Gene Encoding Glutamate Dehydrogenase Research Articles

Transcriptomic response to nitrogen availability reveals signatures of adaptive plasticity during tetraploid wheat domestication.

The domestication of crops, coupled with agroecosystem development, is associated with major environmental changes and provides an ideal model of phenotypic plasticity. Here, we examined 32 genotypes of three tetraploid wheat (Triticum turgidum L.) subspecies, wild emmer, emmer, and durum wheat, which are representative of the key stages in the domestication of tetraploid wheat. We developed a pipeline that integrates RNA-Seq data and population genomics to assess gene expression plasticity and identify selection signatures under diverse nitrogen availability conditions. Our analysis revealed differing gene expression responses to nitrogen availability across primary (wild emmer to emmer) and secondary (emmer to durum wheat) domestication. Notably, nitrogen triggered the expression of twice as many genes in durum wheat compared to that in emmer and wild emmer. Unique selection signatures were identified at each stage: primary domestication mainly influenced genes related to biotic interactions, whereas secondary domestication affected genes related to amino acid metabolism, in particular lysine. Selection signatures were found in differentially expressed genes (DEGs), notably those associated with nitrogen metabolism, such as the gene encoding glutamate dehydrogenase (GDH). Overall, our study highlights the pivotal role of nitrogen availability in the domestication and adaptive responses of a major food crop, with varying effects across different traits and growth conditions.

Improved Glutamic Acid Production Capacity of Corynebacterium glutamicum by the ARTP Mutagenesis Method

Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.

Salt Stress-Related Mechanisms in Leaves of the Wild Barley Hordeum spontaneum Generated from RNA-Seq Datasets.

This study aims to detect salt stress-related genes and mechanisms of the wild barley Hordeum spontaneum. Among the generated RNA-Seq datasets, several regulated transcripts are influenced by levels of cellular carbon, nitrogen and oxygen. Some of the regulated genes act on photorespiration and ubiquitination processes, as well as promoting plant growth and development under salt stress. One of the genes, encoding alanine:glyoxylate aminotransferase (AGT), participates in signaling transduction and proline biosynthesis, while the gene encoding asparagine synthetase (ASN) influences nitrogen storage and transport in plants under stress. Meanwhile, the gene encoding glutamate dehydrogenase (GDH) promotes shoot and root biomass production as well as nitrate assimilation. The upregulated genes encoding alpha-aminoadipic semialdehyde synthase (AASAS) and small auxin-up RNA 40 (SAUR40) participate in the production of proline and signaling compounds, respectively, while the gene encoding E3 ubiquitin-protein ligase regulates the carbon/nitrogen-nutrient response and pathogen resistance, in addition to some physiological processes under biotic and abiotic stresses via signal transduction. The gene encoding the tetratricopeptide repeat (TPR)-domain suppressor of STIMPY (TSS) negatively regulates the carbon level in the cell. In conclusion, this study sheds light on possible molecular mechanisms underlying salt stress tolerance in wild barley that can be utilized further in genomics-based breeding programs of cultivated species.

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

BackgroundItaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy.ResultsIn this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L−1 with a product yield of 81 ± 9 mmol mol−1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L−1 and 116 ± 15 mmol mol−1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L−1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis.ConclusionAs shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Increasing Agmatine Production in Escherichia coli through Metabolic Engineering.

In this study, to obtain higher agmatine yields using the previously developed E. coli strain AUX4 (JM109 ΔspeC ΔspeF ΔspeB ΔargR), the genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), transhydrogenase (pntAB), and biosynthetic arginine decarboxylase (speA) were sequentially overexpressed by replacing their native promoters with the heterologous strong trp, core-trc, or 5Ptacs promoters to generate the plasmid-free E. coli strain AUX11. The fermentation results obtained using a 3-L bioreactor showed that AUX11 produced 2.93 g L-1 agmatine with the yield of 0.29 g agmatine g-1 glucose in the batch fermentation, and the fed-batch fermentation of AUX11 allowed the production of 40.43 g L-1 agmatine with the productivity of 1.26 g L-1 h-1 agmatine. The results showed that the engineered E. coli strain AUX11 can be used for the industrial fermentative production of agmatine.

