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PubMed1997

The adenylate kinase genes of M. voltae, M. thermolithotrophicus, M. jannaschii, and M. igneus define a new family of adenylate kinases.

The adenylate kinase genes (adkA) were cloned from four closely related methanogenic members of the Archaea: the mesophile Methanococcus voltae (Mv), the thermophile M. thermolithotrophicus (Mt) and the hyperthermophiles M. jannaschii (Mj) and M. igneus (Mi). All four genes encode a protein of 192 amino acids (aa), and the four enzymes were closely related, with 68-81% aa identity in pairwise comparisons. It is anticipated that the enzyme set will provide the basis for studies that can establish the structural basis for ADK thermal stability. Mj and Mi contained a gene homologous to M. vannielii sec Y upstream of adkA, while Mv and Mt contained an unidentified, yet conserved, upstream open reading frame (ORF). Mt, Mj and Mi, but not Mv, contained an unidentified, yet highly conserved, ORF directly downstream of adkA. Based on their size, predicted secondary structure and phylogenetic relation to bacterial and eukaryotic adenylate kinases (ADK), it was concluded that the archaeal adkA genes encoded a unique class of ADK, and suggested that Euryarchaeotal and Crenarchaeotal branches of the Archaea contain separate subclasses of the enzyme.

Adenylate Kinase↗
PubMed2002

Intron-dependent stimulation of marker gene expression in cultured insect cells.

We tested in a systematic fashion the effect of an intron on the level of luciferase expression in cultured C6/36 Aedes albopictus cells. The intron was inserted in both orientations, upstream and downstream of the luciferase coding region in two different luciferase expression vectors. The two parental luciferase expression vectors differed only in their promoters, one containing the Drosophila melanogaster actin5C promoter and the other the Autographa californica nuclear polyhedrosis virus hr5/ie1 enhancer/promoter. All resulting intron-containing constructs were tested for their ability to express luciferase in transient assays following electroporation into C6/36 cells. We found that the introns stimulate luciferase expression between twelve and sixtyfold, depending on the promoter. Enhanced expression was only seen when the intron was present in the correct orientation upstream of the luciferase ORF. When the 3' splice sites of the enhanced intron-containing constructs were mutated, the expression level dropped back to below the level of the intronless parental constructs, suggesting that the intron-dependent stimulation of luciferase expression is depending on splicing and is not due to other effects the intron may have on transcription or translation. The luciferase transcripts of all constructs were analysed by reverse transcription, PCR amplification and sequencing, and the results show a perfect correlation between efficient splicing of the intron and elevated levels of luciferase expression. Our findings have the potential to be very useful for boosting expression of foreign proteins in the widely used baculoviral or non-viral systems in insect cells.

Aedes↗
PubMed2002

A single nucleotide polymorphism at the splice donor site of the human MYH base excision repair genes results in reduced translation efficiency of its transcripts.

BACKGROUND: Adenine paired with 8-hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products. RESULTS: An aberrant size of the beta type nuclear form transcript was detected in a lung cancer cell line, VMRC-LCD, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5'-side of the native ORF in the beta type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5'-untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT-PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (approximately 30) than that of the IVS1+5G transcript. CONCLUSIONS: The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells.

Alleles↗
PubMed2006

Recruitment of mRNA cleavage/polyadenylation machinery by the yeast chromatin protein Sin1p/Spt2p.

The yeast chromatin protein Sin1p/Spt2p has long been studied, but the understanding of its function has remained elusive. The protein has sequence similarity to HMG1, specifically binds crossing DNA structures, and serves as a negative transcriptional regulator of a small family of genes that are activated by the SWI/SNF chromatin-remodeling complex. Recently, it has been implicated in maintaining the integrity of chromatin during transcription elongation. Here we present experiments whose results indicate that Sin1p/Spt2 is required for, and is directly involved in, the efficient recruitment of the mRNA cleavage/polyadenylation complex. This conclusion is based on the following findings: Sin1p/Spt2 frequently binds specifically downstream of many ORFs but almost always upstream of the first polyadenylation site. It directly interacts with Fir1p, a component of the cleavage/polyadenylation complex. Disruption of Sin1p/Spt2p results in foreshortened poly(A) tracts on mRNA. It is synthetically lethal with Cdc73p, which is involved in the recruitment of the complex. This report shows that a chromatin component is involved in 3' end processing of RNA.

3' Untranslated Regions↗
PubMed1998

Automatic extraction of motifs represented in the hidden Markov model from a number of DNA sequences.

