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PubMed1995

The molecular basis of pediatric long chain 3-hydroxyacyl-CoA dehydrogenase deficiency associated with maternal acute fatty liver of pregnancy.

Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the heart. Deficiencies of human beta-oxidation enzymes produce sudden, unexplained death in childhood, acute hepatic encephalopathy, skeletal myopathy, or cardiomyopathy. Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.211] catalyzes the third step in beta-oxidation, and this activity is present on the C-terminal portion of the alpha subunit of mitochondrial trifunctional protein. We used single-stranded conformation variance analysis of the exons of the human LCHAD (alpha subunit) gene to determine the molecular basis of LCHAD deficiency in three families with children presenting with sudden unexplained death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome). In all families, the mothers had acute fatty liver and associated sever complications during pregnancies with the affected infants. The analysis in two affected children revealed a G to C mutation at position 1528 (G1528C) of the alpha subunit of the trifunctional protein on both alleles. This is in the LCHAD domain and substitutes glutamine for glutamic acid at position 474 of mature alpha subunit. The third child had this G1528C mutation on one allele and a different mutation (C1132T) creating a premature termination codon (residue 342) on the second allele. Our results demonstrate that mutations in the LCHAD domain of the trifunctional protein alpha subunit in affected offspring are associated with maternal acute fatty liver of pregnancy. This is the initial delineation of the molecular basis of isolated LCHAD deficiency.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1997

Suppression of insulin-like growth factor type I receptor by a triple-helix strategy inhibits IGF-I transcription and tumorigenic potential of rat C6 glioblastoma cells.

Homopurine (AG) and homopyrimidine (CT) oligodeoxyribonucleotides predicted to form triple-helical (triplex) structures have been shown to specifically suppress gene expression when supplied to cultured cells. Here we present evidence that homopurine RNA (effector) sequences designed to form a triplex with a homopurine. homopyrimidine sequence 3' to the termination codon of the insulin-like growth factor type I receptor (IGF-IR) structural gene can efficiently suppress IGF-IR gene transcription. Transfection vectors were constructed to drive transcription of either AG or CT variant triplex-forming strands. To increase the probability of obtaining stable transfectants with adequate expression of effector sequences, these were designed to be transcribed together with cDNA sequences conferring neomycin resistance as a fusion transcript. Rat C6 glioblastoma cells transfected with the AG variant showed dramatic reduction of IGF-IR transcripts compared with untransfected cells. The AG transfectants also exhibited marked down-regulation of the IGF-I, and an enhanced accumulation of serine protease inhibitor nexin-I mRNA. Similar changes in gene expression were observed following transfection of C6 cells with constructs transcribing antisense RNA to IGF-IR transcripts, but were not observed in C6 cells transfected with either the CT triplex variant or with vector lacking triplex-forming sequences. Moreover, C6 cells transfected with AG triplex variant displayed a dramatic inhibition of tumor growth when injected into nude mice. The results suggest that a triple-helix strategy can be used to inhibit transcription elongation of the IGF-IR gene, and emphasize the efficacy of triplex-mediated gene inhibition in an animal model.

Amyloid beta-Protein Precursor↗

PubMed1997

rRNA-like sequences occur in diverse primary transcripts: implications for the control of gene expression.

Many eukaryotic mRNAs contain sequences that resemble segments of 28S and 18S rRNAs, and these rRNA-like sequences are present in both the sense and antisense orientations. Some are similar to highly conserved regions of the rRNAs, whereas others have sequence similarities to expansion segments. In particular, four 18S rRNA-like sequences are found in several hundred different genes, and the location of these four sequences within the various genes is not random. One of these rRNA-like sequences is preferentially located within protein coding regions immediately upstream of the termination codon of a number of genes. Northern blot analysis of poly(A)+ RNA from different vertebrates (chicken, cattle, rat, mouse, and human) revealed that a large number of discrete RNA molecules hybridize at high stringency to cloned probes prepared from the 28S or 18S rRNA sequences that were found to match those in mRNAs. Inhibition of polymerase II activity, which prevents the synthesis of most mRNAs, abolished most of the hybridization to the rRNA probes. We consider the hypotheses that rRNA-like sequences may have spread throughout eukaryotic genomes and that their presence in primary transcripts may differentially affect gene expression.

