scholarly journals Accessibility of the Shine–Dalgarno sequence dictates N-terminal codon bias in E. coli

2017 ◽  
Author(s):  
Sanchari Bhattacharyya ◽  
William M. Jacobs ◽  
Bharat V. Adkar ◽  
Jin Yan ◽  
Wenli Zhang ◽  
...  
Keyword(s):  
Codon Bias ◽  
Mrna Levels ◽  
Coding Region ◽  
Synonymous Substitutions ◽  
E Coli ◽  
Protein Levels ◽  
Shine Dalgarno Sequence ◽  
General Physical ◽  
Molecular Base ◽  
Terminal Codon

AbstractDespite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine–Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E. coli genome specifically implicates avoidance of intra-molecular base-pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes.

scholarly journals Accessibility of the Shine-Dalgarno Sequence Dictates N-Terminal Codon Bias in E. coli

2018 ◽  
Vol 70 (5) ◽  
pp. 894-905.e5 ◽  
Author(s):  
Sanchari Bhattacharyya ◽  
William M. Jacobs ◽  
Bharat V. Adkar ◽  
Jin Yan ◽  
Wenli Zhang ◽  
...  
Keyword(s):  
Codon Bias ◽  
E Coli ◽  
Shine Dalgarno Sequence ◽  
Terminal Codon

Rat Muscle Fructose-1,6-Bisphosphatase: Cloning of the cDNA, Expression of the Recombinant Enzyme, and Expression Analysis in Different Tissues

1999 ◽  
Vol 380 (9) ◽  
pp. 1079-1085 ◽  
Author(s):  
Samiya Al-Robaiy ◽  
Klaus Eschrich
Keyword(s):  
Northern Blotting ◽  
Recombinant Enzyme ◽  
Rat Tissues ◽  
Mrna Levels ◽  
Coding Region ◽  
Total Rna ◽  
E Coli ◽  
Rat Muscle ◽  
Positional Identity ◽  
Fructose 1,6 Bisphosphatase

Abstract The 1282 bp cDNA of an isoenzyme of fructose-1,6- bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose- 1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 μg of total RNA of rat muscle contains 1.7 × 106 molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 × 104 copies of this message were found per μg total RNA of heart and kidney, respectively.

scholarly journals Divergence of the yellow Gene Between Drosophila melanogaster and D. subobscura: Recombination Rate, Codon Bias and Synonymous Substitutions

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 165-175 ◽  
Author(s):  
Agustí Munté ◽  
Montserrat Aguadé ◽  
Carmen Segarra
Keyword(s):  
Drosophila Melanogaster ◽  
Recombination Rate ◽  
Codon Bias ◽  
Regulatory Sequences ◽  
Coding Region ◽  
Recombination Rates ◽  
Fixation Rate ◽  
Synonymous Substitutions ◽  
Cis Acting ◽  
Slightly Deleterious Mutations

Abstract The yellow (y) gene maps near the telomere of the X chromosome in Drosophila melanogaster but not in D. subobscura. Thus the strong reduction in the recombination rate associated with telomeric regions is not expected in D. subobscura. To study the divergence of a gene whose recombination rate differs between two species, the y gene of D. subobscura was sequenced. Sequence comparison between D. melanogaster and D. subobscura revealed several elements conserved in noncoding regions that may correspond to putative cis-acting regulatory sequences. Divergence in the y gene coding region between D. subobscura and D. melanogaster was compared with that found in other genes sequenced in both species. Both, yellow and scute exhibit an unusually high number of synonymous substitutions per site (ps). Also for these genes, the extent of codon bias differs between both species, being much higher in D. subobscura than in D. melanogaster. This pattern of divergence is consistent with the hitchhiking and background selection models that predict an increase in the fixation rate of slightly deleterious mutations and a decrease in the rate of fixation of slightly advantageous mutations in regions with low recombination rates such as in the y-sc gene region of D. melanogaster.

scholarly journals Increased expansion of the lung stimulates calmodulin 2 expression in fetal sheep

2002 ◽  
Vol 282 (3) ◽  
pp. L440-L447 ◽  
Author(s):  
A. M. Gillett ◽  
M. J. Wallace ◽  
M. T. Gillespie ◽  
S. B. Hooper
Keyword(s):  
Northern Blot Analysis ◽  
Fetal Lung ◽  
Mrna Levels ◽  
Lung Growth ◽  
Coding Region ◽  
Fetal Sheep ◽  
Protein Levels ◽  
Lung Expansion ◽  
Rapid Amplification

