Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis

The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.     

Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.     

Identification of amino acid residues in a class I ubiquitin-conjugating enzyme involved in determining specificity of conjugation of ubiquitin to proteins

The ubiquitin pathway is a major system for selective proteolysis in eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities.     

Identification of a novel gene encoding the yeast mitochondrial dicarboxylate transport protein via overexpression, purification, and characterization of its protein product

A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism. Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products. The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions. The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies. Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E. coli culture at a purity of 75%. The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e. Km = 1. 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined. The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported [14C]malonate uptake, whereas other anions tested did not. External substrate competition studies revealed a similar specificity profile. Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters. In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system. In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter. Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family. Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials.     

Isolation and characterization of a cDNA encoding Arabidopsis thaliana mevalonate kinase by genetic complementation in yeast

A 1.64-kb cDNA encoding an Arabidopsis thaliana mevalonate kinase (MK) was cloned by complementation of the erg 12-1 mutation affecting MK in the yeast Saccharomyces cerevisiae, and the nucleotide sequence was determined. The longest open reading frame encodes a protein of 378 amino acids (aa) with a predicted molecular mass of 40,650 Da. A striking feature of the cDNA sequence is a long 5' untranslated region (322 bp). The deduced aa sequence reveals that the plant enzyme shows strong similarities to the yeast and mammalian enzymes, especially the strong hydrophobicity percentage and several conserved regions. Southern analysis suggests that probably only one locus exists in the A. thaliana genome.     

Identification of a self-association region within the SCA1 gene product, ataxin-1

OBJECTIVES: To report the association between the protease inhibitor indinavir and the development of urolithiasis. METHODS: Case reports of three adult patients infected with the human immunodeficiency virus who developed surgical renal stones while being treated with indinavir are presented. RESULTS: Of the 3 patients requiring surgical intervention, stone analyses were available in 2. One stone revealed an inner core of an unidentifiable crystal surrounded by calcium oxalate, and another was found to have indinavir components as determined by thin-layer chromatography and gas chromatography-mass spectrometry. Metabolic evaluation of all 3 patients identified significant hypocitraturia as an isolated finding. CONCLUSIONS: The widely used protease inhibitor indinavir is associated with the development of urolithiasis and may act as a nidus for heterogeneous nucleation leading to the development of mixed urinary stones. Surgical intervention may be necessary in some cases. Underlying metabolic abnormalities may contribute to the increased incidence of stone formation. Urologists and other health care providers should be aware of this association, as combined medical and surgical intervention may be necessary.     

Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.     

Identification and characterization of kraken, a gene encoding a putative hydrolytic enzyme in Drosophila melanogaster

BACKGROUND AND AIMS OF THE STUDY: The study aimed to determine the clinical performance of bovine pericardial aldehyde-treated products alone or in combination with aortic leaflets of porcine origin. These included a composite porcine stentless aortic valve attached to a scalloped pericardial tube (BSAV), and valved and non-valved bovine pericardial conduits for use in left-sided heart lesions (BPG). METHODS: For BSAV grafts, between January 1990 and August 1996, 163 patients (119 males) had their aortic valves replaced by SJM Biocor BASV. Mean age was 37.9 +/- 17.6 years (range: 1 to 76 years). Rheumatic heart disease sequelae (n = 72) and replacement of a prosthetic heart valve (n = 46) were predominant. Preoperative NYHA functional class showed 90 patients (55.2%) in class III and 50 (30.7%) in class IV. BPVC and NVPC grafts were used in 166 patients: acute aortic dissection was the main indication in 52 (31.3%) and chronic in 36 (21/7%). The ascending aorta was involved in 141 patients (84.9%); grafts were seldom used at other sites. In most patients the graft implanted was either a non-valved (n = 79) or a valved (n = 75) pericardial conduit. Twelve patients had a localized lesion and required a patch repair. RESULTS: For BASV grafts, the non-valve-related hospital mortality rate was 4.9%. There were 14.7% non-fatal complications with full recovery of all patients. Mean follow up in 141 patients was 3.0 +/- 1.4 years (range: 1 month to 7.2 years); 14 patients were lost to follow up. Late, non-conduit-related, mortality occurred in seven patients (4.9%). Eight patients underwent reoperation. The current clinical follow up of 127 patients has shown 118 (92.9%) with competent valves and nine (7.0%) with mild stable aortic insufficiency. For BPVC and NVPC grafts, hospital mortality rate was 16.9%, death being related to poor preoperative clinical condition. Postoperative follow up was accomplished in 125 patients; reoperation was necessary in seven patients. Histology showed good tissue preservation up to five years; echocardiography revealed satisfactory findings. No valved conduit had to be reoperated for valve or pericardial tissue wear. CONCLUSIONS: Clinical results of left-sided heterologous pericardial grafts have shown excellent performance over time. The BASV (over seven years) and BPVC and NVPC (eight years) have demonstrated superior results as aortic valves alone or in combination with a pericardial conduit.     

