BAIUCAS: a novel BLAST-based algorithm for the identification of upstream open reading frames with conserved amino acid sequences and its application to the Arabidopsis thaliana genome

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

Download Full-text

Translational Control of Protein Kinase Cη by Two Upstream Open Reading Frames

Molecular and Cellular Biology ◽

10.1128/mcb.01044-09 ◽

2009 ◽

Vol 29 (22) ◽

pp. 6140-6148 ◽

Cited By ~ 15

Author(s):

Hadas Raveh-Amit ◽

Adva Maissel ◽

Jonathan Poller ◽

Liraz Marom ◽

Orna Elroy-Stein ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Translational Control ◽

Translational Regulation ◽

Normal Growth ◽

Amino Acid Starvation ◽

Open Reading Frames ◽

Pkc Isoforms ◽

Upstream Open Reading Frames ◽

Reading Frames

ABSTRACT Protein kinase C (PKC) represents a family of serine/threonine kinases that play a central role in the regulation of cell growth, differentiation, and transformation. Posttranslational control of the PKC isoforms and their activation have been extensively studied; however, not much is known about their translational regulation. Here we report that the expression of one of the PKC isoforms, PKCη, is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation, the latter causing a marked increase in its protein levels. The 5′ untranslated region (5′ UTR) of PKCη is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5′ UTR and show their effect in suppressing the expression of PKCη in MCF-7 growing cells. While the two uORFs function as repressive elements that maintain low basal levels of PKCη in growing cells, they are required for its enhanced expression upon amino acid starvation. We show that the translational regulation during stress involves leaky scanning and is dependent on eIF-2α phosphorylation by GCN2. Our work further suggests that translational regulation could provide an additional level for controlling the expression of PKC family members, being more common than currently recognized.

Download Full-text

Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity

mBio ◽

10.1128/mbio.00844-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 15

Author(s):

Ivaylo P. Ivanov ◽

Jiajie Wei ◽

Stephen Z. Caster ◽

Kristina M. Smith ◽

Audrey M. Michel ◽

...

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Neurospora Crassa ◽

Open Reading Frames ◽

Reading Frame ◽

Coding Sequence ◽

Cell Extracts ◽

Upstream Open Reading Frames ◽

Reading Frames

ABSTRACT Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro . In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression. IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

Download Full-text

A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection

Genome Research ◽

10.1101/gr.5836207 ◽

2007 ◽

Vol 17 (5) ◽

pp. 632-640 ◽

Cited By ~ 95

Author(s):

K. Hanada ◽

X. Zhang ◽

J. O. Borevitz ◽

W.-H. Li ◽

S.-H. Shiu

Keyword(s):

Arabidopsis Thaliana ◽

Purifying Selection ◽

Open Reading Frames ◽

Intergenic Regions ◽

Arabidopsis Thaliana Genome ◽

Reading Frames ◽

Small Open Reading Frames

Download Full-text

Relapsing Fever Spirochetes Contain Chromosomal Genes with Unique Direct Tandemly Repeated Sequences

Infection and Immunity ◽

10.1128/iai.73.5.3025-3037.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 3025-3037 ◽

Cited By ~ 9

Author(s):

Cyril Guyard ◽

Earl M. Chester ◽

Sandra J. Raffel ◽

Merry E. Schrumpf ◽

Paul F. Policastro ◽

...

Keyword(s):

Amino Acid ◽

Human Infection ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Relapsing Fever ◽

Serum Samples ◽

Borrelia Hermsii ◽

Reading Frames ◽

Antigenic Heterogeneity

ABSTRACT Genome sequencing of the relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae identified three open reading frames (ORFs) on the chromosomes that contained internal, tandemly repeated amino acid sequences that were absent in the Lyme disease spirochete Borrelia burgdorferi. The predicted amino acid sequences of these genes (BH0209, BH0512, and BH0553) have hydrophobic N termini, indicating that these proteins may be secreted. B. hermsii transcribed the three ORFs in vitro, and the BH0512- and BH0553-encoded proteins (PBH-512 and PBH-553) were produced in vitro and in experimentally infected mice. PBH-512 and PBH-553 were on the spirochete's outer surface, and antiserum to these proteins reduced the adherence of B. hermsii to red blood cells. PCR analyses of 28 isolates of B. hermsii and 8 isolates of B. turicatae demonstrated polymorphism in each gene correlated with the number of repeats. Serum samples from relapsing fever patients reacted with recombinant PBH-512 and PBH-553, suggesting that these proteins are produced during human infection. These polymorphic proteins may be involved in the pathogenicity of these relapsing fever spirochetes and provide a mechanism for antigenic heterogeneity within their populations.

Download Full-text

Incorporation of iron-sulphur clusters in membrane-bound proteins

Biochemical Society Transactions ◽

10.1042/bst0290418 ◽

2001 ◽

Vol 29 (4) ◽

pp. 418-421 ◽

Cited By ~ 16

Author(s):

A. Seidler ◽

K. Jaschkowitz ◽

M. Wollenberg

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Synechocystis Pcc 6803 ◽

Membrane Bound ◽

Nif Proteins ◽

Reading Frames ◽

Pcc 6803

The completely sequenced genome of the cyano-bacterium Synechocystis PCC 6803 contains several open reading frames, of which the deduced amino acid sequences show similarities to proteins known to be involved in FeS cluster synthesis of nitrogenase (Nif proteins) and other FeS proteins (Isc proteins). In this article, the results of our studies on these proteins are summarized and discussed with respect to their relevance in FeS cluster incorporation in chloroplasts. In cyanobacteria, there appears to exist several pathways for FeS cluster synthesis.

Download Full-text

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria

Applied and Environmental Microbiology ◽

10.1128/aem.66.11.4688-4695.2000 ◽

2000 ◽

Vol 66 (11) ◽

pp. 4688-4695 ◽

Cited By ~ 43

Author(s):

Pornpimon Kiatpapan ◽

Yoshiteru Hashimoto ◽

Hisako Nakamura ◽

Yong-Zhe Piao ◽

Hisayo Ono ◽

...

Keyword(s):

Amino Acid ◽

Shuttle Vector ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Vector System ◽

Propionibacterium Acidipropionici ◽

Rep Protein ◽

Gram Positive Bacteria ◽

High Degree ◽

Reading Frames

ABSTRACT The complete nucleotide sequence of pRGO1, a cryptic plasmid fromPropionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4,orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containingorf1 (repA), orf2(repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichiisubsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp.freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

Download Full-text