scholarly journals Identification and Characterization of alcR, a Gene Encoding an AraC-Like Regulator of Alcaligin Siderophore Biosynthesis and Transport in Bordetella pertussis andBordetella bronchiseptica

1998 ◽  
Vol 180 (4) ◽  
pp. 862-870 ◽  
Author(s):  
Fiona C. Beaumont ◽  
Ho Young Kang ◽  
Timothy J. Brickman ◽  
Sandra K. Armstrong
Keyword(s):  
Bordetella Pertussis ◽  
Transcription Initiation ◽  
Expression System ◽  
Protein A ◽  
Initiation Site ◽  
Open Reading Frame ◽  
Growth Defect ◽  
Nucleotide Sequencing ◽  
Reading Frame ◽  
Siderophore Biosynthesis

ABSTRACT A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels. In-frame alcR deletion mutants of B. pertussisand B. bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned alcR gene provided intrans restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.

scholarly journals Role of a Cytotoxic-T-Lymphocyte Epitope-Defined, Alternative gag Open Reading Frame in the Pathogenesis of a Murine Retrovirus-Induced Immunodeficiency Syndrome

2005 ◽  
Vol 79 (7) ◽  
pp. 4308-4315 ◽  
Author(s):  
Arti Gaur ◽  
William R. Green
Keyword(s):  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Protein S ◽  
Initiation Site ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Ctl Epitope ◽  
Immunodeficiency Syndrome

ABSTRACT LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60 gag ). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.

scholarly journals Chemical modifications of a recombinant bovine stress-inducible 70 kDa heat-shock protein (Hsp70) mimics Hsp70 isoforms from tissues

1995 ◽  
Vol 305 (1) ◽  
pp. 197-203 ◽  
Author(s):  
J A Gutierrez ◽  
V Guerriero
Keyword(s):  
Skeletal Muscle ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Expression System ◽  
Protein A ◽  
Two Dimensional Gel Electrophoresis ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Heat Shock Protein Hsp70 ◽  
Bovine Skeletal Muscle

A cDNA clone for the stress-inducible 70 kDa heat-shock protein (Hsp70) has been isolated from a bovine skeletal-muscle cDNA library. This mRNA encodes a protein with a calculated molecular mass of 70250 Da. The cDNA has one continuous open reading frame capable of encoding a 641-amino-acid protein. Expression of this cDNA in a bacterial expression system produced a protein with a mobility identical with that of the inducible Hsp70 protein from bovine skeletal muscle as determined by SDS/PAGE. Two-dimensional gel electrophoresis demonstrated this protein to have focusing properties identical with that of a minor isoform from bovine skeletal muscle. Upon carbamylation of this bacterially expressed protein, a train of charged proteins with charge differences of -1 were produced. These carbamylated proteins were shown to have similar focusing mobilities to the Hsp70 isoforms isolated from bovine skeletal muscle. These results demonstrate the identification of a skeletal-muscle inducible Hsp70 gene and suggest that the presence of multiple Hsp70 isoforms may be the product of post-translational modifications to the Hsp70 proteins.

scholarly journals Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

Genetics ◽  
1992 ◽  
Vol 131 (3) ◽  
pp. 531-539 ◽  
Author(s):  
C Bornaes ◽  
J G Petersen ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Open Reading Frame ◽  
Predicted Amino Acid Sequence ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Threonine Dehydratase ◽  
Nuclease Mapping

Abstract The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

scholarly journals Regulation and Physiologic Significance of the Agmatine Deiminase System of Streptococcus mutans UA159

2006 ◽  
Vol 188 (3) ◽  
pp. 834-841 ◽  
Author(s):  
Ann R. Griswold ◽  
Max Jameson-Lee ◽  
Robert A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Transcription Initiation ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Initiation Site ◽  
Low Ph ◽  
Pathogenic Potential ◽  
Genome Database ◽  
Reading Frame ◽  
Agmatine Deiminase

