Variants of open reading frame Bm126 in wild-type Bombyx mori nucleopolyhedrovirus isolates exhibit functional differences

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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CZK3, a MAP Kinase Kinase Kinase Homolog in Cercospora zeae-maydis, Regulates Cercosporin Biosynthesis, Fungal Development, and Pathogenesis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.9.760 ◽

2003 ◽

Vol 16 (9) ◽

pp. 760-768 ◽

Cited By ~ 41

Author(s):

Won-Bo Shim ◽

Larry D. Dunkle

Keyword(s):

Map Kinase ◽

Map Kinases ◽

Gray Leaf Spot ◽

Open Reading Frame ◽

Wild Type ◽

Toxic Activity ◽

Reading Frame ◽

Cercospora Zeae Maydis ◽

Map Kinase Kinase Kinase ◽

Map Kinase Kinase

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

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Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2593-2608.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2593-2608 ◽

Cited By ~ 1

Author(s):

D X Tishkoff ◽

A W Johnson ◽

R D Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Open Reading Frame ◽

Strand Exchange ◽

Wild Type ◽

Homologous Pairing ◽

Gene Encoding ◽

Reading Frame ◽

Cloned Gene ◽

Diploid Cells ◽

Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

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Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus

Applied and Environmental Microbiology ◽

10.1128/aem.64.5.1831-1836.1998 ◽

1998 ◽

Vol 64 (5) ◽

pp. 1831-1836 ◽

Cited By ~ 21

Author(s):

Pekka Varmanen ◽

Terhi Rantanen ◽

Airi Palva ◽

Soile Tynkkynen

Keyword(s):

Lactobacillus Rhamnosus ◽

Gene Replacement ◽

Lactobacillus Helveticus ◽

Open Reading Frame ◽

Transcriptional Unit ◽

Wild Type ◽

Reading Frame ◽

Gene Expressing ◽

Produce Acid

ABSTRACT A peptidase gene expressingl-proline-β-naphthylamide-hydrolyzing activity was cloned from a gene library of Lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kbSacI fragment. A sequence analysis of the SacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The clonedpepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates l-leucine-β-naphthylamide andl-phenylalanine-β-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.

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Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively

Journal of General Virology ◽

10.1099/0022-1317-83-6-1477 ◽

2002 ◽

Vol 83 (6) ◽

pp. 1477-1482 ◽

Cited By ~ 40

Author(s):

Kyoji Hagiwara ◽

Shujing Rao ◽

Simon W. Scott ◽

Gerald R. Carner

Keyword(s):

Amino Acids ◽

Bombyx Mori ◽

Nucleotide Sequences ◽

Open Reading Frame ◽

Capsid Proteins ◽

Structural Proteins ◽

Reading Frame ◽

The Family ◽

Molecular Masses ◽

Rice Ragged Stunt Virus

The complete nucleotide sequences of genomic segments S1, S3 and S4 from Bombyx mori cypovirus 1 (BmCPV-1) have been determined. The segments consisted of 4190, 3846 and 3262 nucleotides encoding putative proteins of 1333, 1239 and 1058 amino acids with molecular masses of approximately 148, 140 and 120 kDa (p148, p140 and p120, respectively). All segments possess a single open reading frame. Homology searches showed that all three proteins have homologies to proteins of Rice ragged stunt virus, a member of the genus Oryzavirus within the family Reoviridae. Partial homologies of p140 to structural proteins in other viruses were also found. The predicted molecular masses and the homologies with structural proteins in other viruses lead us to suggest that S1, S3 and S4 encode the capsid proteins VP1, VP3, and VP4, respectively, of BmCPV-1.

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Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2374 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2374-2382 ◽

Cited By ~ 28

Author(s):

M Bernstein ◽

W Hoffmann ◽

G Ammerer ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Gene Fusion ◽

Protein Assembly ◽

Open Reading Frame ◽

Cell Fractionation ◽

Multicopy Plasmid ◽

Wild Type ◽

Cytosol Fraction ◽

Reading Frame

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.

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Deletion of Open Reading Frame UL26 from the Human Cytomegalovirus Genome Results in Reduced Viral Growth, Which Involves Impaired Stability of Viral Particles

Journal of Virology ◽

10.1128/jvi.02585-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5423-5434 ◽

Cited By ~ 47

Author(s):

Kerstin Lorz ◽

Heike Hofmann ◽

Anja Berndt ◽

Nina Tavalai ◽

Regina Mueller ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Transcriptional Activation ◽

Deletion Mutant ◽

Artificial Chromosome ◽

Open Reading Frame ◽

Wild Type Virus ◽

Wild Type ◽

Reading Frame ◽

Transcriptional Activation Domain ◽

Viral Particles

ABSTRACT We previously showed that open reading frame (ORF) UL26 of human cytomegalovirus, a member of the US22 multigene family of betaherpesviruses, encodes a novel tegument protein, which is imported into cells in the course of viral infection. Moreover, we demonstrated that pUL26 contains a strong transcriptional activation domain and is capable of stimulating the major immediate-early (IE) enhancer-promoter. Since this suggested an important function of pUL26 during the initiation of the viral replicative cycle, we sought to ascertain the relevance of pUL26 by construction of a viral deletion mutant lacking the UL26 ORF using the bacterial artificial chromosome mutagenesis procedure. The resulting deletion virus was verified by PCR, enzyme restriction, and Southern blot analyses. After infection of human foreskin fibroblasts, the UL26 deletion mutant showed a small-plaque phenotype and replicated to significantly lower titers than wild-type or revertant virus. In particular, we noticed a striking decrease of infectious titers 7 days postinfection in a multistep growth experiment, whereas the release of viral DNA from infected cells was not impaired. A further investigation of this aspect revealed a significantly diminished stability of viral particles derived from the UL26 deletion mutant. Consistent with this, we observed that the tegument composition of the deletion mutant deviates from that of the wild-type virus. We therefore hypothesize that pUL26 plays a role not only in the onset of IE gene transcription but also in the assembly of the viral tegument layer in a stable and correct manner.

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