Nonoxidative ethanol metabolism: expression of fatty acid ethyl ester synthase-III in cultured neural cells.

Alcohol metabolism in the human brain has been characterized as essentially nonoxidative in nature, with the esterification of ethanol with fatty acids via fatty acid ethyl ester synthase. This pathway of ethanol metabolism is related to end organ damage in the brain but the neural cell type expressing FAEES has not been identified. In this study human and rodent neuroblastoma and glioma cell lines are assayed for fatty acid ethyl ester synthase activity. Cells with neuronal properties demonstrated higher activity than glioma cell lines. We confirmed the presence of the mRNA for one type of synthase, fatty acid ethyl ester synthase-III in three neuronal cell lines--N1E115 cells, PC12 cells, and SK-N-MC cells. These results support the hypothesis that FAEES activity is expressed chiefly in cells with neuronal properties and suggest that non-oxidative ethanol metabolism is potentially related to the toxic effect of ethanol on the human brain.     

Site-specific mutagenesis of two histidine residues in fatty acid ethyl ester synthase-III.

Human myocardial fatty acid ethyl ester synthase-III is a newly described acidic glutathione S-transferase that metabolizes both ethanol and carcinogens. Structure-function studies have not been performed relating these two distinct enzymatic activities. Since there are only two histidine residues in fatty acid ethyl ester synthase-III (His 72 and His 163), the role of each was examined by site-specific mutagenesis. Fatty acid ethyl ester synthase-III mutagenized at position 72 to contain either Gln, Pro or Ala had less than 5% of control glutathione S-transferase activity but retained fatty acid ethyl ester synthase activity under standard assay conditions. In contrast, substitution of histidine 163 with proline had no effect on glutathione S-transferase activity, but it slightly increased synthase activity. Thus, this study indicates that histidine plays a differential role in fatty acid ethyl ester synthase III depending on the nucleophilic substrate.     

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.     

Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase

A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.     

Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.     

优化硫酯酶表达提高大肠杆菌脂肪酸乙酯的生物合成效率

利用基因工程大肠杆菌直接从头生物合成脂肪酸乙酯 (生物柴油) 的相关研究引起了国内外研究人员的广泛关注。在本课题组已经构建的能够从头合成脂肪酸乙酯的大肠杆菌菌株KC3的基础上,通过替换表达不同来源的硫酯酶,发现表达来源于香樟树的硫酯酶Cc FatB1基因能够提高脂肪酸乙酯产量。进一步通过共表达Cc FatB1和大肠杆菌硫酯酶tesA’基因,以及启动子优化,获得了高产脂肪酸乙酯工程菌株KC4。KC4菌株在摇瓶条件和发酵条件下的单位生物量脂肪酸乙酯产率分别为21.4 mg/ (L?OD600)和31.16 mg/ (L?OD600)。该工程菌株的构建进一步提高了脂肪酸乙酯产量,显示了通过基因工程改造大肠杆菌从头合成生物柴油的应用潜力。     

Molecular cloning, sequencing and expression of cDNA encoding a G0-protein from insect

A locust cDNA clone encoding the complete sequence of a guanine nucleotide-binding protein was isolated and its nucleotide sequence determined. Comparing the deduced amino acid sequence with primary structures of other G-proteins revealed striking homologies with the vertebrate G0-protein. The cloned cDNA was expressed and the translation product detected by specific antibodies. Northern blot analysis revealed that the corresponding mRNA exists in two forms, preferentially expressed in the nervous tissue.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954-bp complete coding region for 318 amino acid residues. Homologies between human and rat PRS II were 99% of the amino acids and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme.     

Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm

An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by posttranslational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.     

Molecular cloning, sequencing, and expression of mouse ferrochelatase

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.     

Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence.

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.     

Molecular cloning and sequencing of coho salmon growth hormone cDNA

A cDNA library was constructed using mRNA isolated from coho salmon pituitaries. By employing rainbow trout growth hormone cDNA as a probe, the coho salmon cDNA was isolated and the complete nucleotide (nt) sequence determined. The coding region contains 630 nt while the 5'- and 3'-untranslated regions are 64 and 489 nt in length, respectively. Comparison of the noncoding regions of coho and chum salmon cDNAs reveal identity at the 5' end but significant variation in the 3' end. Chum salmon and rainbow trout have identical amino acid (aa) sequences, but coho salmon growth hormone has a sequence that differs by 6 of the 188 predicted aa. Since salmonids are tetraploid, this difference may be the result of either divergence of the same growth hormone locus or of variation between different loci. Comparisons of the cDNA restriction maps of these three fish species suggest the former possibility.     

