Ts Mutants Research Articles

Identification of a novel alternate promoter element in the pheST operon of Escherichia coli.

Earlier work in this laboratory revealed that fitA was same as pheS as a recombinant clone, pSRJ5R1 harboring pheS+ gene complemented transcription-defective fitA76 Ts (temperature sensitive) mutant. However, this clone lacked the native promoter (NP)of pheST operon. A putative - 10 promoter like element was suggested to act as promoter in this clone. This work investigated the veracity of this putative promoter as well as its downstream regulatory region towards driving the pheS expression. Plasmid clones with promoter-mutations or -deletions were constructed by PCR-based cloning and their ability to complement fitA76 Ts mutant strains was checked. Chromosomal mutations were transferred into various genetic backgrounds via P1-transductions. Relative viability assays were performed to check the extent of complementation. Clones harboring point mutations (PM-pheS) or deletion (PD1-pheS) of - 10 region of the putative promoter did not abolish complementation of the fitA76 Ts phenotype. Subsequently, a novel alternate promoter (AP)was discovered by downstream deletion clone (PD2-pheS) which failed to complement. Keeping PD1-pheS intact but mutating initiation codon of pheS (ATG→TTG) failed to complement. Complementation ability of novel alternate promoter is poor in HfrC strain background unlike native promoter which complements well independent of strain background. A novel alternate-promoter of pheST operon was identified by mutational/deletional analyses and earlier reported putative - 10 promoter was shown to be dispensable. Alternate promoter is relA dependent.

Genetic Evidence of Recessive Epistasis Among Sex Determining silkless1 (sk1) tasselseed1 (ts1) and tasselseed2 (ts2) Genes and Its Utility in Maize (Zea mays L.) Breeding

ABSTRACTIn maize, sex is determined by the formation of staminate and pistillate florets from an initially bisexual floral meristem through action of Silkless1 (Sk1), Tasselseed1 (Ts1) and Tasselseed2 (Ts2) genes. In this study, separate temperate donors having mutant sk1 (EC904829 and EC904831), ts1 (EC904827) and ts2 (EC904824) genes were crossed with sub‐tropical inbreds (HKI323 and HKI1105) to investigate the genetics and interaction of silkless and tasselseed traits. The monogenic and nuclear inheritance of sk1, ts1 and ts2 genes was established in a standard Mendelian principles in both monohybrid and dihybrid crosses irrespective of genetic background. About 444 F2 progenies across digenic population ‐A, ‐B and ‐C segregated as 239 normal: 109 silkless: 96 tasselseed, and 377 progenies of digenic populations ‐D, ‐E and ‐F segregated with 214 normal: 79 silkless: 84 tasselseed plants revealing recessive epistatic interaction (9:3:4) of sk1 with two ts mutations (ts1 and ts2). No progeny with both silkless and tasselseed phenotype was observed. The double mutants (sk1sk1/ts1ts1 and sk1sk1/ts2ts2) were identified through progeny testing. Our study emphasize the untapped potential of these mutants, providing a renewed focus for maize breeders worldwide.

Genetic and molecular characterization of fit95 mutation of Escherichia coli: evidence that fit95 is an allele of pheT.

The originally identified transcription-defective fitA76 temperature-sensitive (Ts) mutation defined an allele of pheS. Both fitA/pheS and fitB/pheT were previously proposed to function as transcription factors. Sequencing pheS region of the fitA76 mutant revealed the same G293→A293 transition found in the translation-defective pheS5 mutant. It was subsequently found that fitA76 harbored a second mutation (fit95) in addition to pheS5 mutation. The fit95 was found to be Ts on -salt media but was found unstable. In this investigation, genetic, physiological and molecular characterization of the fit95 mutation was carried out. The fit95 was genetically re-separated from the pheS5 mutation present in the fitA76 mutant and the same was subsequently mobilized into multiple genetic backgrounds to study its phenotypic modulations by altering the medium and supplements. Based on genetic studies, the unstable -salt Ts phenotype of the fit95 could be stabilized by the presence of rpoB201 mutation. Addition of glucose enhanced Ts phenotype in the presence of rpoB201 mutation, but citrate completely alleviated the Ts phenotype. Further, by series of complementation analyses and molecular cloning, the identity of fit95 was revealed as pheT gene which is part of pheST operon.

CsTs, a C-type lectin receptor-like kinase, regulates the development trichome development and cuticle metabolism in cucumber (Cucumis sativus).