MOTIVATION: Automatic extraction of motifs that occur frequently on a set of unaligned DNA sequences is useful for predicting the binding sites of unknown transcription factors. Several programs for this purpose have been released. However, in our opinion, they are not practical enough to be applied to a large number of upstream sequences. RESULTS: We propose a new program called YEBIS (Yet another Environment for the analysis of BIopolymer Sequences) which is capable of extracting a set of motifs, without any a priori knowledge, from a number of functionally related DNA sequences. Using the hidden Markov model, these motifs are represented in a more general form than other conventional methods, such as the weight matrix method. When applied to several sets of benchmark data, it was found that YEBIS had comparable capability to the existing methods, but was much faster. Moreover, it could extract all known motifs from the LTR sequences (long terminal repeat sequences) in a single run. Finally, it could be successfully applied to approximately 400 human promoter sequences and some of the extracted motifs turned out to be known cis-elements. Therefore, YEBIS could be a practical tool for exploring the upstream sequences of genomic ORFs, some of which are regulated in a similar fashion. AVAILABILITY: YEBIS will be distributed to academic users free of charge. All requests should be sent to the address below. CONTACT: E-MAIL: yada@tokyo.jst.go.jp

Algorithms↗
PubMed1999

Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by paternal deficiency of human chromosome 15q11-q13. There is conflicting evidence from human translocations regarding the direct involvement of SNRPN in the pathogenesis of PWS and it is not known if the phenotypic features result from the loss of expression of a single imprinted gene or multiple genes. In an attempt to dissect genotype/phenotype correlations for the homologous region of mouse chromosome 7C, we prepared three mutant genotypes: (i) mice with a deletion of Snrpn exon 2, which removes a portion of a small, upstream open reading frame (ORF); (ii) mice with double targeting for Snrpn exon 2 and Ube3a; (iii) mice deleted from Snrpn to Ube3a, removing coding exons for both loci and intervening genes. Mice deleted for Snrpn exon 2 have no obvious phenotypic abnormalities and switching of the genomic imprint for the region is conserved. Mice carrying the Snrpn - Ube3a deletion on the paternal chromosome showed severe growth retardation, hypotonia and approximately 80% lethality before weaning. The surviving mice were fertile and were not obese up to 14 months of age. The deletion was transmitted for multiple generations and continued to cause partial lethality when inherited paternally, but not when inherited maternally. The normal imprinted expression and methylation patterns of necdin, a gene outside the deletion region, indicate that the deletion is not an imprinting mutation. The data suggest the presence of a paternally expressed structural gene between Snrpn and Ipw whose deficiency causes lethality, although other possibilities exist, including position effects on expression of imprinted genes or that simultaneous deficiency of both ORFs of Snrpn causes lethality.

Abnormalities, Multiple↗
PubMed2001

Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1.5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5' upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3' end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.

Amino Acid Sequence↗
PubMed2001

Penicillin-binding protein 5 and expression of ampicillin resistance in Enterococcus faecium.

We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW(Efm)) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW(Efm), psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW(Efm) promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF(pCWR558) and D344-SRF(pCWR583) were 64 microg/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.

Ampicillin Resistance↗
PubMed1999

Genetic and biochemical analyses of the tec operon suggest a route for evolution of chlorobenzene degradation genes.

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4, 5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPbeta incompatibility group. PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of Pseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.

Amino Acid Sequence↗
PubMed2003

Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors.

We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

Adhesins, Bacterial↗
PubMed1991

Biosynthesis of human fibroblast growth factor-5.

We have analyzed the biosynthesis of human fibroblast growth factor-5 (FGF-5) at the translational and posttranslational levels. FGF-5 RNA synthesized in vitro can be translated in rabbit reticulocyte lysates to yield a 29,500-Da protein, which is consistent with the molecular weight predicted from the coding sequence. The efficiency of FGF-5 translation is dramatically enhanced if an upstream open reading frame (ORF-1) in the RNA is deleted or if both AUG codons in ORF-1 are destroyed by point mutations, while partial enhancement is achieved by individual mutation of either ORF-1 AUG codon. These data suggest that FGF-5 synthesis requires the scanning of ribosomes past the two ORF-1 AUG codons. The introduction of these ORF-1 mutations into a eukaryotic FGF-5 expression vector increases its capacity to transform mouse NIH 3T3 cells up to 50-fold upon transfection. FGF-5 is secreted from transfected 3T3 cells and from human tumor cells as glycoproteins containing heterogeneous amounts of sialic acid. Glycosidase treatments suggest that the growth factor bears both N-linked and O-linked sugars.

Amino Acid Sequence↗
PubMed2003

Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene.

