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R S Haun

Publications and source records attributed to R S Haun.

At least 19 recordsLinked to original sources
PubMed1999

Cloning and characterization of the human ADP-ribosylation factor 4 gene.

ADP-ribosylation factor 4 (ARF4) is a member of a family of approximately 20 kDa guanine nucleotide-binding proteins that were initially identified by their ability to stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. They have recently been shown to play a role in vesicular trafficking and as activators of phospholipase D. The organization of the human ARF4 gene was determined from a genomic clone isolated from an arrayed PAC genomic library. The gene spans approximately 12 kb and contains six exons and five introns. Translation initiates in exon 1 and terminates in exon 6. Nuclease protection experiments indicated that the major transcription initiation site is located 211 bp 5' to the start of translation. In some cell lines derived from human tissues, however, multiple initiation sites were observed. The proximal 5'-flanking region of the human ARF4 gene lacks a TATA box, is highly GC rich, and contains multiple potential Spl-binding sites. An alignment of the exons for the class I ARF genes (ARF1, ARF2, and ARF3) and class II ARF genes (ARF4 and ARF5) reveals that the members of each class share a common gene organization. The structures of the class I and II ARF genes, however, are quite distinct and support the division of the ARFs into these groups based on deduced amino acid sequence, protein size, phylogenetic analysis, and gene structure.

3' Untranslated Regions↗
PubMed1999

Transcriptional regulation of the human ADP-ribosylation factor 5 (ARF5) gene.

Hybridization of a blot containing 50 human RNAs with an ADP-ribosylation factor 5-specific (ARF5) oligonucleotide probe revealed that the ARF5 gene is expressed in all tissues; however, the level of expression varies significantly with highest levels in pancreas, pituitary gland, and placenta. The 5'-flanking region of the human ARF5 gene lacks a TATA or CAAT box and is highly GC-rich. Primer extension analysis indicates that transcription initiates at a discrete site 62 bp 5' to the start of translation; however, the sequence surrounding the transcription initiation site does not resemble the initiator elements described for other TATA-less genes. Transient transfection of ARF5/luciferase deletion constructs into human IMR-32 neuroblastoma cells revealed that sequences within 169 bp of the transcription initiation site were necessary for full expression. Two GC boxes within this region were modified by site-directed mutagenesis and found to be critical for expression of the reporter constructs. Electrophoretic mobility-shift assays demonstrated specific DNA/protein complexes could be formed with oligonucleotides containing each of the GC boxes and these complexes could be effectively competed by oligonucleotides containing either ARF5 Sp1 site or by an oligonucleotide containing a previously characterized Sp1-binding sequence. The level of ARF5 gene expression, therefore, is dependent upon Sp1 or an Sp1-like factor but does not rely upon a canonical initiator element for accurate transcription initiation.

ADP-Ribosylation Factors↗
PubMed1994

Effect of ADP-ribosylation factor amino-terminal deletions on its GTP-dependent stimulation of cholera toxin activity.

It has been proposed that the amino-terminal domain of ADP-ribosylation factor (ARF) is critical for its stimulation of cholera toxin ADP-ribosyltransferase activity. In this study, recombinant ARF1 (rARF1), r delta 13ARF1 (recombinant ARF1 lacking the first 13 amino acids) and rPKA14ARF1 (recombinant ARF1 in which the first 14 amino acids were replaced by the first 7 amino acids of the cAMP-dependent protein kinase catalytic subunit) were used to assess the effect of the amino terminus on the ability of ARF to enhance ADP-ribosylation of agmatine by the cholera toxin A subunit. The GTP-dependent ARF activities of r delta 13ARF1 and rPKA14ARF1 were similar to that of rARF1, whereas the GTP requirement for half-maximal activation of cholera toxin A, was somewhat higher for rARF1 than it was for r delta 13ARF1 and rPKA14ARF1. These results are consistent with the view that the amino terminus of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin.

