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At least 19 recordsLinked to original sources
PubMed2025

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Schizosaccharomyces↗
PubMed1977

Interaction between mutations of ribosomes and RNA polymerase: a pair of strA and rif mutants individually temperature-insensitive but temperature-sensitive in combination.

A temperature-sensitive lethal mutant of Escherichia coli has been constructed by combining two temperature-insensitive mutations: a rif180 mutation that modifies RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a strA24 mutation that modifies the ribosomal protein S12. The temperature sensitivity is a property of the combination of these two particular alleles; replacement of either of the alleles relieves the temperature sensitivity. An isogenic strain containing a different strA mutation (i.e., rif180 strA11) is not temperature sensitive. Evidently ribosomes modified by the particular strA24 polymerase altered by the rif180 mutation, which suggests that in vivo there may exist some interaction between structures of ribosomes and the RNA polymerase.

Bacterial Proteins↗
PubMed2025

Naturally occurring variation in a cytochrome P450 modifies thiabendazole responses independently of beta-tubulin.

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35D1 and the nuclear hormone receptor nhr-176. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of cyp-35D1 and nhr-176 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive strain, and reciprocal allele replacement strains in different genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele was deleterious, but the lysine allele was selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance.

Animals↗
PubMed1976

The evolution of the cooperative group.

A simple model, illustrating the transition from a population of free swimming, solitary cells (Chlamydomonas-type) to one consisting of small colonies (Gonium-type) serves as a basis to discuss the evolution of the cooperative group. The transition is the result of a mutation of the dynamics of cell division, delayed cell separation leads to colonies of four cells. With this mutation cooperative features appear, such as synchronised cell divisions within colonies and coordinated flagellar function which enables the colony to swim in definite directions. The selective advantages under given, environmental conditions are defined and the periods necessary for complete allelic replacement in small populations are calculated for asexual and sexual reproduction. The assumption of a steady-state population during allelic substitution is critically considered, particularly under conditions of competition. It is shown that density-dependent population control must operate in the process of selection, Sexual reproduction slows down the rate of selection even though all cells are haploid. This phenomenon can be explained in general terms of "organizational dominance', where individual units coordinate the function of their neighbours which may be of a different allelotype. Cooperativity is pointed out as an a priori systemic feature which resides in the sub-units of systems, group formation and coordination appears thus as an almost inevitable event. A particular type of system described as 'closed cycle of positive fitness interaction' is discussed in more detail. It has the remarkable feature that its members cannot compete with each other; selection takes place between whole cycles (in analogy to Eigen's 1971 model). Gonium has a wide spectrum of 'somatic plasticity' which enables it to assume various colonial configurations depending on physiological and environmental conditions. This feature can be explained as the result of dynamic flexibilities on the macro-molecular level. The particular relationship between the vast, molecular complexity and the relative simple dynamics of the cell cycle must lead eventually to the genetic fixation of an environmentally induced phenotype.

Animals↗
PubMed2026

Long-term saline-alkaline selection rewires the growth-survival trade-off in Priestia megaterium.

Saline-alkaline soils impose persistent osmotic, ionic, pH, and nutrient stress on soil microorganisms, but the evolutionary routes by which beneficial bacteria adapt to such conditions remain poorly resolved. We performed adaptive laboratory evolution to examine the adaption of the plant growth-promoting rhizobacterium Priestia megaterium HA22 to long-term oligotrophic saline-alkaline selection. After 175 serial transfers, the evolved lineage proliferated stably at 40 g L-1 Na2SO4 at pH 10.0, whereas the wild-type strain failed to proliferate. Genome resequencing and allelic replacement revealed a 5-bp insertion in spo0A, the master sporulation regulator, as a major adaptive mutation. This mutation abolished sporulation; shortened the lag phase; and enhanced vegetative growth, nutrient uptake, and expression of tricarboxylic acid cycle and nitrogen metabolism gene under saline-alkaline stress. According to untargeted metabolomics, adaptation was accompanied by increased amino acid metabolism and aminoacyl-tRNA biosynthesis, with proline, isoleucine and pantothenic acid functionally promoting growth. A point mutation in ugpB enhanced glycerol-3-phosphate uptake, increased peptidoglycan and wall teichoic acid levels, and partially rescued the survival cost of the spo0A mutation. In greenhouse assays under combined saline-alkaline stress, the evolved strain increased soybean shoot dry weight and root dry weight by 56.08% and 27.02%, respectively. These results indicate that prolonged, predictable saline-alkaline selection can favor active growth rather than dormancy when compensatory cell envelope reinforcement buffers survival costs.