Cucumber (Cucumis sativus) fruit spines are a classic material for researching the development of multicellular trichomes. Some key genes that influence trichome development have been confirmed to be associated with cuticle biosynthesis and secondary metabolism. However, the biological mechanisms underlying trichome development, cuticle biosynthesis, and secondary metabolism in cucumber remain poorly understood. CsTs, a C-type lectin receptor-like kinase gene, reportedly causes a tender trichome phenotype in cucumber when it mutates. In this study, the role of CsTs in cucumber fruit spines morphogenesis was confirmed using gene editing technology. Sectioning and cell wall component detection were used to analyse the main reason of tender fruit spines in the ts mutant. Subsequently, transcriptome data and a series of molecular biology experiments were used to further investigate the relationship between CsTs and cytoskeletal homeostasis in cucumber. CsTs overexpression partially compensated for the abnormal trichome phenotype of an Arabidopsis homolog mutant. Genetic hybridization and metabolic analysis indicated that CsTs and CsMict can affect trichome development and cuticle biosynthesis in the same pathway. Our findings provide important background information for further researching on the molecular mechanism underlying cucumber trichome development and contribute to understanding the biological function of C-type lectin receptor-like kinases.

Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3β-tigloyloxytropane

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3β-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3β-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3β-tigloyloxytropane synthase (TS), catalyzes 3β-tropanol and tigloyl-CoA to form 3β-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3β-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3β-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3β-tigloyloxytropane from tiglic acid and 3β-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3β-tigloyloxytropane.

Abstract 2929: Multiplexed somatic genome editing to model and investigate advanced colorectal cancer

Abstract Colorectal cancer (CRC) is among the deadliest cancers worldwide, with advanced CRC being the leading cause of death. For the development of novel therapy options, it is crucial to investigate mechanisms of advanced CRC in a bona fide way. However, little is known about the impact of distinct genomic alterations on cancer progression. The assessment of the function and role of the putative drivers in tumor progression via sequencing data remains challenging, due to the complexity and possible combinations of the candidates. Hence, genetically engineered mouse models (GEMMs) represent the mainstay for the investigation of the role of oncogenes and tumor suppressor genes in cancer progression. However, particularly in CRC, they are limited by imprecise modeling of cancer-associated changes, challenging scalability, and development of tumors in anatomically incorrect places. To tackle these issues, we developed SOmatic CRISPR-mediated genome Alterations of Tumor suppressors with Exploratory single cell Sequencing (SOCRATES) to analyze advanced CRC. We multiplexed CRISPR-Cas systems, packaged them into lentiviral particles, and delivered them to enterocytes via colonoscopy-guided submucosal injection. With this innovative approach, SOCRATES enables rapid and direct in vivo genome engineering for the modeling of advanced CRC with distinct mutational patterns and the investigation of the cancer cell states and tumor microenvironment (TME) upon editing the genes of interest. Similar to the Greek philosopher Socrates, who established a form of argumentized dialogue between individuals, SOCRATES facilitates the understanding of previously undiscovered communication between cancer cells and TME. We analyzed the influence of the most recurrent tumor suppressor mutations (Fbxw7, Ptprt, Arid1a, Atrx, Atm, Smad4, Pten) on the cancer cell state and TME in advanced CRC. Further, we created a TS-TME interaction atlas and revealed novel mechanistic insights into how the TS mutations rewire cell states and cell-cell communications. With this generated knowledge, SOCRATES will contribute to our understanding of complex tumor ecosystems and foster the improvement of diagnostic and therapeutic approaches. Citation Format: Manuel Mastel, Rene Jackstadt, Jasmin Meier, Gabriele Diamante. Multiplexed somatic genome editing to model and investigate advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2929.

Ribonuclease E strongly impacts bacterial adaptation to different growth conditions

