Retroviral insertions in the murine His-1 locus activate the expression of a novel RNA that lacks an extensive open reading frame

The His-1 locus is a common site of viral insertion in murine myeloid leukemias induced by the wild mouse ecotropic retrovirus, CasBrM. In this report, we describe the cloning of a novel gene at the His-1 locus and show that His-1 expression is associated with the transformed phenotype. Northern (RNA) blot analysis identified His-1 transcripts in four transformed myeloid cell lines but in no normal tissues examined. Two of these cell lines were derived from retrovirus-induced myeloid leukemias that harbor integrated proviruses which drive His-1 gene expression by promoter insertion. The two other cell lines expressed a discrete 3-kb His-1 RNA that is derived from a novel gene consisting of three exons that span 6 kb on mouse chromosome 2. The His-1 gene is conserved as a single-copy sequence in multiple vertebrate species and is expressed as a spliced and polyadenylated RNA. A protein-coding region is not evident from analysis of the His-1 sequence because of the presence of multiple small open reading frames, none of which are greater than 219 bp. This lack of an extensive open reading frame is an unusual feature that is shared by other RNA molecules believed to function in the absence of translation.

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Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.

Genetics ◽

10.1093/genetics/132.1.125 ◽

1992 ◽

Vol 132 (1) ◽

pp. 125-133 ◽

Cited By ~ 1

Author(s):

N L Glass ◽

L Lee

Keyword(s):

Neurospora Crassa ◽

Mating Type ◽

Single Copy ◽

Open Reading Frame ◽

Sexual Cycle ◽

Reading Frame ◽

Heterokaryon Incompatibility ◽

Functional Regions ◽

A Genome ◽

Ascospore Formation

Abstract In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.

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Unexpected Absence of the Epstein-Barr Virus (EBV) lyLMP-1 Open Reading Frame in Tumor Virus Isolates: Lack of Correlation between Met129 Status and EBV Strain Identity

Journal of Virology ◽

10.1128/jvi.77.7.4415-4422.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4415-4422 ◽

Cited By ~ 9

Author(s):

Kimberly D. Erickson ◽

Christoph Berger ◽

William F. Coffin ◽

Edwin Schiff ◽

Dennis M. Walling ◽

...

Keyword(s):

Cell Lines ◽

Epstein Barr Virus ◽

Open Reading Frame ◽

Lytic Cycle ◽

Reading Frame ◽

Barr Virus ◽

Tumor Virus ◽

Epstein Barr ◽

Virus Isolates ◽

Codon 129

ABSTRACT The lytic cycle-associated lytic latent membrane protein-1 (lyLMP-1) of Epstein-Barr virus (EBV) is an amino-terminally truncated form of the oncogenic LMP-1. Although lyLMP-1 shares none of LMP-1's transforming and signal transducing activities, we recently reported that lyLMP-1 can negatively regulate LMP-1-stimulated NF-κB activation. The lyLMP-1 protein encoded by the B95-8 strain of EBV initiates from methionine 129 (Met129) of the LMP-1 open reading frame (ORF). The recent report that Met129 in the B95-8 LMP-1 ORF is not conserved in the Akata strain of EBV prompted us to screen a panel of EBV-positive cell lines for conservation of Met129 and lyLMP-1 expression. We found that 15 out of 16 tumor-associated virus isolates sequenced encoded an ATT or ACC codon in place of ATG in the LMP-1 ORF at position 129, and tumor cell lines harboring isolates lacking an ATG at codon 129 did not express the lyLMP-1 protein. In contrast, we found that EBV DNA from 22 out of 37 healthy seropositive donors retained the Met129 codon. Finally, the lyLMP-1 initiator occurs variably within distinct EBV strains and its presence cannot be predicted by EBV strain identity. Thus, Met129 is not peculiar to the B95-8 strain of EBV, but rather can be found in the background of several evolutionarily distinct EBV strains. Its absence from EBV isolates from tumors raises the possibility of selective pressure on Met129 in EBV-dependent tumors.

