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PubMed1983

Isolation of genomic DNA clones spanning the entire fibronectin gene.

Overlapping recombinant clones that appear to encompass the entire fibronectin gene have been isolated by step-wise screening of a library of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned fibronectin cDNA hybridization probe. The remaining clones were obtained by using defined fragments of this and successive genomic clones as probes. Their relationships and overlaps were determined by electron microscopy, restriction mapping, and heteroduplex analysis. Based on electron microscopic analysis of hybrids between these clones and fibronectin mRNA, the gene is approximately 48 kilobases long, more than 5 times larger than the corresponding mRNA. This large gene contains at least 48 exons interrupted by introns of highly variable size. The total exon size as estimated by R-loop analysis is 8 kilobases, similar to the mRNA for fibronectin. With the exception of the 3'- and 5'-terminal exons, the exons are small and roughly similar in size. The average exon size is 147 +/- 37 base pairs, corresponding to a protein unit of 50 amino acids. The nucleotide sequence of one of these exons was determined. The deduced amino acid sequence has marked homologies with one type of repetitive protein sequence unit known to exist in bovine fibronectin. These results suggest that the gene for fibronectin may have arisen by multiple gene duplications of a primordial gene or genes approximately equal to 150 base pairs long.

Amino Acid Sequence↗

PubMed1982

Molecular evolution in papova viruses and in bacteriophages.

By comparing the DNA sequences of three eukaryotic papova viral genomes, we attempt to show the very close relative phylogeny among the viral species and their host species, and that therefore the viral species appear to have evolved with their hosts. A comparison of the DNA data also reveals that the rate of nucleotide substitutions at the third positions of codons is much faster than at the first and second positions, though the rate varies depending upon the genes. The estimated rates of amino acid substitutions in homologous genes among the three virus species appear to be considerably faster than the rates known for various vertebrate genes. The comparison reveals further that the rate of silent substitutions is faster than that of replacement substitutions. The DNA sequence data on bacteriophages phi X174 and G4 enable us to examine the patterns of nucleotide substitutions in overlapping genes as well as in nonoverlapping genes. It then becomes evident that overlapping genes have a quite different substitutional pattern with respect to the position of nucleotides in codons than do nonoverlapping sequences. In nonoverlapping regions the third positions usually change the fastest among the three codon positions. This pattern does not apply to overlapping genes, which are coded in the same region but with different reading frames. It will be shown that the younger of the two overlapping genes appears to be very tolerant to nucleotide substitutions at any codon position. Further more, the rate of substitution at each nucleotide site appears to be determined by the rate of the corresponding site in the older of the two overlapping genes.

BK Virus↗

PubMed1990

Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.

Amino Acid Sequence↗

PubMed1989

Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.

Amino Acid Sequence↗

PubMed1995

Genomic organization of the human alpha-adducin gene and its alternately spliced isoforms.

The cDNA for the human alpha-adducin gene has been cloned, and different alternately spliced forms have been identified. We report the complete genomic organization of the human alpha-adducin gene and these alternately spliced forms. The human alpha-adducin gene, spanning approximately 85 kb, consists of 16 exons ranging in size from 34 to 1892 bp. One of the spliced forms of the human alpha-adducin gene results from alternate use of the 5' splice donor site for exon 10, while another results in a truncated protein following insertion of 34 bp comprising exon 15, followed by a premature stop codon. This alternate spliced form of alpha-adducin is predicted to result in an altered carboxyl terminus that would eliminate a protein kinase and calmodulin binding site. Seven nucleotide substitutions and 4 insertion/deletions were also identified. The 5' region of the human alpha-adducin gene contains one Sp1 site, two AP2 sites, and two CAAT boxes. No TATA box was apparent, consistent with features of a housekeeping gene. We have mapped another cDNA within the first intron of the human alpha-adducin gene, suggesting overlapping genes in this 4p16.3 genomic region.

Alternative Splicing↗

PubMed2004

The analysis of the complete mitochondrial genome of Lecanicillium muscarium (synonym Verticillium lecanii) suggests a minimum common gene organization in mtDNAs of Sordariomycetes: phylogenetic implications.

