Biomedical subjects

J J Billadello

Publications and source records attributed to J J Billadello.

At least 19 recordsLinked to original sources

PubMed1992

Post-transcriptional regulation of expression of plasminogen activator inhibitor type 1 mRNA by insulin and insulin-like growth factor 1.

In humans and non-human primates, alternative cleavage and polyadenylation of plasminogen activator inhibitor type-1 (PAI-1) pre-mRNA transcripts results in two forms of mature mRNA, an unstable 3.2-kilobase (kb) form, and a relatively more stable 2.2-kb form. Insulin and insulin-like growth factor I (IGF-1) increase steady state levels of PAI-1 mRNA in Hep G2 cells independently and synergistically. In the present study we found that the rate of transcription of the PAI-1 gene is not affected by insulin, IGF-1, or both but that insulin prolongs the half-life of the 3.2-kb PAI-1 mRNA species 2.7-fold without affecting the half-life of the 2.2-kb species. In contrast IGF-1, alone or with insulin, markedly prolongs the half-lives of both species. Our results demonstrate a novel mechanism of regulation of expression of the PAI-1 gene by insulin and IGF-1 operating at the post-transcriptional level.

Animals↗

PubMed1992

The human M creatine kinase gene enhancer contains multiple functional interacting domains.

Cis-elements (-933 to -641) upstream of the human M creatine kinase gene cap site contain an enhancer that confers developmental and tissue-specific expression to the chloramphenicol acetyltransferase gene in C2C12 myogenic cells transfected in culture. Division of the enhancer at -770 into a 5' fragment that includes the MyoD binding sites (-933 to -770) and a 3' fragment that includes the MEF-2 binding site (-770 to -641) resulted in two subfragments that showed minimal activity but in combination interacted in a position- and orientation-independent fashion to enhance activity of the SV40 promoter in transient transfection experiments. A 5' enhancer construct (-877 to -832) including only one (the low affinity) MyoD binding site was active when present in multiple copies. In contrast, a 3' enhancer construct (-749 to -732) including the MEF-2 binding site was inactive even when present in multiple copies. However, if the 5' construct was extended to include the high-affinity MyoD binding site (-877 to -803) the 5' and 3' constructs interacted in a position- and orientation-independent fashion to activate the SV40 promoter. Thus, the human M creatine kinase enhancer comprises multiple functional interacting domains.

3T3 Cells↗

PubMed1991

Multiple positive and negative elements regulate human brain creatine kinase gene expression.

We characterized the developmental expression of the brain creatine kinase (BCK) gene in the C2C12 myogenic cell line with the use of isoenzyme, Western blot, and Northern blot analyses. The results show that both BCK subunit protein and mRNA are upregulated early in myogenesis, and then downregulated in fully differentiated myotubes. To characterize the transcriptional regulatory mechanisms, a chimeric construct containing 1.2 kilobase pairs of 5'-flanking DNA from the human BCK gene placed upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVOCAT was transiently transfected into C2C12 cells. In myoblasts and differentiating myotubes, the time course of expression of the constructs paralleled that of endogenous BCK mRNA. Additional constructs prepared by deleting 5'-flanking DNA were also transfected into C2C12 cells. All constructs were preferentially expressed in myoblasts relative to myotubes with absolute levels of expression increasing with deletion of 5'-flanking DNA. In nonmyogenic cells expression of the plasmids also increased with deletion of 5'-flanking DNA. An element from -1150 to -388 was isolated and found to be capable of suppressing expression of the BCK promoter and of heterologous promoters independent of orientation and position and hence to function as a silencer. Thus, BCK expression is mediated by sequences contained in the 5'-flanking DNA, including negative elements active in both C2C12 cells and nonmyogenic cells and elements that mediate the developmental expression of the BCK gene in C2C12 myogenic cells.

3T3 Cells↗

PubMed1991

Regulatory element analysis and structural characterization of the human sarcomeric mitochondrial creatine kinase gene.

