An On-Demand Metalloprotease from Psychro-Tolerant Exiguobacterium undae Su-1, the Activity and Stability of Which Are Controlled by the Ca2+ Concentration

We reported an on-demand type of metalloprotease from Exiguobacterium undae Su-1. Although this species of bacterium is known to inhabit the permafrost, there are no reports on either strong proteases or peptidases. We found that Su-1 protease is superior to commercially available proteases in proteolytic activity in a lower to normal range of temperature (10–50 °C) as well as in rapid inactivation heat-dependently on the Ca²⁺ concentration. These characteristics meet well with the demands from food processing and manufacturing. Biochemical investigations of the purified enzyme and protein structural analysis after gene cloning confirmed that Su-1 protease conserved high identity in its primary sequence with thermophilic proteases of the M4 family. On the other hand, its flexibility was enhanced when one Ca²⁺ binding site was lost and by replacement for proline and isoleucine residues.     

Characterization of suppressible mutations in the viomycin phosphotransferase gene of the Streptomyces enteric plasmid pVE138.

The viomycin phosphotransferase gene (vph) is expressed and confers resistance to viomycin in both Streptomyces spp. and members of the family Enterobacteriaceae. We report the isolation of UGA (opal) and UAG (amber) mutations in the vph gene of shuttle plasmid pVE138. We found that the five UGA mutations in vph resulted in a temperature-sensitive phenotype in Salmonella typhimurium. Su- strains are Vior at 28 degrees C and Vios at 37 degrees C, whereas Su+UGA strains are Vior at both 28 and 37 degrees C. The single amber mutation isolated was not temperature sensitive and resulted in the expected Vios phenotype in Su- strains and Vior in Su+UAG strains.     

Genetic interactions suggest that Danforth's short tail (Sd) is a gain-of-function mutation

Danforth's short tail (Sd) is a semidominant mutation on mouse chromosome 2 that acts cell autonomously in the notochord and leads to its disintegration, and thus causes severe defects in somite patterning and vertebral column development. The molecular nature of the Sd gene and mutation is unknown, and it is unclear whether Sd is a loss-of-function mutation and the semidominant inheritance of the Sd phenotype is due to haploinsufficiency, or whether Sd represents a gain-of-function mutation in a gene essential for notochord development and maintenance. Here, we report on the genetic interaction between Sd and an insertional mutation called Etl4^lacZ, which provides genetic evidence that Sd is a gain-of-function mutation. Etl4^lacZ is an enhancer trap insertion, which gives rise to lacZ expression in distinct cell types, including the notochord. In homozygosity, the lacZ insertion leads to abnormal vertebrae in the caudal part of the vertebral column. Etl4^lacZ maps approximately 0.75 cM distal to Sd, and in double heterozygotes modifies the Sdphenotype contrarily, depending on the chromosomal configuration of the Sd and Etl4^lacZ mutations: when Etl4^lacZ is present on the chromosome wild type for Sd (Sd +/+ Etl4^lacZ; trans configuration), the Sd phenotype is enhanced, i.e., vertebral malformations extend to more anterior positions and the vertebral body of the axis is further reduced. Conversely, when Etl4^lacZ is present on the same chromosome as Sd (Sd Etl4^lacZ /+ +; cis configuration), the Sd phenotype is attenuated, i.e., vertebral malformations are confined to more posterior levels, and the dens axis, which is severely reduced or absent in Sd heterozygotes, is restored. The different effect of the Etl4^lacZ insertion on Sd, depending on its presence in trans or cis, suggests a direct interaction of the transgene insertion with the Sd gene. Additionally, the attenuation of the Sd phenotype by Etl4^lacZ in cis suggests that Sd is a gain-of-function mutation and lends support to the idea that Etl4^lacZ is a new allele of Sd. Dev. Genet. 23:86–96, 1998. © 1998 Wiley-Liss, Inc.     

Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae

Summary A total of nine regulatory mutations in the nifLA operon of Klebsiella pneumoniae were cloned in the high copy-number plasmid vector pACYC184. The regulatory phenotypes of the resultant clones were then correlated with their restriction maps and their ability to synthesise nifL and nifA polypeptides in vivo. One mutation, nifL2401, was identified as a 400 bp. deletion within the nifL gene. This mutation was non-polar and caused a Nif⁺ phenotype which showed escape from repression by oxygen and low levels of fixed nitrogen. Identification of this deletion allows the first definitive allocation of a mutation with this phenotype to the nifL gene and provides further evidence for the role of the nifL gene product in nif-specific repression.     