ADP-Ribosylation Factor 1↗
PubMed1993

Effects of brefeldin A and accessory proteins on association of ADP-ribosylation factors 1, 3, and 5 with Golgi.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. By cDNA and genomic cloning, at least six mammalian ARFs were identified. Brefeldin A (BFA) disrupts Golgi membranes and inhibits binding of soluble high molecular weight proteins to Golgi fractions. We examined the effects of BFA on binding of ARF1, -3, and -5 to a Golgi fraction in the presence of an ATP-regenerating system and a fraction of soluble, high molecular weight, accessory proteins (SAP), presumably containing complexes identified by others as coatomers that are involved in vesicular transport. ARF binding in all instances was dependent on guanosine 5'-O-(3-thiotriphosphate) and increased by the ATP-regenerating system. Binding of ARF1 and -3, but not ARF5, was enhanced by SAP. BFA inhibited the SAP-dependent, but not the SAP-independent, binding of ARF1 and -3. It had no effect on the increment in binding produced by an ATP-regenerating system. B36, an inactive derivative of BFA, did not inhibit SAP-dependent binding of ARF1 and -3. Binding of ARF5, which was SAP-independent, was not affected by BFA. These observations are consistent with the conclusion that mammalian ARFs differ in their dependence on accessory proteins for interaction with Golgi and, perhaps, other cellular membranes and that BFA specifically inhibits SAP-dependent ARF binding.

ADP-Ribosylation Factor 1↗
PubMed1993

Characterization of the human ADP-ribosylation factor 3 promoter. Transcriptional regulation of a TATA-less promoter.

The 5'-flanking region of the human ADP-ribosylation factor 3 gene contains the features of a housekeeping gene. It lacks a TATA or CAAT box, has several GC boxes within a highly GC-rich region, and utilizes multiple transcription initiation sites. The cis-acting elements involved in regulating expression of the gene were identified by transient transfections of IMR-32 neuroblastoma cells. Reporter plasmids were modified to facilitate construction of defined promoter deletions linked to chloramphenicol acetyltransferase or luciferase using ligation-independent cloning. Transfection analyses indicated that sequences within 58 base pairs of the transcription initiation site were necessary for full expression, in particular a sequence containing the 10-base pair palindrome TCTCGCGAGA. Electrophoretic mobility shift assays performed with IMR-32 nuclear extracts demonstrated that a DNA-binding protein, termed TLTF, bound to an oligonucleotide containing this palindrome. Competition experiments showed that mutations within the core of the palindrome abolished in vitro binding and that the same protein bound to a 5'-proximal sequence. Expression of the promoter containing a mutated palindrome was reduced dramatically, consistent with the conclusion that this region functions in vivo to control expression of the ARF3 gene.

ADP-Ribosylation Factors↗
PubMed1993

Effect of myristoylation on GTP-dependent binding of ADP-ribosylation factor to Golgi.

ADP-ribosylation factors (ARFs), a family of approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro, have been implicated in intracellular protein trafficking and are thought to cycle between cytosolic and membrane compartments. Although isolated predominantly as soluble proteins, ARFs associate with membranes and phospholipids in a GTP-dependent manner. In contrast to other small GTP-binding proteins, ARFs are NH2 terminally myristoylated. Using a bacterial expression system, recombinant myristoylated and non-myristoylated human ARF5 were produced to investigate the role of myristoylation in its association with Golgi. The recombinant ARFs (myristoylated and non-myristoylated) exhibited similar biochemical activity as measured by GTP binding and in vitro activation of cholera toxin. Myristoylated ARF5, however, demonstrated a temperature- and GTP-dependent association with Golgi membranes, whereas non-myristoylated ARF did not bind to Golgi under any of the experimental conditions. These data indicate that myristoylation is necessary, although not sufficient, for membrane attachment, but is not necessary for activation of cholera toxin.