Creatine kinase (EC 2.7.3.2) (CK) isoenzymes are crucial to energy metabolism, particularly in tissues with high energy requirements. Nuclear genes encode four known CK subunits: cytoplasmic muscle, cytoplasmic brain, ubiquitous mitochondrial (uMtCK), and sarcomeric mitochondrial (sMtCK). Herein, we report the isolation and complete structural characterization of the human sMtCK gene. It contains 11 exons and encompasses more than 37 kilobase pairs (kb). The sites of exon localization in the sMtCK-coding region and their precise sizes are identical with the human uMtCK gene. The translation start codon is in the third exon and lies 17 kb from the transcription start site. The human sMtCK gene is located on chromosome 5. Sequence analysis of the sMtCK genomic upstream sequences reveals a typical TATAA box within the 80 base pairs (bp) that, by transfection experiments, are sufficient to promote expression of chimeric plasmids with the chloramphenicol acetyltransferase reporter. Cis-acting sequences in a fragment containing 3360 bp of upstream sequence, the first exon, and 750 bp of the first intron are sufficient to mediate tissue-specific expression. However, these sequences only partially regulate induction of sMtCK expression in differentiating mouse myoblasts. MEF1/MYOD and MEF2 sequence motifs present in the sMtCK gene are not sufficient to regulate differentiation-specific expression. The sMtCK gene contains sequences homologous to several motifs that are shared among some nuclear genes encoding mitochondrial proteins and that may be essential for the coordinated activation of these genes during mitochondrial biogenesis.

Base Sequence↗

PubMed1991

Mediators of induction of augmented expression of plasminogen activator inhibitor type-1 in Hep G2 cells by platelets.

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-beta (TGF-beta) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗

PubMed1991

Multiple transforming growth factor-beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells.

Regulation of plasminogen activation by plasminogen activator inhibitor type-1 (PAI-1) is a critical feature of many biological processes. Transforming growth factor-beta (TGF-beta) induces PAI-1 mRNA and protein in several types of cultured cells, including Hep G2 cells. The present study was performed to define mechanisms by which PAI-1 gene expression is regulated by TGF-beta. Nuclear run-on assays performed on Hep G2 cells stimulated with TGF-beta for 6 h showed a 3.8-fold increase in PAI-1 gene transcription. TGF-beta increased the half-life of PAI-1 mRNA in Hep G2 cells 2.5-fold over control values. To characterize transcriptional regulatory mechanisms, we constructed chimeric genes containing PAI-1 5'-flanking DNA fused upstream of the bacterial chloramphenicol acetyltransferase gene in the vector pSVOCAT and transfected Hep G2 cells. Promoter deletion analysis demonstrated that sequences between -791 and -328 and -328 to -186 base pairs upstream of the PAI-1 gene cap site contain TGF-beta responsive elements that conferred TGF-beta inducibility in an orientation and position-independent manner. Further characterization of the larger TGF-beta-inducible enhancer (-791 to -328) located a TGF-beta-inducible element at nucleotides -791 to -546 upstream of the PAI-1 gene cap site. These results demonstrate that PAI-1 gene regulation by TGF-beta in Hep G2 cells is mediated both at a transcriptional level by two specific inducible elements, as well as by post-transcriptional mechanisms.

Blotting, Northern↗

PubMed1991

Transcriptional regulation of plasminogen activator inhibitor type-1 mRNA in Hep G2 cells by epidermal growth factor.

Secretion of plasminogen activator inhibitor type-1 (PAI-1) by cultured cells is increased after exposure to specific cytokines and growth factors. We have shown previously that incubation of Hep G2 cells with epidermal growth factor (EGF) results in a marked increase in steady state levels of PAI-1 mRNA (Lucore, C.L., et al. (1988) J. Biol. Chem. 263, 15845-15848). The present study was undertaken to determine whether the regulation of expression of PAI-1 mRNA by EGF is mediated at the level of transcription and/or by post-transcriptional mechanisms. The rate of transcription of the PAI-1 gene measured by nuclear run-on assays was found to be increased within 2 h after stimulation of the cells with EGF (5 ng/ml) (3.2 fold increase relative to control, n = 2, range 3.0-3.4). It reached a maximum in 3 h, (9.2 fold increase relative to control, n = 2, range 8.8-9.6) and returned to baseline in 5 h. Exposure of the cells to EGF did not increase the rate of transcription of the glyceraldehyde-3-phosphate dehydrogenase gene. The half life of PAI-1 mRNA in Hep G2 cells was 120 min as determined by RNA blot analysis after exposure of the cells to actinomycin D to inhibit transcription. Stimulation of the cells with EGF did not result in significant change in the half life of PAI-1 mRNA. The results demonstrate that exposure of Hep G2 cells to EGF increases PAI-1 gene transcription.

Cycloheximide↗

PubMed1991

Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats.

