A shared core microbiome in soda lakes separated by large distances.

In alkaline soda lakes, concentrated dissolved carbonates establish productive phototrophic microbial mats. Here we show how microbial phototrophs and autotrophs contribute to this exceptional productivity. Amplicon and shotgun DNA sequencing data of microbial mats from four Canadian soda lakes indicate the presence of > 2,000 species of Bacteria and Eukaryotes. We recover metagenome-assembled-genomes for a core microbiome of < 100 abundant bacteria, present in all four lakes. Most of these are related to microbes previously detected in sediments of Asian alkaline lakes, showing that common selection principles drive community assembly from a globally distributed reservoir of alkaliphile biodiversity. Detection of > 7,000 proteins show how phototrophic populations allocate resources to specific processes and occupy complementary niches. Carbon fixation proceeds by the Calvin-Benson-Bassham cycle, in Cyanobacteria, Gammaproteobacteria, and, surprisingly, Gemmatimonadetes. Our study provides insight into soda lake ecology, as well as a template to guide efforts to engineer biotechnology for carbon dioxide conversion.

An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping.

Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Attachment of nucleosides and other linkers to solid-phase supports for oligonucleotide synthesis.

Specific step-by-step instructions for conversion of 5'-O-(4,4'-dimethoxytrityl)- and base-protected nucleosides and other mono-O-(4,4'-dimethoxytrityl)-protected diols to their hemisuccinate esters and their coupling to CPG (controlled-pore glass) supports bearing aminopropyl or long chain aminoalkyl groups are presented. Additional guidelines are provided for selecting a coupling protocol and performing in-process control.

Nucleoside phosphoramidites containing cleavable linkers.

Phosphoramidite reagents (linker phosphoramidites) containing a cleavable 3'-ester linkage between the nucleoside and the phosphoramidite group can be used to attach the first nucleoside to a solid-phase support. Inexpensive underivatized supports such as LCAA-CPG can then be used as universal supports for oligonucleotide synthesis. No modifications to synthesis coupling conditions and no 3'-dephosphorylation are required. Only oligonucleotides with terminal 3'-OH ends are produced. Phosphoramidites containing both a succinate and a sulfonyldiethanol linkage are particularly useful and create oligonucleotides with both a 3'-OH and 5'-phosphate. In addition, by using these reagents, one oligonucleotide sequence can be added onto the 5'-end of another (tandem synthesis) to produce a string of multiple oligonucleotides linked end-to-end. Deprotection releases the oligonucleotides from each other to yield a mixture of oligonucleotides. This approach is particularly useful for making pairs of PCR primers or both strands of a double-stranded sequence in a single operation.

Tandem oligonucleotide synthesis using linker phosphoramidites.

Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623-631] is added to the 5'-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3'-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5'- and 3'-terminal OH ends and other oligonucleotides with 5'-phosphorylated and 3'-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

Solution structure of a DNA duplex containing an alpha-anomeric adenosine: insights into substrate recognition by endonuclease IV.

The cytotoxic alpha anomer of adenosine, generated in situ by radicals, must be recognized and repaired to maintain genomic stability. Endonuclease IV (Endo IV), a member of the base excision repair (BER) enzyme family, in addition to acting on abasic sites, has the auxiliary function of removing this mutagenic nucleotide in Escherichia coli. We have employed enzymatic, thermodynamic, and structural studies on DNA duplexes containing a central alpha-anomeric adenosine residue to characterize the role of DNA structure on recognition and catalysis by Endo IV. The enzyme recognizes and cleaves our alphaA-containing DNA duplexes at the site of the modification. The NMR solution structure of the DNA decamer duplex establishes that the single alpha-anomeric adenosine residue is intrahelical and stacks in a reverse Watson-Crick fashion consistent with the slight decrease in thermostability. However, the presence of this lesion confers significant changes to the global duplex conformation, resulting from a kink of the helical axis into the major groove and an opening of the minor groove emanating from the alpha-anomeric site. Interestingly, the conformation of the flanking base-paired segments is not greatly altered from a B-type conformation. The global structural changes caused by this lesion place the DNA along the conformational path leading to the DNA structure observed in the complex. Thus, it appears that the alpha-anomeric lesion facilitates recognition by Endo IV.