Phenotypic effect correlating with loss of a novel tumor suppressor gene: towards cloning by complementation

We intend to use a gene complementation approach to clone a tumor suppressor gene on mouse chromosome 2, the loss of which contributes to myeloid leukemia. An in vitro model system has been generated using a clonal cell line, in which tumorigenic chromosomal lesions have been selected along with myeloid differentiation. Among these lesions are deletions of chromosome 2. Comparison of subclones with deleted vs intact chromosomes 2 has allowed the identification of a growth related phenotypic pattern which correlates with the deletion, viz the retention of a marker of immature cells, resistance to inhibition by lipopolysaccharide (LPS), even in the presence of markers of mature myeloid cells, such as resistance to killing by apoptosis-inducing agents. The phenotype is shared by chromosome 2-deleted cell lines derived from conventional tumors. We have begun to investigate the mechanism of the phenotype. The LPS resistance does not correlate with lack of mRNA for CD14, a known cell surface receptor for this agent, or with failure to induce TNF alpha or nitric oxide synthase in response to its binding. The system should allow cloning of the gene using complementation of this phenotype in transfected cell lines.     

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants

A large number of observations suggest that, during Drosophila development there are close functional interactions between the activity of Notch receptor and that of a signaling molecule encoded by wingless gene. In this essay, I summarize these interactions and discuss the possibility that Wingless acts as a ligand for Notch as part of a switch that is iteratively involved in the assignation of cell fates during development.     

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase

The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy.     

RNA binding analysis of yeast REF2 and its two-hybrid interaction with a new gene product, FIR1

The product of the REF2 gene is required for optimal levels of endonucleolytic cleavage at the 3' ends of yeast mRNA, prior to the addition of a poly(A) tail. To test the role of the previously demonstrated nonspecific affinity of REF2 for RNA in this process, we have identified RNA binding mutants in vitro and tested them for function within the cell. One REF2 variant, with an internal deletion of 82 amino acids (269-350), displays a 10-fold reduction in RNA binding, yet still retains full levels of processing activity in vivo. Conversely, a series of carboxyl-terminal deletions that maintain full RNA binding capability have progressively decreasing activity. These results rule out a major role for the central RNA binding domain of REF2 in mRNA 3' end processing and demonstrate the importance of the carboxyl-terminal region. To ask if the stimulatory role of REF2 depends on interactions with other proteins, we used a two-hybrid screen to identify a new protein termed FIR1 (Factor Interacting with REF) encoded on chromosome V. FIR1 interacts with two independent regions of REF2, one of which (amino acids 268-345) overlaps the RNA binding domain and is dispensible for REF2 function, whereas the other (amino acids 391-533) is located within the critical carboxyl-terminus. As with REF2, FIR1 has a small but detectable role in influencing the efficiency of poly(A) site use. Yeast strains containing a disrupted FIR1 gene are slightly less efficient in the use of cryptic poly(A) sites located within the lacZ portion of an ACT1-lacZ reporter construct. Likewise, a double delta ref2, delta fir1 mutant is more defective in processing of a reporter CYC1 poly(A) site than delta ref2 alone. This synergistic response provides additional support for the interaction of FIR1 with REF2 in vivo, and suggests that a number of gene products may be involved in regulating the cleavage reaction in yeast.     

