Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology.

Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single-human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. Although whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study-GDAP1L1-to isolate highly functional live human neurons in vitro.

Draft Genome Sequence of a Single Cell of SAR86 Clade Subgroup IIIa.

SAR86 denotes a 16S clade of gammaproteobacteria that are ubiquitous in ocean surface waters. So far, SAR86 is resistant to cultivation; thus, little is known about the genome contents or physiology of this clade. Recently, four partial genome sequences for SAR86 subclades I and II were published. Here, we present the draft genome sequence of a single cell from SAR86 subgroup IIIa isolated from coastal waters in San Diego, CA.

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification.

We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.

Archaebacterial DNA polymerases tightly bind uracil-containing DNA.

We show that archaebacterial DNA polymerases are strongly inhibited by the presence of small amounts of uracil-containing DNA. Inhibition appears to be competitive, with the DNA polymerase exhibiting approximately 6500-fold greater affinity for binding the inhibitor than a DNase I-activated DNA substrate. All six archaebacterial DNA polymerases tested were inhibited, while no eubacterial, eukaryotic, or bacteriophage enzymes showed this effect. Only a small inhibition resulted when uracil was present as the deoxynucleoside triphosphate, dUTP. The rate of DNA synthesis was reduced by approximately 40% when dUTP was used in place of dTTP for archaebacterial DNA polymerases. Furthermore, an incorporated dUMP served as a productive 3'-primer terminus for subsequent elongation. In contrast, the presence of an oligonucleotide containing as little as a single dUrd residue was extremely inhibitory to DNA polymerase activity on other primer-template DNA.

The dnaB-dnaC replication protein complex of Escherichia coli. I. Formation and properties.

The complex formed between the dnaB and dnaC replication proteins of Escherichia coli is stabilized by ATP binding to dnaC. The dnaB6-dnaC6-ATP6 complex can be maintained without ATP hydrolysis at a concentration as low as 5 x 10(-10) M. The complex is also formed with adenosine 5'-(gamma-thio)triphosphate but generates little or no dnaB activity, suggesting a requirement for ATP hydrolysis in the subsequent stage of binding of the complex to DNA. In this step, dnaC is released, leaving dnaB to function on the associated DNA.

The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions.

The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions. Within the dnaB-dnaC complex, dnaB appears to be inactive but becomes active upon the ATP-dependent release of dnaC from the complex. With adenosine 5'-(gamma-thio)triphosphate substituted for ATP, the dnaB-dnaC complex does not direct dnaB to its targeted actions. Excess dnaC inhibits dna beta actions and augments the ATP gamma S effects. In the dnaA protein-driven initiation of duplex chromosome replication, dnaB is introduced for its essential helicase role via the dnaB-dnaC complex. Similarly, when the dnaA protein interacts nonspecifically with single-stranded DNA, the dnaB-dnaC complex is essential to introduce dnaB for its role in primer formation by primase.

The primosomal protein n' of Escherichia coli is a DNA helicase.

Protein n' of Escherichia coli functions in assembly and translocation of the primosome, a mobile multiprotein complex involved in priming DNA replication (Kornberg, A. (1982) Supplement to DNA Replication, Freeman Publications, San Francisco). By itself, protein n' translocates on single-stranded DNA and destabilizes duplex regions by acting as a DNA helicase, using the energy of ATP or dATP hydrolysis. Single-stranded DNA binding protein was required for melting of duplex regions longer than 40 base pairs. Initial binding of protein n' to a specific site on DNA (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 799-803) is essential for its helicase function. The polarity of protein n' translocation on DNA, in the 3' to 5' direction of the chain, suggests a mechanism for how the primosome may contribute to concurrent replication of both strands at a replication fork.

The beta subunit dissociates readily from the Escherichia coli DNA polymerase III holoenzyme.