Linker phosphoramidite reagents for the attachment of the first nucleoside to underivatized solid-phase supports.

New linker phosphoramidite reagents containing a cleavable 3'-ester linkage are used for attaching the first nucleoside to the surface of a solid- phase support. Inexpensive, underivatized amino supports, such as long chain alkylamine controlled-pore glass, can serve as universal supports. No modifications to phosphoramidite coupling conditions are required and, after synthesis, treatment with NH(4)OH releases the products with 3'-OH ends. No 3'-dephosphorylation is required. Phosphoramidite reagents containing a succinate and sulfonyl diethanol linkage between the nucleoside and phosphoramidite group are particularly advantageous and can be used to create both 3'-OH and 5'-phosphate ends on oligonucleotides. Reproducibility and quality of oligonucleotide synthesis is demonstrated for either column and 96-well plate formats on low-, medium- or high-loading CPG supports.

Structural basis for topoisomerase I inhibition by nucleoside analogs.

Nucleoside analogs such as 1-beta-D-arabinofuranosyl cytidine (AraC) and 2',2'-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2'-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3' endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), an antimetabolite that poisons topoisomerase I.

PURPOSE: Gemcitabine-containing regimens are among standard therapies for the treatment of advanced non-small cell lung,pancreatic, or bladder cancers. Gemcitabine is a nucleoside analogue and its cytotoxicity is correlated with incorporation into genomic DNA and concomitant inhibition of DNA synthesis. However, it is still unclear by which mechanism(s) gemcitabine incorporation leads to cell death. EXPERIMENTAL DESIGN: We used purified oligodeoxynucleotides to study the effects of gemcitabine incorporation on topoisomerase I (top1) activity and tested the role of top1 poisoning in gemcitabine-induced cytotoxicity in cancer cells. RESULTS: We found that top1-mediated DNA cleavage was enhanced when gemcitabine was incorporated immediately 3' from a top1 cleavage site on the nonscissile strand. This position-specific enhancement was attributable to an increased DNA cleavage by top1 and was likely to have resulted from a combination of gemcitabine-induced conformational and electrostatic effects. Gemcitabine also enhanced camptothecin-induced cleavage complexes. We also detected top1 cleavage complexes in human leukemia CEM cells treated with gemcitabine and a 5-fold resistance of P388/CPT45 top1-deficient cells to gemcitabine, indicating that poisoning of top1 can contribute to the antitumor activity of gemcitabine. CONCLUSIONS: The present results extend our recent finding that incorporation of 1-beta-D-arabinofuranosylcytosine into DNA can induce top1 cleavage complexes [P. Pourquier et al. Proc. Natl. Acad. Sci. USA, 97: 1885-1890, 2000]. The enhancement of camptothecin-induced top1 cleavage complexes may, at least in part, contribute to the synergistic or additive effects of gemcitabine in combination with topotecan and irinotecan in human breast or lung cancer cells.

Tandem oligonucleotide synthesis on solid-phase supports for the production of multiple oligonucleotides.

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5'-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3'-O-hydroquinone-O,O'-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5'-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3'- or 5'-end of oligonucleotides. A single NH(4)OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5'- and 3'-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production.

Tandem oligonucleotide synthesis using linker (First Base) phosphoramidites.

First Base linker phosphoramidites add the first nucleoside to underivatized solid-phase supports using a regular phosphoramidite coupling cycle. A cleavable 3'-O-ester linkage allows NH4OH treatment to rapidly generate products with only 3'-OH ends. Either single or multiple sequences linked in tandem can be prepared and no additional deprotection steps are required.