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyzes the reductive amination of 2-oxoglutarate with ammonium and the oxidative deamination of glutamate. In some plants species, GDH is a hexamer and can be separated into seven isoenzymes that are composed of two distinct subunits: α and β. The large number of isoenzymes is the reason why GDH functions are still being intensively researched and widely studied. Until recently, the β subunit of GDH in triticale, a common Polish cereal, was thought to be encoded by the TsGDH1 gene, which can undergo posttranslational modifications and form a heterohexameric enzyme. Here, we report the cloning and molecular characterization of a second glutamate dehydrogenase gene—TsGDH2 (encoding α subunits). The TsGDH2 cDNA contains a 1236-bp open reading frame encoding a 411-amino-acid polypeptide with a calculated molecular mass of 44.5 kDa. To clarify the role of TsGDH2 in triticale, we used triticale GDH α-subunit cDNA to generate transgenic A. thaliana lines with increased and decreased GDH activity via alternation of α-subunit levels.

Glutamate Dehydrogenase Is Required by Mycobacterium bovis BCG for Resistance to Cellular Stress.

We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.

Characterization of natural leaf senescence in tobacco (Nicotiana tabacum) plants grown in vitro.

Leaf senescence is a highly regulated final phase of leaf development preceding massive cell death. It results in the coordinated degradation of macromolecules and the subsequent nutrient relocation to other plant parts. Very little is still known about early stages of leaf senescence during normal leaf ontogeny that is not triggered by stress factors. This paper comprises an integrated study of natural leaf senescence in tobacco plants grown in vitro, using molecular, structural, and physiological information. We determined the time sequence of ultrastructural changes in mesophyll cells during leaf senescence, showing that the degradation of chloroplast ultrastructure fully correlated with changes in chlorophyll content. The earliest degenerative changes in chloroplast ultrastructure coinciding with early chromatin condensation were observed already in mature green leaves. A continuum of degradative changes in chloroplast ultrastructure, chromatin condensation and aggregation, along with progressive decrease in cytoplasm organization and electron density were observed in the course of mesophyll cells ageing. Although the total amounts of endogenous cytokinins gradually increased during leaf ontogenesis, the proportion of bioactive cytokinin forms, as well as their phosphate precursors, in total cytokinin content rapidly declined with ageing. Endogenous indole-3-acetic acid (IAA) levels were strongly reduced in senescent leaves, and a decreasing tendency was also observed for abscisic acid (ABA) levels. Senescence-associated tobacco cysteine proteases (CP, E.C. 3.4.22) CP1 and CP23 genes were induced in the initial phase of senescence. Genes encoding glutamate dehydrogenase (GDH, E.C. 1.4.1.2) and one isoform of cytosolic glutamine synthetase (GS1, E.C. 6.3.1.2) were induced in the late stage of senescence, while chloroplastic GS (GS2) gene showed a continuous decrease with leaf ageing.

Disruption of the gene encoding glutamate dehydrogenase affects growth, amino acids catabolism and survival of Lactobacillus plantarum UC1001

The role played by glutamate dehydrogenase (GDH) in Lactobacillus plantarum was investigated by preparing a GDH-deficient mutant UC1001G. The growth rate and acidification displayed by this mutant were lower than those of the wild-type UC1001. Compared with UC1001G, UC1001 increased the synthesis of proteins that confer a competitive advantage of cell resistance. When both strains were used as adjunct starter for cheese-making, the highest cell survival was found for UC1001. Cheeses made with the adjunct of UC1001 showed the lowest level of free amino acids and the highest content of volatile organic compounds. Both strains produced high level of γ-aminobutyric acid in cheese and under simulated gastrointestinal conditions. The findings of this study provide an evidence of the important role of GDH on growth and survival of Lb. plantarum under different environmental conditions that influences some important functional features of the strain.

Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens

Geobacter species often play an important role in bioremediation of environments contaminated with metals or organics and show promise for harvesting electricity from waste organic matter in microbial fuel cells. The ability of Geobacter species to fix atmospheric nitrogen is an important metabolic feature for these applications. We identified novel regulatory cascades controlling nitrogen-fixation gene expression in Geobacter sulfurreducens. Unlike the regulatory mechanisms known in other nitrogen-fixing microorganisms, nitrogen-fixation gene regulation in G. sulfurreducens is controlled by two two-component His–Asp phosphorelay systems. One of these systems appears to be the master regulatory system that activates transcription of the majority of nitrogen-fixation genes and represses a gene encoding glutamate dehydrogenase during nitrogen fixation. The other system whose expression is directly activated by the master regulatory system appears to control by antitermination the expression of a subset of the nitrogen-fixation genes whose transcription is activated by the master regulatory system and whose promoter contains transcription termination signals. This study provides a new paradigm for nitrogen-fixation gene regulation.