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Lecanicillium muscarium (synonym Verticillium lecanii) with a total size of 24,499-bp has been analyzed. So far, it is the smallest known mitochondrial genome among Pezizomycotina, with an extremely compact gene organization and only one group-I intron in its large ribosomal RNA (rnl) gene. It contains the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, one intronic ORF coding for a possible ribosomal protein (rps), and a set of 25 tRNA genes which recognize codons for all amino acids, except alanine and cysteine. All genes are transcribed from the same DNA strand. Gene order comparison with all available complete fungal mtDNAs-representatives of all four Phyla are included-revealed some characteristic common features like uninterrupted gene pairs, overlapping genes, and extremely variable intergenic regions, that can all be exploited for the study of fungal mitochondrial genomes. Moreover, a minimum common mtDNA gene order could be detected, in two units, for all known Sordariomycetes namely nad1-nad4-atp8-atp6 and rns-cox3-rnl, which can be extended in Hypocreales, to nad4L-nad5-cob-cox1-nad1-nad4-atp8-atp6 and rns-cox3-rnl nad2-nad3, respectively. Phylogenetic analysis of all fungal mtDNA essential protein-coding genes as one unit, clearly demonstrated the superiority of small genome (mtDNA) over single gene comparisons.

Codon↗

PubMed2025

Deciphering estrogen receptor alpha-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape.

OBJECTIVE: To investigate estrogen receptor gene 1 (ESR1) and estrogen-driven transcription in human endometrial stromal cells. DESIGN: RNA sequencing (RNA-seq) and Cleavage Under Targets and Release Using Nuclease (Cut&Run) were performed on telomerase-immortalized human endometrial stromal cells with Clustered Regularly Interspaced Short Palindromic Repeats-mediated ESR1 activation. Hi-C-based chromatin architecture analysis (H3K27ac HiChIP) was conducted in primary endometrial stromal cells. SUBJECTS: Biopsies from two healthy, reproductive-aged volunteers with regular menstrual cycles and no history of gynecological malignancies. EXPOSURE: The ESR1-activated and control endometrial stromal cells were treated with estradiol (E2) or vehicle. Primary endometrial stromal cells were treated with vehicle or a decidualization cocktail. MAIN OUTCOME MEASURES: Differential gene expression analysis (RNA-seq) identified ligand-independent and -dependent ESR1 activity. Cut&Run profiled ESR1 genomic binding in ESR1-activated cells. H3K27ac HiChIP mapped hormone-induced changes in chromatin looping in primary cells. RESULTS: Among seven tested guide RNAs (gRNA), the ESR1-3 gRNA induced robust ESR1 activation and restored E2 responsiveness. Bulk RNA-seq revealed both ligand-dependent and -independent ESR1 transcriptional programs regulating inflammation, proliferation, and cancer-related pathways. Notably, 72% of differentially expressed genes overlapped with genes active in human endometrial tissue during the proliferative estrogen-dominant phase, supporting their physiological relevance. The Cut&Run-seq identified genome-wide ESR1 binding sites, with most binding sites located at distal regulatory elements. Integration of Cut&Run data with H3K27ac HiChIP chromatin loops linked distal ESR1 binding sites to gene promoters, including genes involved in decidualization (e.g., FOXO1) and endometrial cancer (e.g., ERRFI1, NRIP1, and EPAS1). Functional assays showed that ESR1 promotes cell viability and, in the presence of E2, enhances migration. CONCLUSION: The CRISPR-mediated ESR1 activation restores estrogen responsiveness in endometrial stromal cells. Combined transcriptomic, cistromic, and chromatin architecture analyses reveal ESR1's role in regulating decidualization and inflammation-related gene networks, with relevance to endometrial pathologies including endometrial cancer. This model serves as a powerful tool to study estrogen signaling in endometrial stromal cell biology and related pathologies.

Humans↗

PubMed1996

Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides.

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103-108]. Here we describe protocols developed to purify polypeptides alpha and beta together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0 x 10(5) units per mg of protein. The respective denatured molecular masses of polypeptides alpha and beta were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 microM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.

Amino Acid Sequence↗

PubMed2003

Long-term global gene expression patterns in irradiated human lymphocytes.

Radiation-induced chromosomal instability has many features in common with genomic instability of cancer cells. In order to understand the delayed cellular response to ionizing radiation we have studied variations in the patterns of gene expression in primary human lymphocytes at various time points after gamma irradiation in vitro. Cells either exposed to 3 Gy of gamma rays in vitro or unexposed were subjected to long-term growth in bulk culture or as individual T-cell clones. Samples were taken at days 7, 17 or 55 from bulk cultures. The T-cell clones were harvested after 22-46 days. Total RNA was used to generate cDNA probes for hybridization to oligonucleotide arrays containing 12,625 gene templates (Affymetrix). The results showed that: (i) irradiation as well as culture time influence the gene expression patterns, (ii) the number of genes with increased or decreased expression in irradiated cells increases dramatically with increasing culture time, (iii) the changes of gene expression showed a significantly more diversified pattern in the irradiated T-cell clones than in non-irradiated clones. We conclude that the diversification of the transcriptome associated with radiation exposure reflects subtle changes of expression in many genes, rather than being the result of major changes in a few genes. Finally, (iv) we sorted out a set of genes whose change of expression correlates with radiation exposure in both bulk cultures and T-cell clones. Very few of these genes overlap with genes that change during the acute response to radiation. This set of genes may be regarded as a starting point for further studies of the cellular phenotype associated with radiation-induced genomic instability.