ADP-Ribosylation Factors↗
PubMed1992

Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro.

ADP-Ribosylation Factors↗
PubMed1992

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.

ADP-Ribosylation Factor 6↗
PubMed1992

Characterization of the human gene encoding ADP-ribosylation factor 1, a guanine nucleotide-binding activator of cholera toxin.

Mammalian ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin ADP-ribosyltransferase activity, were grouped into three classes based on deduced amino acid sequence. Human ARF 1, a class I ARF, is identical with its bovine counterpart, has a distinctive pattern of tissue and developmental expression, and is encoded by a approximately 1.9-kilobase mRNA. ARF 1 cDNAs were isolated from a human fibroblast cDNA library; one arose via an alternative polyadenylation signal (AA-TACA) 84 nucleotides 5' to the polyadenylation signal (AATAAA) used in the 1815-base pair cDNA. The polyadenylation signals, their respective locations, and the surrounding nucleotide sequences are conserved in human and rat. The human ARF 1 gene, with four introns, spans approximately 16.5 kilobases. Exon 1 (46 base pairs) contains only untranslated sequence. Translation initiates in exon 2, which encodes the sequence GXXXXGK involved in phosphate binding (GTP hydrolysis). The sequence DVGG is encoded in exon 3, and NKQD, which is involved in the interaction with the guanine ring, is interrupted following the codon for Q by intron 4. The carboxyl-terminal 53 amino acids and greater than 1110 base pairs of 3'-untranslated region are encoded in exon 5. Primer extension and mung bean and S1 nuclease mapping indicated multiple transcription initiation sites and were consistent with Northern analyses. The 5'-flanking region has a high GC content but no TATA or CAAT box, as found in housekeeping genes. In addition, the two human class I ARF genes, ARF 1 and ARF 3, have similar exon/intron organizations and use GC-rich promoters.

ADP-Ribosylation Factors↗
PubMed1992

Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli.

A plasmid vector has been constructed that allows the ligation-independent cloning of cDNAs in any reading frame and directs their synthesis in Escherichia coli as glutathione S-transferase-linked fusion proteins. The cloning procedure does not require restriction enzyme digestion of the target sequence and does not introduce any additional sequences between the thrombin cleavage site and the foreign protein. Extended single-stranded tails complementary between the vector and insert, generated by the (3'----5') exonuclease activity of T4 DNA polymerase, obviate the need for in vitro ligation prior to bacterial transformation. This cloning procedure is rapid and highly efficient, and has been used successfully to construct a series of fusion proteins to investigate the sequence requirements for efficient thrombin cleavage.

ADP-Ribosylation Factors↗
PubMed1991

Isolation and characterization of the human gene for ADP-ribosylation factor 3, a 20-kDa guanine nucleotide-binding protein activator of cholera toxin.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. Five different human ARFs have been identified by cDNA cloning. Northern analysis using ARF 3-specific oligonucleotides identified two mRNAs of 3.7 and 1.2 kilobases (kb). We report here the complete nucleotide sequence of the 3.7-kb ARF 3 mRNA derived from three overlapping cDNAs isolated from human hippocampus and fetal brain cDNA libraries, as well as the structure of human ARF 3 gene. Sequences of two overlapping genomic clones indicated that the ARF 3 gene spans approximately 18.3 kb and contains five exons and four introns. The conserved amino acid sequences involved in guanine nucleotide binding by ARF 3 are distributed among separate exons, as found in other GTP-binding protein genes. Translation initiates in exon 2 which includes the sequence GXXXXGK that probably participates in phosphate binding and GTP hydrolysis. The sequence DVGG in exon 3 coordinates binding of Mg2+ and the beta-phosphate of GDP. In the ARF 3 gene in contrast to those of other GTP-binding proteins, the sequence NKXD (which is thought to contribute to the specificity of interaction with the guanine ring) is divided between exons 4 and 5. The latter encodes the COOH-terminal 53 amino acids of ARF 3 and contains greater than 2500 base pairs of untranslated DNA. The sequence AATTAA is 19 bases 5' to the polyadenylation addition site of the 3.7-kb mRNA. Multiple transcription start sites were identified by primer extension and S1 and mung bean nuclease analyses. The 5'-flanking region of exon 1 contains neither a TATA nor a CAAT box, but is high in GC content (greater than 70%) and includes three potential Sp1-binding sites (GC box), consistent with the promoters described for several housekeeping genes. The 1.2-kb ARF 3 mRNA is shown to arise by use of an alternative polyadenylation signal (AACAAA) at nucleotide 1091 within the ARF 3 cDNA.