Control of the Synthesis and Subcellular Targeting of the Two GDH Genes Products in Leaves and Stems of Nicotiana plumbaginifolia and Arabidopsis thaliana

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Ammonia metabolism in Aedes aegypti

We investigated the mechanisms by which Aedes aegypti mosquitoes are able to metabolize ammonia. When females were given access to solutions containing NH 4Cl or to a blood meal, hemolymph glutamine and proline concentrations increased markedly, indicating that ammonium/ammonia can be removed from the body through the synthesis of these two amino acids. The importance of glutamine synthetase was shown when an inhibitor of the enzyme was added to the meal causing the glutamine concentration in hemolymph to decrease significantly, while the proline concentration increased dramatically. Unexpectedly, we found an important role for glutamate synthase. When mosquitoes were fed azaserine, an inhibitor of glutamate synthase, the glutamine concentration increased and the proline concentration decreased significantly. This confirms the presence of glutamate synthase in mosquitoes and suggests that this enzyme contributes to the production of glutamate for proline synthesis. Several key enzymes related to ammonium/ammonia metabolism showed activity in homogenates of mosquito fat body and midgut. The mosquito genes encoding glutamate dehydrogenase, glutamine synthetase, glutamate synthase, pyrroline-5-carboxylate synthase were cloned and sequenced. The mRNA expression patterns of these genes were examined by a real-time RT-PCR in fat body and midgut. The results show that female mosquitoes have evolved efficient mechanisms to detoxify large loads of ammonium/ammonia.

Molecular Properties and Enhancement of Thermostability by Random Mutagenesis of Glutamate Dehydrogenase fromBacillus subtilis

The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD+ as cofactors respectively. The enzyme showed low thermostability with T(m) = 41 degrees C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their T(m) values were 61 degrees C and 49 degrees C respectively. Furthermore, Q144R had a remarkably high k(cat) value (435 s(-1)) for amination reaction at 37 degrees C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use.

Thermostable glutamate dehydrogenase from a commensal thermophile, Symbiobacterium toebii; overproduction, characterization, and application

A gene encoding glutamate dehydrogenase (GDH) was found in the genome sequence of a commensal thermophile, Symbiobacterium toebii. The amino acid sequence deduced from the gdh I of S. toebii was well conserved with other thermostable GDHs. The gdh I which encodes GDH consisting of 409 amino acids was cloned and expressed in E. coli DH5α under the control of a highly constitutive expression (HCE) promoter in a pHCE system. The recombinant GDH was expressed without addition of any inducers in a soluble form. The molecular mass of the GDH was estimated to be 263 kDa by Superose 6 HR gel filtration chromatography and 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicating that the GDH was composed of hexameric form. The optimal temperature and pH of the purified enzyme were 60 °C and 9.0, respectively, and the purified GDH retained more than 75% of its original activity after an incubation at 70 °C for 30 min. Although NADP(H) was the preferred cofactor, S. toebii GDH was able to utilize either NADP(H) or NAD(H) as coenzyme.

Characterization of the gene encoding glutamate dehydrogenase ( gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1.

The gene encoding glutamate dehydrogenase ( gdhA) in the ruminal bacterium Ruminococcus flavefaciens FD-1 was cloned. A degenerate primer based on the N-terminal amino acid sequence of the purified protein was used in conjunction with genome walking to obtain the complete ORF of 1,365 bp, capable of encoding a polypeptide of 455 amino acid residues. The translated ORF contained the amino acid motifs characteristic of the subfamily GDH S_50(I) small glutamate dehydrogenases, including the catalytic site, and matched the originally deduced N-terminal amino acid sequence. BLAST search yielded high scores with other GdhA sequences from a variety of organisms, the closest match being with the GdhA sequence of Corynebacterium glutamicum (63% amino acid identity). Classification of the GdhA enzyme from R. flavefaciens FD-1 as a GDH S_50(I) subfamily member was further supported by phylogenetic analysis. The transcript size determined by Northern blot analysis was in good agreement with the putative regulatory region of the gene and confirmed its monocistronic structure. R. flavefaciens GdhA activity appears to be regulated primarily at the level of transcription. Brief exposure to 20 mM NH(4)Cl prior to extraction did not alter the level of activity. Transcriptional regulation, studied with quantitative real-time RT-PCR, demonstrated a three-fold increase of the gdhA transcript concentration in ammonia-limited cells in comparison with an excess of ammonia in the medium. This is in agreement with the enzyme activity data obtained under ammonia- and carbon-limited growth conditions.

