A lack of genetic diversity and minimal adaptive evolutionary divergence in introduced Mysis shrimp after 50 years.

The successes of introduced populations in novel habitats often provide powerful examples of evolution and adaptation. In the 1950s, opossum shrimp (Mysis diluviana) individuals from Clearwater Lake in Minnesota, USA were transported and introduced to Twin Lakes in Colorado, USA by fisheries managers to supplement food sources for trout. Mysis were subsequently introduced from Twin Lakes into numerous lakes throughout Colorado. Because managers kept detailed records of the timing of the introductions, we had the opportunity to test for evolutionary divergence within a known time interval. Here, we used reduced representation genomic data to investigate patterns of genetic diversity, test for genetic divergence between populations, and for evidence of adaptive evolution within the introduced populations in Colorado. We found very low levels of genetic diversity across all populations, with evidence for some genetic divergence between the Minnesota source population and the introduced populations in Colorado. There was little differentiation among the Colorado populations, consistent with the known provenance of a single founding population, with the exception of the population from Gross Reservoir, Colorado. Demographic modeling suggests that at least one undocumented introduction from an unknown source population hybridized with the population in Gross Reservoir. Despite the overall low genetic diversity we observed, F ST outlier and environmental association analyses identified multiple loci exhibiting signatures of selection and adaptive variation related to elevation and lake depth. The success of introduced species is thought to be limited by genetic variation, but our results imply that populations with limited genetic variation can become established in a wide range of novel environments. From an applied perspective, the observed patterns of divergence between populations suggest that genetic analysis can be a useful forensic tool to determine likely sources of invasive species.

Structural basis of archaeal FttA-dependent transcription termination.

The ribonuclease FttA mediates factor-dependent transcription termination in archaea 1-3 . Here, we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, reveal functional homology between archaeal and eukaryotic factor-dependent termination, and reveal fundamental mechanistic similarities in factor-dependent termination in the three domains of life: bacterial, archaeal, and eukaryotic. One sentence summary: Cryo-EM reveals the structure of the archaeal FttA pre-termination complex.

Histones direct site-specific CRISPR spacer acquisition in model archaeon.

CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.

Insect Freeze-Tolerance Downunder: The Microbial Connection.

Insects that are freeze-tolerant start freezing at high sub-zero temperatures and produce small ice crystals. They do this using ice-nucleating agents that facilitate intercellular ice growth and prevent formation of large crystals where they can damage tissues. In Aotearoa/New Zealand the majority of cold adapted invertebrates studied survive freezing at any time of year, with ice formation beginning in the rich microbiome of the gut. Some freeze-tolerant insects are known to host symbiotic bacteria and/or fungi that produce ice-nucleating agents and we speculate that gut microbes of many New Zealand insects may provide ice-nucleating active compounds that moderate freezing. We consider too the possibility that evolutionary disparate freeze-tolerant insect species share gut microbes that are a source of ice-nucleating agents and so we describe potential transmission pathways of shared gut fauna. Despite more than 30 years of research into the freeze-tolerant mechanisms of Southern Hemisphere insects, the role of exogenous ice-nucleating agents has been neglected. Key traits of three New Zealand freeze-tolerant lineages are considered in light of the supercooling point (temperature of ice crystal formation) of microbial ice-nucleating particles, the initiation site of freezing, and the implications for invertebrate parasites. We outline approaches that could be used to investigate potential sources of ice-nucleating agents in freeze-tolerant insects and the tools employed to study insect microbiomes.

The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.

Small Peptide Derivatives Within the Carbohydrate Recognition Domain of SP-A2 Modulate Asthma Outcomes in Mouse Models and Human Cells.

