Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects. Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1. The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails. Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway. We show that recruitment of Htz1 to chromatin requires the SWR-C. Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.
The classic murine muscular dystrophy strain, dy, was first described almost 40 years ago. We have identified the molecular basis of an allele of dy, called dy2J, by detecting a mutation in the laminin alpha 2 chain gene--the first identified mutation in laminin-2. The G to A mutation in a splice site consensus sequence causes abnormal splicing and expression of multiple mRNAs. One mRNA is translated into an alpha 2 polypeptide with a deletion in domain VI. The truncated protein apparently lacks important qualities of the wild type protein and is unable to provide sufficient muscle stability.
Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into E. coli a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene rnlB produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications.
Diurnal and seasonal variation, intensity, and structure of deep convective systems (DCSs; with 20-dBZ echo tops exceeding 14 km) over the Tibetan Plateau–South Asian monsoon region from the Tibetan Plateau (TP) to the ocean are investigated using 14 yr of Tropical Rainfall Measuring Mission (TRMM) data. Four unique regions characterized by different orography are selected for comparison, including the TP, the southern Himalayan front (SHF), the South Asian subcontinent (SAS), and the ocean. DCSs and intense DCSs (IDCSs; with 40-dBZ echo tops exceeding 10 km) occur more frequently over the continent than over the ocean. About 23% of total DCSs develop into IDCSs in the SHF, followed by the TP (21%) and the SAS (15%), with the least over the ocean (2%). The average 20-dBZ echo-top height of IDCSs exceeds 16 km and 9% of them even exceed 18 km. DCSs and IDCSs are the most frequent over the SHF, especially in the westernmost SHF, where the intensity—in terms of strong radar echo-top (viz., 40 dBZ) height, ice-particle content, and lightning flash rate—is the strongest. DCSs over the TP are relatively weak in convective intensity and small in size but occur frequently. Oceanic DCSs possess the tallest cloud top (which mainly reflects small ice particles) and the largest size, but their convective intensity is markedly weaker. DCSs and IDCSs show a similar diurnal variation, mainly occurring in the afternoon with a peak at 1600 local time over land. Although most of both DCSs and IDCSs occur between April and October, DCSs have a peak in August, whereas IDCSs have a peak in May.
Background: Yeast RNR small subunit is an Rnr2-Rnr4 heterodimer; only Rnr2 contains a cluster. Results: rnr4 and dre2 mutants are defective in Rnr2 cluster formation and display synthetic growth defects with grx3/4. Conclusion: Rnr4 stabilizes Rnr2 for cluster assembly via a pathway dependent on monothiol glutaredoxins Grx3/Grx4 and Fe-S cluster protein Dre2. Significance: Understanding RNR cluster assembly may provide new cancer therapeutic strategy.
Fidelity in DNA replication and repair requires adequate and balanced deoxyribonucleotide pools that are maintained primarily by regulation of ribonucleotide reductase (RNR). RNR is controlled via transcription, protein inhibitor association, and subcellular localization of its two subunits, R1 and R2. Saccharomyces cerevisiae Sml1 binds R1 and inhibits its activity, while Schizosaccharomyces pombe Spd1 impedes RNR holoenzyme formation by sequestering R2 in the nucleus away from the cytoplasmic R1. Here we report the identification and characterization of S. cerevisiae Dif1, a regulator of R2 nuclear localization and member of a new family of proteins sharing separate homologous domains with Spd1 and Sml1. Dif1 is localized in the cytoplasm and acts in a pathway different from the nuclear R2-anchoring protein Wtm1. Like Sml1 and Spd1, Dif1 is phosphorylated and degraded in cells encountering DNA damage, thereby relieving inhibition of RNR. A shared domain between Sml1 and Dif1 controls checkpoint kinase-mediated phosphorylation and degradation of the two proteins. Abolishing Dif1 phosphorylation stabilizes the protein and delays damage-induced nucleus-to-cytoplasm redistribution of R2. This study suggests that Dif1 is required for nuclear import of the R2 subunit and plays an essential role in regulating the dynamic RNR subcellular localization.Maintenance of genomic stability depends on faithful replication of DNA and repair of lesions after damage. Fidelity of both DNA replication and repair is influenced by perturbation in the sizes and relative ratios of cellular deoxynucleotide triphosphate (dNTP) pools. Ribonucleotide reductase (RNR) catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms and is largely responsible for maintaining cellular dNTP pools (34). As maximal DNA synthesis requires high concentrations of dNTPs, the RNR activity plays an important role in cell proliferation (31). On the other hand, increased RNR activity has been associated with malignant transformation and resistance to chemotherapy (12,14,25,56).The class I RNR holoenzymes are commonly found in eukaryotes and eubacteria and comprise two subunits, R1 and R2 (34). The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21,37,47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe) 2 -Y ⅐ ] essential for nucleotide reduction (36,40,42). Mammalian genomes contain a single R1 gene and two R2 genes; the cell cycle-regulated RRM2 is responsible for providing dNTPs in actively dividing cells, and the DNA damage-inducible p53R2 is required for replenishing dNTP pools in cells under genotoxic stress (9,22,44). Loss of p53R2 causes mitochondrial DNA depletion and increased apoptosis (22). The budding yeast Saccharomyces cerevisiae has two R1 genes, RNR1 and RNR3. RNR1 is essential for mitotic growth, while RNR3 is high...
Phages show an elevated mutation rate and remarkably rapid evolution when attacked by the bacterial CRISPR/Cas system.
The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative “linker” between Ca2+ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca2+ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca2+-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca2+-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.
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