Glucagon gene transcription in the endocrine pancreas is regulated by at least four cis-acting DNA control elements. We showed previously that G1 is critical for alpha cell-specific expression. G1 contains three AT-rich sequences important for promoter function, which represent candidate binding sites for homeodomain transcription factors. Performing reverse transcription-polymerase chain reaction amplifications with degenerate oligonucleotide primers homologous to the Antennapedia homeobox, cDNA clones corresponding to the caudal-related gene cdx-2/3 were predominantly obtained from glucagon-producing cells and primary non-beta cells. From RNase protection and polymerase chain reaction analyses, cdx-2/3 turned out to be the only caudalrelated gene that is expressed at significant levels in cells of the endocrine pancreas. Cdx-2/3 binds with high affinity to an AT-rich motif of G1, which matches the consensus binding site of caudal-related proteins. In the glucagon-producing hamster cell line InR1G9, Cdx-2/3 is a subunit of complex B3 formed on G1. Alternative splicing generates two cdx-2/3 transcripts in islet cells, coding for a full-length protein and an amino-terminally truncated isoform. Although both isoforms bind G1 with similar affinity, only the full-length Cdx-2/3 A protein activates glucagon gene transcription in non-glucagonproducing cells, transcriptional activation being dosedependent. We therefore conclude that the caudal-related gene cdx-2/3 is implicated in the transcriptional control of glucagon gene expression in the alpha cells of the islets of Langerhans.Glucagon and the glucagon-like peptides are synthesized as a common precursor, preproglucagon, encoded by the glucagon gene and are involved in the control of glucose homeostasis.
The insulin and glucagon genes are expressed in the beta and alpha cells of the islets of Langerhans, respectively. The factors controlling their cell-and islet-specific expression are poorly known. Insulin-enhancer factor-1 (IEF1) has previously been shown to interact with the E boxes of the rat insulin I and II genes and was proposed to play a critical role in beta cell-specific expression. BETA2, a recently identified basic helix-loophelix (bHLH) protein, binds with high affinity and transactivates the rat insulin II gene upon dimerization with the ubiquitous bHLH protein E47. We show here that the heterodimer E47/BETA2 also binds and transactivates the rat insulin I and glucagon genes and exhibits the same characteristics as IEF1. In transfection experiments, the E boxes of the insulin I and glucagon genes confer transcriptional activity in both insulin-and glucagon-producing cells, which is increased by overexpression of E47 and BETA2. However, overexpression of E47 inhibits only E box-mediated glucagon gene expression, whereas it activates insulin gene transcription, indicating that the E boxes of the insulin and glucagon genes display gene-specific characteristics. We conclude that the heterodimer E47/BETA2 represents an islet-specific factor that controls both insulin and glucagon gene transcription and that the E47/BETA2 ratio may be important for regulated gene expression.Glucagon and insulin are two major antagonist hormones secreted by the endocrine pancreas which control glucose homeostasis. Glucagon gene expression in the adult is restricted to the alpha cells of the pancreas, the L-cells of the intestine, and some specific cell types in the central nervous system, whereas insulin gene expression is limited to pancreatic beta cells. The regulation as well as the cell-specific expression of both genes rely on specific interactions between cis-acting DNA sequences of their promoters and trans-acting factors (1, 2).Four DNA control elements have been defined within the first 300 bp 1 of the rat glucagon gene promoter which function as islet-or cell-specific cis-acting elements (3, 4). G1 is a proximal promoter element located upstream of the TATA box and involved in cell-specific expression (5). G2 and G3 are distal enhancers, whereas G4 is located in the proximal promoter just upstream of G1 (4, 6). The G4 element contains two palindromic consensus sequence CANNTG motifs, one of which (E3) functions as an E box, separated by an intervening sequence. The complete element acts as a mini-enhancer in glucagonproducing cells (6). E box motifs bind factors which belong to the basic helixloop-helix (bHLH) family of transcription factors. These factors have been shown to participate in the regulation of several celland tissue-specific genes (7-11). The bHLH transcription factor family is divided into three groups according to their structure, DNA-binding ability, and cell distribution. Class A factors, such as USF or E47, are ubiquitous and bind DNA as homo-or heterodimers, whereas class B factors are tissue...
Triticale, an intergeneric hybrid crop-plant, is generated when female wheat lines are fertilised with pollen from rye. We have investigated the mitochondrial DNA organisation and the expression of a total of 11 different triticale genotypes, varying in their nuclear and cytoplasmic backgrounds. In Southern hybridisations using probes homologous to the upstream flanking sequences, mtDNA fragments characteristic of both wheat and rye mtDNA can be detected in all triticale lines analysed. In addition, clones isolated fom a triticale lambda library exhibit either a maternal-like or paternal-like organisation of the orf25 gene region. By PCR cloning, four different orf25 gene copies were identified in triticale, three of which correspond to maternal (85%) or paternal (12%) orf25 sequences. Three percent of all clones represent a novel type, that might have arisen by homologous recombination. Although these data suggest biparental inheritance of mtDNA in wheat/rye crosses, paternal-like gene copies can also be detected in maternal wheat mitochondria. Their stoichiometry as assayed by competitive PCR is about 0.1% of total orf25 gene copies. The high abundance of paternal-like sequences in the F1 hybrid might therefore be due to either the transmission of rye mtDNA in the intergeneric cross and/or the amplification of sequences in triticale that persist in sub-stoichiometric amounts in wheat. These data suggest that amplification and recombination of sub-genomic mitochondrial molecules are affected by different nuclear genotypes. Interestingly, sequence analysis of triticale RT-PCR clones indicates a selective transcription of maternal-like orf25 gene copies in triticale. Mitochondrial gene expression may therefore possess mechanisms to compensate for the variation of mtDNA organisation.