Amanitins↗

PubMed1997

Triplet repeat polymorphism in the transmembrane region of the MICA gene: a strong association of six GCT repetitions with Behçet disease.

A member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), MICA, has been recently identified near the HLA-B gene on the short arm of human chromosome 6. The predicted amino acid sequence of the MICA chain suggests that it folds similarly to typical class I chains and may have the capacity to bind peptides or other short ligands. Therefore, MICA is predicted to have a specialized function in antigen presentation or T cell recognition. During nucleotide sequence analyses of the MICA genomic clone, we found a triplet repeat microsatellite polymorphism of (GCT/AGC)n in the transmembrane (TM) region of the MICA gene. In 68 HLA homozygous B cell lines, 5 distinct alleles of this microsatellite sequence were detected. One of them contained an additional one base insertion that created a frameshift mutation resulting in a premature termination codon in the TM region. This particular allele may encode a soluble, secreted form of the MICA molecule. In addition, we have investigated this microsatellite polymorphism in 77 Japanese patients with Behcet disease, which is known to be associated with HLA-B51. The microsatellite allele consisting of 6 repetitions of GCT/AGC was present at significantly higher frequency in the patient group (Pc = 0.00055) than in a control population. Furthermore, the (GCT/AGC)6 allele was present in all B51 positive patients and in an additional 13 B51 negative patients. These results suggest the possibility of a primary association of Behcet disease with MICA rather than HLA-B.

Asian People↗

PubMed1997

Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice.

The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCID-responsible gene. We isolated an ORF of the murine DNA-PKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration.

Amino Acid Sequence↗

PubMed1997

Creation of libraries with long ORFs by polymerization of a microgene.

We describe a novel method for constructing pools of DNA sequences that encode large proteins with molecular diversity. Sets of primer pairs that form 8 to 10 complementary base pairs in the 3' region and have double mismatch pairs at their 3'-OH ends were designed so that primer dimers recreated short stretches of DNA (microgenes) devoid of termination codons. Cycles of denaturation and elongation reactions with a pair of primers, four dNTPs, and 3'-5' exo+ thermostable DNA polymerase gave head-to-tail polymers of the primer dimer unit (microgene) whose sizes exceeded 12 kb. No template was required in this reaction, but mismatched nucleotides at 3'-OH ends of the primers were critical for efficient polymerization. At end-joining junctions of a microgene, nucleotide insertions and deletions randomly occurred, resulting in combinatorial libraries of three reading frames from a single microgene. Further molecular diversity could be incorporated by using a mixture of primers. The resultant polymers have long ORFs whose products have a repetitious nature that could facilitate the formation of higher structures of translated products. Thus, microgene polymers may be used as a source of libraries for in vitro protein evolution experiments. Ligation of a microgene is apparently related to the nonhomologous recombination of double-strand breaks in DNA that has been shown to be catalyzed by DNA polymerases. We named this polymerization reaction the "microgene polymerization reaction."

Amino Acid Sequence↗

PubMed1999

Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX.

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes, HYAL1 and HYAL2, encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the HYAL1 alleles of the patient, a 1412G --> A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene, HYAL3, have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.

Amino Acid Sequence↗

PubMed1995

Studies on the translocation of the amino terminus of apolipoprotein B into the endoplasmic reticulum.