Pressure overload of the left ventricle induces synthesis of creatine kinase isoenzymes. To determine whether this response is associated with an altered pattern of creatine kinase gene expression, we induced arterial hypertension in rats by suprarenal aortic banding. After 4 days, left ventricular myocardium from hypertensive (n = 7) and normotensive, sham-operated (n = 5) rats was analyzed for isoenzyme activities by chromatography; M and B creatine kinase subunit protein by Western blot; and M, B, and mitochondrial creatine kinase mRNA by Northern blot. Although total creatine kinase activity increased in hypertensive (1,096 +/- 214 IU/g left ventricle) compared with normotensive rats (648 +/- 81 IU/g left ventricle, p less than 0.01), the relative proportions of the cytoplasmic and mitochondrial isoenzymes did not change. The mass of M and B subunits increased 1.9- and 2.7-fold, respectively, in hypertensive compared with control rats. Similarly, the mRNA for M and B subunits as well as mitochondrial creatine kinase increased 2.6-, 1.6-, and 1.8-fold, respectively, in hypertensive rats compared with control rats. Thus, increased energy requirements in acute pressure overload are met by generalized induction of creatine kinase mRNA and subunit protein and not by an isoenzyme switch.

Animals↗

PubMed1990

Induction of synthesis of plasminogen activator inhibitor type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells.

Plasminogen activator inhibitor type-1 (PAI-1) can modify fibrinolytic activity in vitro and in vivo. The present study was performed to determine whether pharmacologic concentrations of tissue-type plasminogen activator (t-PA) can initiate negative feedback by stimulating PAI-1 synthesis. In both human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVEC), t-PA increased the total concentrations and appearance of newly synthesized protein in conditioned media of free PAI-1 and PAI-1 complexed with t-PA in a dose and time dependent fashion judging from results after immunoprecipitation of metabolically labeled PAI-1. The t-PA effect was not attributable simply to release of stored or matrix-bound PAI-1. In HUVEC, Northern blot analyses indicated that t-PA increased steady-state levels of PAI-1 mRNA two-fold. In contrast PAI-1 mRNA expression was not increased in Hep G2 cells. Thus, mechanisms of stimulation appeared to differ in the two cell lines. The results obtained are consistent with the hypothesis that increased PAI-1 synthesis and secretion in response to t-PA may limit or attenuate fibrinolysis locally or systemically in vivo.

Cells, Cultured↗

PubMed1990

Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells.

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.

Binding Sites↗

PubMed1990

Tissue-specific distribution and developmental regulation of M and B creatine kinase mRNAs.

To characterize the tissue-specific distribution and developmentally regulated expression of M and B creatine kinase mRNA in rats, total cellular RNA was isolated from adult rat tissues and from skeletal muscle, heart, brain and intestine at selected stages of development. Northern blots were prepared and hybridized with M and B subunit-specific probes derived from the 3'-untranslated region. M creatine kinase mRNA was expressed abundantly in heart and skeletal muscle, and less abundantly in lung. B creatine kinase mRNA was found in all tissues examined except liver and was abundant in brain, heart and intestine. The developmentally regulated expression of M and B creatine kinase mRNA was determined in skeletal muscle, heart, brain and intestine. The developmental program of B creatine kinase mRNA was different for each tissue examined. During development, M creatine kinase mRNA was up-regulated in both heart and skeletal muscle with a different regulatory program. This resulted in replacement of B mRNA by M mRNA as the predominant species at an earlier developmental stage in heart when compared to skeletal muscle.

Animals↗

PubMed1990

Labeling of human clots in vitro with an active-site mutant of t-PA.

Prompt detection of acute thrombosis and its response to treatment with thrombolytic agents generally require angiography. Scintigraphic approaches with labeled antibodies to or components of the coagulation and fibrinolytic systems have been disappointing because of prolonged circulating half-lives of tracers and relatively slow or limited binding to thrombi. Accordingly, we developed and characterized a thrombolytically inactive, active-site mutant (Ser-478----Thr) of tissue-type plasminogen activator (t-PA) designed to detect thrombi in vivo. Binding of iodine-125-(125I) labeled Ser----Thr t-PA to thrombi in vitro was time- and concentration-dependent, and specific judging from inhibition by pre-incubation with anti-t-PA IgG. Clearance of 125I-labeled mutant t-PA in rabbits was rapid and biexponential (alpha t1/2 = 1.9 +/- 0.4 min, beta t1/2 = 39.8 +/- 11.2 min). Thus, the amidolytically inactive mutant of t-PA designed binds rapidly and specifically to human thrombi in vitro and is cleared rapidly from the circulation in vivo--properties rendering it attractive as a potentially useful clot imaging agent.

Humans↗

PubMed1989

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1.

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.

Base Sequence↗

PubMed1989

Potential attenuation of fibrinolysis by growth factors released from platelets and their pharmacologic implications.