Cells, Cultured↗

PubMed2006

A genome-wide linkage scan for genes controlling variation in renal function estimated by serum cystatin C levels in extended families with type 2 diabetes.

We performed a variance components linkage analysis of renal function, measured as glomerular filtration rate (GFR), in 63 extended families with multiple members with type 2 diabetes. GFR was estimated from serum concentrations of cystatin C and creatinine in 406 diabetic and 428 nondiabetic relatives. Results for cystatin C were summarized because they are superior to creatinine results. GFR aggregates in families with significant heritability (h(2)) in diabetic (h(2) = 0.45, P < 1 x 10(-5)) and nondiabetic (h(2) = 0.36, P < 1 x 10(-3)) relatives. Genetic correlation (r(G) = 0.35) between the GFR of diabetic and nondiabetic relatives was less than one (P = 0.01), suggesting that genes controlling GFR variation in these groups are different. Linkage results supported this interpretation. In diabetic relatives, linkage was strong on chromosome 2q (logarithm of odds [LOD] = 4.1) and suggestive on 10q (LOD = 3.1) and 18p (LOD = 2.2). In nondiabetic relatives, linkage was suggestive on 3q (LOD = 2.2) and 11p (LOD = 2.1). When diabetic and nondiabetic relatives were combined, strong evidence for linkage was found only on 7p (LOD = 4.0). In conclusion, partially distinct sets of genes control GFR variation in relatives with and without diabetes on chromosome 2q, possibly on 10q and 18p in the former, and on 7p in both. None of these genes overlaps with genes controlling variation in urinary albumin excretion.

Adult↗

PubMed2006

Effect of fescue toxicosis on hepatic gene expression in mice.

Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endophytic fungus Neotyphodium coenophialum. Signs of fescue toxicosis include increased core body temperature and respiration rate and decreased milk yield and reproductive performance. Laboratory mice also exhibit symptoms of fescue toxicosis, as indicated by reduced growth rate and reproductive performance. Mice were used to study the effects of fescue toxicosis on hepatic gene expression. Twenty-seven mice were randomly allocated to a diet containing either 50% endophyte-infected (E+; 6 ppm ergovaline) or endophyte-free (E-) fescue seed for 2 wk under thermoneutral conditions. Liver genes differentially expressed due to fescue toxicosis were identified using DNA microarray. A 2-stage ANOVA of microarray data identified 36 differentially expressed genes between mice fed E+ and E- diets. Another analysis method, significance analysis of microarray, identified 9 genes as differentially expressed between treatment groups, and some genes overlapped with genes identified by ANOVA. Hierarchical clustering of 36 genes identified by ANOVA clearly separated the mice by diet, with 100% confidence as computed by bootstrap analysis. Expression of 11 genes was verified using quantitative real-time PCR. The E+ diet resulted in downregulation of genes involved in the sex-steroid metabolism pathway and genes involved in cholesterol and lipid metabolism. Genes coding for ribosomes and protein synthesis were upregulated by the E+ diet. Genes identified in the present analysis indicate some of the mechanisms by which fescue toxicosis occurs in animals.

Animals↗

PubMed1987

Isolation and structural characterization of the rat acyl-CoA oxidase gene.

Overlapping genomic clones containing the entire sequence of the rat acyl-CoA oxidase gene were isolated and characterized. This gene spans about 25 kilobases, and there are 14 exons and 13 introns. All of the exon-intron junction sequences agree with the GT/AG rule. The results of Southern blot analyses indicated that the gene occurs once per haploid genome. In a preceding paper (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furuta, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137), we described the presence of two species of acyl-CoA oxidase mRNA which have different sequences, only in a small region. We identified two exons corresponding to this region. The above two mRNA species are produced by alternative splicing of these exons. Several mRNA cap sites were detected by S1 mapping and by the primer extension method. The principal one was mapped at 75 nucleotides upstream of the initiator methionine codon. The 5'-flanking region of the acyl-CoA oxidase gene was sequenced up to about 1300 nucleotides, upstream of the cap sites. There was neither a "TATA" box nor a typical "CCAAT" box sequence, at least up to nucleotide -500, rather, this region is G + C-rich (65%). "GC" box hexanucleotides (GGGCGG or CCGCCC) repeat six times in this region. Two of them, located nearest the cap sites, are in a complete 11-base pair direct repeat, 5'-GGGCGGGGCCG-3'. The features of the 5'-flanking sequence of this gene resemble those of the gene for rat enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The possible functional significance of the characteristic 5'-flanking sequence elements in the transcriptional regulation of acyl-CoA oxidase is given attention.