Obstruction of the fetal trachea causes the lungs to expand with accumulated liquid. Although this is a potent stimulus for lung growth, the mechanisms involved are unknown. Our aim was to identify genes that are differentially expressed as a result of increased fetal lung expansion. Using differential display RT-PCR, we isolated a cDNA fragment partially encoding calmodulin 2 (CALM2) and identified the remainder of the coding region by 5′-rapid amplification of cDNA ends. Differential expression of CALM2 was confirmed by Northern blot analysis; CALM2 mRNA levels were increased to 161 ± 5% of control at 2 days of increased lung expansion, induced by tracheal obstruction (TO), and had returned to control levels at days 4 and 10. Using in situ hybridization analysis, we found that the proportion of CALM2-labeled cells increased from 10.3 ± 1.0% to 21.4 ± 6.8% by 2 days of TO. This increase in CALM2 expression was reflected by a tendency for calmodulin protein levels to increase from 122.7 ± 17.3 to 156.5 ± 17.7 at 2 days of TO. Thus increases in fetal lung expansion result in time-dependent changes in CALM2 mRNA levels, which closely parallels the changes in lung DNA synthesis rates. As calmodulin is essential for cell proliferation, increased CALM2 mRNA levels may reflect an important role for calmodulin in expansion-induced fetal lung growth.

scholarly journals cDNA sequence, deduced amino acid sequence, predicted gene structure and chemical regulation of mouse Cyp2e1

1992 ◽  
Vol 281 (3) ◽  
pp. 689-695 ◽  
Author(s):  
J E Freeman ◽  
D Stirling ◽  
A L Russell ◽  
C R Wolf
Keyword(s):  
Sequence Similarity ◽  
Mrna Translation ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Coding Region ◽  
Cdna Sequences ◽  
Protein Levels ◽  
Cyp2e1 Gene ◽  
Wide Range ◽  
Dependent Protein

The cDNA encoding the mouse Cyp2e1 protein has been isolated and sequenced, and shown to share 92%, 79%, 80% and 79% sequence similarity over the coding region with rat, human, rabbit 1 and rabbit 2 CYP2E1 cDNA sequences respectively. The predicted Cyp2e1 protein contains 493 amino acids, with a molecular mass of 56781 Da. The protein contains many features common to other cytochrome P450s, including a potentially phosphorylatable serine residue at position 129 within a canonical cyclic AMP-dependent protein kinase site. Southern blot analysis of genomic DNA prepared from C57BL/6 and DBA/2N mice suggests the presence of only a single Cyp2e1 gene. The Cyp2e1 gene was isolated and its organization was established by PCR using oligonucleotides to its predicted intron/exon boundaries. These results showed that the mouse Cyp2e1 gene is approx. 11,000 bp in length and has a similar structure to the human and rat CYP2E1 genes. Cyp2e1 protein expression was studied in a variety of tissues and a sexual dimorphism in its levels in some tissues was noted. Acetone treatment induced the Cyp2e1 protein in all of the tissues studied in both sexes, but this Cyp2e1 protein induction was not accompanied by an increase in Cyp2e1 mRNA levels. Indeed, mRNA levels were seen to be decreased on treatment, suggesting that acetone administration affects either mRNA translation efficiency or protein stability. Of a wide range of drugs known to modify other cytochrome P450 levels only diethylnitrosamine had a significant effect on Cyp2e1, causing a decrease in protein levels.

The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

1992 ◽  
Vol 68 (01) ◽  
pp. 040-047 ◽  
Author(s):  
C Scott Jamison ◽  
Bryan F Burkey ◽  
Sandra J Friezner Degen
Keyword(s):  
Total Protein ◽  
Hepg2 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Analysis ◽  
Secreted Protein ◽  
Mrna Levels ◽  
Vitamin K1 ◽  
Maximal Increase ◽  
Protein Levels ◽  
Warfarin Treatment

SummaryCultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 5-12
Author(s):  
Eric Alani ◽  
Nancy Kleckner
Keyword(s):  
Gene Fusion ◽  
Enzyme Assay ◽  
Decarboxylase Activity ◽  
Lacz Gene ◽  
Coding Region ◽  
Ura3 Gene ◽  
Promoter Sequences ◽  
E Coli ◽  
New Type ◽  
Fusion Analysis

ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

scholarly journals Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lidan Liu ◽  
Chaim Z. Aron ◽  
Cullen M. Grable ◽  
Adrian Robles ◽  
Xiangli Liu ◽  
...  
Keyword(s):  
Proinflammatory Cytokine ◽  
Inflammatory Cytokine ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Mrna Levels ◽  
Wild Type ◽  
Cytokine Mrna ◽  
Protein Levels ◽  
Cytokine Levels

AbstractLevels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

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