The number of V1b vasopressin receptors (V1bR) in the anterior pituitary plays an important role during adaptation of the hypothalamic-pituitary-adrenal axis to stress in rats. Regulation of V1bR expression involves transcriptional and translational mechanisms. One of the elements mediating transcriptional activation of the rat V1bR gene is a long stretch of GAGA repeats (GAGA box) in the promoter located near the transcription start point capable of binding a protein complex of 127 kDa present in pituitary nuclear extracts. There is a lack of correlation between changes in V1bR mRNA and the number of VP binding sites, suggesting that V1bR expression depends on the efficiency of V1b R mRNA translation into protein. Two mechanisms by which the 5' untranslated region (5'-UTR) of the rat V1bR mRNA can mediate either inhibition or activation of V1bR mRNA translation have been identified. First, upstream open reading frames (ORF) present in the 5'-UTR repress translation of the major ORF encoding the V1b receptor, and secondly, an internal ribosome entry site (IRES) activates V1bR translation. Stimulation of IRES activity through protein kinase C-mediated pathways results in V1bR mRNA translation increasing V1bR protein levels. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

5' Untranslated Regions↗
PubMedDate not supplied

Characterization of the regulatory region of Adra2c, the gene encoding the murine alpha2C adrenoceptor subtype.

The 5' flanking sequence (3,227 base pairs, bp) of the mouse Adra2c subtype gene was determined and characterized. The transcription start site was mapped to nucleotide 'A' of two initiator motifs in tandem array, i.e. 1,159 and 1,153 bp upstream from the initiation codon of the open reading frame (ORF) of Adra2c, respectively. One structural feature salient to the 5' regulatory region of Adra2c is present in the sequence 1 kb immediately upstream from the receptor ORF, which is highly enriched in GC content (76%) and CpG island counts (i.e. CpG/GpC, 146:177), and thus rich in Sp1-binding motifs. At the 3' flanking region, the polyadenylation signal was mapped to 481 bp downstream from the termination codon. The transcript defined by sequence data thereby is consistent with a size of 3 kb (brain form) determined by Northern blot analysis. The transgene, Adra2c-NN- lacZ, which links the promoter region of Adra2c to the lacZ reporter gene, was constructed in order to evaluate the functional capacity of the promoter and the putative motifs residing within the defined regulatory region (1.9 kb upstream from the ORF) in directing the reporter gene expression in vitro in transiently transfected cells and in vivo in transgenic (Tg) mice. Permissive cell types to Adra2c-NN include those derived from neural and kidney lineages. Significant Adra2c-NN-driven reporter expression in Tg mice established suggests that alpha2C adrenoceptor expression is permissive under Adra2c-NN in central (cerebral cortex, hippocampus, subthalamus, hypothalamus, superior colliculus, cerebellum, and brain stem) and peripheral (pancreatic beta-islets) tissues.

Amino Acid Motifs↗
PubMed2000

Genes involved in anaerobic metabolism of phenol in the bacterium Thauera aromatica.

Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor ("phenylphosphate synthase") and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate ("phenylphosphate carboxylase"). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcus strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.

Amino Acid Sequence↗
PubMed2002

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27.

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.

Base Sequence↗
PubMed1997

The apocytochrome-b gene in Chlorogonium elongatum (Chlamydomonadaceae): an intronic GIY-YIG ORF in green algal mitochondria.

The mitochondrial cob gene from the green alga Chlorogonium elongatum (Chlamydomonadaceae) is interrupted by two group-I introns each containing an open reading frame in-phase with the upstream exon. One of these ORFs belongs to the LAGLI-DADG family, the other to the GIY-YIG family. The latter has not yet been identified in any mitochondrial genome except those from fungi. The Chlorogonium ORFs are similar to ORFs encoded by fungal introns that are located at an identical position within the gene, and to the ORF encoded by the mobile intron in the Chlamydomonas smithii cob gene.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence and genetic organization of peanut clump virus RNA 2 and partial characterization of deleted forms.

The complete nucleotide sequence of peanut clump virus RNA 2 has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 4503 residues in length and contains six long open reading frames (ORFs). The cistron for the coat protein (ORF 1) is located nearest the 5' end of the RNA and is separated from the 5' extremity by a noncoding region of 389 nucleotides. ORF 2 starts one nucleotide upstream of the first residue of the UGA termination codon of the coat protein cistron and encodes a polypeptide of 39300 M(r). Four additional ORFs with the potential to encode polypeptides with approximate M(r)'s of 51,000 (ORF 3), 13,800 (ORF 4), 9400 (ORF 5), and 6800 (ORF 6) are found on the 3' proximal half of the RNA. ORFs 3, 4, and 5 resemble the Triple Gene Block found in potexviruses, carlaviruses, a hordeivirus, and a furovirus. The 276 nucleotide 3' noncoding region of RNA 2 displays extensive sequence homology with the 3' noncoding region of RNA 1. Several peanut clump virus isolates propagated on Nicotiana benthamania contain RNA 2's significantly shorter than that of the type isolate. Partial characterization of two such isolates reveals that their RNA 2's have undergone deletions within ORF 2.

Amino Acid Sequence↗
PubMed1997

A rubrerythrin operon and nigerythrin gene in Desulfovibrio vulgaris (Hildenborough).

Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp Sal1 fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe3+(SCys)4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.

Amino Acid Sequence↗