铜绿假单胞菌二鸟苷酸环化酶SiaD突变体的功能研究

【目的】铜绿假单胞菌(Pseudomonas aerugionsa)二鸟苷酸环化酶SiaD调控着铜绿假单胞菌的生物被膜形成等表型。在研究过表达siaD对生物被膜的调控作用时发现,与野生型siaD基因回补菌株相比,一株回补菌株的生物被膜产量显著升高。本文的目的即是探究该菌株生物被膜产量升高的原因,并对该菌株的其他表型进行研究。【方法】通过测序确定突变位点;利用生物被膜定性和定量实验对发生点突变的菌株表型进行分析;利用Western blotting实验检测SiaD^R119M蛋白表达水平;利用GST-pulldown实验检测SiaC蛋白与SiaD^R119M蛋白在体外的结合能力;针对siaD^R119M点突变基因进行融合蛋白表达载体的构建,表达并纯化该蛋白,利用高效液相色谱检测SiaD^R119M的酶活;为了进一步研究c-di-GMP与细菌运动能力的关系,对细菌的运动能力进行检测。【结果】测序比对结果显示,序列的第119个氨基酸发生了突变,由精氨酸突变成了甲硫氨酸。生物被膜定性和定量实验显示,与野生型siaD...     

Characterization of Mutations at the Mouse Phenylalanine Hydroxylase Locus

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. InPAH^ENU1,the phenotype is mild. ThePah^enu1mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. InPAH^ENU2,the phenotype is severe. ThePah^enu2mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. InPAH^ENU2,the sequence information was used to devise a direct genotyping system based on the creation of a newAlw26I restriction endonuclease site.     

Serological and gene sequencing analysis of a case of para-Bombay phenotype Amh

The paper aims to study the serological and genetic characteristics of a case of para-Bombay Am^h. The serological method was applied to identify the proband's ABO phenotype and PCR-SSP assay was used to analyze the genotype of the para-Bombay blood. DNA sequencing of the PCR products of the first exon of FUT1 gene was used to analyze the genotype and nucleic acid sequence mutation. The serological results showed that the ABO phenotype of the proband was O-type. However, while after absorption-elution test, the ABO phenotype showed weak A-type. The serological test also showed that the irregular antibody anti-H was positive. PCR-SSP assay showed that the proband was h4 para-Bombay type and sequence analysis showed a point mutation c.35C > T of FUT1 gene. The study suggests that genetic analysis is necessary for blood typing in those who have elusive immunological typing results.     

Serological and gene sequencing analysis of a case of para-Bombay phenotype Amh

The paper aims to study the serological and genetic characteristics of a case of para-Bombay Am^h . The serological method was applied to identify the proband''s ABO phenotype and PCR-SSP assay was used to analyze the genotype of the para-Bombay blood. DNA sequencing of the PCR products of the first exon of FUT1 gene was used to analyze the genotype and nucleic acid sequence mutation. The serological results showed that the ABO phenotype of the proband was O-type. However, while after absorption-elution test, the ABO phenotype showed weak A-type. The serological test also showed that the irregular antibody anti-H was positive. PCR-SSP assay showed that the proband was h4 para-Bombay type and sequence analysis showed a point mutation c.35C>T of FUT1 gene. The study suggests that genetic analysis is necessary for blood typing in those who have elusive immunological typing results.     

The chronic proliferative dermatitis mouse mutation (cpdm): mapping of the mutant gene locus

The chronic proliferative dermatitis (cpdm) mouse mutation was mapped to mouse Chromosome 15 using an intraspecific cross between C57BL/KaLawRij cpdm/cpdm and BALB/cJ mice. A second autosomal recessive mutation that resulted in a phenotype similar to that of cpdm arose spontaneously in a colony of OcB-3/Dem recombinant congenic mice. This new mutation was found to be allelic with cpdm. Therefore, the gene symbol for the allelic mutation is cpdm^Dem. The phenotype of these mutant mice consists of eosinophil-driven severe and progressive inflammatory changes in multiple organs including the skin and lungs.     

Characterization of a dominant mutation for the liguleless trait: Aegilops tauschii liguleless (Lgt)

Background

Leaves of Poaceae have a unique morphological feature: they consist of a proximal sheath and a distal blade separated by a ligular region. The sheath provides structural support and protects young developing leaves, whereas the main function of the blade is photosynthesis. The auricles allow the blade to tilt back for optimal photosynthesis and determine the angle of a leaf, whereas the ligule protects the stem from the entry of water, microorganisms, and pests. Liguleless variants have an upright leaf blade that wraps around the culm. Research on liguleless mutants of maize and other cereals has led to identification of genes that are involved in leaf patterning and differentiation.

