Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G₁₇ and A₁₇) and a dinucleotide [(CA)₁₇] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR⁻) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5′ end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.     

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells – spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.     

A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

灰黄霉素产生菌耐前体变株F-1012的选育及其发酵特性

以灰黄霉素产生菌D-756为出发菌株,经过三代的紫外线+氯化锂的复合诱变处理,采用快速筛选方法,获得了耐前体变株F-1012。对该变株的耐氯特性和发酵特性进行研究,结果表明,把发酵培养基中的氯化物浓度提高到2.0%,大米粉量提高到18%,该变株发酵单位最高。发酵最适条件,起始pH自然(约5.7);移种量为15%;装量20 ml/250 ml三角瓶;发酵周期为288小时。     

Gaining insights into relevance across cancers based on mutation features of TP53 gene

The tumor suppressor gene TP53, one of the most frequently mutated genes, is recognized as the guardian of genome and can provide a significant barrier to neoplastic transformation and tumor progression. Traditional theory believes that TP53 mutations are equal among cancer types. However, to date, no study has explored the TP53 mutation profile from a holistic and systematic standpoint to discovery its relevance and feature with cancers. Mutation signature, an unbiased approach to identify the mutational processes, can be a potent indicator for exploring mutation-driven tumor occurrence and progression. In this research, several features such as hotspots, mutability and mutation signature of somatic TP53 mutations derived from 18 types of cancer tissues from cBioPortal were analyzed and manifested the organizational preference among cancers. Mutation signatures found in almost all cancer types were Signature 6 related to mismatch repair deficiency, and Signature 1 that reflects the natural decomposition of 5-methylcytosine into thymine associated with aging. Meanwhile, several signatures of TP53 mutations displayed tissue-selective. Mutations enriched in bladder, skin, lung cancer were associated with signatures of APOBEC activity (Signature 2 and 13), alkylating agents (Signature 11), and tobacco smoke (Signature 4), respectively. Moreover, Signature 4 and 29 associated with tobacco smoking or chewing found in lung, sarcoma, esophageal, and head and neck cancer may be related to their smoking history. In addition, several digestive cancers, including colorectal, stomach, pancreatic and esophageal cancers, showed the high correlation in context and mutation signature profiles. Our study suggests that the tissue-selective activity of mutational processes would reflect the tissue-specific enrichment of TP53 mutations and provides a new perspective to understand the relevance of diverse diseases based on the spectrum of TP53 mutations.     

A Novel Mitochondrial Heteroplasmic C13806A Point Mutation Associated with Iranian Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by decreased expression of the protein Frataxin. Frataxin deficiency leads to excessive free radical production and dysfunction of chain complexes. Mitochondrial DNA (mtDNA) could be considered a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. It prompted us to focus on the mtDNA and monitor the nucleotide changes of genome which are probably the cause of respiratory chain defects and reduced ATP generation. We searched about 46% of the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE) and DNA fragments showing abnormal banding patterns were sequenced for the identification of exact mutations. In 18 patients, for the first time, we detected 26 mtDNA mutations; of which 5 (19.2%) was novel and 21 (80.8%) have been reported in other diseases. Heteroplasmic C13806A polymorphisms were associated with Iranian FRDA patients (55.5%). Our results showed that NADH dehydrogenase (ND) genes mutations in FRDA samples were higher than normal controls (P < 0.001) and we found statistically significant inverse correlation (r = −0.8) between number of mutation in ND genes and age of onset in FRDA patients. It is possible that mutations in ND genes could constitute a predisposing factor which in combination with environmental risk factors affects age of onset and disease progression.