ABSTRACT We previously demonstrated that Streptococcus mutans expresses a functional agmatine deiminase system (AgDS) encoded by the agmatine-inducible aguBDAC operon (A. R. Griswold, Y. Y. Chen, and R. A. Burne, J. Bacteriol. 186:1902-1904, 2004). The AgDS yields ammonia, CO2, and ATP while converting agmatine to putrescine and is proposed to augment the acid resistance properties and pathogenic potential of S. mutans. To initiate a study of agu gene regulation, the aguB transcription initiation site was identified by primer extension and a putative σ70-like promoter was mapped 5′ to aguB. Analysis of the genome database revealed an open reading frame (SMU.261c) encoding a putative transcriptional regulator located 239 bases upstream of aguB. Inactivation of SMU.261c decreased AgD activity by sevenfold and eliminated agmatine induction. AgD was also found to be induced by certain environmental stresses, including low pH and heat, implying that the AgDS may also be a part of a general stress response pathway of this organism. Interestingly, an AgDS-deficient strain was unable to grow in the presence of 20 mM agmatine, suggesting that the AgDS converts a growth-inhibitory substance into products that can enhance acid tolerance and contribute to the competitive fitness of the organism at low pH. The capacity to detoxify and catabolize agmatine is likely to have major ramifications on oral biofilm ecology.

Expression of tissue-specific Ren-1 and Ren-2 genes of mice: comparative analysis of 5'-proximal flanking regions

1984 ◽  
Vol 4 (11) ◽  
pp. 2321-2331
Author(s):  
L J Field ◽  
W M Philbrick ◽  
P N Howles ◽  
D P Dickinson ◽  
R A McGowan ◽  
...  
Keyword(s):  
Structural Gene ◽  
Transcription Initiation ◽  
Submaxillary Gland ◽  
Inbred Strains ◽  
Initiation Site ◽  
Sequence Motifs ◽  
Specific Expression ◽  
Tissue Specific ◽  
Reading Frame ◽  
A Minor

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed submaxillary gland renin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue-specific expression. In an effort to understand the molecular basis for this differential expression, detailed analysis of the genomic sequences corresponding to the Ren-1 and Ren-2 genes, and the transcripts originating from these loci, was undertaken. Sequence analysis of regions proximal to the structural genes indicated the presence of eucaryotic consensus sequences for transcription. These sequence motifs were strongly conserved between Ren-1 and Ren-2. Approximately 150 bases upstream from the major transcription initiation site, significant differences between these genes were apparent, including the presence of a repetitive DNA element in the Ren-2 copy as well as other breaks in homology and sequence curiosities. Strong homology between Ren-1 and Ren-2 resumed at a point ca. 200 bases further upstream on Ren-1. S1 analysis of submaxillary gland and kidney RNA populations indicated that the majority of transcripts initiate at homologous positions on Ren-1 and Ren-2. On a per cell basis, the accumulation of Ren-1 transcripts in the kidney and Ren-2 transcripts in the submaxillary gland are probably equivalent. These results suggest that it is tissue-specific utilization of the homologous start sites that is critical to their differential patterns of expression. Models which can account for this observation are presented. Interestingly, we found a minor fraction of transcripts initiating 5' to the major transcription start site. These transcripts encoded an open reading frame which may add an additional 23 amino acids to the N-terminus of the renin precursor.

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison

Genome ◽  
1991 ◽  
Vol 34 (1) ◽  
pp. 6-12 ◽  
Author(s):  
Shiv S. Prasad ◽  
Linda J. Harris ◽  
David L. Baillie ◽  
Ann M. Rose
Keyword(s):  
Amino Acid ◽  
Transposable Element ◽  
Sequence Comparison ◽  
Horizontal Transmission ◽  
Protein A ◽  
Open Reading Frame ◽  
Single Open Reading Frame ◽  
Major Open Reading Frame ◽  
Reading Frame ◽  
Caenorhabditis Species

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150–200 and 1421–1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.Key words: Caenorhabditis, transposable element, sequence comparison.

scholarly journals Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats

2005 ◽  
Vol 86 (3) ◽  
pp. 561-574 ◽  
Author(s):  
Heather M. Coleman ◽  
Stacey Efstathiou ◽  
Philip G. Stevenson
Keyword(s):  
Transcription Initiation ◽  
Repeat Unit ◽  
Initiation Site ◽  
Reading Frame ◽  
Left Hand ◽  
Murine Gammaherpesvirus ◽  
Terminal Repeats ◽  
Gammaherpesvirus 68 ◽  
The Right ◽  
Murine Gammaherpesvirus 68