ABSTRACT Adaptation of bacteria to changes in their environment is often accomplished by changes of the transcriptome. While we learned a lot on the impact of transcriptional regulation in bacterial adaptation over the last decades, much less is known on the role of ribonucleases. This study demonstrates an important function of the endoribonuclease RNase E in the adaptation to different growth conditions. It was shown previously that RNase E activity does not influence the doubling time of the facultative phototroph Rhodobacter sphaeroides during chemotrophic growth, however, it has a strong impact on phototrophic growth. To better understand the impact of RNase E on phototrophic growth, we now quantified gene expression by RNA-seq and mapped 5’ ends during chemotrophic growth under high oxygen or low oxygen levels and during phototrophic growth in the wild type and a mutant expressing a thermosensitive RNase E. Based on the RNase E-dependent expression pattern, the RNAs could be grouped into different classes. A strong effect of RNase E on levels of RNAs for photosynthesis genes was observed, in agreement with poor growth under photosynthetic conditions. RNase E cleavage sites and 5’ ends enriched in the rne ts mutant were differently distributed among the gene classes. Furthermore, RNase E affects the level of RNAs for important transcription factors thus indirectly affecting the expression of their regulons. As a consequence, RNase E has an important role in the adaptation of R. sphaeroides to different growth conditions.

Functional analysis of the dehydratase domains of the PUFA synthase from Emiliania huxleyi in Escherichia coli and Arabidopsis thaliana

BackgroundPolyunsaturated fatty acid (PUFA) synthase is a multi-domain mega-enzyme that effectively synthesizes a series of PUFAs in marine microorganisms. The dehydratase (DH) domain of a PUFA synthase plays a crucial role in double bond positioning in fatty acids. Sequencing results of the coccolithophore Emiliania huxleyi (E. huxleyi, Eh) indicated that this species contains a PUFA synthase with multiple DH domains. Therefore, the current study, sought to define the functions of these DH domains (EhDHs), by cloning and overexpressing the genes encoding FabA-like EhDHs in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana).ResultsA complementation test showed that the two FabA-like DH domains could restore DH function in a temperature-sensitive (Ts) mutant. Meanwhile, overexpression of FabA-like EhDH1 and EhDH2 domains increased the production of unsaturated fatty acids (UFAs) in recombinant E. coli by 43.5–32.9%, respectively. Site-directed mutagenesis analysis confirmed the authenticity of active-site residues in these domains. Moreover, the expression of tandem EhDH1-DH2 in A. thaliana altered the fatty acids content, seed weight, and germination rate.ConclusionsThe two FabA-like DH domains in the E. huxleyi PUFA synthase function as 3-hydroxyacyl-acyl carrier protein dehydratase in E. coli. The expression of these domains in E. coli and A. thaliana can alter the fatty acid profile in E. coli and increase the seed lipid content and germination rate in A. thaliana. Hence, introduction of DH domains controlling the dehydration process of fatty acid biosynthesis in plants might offer a new strategy to increase oil production in oilseed plants.

Single amino acid substitutions in hydrophobic cores at a head-coiled coil junction region of cohesin facilitate its release of DNA during anaphase.

Cohesin holds sister chromatids together and is cleaved by separase/Cut1 to release DNA during the transition from mitotic metaphase to anaphase. The cohesin complex consists of heterodimeric structural maintenance of chromosomes (SMC) subunits (Psm1 and Psm3), which possess a head and a hinge, separated by long coiled coils. Non-SMC subunits (Rad21, Psc3 and Mis4) bind to the SMC heads. Kleisin/Rad21's N-terminal domain (Rad21-NTD) interacts with Psm3's head-coiled coil junction (Psm3-HCJ). Spontaneous mutations that rescued the cleavage defects in temperature-sensitive (ts) separase mutants were identified in the interaction interface, but the underlying mechanism is yet to be understood. Here, we performed site-directed random mutagenesis to introduce single amino acid substitutions in Psm3-HCJ and Rad21-NTD, and then identified 300 mutations that rescued the cohesin-releasing defects in a separase ts mutant. Mutational analysis indicated that the amino acids involved in hydrophobic cores (which may be in close contact) in Psm3-HCJ and Rad21-NTD are hotspots, since 80 mutations (approx. 27%) were mapped in these locations. Properties of these substitutions indicate that they destabilize the interaction between the Psm3 head and Rad21-NTD. Thus, they may facilitate sister chromatid separation in a cleavage-independent way through cohesin structural re-arrangement.

Refined Capsid Structure of Human Adenovirus D26 at 3.4 Å Resolution.