Apolipoprotein (apo) B is either co-translationally assembled into lipoproteins, or becomes associated with the membrane of the endoplasmic reticulum (ER) and is subsequently degraded. It has been proposed that apoB undergoes a novel process of translocation which generates cytoplasmically exposed apoB in the ER of hepatic and non-hepatic cells. Transmembrane forms of apoB can also be generated by in vitro translation (Chuck, S. L., and Lingappa, V. R. (1992) Cell 68, 9-21), which might explain the origin of untranslocated apoB in vivo. Here we have investigated a protocol which generates transmembrane forms of apoB during in vitro translation of truncated RNA transcripts. We observe that apoB can become transmembrane at sites of ribosome pausing and be held in this configuration by persistence of tRNA on the peptide chains. Ribosome pausing also occurs at these same sites in the absence of acceptor microsomes. Transmembrane topology can be generated at sites of ribosome pausing in a cytosolic protein, sea urchin cyclin when fused to a signal sequence. Mapping of the ribosome pause sites in apoB and in cyclin revealed no amino acid sequence homology. Chimeric constructs with engineered downstream glycosylation sites showed no evidence that ribosome pause sequences affect translocation of transcripts with termination codons. Transmembrane forms of apoB and cyclin were not generated during translocation into the ER in transfected COS cells.

Amino Acid Sequence↗

PubMed1995

Cloning and functional expression of a thyrotropin receptor cDNA from rat fat cells.

Thyrotropin receptor (TSH-R) has been thought to be thyroid-specific, but, by Northern blot analysis, we found that rat adipose tissue expressed TSH-R mRNAs in amounts approaching those in the thyroid. To investigate the function of TSH-R from adipose tissue, we screened a rat fat cell lambda gt11 cDNA library for TSH-R sequences using a 32P-labeled rat thyroid TSH-R cDNA as a probe. Among 10(6) plaques, we obtained four positive clones. Sequencing of these cDNAs has revealed that two of them (F alpha and F beta) contained both initiation and termination codons. Comparison of F alpha with the thyroid TSH-R cDNA sequence revealed that F alpha was almost identical to the thyroid TSH-R, except that nucleotides 1041 and 1277 were changed from A to G and from C to T, respectively. In contrast, we found that F beta contained 21 novel nucleotides between nucleotides 467 and 468 of the thyroid TSH-R cDNA, encoding an additional 7 amino acids. However, when we prepared mRNA from adipose tissue and transcribed it into cDNA, we failed to amplify the F beta type of TSH-R cDNA by polymerase chain reaction, suggesting that F beta mRNAs are rare in the tissue. We then ligated F cDNAs into pSG5 and transfected them with pSV2-neo into Chinese hamster ovary (CHO)-K1 cells. TSH stimulated cAMP formation in CHO-F alpha cells in a manner similar to that in CHO cells transfected with thyroid TSH-R cDNA. In contrast, no increase of cAMP was observed in CHO-F beta cells. IgG from patients with Graves' disease (n = 4) showed thyroid-stimulating antibody activity only in CHO-F alpha cells (1288-4582%). In addition, CHO-F alpha cells and CHO cells transfected with thyroid TSH-R showed similar 125I-TSH binding activity. These results indicate that the fat cell expresses high levels of a TSH-R whose function is indistinguishable from that in the thyroid and suggest that the TSH-R autoantibody plays an important role in the pathogenesis of the extrathyroidal manifestations of Graves' disease.

Adipose Tissue↗

PubMed1995

Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor and endothelial cell-specific mitogen, is up-regulated by hypoxia. However, the mechanism(s) responsible for hypoxic induction of VEGF has not been clearly delineated. We report that the steady state VEGF mRNA levels are increased 12 +/- 0.6-fold, but the transcriptional rate for VEGF is increased only 3.1 +/- 0.6-fold by hypoxia in PC12 cells. In order to investigate cis-regulatory sequences which mediate this response to hypoxia, we cloned the rat genomic sequences encoding VEGF and identified a 28-base pair element in the 5' promoter that mediates hypoxia-inducible transcription in transient expression assays. This element has sequence and protein binding similarities to the hypoxia-inducible factor 1 binding site within the erythropoietin 3' enhancer. Post-transcriptional mechanisms have also been suggested to play a role in the hypoxic induction of VEGF. Evidence is provided that a frequently used polyadenylation site is 1.9 kilobases downstream from the translation termination codon for rat VEGF. This site is 1.5 kilobases further downstream from the polyadenylation site previously reported for VEGF. This new finding reveals sequence motifs in the 3'-untranslated region that may mediate VEGF mRNA stability.