Surfactant Protein-A (SP-A) is an innate immune modulator that regulates a variety of pulmonary host defense functions. We have shown that SP-A is dysfunctional in asthma, which could be partly due to genetic heterogeneity. In mouse models and primary bronchial epithelial cells from asthmatic participants, we evaluated the functional significance of a particular single nucleotide polymorphism of SP-A2, which results in an amino acid substitution at position 223 from glutamine (Q) to lysine (K) within the carbohydrate recognition domain (CRD). We found that SP-A 223Q humanized mice had greater protection from inflammation and mucin production after IL-13 exposure as compared to SP-A-2 223K mice. Likewise, asthmatic participants with two copies the major 223Q allele demonstrated better lung function and asthma control as compared to asthmatic participants with two copies of the minor SP-A 223K allele. In primary bronchial epithelial cells from asthmatic participants, full-length recombinant SP-A 223Q was more effective at reducing IL-13-induced MUC5AC gene expression compared to SP-A 223K. Given this activity, we developed 10 and 20 amino acid peptides of SP-A2 spanning position 223Q. We show that the SP-A 223Q peptides reduce eosinophilic inflammation, mucin production and airways hyperresponsiveness in a house dust mite model of asthma, protect from lung function decline during an IL-13 challenge model in mice, and decrease IL-13-induced MUC5AC gene expression in primary airway epithelial cells from asthmatic participants. These results suggest that position 223 within the CRD of SP-A2 may modulate several outcomes relevant to asthma, and that short peptides of SP-A2 retain anti-inflammatory properties similar to that of the endogenous protein.

CC16 Deficiency in the Context of Early-Life Mycoplasma pneumoniae Infection Results in Augmented Airway Responses in Adult Mice.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16-/-) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults (early life infection model; ELIM) and compared to adult mice infected for only 3 days (adult infection model; AIM). CC16-/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16-/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16-/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16-/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16-/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Molecular Patterns in Acute Pancreatitis Reflect Generalizable Endotypes of the Host Response to Systemic Injury in Humans.

OBJECTIVE: Acute Pancreatitis (AP) is sudden onset pancreas inflammation that causes systemic injury with a wide and markedly heterogeneous range of clinical consequences. Here, we hypothesized that this observed clinical diversity corresponds to diversity in molecular subtypes that can be identified in clinical and multiomics data. SUMMARY BACKGROUND DATA: Observational cohort study. n = 57 for the discovery cohort (clinical, transcriptomics, proteomics, and metabolomics data) and n = 312 for the validation cohort (clinical and metabolomics data). METHODS: We integrated coincident transcriptomics, proteomics, and metabolomics data at serial time points between admission to hospital and up to 48 hours after recruitment from a cohort of patients presenting with acute pancreatitis. We systematically evaluated 4 different metrics for patient similarity using unbiased mathematical, biological, and clinical measures of internal and external validity.We next compared the AP molecular endotypes with previous descriptions of endotypes in a critically ill population with acute respiratory distress syndrome (ARDS). RESULTS: Our results identify 4 distinct and stable AP molecular endotypes. We validated our findings in a second independent cohort of patients with AP.We observed that 2 endotypes in AP recapitulate disease endotypes previously reported in ARDS. CONCLUSIONS: Our results show that molecular endotypes exist in AP and reflect biological patterns that are also present in ARDS, suggesting that generalizable patterns exist in diverse presentations of critical illness.

A genetically based ecological trade-off contributes to setting a geographic range limit.

Understanding the ecological factors that shape geographic range limits and the evolutionary constraints that prevent populations from adaptively evolving beyond these limits is an unresolved question. Here, we investigated why the euryhaline fish, Poecila reticulata, is confined to freshwater within its native range, despite being tolerant of brackish water. We hypothesised that competitive interactions with a close relative, Poecilia picta, in brackish water prevents P. reticulata from colonising brackish water. Using a combination of field transplant, common garden breeding, and laboratory behaviour experiments, we find support for this hypothesis, as P. reticulata are behaviourally subordinate and have lower survival in brackish water with P. picta. We also found a negative genetic correlation between P. reticulata growth in brackish water versus freshwater in the presence of P. picta, suggesting a genetically based trade-off between salinity tolerance and competitive ability could constrain adaptive evolution at the range limit.

Archaeal DNA Repair Mechanisms.

Archaea often thrive in environmental extremes, enduring levels of heat, pressure, salinity, pH, and radiation that prove intolerable to most life. Many environmental extremes raise the propensity for DNA damaging events and thus, impact DNA stability, placing greater reliance on molecular mechanisms that recognize DNA damage and initiate accurate repair. Archaea can presumably prosper in harsh and DNA-damaging environments in part due to robust DNA repair pathways but surprisingly, no DNA repair pathways unique to Archaea have been described. Here, we review the most recent advances in our understanding of archaeal DNA repair. We summarize DNA damage types and their consequences, their recognition by host enzymes, and how the collective activities of many DNA repair pathways maintain archaeal genomic integrity.

