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PubMed1993

Assembly of fibronectin molecules with mutations or deletions of the carboxyl-terminal type I modules.

Fibronectin is a large modular protein that is assembled into fibrils in a stepwise process that involves the binding of soluble fibronectin to the cell surface and formation of fibronectin multimers that are stabilized by disulfides. Fibronectin contains two types of disulfide-containing repeat modules, types I and II. The type I modules form units that mediate binding to assembly sites (I-1 through I-5), mediate binding to gelatin (I-6 through I-9 plus the type II modules), or have no known function other than fibrin binding (I-10 through I-12). All type I modules contain four cysteines that are disulfide-linked in a 1-3, 2-4 arrangement, except for I-12 that contains six cysteines disulfide-bonded in an unknown arrangement. I-12 contains the consensus sequence Cys-Xaa-Yaa-Cys found in a number of proteins involved in disulfide exchange reactions [Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237; Boniface, J. J., & Reichert, L. E., Jr. (1990) Science 247, 61]. We explored the role of I-12 and adjacent type I modules of fibronectin in matrix assembly. We generated mutant fibronectins in which the second and sixth or fifth and sixth cysteine residues in I-12 were changed to serines (CS mutants) or that contained deletions of the 12th (delta 12) or 10th through 12th (delta 10-12) type I modules. Expression of I-12 as a fusion protein with the gelatin binding part of fibronectin indicated that this module folds independently and that the most likely disulfide pairing is 1-4, 2-6, 3-5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2002

Testing for recombination in a short nuclear DNA sequence of the European meadow grasshopper, Chorthippus parallelus.

Single-copy nuclear DNA sequences have high potential as a source of genetic markers for population analyses. However, the difficulties that arise when haplotypes that are the product of recombinational rearrangements are present require additional consideration. Two statistical methods for identifying potential recombinants by detecting anomalies in the distribution of variable sites along sequences were used to screen sequences from a single-copy nuclear DNA fragment, cpnl-1, of the European meadow grasshopper (Chorthippus parallelus). Five of the 71 haplotypes in the cpnl-1 data set showed nonrandom distribution of polymorphic sites using both methods. The second method pinpointed an additional four haplotypes. Estimates of the rate of recombination in the entire data set were obtained using standard methods. It is concluded that cpnl-1 haplotypes have been involved in recombination or gene conversion events at a rate more than twice the mutation rate. This confirms that recombination and gene conversion are significant factors in the generation of haplotype variation in nuclear gene sequences. The cpnl-1 haplotypes identified by the tests were present only in populations that have had recent contact; the Balkan and Turkish refugial populations and their post-glacial colonies to the north. This is discussed in relation to the phylogenetic inferences drawn from the same data in a previous report.

Animals↗

PubMed1988

Structure and evolution of the Adh genes of Drosophila mojavensis.

The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.

Alcohol Dehydrogenase↗

PubMed2025

Methylome profiling of cell-free DNA during the early life course in (un)complicated pregnancies using MeD-seq: Protocol for a cohort study embedded in the prospective Rotterdam periconception cohort.

INTRODUCTION: Placental DNA methylation differences have been associated with timing in gestation and pregnancy complications. Maternal cell-free DNA (cfDNA) partly originates from the placenta and could enable the minimally invasive study of placental DNA methylation dynamics. We will for the first time longitudinally investigate cfDNA methylation during pregnancy by using Methylated DNA Sequencing (MeD-seq), which is compatible with low cfDNA levels and has an extensive genome-wide coverage. We aim to investigate DNA methylation in placental tissues and cfDNA during different trimesters in uncomplicated pregnancies, and in pregnancies with placental-related complications, including preeclampsia and fetal growth restriction. Identified gestational-age and disease-specific differentially methylated regions (DMRs) could lead to numerous applications including biomarker development. METHODS AND ANALYSIS: Our study design involves three sub-studies. Sub-study 1 is a single-centre prospective, observational subcohort embedded within the Rotterdam Periconception cohort (Predict study). We will longitudinally collect maternal plasma in each trimester and during delivery, and sample postpartum placentas (n = 300). In sub-study 2, we will prospectively collect first and second trimester placental tissues (n = 10 per trimester). In sub-study 3 we will retrospectively collect plasma after non-invasive prenatal testing (NIPT) in an independent validation case-control cohort (n = 30-60). A methylation-dependent restriction enzyme (LpnPI) will be used to generate DNA fragments followed by sequencing on the Illumina NextSeq2000 platform. DMRs will be identified in placental tissues and cell types, and in cfDNA related to gestational-age or placental-related complications. (Paired) placental methylation profiles will be correlated to DMRs in cfDNA to aid tissue-of-origin analysis. We will establish a methylation score to predict associated diseases. DISCUSSION: This study will provide insights in placental DNA methylation dynamics in health and disease, and could lead to clinical relevant biomarkers.

