scholarly journals Identification of ccdA inParacoccus pantotrophus GB17: Disruption ofccdA Causes Complete Deficiency inc-Type Cytochromes

2001 ◽  
Vol 183 (1) ◽  
pp. 257-263 ◽  
Author(s):  
Frank Bardischewsky ◽  
Cornelius G. Friedrich
Keyword(s):  
Sulfur Oxidation ◽  
Open Reading Frames ◽  
Redox Mediators ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Structural Identity ◽  
Paracoccus Pantotrophus ◽  
Type Gene ◽  
Extension Analysis ◽  
Reading Frames

ABSTRACT A transposon Tn5-mob insertional mutant ofParacoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently. ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn5-mob was inserted into ccdA. The deduced ORFA product showed no similarity to any protein in databases. However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of P. pantotrophus is 42% identical to a protein predicted by ORF2, and its location within thesox gene cluster coding for lithotrophic sulfur oxidation suggested a different function.

scholarly journals Identification and Sequencing of β-Myrcene Catabolism Genes from Pseudomonas sp. Strain M1

1999 ◽  
Vol 65 (7) ◽  
pp. 2871-2876 ◽  
Author(s):  
Sandra Iurescia ◽  
Andrea M. Marconi ◽  
Daniela Tofani ◽  
Augusto Gambacorta ◽  
Annalisa Paternò ◽  
...  
Keyword(s):  
Genomic Library ◽  
Enrichment Culture ◽  
Open Reading Frames ◽  
Gas Chromatography Mass Spectrometry ◽  
Complete Agreement ◽  
Wild Type ◽  
Genomic Insert ◽  
The One ◽  
Acyl Coenzyme A ◽  
Reading Frames

ABSTRACT The M1 strain, able to grow on β-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One β-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique β-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on β-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA,myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, andmyrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. ThemyrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for β-myrcene catabolism in Pseudomonas sp. strain M1.

A plasmid from a non-insect-transmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line

Gene ◽  
2002 ◽  
Vol 298 (2) ◽  
pp. 195-201 ◽  
Author(s):  
Hisashi Nishigawa ◽  
Kenro Oshima ◽  
Shigeyuki Kakizawa ◽  
Hee-Young Jung ◽  
Tsutomu Kuboyama ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Wild Type ◽  
Wild Type Line ◽  
Reading Frames

scholarly journals Cloning of two Bombyx homologues of the Drosophila rosy gene and their relationship to larval translucent skin colour mutants

1998 ◽  
Vol 71 (1) ◽  
pp. 11-19 ◽  
Author(s):  
YUJI YASUKOCHI ◽  
TOSHIO KANDA ◽  
TOSHIKI TAMURA
Keyword(s):  
Tissue Specificity ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Striking Similarity ◽  
Wild Type ◽  
Rt Pcr ◽  
Significant Difference ◽  
Phage Dna ◽  
Polymerase Chain ◽  
Reading Frames

To clone the Bombyx xanthine dehydrogenase (XDH) gene as a dominant marker for silkworm transgenesis, we performed nested reverse transcriptase–polymerase chain reaction (RT-PCR) using embryonic mRNA and primers designed from the conserved region of Drosophila and rat XDH genes. Sequencing of amplified 180 bp fragments showed that two different sequences were present in the fragments. Since both possessed striking similarity to XDH genes of other organisms, we considered these to be portions of silkworm XDH genes and designated them BmXDH1 and BmXDH2. Subsequently we cloned separately the entire region of the two cDNAs by PCR using phage DNA of an embryonic cDNA library and sequenced them. The two cDNAs were around 4 kb in size and possessed complete open reading frames. The deduced amino acid sequences of the two BmXDHs were very similar to each other and to those of other organisms. The expression pattern of wild-type larvae basically followed the tissue specificity of the enzyme and no significant difference was observed between the two XDH genes. The expression of both genes was detected in the XDH-deficient mutants, oq and og, but non-synonymous substitutions were specifically detected in the BmXDH1 of the oq mutant. In addition, a length polymorphism of the second intron of the BmXDH1 co-segregated with the oq translucent phenotype, suggesting that deficiency in BmXDH1 is the cause of the oq translucent phenotype.

