Mapping of I gene mutations which lead to repressors with increased affinity for lac operator

A genetic mapping system is used to locate mutations on the lac repressor gene (I) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (I^tb) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. I^tb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which I^tb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc,En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis

Marfan syndrome has been associated with approximately 562 mutations in the fibrillin-1 (FBN1) gene. Mutation scanning of the FBN1 gene with DNA direct sequencing is time-consuming and expensive because of its large size. This study analyzed the diagnostic value of high-resolution melting analysis as an alternative method for scanning of the FBN1 gene. A total of 75 polymerase chain reaction (PCR) amplicons (179-301 bp, average 256 bp) that covered the complete coding regions and splicing sites were evaluated on the 96-well LightCycler system. Melting curves were analyzed as fluorescence derivative plots (−dF/dT vs. temperature). To determine the sensitivity of this method, a total of 82 samples from patients with Marfan syndrome and 50 unaffected individuals were analyzed. All mutations reported in this study had been confirmed previously by direct sequencing analysis. Melting analysis identified 48 heterozygous variants. The variant c.3093 G>T (exon 25) was incorrectly identified by melting curve analysis. The sensitivity of the technique in this sample was 98.78% (81/82). This study demonstrated that high-resolution melting analysis is a reliable gene scanning method with greater speed than DNA sequencing. Our results support the use of this technology as an alternative method for the diagnosis of Marfan syndrome as well as its suitability for high-throughput mutation scanning of other large genes.     

O0 and strong-polar mutations in the gal operon are insertions

Summary Three dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal ⁺ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.     

Detection of simple mutations and polymorphisms in large genomic regions

We have developed a novel technology that makes it possible to detect simple nucleotide polymorphisms directly within a sample of total genomic DNA. It allows, in a single Southern blot experiment, the determination of sequence identity of genomic regions with a combined length of hundreds of kilobases. This technology does not require PCR amplification of the target DNA regions, but exploits preparative size-fractionation of restriction-digested genomic DNA and a newly discovered property of the mismatch-specific endonuclease CEL I to cleave heteroduplex DNA with a very high specificity and sensitivity. We have used this technique to detect various simple mutations directly in the genomic DNA of isogenic pairs of recombinant Pseudomonas aeruginosa, Escherichia coli and Salmonella isolates. Also, by using a cosmid DNA library and genomic fractions as hybridization probes, we have compared total genomic DNA of two clinical P.aeruginosa clones isolated from the same patient, but exhibiting divergent phenotypes. The mutation scan correctly detected a GA insertion in the quorum-sensing regulator gene rhlR and, in addition, identified a novel intragenomic polymorphism in rrn operons, indicating very high stability of the bacterial genomes under natural non-mutator conditions.     

Insertion mutations in the control region of the galactose operon of E. coli. II. Physical characterization of the mutations

Summary Four gal negative mutations, which affect the expression of the gal operon severely as described in the preceding paper (Saedler et al.), are characterized as insertions of DNA by CsCl density gradient centrifugation of transducing phages carrying the mutations and by electron microscopy of hybrid DNA molecules in which the insertion forms a singlestranded loop.Mutation galOP _in 308 is shown by both procedures to be about twice as large as the three other insertions, which are similar in size. The length of the insertions as determined by electron microscopy corresponds to about 1500 nucleotide pairs galOP _in 308 and 800, 700, and 700 nucleotide pairs for galOP _in 128, 141, and 306 respectively. Single-stranded regions are seen in hybrid molecules prepared between DNAs from galOP _in 306 and 128, 141 or 308 as well as from galOP _in 308 and 128. No such single-stranded regions are observed in hybrid molecules between DNAs from galOP _in 128 and 141.Thus, at least three of the four insertions are not identical.     

Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: characterization of neuroendocrine phenotypes and novel mutations

Context

TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway.

Objective

To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations.

Results

From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%). We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants) found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn) probably caused by the misfolding of the mutated NK3R protein.We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio.

Conclusion

The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations.     

Point mutations in the DNA binding domain of p53 contribute to glioma progression and poor prognosis

TP53 mutations play a significant role in glioma tumorigenesis. When located in in the DNA binding domain, these mutations can perturb p53 protein conformation and its function, often culminating in altered downstream signaling. Here we describe prevalent pattern of TP53 point mutations in a cohort of 40 glioma patients and show their relevance to gliomagenesis. Point mutations in exon 5–9 of TP53 gene were detected by DNA sequencing. Possible influence of identified mutations at the function of p53 was studied computationally and correlated with the survival. Point mutations in TP53 were detected in 10 glioma samples (25%), out of which 70% were from high grade glioma. A total of 19 TP53 point mutations were identified, out of which 42% were found to be in the DNA binding region of p53. Computational analysis predicted 87.5% of these mutations to be “probably damaging”. In three patients with tumors possessing point mutations R273H, R248Q, Y163H and R175H and poor survival times, structural analysis revealed the nature of these mutations to be disruptive and associated with high risk for cancer progression. In high grade glioma, recurrent TP53 point mutations may be the key to tumor progression, thus, emphasizing their significance in gliomagenesis.     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.     