Subunits a and nine are components from two different portions of the mt H+-ATP synthase enzyme, namely the soluble catalytic (F,) and the membranous proton-conduction (F,-ATPase) complexes. In wheat and triticale the genes for both subunits are located adjacently in the mt genome, irrespective of whether these lines are carrying the aestivum, durum, or timopheevi cytoplasmic male sterility cytoplasm. However, in the timopheevi mt genome the atpA/ atp9 cistron is part of a large DNA repeat and thus present in two different genomic environments. The repeat comprises at least 9 kb that are delimited 941 bp downstream of the atp9 gene. The 2.7-kb triticale atpAlatp9 sequence shows 100% homology with a Triticum aestivum sequence (Schulte et al., 1989), containing both reading frames, the intergenic region, and at least the first 400 bp upstream and the first 46 bp downstream of the atpAlatp9 gene region.Transcription of the atpAlatp9 cistron in triticale gives rise to a co-transcript of 2.6 kb, which is posttranscriptionally modified by 5' processing. To investigate whether further RNA modifications occur, we have analyzed this triticale transcript for RNA editing (Table I). To date, editing of an atpA transcript has been described in only two dicotyledonous species (Schuster et al., 1991; Senda et al., 1993). Overlapping cDNA clones covering the completely transcribed gene region were generated by reverse transcription PCR, and 10 individual clones were subsequently sequenced. In total, 17 RNA-editing sites could be identified, a11 of which represent cytidine to uridine transitions. The open reading frame of the atpA transcript carries five nonsilent and two silent RNA-editing sites. Equivalent alterations have been detected in Oenothera (amino acid 497) and sugar beet (amino acids 393 and 431), while a11 remaining sites are pre-edited in these two dicotyledonous species. Modified codons are located preferentially in the carboxy-terminal region of the protein, while silent RNAediting sites are found exclusively at the amino terminus of the a subunit. Enzymatic studies with peptides derived by limited proteolysis from the a subunit suggest that the Table 1. Characteristics of the atpA a n d atp9 genes a n d their derived cDNA from triticale Organism:Triticale (X Triticosecale Wittmack; alloplasmic Triticum durum D30, timopheevi cytoplasm X Secale cereale L301). Function:The atpA and atp9 genes code for subunits of the soluble catalytic portion (F,) and the membranous proton-conducting portion ( F, ) of the m t ATP synthase complex, respectively. Cloning Techniques: D N A cloning: A genomic library in EMBL4 (Mohr et al., 1993) was screened with an oligonucleotide specific for the atpA gene. Restriction fragments were subcloned into the p U C l 9 or pBluescript vectors. cDNA cloning: cDNA synthesis was performed using DNaseltreated total RNA from triticale and five specific antisense oligonucleotides. Subsequent PCR amplification was done according to standard protocols using Replitherm (Biozym, Hameln, Cermany)...
The gene region coding for subunits alpha and 9 of the mitochondrial ATP synthase exhibit an identical DNA sequence in wheat, rye, and the intergeneric hybrid triticale (xTriticosecale Wittmack). However, co-transcripts containing both genes show different sizes depending on the nuclear genotype. To investigate nuclear-mitochondrial interactions leading to this variation, we performed a comparative transcript analysis with various lines carrying defined nuclear and cytoplasmic genotypes. Northern analyses showed that all wheat lines investigated possess a single atpA/atp9 mRNA of 2.6kb, whereas in rye and five independent triticale lines an additional transcript of 2.35kb appeared. Primer-extension and RNase-protection analyses indicate that the co-transcripts of this gene have staggered 5' termini in some lines, whereas the 3' termini seem to be similar in wheat, rye, and triticale. Transcription is initiated at position -338/-339 upstream of the atpA gene in all lines investigated, giving rise to a 2.6-kb mRNA. In rye and triticale, staggered 5' termini were observed closer to the translational start. The DNA sequences upstream of these termini exhibit homology to plant mitochondrial-processing sites, therefore the proximal 5' ends are most probably generated by RNA processing. As the processing event occurs more frequently in triticale carrying the Triticum timopheevi cytoplasm, trans-acting factors from rye are likely to interact with other cytoplasmic factors resulting in the observed RNA modification. Most interestingly, the T. timopheevi cytoplasm inducing male sterility in alloplasmic wheat, fails to generate the CMS phenotype in triticale. The data support our hypothesis that nuclear factors affect mitochondrial gene expression and thus control sexual fertility in wheat and triticale.
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