scholarly journals Construction and Characterization of Murine Cytomegaloviruses That Contain Transposon Insertions at Open Reading Frames m09 and M83

2000 ◽  
Vol 74 (16) ◽  
pp. 7411-7421 ◽  
Author(s):  
Xiaoyan Zhan ◽  
Manfred Lee ◽  
Jianqiao Xiao ◽  
Fenyong Liu
Keyword(s):  
Viral Replication ◽  
Scid Mice ◽  
Open Reading Frames ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Insertion Mutation ◽  
Type Virus ◽  
Wild Type ◽  
Reading Frames

ABSTRACT A transposon derived from Escherichia coliTn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.

scholarly journals Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR

2004 ◽  
Vol 186 (20) ◽  
pp. 6714-6720 ◽  
Author(s):  
Christopher D. Herring ◽  
Frederick R. Blattner
Keyword(s):  
Minimal Medium ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Oligonucleotide Microarrays ◽  
Wild Type ◽  
Suppressor Trna ◽  
Cellular Physiology ◽  
Putative Operon ◽  
Amber Suppressor ◽  
Reading Frames

ABSTRACT Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.

scholarly journals Vibrio vulnificus Has the Transmembrane Transcription Activator ToxRS Stimulating the Expression of the Hemolysin GenevvhA

2000 ◽  
Vol 182 (12) ◽  
pp. 3405-3415 ◽  
Author(s):  
Shee Eun Lee ◽  
Sung Heui Shin ◽  
Soo Young Kim ◽  
Young Ran Kim ◽  
Dong Hyeon Shin ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Wild Type ◽  
Transcription Activator ◽  
Reading Frame ◽  
Type Gene ◽  
Virulence Regulator

ABSTRACT In an attempt to dissect the virulence regulatory mechanism inVibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS(toxRS Vc) homologs in V. vulnificus. By comparing the sequences of toxRS ofV. cholerae and V. parahaemolyticus(toxRS Vp), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kbBglII-HindIII fragment and a 1.2-kbHindIII fragment containing two complete open reading frames and one partial open reading frame attributable totoxR Vv, toxS Vv, andhtpG Vv were cloned. ToxRVv shared 55.0 and 63.0% sequence homology with ToxRVc and ToxRVp, respectively. ToxSVv was 71.5 and 65.7% homologous to ToxSVc and ToxSVp, respectively. The amino acid sequences of ToxRSVv showed transmembrane and activity domains similar to those observed in ToxRSVc and ToxRSVp. Western blot analysis proved the expression of ToxRVv in V. vulnificus. ToxRSVv enhanced, in an Escherichia coli background, the expression of the V. vulnificushemolysin gene (vvhA) fivefold. ToxRSVv also activated the ToxRVc-regulated ctx promoter incorporated into an E. coli chromosome. AtoxR Vv null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. ThetoxR Vv mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxRVv may regulate the virulence expression of V. vulnificus.

scholarly journals Promiscuous Behavior of HPV16E6 Specific T Cell Receptor Beta Chains Hampers Functional Expression in TCR Transgenic T Cells, Which Can Be Restored in Part by Genetic Modification

2010 ◽  
Vol 32 (1-2) ◽  
pp. 43-56
Author(s):  
Kirsten B. J. Scholten ◽  
Janneke J. Ruizendaal ◽  
Marcus Graf ◽  
Thomas Schoedl ◽  
Duco Kramer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Receptor Gene ◽  
Careful Analysis ◽  
Cell Receptor ◽  
Open Reading Frames ◽  
Wild Type ◽  
Human T Cells ◽  
Reading Frames

