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D J Bergsma

Publications and source records attributed to D J Bergsma.

At least 55 records · Page 3Linked to original sources
PubMed1991

The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms.

Cyclophilin (CyP), a major cytosolic protein possessing peptidyl-prolyl cis-trans isomerase activity, has been implicated as the specific receptor of the immunosuppressive drug cyclosporin A (CsA). To identify other potential CsA receptors related to CyP, two human cDNA libraries were screened under low stringency conditions using human CyP cDNA (encoding hCyP1) as a probe. Two cDNAs were identified which encode distinct proteins related to human hCyP1. These two novel proteins, designated hCyP2 and hCyP3, share 65 and 76% amino acid sequence homology with hCyP1, respectively. Both hCyP2 and hCyP3 contain NH2-terminal hydrophobic extensions of 32 and 42 amino acids, respectively. Protein-specific antibodies revealed the predominant association of hCyP2 and hCyP3 with membranes and subcellular organelles, which suggests that the amino-terminal leader sequences of the two CyP isoforms may act as signal peptides. In contrast to the results with hCyP1, Southern blot analysis indicated that both hCyP2 and hCyP3 gene sequences are represented infrequently in the human genome. Northern and Western blot analysis showed that the distribution of mRNA and proteins of the three hCyPs in differing tissues and cell types was similar. Each hCyP protein was expressed in Escherichia coli, purified, and shown to be an active peptidyl-prolyl isomerase. Substrate specificity was examined with 11 synthetic peptides (Suc-Xaa-Yaa-Pro-Phe-4-nitroanilide), and inhibition of the peptidyl-prolyl isomerase activities associated with hCyP1, hCyP2, and hCyP3 was studied with CsA, MeAla6-CsA and MeBm2t1-CsA. From both equilibrium considerations and the results of kinetic characterizations it is proposed that of these three CyP proteins, hCyP1 is the most likely intracellular target for CsA.

Amino Acid Isomerases↗
PubMed1991

The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase with an N-terminal signal sequence.

Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene [Haendler et al., Gene 83 (1989) 39-46]. We recently identified a second Cyp-related gene in yeast, CYP2 [Koser et al., Nucleic Acids Res. 18 (1990) 1643] which predicts a protein with a hydrophobic leader sequence. A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli. The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA. The CYP2 gene is genetically unlinked to CYP1. As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability. Disruption of all three of the known yeast PPIase-encoding genes [CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence. Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity. This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast. Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.

Amino Acid Isomerases↗
PubMed1991

A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity.

Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA). To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E. coli. The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP. Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis. The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E. coli (Liu, J. & Walsh, C.T. (1990) Proc. Natl. Acad. Sci. USA 87, 4028-4032).

Alanine↗
PubMed1991

Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Amino Acid Isomerases↗
PubMed1990

A Candida albicans homolog of a human cyclophilin gene encodes a peptidyl-prolyl cis-trans isomerase.

A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.

Amino Acid Isomerases↗
PubMed1990

Analysis of a CR1 (chicken repeat) sequence flanking the 5' end of the gene encoding alpha-skeletal actin.

Genomic Southern blots of chicken DNA, probed with radiolabeled DNA fragments that flank the 5'-end of the asa gene encoding chicken alpha-skeletal actin, indicate the presence of repetitive nucleotide sequences. Sequence analysis of this region reveals a member of the CR1 family of middle-repetitive elements. A 186-bp restriction fragment carrying the 3'-end of this CR1 element binds factor(s) present in nuclear extracts, as assayed by band-shift electrophoresis. However, the CR1 repeat has limited influence on transcription from the alpha-skeletal actin promoter, as assessed by CAT assays of transfected chicken myoblasts and fibroblasts.

Actins↗
PubMed1989

Transient production and secretion of human transforming growth factor TGF-beta 2.

Transient transfection of simian COS cells with a recombinant plasmid encoding the human transforming growth factor TGF-beta 2 precursor protein results in the production of a latent, biologically inactive protein. Upon acidification, recombinant TGF-beta 2 exhibits full biological activity, including inhibition of mink lung epithelial cell growth, stimulation of anchorage-independent growth of murine embryonic fibroblasts, and competition for TGF-beta receptor binding. Further analysis of conditioned media with antiserum to either a pro- [amino acid (aa) residues 1-220] or mature [aa 297-414] peptide of the TGF-beta 2 precursor suggests that TGF-beta 2, similar to TGF-beta 1 production in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol. 7 (1987) 3418-3427], is initially synthesized as a larger precursor protein which is proteolytically cleaved to yield the mature 112-aa transforming growth factor.

Animals↗
PubMed1988

A 29-nucleotide DNA segment containing an evolutionarily conserved motif is required in cis for cell-type-restricted repression of the chicken alpha-smooth muscle actin gene core promoter.