Acyl-CoA Oxidase↗

PubMed1995

Complete nucleotide sequences of Marburg virus genes 5 and 6 encoding VP30 and VP24 proteins.

Nucleotide sequences of the genes 5 and 6 of the Marburg virus, Popp strain, were determined. ORFs encoding polypeptides VP30 (281 a.a., MW 32,640) and VP24 (253 a.a., MW 28,621) were found. The putative transcription start and stop signals for viral RNA-dependent RNA polymerase were revealed for both genes. Overlapping of genes 5 and 6 was shown. The deduced amino acid sequences of VP30 and VP24 proteins displayed significant homology with the analogous proteins of another filovirus, the Ebola virus (33% and 37%, respectively). The VP24 appeared to have a hydrophobic amino acid composition; content of hydrophobic amino acids was 40.7%. Model of VP24 location in the virion was suggested.

Amino Acid Sequence↗

PubMed1997

[Mutations and their significance in the corepromoter region of hepatitis B virus].

OBJECTIVE: To study the mutations in the core promoter (CP) region of hepatitis B virus (HBV) in Chinese viral hepatits B patients. METHODS: CP regions of 48 HBV strains were analysed by polymerase chain reaction (PCR) direct sequencing approach. RESULTS: 67% of the samples were detected to have point mutations in the CP region. The hot spots located in nt 1754-1766 and nt 1801-1811 while nucleotides in nt 1777-1800 and nt 1812-1836 were highly conserved. nt. 1764 mutation was present in HBeAg negative patients. There were 16 point mutations in region overlapping X gene, 9 of which lead to amino acid change. CONCLUSION: Though mutations in CP region of HBV appear frequently, the sequences associated with viral transcription are rarely changed. Point mutation at nt 1764 is related to HBeAg negative phenotype, but it is not the specific mutation of fulminant hepatitis. The importance of mutations in the X gene overlapping region needs to be further investigated.

Genetic Variation↗

PubMed2005

Profiling estrogen-regulated gene expression changes in normal and malignant human ovarian surface epithelial cells.

Estrogens regulate normal ovarian surface epithelium (OSE) cell functions but also affect epithelial ovarian cancer (OCa) development. Little is known about how estrogens play such opposing roles. Transcriptional profiling using a cDNA microarray containing 2400 named genes identified 155 genes whose expression was altered by estradiol-17beta (E2) in three immortalized normal human ovarian surface epithelial (HOSE) cell lines and 315 genes whose expression was affected by the hormone in three established OCa (OVCA) cell lines. All but 19 of the genes in these two sets were different. Among the 19 overlapping genes, five were found to show discordant responses between HOSE and OVCA cell lines. The five genes are those that encode clone 5.1 RNA-binding protein (RNPS1), erythrocyte adducin alpha subunit (ADD1), plexin A3 (PLXNA3 or the SEX gene), nuclear protein SkiP (SKIIP), and Rap-2 (rap-2). RNPS1, ADD1, rap-2, and SKIIP were upregulated by E2 in HOSE cells but downregulated by estrogen in OVCA cells, whereas PLXNA3 showed the reverse pattern of regulation. The estrogen effects was observed within 6-18 h of treatment. In silicon analyses revealed presence of estrogen response elements in the proximal promoters of all five genes. RNPS1, ADD1, and PLXNA3 were underexpressed in OVCA cell lines compared to HOSE cell lines, while the opposite was true for rap-2 and SKIIP. Functional studies showed that RNPS1 and ADD1 exerted multiple antitumor actions in OVCA cells, while PLXNA3 only inhibited cell invasiveness. In contrast, rap-2 was found to cause significant oncogenic effects in OVCA cells, while SKIIP promotes only anchorage-independent growth. In sum, gene profiling data reveal that (1) E2 exerts different actions on HOSE cells than on OVCA cells by affecting two distinct transcriptomes with few overlapping genes and (2) among the overlapping genes, a set of putative oncogenes/tumor suppressors have been identified due to their differential responses to E2 between the two cell types. These findings may explain the paradoxical roles of estrogens in regulating normal and malignant OSE cell functions.

Cell Line↗

PubMed1991

Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene.