ADP-Ribosylation Factors↗
PubMed1991

Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase.

This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.

Amino Acid Sequence↗
PubMed1990

A transcriptional enhancer essential for the expression of the rat cholecystokinin gene contains a sequence identical to the -296 element of the human c-fos gene.

We report the identification and characterization of the cis-acting elements responsible for the expression of the rat cholecystokinin (CCK) gene. Deletion mutations were constructed by linking variable amounts of the 5'-flanking region of the CCK gene to the bacterial chloramphenicol acetyltransferase reporter gene. The transcriptional activity of the CCK promoter deletion constructs was measured by monitoring chloramphenicol acetyltransferase enzyme activity after transient transfections. It is shown that sequences within 102 base pairs of the cap site are required for the expression from this promoter. This region contains a sequence that is identical to the -296 element of the human c-fos gene and is homologous with the polyoma enhancer and the cAMP- and 12-O-tetradecanoylphorbol-13-acetate-responsive elements described for several genes. In addition, the -119 to -81 fragment of the CCK promoter contains a transcriptional enhancer that potentiates the transcription from the herpes simplex virus thymidine kinase promoter in a position- and orientation-independent manner. DNase I protection and gel retardation experiments indicated the ability of several trans-acting factors found in nuclear extracts to bind specifically to regions of the CCK promoter. In particular, two complexes formed adjacent to the CCK enhancer region. One complex, CCK-1a, formed with sequences 5' to the enhancer whereas the other complex, CCK-1b, formed with the sequences identified by DNase I footprinting, 3' to the enhancer. Oligonucleotide competition experiments indicated that these complexes are formed by the same transacting factor or factors with similar binding specificities.

Animals↗
PubMed1990

Cloning and expression of a protein-tyrosine-phosphatase.

A rat brain cDNA library was screened by using a mixture of oligonucleotides whose sequences were deduced from the amino acid sequence of a human placental protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) reported by Charbonneau et al. [Charbonneau, H., Tonks, N. K., Walsh, K. A. & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. USA 85, 7182-7186]. The isolated clones encode a PTPase of 432 amino acids having a mass of 49,679 daltons and showing 97% sequence identity to the corresponding 321 residues of the placental enzyme. The coding sequence of the PTPase was placed behind a bacteriophage T7 promoter and the protein was expressed in Escherichia coli. The recombinant protein had a molecular weight of 50,000 by SDS/PAGE analysis and showed an absolute specificity for phosphotyrosine-containing substrates. Northern analysis documented that there were two sizes of RNA, 4.3 and 2.0 kilobases, which encode the PTPase. Both transcripts were present in a number of tissues, and the smaller RNA appears to arise by the use of an alternative polyadenylylation signal. The PTPase was also localized by in situ hybridization in the rat central nervous system. A diffuse pattern of hybridization signal is seen in a number of brain areas, with the hippocampus showing the highest levels of mRNA. Sequences located at the C terminus of the rat brain PTPase contain possible sites for phosphorylation as well as signals which could serve for membrane attachment.

Animals↗
PubMed1989

Postnatal development of cholecystokinin-like immunoreactivity and its mRNA level in rat brain regions.

Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were very low at birth. Excluding the cerebellum, the levels in these regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