Results

We characterized an induced liguleless mutant (LM) of Aegilops tauschii Coss., a donor of genome D of bread wheat Triticum aestivum L.. The liguleless phenotype of LM is under dominant monogenic control (Lg^t). To determine precise position of Lg^t on the Ae. tauschii genetic map, highly saturated genetic maps were constructed containing 887 single-nucleotide polymorphism (SNP) markers derived via diversity arrays technology (DArT)seq. The Lg^t gene was mapped to chromosome 5DS. Taking into account coordinates of the SNP markers, flanking Lg^t, on the pseudomolecule 5D, a chromosomal region that contains this gene was determined, and a list of candidate genes was identified. Morphological features of the LM phenotype suggest that Lg^t participates in the control of leaf development, mainly, in leaf proximal–distal patterning, and its dominant mutation causes abnormal ligular region but does not affect reproductive development.

Conclusions

Here we report characterization of a liguleless Ae. tauschii mutant, whose phenotype is under control of a dominant mutation of Lg^t. The dominant mode of inheritance of the liguleless trait in a Triticeae species is reported for the first time. The position of the Lg^t locus on chromosome 5DS allowed us to identify a list of candidate genes. This list does not contain Ae. tauschii orthologs of any well-characterized cereal genes whose mutations cause liguleless phenotypes. Thus, the characterized Lg^t mutant represents a new model for further investigation of plant leaf patterning and differentiation.

     

Generation of precise point mutation mice by footprintless genome modification

Point mutation mice are a key tool in the study of biological functions of genomic DNA sequences and the creation of human disease models. These mice are produced by homologous recombination combined with site‐specific recombinase, which allows removal of drug selection cassettes. However, the methods currently available leave ectopic sequences in the “inactive” intron region of the targeted genome in addition to the desired mutation. Since recent research suggests that the intron region may actually have some functionality, these sequences could potentially interfere with neighboring gene expression and, as a result, affect the mouse phenotype. To completely avoid this issue, we used the PiggyBac transposon to remove selection cassettes and achieve precise genome modification without leaving behind a footprint. This PiggyBac system allowed us to successfully generate mice carrying an artificially introduced W^v point mutation in the Kit gene, and these mice were confirmed to have phenotypes identical to spontaneous W^v mutation mice. Generation of W^v‐mutation corrected mice was also possible, and phenotypes were completely restored. Our footprintless genome modification technology can generate precise point mutation mice with only the desired mutation, and they reflect an accurate phenotype that makes these mice a reliable and “worry‐free” research resource. 52:68–77, 2014. © 2013 Wiley Periodicals, Inc.     

Molecular Basis for the rhino (hr)Phenotype: A Nonsense Mutation in the MousehairlessGene

The hairless (hr) and rhino (hr^rh) mutations are autosomal recessive allelic mutations that map to mouse Chromosome 14. Both hairless and rhino mice have a number of skin and nail abnormalities and develop a striking form of total alopecia at approximately 3–4 weeks of age. The molecular basis of the hairless mouse phenotype was previously found to be the result of a murine leukemia proviral insertion in intron 6 of thehrgene that resulted in aberrant splicing. In this study, we report a 2-bp substitution in exon 4 of thehrgene in a second allele ofhr,rhino 8J (hr^rh-8J), leading to a nonsense mutation. These findings document the molecular basis of the rhino phenotype for the first time and suggest that rhino is a functional knock-out of thehrgene.     

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm^S) to its mutant phenotype (Sm^R). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.     

The role of the functional sites of the merlin tumor suppressor in <Emphasis Type="Italic">Drosophila Spermatogenesis</Emphasis>

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene, an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr⁵⁵⁹ residue of the Drosophila Merlin protein was homologous to Ser⁵¹⁸ of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr⁵⁵⁹ of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT^559D (an analog of the phosphorylated form) and MerT^559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression, for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT^559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT^559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT^559A mutant form, and mycMer^345–635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case of ectopic expression of the Mer^T559D analog of the phosphorylated form. Ectopic expression of the mycMer³, mycMer^ΔBB, and mycMer^1–379 truncate variants led to disturbance of meiotic cytokinesis.     