Overlapping genomic clones containing the entire sequence of the human angiotensin I-converting enzyme (ACE) gene were isolated from a lamda phage human DNA library. This gene spans 21 kilobases (kb) and comprises 26 exons, ranging in size from 88 to 481 base pairs. Intron-exon boundaries were sequenced and the relative positions of the exons were mapped. The two different mRNAs transcribed from the ACE gene were assigned to their respective exons. The large endothelial type ACE mRNA (4.3 kb long) is transcribed from exon 1 to exon 26, excluding exon 13. The 3-kb long testicular ACE mRNA is transcribed from exon 13 to exon 26. Exon 13 encodes for the 67 amino acids of the NH2-terminal region of the testicular ACE, whereas downstream exons encode a sequence common to both isozymes. The gene duplication suggested by the internal homology of the endothelial ACE mRNA is now confirmed by the presence of two homologous clusters of eight exons (exons 4-11 and exons 17-24) having similar sizes and codon phases at exon-intron boundaries. The presence of two alternate promoters was investigated by ribonuclease protection assays. The different 5' ends of the two ACE transcripts revealed a promoter for the endothelial ACE mRNA in the 5'-flanking region of the first exon and a promoter for the testicular ACE mRNA situated in intron 12.

Bacteriophage lambda↗

PubMed1999

Model for polymerase access to the overlapped L gene of respiratory syncytial virus.

The last two genes of respiratory syncytial virus (RSV), M2 and L, overlap by 68 nucleotides, an arrangement which has counterparts in a number of nonsegmented negative-strand RNA viruses. Thus, the gene-end (GE) signal of M2 lies downstream of the L gene-start (GS) signal, separated by 45 nucleotides. Since RSV transcription ostensibly is sequential and unidirectional from a single promoter within the 3' leader region, it was unclear how the polymerase accesses the L GS signal. Furthermore, it was previously shown that 90% of transcripts which are initiated at the L GS signal are polyadenylated and terminated at the M2 GE signal, yielding a short, truncated L mRNA as the major transcription product of the L gene. Despite these apparent down-regulatory features, we show that the accumulation of full-length L mRNA during RSV infection is only sixfold less than that of its upstream neighbor, M2. We used cDNA-encoded genome analogs in an intracellular transcription assay to investigate the mechanism of transcription of the overlapped genes. Expression of L was found to be dependent on sequential transcription from the 3' end of the genome. Apart from the L GS signal, the only other strict requirement for initiation at L was the M2 GE signal. This implies that the polymerase accesses the L GS signal only following arrival at the M2 GE signal. Thus, polymerase which terminates at the M2 GE signal presumably scans upstream to initiate at the L GS signal. This also would provide a mechanism whereby polymerase which terminates prematurely during transcription of L could recycle from the M2 GE signal to the L GS signal, thereby accounting for the unexpectedly high level of synthesis of full-length L mRNA. The sequence and spacing between the two signals were not critical. Furthermore, the polymerase also was capable of efficiently transcribing from an L GS signal placed downstream of the M2 GE signal, implying that the overlapping arrangement is not obligatory. When copies of the L GS signal were placed concurrently upstream and downstream of the M2 GE signal, both were utilized. This finding indicates that a polymerase situated at a GE signal is capable of scanning for a GS signal in either the upstream or downstream direction and thereafter initiating transcription.

Base Sequence↗

PubMed2001

The formamidase gene of Aspergillus nidulans: regulation by nitrogen metabolite repression and transcriptional interference by an overlapping upstream gene.

The ability to utilize formamide as a sole nitrogen source has been found in numerous fungi. We have cloned the fmdS gene encoding a formamidase from Aspergillus nidulans and found that it belongs to a highly conserved family of proteins separate from the major amidase families. The expression of fmdS is primarily regulated via AreA-mediated nitrogen metabolite repression and does not require the addition of exogenous inducer. Consistent with this, deletion analysis of the 5' region of fmdS has confirmed the presence of multiple AreA-binding sites containing a characteristic core GATA sequence. Under carbon starvation conditions the response to nitrogen starvation is eliminated, indicating that the lack of a carbon source may result in inactivation of AreA. Sequence analysis and isolation of cDNAs show that a gene of unknown function lies directly 5' of fmdS with its transcript overlapping the fmdS coding region. Disruption of the 5' gene and analysis of the effects of overexpression of this gene on fmdS expression has shown that expression of this upstream gene interferes with fmdS transcription, resulting in a strong dependence on AreA activation for expression. Therefore the relative position of these two genes is essential for normal regulation of fmdS.

Amidohydrolases↗