Various adenoviruses are being used as viral vectors for the generation of vaccines against chronic and emerging diseases (e.g., AIDS, COVID-19). Here, we report the improved capsid structure for one of these vectors, human adenovirus D26 (HAdV-D26), at 3.4 Å resolution, by reprocessing the previous cryo-electron microscopy dataset and obtaining a refined model. In addition to overall improvements in the model, the highlights of the structure include (1) locating a segment of the processed peptide of VIII that was previously believed to be released from the mature virions, (2) reorientation of the helical appendage domain (APD) of IIIa situated underneath the vertex region relative to its counterpart observed in the cleavage defective (ts1) mutant of HAdV-C5 that resulted in the loss of interactions between the APD and hexon bases, and (3) the revised conformation of the cleaved N-terminal segments of pre-protein VI (pVIn), located in the hexon cavities, is highly conserved, with notable stacking interactions between the conserved His13 and Phe18 residues. Taken together, the improved model of HAdV-D26 capsid provides a better understanding of protein–protein interactions in HAdV capsids and facilitates the efforts to modify and/or design adenoviral vectors with altered properties. Last but not least, we provide some insights into clotting factors (e.g., FX and PF4) binding to AdV vectors.

Functional divergence of orthologous temperature-sensitive mutations in C. elegans and C. briggsae.

To learn if orthologous mutations are temperature-sensitive in related species, we studied four C. briggsae mutations orthologous to alleles of important C. elegans genes. Both Cel-glp-4(bn2) and Cbr-glp-4(v473) are temperature-sensitive, causing sterility at 25°C. By contrast, Cel-fog-1 ( q253) is strongly ts , but its ortholog Cbr-fog-1(v442) causes a loss-of-function at all temperatures. Finally, the C. elegans glp-1 alleles bn18 and e2141 are ts sterile. However, their C. briggsae orthologs, Cbr-glp-1(v429) and Cbr-glp-1(v438) respectively, are wild-type at all temperatures. Thus, a ts mutation in one species provides clues about how to design ts alleles in another, but all theoretical outcomes are possible.

Hypothermia Is a Potential New Therapy for a Subset of Tumors with Mutant p53.

The tumor suppressor p53 gene is mutated in approximately 50% of all human tumors. Many tumor-associated mutant p53 proteins misfold into a common, denatured conformation and accumulate to high levels in human tumors. In such tumors, these mutant forms of p53 provide a "gain of function" to promote tumor progression. Therefore, targeting mutant p53 has become an attractive approach for cancer therapy. In this issue, the study by Lu and colleagues supports the premise that certain forms of mutant p53 are temperature sensitive in conformation; these forms of p53 are mutant in conformation at physiologic temperature, but can refold into a normal, or "wild-type" conformation at lower temperature (32°C to 34°C). Notably, these temperature-sensitive mutants account for up to 7.5% of all human tumors that carry mutant p53, so this fraction of patients is estimated to be quite significant. Results from this study show that employing therapeutic hypothermia to reduce the core temperature of mice bearing tumors with these temperature-sensitive mutant forms of p53 (ts mutant p53) causes ts mutant p53 to switch to a wild-type conformation in tumors, inhibiting tumor growth. Moreover, combining hypothermia with chemotherapy leads to durable remission of such tumors, with no obvious toxicity to normal tissues.See related article by Lu et al., p. 3905.

Reverse genetic engineering of simian rotaviruses with temperature-sensitive lesions in VP1, VP2, and VP6

Rotaviruses are 11-segmented double-stranded RNA viruses and important causes of acute gastroenteritis in young children. To investigate the functions of specific viral proteins during the rotavirus lifecycle, temperature-sensitive (ts) mutants were previously created using a cultivatable simian strain (SA11) and chemical mutagenesis. These ts SA11 mutants replicate more efficiently at the permissive temperature of 31 °C than at the non-permissive temperature of 39 °C. Prototype strains SA11-tsC, SA11-tsF, and SA11-tsG were mapped to the genes encoding structural proteins VP1, VP2, and VP6, respectively, and putative ts lesions were identified using Sanger sequencing. However, additional background mutations in their genomes had hampered validation of the ts lesions and confounded their use in mechanistic studies. Here, we employed plasmid only-based reverse genetics to engineer recombinant (r) SA11 rotaviruses containing only the putative ts lesions of SA11-tsC (L138P change in VP1), SA11-tsF (A387D change in VP2) or SA11-tsG (S10T, D13H, and A121G changes in VP6). For simplicity, we refer to these newly-engineered, isogenic viruses as rSA11-tsVP1, rSA11-tsVP2, and rSA11-tsVP6. Single-cycle growth assays revealed that these mutants indeed exhibit ts phenotypes with significantly diminished titers (>1.5-logs) at 39 °C versus 31 °C. The rSA11 ts mutants proved genetically stable at the population-level following 3 sequential passages at 39 °C, but individual revertant clones were detected in plaque assays. Heat sensitivity experiments showed that pre-incubation of rSA11-tsVP1 or rSA11-tsVP2, but not rSA11-tsVP6, at 39 °C diminished replication at 31 °C. This result indicates that the ts lesions in VP1 and VP2 affect the incoming virion but those in VP6 affect a later stage of the viral lifecycle. In silico molecular dynamics simulations predicted temperature-dependent, long-range effects of the S10T, D13H, and/or A121G changes on the VP6 structure. Altogether, our results confirm the ts lesions of the original SA11-tsC, SA11-tsF, and SA11-tsG mutants, provide a new set of isogenic strains for investigating aspects of rotavirus replication, and shed light on how the ts lesions might impact VP1, VP2, or VP6 functions.

CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.

Hypothermia Effectively Treats Tumors with Temperature-Sensitive p53 Mutations.

The p53 tumor suppressor is frequently inactivated by mutations in cancer. Most p53 mutations are located in the DNA-binding domain, causing local disruption of DNA-binding surface or global misfolding. Rescuing the structural defect of mutant p53 is an attractive therapeutic strategy, but its potential remains unproven due to a lack of drugs capable of efficiently rescuing misfolded p53. Although mutant p53 in tumors is inactive at 37°C, approximately 15% are temperature sensitive (ts) and regain DNA-binding activity at 32°C to 34°C (ts mutants). This temperature is achievable using a therapeutic hypothermia procedure established for resuscitated cardiac arrest patients. To test whether hypothermia can be used to target tumors with ts p53 mutations, the core temperature of tumor-bearing mice was lowered to 32°C using the adenosine A1 receptor agonist N6-cyclohexyladenoxine that suppresses brain-regulated thermogenesis. Hypothermia treatment (32 hours at 32°C × 5 cycles) activated endogenous ts mutant p53 in xenograft tumors and inhibited tumor growth in a p53-dependent fashion. Tumor regression and durable remission in a ts p53 lymphoma model was achieved by combining hypothermia with chemotherapy. The results raise the possibility of treating tumors expressing ts p53 mutations with hypothermia. SIGNIFICANCE: Pharmacologic inhibition of brain-regulated thermogenesis and induction of 32°C whole-body hypothermia specifically targets tumors with temperature-sensitive p53 mutations, rescuing p53 transcriptional activity and inducing tumor regression.See related commentary by Hu and Feng, p. 3762.

Knockdown of the mRNA encoding the ribosomal protein eL38 in mammalian cells causes a substantial reorganization of genomic transcription

The ribosomal protein eL38 is a component of the mammalian translation machine. The deletion of the Rpl38 locus in mice results in the Tail-short (Ts) mutant phenotype characterized by a shortened tail and other defects in the axial skeleton development. Here, using the next-generation sequencing of total RNA from HEK293 cells knocked down of eL38 mRNA by transfection with specific siRNAs, we examined the effect of reduced eL38 content on genomic transcription. An approximately 4-fold decrease in the level of eL38 was shown to cause changes in the expression of nearly 1500 genes. Among the down-regulated genes, there were those responsible for p53 activity, Ca2+ metabolism and several signaling processes, as well as genes involved in the organization and functioning of the cytoskeleton. The genes related to rRNA processing and translation, along with many others, including those whose dysregulation is associated with developmental disorders, turned out to be up-regulated. Thus, we demonstrated that the decreased RPL38 expression leads to a significant reorganization of genomic transcription. Our findings suggest a possible link between the balance of eL38 and genes implicated in osteogenesis, thereby contributing to the elucidation of the reasons for the appearance of the above Ts mutant phenotype in animals.

A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453 ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

Loss of DIAPH3, a Formin Family Protein, Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells.

Cell division is essential for the maintenance of life and involves chromosome segregation and subsequent cytokinesis. The processes are tightly regulated at both the spatial and temporal level by various genes, and failures in this regulation are associated with oncogenesis. Here, we investigated the gene responsible for defects in cell division by using murine temperature-sensitive (ts) mutant strains, tsFT101 and tsFT50 cells. The ts mutants normally grow in a low temperature environment (32 °C) but fail to divide in a high temperature environment (39 °C). Exome sequencing and over-expression analyses identified Diaph3, a member of the formin family, as the cause of the temperature sensitivity observed in tsFT101 and tsFT50 cells. Interestingly, Diaph3 knockout cells showed abnormality in cytokinesis at 39 °C, and the phenotype was rescued by re-expression of Diaph3 WT, but not Diaph1 and Diaph2, other members of the formin family. Furthermore, Diaph3 knockout cells cultured at 39 °C showed a significant increase in the level of acetylated α-tubulin, an index of stabilized microtubules, and the level was reduced by Diaph3 expression. These results suggest that Diaph3 is required for cytokinesis only under high temperature conditions. Therefore, our study provides a new insight into the mechanisms by which regulatory factors of cell division function in a temperature-dependent manner.