The Role of Archaeal Chromatin in Transcription.

Genomic organization impacts accessibility and movement of information processing systems along DNA. DNA-bound proteins dynamically dictate gene expression and provide regulatory potential to tune transcription rates to match ever-changing environmental conditions. Archaeal genomes are typically small, circular, gene dense, and organized either by histone proteins that are homologous to their eukaryotic counterparts, or small basic proteins that function analogously to bacterial nucleoid proteins. We review here how archaeal genomes are organized and how such organization impacts archaeal gene expression, focusing on conserved DNA-binding proteins within the clade and the factors that are known to impact transcription initiation and elongation within protein-bound genomes.

TFS and Spt4/5 accelerate transcription through archaeal histone-based chromatin.

RNA polymerase must surmount translocation barriers for continued transcription. In Eukarya and most Archaea, DNA-bound histone proteins represent the most common and troublesome barrier to transcription elongation. Eukaryotes encode a plethora of chromatin-remodeling complexes, histone-modification enzymes and transcription elongation factors to aid transcription through nucleosomes, while archaea seemingly lack machinery to remodel/modify histone-based chromatin and thus must rely on elongation factors to accelerate transcription through chromatin-barriers. TFS (TFIIS in Eukarya) and the Spt4-Spt5 complex are universally encoded in archaeal genomes, and here we demonstrate that both elongation factors, via different mechanisms, can accelerate transcription through archaeal histone-based chromatin. Histone proteins in Thermococcus kodakarensis are sufficiently abundant to completely wrap all genomic DNA, resulting in a consistent protein barrier to transcription elongation. TFS-enhanced cleavage of RNAs in backtracked transcription complexes reactivates stalled RNAPs and dramatically accelerates transcription through histone-barriers, while Spt4-Spt5 changes to clamp-domain dynamics play a lesser-role in stabilizing transcription. Repeated attempts to delete TFS, Spt4 and Spt5 from the T. kodakarensis genome were not successful, and the essentiality of both conserved transcription elongation factors suggests that both conserved elongation factors play important roles in transcription regulation in vivo, including mechanisms to accelerate transcription through downstream protein barriers.

A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10-4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.

In Niemann-Pick C1 mouse models, glial-only expression of the normal gene extends survival much further than do changes in genetic background or treatment with hydroxypropyl-beta-cyclodextrin.

The Npc1nmf164 allele of Npc1 provides a mouse model for Niemann-Pick disease type C1 (NPC1), a genetic disease known to have a widely variable phenotype. The transfer of the Npc1nmf164 mutation from the C57BL/6J inbred strain to the BALB/cJ inbred strain increased the mean lifespan from 117.8days to 153.1days, confirming that the severity of the NPC1 phenotype is strongly influenced by genetic background. The transfer of another Npc1 allele, Npc1nih, to this background also extended survival of the homozygotes indicating that the modifying effect of BALB/cJ is not limited to a single allele of Npc1. The increased longevity due to the BALB/cJ background did not map to a previously mapped modifier on chromosome 19, indicating the presence of additional genes impacting disease severity. The previously studied Glial Fibrillary Acidic Protein promoter-Npc1 cDNA transgene (GFAP-Npc1) which only expresses NPC1 in astrocytes further extended the lifespan of Npc1nmf164 homozygotes on a BALB/cJ background (up to 600days). Hydroxypropyl-ß-cyclodextrin (HPßCD) treatment, not previously tested in the Npc1nmf164 mutant, extended life in the Npc1nmf164 homozygotes but not the transgenic, Npc1nmf164 mice on the BALB/cJ background. In all cases, lack of weight gain and early cerebellar symptoms of loss of motor control were found. At termination, the one mouse sacrificed for histological studies showed severe, diffuse pulmonary alveolar proteinosis suggesting that pulmonary abnormalities in NPC1 mouse models are not unique to the Npc1nih allele.