Humans↗

PubMed1982

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.

Animals↗

PubMed1991

The C-terminal domain of RNA polymerase II of the malaria parasite Plasmodium berghei.

The C-terminal domain (CTD) of RNA polymerase II (RNAP) has an essential function in the regulation of transcription. The CTD of the human malaria parasite, Plasmodium falciparum, differs dramatically from that of higher eukaryotes. To determine whether this is a general feature of malarial parasites, we have analysed the CTD of the distantly related rodent malaria parasite P.berghei. The CTDs of the two parasites enzymes are very similar in amino acid composition and contain the basic structure of most eukaryotic CTDs, which is a tandem repeat of a heptapeptide (SPTSPSY). The CTD of P.berghei differs, however, in three aspects from the CTD of P.falciparum and other eukaryotes. First, both domains show a divergence from the consensus sequence at position 6 of the heptapeptide repeat. The Ser6 is always substituted, with a bias for lysine. The latter substitution might increase the binding efficiency to the DNA template. Second, the rodent and human malarial CTDs contain a 3' extension of, respectively, 66 or 67 amino acid residues. This tail-piece is unique among eukaryotes. Third, the enlargement of the CTD of the human parasite by six heptapeptide repeats is most likely generated by a recent amplification of a specific repeat unit.

Amino Acid Sequence↗

PubMed2002

Study of the visual evoked magnetic field with the m-sequence technique.

PURPOSE: Multifocally stimulated visual evoked magnetic field (VEF) examination with an m-sequence technique (multifocal VEF; mVEF) was studied, and the neural generators at peaks of mVEF were estimated in the visual cortex. METHODS: Visual field stimulation was generated by a multifocal testing system with use of the m-sequence technique. The stimulation pattern covered a central area extending from 0.6 degrees to 10 degrees in radius outward from the center of four visual-field quadrants. The stimulation pattern was projected onto a screen by a liquid crystal projector. VEFs of 14 healthy adults were recorded with a 160-channel, whole-head-type magnetoencephalography (MEG) system. The output signals of 16 selected MEG sensors covering the occipital region were recorded for each subject with the multifocal testing system, and the second-order responses were calculated. The analyzed response data files were transferred to the MEG system, a single equivalent current dipole (ECD) was estimated to locate the neural generator, and the localization was superimposed onto the corresponding brain magnetic resonance image of the subject. RESULTS: mVEFs showed three peak waves (N75m, P100m, N145m) in 75% of the subjects and two peak waves (N75m, N145m) in 25%. (N, P and m denote negative, positive, and magnetic fields, respectively.) Latencies of the first and the last peak were similar between the two kinds of peak waves. ECD examination showed more than 97% of goodness of fit at all peaks, and the relation between EDCs and the stimulated visual field coincided with a retinotopic organization that fit a cruciform model in all subjects. ECD depths from the occipital pole were similar to the depth expected from the human linear cortical magnification factor model in all subjects. Main neural generators of all mVEF components (N75m, P100m, N145m) were shown in the striate cortex (V1). CONCLUSIONS: Testing the VEF with an m-sequence technique showed stable responses to simultaneous stimulation of four visual-field quadrants. Consistency of correlation of the estimated ECD with the known cortical organization of the primary visual cortex confirmed the reliability of this examination. The three mVEF peaks were thought to derive mainly from V1 activity.

Adult↗

PubMed1988

Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus.

The p53 gene is rearranged in a high proportion of erythroleukemic cell lines derived from the spleens of mice infected with Friend leukemia virus. These rearrangements result in either the synthesis of a truncated protein or the inactivation of the p53 gene. Here we have molecularly characterized the rearrangements in two murine erythroleukemic cell lines induced by Friend leukemia virus, DP20-1 and CB3, that contain a rearranged p53 gene and fail to express p53 protein. The rearrangement in the DP20-1 cell line is due to the insertion of Friend spleen focus-forming provirus (SFFV) in the 3' end of the p53 gene in intron sequences between exons 9 and 10. Transfection of molecular clones of this SFFV provirus into NIH3T3 cells results in the generation of infectious virus as determined by its ability, in the presence of helper virus, to induce rapid splenomegaly and polycythemia when injected into adult DBA/2J mice. Insertion of SFFV in DP20-1 cells resulted in the expression of an aberrant 2.9 kb RNA species. Analysis of a molecular clone of the rearranged p53 gene in a second cell line, CB3, revealed that the p53 gene in this clone has sustained a large deletion within the p53 gene resulting in the loss of coding sequences between exons 4 and 8. The 5' end of the deletion originates within exon 4 and extends 3' to within the eighth intron. The significance of these findings with regard to the multi-stage nature of Friend virus induced erythroleukemia is discussed.