Adaptive laboratory evolution↗
PubMed2025

Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection.

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual life cycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection-selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

Cryptosporidium parvum↗
PubMed2026

AAV-mediated CBLN1 replacement rescues hereditary ataxia caused by bi-allelic CBLN1 variants.

Cbln1 is a secreted synaptic organizer required for parallel fiber-Purkinje cell (PF-PC) synapse integrity, climbing fiber (CF) refinement, and cerebellar motor learning but has not previously been implicated in human disease. We identified bi-allelic CBLN1 missense variants (A63P and Y112C) in two unrelated families with early-onset cerebellar ataxia accompanied by oculomotor abnormalities, cerebellar atrophy, and variable cognitive delay. In heterologous cells, both variants showed reduced steady-state protein abundance, impaired maturation through the early secretory pathway, and little or no detectable secretion, resulting in markedly reduced extracellular CBLN1 availability. Consistently, cerebellar granule cells expressing CBLN1-Y112C failed to induce excitatory synapses onto glutamate receptor δ2 (GluD2)-expressing cells in vitro. A knockin mouse harboring Y112C lacked synaptic Cbln1 and recapitulated key features of Cbln1 deficiency, including disrupted PF-PC synapse organization, persistent CF multi-innervation, impaired PF-PC transmission, and long-term depression, and deficits in motor coordination and oculomotor learning. Notably, systemic delivery of an astrocyte-targeted adeno-associated virus expressing wild-type CBLN1 in adult mutant mice restored synaptic CBLN1 localization, cerebellar synaptic function, plasticity, and behavior. These findings establish CBLN1 deficiency as a cause of hereditary ataxia and identify extracellular CBLN1 replacement as a therapeutic strategy for a reversible cerebellar synaptopathy.

CBLN1↗
PubMed2026

Transcriptomic shift in ethanol and amino acid metabolic genes regulated by Med15 during alcoholic fermentation.

Organisms that thrive in extreme environments provide natural experiments in evolution, revealing the genetic regulators that orchestrate complex phenotypic change. Wine yeast (WY) are specialized strains that are adapted to survive in the wine making environment while producing high concentrations of ethanol. In addition to large genomic changes that differentiate WY from yeast used in other industries, SNP and polyglutamine tract polymorphism in the transcriptional regulator Med15 are associated with the fermentation efficiency and stress response phenotypes of WY. In this study, we investigated the transcriptional differences during wine fermentation in transgenic lab strain yeast having integrated WY MED15 alleles. Compared to the unmodified lab strain (MED15 LAB), the same strain in which the MED15 locus was replaced with a MED15 allele from yeast isolated from palm wine, the fermented sap of palm (oil, date, coconut) trees (MED15 WY23), exhibited enhanced expression of amino acid biosynthesis genes as well as stress resistance and metabolic adaptation genes. Our experimental data confirm the role of arginine in efficient fermentation and suggest that certain MED15 alleles alter the expression patterns of arginine pathway genes in some cases improving carbon flux under nitrogen stress. The global benefits conferred by natural polymorphisms in a single transcriptional regulator highlight Med15 as a target for engineering of strains devoted to various types of alcohol production.

Ethanol↗
PubMed2026

Transcriptomic shift in ethanol and amino acid metabolic genes regulated by Med15 during alcoholic fermentation.

Organisms that thrive in extreme environments provide natural experiments in evolution, revealing the genetic regulators that orchestrate complex phenotypic change. Wine yeast are specialized strains that are adapted to survive in the wine making environment while producing high concentrations of ethanol. In addition to large genomic changes that differentiate wine yeast from yeast used in other industries, single nucleotide and polyglutamine tract polymorphisms in the transcriptional regulator Med15 are associated with the fermentation efficiency and stress response phenotypes of wine yeast. In this study we investigated the transcriptional differences during wine fermentation in transgenic lab strain yeast having integrated wine yeast MED15 alleles. Compared to the unmodified lab strain (LAB or MED15 LAB ), the same strain in which the MED15 locus was replaced with a MED15 allele from yeast isolated from palm wine, the fermented sap of palm (oil, date, coconut) trees, (WY23, or MED15 WY23 ) exhibited enhanced expression of glycolytic, fermentation, and amino acid biosynthesis genes. Our experimental data confirms the importance of arginine biosynthetic genes during the fermentation process and suggests that the improvement in fermentation efficiency in strains with MED15 alleles from some wine yeast strains may be related to the role of Med15 in expression of the genes of the arginine biosynthetic pathway. The global benefit conferred by polymorphisms in a single transcriptional regulator, makes Med15 a prime target for engineering of strains devoted to various types of alcohol production.