Glutamate dehydrogenase mRNA is immediately induced after phencyclidine treatment in the rat brain

To clarify the molecular mechanism of phencyclidine (PCP)-induced schizophreniform psychosis in humans and of behavioral abnormalities in experimental animals, we used differential screening of a cDNA library from the cerebral cortex of rats treated with PCP. We identified a PCP-induced cDNA clone as the gene encoding glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism. GDH mRNA levels significantly increased as early as 15 min following PCP administration in both the cerebral cortex and the cerebellum. This effect was observed even in the presence of a protein synthesis inhibitor, cycloheximide. In contrast to a transient increase in c- fos expression, the elevation of GDH mRNA levels lasted up to 8 days after a single PCP injection. These results suggest that GDH mRNA induction may be involved in the pathology of PCP-induced psychosis, and that GDH may be one of the candidate genes that are vulnerable in subjects with schizophrenia.

A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism.

In Bacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducible beta-galactosidase activity. One such isolate was obtained. The region upstream of lacZ was subcloned in Escherichia coli in such a way that it could be replaced in the B. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3' end of an ORF homologous to gdh genes encoding glutamate dehydrogenase, with highest homology to the homologue from Clostridium difficile; the 5' end of an ORF homologous to a Saccharomyces cerevisiae gene encoding delta 1-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosynthetic argCJBD-cpa-F cluster. The same region has also been sequenced by others as part of the B. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termed rocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 bp AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to bp -16/-22 to +1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

PCR-mediated cloning and sequencing of the gene encoding glutamate dehydrogenase from the archaeon Sulfolobus shibatae: identification of putative amino-acid signatures for extremophilic adaptation

Highly degenerate oligodeoxyribonucleotides (oligos) were used to PCR amplify the most conserved region of the glutamate dehydrogenase (GDH)-encoding gene from the extreme thermophilic archaeon, Sulfolobus shibatae. The amplified fragment was cloned and sequenced, and then used as a homologous probe to clone a genomic restriction fragment containing the near-complete gdhA gene. The deduced amino acid (aa) sequence shows a very high degree of similarity with the aa sequence previously determined by direct sequencing of the purified enzyme from Sulfolobus solfataricus [Maras et al., Eur. J. Biochem. 203 (1992) 81–87]. The introduction of this new sequence into our GDH phylogenetic trees [Benachenhou-Lahfa et al., J. Mol. Evol. 35 (1993) 335–346] showed that it is a new member of hexameric GDH family II, and did not modify the topology of the trees. Comparison of the primary structures of extremophilic GDH enzymes (halophilic, thermophilic and hyperthermophilic) with those of their non-halophilic and mesophilic counterparts in this family II led us to identify a few aa changes which seem to be specific either to hyperthermophilic or halophilic adaptation.

Isolation and characterization of the rat gene encoding glutamate dehydrogenase

The concentration of glutamate dehydrogenase (GDH) varies strongly between different organs and between different regions within organs. To permit further studies on the regulation of GDH expression, we isolated and characterized the rat gene encoding the GDH protein. This gene contains 13 exons and spans approximately 34 kbp. The GDH gene is present as a single, autosomally located copy in the Wistar rat genome, but shows an extensive restriction-fragment-length polymorphism for several enzymes. Promoter activity of the 5'-flanking sequence is shown by transient transfection experiments. The 5'-flanking sequence contains a TTAAAA sequence at position -29, instead of a consensus TATA box and, like many other TATA-less promoters, is characterized by a very high G + C content. In addition, consensus sequences for the binding sites of the transcription factors Sp1 and Zif268 are present in the G + C-rich upstream region.

Accurate transcription of cloned Neurospora RNA polymerase II-dependent genes in vitro by homologous soluble extracts.

We have developed soluble extracts from Neurospora crassa capable of accurately initiating the transcription of cloned Neurospora protein-encoding genes by RNA polymerase II in vitro. The genes encoding glutamate dehydrogenase (am) and histones H3 and H4 were transcribed by the extracts, and transcription was sensitive to alpha-amanitin at 1 mg/ml. The 5' heterogeneity of the in vitro initiation reactions was highly specific. Of the 17 transcription initiation sites within the inducible qa gene cluster, only one minor site was used in vitro, suggesting that, in general, transcription from qa gene promoters requires at least one different protein from those required for transcription of the am and histone genes.