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Pax-6, a murine paired box gene, is expressed in the developing CNS

Development ◽

10.1242/dev.113.4.1435 ◽

1991 ◽

Vol 113 (4) ◽

pp. 1435-1449 ◽

Cited By ~ 126

Author(s):

C. Walther ◽

P. Gruss

Keyword(s):

Amino Acids ◽

Neural Tube ◽

Multigene Family ◽

Ventricular Zone ◽

Open Reading Frame ◽

Cdna Clones ◽

Coding Region ◽

Paired Domain ◽

Reading Frame ◽

Pax Genes

A multigene family of paired-box-containing genes (Pax genes) has been identified in the mouse. In this report, we describe the expression pattern of Pax-6 during embryogenesis and the isolation of cDNA clones spanning the entire coding region. The Pax-6 protein consists of 422 amino acids as deduced from the longest open reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with day 8 of gestation, Pax-6 is expressed in discrete regions of the forebrain and the hindbrain. In the neural tube, expression is mainly confined to mitotic active cells in the ventral ventricular zone along the entire anteroposterior axis starting at day 8.5 of development. Pax-6 is also expressed in the developing eye, the pituitary and the nasal epithelium.

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Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame

Hepatology ◽

10.1002/hep.1840360620 ◽

2002 ◽

Vol 36 (6) ◽

pp. 1431-1438 ◽

Cited By ~ 12

Author(s):

Hideki Aizaki ◽

Takashi Harada ◽

Motoyuki Otsuka ◽

Naohiko Seki ◽

Mami Matsuda ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Liver Cell ◽

Expression Profiling ◽

Open Reading Frame ◽

Reading Frame

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Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

Journal of Virology ◽

10.1128/jvi.00743-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 9

Author(s):

Benjamin Brennan ◽

Veronica V. Rezelj ◽

Richard M. Elliott

Keyword(s):

Virus Infection ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Nonstructural Protein ◽

Transcription Termination ◽

Severe Disease ◽

Open Reading Frame ◽

Coding Region ◽

Reading Frame ◽

Green Fluorescent

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA.

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Proliferation Response to Interleukin-2 and Jak/Stat Activation of T Cells Immortalized by Human T-Cell Lymphotropic Virus Type 1 Is Independent of Open Reading Frame I Expression

Journal of Virology ◽

10.1128/jvi.73.11.9642-9649.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9642-9649 ◽

Cited By ~ 31

Author(s):

Nathaniel D. Collins ◽

Celine D’Souza ◽

Björn Albrecht ◽

Michael D. Robek ◽

Lee Ratner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Virus Type ◽

Interleukin 2 ◽

Receptor Expression ◽

Open Reading Frame ◽

Reading Frame ◽

Human T Cell

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12I has a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (α, β, or γc) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.

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Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2435-2443.1987 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2435-2443

Author(s):

I L Andrulis ◽

J Chen ◽

P N Ray

Keyword(s):

Cell Lines ◽

Untranslated Region ◽

Asparagine Synthetase ◽

Synthetase Activity ◽

Coding Region ◽

Protein Coding ◽

Reading Frame ◽

Amino Terminal ◽

Multiple Copies ◽

Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

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Targeted Inactivation of a Tobacco Intron–containing Open Reading Frame Reveals a Novel Chloroplast-encoded Photosystem I–related Gene

The Journal of Cell Biology ◽

10.1083/jcb.139.1.95 ◽

1997 ◽

Vol 139 (1) ◽

pp. 95-102 ◽

Cited By ~ 85

Author(s):

Stephanie Ruf ◽

Hans Kössel ◽

Ralph Bock

Keyword(s):

Photosystem I ◽

Reverse Genetics ◽

Higher Plants ◽

Single Copy ◽

Open Reading Frame ◽

Higher Plant ◽

Reading Frame ◽

Chloroplast Genomes ◽

Plant Chloroplast ◽

Targeted Inactivation

The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Δycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Δycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

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Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2435 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2435-2443 ◽

Cited By ~ 56

Author(s):

I L Andrulis ◽

J Chen ◽

P N Ray

Keyword(s):

Cell Lines ◽

Untranslated Region ◽

Asparagine Synthetase ◽

Synthetase Activity ◽

Coding Region ◽

Protein Coding ◽

Reading Frame ◽

Amino Terminal ◽

Multiple Copies ◽

Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

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