Background: T cell receptor gene transfer is a promising strategy to treat patients suffering from HPV induced malignancies. Therefore we isolated the TCRαβ open reading frames of an HPV16E6 specific CTL clone and generated TCR transgenic T cells. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. Here we addressed both issues employing three different expression platforms.Methods: We isolated the HVP16E6 specific TCRα and TCRβ open reading frames and retrovirally transduced human T cells with either wild-type (wt), or codon-modified (cm) chains to achieve enhanced TCR expression levels, or used codon-modification in combination with cysteinization (cmCys) of TCRs to facilitate preferential pairing of the introduced TCRα and TCRβ chains.Results: Careful analysis of recipient T cells carrying the HPV16E6 TCRβ and endogenous TCR chains revealed the transgenic TCRβ chain to behave very promiscuously. Further analysis showed that the percentage of tetramer positive T cells in codon-modified/cysteinized TCR transgenic T cells was four-fold higher compared to wild-type and two-fold higher compared to codon-modification only. Functional activity, as determined by IFN-γ production, was high in cmCysTCR transgenic T cells, where it was low in cm and wt TCR transgenic T cells. Recognition of endogenously processed HPV16E6 antigen by cmCysTCR transgenic T cells was confirmed in a cytotoxicity assay.Conclusions: Promiscuous behavior of the HPV16E6 specific TCRβ chain can in part be forced back into specific action in TCR transgenic T cells by codon modification in combination with the inclusion of an extra cysteine in the TCR chains.

scholarly journals An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby

Genome ◽  
2022 ◽  
Author(s):  
Sakura Hayashi ◽  
Konami Shimizu ◽  
Yusuke Honda ◽  
Yukako Katsura ◽  
Akihiko Koga
Keyword(s):  
Endogenous Retrovirus ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Length Variation ◽  
Wild Type ◽  
Long Terminal Repeats ◽  
Recent Event ◽  
Melanin Biosynthesis ◽  
Dna Fragment ◽  
Reading Frames

An albino infant wallaby was born to a mother with the wild-type body color. PCR and sequencing analyses of <i>TYR</i> (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Because the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named <i>walb</i>. We cloned other <i>walb</i> copies residing in the genomes of this species and another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of <i>gag</i>, <i>pol</i>, and <i>env</i> of retroviruses. It is not known through which of the following likely processes the walb copy was inserted into <i>TYR</i>: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into <i>TYR</i> is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances. 

scholarly journals A TRAP Transporter for Pyruvate and Other Monocarboxylate 2-Oxoacids in the Cyanobacterium Anabaena sp. Strain PCC 7120

2010 ◽  
Vol 192 (22) ◽  
pp. 6089-6092 ◽  
Author(s):  
Rafael Pernil ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Open Reading Frames ◽  
Wild Type ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Significant Uptake ◽  
Pcc 7120 ◽  
Reading Frames ◽  
T System

ABSTRACT In the cyanobacterium Anabaena sp. strain PCC 7120, open reading frames (ORFs) alr3026, alr3027, and all3028 encode a tripartite ATP-independent periplasmic transporter (TRAP-T). Wild-type filaments showed significant uptake of [14C]pyruvate, which was impaired in the alr3027 and all3028 mutants and was inhibited by several monocarboxylate 2-oxoacids, identifying this TRAP-T system as a pyruvate/monocarboxylate 2-oxoacid transporter.

scholarly journals Succinate:Quinol Oxidoreductases in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Presence and Function in Metabolism and Electron Transport

2000 ◽  
Vol 182 (3) ◽  
pp. 714-722 ◽  
Author(s):  
Jason W. Cooley ◽  
Crispin A. Howitt ◽  
Wim F. J. Vermaas
Keyword(s):  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Double Mutant ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Growth Media ◽  
Wild Type ◽  
Succinate Dehydrogenase Activity ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT The open reading frames sll1625 andsll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the Δsll1625 and Δsll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Δsll1625 Δsll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.

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