Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo

1992 ◽  
Vol 22 (6) ◽  
pp. 507-509 ◽  
Author(s):  
M. Rohe ◽  
F. Meinhardt
Keyword(s):  
Open Reading Frames ◽  
Linear Plasmid ◽  
Morchella Conica ◽  
Reading Frames

scholarly journals Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion

2008 ◽  
Vol 190 (18) ◽  
pp. 6111-6118 ◽  
Author(s):  
P. Rousseau ◽  
C. Loot ◽  
C. Turlan ◽  
S. Nolivos ◽  
M. Chandler
Keyword(s):  
Insertion Sequence ◽  
Regulatory Protein ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
Functional Differences ◽  
Left And Right ◽  
Ordered Assembly ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

scholarly journals Genomic Sequence of Rhesus Cytomegalovirus 180.92: Insights into the Coding Potential of Rhesus Cytomegalovirus

2006 ◽  
Vol 80 (8) ◽  
pp. 4179-4182 ◽  
Author(s):  
Pierre Rivailler ◽  
Amitinder Kaur ◽  
R. Paul Johnson ◽  
Fred Wang
Keyword(s):  
Genomic Sequence ◽  
Open Reading Frames ◽  
Rhesus Cytomegalovirus ◽  
Human Cmv ◽  
Coding Capacity ◽  
Coding Potential ◽  
Reading Frames ◽  
Genetic Rearrangements

ABSTRACT A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence

1988 ◽  
Vol 8 (12) ◽  
pp. 5439-5447
Author(s):  
P P Mueller ◽  
B M Jackson ◽  
P F Miller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequences ◽  
Lacz Fusions ◽  
Upstream Open Reading Frames ◽  
Reading Frames ◽  
Starvation Conditions

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

E1−E4+ Adenoviral Gene Transfer Vectors Function as a “Pro-Life” Signal to Promote Survival of Primary Human Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2936-2944 ◽  
Author(s):  
Ramachandran Ramalingam ◽  
Shahin Rafii ◽  
Stefan Worgall ◽  
Douglas E. Brough ◽  
Ronald G. Crystal
Keyword(s):  
Endothelial Cells ◽  
Open Reading Frames ◽  
Adenoviral Gene Transfer ◽  
Vector Genome ◽  
Transfer Vectors ◽  
Reading Frames ◽  
Human Endothelium ◽  
Pro Life

Abstract Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1−E4+ replication–deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1−E4+ Ad vectors provide an “antiapoptotic” signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of “suspended animation,” remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a “pro-life” program that modifies cultured endothelial cells to survive for prolonged periods.

scholarly journals Characterization of Endogenous Retroviruses in Sheep

2003 ◽  
Vol 77 (20) ◽  
pp. 11268-11273 ◽  
Author(s):  
Nikolai Klymiuk ◽  
Mathias Müller ◽  
Gottfried Brem ◽  
Bernhard Aigner
Keyword(s):  
Endogenous Retrovirus ◽  
Ovis Aries ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
In Vivo Experiments ◽  
Pregnant Sheep ◽  
Reading Frames

ABSTRACT Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

scholarly journals Accurate detection of short and long active ORFs using Ribo-seq data

2019 ◽  
Vol 36 (7) ◽  
pp. 2053-2059 ◽  
Author(s):  
Saket Choudhary ◽  
Wenzheng Li ◽  
Andrew D. Smith
Keyword(s):  
Caenorhabditis Elegans ◽  
Deep Sequencing ◽  
Open Reading Frames ◽  
Supplementary Information ◽  
Supplementary Data ◽  
Accurate Detection ◽  
Short Orfs ◽  
Coding Potential ◽  
Reading Frames

Abstract Motivation Ribo-seq, a technique for deep-sequencing ribosome-protected mRNA fragments, has enabled transcriptome-wide monitoring of translation in vivo. It has opened avenues for re-evaluating the coding potential of open reading frames (ORFs), including many short ORFs that were previously presumed to be non-translating. However, the detection of translating ORFs, specifically short ORFs, from Ribo-seq data, remains challenging due to its high heterogeneity and noise. Results We present ribotricer, a method for detecting actively translating ORFs by directly leveraging the three-nucleotide periodicity of Ribo-seq data. Ribotricer demonstrates higher accuracy and robustness compared with other methods at detecting actively translating ORFs including short ORFs on multiple published datasets across species inclusive of Arabidopsis, Caenorhabditis elegans, Drosophila, human, mouse, rat, yeast and zebrafish. Availability and implementation Ribotricer is available at https://github.com/smithlabcode/ribotricer. All analysis scripts and results are available at https://github.com/smithlabcode/ribotricer-results. Supplementary information Supplementary data are available at Bioinformatics online.

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 Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

2014 ◽  
Vol 13 (5) ◽  
pp. 664-674 ◽  
Author(s):  
Bhaskar Anand Jha ◽  
Abeer Fadda ◽  
Clementine Merce ◽  
Elisha Mugo ◽  
Dorothea Droll ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Mrna Levels ◽  
Coding Region ◽  
Content Type ◽  
Puf Proteins ◽  
Reading Frames

ABSTRACT Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.

scholarly journals An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (7) ◽  
pp. 4485-4492 ◽  
Author(s):  
B A Dombroski ◽  
Q Feng ◽  
S L Mathias ◽  
D M Sassaman ◽  
A F Scott ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Consensus Sequence ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Long Terminal Repeats ◽  
Base Pairs ◽  
In Vivo Assay ◽  
Reading Frames

L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.

scholarly journals Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis inStreptomyces griseus DSM40695

2000 ◽  
Vol 44 (7) ◽  
pp. 1809-1817 ◽  
Author(s):  
Wyatt C. Smith ◽  
Longkuan Xiang ◽  
Ben Shen
Keyword(s):  
Polyketide Synthase ◽  
Isotope Labeling ◽  
Streptomyces Griseus ◽  
Open Reading Frames ◽  
A Cell ◽  
Cyclic Polyethers ◽  
The Difference ◽  
Reading Frames

ABSTRACT The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR andnonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of thenonS mutant by the expression of nonS intrans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of thenonS mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonSgene and fermentation of the nonS mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis inS. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

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