Purification and characterization of fatty acid ethyl ester synthase-II from human myocardium.

Fatty acid ethyl ester synthases metabolize ethanol nonoxidatively in those extrahepatic organs most commonly damaged by alcohol abuse. This study was designed to isolate and purify human myocardial synthase-II, one of the enzymes responsible for catalyzing the formation of fatty acid ethyl esters. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase-I, synthase-II, and synthase-III activities, eluting at conductivities of 5, 7, and 11 mS, respectively. From this elution profile, fatty acid ethyl ester synthase-II accounts for up to 50% of total synthesis in the human heart. This enzyme species was purified over 2200-fold to homogeneity after chromatography over hydroxylapatite, CM-cellulose, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this homogeneous species showed a single band at 65 kDa which corresponded to its molecular weight determined by gel filtration. This molecular weight and its lack of glutathione transferase activity indicate that this species is not related to synthase-I and -III. Homogeneous synthase-II has a Vmax for palmitate, stearate, oleate, and linoleate of 70, 80, 140, and 120 nmol/mg/h, respectively. The Km for palmitate, stearate, oleate, and linoleate is 0.19, 0.12, 0.10, and 0.18 mM, respectively. The substrate specificity with respect to alcohol chain length was also investigated in the presence of 0.65 mM [14C]oleic acid. The Vmax for methanol, ethanol, propanol, and butanol was 180, 100, 280, and 410 nmol/mg/h, respectively. The Km for methanol, ethanol, propanol, and butanol was 1.16, 1.04, 0.58, and 0.33 M, respectively. The N-terminal 17-amino acid sequence of human synthase-II does not correspond to any known N-terminal amino acid sequence, indicating that this may be a novel protein. However, it has over 70% homology to a sequence close to the C terminus of rabbit cytochrome P-450IIC1 and over 50% homology to a sequence of human hemopexin starting at residue 16. Synthase-II does not cross-react with human hemopexin antibody and rat cytochrome P-450C antibody. Thus, this study provides evidence that synthase-II is a novel protein, distinct from synthase-I and -III, and it also provides a foundation for subsequent cloning and genetic studies of fatty acid ethyl ester synthase-II in man.     

Molecular cloning and expression of Arabidopsis fatty acid hydroperoxide lyase.

Fatty acid hydroperoxide lyase (HPOL), an enzyme of the octadecanoid pathway that forms carbon-6 aldehydes such as n-hexanal or (Z)-3-hexenal, was cloned from Arabidopsis thaliana as a full-length cDNA. The HPOL activity obtained by expressing the cDNA in Escherichia coli formed n-hexanal from linoleic acid 13-hydroperoxide, whereas linoleic acid 9-hydroperoxide was not a substrate for the enzyme. The HPOL mRNA is expressed at low level in leaves; however, its accumulation can be found in the inflorescence. Wounding or methyl jasmonate treatments increase the mRNA level in leaves. These results indicate that the HPOL gene is up-regulated in leaves in response to wounding and that the enzyme may be an active component of the octadecanoid defense response.     

Molecular cloning of human preproacrosin cDNA

Summary Complementary DNA-clones for human preproacrosin have been isolated from a human testis cDNA library in gt11. The nucleotide sequence of the 1402bp cDNA insert includes a 20 bp 5 noncoding region, an open reading frame of 1263bp corresponding to 421 amino acids (45.9 kdalton), and a 105 bp 3 untranslated region. The deduced amino acid sequence is compared with that recently evaluated from a cDNA clone for boar preproacrosin. The sequence identity is 70%; the leader sequence, the catalytic triad (His, Asp, Ser; which is characteristic for serine proteinases) and the positions of the cysteine residues crosslinking the light and the heavy chain of the active enzyme, acrosin, are conserved in both species. At the C-terminal end, a proline-rich sequence is present in both species; this may represent the species-specificity of acrosin.     

Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed. Northern blot analysis indicated that cathepsin H is predominantly synthesized in kidney. A high degree of sequence homology was observed with rat cathepsin H, especially within the propeptide. The part of the prosequence coding for the "minichain" is conserved in the prosequence of aleurain, a plant thiol protease.