scholarly journals Efficiency of T4 Gene 60Translational Bypassing

1998 ◽  
Vol 180 (7) ◽  
pp. 1822-1830 ◽  
Author(s):  
Rafael Maldonado ◽  
Alan J. Herr
Keyword(s):  
Stop Codon ◽  
Bacteriophage T4 ◽  
Landing Site ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Mrna Levels ◽  
Amino Terminal ◽  
Nascent Peptide ◽  
Reporter Systems ◽  
Internal Translation

ABSTRACT Ribosomes translating bacteriophage T4 gene 60 mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47. A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff. Here we show that this signal is insensitive to the addition of coding information at its N terminus. In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene 60 mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding. Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion). Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels. An internal translation initiation site at Met12 of gene 60(which eliminates part of the essential nascent peptide) also distorts these estimates. Together, these results support an efficiency estimate of ∼50%, less than previously reported. This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.

scholarly journals utR.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation

2021 ◽  
Author(s):  
Yating Liu ◽  
Joseph Dougherty
Keyword(s):  
Stop Codon ◽  
R Package ◽  
Initiation Site ◽  
Open Reading Frames ◽  
Start Codon ◽  
Translation Initiation Site ◽  
Mouse Species ◽  
Translational Regulators ◽  
Post Transcriptional Regulation ◽  
Annotated Translation

Whole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs modulate their function. utR.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utR.annotation provides information of each variant on whether and how it alters known translational regulators including:upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, the Kozak sequence at the annotated translation initiation site, start codon, and stop codon, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model from empirical data.

91 DISRUPTION OF TET1 DURING PORCINE EMBRYOGENESIS USING CRISPR/Cas9 SYSTEM

2017 ◽  
Vol 29 (1) ◽  
pp. 153
Author(s):  
K. Uh ◽  
J. Ryu ◽  
C. Ray ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Translation Initiation ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Epigenetic Marks ◽  
Hydroxymethyl Cytosine ◽  
Biallelic Mutations

Ten-eleven translocation (TET) enzymes catalyse oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine. This TET-mediated conversion of 5-methylcytosine to 5-hydroxymethyl cytosine is implicated in initiating the DNA demethylation process, observed post-fertilization. Three members (TET1–3) of the TET family are differentially expressed during embryo development and appear to have different roles. Previous studies in mice suggest that TET1 is a key regulator in maintaining pluripotency in embryonic stem cells by managing epigenetic marks such as DNA methylation. This would imply that TET1 should be a regulator of epigenetic marks during embryo development, although this has not been demonstrated. Previously, we have cloned porcine TET1 from blastocysts (GenBank accession number KC137683) and demonstrated that the level of TET1 (mRNA and protein) was high in blastocysts. The protein level was greater in the inner cell mass compared with the trophectoderm. In this study, we generated TET1 knockout porcine embryos using CRISPR/Cas9 system to study the role of TET1 in controlling epigenetic marks during porcine embryo development. First, 2 sgRNA, immediately downstream of the presumable translation initiation site, were designed and synthesised; location of the sgRNA were nucleotide position at 2 to 21 bp and 23 to 42 bp, respectively (KC137683). Then, sgRNA (10 ng μL−1 each) and Cas9 mRNA (20 ng μL−1) were injected into the cytoplasm of IVF zygotes, and Day 7 blastocysts were genotyped. All embryos carried mutations on both alleles of TET1 (10/10), one homozygous and 9 biallelic mutations. However, immunocytochemistry analysis of other CRISPR/Cas9 injected embryos revealed that TET1 was not removed (10/10), indicating that the sgRNA may have not introduced a premature stop codon 3′ to the presumable translation initiation site. Therefore, 2 new sgRNA were designed to generate a premature stop codon at the 5′ side of a key functional domain, the 2-oxoglutarate-Fe(II)-dependent oxygenase domain (4690 to 5160 bp); the locations of the 2 sgRNA were 4450 to 4469 bp and 4501 to 4520 bp, respectively. Similarly, all of the embryos carried mutations in TET1 (7/7), 2 homozygous and 5 biallelic mutations. In addition, TET1 proteins were not detected in 11 of 16 blastocysts, confirmed by immunocytochemistry. In this study, we successfully generated embryos lacking TET1 by introducing designed CRISPR/Cas9 system during embryogenesis. Presence of TET1 from the first injection experiment suggests that the presumable translation initiation site is not accurate. Discrepancy between genotyping and immunocytochemistry results from the second injection experiment indicates that embryos possessing TET1 protein probably have mutations in triplets, thus no premature stop codon was synthesised. Further studies will focus on identifying the role of TET1 in maintaining pluripotency and epigenetic modification during pre-implantation stage using these embryos.

