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Biomedical subjects

B J Thiele

Publications and source records attributed to B J Thiele.

At least 19 recordsLinked to original sources
PubMed2004

Controlling the release and production of renin.

The renin-angiotensin system (RAS) plays a pivotal role for a variety of cardiovascular functions. The diversity of renin actions is reflected by its complex control. The major stimulus for the release of renin from the vesicles in juxtaglomerular cells is determined by stretch, as induced by changes in arterial pressure. The production of renin underlies a vastly complex control network, which takes place at different levels, such as transcription and translation. With regard to transcription, important regions for binding transcription factors have been identified several years ago, but the conservation of nucleotide sequences throughout different species suggests that there might be additional binding regions of importance. At the post-transcriptional level, the modulation of renin mRNA stability is seems pivotal. The half-life of renin mRNA appears to be controlled by the interaction between several regulatory proteins, most of which are well known in other systems. Moreover, in addition to the modulation of mRNA stability, the translation efficiency seems to play a key role in determining the amount of renin to be produced.

Humans↗
PubMed2000

Expression of the gene and processed pseudogenes encoding the human and rabbit translationally controlled tumour protein (TCTP).

In humans and rabbits, the TPT1 gene encoding the translationally controlled tumour protein TCTP generates two mRNAs (TCTP mRNA1 and TCTP mRNA2) which differ in the length of their 3' untranslated regions. The distribution of these mRNAs was investigated in 10 rabbit and 50 human tissues. They were transcribed in all tissues investigated, but differed considerably in their quantity and ratio of expression. This indicates an extensive transcriptional control and involvement of tissue-specific factors. In the rabbit genome numerous processed, intronless pseudogenes were detected. Four, corresponding to both types of mRNAs, were sequenced and analysed in detail; all displayed only few mutations and were either preserved completely in the original amino acid sequence of the intron containing gene, or contained only minor mutations in the coding region which did not interrupt the open reading frame. In the mRNA population of rabbit reticulocytes two additional TCTP RNAs of the TCTP mRNA2 type were detected, which have the characteristics of pseudogene transcripts. Pseudogene transcription was supported further by CAT reporter gene assays showing substantial promoter activity of 5'-flanking regions of two TPT1 pseudogenes.

Animals↗
PubMed1999

The diversity of the lipoxygenase family. Many sequence data but little information on biological significance.

Lipoxygenases form a family of lipid peroxidising enzymes, which oxygenate free and esterified polyenoic fatty acids to the corresponding hydroperoxy derivatives. They are widely distributed in both the plant and animal kingdoms. During the last couple of years more and more lipoxygenase isoforms have been discovered but for most of them the biological significance remains unclear. This review attempts to classify the currently known mammalian lipoxygenase isoforms and critically reviews the concepts for their biological importance.

Animals↗
PubMed1999

Tissue-specific translational regulation of alternative rabbit 15-lipoxygenase mRNAs differing in their 3'-untranslated regions.

By screening a rabbit reticulocyte library, an alternative 15-LOX transcript of 3.6 kb (15-LOX mRNA2) was detected containing a 1019 nt longer 3'-untranslated region (UTR2) than the main 2.6 kb mRNA (15-LOX mRNA1). In anaemic animals, northern blotting showed that 15-LOX mRNA2 was predominantly expressed in non-erythroid tissues, whereas 15-LOX mRNA1 was exclusively expressed in red blood cells and bone marrow. The 15-LOX 3'-UTR2 mRNA2 contained a novel 8-fold repetitive CU-rich motif, 23 nt in length (DICE2). This motif is related but not identical to the 10-fold repetitive differentiation control element (DICE1) of 19 nt residing in the 15-LOX UTR1 mRNA1. DICE1 was shown to interact with human hnRNP proteins E1 and K, thereby inhibiting translation. From tissues expressing the long 15-LOX mRNA2, two to three unidentified polypeptides with molecular weights of 53-55 and 90-93 kDa which bound to DICE2 were isolated by RNA affinity chromatography. A 93 kDa protein from lung cytosol, which was selected by DICE2 binding, was able to suppress translational inhibition of 15-LOX mRNA2, but not of 15-LOX mRNA1, by hnRNP E1. A possible interaction between DICE1/DICE2 cis / trans factors in translational control of 15-LOX synthesis is discussed. Furthermore, the 3'-terminal part of the highly related rabbit leukocyte-type 12-LOX gene was analysed. Very similar repetitive CU-rich elements of the type DICE1 (20 repeats) and DICE2 (nine repeats) were found in the part corresponding to the 3'-UTR of the mRNA.