Animals↗

PubMed2003

Versatility of selenium catalysis in PHGPx unraveled by LC/ESI-MS/MS.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx; EC 1.11.1.12), a broad-spectrum thiol-dependent peroxidase, deserves renewed interest as a regulatory factor in various signaling cascades and as a structural protein in sperm cells. We present a first attempt to identify catalytic intermediates and derivatives of the selenoprotein by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/ESI-MS/MS) and to explain observed specificities by molecular modeling. The ground state enzyme E proved to correspond to position 3-170 of the deduced porcine sequence with selenium being present as selenocysteine at position 46. The selenenic acid form, which is considered to be the first catalytic intermediate F formed by reaction with hydroperoxide, could not be identified. The second catalytic intermediate G was detected as Se-glutathionylated enzyme. This intermediate is generated in the reverse reaction where the active site selenol interacts with glutathione disulfide (GSSG). According to molecular models, specific binding of reduced glutathione (GSH) and of GSSG is inter alia facilitated by electrostatic attraction of Lys-48 and Lys-125. Polymerization of PHGPx is obtained under oxidizing conditions in the absence of low molecular weight thiols. Analysis of MS spectra revealed that the process is due to a selective reaction of Sec-46 with Cys-148' resulting in linear polymers representing dead-end intermediates (G'). FT Docking of PHGPx molecules allowed reactions of Sec-46 with either Cys-66', Cys-107', Cys-168' or Cys-148', the latter option being most likely as judged by the number of proposed intermediates with reasonable hydrogen bonds, interaction energies and interface areas. We conclude that the same catalytic principles, depending on the conditions, can drive the diverse actions of PHGPx, i.e. hydroperoxide reduction, GSSG reduction, S-derivatization and self-incorporation into biological structures.

Amino Acid Sequence↗

PubMed1997

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Amino Acid Sequence↗

PubMed1991

The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence.

Synthetic tandem repeats (STRs) of oligonucleotides have previously been shown to detect polymorphic loci in the human genome. Here, we report results from the use of three such probes to screen a human cosmid library. Nine of the 45 positive clones that were analyzed appear to contain highly polymorphic minisatellite or VNTR loci. The degree of enrichment for minisatellite sequences varied with the choice of STR: one provided a 15- to 20-fold enrichment (4 polymorphic loci among 10 clones), whereas 2 others gave a 3- to 5-fold enrichment (5 polymorphic probes in a total of 35 clones) compared to random screening. The 9 VNTR markers have been localized by linkage analysis in the CEPH panel and/or by in situ hybridization. Eight probes identify new loci, one of which maps to an interstitial region. One of the VNTR loci (identified by probe CEB1) was found to be hypermutable, with 52 mutation events identified among 310 children characterized in 40 CEPH families. The parental origin of the mutation could be identified in all instances, and only one mutation was found to be of maternal origin. The mutation rate in males was estimated to be approximately 15%. Segregation analysis of flanking markers suggests that mutations are not associated with crossing over. As the only previously described hypermutable minisatellite loci in humans have equal rates of male and female mutations, these observations establish that a second type of hypermutable minisatellite exists in the human genome. In neither case does the generation of new alleles appear to be associated with unequal crossing over.

Base Sequence↗

PubMed2000

Neuroendocrine secretory protein 55 (NESP55): alternative splicing onto transcripts of the GNAS gene and posttranslational processing of a maternally expressed protein.

Recent studies established a novel genomically imprinted gene located 45 kb upstream of the human GNAS1 locus. This locus encoded for the Neuroendocrine Secretory Protein with an apparent molecular weight of 55,000 (NESP55), which is transcribed exclusively from the maternal allele. We sequenced rat and human NESP55 and investigated tissue-specific splicing of its mRNA and posttranslational modifications of the protein in various tissues. Alternative mRNA splicing of NESP55 was analyzed by sequencing of cDNA clones, RT-PCR and Northern blotting. Two main splice variants, which were generated in a tissue-specific manner, were identified: The open reading frame encoding NESP55 was spliced onto exons 2-13 of Gsalpha in the adrenal medulla, pituitary and the brain. In addition, in the pituitary a second shorter, prominent mRNA transcript was generated by splicing of NESP55 onto exons 2, 3 and N1 of Gsalpha. Several of the cDNA clones isolated contained inverted repeats of 50-150 bp at their 5' or 3' termini, which might form hairpin stems and thus alter mRNA stability. The NESP55 open reading frame encoded a hydrophilic protein of 28,018 Da (human) and 29,218 Da (rat), respectively, which resembled the class of acidic, neuroendocrine secretory proteins collectively called chromogranins. NESP55 is highly conserved among mammalian species. It is posttranslationally acidified by the addition of keratan sulfate glycosaminoglycan chains and differentially processed by endopeptidases in various endocrine and neuronal tissues.