Trithorax: A new homoeotic mutation of Drosophila melanogaster causing transformations of abdominal and thoracic imaginal segments

Summary A new homoeotic mutation, trithorax ¹ (trx ¹), of Drosophila melanogaster is described which causes intersegmental transformations in both the adult thorax and abdomen. Specifically, the metathorax and ventral prothorax are partially transformed to mesothroax, whilst abdominal segments are partially transformed towards the first abdominal segment. Three variables are shown to influence the expressivity of the mutant phenotype: (1) presence of wild type copies of the gene in the female parent; (2) culture temperature during the first four hours of embryogenesis; (3) exposure of embryos to ether vapour at the blastoderm stage. In addition it is shown that phenocopies of the mutant phenotype can be induced by ether treatment of heterozygous trx ¹ embryos with a frequency higher than that of bithorax phenocopies in wild type embryos.These findings are together taken to imply that the mutated gene functions early during embryogenests, and that a common mechanism underlies the determination of all the thoracic and abdominal segments.     

SLC4A11 mutations causative of congenital hereditary endothelial dystrophy (CHED) progressing to Harboyan syndrome in consanguineous Pakistani families

Background

Autosomal recessive corneal hereditary endothelial dystrophy (CHED) is a rare congenital disorder of cornea. Mutations in SLC4A11 gene are associated with CHED phenotype. CHED is also an early feature of Harboyan syndrome. The aim of the present study was to identify genetic mutations in the SLC4A11 gene in CHED cases belonging to inbred Pakistani families. Furthermore, all homozygous mutation carriers were investigated for hearing deficit.

Methods and results

This study included consanguineous CHED families presented at Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan from June 2018 to September 2018. DNA was extracted from blood samples. Direct sequencing of SLC4A11 gene was performed. All identified variants were evaluated by in silico programs i.e., SIFT, PolyPhen‐2, and MutationTaster. Pathogenicity of the two identified splice site variants was analyzed by Human Splicing Finder and MaxEnt Scan. Screening of five CHED families revealed a total of three previously un reported (p.Arg128Gly, c.2241-2A?>?T and c.1898-2A?>?C in family CHED19, CHED22 and CHED26 respectively) and two already reported homozygous disease causing variants (p.Arg869Cys and p.Val824Met in family CHED24 and CHED25 respectively) as predicted by mutation taster. All of these variants segregated with disease phenotype and were not detected in controls.

Conclusion

Affected individuals of the five CHED families screened in this study had the disease due to SLC4A11 mutations and progressing to Harboyan syndrome. Identification of previously unreported mutations aid to heterogeneity of SLC4A11 and CHED pathogenesis as well as helped to provide genetic counseling to affected families.

     

Phenotypic suppression of the Drosophila mitochondrial disease-like mutant tko by duplication of the mutant gene in its natural chromosomal context

A mutation in the Drosophila gene technical knockout (tko^25t), encoding mitoribosomal protein S12, phenocopies human mitochondrial disease. We isolated three spontaneous X-dominant suppressors of tko^25t (designated Weeble), exhibiting almost wild-type phenotype and containing overlapping segmental duplications including the mutant allele, plus a second mitoribosomal protein gene, mRpL14. Ectopic, expressed copies of tko^25t and mRpL14 conferred no phenotypic suppression. When placed over a null allele of tko, Weeble retained the mutant phenotype, even in the presence of additional transgenic copies of tko^25t. Increased mutant gene dosage can thus compensate the mutant phenotype, but only when located in its normal chromosomal context.     

Interaction of an antimutator gene with DNA repair pathways in Escherichia coli K-12

Summary A mutation in the purB gene of E. coli K-12, isolated and partially characterized by Geiger and Speyer (1977), confers a temperature sensitive requirement for adenine and an antimutator phenotype at 30°C. Several hypotheses about the mechanism of action of this mutation, named mud for mutation defective, were tested in the present work. The mud mutation has no effect upon the induction of the SOS response, so the antimutator phenotype is unlikely to be due to repression of mutagenic repair. Mud cells are resistant to the cytotoxic and mutagenic effects of alkylating agents such as MNNG, but this resistance is not due simply to derepression of the adaptive response. DNA isolated from mud cells is not undermethylated relative to DNA from purB ⁺ cells, so the antimutator phenotype of mud cannot be due to reduced hotspot base-substitution mutation at methylated cytosine residues. Nor is there a longer lag in post-replicative DNA methylation, which indicates that there is no enhancement of mismatch repair resulting from an extended time window for strand discrimination. Measurement of nucleotide pool levels demonstrated an elevation of dCTP in mud cells and a reduction of all other nucleoside triphosphates.This work was supported in part by Public Health Service grants numbers GM15697 and CA32182