Formation Of Premature Stop Codon Research Articles

Molecular Genetic Characteristics of the Mutant Strain B-162/2 of the Bacteria <i>Pseudomonas chlororaphis</i> subsp. <i>aurantiaca</i>

The production of microorganisms that produce biologically active compounds for agriculture, the chemical, veterinary and pharmaceutical industries as well as for environmental protection continues to be an important direction of microbial biotechnology. One of the most effective approaches to the production of producers is chemical mutagenesis, which, in combination with the right breeding strategy, makes it possible to obtain highly productive strains. A significant disadvantage of chemical mutagenesis is the large number of induced mutations in the genomes of mutant strains, which makes it difficult to identify genes and, accordingly, biosynthetic pathways involved in the production of a given compound. The solution to this problem is modern technologies of genome sequencing and analysis, which make it possible to identify new genes and unknown biochemical pathways involved in the formation of biologically active compounds. The aim of the work was to analyse the genome of the mutant strain B-162/2 of the bacterium Pseudomonas chlororaphis subsp. aurantiaca, which is capable of increased production of biologically active compounds of the phenazine series and is resistant to hydrogen peroxide. When analysing the genome of strain B-162/2 in full size, 6482 coding sequences and 64 coding RNA sequences were identified. Comparison of the genome of the B-162/2 strain with the genome of the wild type B-162 allowed the identification of 39 mutations, 5 of which are localised in intergenic regions, and 34 affected coding sequences. Of the mutations detected, 14 led to a radical amino acid substitution in the proteins and 2 led to the formation of premature stop codons (methyl group sensor and MFS-type transporter). Several substitutions with high values of the Grantham coefficient were found, which could possibly lead to a change in the activity of the proteins concerned. The presence of three regions with phage genes in the genome of the B-162/2 strain was detected.

Complete genomes of the eukaryotic poultry parasite Histomonas meleagridis: linking sequence analysis with virulence / attenuation

BackgroundHistomonas meleagridis is a protozoan parasite and the causative agent of histomonosis, an important poultry disease whose significance is underlined by the absence of any treatment and prophylaxis. The recent successful in vitro attenuation of the parasite urges questions about the underlying mechanisms.ResultsWhole genome sequence data from a virulent and an attenuated strain originating from the same parental lineage of H. meleagridis were recruited using Oxford Nanopore Technology (ONT) and Illumina platforms, which were combined to generate megabase-sized contigs with high base-level accuracy. Inspecting the genomes for differences identified two substantial deletions within a coding sequence of the attenuated strain. Additionally, one single nucleotide polymorphism (SNP) and indel targeting coding sequences caused the formation of premature stop codons, which resulted in the truncation of two genes in the attenuated strain. Furthermore, the genome of H. meleagridis was used for characterizing protein classes of clinical relevance for parasitic protists. The comparative analysis with the genomes of Trichomonas vaginalis, Tritrichomonas foetus and Entamoeba histolytica identified ~ 2700 lineage-specific gene losses and 9 gene family expansions in the H. meleagridis lineage.ConclusionsTaken as a whole, the obtained data provide the first hints to understand the molecular basis of attenuation in H. meleagridis and constitute a genomics platform for future research on this important poultry pathogen.

TA, GT and AC are significantly under-represented in open reading frames of prokaryotic and eukaryotic protein-coding genes.

Genomes can be considered a combination of 16 dinucleotides. Analysing the relative abundance of different dinucleotides may reveal important features of genome evolution. In present study, we conducted extensive surveys on the relative abundances of dinucleotides in various genomic components of 28 bacterial, 20 archaean, 19 fungal, 24 plant and 29 animal species. We found that TA, GT and AC are significantly under-represented in open reading frames of all organisms and in intergenic regions and introns of most organisms. Specific dinucleotides are of greatly varied usage at different codon positions. The significantly low representations of TA, GT and AC are considered the evolutionary consequences of preventing formation of pre-mature stop codons and of reducing intron-splicing options in candidate primary mRNA sequences. These data suggest that a reduction of TA and GT occurred on both strands of the DNA sequence at an early stage of de novo gene birth. Interestingly, GT and AC are also significantly under-represented in current prokaryotic genomes, suggesting that ancient prokaryotic protein-coding genes might have contained introns. The greatly varied usages of specific dinucleotides at different codon positions are considered evolutionary accommodations to compensate the unavailability of specific codons and to avoid formation of pre-mature stop codons. This is the first report presenting data of dinucleotide relative abundance to indicate the possible existence of spliceosomal introns in ancient prokaryotic genes and to hypothesize early steps of de novo gene birth.

