scholarly journals Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080)

1994 ◽  
Vol 298 (1) ◽  
pp. 31-37 ◽  
Author(s):  
A Fertala ◽  
A L Sieron ◽  
A Ganguly ◽  
S W Li ◽  
L Ala-Kokko ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Tumour Cell ◽  
Copy Number ◽  
Gene Copy Number ◽  
Basement Membranes ◽  
Gene Copy ◽  
Nucleotide Sequencing ◽  
Tumour Cell Line ◽  
Col2a1 Gene

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.

scholarly journals Nongenotoxic ABCB1 activator tetraphenylphosphonium can contribute to doxorubicin resistance in MX-1 breast cancer cell line

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raimonda Kubiliute ◽  
Indre Januskeviciene ◽  
Ruta Urbanaviciute ◽  
Kristina Daniunaite ◽  
Monika Drobniene ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Protein Level ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Common Mechanism ◽  
Dna Hypomethylation ◽  
Mesenchymal Transition ◽  
Molecular Features

AbstractHyperactivation of ABC transporter ABCB1 and induction of epithelial–mesenchymal transition (EMT) are the most common mechanism of acquired cancer chemoresistance. This study describes possible mechanisms, that might contribute to upregulation of ABCB1 and synergistically boost the acquisition of doxorubicin (DOX) resistance in breast cancer MX-1 cell line. DOX resistance in MX-1 cell line was induced by a stepwise increase of drug concentration or by pretreatment of cells with an ABCB1 transporter activator tetraphenylphosphonium (TPP+) followed by DOX exposure. Transcriptome analysis of derived cells was performed by human gene expression microarrays and by quantitative PCR. Genetic and epigenetic mechanisms of ABCB1 regulation were evaluated by pyrosequencing and gene copy number variation analysis. Gradual activation of canonical EMT transcription factors with later activation of ABCB1 at the transcript level was observed in DOX-only treated cells, while TPP+ exposure induced considerable activation of ABCB1 at both, mRNA and protein level. The changes in ABCB1 mRNA and protein level were related to the promoter DNA hypomethylation and the increase in gene copy number. ABCB1-active cells were highly resistant to DOX and showed morphological and molecular features of EMT. The study suggests that nongenotoxic ABCB1 inducer can possibly accelerate development of DOX resistance.

scholarly journals Long-term reduction of amplified ornithine decarboxylase sequences in human myeloma cells

1995 ◽  
Vol 310 (1) ◽  
pp. 299-303
Author(s):  
J Wahlfors ◽  
S Myöhänen ◽  
V P Korhonen ◽  
L Alhonen ◽  
J Jänne
Keyword(s):  
Enzyme Activity ◽  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Copy Number ◽  
Methylation Status ◽  
Gene Copy Number ◽  
Myeloma Cell ◽  
Gene Copy ◽  
Myeloma Cell Line ◽  
Myeloma Cells

(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Co-adaption of tRNA Gene Copy Number and Amino Acid Usage Influences Translation Rates in Three Life Domains

2017 ◽  
Author(s):  
Meng-Ze Du ◽  
Wen Wei ◽  
Lei Qin ◽  
Shuo Liu ◽  
An-Ying Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Copy Number ◽  
Trna Gene ◽  
Fluorescent Protein ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Translation Rate ◽  
Translation Speed ◽  
Fast Growing

AbstractThe cellular translation process should obey the principle of maximizing efficiency and minimizing resource and energy costs. Here, we validated this principle by focusing on the basic translation components of tRNAs and amino acids. To most efficiently utilize these components, we reasoned that the quantities of the 20 tRNAs and their corresponding amino acids would be consistent in an organism. The two values should match at both the organismal and protein scales. For the former, they co-vary to meet the need to translate more proteins in fast-growing or larger cells. For the latter, they are consistent to different extents for various proteins in an organism to comply with different needs of translation speed. In this work, 310 out of 410 genomes in three domains had significant co-adaptions between the tRNA gene copy number and amino acid composition, and thus validating the principle at the organism scale. Furthermore, fast-growing bacteria co-adapt better than slow-growing ones. Highly expressed proteins and those connected to acute responses have better co-adaption, illustrating the principle at the individual protein scale. Experimentally, manipulating the tRNA gene copy number to optimize co-adaption between enhanced green fluorescent protein (EGFP) and tRNA gene set of Escherichia coli indeed lifted the translation rate (speed). Our results also contribute to revealing a translation rate-associated factor with universal and global effects. From a practical perspective, our findings suggest a strategy to increase the expression of target proteins and have implications for designing chassis cells in the field of synthetic biology field.