Use of a transposon-encoded site-specific resolution system for construction of large and defined deletion mutations in bacterial chromosome

A class II transposon, Tn1722, encodes a site-specific resolution system, in which the resolvase (TnpR) efficiently catalyzes intramolecular recombination between the two directly oriented copies of the resolution site (res), leading to precise excision of the intervening DNA region. This property was exploited to develop the general strategies to introduce the large and defined deletion mutations into the bacterial chromosome. The Tn1722 res site was inserted into the plasmid carrying a cloned chromosomal fragment, and the resulting plasmid was integrated into a Tn1722-containing target chromosome by single crossover-mediated homologous recombination. The plasmid integrant carrying the two copies of the res site in the same orientation could efficiently excise the chromosomal region locating between the two res sites by means of the site-specific resolution system. Such site-specific deletion could be also detected by appropriate integration of the res–tnpR-containing plasmid into the chromosome in which another copy of the res site had been inserted through allelic exchange. This latter strategy was further modified to isolate the deletion mutations that were free of the resistance markers used for introduction of the res site and the res–tnpR block into the target chromosome. The deletion systems were applied to analyze the 103-kb pvd region of Pseudomonas aeruginosa PAO carrying most of the pyoverdin biosynthetic genes. Successful isolation of the mutation lacking more than a 100-kb fragment in the pvd region indicated that this region did not carry any essential genes.     

Induction of base-pair substitution and prameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD3052. We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-induced base-pair substitution mutations arise by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.     

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

A novel familial case of diffuse leukodystrophy related to NDUFV1 compound heterozygous mutations

NDUFV1 mutations have been related to encephalopathic phenotypes due to mitochondrial energy metabolism disturbances. In this study, we report two siblings affected by a diffuse leukodystrophy, who carry the NDUFV1 c.1156C>T (p.Arg386Cys) missense mutation and a novel 42-bp deletion. Bioinformatic and molecular analysis indicated that this deletion lead to the synthesis of mRNA molecules carrying a premature stop codon, which might be degraded by the nonsense-mediated decay system. Our results add information on the molecular basis and the phenotypic features of mitochondrial disease caused by NDUFV1 mutations.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

Identification of drug resistance mutations among Mycobacterium bovis lineages in the Americas

Identifying the Mycobacterium tuberculosis resistance mutation patterns is of the utmost importance to assure proper patient’s management and devising of control programs aimed to limit spread of disease. Zoonotic Mycobacterium bovis infection still represents a threat to human health, particularly in dairy production regions. Routinary, molecular characterization of M. bovis is performed primarily by spoligotyping and mycobacterial interspersed repetitive units (MIRU) while next generation sequencing (NGS) approaches are often performed by reference laboratories. However, spoligotyping and MIRU methodologies lack the resolution required for the fine characterization of tuberculosis isolates, particularly in outbreak settings. In conjunction with sophisticated bioinformatic algorithms, whole genome sequencing (WGS) analysis is becoming the method of choice for advanced genetic characterization of tuberculosis isolates. WGS provides valuable information on drug resistance and compensatory mutations that other technologies cannot assess. Here, we performed an analysis of the most frequently identified mutations associated with tuberculosis drug resistance and their genetic relationship among 2,074 Mycobacterium bovis WGS recovered primarily from non-human hosts. Full-length gene sequences harboring drug resistant associated mutations and their phylogenetic relationships were analyzed. The results showed that M. bovis isolates harbor mutations conferring resistance to both first- and second-line antibiotics. Mutations conferring resistance for isoniazid, fluoroquinolones, streptomycin, and aminoglycosides were identified among animal strains. Our findings highlight the importance of molecular surveillance to monitor the emergence of mutations associated with multi and extensive drug resistance in livestock and other non-human mammals.     

Spontaneous and induced mutability or frameshift strains of Salmonella typhimurium carrying uvrB and polA mutations

Three strains Salmonella typhimurium carrying frameshift mutations affecting the histidine genes (hisC3076, hisD3052 and hisC207) showed increased sensitivity to mutagenesis by ICR-191 (as judged by measuring back mutation to prototrophy), if they were made deficient in excision repair by deleting the uvrB gene. One frameshift strain, hisC3076, also showed increased sensitivity to mutagenesis by ICR-191 when it carried either of two different polA alleles, whereas the hidD305 and hisD207 frameshifts reduced sensitivity to mutagenesis in the presence of these alleles. Studies of spontaneous back mutation to prototrophy revealed siginificant mutator effects of the polA1 mutation on reversion of the hisD3052 frameshift and of the polA3 mutation on reversion of the hisC3076 frameshift. Other smaller mutator effects of the polA alleles on reversion of the his mutations may also be present. In an attempt to explain the complex interactions between different polA alleles and different frameshift mutations, it is tentatively suggested that deletion frameshift may arise mainly during DNA replication, while addition frameshifts may arise mainly during post-replication repair.