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

scholarly journals Evolutionary tinkering: birth of a novel chloroplast protein

2007 ◽  
Vol 403 (3) ◽  
pp. e13-e14 ◽  
Author(s):  
Tatjana Kleine ◽  
Dario Leister
Keyword(s):  
Stress Responses ◽  
Initiation Site ◽  
Flowering Plant ◽  
Translation Initiation Site ◽  
Novel Proteins ◽  
Evolutionary Innovation ◽  
Biochemical Journal ◽  
Mrna Variants ◽  
Plastid Protein ◽  
Internal Translation

The term ‘evolutionary tinkering’ refers to evolutionary innovation by recombination of functional units, and includes the creation of novel proteins from pre-existing modules. A novel instance of evolutionary tinkering was recently discovered in the flowering plant genus Nicotiana: the conversion of a nuclear transcription factor into the plastid-resident protein WIN4 (wound-induced clone 4) involved in environmental stress responses. In this issue of the Biochemical Journal, Kodama and Sano now show that two steps are necessary for the establishment of the novel plastid protein: the acquisition of an internal translation initiation site and the use of multiple transcription starts to produce short mRNA variants that encode the plastid-targeted protein form.

scholarly journals SUN-085 Clinical and Hormonal Features of 37 Families with Central Precocious Puberty Due to MKRN3 Loss-Of -Function Mutations

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Carlos Eduardo Seraphim ◽  
Ana Pinheiro Machado Canton ◽  
Luciana Ribeiro Montenegro ◽  
Maiara Ribeiro Piovesan ◽  
Tabata Mariz Bohlen ◽  
...  
Keyword(s):  
Precocious Puberty ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Central Precocious Puberty ◽  
Time Interval ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Missense Variants ◽  
Amino Terminal ◽  
Mediterranean Countries

Abstract Context: Loss-of-function mutations in the maternally imprinted Makorin RING-finger 3 (MKRN3) gene (15q11.2) are the most prevalent cause of familial central precocious puberty (CPP). Objectives: To analyze the phenotypes of a large cohort of children with CPP due to MKRN3 mutations and to compare them with the phenotypes of idiopathic CPP. Setting and Participants: We studied 73 individuals from 37 families with mutations in MKRN3 originating from nine different countries. The phenotypes of these patients at initial diagnosis were compared to a cohort of 124 patients with idiopathic CPP. Additionally, expression of nine different genes implicated with pubertal timing, including MKRN3, was performed in the hypothalamus of female mice in different phases of sexual maturation. Results: Nineteen different heterozygous, paternally inherited mutations in MKRN3 were identified in 73 patients with CPP (48 girls and 25 boys). Six MKRN3 mutations were frameshifts, one introduced a premature stop codon, 11 were missense mutations predicted to be pathogenic, and one was a deletion in the promoter region. A frameshift mutation affecting codon 161 in the amino terminal region of the protein was the most frequent MKRN3 defect (46%), representing a hotspot region. Among the cohort with MKRN3 mutations, first pubertal signs occurred at 6·2 ± 1·2 years in girls and 7·6 ± 1·4 years in boys. Patients harboring severe frameshift/nonsense mutations did not differ significantly in any clinical or hormonal parameters compared to the 20 patients with missense variants. However, when the 48 girls with MKRN3 mutations were compared with 124 idiopathic CPP girls, some parameters could be considered as possible predictors of the genetic cause: a lower age at first medical appointment (7·1 ± 1·1 in the MKRN3 group vs. 8·0 ± 2 years in the idiopathic group; p< 0.001) and a shorter time interval between puberty onset and medical assistance (0·8 ± 0·8 vs 2·2 ± 2·1 years; p< 0.001). Interestingly, the other predictor of MKRN3 mutations was a higher basal FSH level (5·1 ± 2·3 vs 3·9 ± 2·7 IU/L; p = 0.017) at first evaluation, although no cutoff value yielded good accuracy. Patients originating from European/Mediterranean countries were more likely to have missense variants (56% of all mutations) than North American and South American (23%) counterparts (p <0.001). Mouse Mkrn3 mRNA levels in the arcuate nucleus were highest in the prepubertal phase when compared with expression of other genes and Mkrn3 decreased progressively through puberty and adult ages. Conclusions: Different types of loss-of-function MKRN3 mutations were associated with premature sexual development in both sexes. Their phenotypes were relatively uniform, regardless of the mutation type. Clinical features of children with MKRN3 mutations were similar to the idiopathic CPP group.

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