Purified DNA polymerase III holoenzyme (holoenzyme) was separated by glycerol gradient sedimentation into the beta subunit and the subassembly that lacks it (pol III). In the presence of ATP, beta subunit dimer dissociated from holoenzyme with a KD of 1 nM; in the absence of ATP, the KD was greater than 5 nM. The beta subunit was known to remain tightly associated in the holoenzyme upon formation of an initiation complex with a primed template and during the course of replication. With separation from the template, holoenzyme dissociated into beta and pol III. Cycling to a new template depended on the reformation of holoenzyme. Holoenzyme was in equilibrium with pol III and the beta subunit in crude enzyme fractions as well as in pure preparations.

A fidelity assay using "dideoxy" DNA sequencing: a measurement of sequence dependence and frequency of forming 5-bromouracil X guanine base mispairs.

DNA replication fidelity has been assayed by using a modified DNA sequencing reaction. In one experimental approach, dideoxycytidine 5'-triphosphate (ddCTP) was used as a chain terminator during replication of M13 phage DNA by the large fragment of DNA polymerase I. The deoxyribonucleotide analogue BrdUTP was used to compete against ddCTP-induced chain terminations as an assay for B X G base mispairing (B represents bromodeoxyuridine when the analogue is present as a base pair or base mispair). By comparing BrdUTP to dCTP for competition against ddCTP, an average misincorporation frequency for BrdUMP of 0.2% was found. A similar average misincorporation frequency has been measured previously for the incorporation of radioactively labeled BrdUMP and dCMP into the synthetic template-primer poly-[d(G,T)] X oligo(dA). The advantage of the sequencing method is that an error frequency is determined for each template guanine in a defined DNA sequence, thus providing information on the effect of neighboring base sequences on fidelity. Misincorporation frequencies varied no more than 5-fold among 50 template guanines tested. The approach used here is not limited for use with nucleotide analogues but is generally applicable in determining misincorporation frequencies and sequence specificities for any deoxynucleoside triphosphate substrate. In a second experimental approach, base mispairing between bromouracil and guanine was demonstrated directly by using 5-bromodideoxyuridine 5'-triphosphate (BrddUTP). A comparison of chain terminations attributable to BrddUTP and to dideoxythymidine 5'-triphosphate (ddTTP) revealed that B X A and T X A base pairs formed at about the same rate, whereas B X G mispairs occurred 4-10 times more frequently than T X G. The elevation in the frequency of B X G over T X G mispairs is consistent with the mutagenic behavior of the base analogue.

The biochemical basis of 5-bromouracil-induced mutagenesis. Heteroduplex base mispairs involving bromouracil in G x C----A x T and A x T----G x C mutational pathways.

We have investigated the mechanism of bromouracil-induced transition mutations in vitro using synthetic DNA templates and purified T4 DNA polymerase. Evidence is presented for the occurrence of bromouracil-guanine base pairs in product DNA in the G x C----A x T pathway where guanine is present in the DNA template and bromouracil is present as the deoxynucleoside triphosphate substrate 5-bromodeoxyuridine triphosphate. This finding supports a widely known but as yet untested model proposed by Freese (Freese, E. (1959) J. Mol. Biol. 1, 87-105) in which bromouracil-guanine base pairs are intermediates in 5-bromodeoxyuridine-induced transition mutation pathways. We find that the newly formed B x G base pairs are proofread with an efficiency of 75-85% by the 3' -exonuclease of T4 polymerase. The insertion of bromouracil occurring in direct competition with cytosine deoxyribonucleotides opposite template guanine sites is 1.1 +/- 0.14% (mean +/- S.E.), and the misincorporation ratio, inc(B)/inc(C), is reduced 6-fold by the action of the proofreading exonuclease to 0.16 +/- 0.02% (mean +/- S.E.). A previous study by Trautner et al. (Trautner, T. A., Swartz, M. N., and Kornberg, A. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 449-455) suggested that, while template bromouracil stimulates incorporation of dGMP in the A x T----G x C transition mutation pathway, it may not be occurring exclusively by the pathway proposed by Freese. We concur with these earlier results, and, in addition, we find the surprising result that the 3'-exonuclease activity of wild-type T4 polymerase removes little or no incorporated dGMP on bromouracil-containing templates.