Journal Article↗
PubMed1977

Heterozygote advantage for the phenylketonuria allele.

Mean weight at birth of unaffected (normal homozygous and PKU heterozygous) offspring of parents heterozygous for the phenylketonuria (PKU) allele averages significantly above that of Norwegian neonates, with no significant difference in mean age of mothers or in mean parity. It approaches the optimal birthweight--that which confers the minimum overall mortality in the pre-, peri-, and postnatal periods. This near-optimal birthweight together with the possibly higher effective fertility observed in PKU heterozygous couples (at least in those who married before 1940), has apparently more than outweighed the disadvantages of the allele in PKU homozygous offspring as shown, for example, in an excessive number of pre- and perinatal deaths among the total offspring of PKU heterozygotes, to say nothing of the PKU survivors who, often, used to die young. The two effects--fertility and viability--apparently both contribute in the same direction, to give a biological fitness in excess of 1 for the heterozygote. This heterozygote advantage presumably explains the presence of the allele at frequencies above those to be expected from the simple replacement of a homozygously-lethal allele by mutation alone.

Alleles↗
PubMed1978

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.

Alleles↗
PubMed1975

Genetic position and amino acid replacements of several mutations in ribosomal protein S5 from Escherichia coli.

The relative genetic position of the following four mutations of ribosomal protein S5 has been determined: spc-13, a mutation to spectinomycin resistance; stri N421 and strid1023, mutations suppressing dependence on streptomycin and sup0-1, a mutation suppressing partially the temperature-sensitive phenotype of an alanyl-tRNA synthetase mutation. The transduction experiments performed indicate that the spc-13 site is located in the S5 cistron proximal to the strA locus, that sup0-1 maps proximal to the aroE gene and that the striN421 and strid1023 loci are located between these two mutational sites. Proteinchemical analysis of the amino acid replacement in protein S5 of strain N421 (carrying the striN421 allele) has shown that an arginine residue is replaced by leucine which results in the appearance of a trypsin intensitive bond between the tryptic peptides T2 and T16. The same alteration has been previously found by Itoh and Wittmann (1973) in the S5 protein of strain d1023. Determination of the alteration of ribosomal protein S5 of strain 0-1 (sup0-1 allele) revealed that the C-terminal tryptic peptide is altered. It differs from that of the wild-type protein by the lack of five amino acids and the appearance of a C-terminal glycine residue instead of a lysine residue. This change can be explained by the deletion of eleven nucleotides in the S5 cistron of strain 0-1. The recent determination of the primary structure of ribosomal protein S5 (Wittmann-Liebold and Greuer, 1975) allows the ordering of the S5 alterations employed: The order is spc-13-strid1023 (striN421)-sup0-1 with the spc-13 amino acid replacement being located at the NH2-terminal portion of the S5 sequence and the alteration of strain 0-1 at the COOH-terminal end. The proteinchemical results are therefore in full agreement with the genetic data and unambiguously allow the conclusion that the S5 cistron is transcribed counterclock-wise on the Escherichia coli chromosome.

Amino Acid Sequence↗
PubMed1976

The mucopolysaccharidoses (a review).

The mucopolysaccharidoses are a group of genetic diseases characterized by storage of incompletely degraded glycosaminoglycans. Such storage causes marked distortion of many tissues with consequent severe somatic changes and mental retardation. Storage of glycosaminoglycans results from markedly diminished activity of specific hydrolases requisite for the normal degradation of glycosaminoglycans. The specific enzymic defects have been identified in nine different diseases. In some cases evidence has been obtained indicating the existence of additional allelic diseases based on the same enzyme. The knowledge obtained from these studies has made prenatal diagnosis possible and has led to the possibility that therapy may be undertaken utilizing enzyme replacement.