Refinement of IKZF1 Genomic Status in Pediatric Philadelphia Positive Acute Lymphoblastic Leukemia

Background:IKZF1 deletions are the most frequent secondary genetic alterations with prognostic impact in pediatric Philadelphia-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCP-ALL). Studies of IKZF1 status have mainly focused on deletions of the gene whereas very little is known about other genetic alterations in the IKZF1 locus.Aim: Using an amplicon deep sequencing approach to investigate IKZF1 mutations occurrence in pediatric Ph+ BCP-ALL reported as IKZF1wildtype (WT) for deletions by MLPA analysis.Methods: Six European centres participated in this study with a total of 98 patients.The 454 GS Junior system (Roche) was used to sequence the coding regions of IKZF1, Exon1 - Exon7 (Mullighan C. et al, 2009). An average depth of 250 reads (minimum of 102 reads, maximum of 853 reads) per amplicon was achieved. Quality control analysis excluded variants coverage of <50 reads in both forward and reverse directions. Variant analyses were performed using Amplicon Variant Analyzer (AVA) software (Roche). Variants at intronic regions or in homopolymeric stretches were excluded from further analysis, and a cut-off ≥10% mutated reads was applied to define mutations. Finally, variants described as SNPs or silent mutations were excluded.Results: In total, 882 amplicons were screened obtaining 337 variants. Of these, 14 variants corresponding to mutations with predicted deleterious impact on the function of IKZF1 passed the filters of our analysis pipeline. Twelve out of 98 (12%) IKZF1 non-deleted patients were mutated and 2 of them carried double mutations. Both cases showed different mutant allele frequencies of the 2 mutations, suggesting the presence of leukemic sub-populations with different IKZF1 status. Mutations can be subdivided in 2 categories: 5 miss-sense point mutations localized at the DNA binding domain with a dominant negative effect and 7 frameshift mutations leading to haploinsufficiency and impairing also the dimerization activity of IKZF1. The 5 point mutations were located in exon4 and 4 out of 5 were localized in the coding region of zinc finger 2 (ZF2), essential for DNA-protein interaction. Three patients carried the same c.698A>G substitution, that caused the change of Asparagine 159 into Serine. The 7 frameshift mutations (2 deletions, 4 insertions and 1 InDel) were identified all along the IKZF1locus. These nucleotide variations caused a shift in the reading frame and a consequent formation of premature stop codons depleting the C-terminal dimerization domain.Six out of 12 patients carrying IKZF1 mutations experienced an event (either relapse or death in CCR): 6 patients were treated in the pre-TKI (tyrosine kinase inhibitor) era and had 5 events while the remaining 6 patients received imatinib and showed a more favourable outcome with only one patient who died in CCR. This trend is in line with the findings recently reported by Van der Veer et al. (2014). In this study on IKZF1 deletions were identified in 126 of 191 patients, while 65 were defined IKZF1 WT on the basis of deletions analysis alone. Our study revealed that 6 of these 65 IKZF1 WT patients carried IKZF1 mutations (3 in the pre-TKI and 3 in the EsPhALL cohort). The outcome of mutated patients and deleted patients taken together (IKZF1 aberrant) is consistent with results reported in the Van der Veer study, both for the pre-TKI cohort [(4-year disease free survival (DFS) was 28.1% [SE 6.4] for IKZF1 aberrant vs 64.3% [SE 9.7] for IKZF1 WT, P=0.0036); Van der Veer study (30.0% [SE 6.8] for IKZF1 deleted vs 57.5% [SE 9.4] for IKZF1 WT, P=0.013)] and for the EsPhALL cohort [(4-year DSF was 55.7% [SE 6.8] for IKZF1 aberrant vs 59.8% [SE 11.6] for IKZF1 WT (P=0.348); Van der Veer study (4-year DFS was 53.6 [SE 7.0] for IKZF1 deleted vs 63.1 [SE 11.0] for IKZF1WT, P=0.1676].Conclusion: This work revealed IKZF1 mutations in >10% of Ph+ BCP-ALL pediatric patients previously classified as IKZF1 WT on the basis of deletions analysis. Our results, together with data of IKZF1 deletions, highlight a 70-75% incidence of IKZF1 aberrations in pediatric Ph+ patients. Exon4 carried 9 of 14 aberrations: 7 point mutations and 2 deletions, suggesting a hotspot region for somatic mutations acquisition. The outcome of mutated patients is in line with the data previously published on the impact of IKZF1 deletions in pre and post TKI cohorts, suggesting a similar impact of mutations and deletions on clinical outcome of pediatric Ph+ patients. DisclosuresNo relevant conflicts of interest to declare.