3' Untranslated Regions↗
PubMed1999

Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH-GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH-GPx is less well investigated. In this study we cloned the PH-GPx cDNA from a mouse fibroblast cDNA library and the PH-GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5'-flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies.

Animals↗
PubMed1999

Expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits.

From a rabbit reticulocyte library a full length cDNA was isolated which predicted a novel lipoxygenase (LOX) sharing 99% identical amino acids with the rabbit 15-lipoxygenase. HPLC product analysis of the bacterially expressed protein identified it as a leukocyte-type 12-lipoxygenase (1.12-LOX). This proves the co-expression of a 15-lipoxygenase and a 1.12-lipoxygenase in one mammalian species. Among the six amino acids that are different to rabbit 15-lipoxygenase, leucine 353 is shown to be the primary determinant for 12-positional specificity. In the 3'-untranslated region of the 12-LOX-mRNA a CU-rich, 20-fold repetitive element has been found, closely related to the differentiation control element (DICE) of the rabbit 15-LOX-mRNA which is organized by ten repeats of 19 bases. By genomic PCR the 3'-terminal part of the gene for the novel 12-lipoxygenase containing the introns 10-13 has been amplified and sequenced. The introns were very similar in length to the corresponding 15-lipoxygenase introns with 89% to 95% identical nucleotide sequences. By screening a rabbit reticulocyte library an alternative 15-lipoxygenase transcript of 3.6 kb has been detected containing a 1019 nucleotides longer 3'-untranslated region (UTR2) than the main 2.6 kb mRNA. The determination of the tissue distribution by Northern blotting showed that the 3.6 kb mRNA2 was only expressed in non-erythroid tissues, whereas the 2.6 kb mRNA1 was exclusively expressed in reticulocytes. The only cell type which has been found to express the 1.12-lipoxygenase abundantly are monocytes. The results indicate that the expression of 1.12-lipoxygenase and 15-lipoxygenase is highly regulated. The UTR2 of the 15-LOX-mRNA2 contained a novel eight-fold repetitive CU-rich motif of 23 bases length which is related but not identical to the DICE of 19 bases in the UTR1. The analysis of a genomic recombinant of the complete 9.0 kb Alox15 gene confirmed that UTR1 and UTR2 are not interrupted by an additional intron.

Alternative Splicing↗
PubMed1998

Structure of the promoter and complete sequence of the gene coding for the rabbit translationally controlled tumor protein (TCTP) P23.

We have isolated genomic recombinants containing the complete gene coding for the rabbit translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF) P23. The gene is organized into five introns and six exons and its total length amounts to 3819 nucleotides. All intron/exon boundaries are in accordance with the GT/AG rule. Transcription of the gene generates two mRNAs of 843 and 1163 nucleotides differing in the length of their 3'-untranslated regions. They are formed by alternative polyadenylation. The transcription initiation site has been determined by comparison of sequences of the gene and several processed TCTP pseudogenes. The full-length 5'-untranslated region comprises 116 nucleotides and starts with an oligopyrimidine tract important for translational regulation. Additionally 1.2 kb of the 5'-flanking promoter region has been sequenced. The promoter contains a TATA box at -30 and potential binding sites for transcription factors such as stimulating protein 1 (Sp1), nuclear factor 1 (NF1), activator protein 1 (AP1), c-Ets1, cAMP-response element (CP2), myeloid-specific zinc finger protein 1 (MZF1) and others. For functional analysis 5'-flanking sequences up to -918 were fused to the chloramphenicol acetyltransferase (CAT) gene and tested using a rabbit aortic smooth-muscle cell line by cell transfection and CAT assays. The results confirm that the analyzed gene is the actively transcribed TCTP gene.

Amino Acid Sequence↗
PubMed1998

Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits.

In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis.

Amino Acid Sequence↗
PubMed1997

mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end.

Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3' untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K and hnRNP E1 into HeLa cells specifically silenced the translation of reporter mRNAs bearing a differentiation control element in their 3' untranslated region. Silenced LOX mRNA in rabbit reticulocytes specifically coimmunoprecipitated with hnRNP K. In a reconstituted cell-free translation system, addition of recombinant hnRNP K and hnRNP E1 recapitulates this regulation via a specific inhibition of 80S ribosome assembly on LOX mRNA. Both proteins can control cap-dependent and internal ribosome entry site-mediated translation by binding to differentiation control elements. Our data suggest a specific cytoplasmic function for hnRNPs as translational regulatory proteins.

Amino Acid Sequence↗
PubMed1997

Molecular analysis of 29 pyruvate kinase-deficient patients from central Europe with hereditary hemolytic anemia.

We investigated the DNA of 29 unrelated pyruvate kinase (PK) deficiency (PKD) patients from Central Europe with hereditary nonspherocytic hemolytic anemia for mutations in the PK-L/R gene. Among 58 potentially affected alleles, 53 mutations were identified, of which 17 were different from each other. Of these 17 mutations, 13 were single-nucleotide (nt) substitutions resulting in amino acid exchanges, G787A (Gly263-Arg), G994A (Gly332-Ser), G1006T (Ala336-Ser), G1010A (Arg337-Gln), A1081G (Asn361-Asp), G1127T (Ser376-Ile), G1174A (Ala392-Thr), G1281T (Glu427-Asp), C1454T (Ser485-Phe), C1456T (Arg486-Trp), G1493A (Arg498-His), G1529A (Arg510-Gin), and C1594T (Arg532-Trp); 1 in-frame triplet deletion, 1060delAAG (delLys354); 1 in-frame triplet insertion, 1203insAGC (insSer after Cys401); 1 splicesite mutation, 101-1G-A; and 1 frameshift deletion, 628delGT. Six mutations, 628delGT, G787A, G1010A, G1127T, G1281T, and C1454T, are described for the first time. To test the hypothesis of a single origin of the most common PK mutation in the European population, G1529A, we investigated all patients at four polymorphic sites in the PK-L/R gene: C/A at nt 1705, C/T at nt 1992, the (ATT)n microsatellite in intron J, and a polymorphism (T)10/(T)19 in intron I. Nine patients homozygous for mutation G1529A were consistent in all four markers. In the group of patients homozygous for mutation G1529A, the hematologic parameters and clinical manifestations have been studied in detail. Although having an identical mutation in the PK-L/R gene, the patients are affected differently. Their appearance ranges from a very mild compensated hemolysis to a severe anemia. Possible molecular explanations are discussed.

Anemia, Hemolytic↗
PubMed1997

Complete genomic sequence of the human PK-L/R-gene includes four intragenic polymorphisms defining different haplotype backgrounds of normal and mutant PK-genes.

The human pyruvate kinase L/R-gene has been completely sequenced in unrelated normal individuals and in pyruvate kinase-deficient patients by a PCR-based direct genomic sequencing approach and analyzed for polymorphisms. The total length of the gene is 8409 nucleotides. Four polymorphic sites have been detected: C/A1705 and C/T1992 in exon 12, a T-stretch in intron 1 occurring in the two polymorphic forms (T)10 and (T)19 and an (ATT)n microsatellite in intron J which has been found in the variation (ATT)11-17. Haplotype analysis using these four markers has been applied to trace the genetic background in PK-deficiencies. The results support the idea of a single origin of most of the individual PK-mutations.

Anemia, Hemolytic↗
PubMed1994

Mutations in the pyruvate kinase L gene in patients with hereditary hemolytic anemia.