Increased concentrations of the fast-acting tissue-type plasminogen activator (t-PA) inhibitor attenuate the fibrinolytic activity of pharmacologically administered activators of the fibrinolytic system such as t-PA. Accordingly, it was hypothesized that augmentation of synthesis and elaboration of inhibitor from the liver, leading to increased concentrations of inhibitor in plasma, or from endothelial cells in the vicinity of thrombi undergoing lysis, leading to increased concentrations locally, may contribute to failure of pharmacologically induced thrombolysis or to early reocclusion. Because platelets are rich in transforming growth factor beta and epidermal growth factor-like activity, it was thought that release of growth factors from platelets activated in vivo could mediate increases of the inhibitor in plasma by stimulating its formation in the liver and its local release from endothelial cells in the vicinity of thrombi. If so, fibrinolysis might be rendered more effective by concomitant prevention of platelet growth factor release. Transforming growth factor beta, a major constituent of platelets, increased concentrations of the t-PA inhibitor messenger ribonucleic acid (mRNA) in human hepatoma cells in a specific and dose-dependent manner. A peak effect was seen with 5 ng/ml and a 10-fold increase in 6 hours. Release of inhibitor protein into conditioned media increased as well. Induction of the inhibitor mRNA increase was elicited by exposure as brief as 30 minutes. Cycloheximide, an inhibitor of protein synthesis, was not inhibitory. The mechanisms responsible differed from those seen with epidermal growth factor, shown previously in the laboratory to increase inhibitor mRNA. In addition, the 2 factors were synergistic. Platelet lysates elicited effects simulating those of the purified growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.

Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.

Amino Acid Sequence↗

PubMed1988

Developmental regulation and tissue-specific expression of the human muscle creatine kinase gene.

To define mechanisms regulating expression of M creatine kinase, the human gene including 5'-flanking DNA was cloned, characterized, and partially sequenced. The gene contains 8 exons interrupted by 7 introns spanning 17.5 kilobase pairs of DNA. The intron-exon splice sites were identified and conform to the GT-AG consensus rule. The TATA and CAAT boxes are located at positions -31 and -56 upstream of the transcription start site as determined by primer extension. The 5'-untranslated region is interrupted with the translation start codon located in the second exon. To determine whether sequences within the 5'-upstream DNA confer tissue-specific expression and developmental regulation, constructs containing 2620 base pairs of human M creatine kinase 5'-flanking DNA fused upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVO-CAT were transfected into cultured C2C12 myoblasts. There was 17-fold induction of chloramphenicol acetyltransferase activity during differentiation as C2C12 myoblasts fused to form myotubes. The M creatine kinase fusion construct was not expressed in transfected nonmuscle cell lines, COS-7 and NIH/3T3. Thus, cis-acting sequences within 2620 base pairs of the cap site are sufficient to direct developmental regulation and tissue-specific expression of the human M creatine kinase gene.

Base Sequence↗

PubMed1988

Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor.

To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells. Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells. Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases). Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h. Factors responsible were found to be trypsin-sensitive and dialysis-resistant. Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response. EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner. Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h. The response was not attenuated by cycloheximide (25 micrograms/ml). Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase. Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold). We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.

Carcinoma, Hepatocellular↗

PubMed1987

Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue.

Medium-chain acyl-CoA dehydrogenase (MCAD; acyl-CoA: (acceptor) 2,3-oxidoreductase, EC 1.3.99.3) is one of three similar enzymes that catalyze the initial step of fatty acid beta-oxidation. Definition of the primary structure of MCAD and the tissue distribution of its mRNA is of biochemical and clinical importance because of the recent recognition of inherited MCAD deficiency in humans. The MCAD mRNA nucleotide sequence was determined from two overlapping cDNA clones isolated from human liver and placental cDNA libraries, respectively. The MCAD mRNA includes a 1263-base-pair coding region and a 738-base-pair 3'-nontranslated region. A partial amino acid sequence (137 residues) determined on peptides derived from MCAD purified from porcine liver confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human MCAD cDNA with the partial protein sequence of the porcine MCAD revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis shows that MCAD mRNA is expressed in a variety of rat (2.2 kilobases) and human (2.4 kilobases) tissues. Blot hybridization of RNA prepared from cultured skin fibroblasts from a patient with MCAD deficiency disclosed that mRNA was present and of similar size to MCAD mRNA derived from control fibroblasts. The isolation and characterization of MCAD cDNA is an important step in the definition of the defect underlying MCAD deficiency and in understanding its metabolic consequences.

Acyl-CoA Dehydrogenase↗