Gammaherpesviruses persist as latent episomes in a dynamic lymphocyte pool. The regulated production of an episome maintenance protein is therefore crucial to their survival. The transcription initiation site of the murine gammaherpesvirus 68 episome maintenance protein, ORF73, was mapped to the viral terminal repeats, more than 10 kb distant from the open reading frame (ORF) itself. A 5′ non-coding exon in the terminal repeats was spliced to the right end of the viral unique sequence, and then across ORFs 75a, 75b, 75c and 74 to ORF73. The right-hand portion of a single repeat unit was sufficient for constitutive promoter activity. The unique left end of the viral genome further enhanced ORF73 transcription. This, together with the large size of the predominant ORF73 mRNA, suggested that transcription initiates in distal repeat units and then splices between repeats to generate an extensive 5′ untranslated region. A second promoter in the left-hand portion of the proximal terminal repeat unit generated a transcript which overlapped that of ORF73, but failed to splice to the ORF73 coding exon and so transcribed ORF75a. In distal repeat copies, however, transcription from this promoter would enter the next repeat unit to become an ORF73 mRNA. There was a third promoter just upstream of ORF73 itself. These data indicate that ORF73 transcription is highly complex, and support the idea that the terminal repeats of gamma-2-herpesviruses constitute a vital component of episomal persistence.

scholarly journals Structure and expression of the human immunoglobulin lambda genes.

1990 ◽  
Vol 172 (2) ◽  
pp. 609-620 ◽  
Author(s):  
T J Vasicek ◽  
P Leder
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Rna Splicing ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Enhancer Activity ◽  
Initiation Point ◽  
Reading Frame ◽  
Duplication Events

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

scholarly journals A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

1998 ◽  
Vol 180 (9) ◽  
pp. 2522-2530 ◽  
Author(s):  
Sergio L. Fuenmayor ◽  
Mark Wild ◽  
Alastair L. Boyes ◽  
Peter A. Williams
Keyword(s):  
Single Gene ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Gas Chromatography Mass Spectrometry ◽  
Nucleotide Sequencing ◽  
Naphthalene Dioxygenase ◽  
Sequence Comparisons ◽  
Reading Frame ◽  
Selective Enrichment ◽  
E Coli

ABSTRACT Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kbBamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb(for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalenecis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAbwere two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designatednagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together inE. coli, nagG, nagH, andnagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts ofnagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene ordernagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.

urbs1, a gene regulating siderophore biosynthesis in Ustilago maydis, encodes a protein similar to the erythroid transcription factor GATA-1

1993 ◽  
Vol 13 (11) ◽  
pp. 7091-7100
Author(s):  
C Voisard ◽  
J Wang ◽  
J L McEvoy ◽  
P Xu ◽  
S A Leong
Keyword(s):  
Transcription Factor ◽  
Screening Method ◽  
Ustilago Maydis ◽  
Suppressor Gene ◽  
Open Reading Frame ◽  
Wild Type ◽  
Induced Mutations ◽  
Iron Starvation ◽  
Reading Frame ◽  
Siderophore Biosynthesis

Ustilago maydis secretes ferrichrome-type siderophores, ferric-ion-binding compounds, in response to iron starvation. TA2701, a non-enterobactin-producing, non-ferrichrome-utilizing mutant of Salmonella typhimurium LT-2, was employed as a biological indicator in a novel screening method to isolate three N-methyl-N'-nitro-N-nitrosoguanidine-induced U. maydis mutants defective in the regulation of ferrichrome-type siderophore biosynthesis. These mutants displayed a constitutive phenotype; they produced siderophores in the presence of iron concentrations that would typically repress siderophore synthesis in wild-type strains. A 4.8-kb fragment of U. maydis genomic DNA capable of restoring normal regulation of siderophore biosynthesis in the constitutive mutants was identified. This segment of DNA contains an intronless open reading frame that specifies a protein of 950 amino acids containing two finger motifs similar to those found in the erythroid transcription factor GATA-1. Disruption of this open reading frame in a wild-type strain gave rise to cells that produced siderophores constitutively. Genetic studies indicated that the disruption mutation was allelic to the chemically induced mutations, confirming that the structural gene for a regulator rather than a suppressor gene had been cloned. Northern (RNA) analysis of the gene revealed a 4.2-kb transcript that is expressed constitutively at low levels in wild-type cells. The data support the hypothesis that this gene, which we designate urbs1 (Ustilago regulator of biosynthesis of siderophores), acts directly or indirectly to repress biosynthesis of siderophores in U. maydis.

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