Acetylglucosaminidase↗
PubMed2026

Emerging Therapies for Angelman Syndrome.

Angelman syndrome (AS) is a complex neurogenetic disorder characterized by severe global developmental delay, motor dysfunction, and epilepsy, primarily resulting from the lack of functional ubiquitin protein ligase E3A (UBE3A) protein expression in neurons. While current management remains largely symptomatic, the therapeutic landscape for AS is rapidly evolving. Emerging strategies aim to restore UBE3A function through upstream interventions, such as gene replacement therapy or unsilencing of the imprinted paternal allele, which is present but transcriptionally silenced in neurons due to genomic imprinting. This imprinting is mediated by the distal portion of a long non-coding RNA known as the UBE3A-antisense transcript (UBE3A-ATS). This UBE3A-ATS has become a key therapeutic target, with several approaches developed to unsilence the paternal allele, including antisense oligonucleotides (ASOs), CRISPR-based editing, synthetic microRNA, and other modalities. To date, three ASO programs have demonstrated promising signals in early clinical development, with reported improvements in clinical outcomes and electroencephalography (EEG) biomarkers. Given the potential for improved outcomes with early intervention, the inclusion of AS in broader genomic newborn screening programs is currently being explored. An early-intervention approach, or combination of approaches, holds significant promise for transforming the lives of individuals affected by AS with outcomes dependent on their age or genotype.

Humans↗
PubMed2025

Substitution of Glutamic Acid at Position 71 of DRβ1*04:01 and Collagen-Specific Tolerance Without Alloreactivity.

OBJECTIVE: The DRB1 locus is strongly associated with both susceptibility and resistance to rheumatoid arthritis (RA). DRB1 alleles encoding the VKA or VRA epitope in positions 11, 71, and 74 confer the highest risk of developing RA, whereas the allele encoding VEA is protective. We therefore investigated the feasibility of creating antigen-specific tolerance without inducing alloreactivity by replacing lysine with glutamic acid at position 71 in DRβ1*04:01. METHODS: Individual DRB1 alleles and the DRB1*04:01K71E allele were cloned into T2 cell lines to measure binding of biotinylated peptides. Transgenic animals expressing DRB1*04:01, DRB1*01:01, or DRB1*04:01K71E were injected with collagen to measure T cell proliferation. Skin and bone marrow transplants between DRB1*04:01K71E and DRB1*04:01 mice were performed to determine if the single amino acid change at position 71 would be recognized as foreign. DRB1*04:01 mice transplanted with DRB1*04:01K71E bone marrow were injected with collagen to test if resistance to collagen sensitization could be transferred. RESULTS: Replacing lysine (K) at position 71 in DRβ1*04:01 with glutamic acid (E) blocked collagen peptide binding and rendered the DRB1*04:01K71E mice resistant to collagen sensitization. Skin and bone marrow transplants from DRB1*04:01K71E mice were not rejected by DRB1*04:01 mice, suggesting the single E71 difference was not recognized as allogeneic. Bone marrow from DRB1*04:01K71E mice adoptively transferred antigen-specific tolerance to collagen to DRB1*04:01 mice. CONCLUSION: These studies demonstrate that editing a single amino acid in DRβ1*04:01 blocks collagen peptide binding without inducing alloreactivity and could therefore represent a gene therapy approach to induce antigen-specific passive tolerance.

Animals↗
PubMed1976

[Mutator effect of suppressed amber-alleles of early genes of bacteriophage T4].

The mutator effect of amber alleles of three early genes (43, 32, 47), which were suppressed by the bacterial suppressor gene, was studied. There are some advantages in using the suppressed amber alleles instead of ts, because in this case definite amino acid substitutions take place in the protein due to certain suppressor gene effect. For example, in Escherichia coli CR63(Su+-1) the replacement of the original amino acid by serine takes place. Studying the mutator effect of 43 alleles of DNA polymerase gene of phage T4 with tester mutant r131 showed that in condition of suppression by the gene Su+ -1 only 13,9% of alleles possessed the mutator activity. In the same experiments with mutants of genes 32 and 47 the mutator effect was not observed.

Alleles↗
PubMed2024

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Animals↗
PubMed2024

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during mid-embryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these 2 paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Preprint↗