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Nature Communications Aug 2023Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the...
Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.
Topics: Humans; Escherichia coli; High-Throughput Nucleotide Sequencing; RNA-Seq; Anti-Bacterial Agents; DNA Primers; RNA, Ribosomal
PubMed: 37612289
DOI: 10.1038/s41467-023-40137-9 -
Molecular Cell Aug 2023During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during...
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Replication; DNA Helicases; Saccharomyces cerevisiae; DNA, Single-Stranded
PubMed: 37506699
DOI: 10.1016/j.molcel.2023.06.035 -
Nucleic Acids Research Aug 2023The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as...
The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.
Topics: DNA-Directed DNA Polymerase; HIV Reverse Transcriptase; Introns; Retroviridae; RNA; RNA-Directed DNA Polymerase; DNA Replication
PubMed: 37279911
DOI: 10.1093/nar/gkad478 -
Molecular Aspects of Medicine Jun 2024Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time... (Review)
Review
Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.
Topics: Nucleic Acid Denaturation; Humans; DNA; Polymerase Chain Reaction
PubMed: 38489863
DOI: 10.1016/j.mam.2024.101268 -
Bioscience Reports Jul 2023To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these... (Review)
Review
To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.
Topics: DNA Primase; DNA-Directed DNA Polymerase; DNA Replication; Catalytic Domain; DNA
PubMed: 37358261
DOI: 10.1042/BSR20221986 -
BMC Ecology and Evolution May 2024Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for... (Comparative Study)
Comparative Study
Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.
Topics: Mollusca; Animals; DNA Barcoding, Taxonomic; DNA, Environmental; DNA Primers; Biodiversity
PubMed: 38822255
DOI: 10.1186/s12862-024-02265-8 -
Molecular Ecology Jun 2024Molecular tools are an indispensable part of ecology and biodiversity sciences and implemented across all biomes. About a decade ago, the use and implementation of... (Review)
Review
Molecular tools are an indispensable part of ecology and biodiversity sciences and implemented across all biomes. About a decade ago, the use and implementation of environmental DNA (eDNA) to detect biodiversity signals extracted from environmental samples opened new avenues of research. Initial eDNA research focused on understanding population dynamics of target species. Its scope thereafter broadened, uncovering previously unrecorded biodiversity via metabarcoding in both well-studied and understudied ecosystems across all taxonomic groups. The application of eDNA rapidly became an established part of biodiversity research, and a research field by its own. Here, we revisit key expectations made in a land-mark special issue on eDNA in Molecular Ecology in 2012 to frame the development in six key areas: (1) sample collection, (2) primer development, (3) biomonitoring, (4) quantification, (5) behaviour of DNA in the environment and (6) reference database development. We pinpoint the success of eDNA, yet also discuss shortfalls and expectations not met, highlighting areas of research priority and identify the unexpected developments. In parallel, our retrospective couples a screening of the peer-reviewed literature with a survey of eDNA users including academics, end-users and commercial providers, in which we address the priority areas to focus research efforts to advance the field of eDNA. With the rapid and ever-increasing pace of new technical advances, the future of eDNA looks bright, yet successful applications and best practices must become more interdisciplinary to reach its full potential. Our retrospect gives the tools and expectations towards concretely moving the field forward.
Topics: Biodiversity; DNA Barcoding, Taxonomic; DNA, Environmental; Ecology; Ecosystem; Environmental Monitoring; History, 21st Century
PubMed: 38624076
DOI: 10.1111/mec.17355 -
Molecular Ecology Dec 2023Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different... (Review)
Review
Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different trophic levels, and to evaluate impacts on interspecific interactions. Molecular approaches such as DNA metabarcoding are boosting our ability to perform biodiversity inventories. Nevertheless, even though a few studies have recently attempted exhaustive reconstructions of communities, holistic assessments remain rare. The majority of metabarcoding studies published in the last years used just one or two markers and analysed a limited number of taxonomic groups. Here, we provide an overview of emerging approaches that can allow all-taxa biological inventories. Exhaustive biodiversity assessments can be attempted by combining a large number of specific primers, by exploiting the power of universal primers, or by combining specific and universal primers to obtain good information on key taxa while limiting the overlooked biodiversity. Multiplexes of primers, shotgun sequencing and capture enrichment may provide a better coverage of biodiversity compared to standard metabarcoding, but still require major methodological advances. Here, we identify the strengths and limitations of different approaches, and suggest new development lines that might improve broad scale biodiversity analyses in the near future. More holistic reconstructions of ecological communities can greatly increase the value of metabarcoding studies, improving understanding of the consequences of ongoing environmental changes on the multiple components of biodiversity.
Topics: Biodiversity; DNA; DNA Barcoding, Taxonomic; DNA Primers; Ecology
PubMed: 36762839
DOI: 10.1111/mec.16881 -
Biochemical Society Transactions Jun 2024Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the... (Review)
Review
Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.
Topics: DNA, Mitochondrial; DNA Replication; Animals; Mitochondria; Humans; DNA-Directed RNA Polymerases; Transcription, Genetic; Drosophila; Exoribonucleases; Drosophila melanogaster
PubMed: 38884788
DOI: 10.1042/BST20230952