Alternative Splicing↗

PubMed2001

Somatic diversity of the immunoglobulin repertoire is controlled in an isotype-specific manner.

We have studied two aspects of the IgE immune response. First, we have compared the kinetics of the IgE response to the T cell-dependent antigen ph-Ox coupled to ovalbumin with that of the IgG1 response and we have assessed the quality of the IgE response. Second, we have studied the generation of somatic diversity, understood as the combined effect of somatic mutation and the selection of D(iversity) and J(oining) elements, in germinal center B cells at the molecular level, using the germ-line sequence of the prototype anti-ph-Ox heavy chain variable element V(H)Ox1 as reference. We evaluated sequences derived from mu-, gamma 1- and epsilon-variable elements and showed that somatic diversification was different for all isotypes studied. We further compared the IgE responses of wild-type mice with those of mice expressing a truncated cytoplasmic IgE tail (IgE(KVK Delta tail)). IgE(KVK Delta tail) mice showed a more diverse sequence pattern. We corroborated previous results suggesting that short CDR3 regions are indicative for high-affinity antibodies by measuring relative affinities of phage-expressed Fab fragments with prototype long and short CDR3 regions. Therefore, the composition of the antigen-receptor is responsible for the selection process and the expansion of antigen-specific cells, leading to an isotype-specific antibody repertoire.

Amino Acid Sequence↗

PubMed1994

T cell epitopes encompassing the mutational hot spot position 61 of p21 ras. Promiscuity in ras peptide binding to HLA.

Activated ras carry a point mutation either in codon 12, 13 or 61 which is tumor specific. Peptides derived from this oncoprotein are therefore potential tumor antigens. Essential for the feasibility of using ras-derived peptides in therapy of cancer is whether p21 ras-derived peptides can be processed, bind to human histocompatibility leukocyte antigen (HLA) and be recognized by T cells. Here we report the fine specificity and HLA restriction of several T lymphocyte clones (TLC) specific for a peptide which is derived from the second mutational hot spot in ras encoding residue 61. These TLC were generated from memory T cells present in the blood of a cancer patient and recognized a ras-derived peptide carrying Leu instead of Gln at residue 61. By sequencing of the T cell receptor (TcR) genes three sets of "sister" TLC carrying highly different TcR were identified. Two of the TLC recognized a peptide carrying the 61 Leu mutation presented by HLA-DQ8 and one recognized the same peptide presented by HLA-DQ4. By using truncated peptides derived from residues 51 to 69 of p21 ras, partially overlapping minimal epitopes could be defined. All three TLC recognized the corresponding recombinant mutant p21 ras oncoprotein carrying Leu at residue 61 presented by autologous B-lymphoblastoid cell lines (B-LCL). This demonstrates that naturally derived ras peptides from this region of p21 ras encompass the three epitopes recognized by the TLC. These results indicate that immunogenic ras-derived peptides may be used in immunotherapy of cancer where transforming ras oncoproteins are involved.

Amino Acid Sequence↗

PubMed1993

Structural heterogeneity of Caucasian N-acetyltransferase at the NAT1 gene locus.

The human N-acetylation polymorphism is a genetic trait phenotypically reflected by differences in N-acetyltransferase (NAT) activity with therapeutic agents (rapid and slow acetylation), but a genetic invariability in N-acetylation of some arylamine drugs is also known. There are two highly similar human NAT genes: NAT1 is thought to encode a genetically invariant protein, whereas NAT2 has conclusively been shown to represent a polymorphic locus. This study demonstrates the presence of discrete NAT1 structural variants among Caucasians. These were detected by direct sequencing of 1.6-kilobase NAT1 fragments generated by the polymerase chain reaction with liver and leukocyte DNA from 13 subjects of established acetylator phenotype and NAT2 genotype. A prominent alteration in one of the variants was obliteration of the consensus polyadenylation signal (AATAAA-->AAAAAA). Several mutations were discernible in all regions of the second variant allele, including silent (codon 153) and nonsilent (Ser-214-->Ala) substitutions in the coding region and deletion of nine bases from an AT-rich segment in the 3' untranslated region. One-half of the unrelated subjects were either homozygous or heterozygous for the mutant NAT1 alleles, both of which obeyed a Mendelian inheritance pattern. These novel results unambiguously show that human NAT1, like NAT2, is a polymorphic locus.

Acetylation↗

PubMed1993

ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity.

The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTP gamma S-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.

ADP-Ribosylation Factor 1↗

PubMed1985

DNA ligase-AMP adducts: identification of yeast DNA ligase polypeptides.

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.

Adenosine Monophosphate↗

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↗