Animals↗

PubMed1995

Sequence, genomic organization, and expression of the novel homeobox gene Hesx1.

Extensive analyses of homeobox gene expression and function during murine embryogenesis have demonstrated that homeobox gene products are key components in the establishment of pattern formation and regional identity during development. In this paper we report the molecular characterization and expression of a novel murine homeobox sequence, Hesx1, isolated from pluripotent embryonic stem cells. Hesx1 is expressed as two transcripts of 1.0 and 1.2 kilobases which encode an identical 185 amino acid open reading frame. The transcripts differ in the 3'-untranslated region due to the differential utilization of a weak splice donor site located immediately downstream of the translation termination codon. The Hesx1 homeodomain shared 80% identity with the Xenopus homeoprotein XANF-1 and was less than 50% related to other homeodomain sequences. Hesx1 and XANF-1 therefore constitute the founder members of a new homeodomain class. Hesx1 expression was down-regulated during embryonic stem cell differentiation and was detected in tissue-specific RNA samples derived from the embryonic liver, and at lower levels in viscera, amnion, and yolk sac. Expression in adult mice was not detected. These sites of expression are consistent with a role for Hesx1 in the regulation of developmental decisions in the early mouse embryo and during fetal hematopoiesis.

Amino Acid Sequence↗

PubMed1996

Molecular genetic analysis of the human Lewis histo-blood group system. II. Secretor gene inactivation by a novel single missense mutation A385T in Japanese nonsecretor individuals.

The Lewis histo-blood group system comprises two major antigens, Lewis a and Lewis b. The Lewis b antigen is a product of two fucosyltransferases, the alpha(1,3/1,4)fucosyltransferase (Lewis enzyme; Fuc-TIII) encoded by the Lewis gene and an alpha(1,2)fucosyltransferase which is not required for synthesis of Lewis a antigen. An enzyme responsible for secreting ABH antigens into body secretions (secretor enzyme) is also one of alpha(1,2)fucosyltransferases. A candidate gene encoding secretor enzyme Sec2 gene was recently cloned by Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D. ((1995) J. Biol. Chem. 270, 4632-4639) and Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. ((1995) J. Biol. Chem. 270, 4640-4649) who demonstrated a G428A nonsense mutation (Trp143 to terminal codon) in Sec2 of nonsecretors. However, the G428A nonsense mutation discovered in the Sec2 gene of nonsecretors in an ethnic group other than Japanese was not found in any of 45 Japanese nonsecretors, whereas one Filipino who had been erroneously registered as a Japanese possessed the G428A mutation heterozygously. In order to explore the Sec2 gene of a Japanese population, we performed a molecular genetic analysis of the Sec2 gene on 226 Japanese individuals, 21 in a family study and 205 in a random sampling study. We discovered two novel mutations in the Sec2 gene, an A385T missense mutation (Ile129 to Phe) that results in inactivation of Sec2-encoded alpha(1,2)fucosyltransferase and a C357T silent mutation which is irrelevant to amino acid substitution, in Japanese nonsecretors. The analysis of Japanese individuals using the polymerase chain reaction-restriction fragment length polymorphism method found three alleles in the Sec2 gene, the first having no mutation, the second having a C357T mutation, and the third having both C357T and A385T mutations, which we designated as Se1, Se2, and sej, respectively. Among 226 Japanese individuals, 40 having a Le(a+b-) phenotype and 5 having a Le(a-b-) nonsecretor phenotype were homozygous for sej/sej, whereas 149 having a Le(a-b+) phenotype and 32 having a Le(a-b-)-secretor phenotype possessed at least one Se1 or Se2. The frequencies of occurrence of Se1, Se2, and sej among 410 alleles examined in a random sample of 205 Japanese individuals were 15, 46, and 39%, respectively, indicating a rather wide distribution of the sej allele in the Japanese population. The results show that the Sec2 gene really encodes the secretor enzyme alpha(1,2)fucosyltransferase and indicate that a ethnic group-specific nonsense or missense point mutation in the Sec2 gene determines nonsecretor status. The phylogenic aspect and biological significance of the Se and Le genes are discussed.

