Structural Organization of the Gene for the E1α Subunit of the Human Pyruvate Dehydrogenase Complex

Maragos, C.; Hutchison, Wendy; Hayasaka, Kiyoshi; Brown, Garry K.; Dahl, Hans Henrik M.

doi:10.1016/s0021-9258(18)63857-8

Cited by 50 publications

(7 citation statements)

References 25 publications

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“…1993 American Chemical Society amplification in the presence of specific primers. The sense primer spanned bases -763 to -743 (5'-CCCCCTTAAAAT-GTGGAGCCC-3') from the putative transcription start site of the human Eia gene (Maragos et al, 1989). Antisense primer (5'-CTTCAGGTGCCCCAGCAGA-3') was complementary to bases +15 to +33 (Maragos et al, 1989) of the human Eia gene.…”

Section: Methodsmentioning

confidence: 99%

“…The sense primer spanned bases -763 to -743 (5'-CCCCCTTAAAAT-GTGGAGCCC-3') from the putative transcription start site of the human Eia gene (Maragos et al, 1989). Antisense primer (5'-CTTCAGGTGCCCCAGCAGA-3') was complementary to bases +15 to +33 (Maragos et al, 1989) of the human Eia gene. The amplification mixture (100 ^L) contained 2.5 units of Thermus aquaticus (Taq) polymerase, 100 pmol each of the sense and antisense primers, 200 nmol of each dNTP, and 3 pg of genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

“…The level of Eia mRNA is regulated by the glucose concentration in cultured pancreatic islets and by fasting in isolated islet preparations (MacDonald et al, scription has not been examined. The genomic structure and the 5'-flanking sequence of the human Eja gene have been reported (Maragos et al, 1989). This information provides the basis for further investigation of the mechanisms underlying regulated expression of this gene.…”

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confidence: 94%

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Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase .alpha.-subunit gene

Chang¹,

Naik²,

Johanning³

et al. 1993

Biochemistry

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We have characterized the 5'-flanking region of the alpha-subunit gene of the human pyruvate dehydrogenase (E1). DNase I footprinting with rat liver nuclear extracts identified 7 major protein-binding domains termed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 to +33). P1 through P4 are clustered in the -221/+33 region. These protein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been implicated in the constitutive transcription of several genes. Oligonucleotide competition studies indicate that oligonucleotides specific for CTF/NF-1 and Sp1 displaced the nuclear proteins bound to the CAAT box (within P3) and an Sp1 site (within P4), respectively. Several other well-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, AP-2, and Sp1) have been shown to bind to the -221/+33 region. Other elements located upstream of the -221/+33 region, which includes nuclease protection domains P5-P7, are required for enhanced promoter activity of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to deletion fragments of the 5'-flanking region. Crucial element(s) for promoter activity and complex DNA-nuclear protein interactions were confined within a region spanning -221/+33. This region also retained more than 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and housekeeping gene promoters, suggesting complex transcriptional regulation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 94%

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Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase .alpha.-subunit gene

Chang¹,

Naik²,

Johanning³

et al. 1993

Biochemistry

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“…The Ei enzyme is a heterotetramer of 2a and 2(3subunits, but so far only mutations in the E{a gene have been found. This gene is approximately 17 kilobase (kb) long, contains 11 exons, and is localized on the X-chromosome in band Xp22.1 (Koike et al, 1990;Maragos et al, 1989;Brown et alM 1989), mRNA is expressed in all tissues and is approximately 1.5 kb long, encoding a protein of 390 amino acids (Dahl et al, 1987;De Meirleir et al, 1988;Koike et al, 1988;Ho et ah, 1989).…”

Section: Introductionmentioning

confidence: 99%

Mutation analysis of the pyruvate dehydrogenase E1α gene in eight patients with a pyruvate dehydrogenase complex deficiency

Lissens¹,

Meırleır²,

Seneca³

et al. 1996

Hum. Mutat.

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Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X-linked E ^ gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single-strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA* The 4 female patients were shown to carry both a normal and a mutated Exa gene.

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“…

Ho & Patel, 1990). The DNA sequences of the human Ela and El/3 genes have also been reported (Maragos et al, 1989;Koike et al, 1990). The protein-binding regions and functional cis elements in the 5'-flanking region of the a subunit gene have been reported (Chang et al, 1993), but such information is not available on the human El/3 promoter-regulatory region.

…”

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confidence: 99%

Characterization of the Promoter Regulatory Region of the Human Pyruvate Dehydrogenase .beta. Gene

Madhusudhan¹,

Naik²,

Patel³

1995

Biochemistry

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A genomic clone (19 kb) harboring the intron-exon sequences and the promoter-regulatory region of the E1 beta gene of human pyruvate dehydrogenase complex was isolated by screening a placental genomic library. The nucleotide sequence of the promoter region (1245 bp) showed 18 differences (including mismatches, insertions, and deletions) as compared to the published sequence [Koike et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5594-5597]. The E1 beta promoter lacked a TATA box homology but contained initiator sequences (two) and Sp1 sites (three) which are frequently found in TATA-less promoters. The DNase I footprinting pattern of the promoter region with crude rat liver nuclear extracts showed at least seven regions of protein binding and nuclease protection (P1-P7). The DNase I protected regions contained consensus nucleotide sequences recognized by GATA-1, Sp1, IgNF-A, Lva, bicoid Q9, NF-kB, HNF-5, H4TF-1, WAP5, and ADH transription factors. Transient expression of chloramphenicol acetyltransferase (CAT) suggested the possible presence of negative elements located within the sequence from -2316 to -930, whereas deletion constructs containing -929 to +32 and -98 to +32 DNA sequences showed approximately 7- and 20-fold increases in CAT activity over the basal CAT activity. Additional studies indicated the presence of an orientation-dependent cis element (or elements) within the region from -282 to -397 that acts as an enhancer or a repressor upon a heterologous thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

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Structural Organization of the Gene for the E1α Subunit of the Human Pyruvate Dehydrogenase Complex

Cited by 50 publications

References 25 publications

Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase .alpha.-subunit gene

Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase .alpha.-subunit gene

Mutation analysis of the pyruvate dehydrogenase E1α gene in eight patients with a pyruvate dehydrogenase complex deficiency

Characterization of the Promoter Regulatory Region of the Human Pyruvate Dehydrogenase .beta. Gene

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