From Selective Full-Length Genes Isolation by TAR Cloning in Yeast to Their Expression from HAC Vectors in Human Cells

2014 ◽  
pp. 3-26 ◽  
Author(s):  
Natalay Kouprina ◽  
Nicholas C. O. Lee ◽  
Artem V. Kononenko ◽  
Alexander Samoshkin ◽  
Vladimir Larionov
Keyword(s):  
Human Cells ◽  
Full Length ◽  
Tar Cloning

scholarly journals Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

2021 ◽  
Author(s):  
Evangelos D. Karousis ◽  
Foivos Gypas ◽  
Mihaela Zavolan ◽  
Oliver Muehlemann
Keyword(s):  
Regulation Of Gene Expression ◽  
Degradation Pathway ◽  
Human Cells ◽  
Full Length ◽  
Main Function ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Short Read ◽  
Expression Levels ◽  
Short Read Sequencing

Background: Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. Results: To identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD still seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. Conclusions: Long-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

scholarly journals Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0121055 ◽  
Author(s):  
Bin Huang ◽  
Tanja Lucas ◽  
Claudia Kueppers ◽  
Xiaomin Dong ◽  
Maike Krause ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Human Cells ◽  
Full Length ◽  
Mutant Huntingtin ◽  
Scalable Production

scholarly journals Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Evangelos D. Karousis ◽  
Foivos Gypas ◽  
Mihaela Zavolan ◽  
Oliver Mühlemann
Keyword(s):  
Regulation Of Gene Expression ◽  
Degradation Pathway ◽  
Human Cells ◽  
Full Length ◽  
Main Function ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Short Read ◽  
Expression Levels ◽  
Short Read Sequencing

Abstract Background Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. Results To identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. Conclusions Long-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

scholarly journals Mechanistic constraints on diversity in human V(D)J recombination.

1996 ◽  
Vol 16 (1) ◽  
pp. 258-269 ◽  
Author(s):  
G H Gauss ◽  
M R Lieber
Keyword(s):  
Statistical Analysis ◽  
Base Composition ◽  
Human Cells ◽  
Full Length ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Inverted Repeats ◽  
Stacking Interactions ◽  
Large Collection ◽  
Significant Frequency ◽  
End Sequences

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.

scholarly journals Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

2019 ◽  
Vol 295 (4) ◽  
pp. 1105-1119 ◽  
Author(s):  
Mukul Sherekar ◽  
Sae-Won Han ◽  
Rodolfo Ghirlando ◽  
Simon Messing ◽  
Matthew Drew ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Analytical Ultracentrifugation ◽  
Small Gtpases ◽  
Human Cells ◽  
Full Length ◽  
Mitogen Activated Protein Kinase ◽  
Size Exclusion ◽  
Type I ◽  
High Affinity

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

scholarly journals Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells

2002 ◽  
Vol 83 (2) ◽  
pp. 439-442 ◽  
Author(s):  
Lars Aagaard ◽  
Jacob Giehm Mikkelsen ◽  
Søren Warming ◽  
Mogens Duch ◽  
Finn Skou Pedersen
Keyword(s):  
Mammalian Cells ◽  
Human Cells ◽  
Full Length

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1 b cells with respect to infection though the ecotropic receptor pathway.

scholarly journals Dynamics of the Full Length and Mutated Heat Shock Factor 1 in Human Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (7) ◽  
pp. e67566 ◽  
Author(s):  
Gaëtan Herbomel ◽  
Meike Kloster-Landsberg ◽  
Eric G. Folco ◽  
Edwige Col ◽  
Yves Usson ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Factor ◽  
Human Cells ◽  
Full Length ◽  
Heat Shock Factor 1

mRNA transcripts related to full-length endogenous retroviral DNA in human cells

Nature ◽  
1983 ◽  
Vol 306 (5943) ◽  
pp. 604-607 ◽  
Author(s):  
Arnold B. Rabson ◽  
Paul E. Steele ◽  
Claude F. Garon ◽  
Malcolm A. Martin
Keyword(s):  
Human Cells ◽  
Full Length ◽  
Mrna Transcripts

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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