Amino Acid Sequence↗

PubMed1996

Extensive alternative splicing within the amino-propeptide coding domain of alpha2(XI) procollagen mRNAs. Expression of transcripts encoding truncated pro-alpha chains.

Heterogeneity in type XI procollagen structure is extensive because all three alpha(XI) collagen genes undergo complex alternative splicing within the amino-propeptide coding domain. Exon 7 of the human and exons 6-8 of the mouse alpha2(XI) collagen genes, encoding part of the amino-propeptide variable region, have recently been shown to be alternatively spliced. We show that exon 6-containing mRNAs for human alpha2(XI) procollagen are expressed at 28 weeks in fetal tendon and cartilage but not at 38-44 days or 11 weeks. In the mouse, exon 6 is expressed in chondrocytes from 13.5 days onward. We recently identified conserved sequences within intron 6 of the human and mouse alpha2(XI) collagen genes, containing additional consensus splice acceptor and donor sites that potentially increase the size of exon 7, dividing it into three parts, designated 7A, 7B, and 7C. We show by reverse transcription polymerase chain reaction and in situ hybridization that these potential splice sites are used to yield additional alpha2(XI) procollagen mRNA splice variants that are expressed in fetal tissues. In human, expression of exon 7B-containing transcripts may be developmental stage-specific. Interestingly, inclusion of exon 7A or exon 7B in human and mouse alpha2(XI) procollagen mRNAs, respectively, would result in the insertion of an in-frame termination codon, suggesting that some of the additional splice variants encode a truncated pro-alpha2(XI) chain.

Alternative Splicing↗

PubMed1996

Functional Re-expression of laminin-5 in laminin-gamma2-deficient human keratinocytes modifies cell morphology, motility, and adhesion.

Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by a reduced adherence of keratinocytes consequent to deficient expression of the extracellular adhesive ligand laminin-5. To complement the genetic defect causing H-JEB, we transferred an eukaryotic cassette expressing the cDNA for the gamma2 chain of laminin-5 into H-JEB keratinocytes in which the expression of the polypeptide is hampered by a homozygous mutation generating a premature termination codon. Transfection using adenovirus-polylysin-transferrin-DNA complexes resulted in a transient synthesis of the recombinant laminin gamma2 chain that associated with the endogenous alpha3 and beta3 chains to form laminin-5 molecules readily deposited on the tissue culture substrate. Furthermore, retroviral-mediated transduction of the gamma2 cDNA yielded persistent expression and polarized secretion of laminin-5. The protein incorporated into the basement membrane produced by the revertant cells inoculated subcutaneously in nude mice. In these transfectants, re-expression of laminin-5 induced changes in cell morphology and reorganization of focal adhesions that assumed the shape and distribution of the counterparts detected in normal keratinocytes. These observations correlated with an enhanced cell-substrate adhesion and a reduced motility of the transfected cells. Our results demonstrate that a restored expression of laminin-5 induces a phenotypic reversion of genetically altered H-JEB keratinocytes and open new perspectives to the analysis of the mechanisms regulating adhesion of epithelial cells.

Animals↗

PubMed1996

Analysis of abrB mutations, mutant proteins, and why abrB does not utilize a perfect consensus in the -35 region of its sigma A promoter.

The Bacillus subtilis global regulator AbrB is a DNA-binding protein composed of six identical monomers of 96 amino acids that shows specificity to the promoter regions of its target genes including its own. We have sequenced thirteen previously uncharacterized abrB mutations. Four mutant AbrB proteins were purified, and their DNA-binding properties and multimeric structures were examined. AbrB23 (R25S) had no appreciable DNA binding activity but retained a hexameric structure, indicating that Arg25 is important in DNA interactions. Three other mutant proteins, AbrB1 (C56Y), AbrB19 (Gln83-->termination codon), and AbrB100 (L69P), showed decreased DNA binding and altered multimeric interactions. Analysis of the expression and AbrB binding affinities of mutant abrB promoters demonstrated that a consensus -35 region is incompatible with proper autoregulation of the abrB gene.