A series of 5' deletion mutations of the upstream flanking sequences of the chicken alpha-smooth muscle (aortic) actin gene was prepared and inserted into the chloramphenicol acetyltransferase expression vector pSV0CAT. Deletion recombinants were transfected into fibroblasts, which actively express the alpha-smooth muscle actin gene, and primary myoblast cultures, which accumulate much lower quantities of alpha-smooth muscle actin mRNAs. The first 122 nucleotides of 5'-flanking DNA were found to contain a "core" promoter capable of accurately directing high levels of transcription in both fibroblasts and myotubes. The activity of this core promoter is modulated in fibroblasts by a "governor" element(s) located at least in part between nucleotides -257 and -123. This region contains sequences potentially conserved between mammalian and avian alpha-smooth muscle actin genes as well as one of a pair of 16-base-pair inverted CCAAT box-associated repeats which are conserved among all chordate muscle actin genes examined to date. A smaller DNA segment (-151 to -123) containing these upstream CCAAT box-associated repeats was sufficient to suppress expression of the core promoter in muscle cultures, suggesting that the upstream CCAAT box-associated repeats play a negative role in the alpha-smooth muscle actin gene promoter.

Actins↗
PubMed1986

Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs.

The alpha-smooth muscle (aortic) actin gene is a distinct member of the actin multigene family which is expressed in vascular smooth muscle cells. We have determined the complete nucleotide sequence of 11 kilobase pairs of genomic DNA encoding the chicken alpha-smooth muscle actin gene. This single copy gene specifies a protein identical in sequence to the major alpha-actin from bovine aorta. The protein-coding sequences are interrupted by seven introns which are at codons specifying amino acid residues 41/42, 84/85, 121/122, 150, 204, 267, and 327/328. An eighth intron was found in the mRNA 5' untranslated region. The 5' flanking sequences contain elements which are conserved in other chicken muscle actin genes. Additional sequences at the 5' end of the gene may be conserved in at least one human actin gene. We have identified at least four messenger RNAs ranging in size from approximately 1370 to 2700 nucleotides (excluding poly(A) tails) which are transcribed from the alpha-smooth muscle actin gene. These RNAs differ in the length of their 3' untranslated regions, probably as a result of the utilization of alternative polyadenylation signals. This is the first report of an actin gene with multiple mRNA transcripts.

Actins↗
PubMed1986

Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5' end of the chicken alpha-skeletal actin gene.

alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression.

Acetyltransferases↗
PubMed1986

Delimitation and characterization of cis-acting DNA sequences required for the regulated expression and transcriptional control of the chicken skeletal alpha-actin gene.

We have previously observed that DNA sequences within the 5'-flanking region of the chicken skeletal alpha-actin gene harbor a cis-acting regulatory element that influences cell type and developmental stage-specific expression (J. M. Grichnik, D. J. Bergsma, and R. J. Schwartz, Nucleic Acids Res 14:1683-1701, 1986). In this report we have constructed unidirectional 5'-deletion and region-specific deletion-insertion mutations of the chicken skeletal alpha-actin upstream region and inserted these into the chloramphenicol acetyltransferase expression vector pSV0CAT. These constructions were used to locate DNA sequences that are required for developmental modulation of expression when transfected into differentiating myoblasts. With this assay we have delimited the 5' boundary of a cis-acting regulatory element to ca. 200 base pairs upstream of the mRNA cap site. In addition, we have preliminarily identified DNA sequences that may be important subcomponents within this element. A second major focus of this study was to identify those DNA signals within the regulatory element that control transcription. Toward this end, the expression phenotypes of progressive 5'-deletion and deletion-insertion mutants of the 5'-flanking region of the chicken skeletal alpha-actin gene were assayed in microinjected Xenopus laevis oocytes. These experiments defined a cis-acting transcriptional control region having a 5' border 107 base pairs preceding the alpha-actin RNA cap site. Proximal and distal functionally important regions of DNA were identified within this element. These DNA signals included within their DNA sequences the "CCAAT" and "TATA" box homologies.

Actins↗
PubMed1985

Novel chicken actin gene: third cytoplasmic isoform.

We identified a novel chicken actin gene. The actin protein deduced from its nucleotide sequence very closely resembles the vertebrate cytoplasmic actins; accordingly, we classified this gene as a nonmuscle type. We adopted the convention for indicating the nonmuscle actins of the class Amphibia (Vandekerckhove et al., J. Mol. Biol. 152:413-426) and denoted this gene as type 5. RNA blot analysis demonstrated that the type 5 actin mRNA transcripts accumulate in adult tissues in a pattern indicative of a nonmuscle actin gene. Genomic DNA blots indicated that the type 5 actin is a single copy gene and a distinct member of the chicken actin multigene family. Inspection of the nucleotide sequence revealed many features that distinguished the type 5 gene from all other vertebrate actin genes examined to date. These unique characteristics include: (i) an initiation Met codon preceding an Ala codon, a feature previously known only in plant actins, (ii) a single intron within the 5' untranslated region, with no interruptions in the coding portion of the gene, and (iii) an atypical Goldberg-Hogness box (ATAGAA) preceding the mRNA initiation terminus. These unusual features have interesting implications for actin gene diversification during evolution.

Actins↗
PubMed1984

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Actins↗
PubMed1983

Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.

Animals↗