Isolation of a gene product expressed by a subpopulation of human lung fibroblasts by differential display

Fibroblasts are the major cell type responsible for synthesizing matrix constituents in lung and other connective tissues. Evidence indicates that fibroblasts are heterogeneous, and that subpopulations with some distinct properties are clonally selected and expanded in fibrotic diseases. However, few distinct markers capable of demonstrating the presence of fibroblast subpopulations in tissues have been isolated so far. With the objective of identifying proteins that could detect fibroblast subpopulations, we compared the messenger RNA (mRNA) expression of two cultured human lung fibroblast subpopulations by differential display. Total RNA was obtained, complementary DNA (cDNA) was synthesized, and the polymerase chain reaction (PCR) products obtained with several primer pairs were compared. One 724-bp product, which was strongly expressed by one human lung fibroblast subpopulation, was identified and cloned. This product was poorly expressed by the other lung fibroblast subpopulation. The mRNA for the gene encoding this product was not detectable in human smooth-muscle cells, endothelial cells, or epithelial cells, although it was present in dermal fibroblasts. The mRNA was detected in normal and fibrotic human lungs. Search of the National Center for Biotechnology (NCBI) GenBank DNA database with the sequence obtained from this clone revealed no significant matches. However, a search of the NCBI database of expressed sequence tags (dBEST) revealed five different human expressed sequence tag (EST) clones corresponding to the LR8 cDNA sequence. Six additional mouse and one pig EST clones were identified that showed significant similarity to the human fibroblast cDNA. Composites of the entire coding sequences for the human fibroblast gene product and the mouse homologue were assembled from the respective overlapping EST sequences. The open reading frame identified for each composite sequence predicted protein products of 270 and 263 amino acids for the human and mouse sequences, respectively, which were 52% identical, with three gaps. At the amino acid level, no significant sequence similarity was detected with any other sequences in exhaustive searches of the NCBI DNA and protein databases or the Blocks databases. A PCR product with predicted length and sequence was obtained by using a sense primer upstream to LR8 and an antisense primer within LR8. Our results indicate that this differentially displayed product represents a previously undescribed protein that could be useful for distinguishing fibroblasts, and possibly fibroblast subpopulations, from other cell types in lungs and other tissues.     

The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice

Angiotensin-converting enzyme (ACE) generates the vasoconstrictor angiotensin II, which plays a critical role in maintenance of blood pressure in mammals. Although significant ACE activity is found in plasma, the majority of the enzyme is bound to tissues such as the vascular endothelium. We used targeted homologous recombination to create mice expressing a form of ACE that lacks the COOH-terminal half of the molecule. This modified ACE protein is catalytically active but entirely secreted from cells. Mice that express only this modified ACE have significant plasma ACE activity but no tissue-bound enzyme. These animals have low blood pressure, renal vascular thickening, and a urine concentrating defect. The phenotype is very similar to that of completely ACE-deficient mice previously reported, except that the renal pathology is less severe. These studies strongly support the concept that the tissue-bound ACE is essential to the control of blood pressure and the structure and function of the kidney.     

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.     

Identification of Asp258 as the metal coordinate of pigeon liver malic enzyme by site-specific mutagenesis

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had < 0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe(2+)-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of semaphorin E as a non-MDR drug resistance gene of human cancers

To improve cancer chemotherapy, a better understanding of the molecular mechanisms of drug resistance is essential. To identify the molecules responsible for drug resistance that is unrelated to MDR1 or MRP gene products, a eukaryotic expression cDNA library of cis-diamminedichloroplatinum(II) (CDDP)-resistant ovarian cancer TYKnuR cells was introduced into Cos-7 cells. After repeated CDDP selection, cDNA homologous to murine semaphorin E was isolated from surviving cells. Human semaphorin E (H-sema E) was overexpressed in CDDP-resistant cell lines and was readily induced not only by diverse chemotherapeutic drugs but also by x-ray and UV irradiation. Transfection of H-sema E conferred a drug-resistant phenotype to CDDP-sensitive cells. In addition, the aberrant expression of H-sema E protein was detected immunohistochemically in 14 of 42 (33.3%) recurrent squamous cell carcinomas removed at autopsy after extensive radiochemotherapy. Recently, another member of the semaphorin family, CD100, was shown to significantly improve the viability of B lymphocytes. These results suggest the involvement of semaphorins in diverse cell survival mechanisms.     

Dual functions of bacteriophage T4D gene 28 product: structural component of the viral tail baseplate central plug and cleavage enzyme for folyl polyglutamates. I. Identification of T4D gene 28 product in the tail plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28(ts) phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28(ts) and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28(ts) and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28(ts) particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28(ts) particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28(ts) revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.