a CCK-33-based probe identified multiple bands, only one of those candidate proteins was predominantly labeled in the present work (Mr 100,000) by using a probe that is cross-linked through a site in greater proximity to the receptor-binding domain. Labeling was inhibited in a concentration-dependent manner by CCK-8 but not by structurally unrelated ligands. Although endo-beta-N-acetyl-glucosaminidase F digestion shifted this band by Mr 5,000, demonstrating that it was a glycoprotein, the deglycosylation product was very different from other CCK receptors studied. Also, unlike pancreatic and gallbladder CCK receptors, affinity labeling of this receptor was not affected by L364,718. These observations confirm that the gastric smooth muscle tumor CCK receptor represents a receptor subtype that is distinct from other peripheral CCK receptors, biochemically as well as functionally.

Adult↗

PubMed1987

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor.

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking, and rendered the peptide resistant to aminopeptidases. The decapeptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr = 85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.

Affinity Labels↗

PubMed1987

Fatal soft-tissue infection complicating pulmonary arteriovenous fistulae.

A patient with pulmonary arteriovenous (AV) fistulae presented with an ultimately fatal anaerobic soft tissue infection. This represents the first report of such a complication, but the association of intracerebral anaerobic abscess with pulmonary AV fistulae suggests the possibility of a common pathophysiologic mechanism.

Abscess↗

PubMed1987

Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes.

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[125I]iodobenzimidate and N-[4-(4'-azido-3'-[125I]iodophenylazo)benzoyl]-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2,000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using 125I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone.

Affinity Labels↗

PubMed1986

Preparation and characterization of a new cholecystokinin receptor probe that can be oxidatively radioiodinated.

Oxidative iodination is the simplest, most efficient, and least expensive method for radiolabeling peptide hormones. It has not been applied to cholecystokinin (CCK), however, because this peptide lacks an unsubstituted tyrosine residue and oxidation abolishes its biologic activity. We report the synthesis, purification, and characterization of the first derivative of CCK that can be radioiodinated with an oxidative method while maintaining full biologic activity and receptor binding affinity. Boc-Tyr-[(Thr28, Nle31)CCK-25-33] was synthesized, iodinated using a solid-phase oxidant, and purified on reverse-phase high-pressure liquid chromatography to a specific activity of 2125 Ci/mmol. Both native and iodinated Boc-Tyr-[(Thr28, Nle31)CCK-25-33] exhibited efficacy and potency for stimulation of in vitro pancreatic enzyme secretion that were identical to CCK-8. Binding of Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] to rat pancreatic plasma membranes was rapid, reversible, temperature-dependent, saturable, and specific. The abilities of various molecular forms of CCK to compete for this binding paralleled their potencies for stimulation of pancreatic secretion (Kd: CCK-8, 0.8 nM; CCK-33, 3 nM; CCK-8DS, 1 microM; CCK-4, 50 microM), whereas structurally unrelated pancreatic ligands (1 microM) demonstrated no significant competition. These findings suggest that Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] interacts with the same high-affinity pancreatic receptor as the CCK receptor probes previously reported, i.e., the Bolton-Hunter-labeled CCK-33 and CCK-8. This probe also possesses the major advantages of simplicity, efficiency, and cost of the labeling procedure, and the stability and relative oxidation-resistance of the product. Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] will be useful for the characterization of binding interactions between CCK and its target tissues.

Animals↗