Greater than pH 8: The pH dependence of EDTA as a preservative of high molecular weight DNA in biological samples.

Ethylenediaminetetraacetic acid (EDTA) is a divalent cation chelator and chemical preservative that has been shown to be the active ingredient of the popular DNA preservative DESS. EDTA may act to reduce DNA degradation during tissue storage by sequestering divalent cations that are required by nucleases naturally occurring in animal tissues. Although EDTA is typically used between pH 7.5 and 8 in preservative preparations, the capacity of EDTA to chelate divalent cations is known to increase with increasing pH. Therefore, increasing the pH of EDTA-containing preservative solutions may improve their effectiveness as DNA preservatives. To test this hypothesis, we stored tissues from five aquatic species in 0.25 M EDTA adjusted to pH 8, 9, and 10 for 12 months at room temperature before DNA isolation. For comparison, tissues from the same specimens were also stored in 95% ethanol. DNA extractions performed on tissues preserved in EDTA pH 9 or 10 resulted in as great or greater percent recovery of high molecular weight DNA than did extractions from tissues stored at pH 8. In all cases examined, percent recovery of high molecular weight DNA from tissues preserved in EDTA pH 10 was significantly better than that observed from tissues preserved in 95% ethanol. Our results support the conclusion that EDTA contributes to DNA preservation in tissues by chelating divalent cations and suggest that preservative performance can be improved by increasing the pH of EDTA-containing DNA preservative solutions.

DESS deconstructed: Is EDTA solely responsible for protection of high molecular weight DNA in this common tissue preservative?

DESS is a formulation widely used to preserve DNA in biological tissue samples. Although it contains three ingredients, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA) and sodium chloride (NaCl), it is frequently referred to as a DMSO-based preservative. The effectiveness of DESS has been confirmed for a variety of taxa and tissues, however, to our knowledge, the contributions of each component of DESS to DNA preservation have not been evaluated. To address this question, we stored tissues of three aquatic taxa, Mytilus edulis (blue mussel), Faxonius virilis (virile crayfish) and Alitta virens (clam worm) in DESS, each component of DESS individually and solutions containing all combinations of two components of DESS. After storage at room temperature for intervals ranging from one day to six months, we extracted DNA from each tissue and measured the percentage of high molecular weight (HMW) DNA recovered (%R) and normalized HMW DNA yield (nY). Here, HMW DNA is defined as fragments >10 kb. For comparison, we also measured the %R and nY of HMW DNA from extracts of fresh tissues and those stored in 95% EtOH over the same time intervals. We found that in cases where DESS performed most effectively (yielding ≥ 20%R of HMW DNA), all solutions containing EDTA were as or more effective than DESS. Conversely, in cases where DESS performed more poorly, none of the six DESS-variant storage solutions provided better protection of HMW DNA than DESS. Moreover, for all taxa and storage intervals longer than one day, tissues stored in solutions containing DMSO alone, NaCl alone or DMSO and NaCl in combination resulted in %R and nY of HMW DNA significantly lower than those of fresh tissues. These results indicate that for the taxa, solutions and time intervals examined, only EDTA contributed directly to preservation of high molecular weight DNA.

Development of smart anti-glycan reagents using immunized lampreys.

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. We identified a wide variety of glycan-specific VLRBs detectable in lamprey plasma after immunization with whole fixed cells, tissue homogenates, and human milk. The cDNAs from lamprey lymphocytes were cloned into yeast surface display (YSD) libraries for enrichment by multiple methods. We generated VLRB-Ig chimeras, termed smart anti-glycan reagents (SAGRs), whose specificities were defined by microarray analysis and immunohistochemistry. 15 VLRB antibodies were discovered that discriminated between linkages, functional groups and unique presentations of the terminal glycan motif. The development of SAGRs will enhance future studies on glycan expression by providing sequenced, defined antibodies for a variety of research applications.

Generation of a Xenopus laevis F1 albino J strain by genome editing and oocyte host-transfer.

Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs.

Luteinizing Hormone is an effective replacement for hCG to induce ovulation in Xenopus.

Injection of human Chorionic Gonadotropin (hCG) directly into the dorsal lymph sac of Xenopus is a commonly used protocol for induction of ovulation, but recent shortages in the stocks of commercially available hCG as well as lack of a well tested alternative have resulted in frustrating experimental delays in laboratories that predominantly use Xenopus in their research. Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as hCG; this suggests that LH may serve as a good alternative to hCG for promoting ovulation in Xenopus. LH has been found to induce maturation of Xenopus oocytes in vitro, but whether it can be used to induce ovulation in vivo has not been examined. Here we compared the ability of four mammalian LH proteins, bovine (bLH), human (hLH), ovine (oLH), porcine (pLH), to induce ovulation in Xenopus when injected into the dorsal lymph sac of sexually mature females. We find that both ovine and human LH, but not bovine or porcine, are good substitutes for hCG for induction of ovulation in WT and J strain Xenopus laevis and Xenopus tropicalis.

Niche divergence by deep-sea octocorals in the genus Callogorgia across the continental slope of the Gulf of Mexico.

Environmental variables that are correlated with depth have been suggested to be among the major forces underlying speciation in the deep sea. This study incorporated phylogenetics and ecological niche models (ENM) to examine whether congeneric species of Callogorgia (Octocorallia: Primnoidae) occupy different ecological niches across the continental slope of the Gulf of Mexico (GoM) and whether this niche divergence could be important in the evolution of these closely related species. Callogorgia americana americana, Callogorgia americana delta and Callogorgia gracilis were documented at 13 sites in the GoM (250-1000 m) from specimen collections and extensive video observations. On a first order, these species were separated by depth, with C. gracilis occurring at the shallowest sites, C. a. americana at mid-depths and C. a. delta at the deepest sites. Callogorgia a. delta was associated with areas of increased seep activity, whereas C. gracilis and C. a. americana were associated with narrow, yet warmer, temperature ranges and did not occur near cold seeps. ENM background and identity tests revealed little to no overlap in ecological niches between species. Temporal calibration of the phylogeny revealed the formation of the Isthmus of Panama was a vicariance event that may explain some of the patterns of speciation within this genus. These results elucidate the potential mechanisms for speciation in the deep sea, emphasizing both bathymetric speciation and vicariance events in the evolution of a genus across multiple regions.