Cytochrome P450 2B diversity in a dietary specialist—the red tree vole (Arborimus longicaudus)

2018 ◽  
Vol 99 (3) ◽  
pp. 578-585 ◽  
Author(s):  
Smiljka Kitanovic ◽  
Chad A Marks-Fife ◽  
Quincy A Parkes ◽  
P Ross Wilderman ◽  
James R Halpert ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Copy Number ◽  
Plant Secondary Metabolites ◽  
Gene Copy Number ◽  
Douglas Fir ◽  
Amino Acid Sequences ◽  
Gene Copy ◽  
Amino Acid Residues ◽  
Herbivorous Species

Abstract Although herbivores rely on liver enzymes to biotransform plant secondary metabolites ingested in plant-based diets, only a few enzymes from a handful of species have been characterized at the genomic level. In this study, we examined cytochrome P450 2B (CYP2B) sequence diversity and gene copy number in a conifer specialist, the red tree vole (Arborimus longicaudus). We fed captive individuals exclusively Douglas-fir (Pseudotsuga menziesii) foliage, cloned and sequenced their liver CYP2B cDNA, and estimated CYP2B gene copy number. We identified 21 unique CYP2B nucleotide sequences, and 20 unique CYP2B amino acid sequences. Gene copy number of CYP2B was estimated at 7.7 copies per haploid genome. We compared red tree vole CYP2B with CYP2B sequences of a generalist, the prairie vole (Microtus ochrogaster), found in GenBank. Our study revealed that the CYP2B enzymes of red tree voles possess unique sequences compared to CYP2B enzymes of other herbivorous species. The unique combination of amino acid residues at key substrate recognition sites of CYP2B enzymes may underlie the ability of the red tree vole to specialize on a highly toxic diet of Douglas-fir.

scholarly journals Brachyury gene copy number gain and activation of the PI3K/Akt pathway: association with upregulation of oncogenic Brachyury expression in skull base chordoma

2018 ◽  
Vol 128 (5) ◽  
pp. 1428-1437 ◽  
Author(s):  
Ryohei Otani ◽  
Akitake Mukasa ◽  
Masahiro Shin ◽  
Mayu Omata ◽  
Shunsaku Takayanagi ◽  
...  
Keyword(s):  
Cell Line ◽  
Copy Number ◽  
Growth Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Copy Number ◽  
Copy Number Gain ◽  
High Expression ◽  
Gene Copy ◽  
Akt Pathway ◽  
Expression Levels

OBJECTIVEChordoma is a slow-growing but clinically malignant tumor, and the prognosis remains poor in many cases. There is a strong impetus to develop more effective targeted molecular therapies. On this basis, the authors investigated the potential of Brachyury, a transcription factor involved in notochord development, as a candidate molecular target for the treatment of chordoma.METHODSBrachyury gene copy number and expression levels were evaluated by quantitative polymerase chain reaction in 27 chordoma samples, and the transcriptomes of Brachyury high-expression tumors (n = 4) and Brachyury low-expression tumors (n = 4) were analyzed. A chordoma cell line (U-CH2) was used to investigate the signaling pathways that regulate Brachyury expression.RESULTSAll chordoma specimens expressed Brachyury, and expression levels varied widely. Patients with higher Brachyury expression had significantly shorter progression-free survival (5 months, n = 11) than those with lower expression (13 months, n = 16) (p = 0.03). Somatic copy number gain was confirmed in 12 of 27 (44%) cases, and copy number was positively correlated with Brachyury expression (R = 0.61, p < 0.001). Expression of PI3K/Akt pathway genes was upregulated in Brachyury high-expression tumors, and suppression of PI3K signaling led to reduced Brachyury expression and inhibition of cell growth in the U-CH2 chordoma cell line.CONCLUSIONSActivation of the PI3K/Akt pathway and Brachyury copy number gain are strongly associated with Brachyury overexpression, which appears to be a key event in chordoma growth regulation. These findings suggest that targeting Brachyury and PI3K/Akt signaling may be an effective new approach for treating chordoma.

scholarly journals Co-adaption of tRNA gene copy number and amino acid usage influences translation rates in three life domains

DNA Research ◽  
2017 ◽  
Vol 24 (6) ◽  
pp. 623-633 ◽  
Author(s):  
Meng-Ze Du ◽  
Wen Wei ◽  
Lei Qin ◽  
Shuo Liu ◽  
An-Ying Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Copy Number ◽  
Trna Gene ◽  
Gene Copy Number ◽  
Amino Acid Usage ◽  
Gene Copy ◽  
Life Domains

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

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