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.

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 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.

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 Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

1989 ◽  
Vol 9 (2) ◽  
pp. 541-550 ◽  
Author(s):  
S E Porter ◽  
J J Champoux
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Unknown Origin ◽  
Simian Virus ◽  
Asymmetric Distribution ◽  
Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

scholarly journals Ancient Adversary – HERV-K (HML-2) in Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Eoin Dervan ◽  
Dibyangana D. Bhattacharyya ◽  
Jake D. McAuliffe ◽  
Faizan H. Khan ◽  
Sharon A. Glynn
Keyword(s):  
Tumour Progression ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
Human Endogenous Retroviruses ◽  
Viral Particles ◽  
Metastatic Capacity ◽  
Coding Capacity ◽  
Reading Frames

Human endogenous retroviruses (HERV), ancient integrations of exogenous viruses, make up 8% of our genome. Long thought of as mere vestigial genetic elements, evidence is now accumulating to suggest a potential functional role in numerous pathologies including neurodegenerative diseases, autoimmune disorders, and multiple cancers. The youngest member of this group of transposable elements is HERV-K (HML-2). Like the majority of HERV sequences, significant post-insertional mutations have disarmed HERV-K (HML-2), preventing it from producing infectious viral particles. However, some insertions have retained limited coding capacity, and complete open reading frames for all its constituent proteins can be found throughout the genome. For this reason HERV-K (HML-2) has garnered more attention than its peers. The tight epigenetic control thought to suppress expression in healthy tissue is lost during carcinogenesis. Upregulation of HERV-K (HML-2) derived mRNA and protein has been reported in a variety of solid and liquid tumour types, and while causality has yet to be established, progressively more data are emerging to suggest this phenomenon may contribute to tumour growth and metastatic capacity. Herein we discuss its potential utility as a diagnostic tool and therapeutic target in light of the current in vitro, in vivo and clinical evidence linking HERV-K (HML-2) to tumour progression.

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

scholarly journals Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs.

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378 ◽  
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

scholarly journals Complete nucleotide sequence of a mouse VL30 retro-element.

1988 ◽  
Vol 8 (8) ◽  
pp. 2989-2998 ◽  
Author(s):  
S E Adams ◽  
P D Rathjen ◽  
C A Stanway ◽  
S M Fulton ◽  
M H Malim ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Complete Nucleotide Sequence ◽  
Leukemia Virus ◽  
Open Reading Frames ◽  
Long Terminal Repeats ◽  
Base Pairs ◽  
Viral Packaging ◽  
Protein Functions ◽  
Active Promoter ◽  
Reading Frames

The complete nucleotide sequence of a mouse retro-element is presented. The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp. The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements. However, some regions of the genome showed some homology to retroviral gag and pol open reading frames. There was no region in VL30 corresponding to a retroviral env gene. This implies that VL30 is related to retrotransposons rather than to retroviruses. The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites. These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission.

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules

1989 ◽  
Vol 9 (2) ◽  
pp. 541-550
Author(s):  
S E Porter ◽  
J J Champoux
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Unknown Origin ◽  
Simian Virus ◽  
Asymmetric Distribution ◽  
Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

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