We have completely sequenced the introns of the human L-type pyruvate kinase (PK) gene using the published cDNA sequence. Subsequently, DNA from 12 unrelated PK deficiency (PKD) patients of Central European origin was investigated for mutations in this gene by solid-phase sequencing. We detected 10 different mutations, 9 of which result in single amino acid alterations, whereas the tenth destroys a splice site. Eight of the 10 mutations have not been described before. We found 7 missense mutations: G994-->A (Gly-332-->Ser), G1006-->T (Ala-336-->Ser), A1081-->G (Asn-361-->Asp), G1174-->A (Ala-392-->Thr), G1493-->A (Arg-498-->His), G1529-->A (Arg-510-->Gln), C1594-->T (Arg-532-->Trp), one in-frame triplet deletion (del) as well as one insertion (ins): del AAG1060-62 (del Lys-354), ins AGC after C1203 (ins Ser after Cys-401), and one splice-site mutation at the border of intron A to exon 3: g/G283-->a/G. Although the enzymatic properties are substantially changed in all PK mutations, only two affected amino acid positions are in or close to the active site. Mutations C1594-->T, G994-->A, del AAG1060-62 and the splice-site mutation g/G283-->a/G have been detected in two different patients each. Mutation G1529-->A was found in five different alleles. Haplotype analysis with the A/C polymorphism at position 1705 gave evidence for a single origin of this most frequent mutation in PKD as suggested by Baronciani and Beutler (Proc Natl Acad Sci USA 90:4324, 1993). Carrier detection and prenatal diagnosis are now feasible for the affected families.

Anemia, Hemolytic, Congenital↗
PubMed1994

Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region.

During red blood cell differentiation, the mRNA encoding rabbit erythroid 15-lipoxygenase (LOX) is synthesized in the early stages of erythropoiesis, but is only activated for translation in peripheral reticulocytes. Erythroid LOX, which like other lipoxygenases catalyses the degradation of lipids, is unique in its ability to attack intact phospholipids and is the main factor responsible for the degradation of mitochondria during reticulocyte maturation. Strikingly, rabbit erythroid LOX mRNA has 10 tandem repeats of a slightly varied, pyrimidine-rich 19 nt motif in its 3'-untranslated region (3'-UTR). In this study we demonstrate, using gel retardation and UV-crosslinking assays, that this 3'-UTR segment specifically binds a 48 kDa reticulocyte protein. Furthermore, the interaction between the 3'-UTR LOX repeat motif and the 48 kDa protein, purified to homogeneity by specific RNA chromatography, is shown to be necessary and sufficient for specific translational repression of LOX as well as reporter mRNAs in vitro. To our knowledge this is the first case in which translation, presumably at the initiation step, is regulated by a defined protein-RNA interaction in the 3'-UTR.

Animals↗
PubMed1993

Bacterial expression, purification and partial characterization of recombinant rabbit reticulocyte 15-lipoxygenase.

The recombinant rabbit reticulocyte 15-lipoxygenase has been expressed in E. coli with a yield of about 50-70 micrograms pure lipoxygenase protein per 1 of liquid culture. The enzyme has been purified to apparent homogeneity from the bacteria lysis supernatant by ammonium sulfate precipitation, and two consecutive steps of anion exchange chromatography on a Mono Q column. As the native enzyme the recombinant lipoxygenase has a molecular mass of 75 kDa, an isoelectric point of 5.5 and oxygenates both linoleic acid (formation of 13S-hydroperoxy-9Z,13E-octadecadienoic acid) and arachidonic acid. With the latter substrate it exhibits a dual positional specificity (formation of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetranoic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid in a ratio of 12:1). Furthermore, the enzyme is capable of oxygenating biomembranes, as indicated by HPLC analysis of esterified oxygenated polyenoic fatty acids.

Animals↗
PubMed1990

Structure of the mRNA and of the gene coding for the rabbit erythroid 15-lipoxygenase.

The complete structure of the rabbit erythroid cell-specific 15-lipoxygenase mRNA and its gene was established by sequencing cDNA and genomic recombinants. The transcription initiation site was obtained by primer-extension sequencing. A presumptive promoter structure was characterized by sequencing 0.5 kb 5' to the transcription initiation site and by transfection experiments using constructs with the chloramphenicol transferase gene. The mRNA codes for a polypeptide of 662 amino acids. Its 3' untranslated region contains an intriguing repeated sequence of 10 copies with the consensus C4PuC3TCTTC4AAG which may be involved in its regulation during reticulocyte maturation. The transcription unit consists of 8.0 kb and is split like the gene for the leukocyte 5' lipoxygenase by 13 introns. Another interesting aspect in the structure of the 15-LOX gene is a highly conserved repeat in intron seven consisting of a unit of 54 nucleotides which is repeated eight times. Comparing the predicted amino acid sequence with those from other lipoxygenases published recently shows that lipoxygenases are a related group of enzymes which may have arisen from a common ancestral gene.

Amino Acid Sequence↗
PubMed1989

The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase.

We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5' fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.

Animals↗