A new β0‐thalassemia frameshift mutation [β 48 (‐T)] in a Uruguayan family

SummaryWe describe here a new frameshift mutation of β‐thalassemia in a Uruguayan family with Italian ancestry [β48 (‐T); HBB:c.146delT]. This frameshift results in formation of premature stop codon (TGA) 40 bp downstream and in a short unstable product that is degraded in the cell.

Genetic analysis of variegate porphyria (VP) in Italy: identification of six novel mutations in the protoporphyrinogen oxidase (PPOX) gene.

Variegate Porphyria (VP) is one of the acute hepatic porphyrias, and is clinically characterised by skin lesions and acute neuropsychiatric/visceral attacks that occur separately or together. The disorder is caused by a partial deficiency of protoporphyrinogen oxidase, the penultimate enzyme in the heme biosynthetic pathway, and a number of mutations have been described for the corresponding gene (PPOX). Here we report a genetic analysis of VP in Italy, and the identification of six novel and three previously characterised mutations from nine affected individuals and families. Among those newly identified, two mutations were small deletions (c.418_419delAA; c.759delA), leading to the formation of premature stop codons, two were splicing defects (IVS10+2T>G; IVS12+1G>C), one was a nonsense (c.384G>A=p.W128X) and one a missense mutation (c.848T>A=I283N). This is the first study of the molecular genetics of Variegate Porphyria in patients of Italian origin, and the finding of six novel mutations out of nine identified confirms the genetic heterogeneity observed for this disorder.

APC germline mutations identified in Czech patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited predisposition to colorectal cancer, which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. The APC mutations have been investigated in 46 Czech unrelated FAP families and 9 suspected FAP families using DGGE analysis and direct DNA sequencing. We found 25 germline APC mutations and identified 11 which were not previously reported. Of the identified mutations, 10 were 1 to 5 bp deletions, four were 1 bp insertions and six were nonsense, all leading to the formation of premature stop codon. In addition, we detected two mutations in the splice-donor region of APC intron 11, one missense and two samesense mutations. Phenotypes of patients with known and novel types of mutations are presented and discussed.

Molecular analysis of the hydroxymethylbilane synthase (HMBS) gene in Italian patients with acute intermittent porphyria: report of four novel mutations.

Acute intermittent porphyria (AIP) is an autosomal disorder caused by molecular abnormalities in the hydroxymethylbilane synthase (HMBS) gene coding for the third enzyme in the heme biosynthetic pathway. So far, more than 160 different mutations responsible for AIP have been identified in this gene. We have now identified seven mutations in eight unrelated Italian patients with AIP: two splicing defects (IVS7+2T-->C, 612G-->T), three small deletions (308-309delTG, 730-731delCT, 182delA) and two missense mutations (134C-->A, 541C-->T). The splicing defects were responsible for activation of splicing cryptic sites respectively within intron 7 (15 bp insertion) and exon 10 (9 bp deletion). The small deletions resulted in frameshifts leading to the formation of premature stop codons. The 134C-->A and 541C-->T mutations caused the formation of stop codons likely to be responsible for drastic disruption of the HMBS structure (Ser45Ter, Gln181Ter). This is the first molecular study in AIP patients of Italian origin leading to the identification of four new mutations and three molecular defects that have already been described.