Examining immune arms in mice immunized with site-specific influenza virus mutants

Site-specific mutants as candidates for live influenza vaccines were resulted from directly introducing into the genome of the pathogenic influenza virus A/WSN/33 (H1N1) strain ts mutations derived from the genes encoding the polymerase complex proteins from some cold-adapted strains serving as attenuation donor. Here we present the data of a comparative study examining immune system arms in mice immunized intranasally with influenza virus mutants and classical cold-adapted reassortant obtained by crossing cold-adapted strain Donor A/Krasnodar/101/35/59 (H2N2) with strain A/WSN/33 (H1N1) bearing surface antigens (hemagglutinin and neuraminidase) similar to mutants. Immunophenotyping mononuclear leukocytes from immunized mice indicated at moderate suppressive effect after using site-specific mutant and the HA reassortant viruses on some immune cell subsets. All viruses in immunized mice resulted in activation of certain lymphocyte subsets including MHC II-positive cells, CD45+/CD19+ B lymphocytes and natural killer cells (CD16/32+/CD3–). Timescale and magnitude of activation markedly differed for each cell subsets. Mice immunized with mutants M26 and U2 peaked with count of CD16/32+/CD3– expressing cells on day 2 after the second immunization compared with control (p 0.05) that may suggest about an important role for NK cells in activating immune response. In contrast, no significant changes were observed during the study in percentage of CD4+/CD25+/Fox P3 regulatory T cells, CD4+ T helpers and CD8+ cytotoxic cells, except for a sharply decreased count of activated CD4+/CD25+ cells (4-fold) on day 7 after immunization with mutant virus M26. Moreover, mutants U2 and M26 more moderately increased percentage of TLR2- and TLR4-positive cells. The viruses studied ambiguously affected count of TLR9-expressing cells in immunized animals. All viruses increased phagocytic activity in monocytes, but not neutrophils. Despite the moderate activation of innate and adaptive immunity arms, site-specific mutants more profoundly affected humoral reactions inducing increased antibody titers, so that immunogenicity of mutant viruses was higher than that of the cold-adapted reassortant. Thus, the findings hold a promise of using site-specific mutants as live influenza vaccines.

The Search for Novel Splicing Factors: Mapping Mutations in a Temperature‐Sensitive Schizosaccharomyces pombe Library

Gene expression, starting with the transcription of DNA to messenger RNA (mRNA) and the subsequent translation of mRNA transcripts to form proteins is a vital process for all living cells. Newly transcribed RNAs must be processed before they can serve as templates for protein synthesis. RNA splicing is a key step in RNA processing. Defective splicing disrupts the production of functional proteins and is linked to debilitating human diseases such as cancer, frontotemporal dementia, and Duchenne muscular dystrophy. However, the full set of factors involved in the splicing pathway remain unknown. Hence, uncovering the complete set of splicing factors is critical for the development of new therapeutic interventions.A recent sequencing‐based genetic screen 1, 2 of a temperature‐sensitive (ts) Schizosaccharomyces pombe library 3 identified a subset of mutants that displayed global splicing defects, which may highlight essential genes with potentially novel implications in splicing. The identities of the specific mutations for each of the strains in this ts library (N = ~2300) have never been previously mapped. Therefore, we aim to map the mutations in this library and quantify their impacts on RNA splicing, allowing us to identify the genes detected in the recent screen and possibly reveal additional novel splicing factors and functional connections. Here we use Tn5 tagmentation for genome fragmentation and Gibson assembly for the high‐throughput synthesis of a wild‐type (WT) cloning library, which will then be transformed into each mutant strain to identify the mutant alleles via complementation testing. Once the alleles have been identified, we will characterize their global splicing deficiencies by sequencing methods to measure the ratio of unspliced to spliced RNA transcripts for all introns. Our efforts will annotate the ts mutant library for general use and contribute to the knowledge of the elements involved in RNA splicing.