Amino Acid Sequence↗

PubMed1998

c-myc mRNA is down-regulated during myogenic differentiation by accelerated decay that depends on translation of regulatory coding elements.

Murine C2C12 myoblasts induced to differentiate into multinucleated myotubes decrease their levels of c-myc mRNA 3-10-fold through posttranscriptional mechanisms that recognize regulatory elements contained in protein-coding sequences in exons 2 and 3 of the mRNA. To determine the mechanism by which these elements mediate c-myc mRNA down-regulation, we examined the regulation of mutant MYC and human beta-globin-MYC fusion mRNAs. Regulation of mRNAs containing MYC exon 2 or 3 is abolished by insertion of an upstream termination codon indicating that regulatory function depends on their translation. Exploiting this translation dependence, we show that pharmacologic inhibition of translation with cycloheximide abolishes the down-regulation of regulated MYC and globin-MYC mRNAs and induces their levels in differentiating C2C12 cells. We exclude the possibility that this induction in mRNA levels results from cycloheximide effects on transcription or processing of parts of the RNA other than the regulatory elements, leading to the conclusion that cycloheximide induction results from mRNA stabilization. We show that the magnitude of cycloheximide induction can be used to estimate turnover rates of mRNAs whose decay is translation-dependent. By using cycloheximide inducibility to examine turnover rates of MYC and globin-MYC mRNAs, we show that the MYC exon 2 and exon 3 regulatory elements, but not MYC 3'-untranslated region or chloramphenicol acetyltransferase coding sequences, mediate accelerated mRNA decay in differentiating, but not undifferentiated, C2C12 cells. We show that these regulatory elements must be translated to confer accelerated mRNA decay and that increased turnover occurs in the cytoplasm and not in the nucleus. Finally, using cycloheximide induction to examine mRNA half-lives, we show that mRNA turnover is increased sufficiently by mechanisms targeting the exon 2 and 3 regulatory elements to account for the magnitude of c-myc mRNA down-regulation during differentiation. We conclude from these results that c-myc mRNA down-regulation during myogenic differentiation is due to translation-dependent mechanisms that target mRNAs containing myc exon 2 and 3 regulatory elements for accelerated decay.

Animals↗

PubMed1999

Target and specificity of a nuclear gene product that participates in mRNA 3'-end formation in Chlamydomonas chloroplasts.

Chloroplast mRNA maturation is catalyzed by nucleus-encoded processing enzymes. We previously described a recessive nuclear mutation (crp3) that affects 3'-end formation of several chloroplast mRNAs in Chlamydomonas reinhardtii (Levy, H., Kindle, K. L., and Stern, D. B. (1997) Plant Cell 9, 825-836). In the crp3 background, atpB mRNA lacking a 3'-inverted repeat normally required for stability accumulates as a discrete transcript. The mutation also affects the atpA gene cluster; polycistronic mRNAs with psbI or cemA 3'-ends accumulate to a lower level in the crp3 background. Here, we demonstrate that the crp3 mutation also alters 3'-end formation of psbI mRNA and cemA-containing mRNAs. A novel 3'-end is formed in monocistronic psbI transcripts, and this is the only terminus observed when the psbI 3'-untranslated region is fused to an aadA reporter gene. Accumulation of mRNAs with 3'-ends between cemA and atpH, which is immediately downstream, was reduced. However, this sequence was not recognized as a 3'-end formation element in chimeric genes. The crp3 mutation was able to confer stability to three different atpB 3'-stem-loop-disrupting mutations that lack sequence similarity, but are located at a similar distance from the translation termination codon. We propose that the wild-type CRP3 gene product is part of the general 3' --> 5' processing machinery.

3' Untranslated Regions↗

PubMed2000

Abnormal sodium stimulation of carnitine transport in primary carnitine deficiency.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na(+)/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K(Na)) from the physiological value of 11.6 to 187 mm. Substitution of Glu(452) with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K(Na). Carnitine transport decreased exponentially with increased K(Na). The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer.

Animals↗