Investigating trehalose synthesis genes after cold acclimation in the Antarctic nematode Panagrolaimus sp. DAW1.

Panagrolaimus sp. DAW1 is a freeze-tolerant Antarctic nematode which survives extensive intracellular ice formation. The molecular mechanisms of this extreme adaptation are still poorly understood. We recently showed that desiccation-enhanced RNA interference (RNAi) soaking can be used in conjunction with quantitative polymerase chain reaction (qPCR) to screen for phenotypes associated with reduced expression of candidate genes in Panagrolaimus sp. DAW1. Here, we present the use of this approach to investigate the role of trehalose synthesis genes in this remarkable organism. Previous studies have shown that acclimating Panagrolaimus sp. DAW1 at 5°C before freezing or desiccation substantially enhances survival. In this study, the expression of tps-2 and other genes associated with trehalose metabolism, as well as lea-1, hsp-70 and gpx-1, in cold-acclimated and non-acclimated nematodes was analyzed using qPCR. Pd-tps-2 and Pd-lea-1 were significantly upregulated after cold acclimation, indicating an inducible expression in the cold adaptation of Panagrolaimus sp. DAW1. The role of trehalose synthesis genes in Panagrolaimus sp. DAW1 was further investigated by RNAi. Compared to the controls, Pd-tps-2a(RNAi)-treated and cold-acclimated nematodes showed a significant decrease in mRNA, but no change in trehalose content or freezing survival. The involvement of two other trehalose synthesis genes (tps-2b and gob-1) was also investigated. These findings provide the first functional genomic investigation of trehalose synthesis genes in the non-model organism Panagrolaimus sp. DAW1. The presence of several trehalose synthesis genes with different RNAi sensitivities suggests the existence of multiple backup systems in Panagrolaimus sp. DAW1, underlining the importance of this sugar in preparation for freezing.

Imaging of Vanadium in Microfossils: A New Potential Biosignature.

The inability to unambiguously distinguish the biogenicity of microfossil-like structures in the ancient rock record is a fundamental predicament facing Archean paleobiologists and astrobiologists. Therefore, novel methods for discriminating biological from nonbiological chemistries of microfossil-like structures are of the utmost importance in the search for evidence of early life on Earth. This, too, is important for the search for life on Mars by in situ analyses via rovers or sample return missions for future analysis here on Earth. Here, we report the application of synchrotron X-ray fluorescence imaging of vanadium, within thermally altered organic-walled microfossils of bona fide biological origin. From our data, we demonstrate that vanadium is present within microfossils of undisputable biological origin. It is well known in the organic geochemistry literature that elements such as vanadium are enriched and contained within crude oils, asphalts, and black shales that have been formed by diagenesis of biological organic material. It has been demonstrated that the origin of vanadium is due to the diagenetic alteration of precursor chlorophyll and heme porphyrin pigment compounds from living organisms. We propose that, taken together, microfossil-like morphology, carbonaceous composition, and the presence of vanadium could be used in tandem as a biosignature to ascertain the biogenicity of putative microfossil-like structures. Key Words: Microfossils-Synchrotron micro-X-ray fluorescence-Vanadium-Tetrapyrrole-Biosignature. Astrobiology 17, 1069-1076.

Molecular snapshot of an intracellular freezing event in an Antarctic nematode.

The Antarctic nematode, Panagrolaimus sp. DAW1 (formerly called Panagrolaimus davidi), is the best documented example of an organism able to survive intracellular ice formation in all of its compartments. Not only is it able to survive such extreme physiological disruption, but it is able to produce progeny once thawed from such a state. In addition, under slower rates, or less extreme degrees, of cooling, its body remains unfrozen and the vapour pressure difference between the supercooled body fluids and the surrounding ice leads to a process termed cryoprotective dehydration. In contrast to a fairly large body of work in building up our molecular understanding of cryoprotective dehydration, no comparable work has been undertaken on intracellular freezing. This paper describes an experiment subjecting cultures of Panagrolaimus sp. DAW1 to a range of temperatures including a rapid descent to -10 °C, in a medium just prior to, and after, freezing. Through deep sequencing of RNA libraries we have gained a snapshot of which genes are highly abundant when P. sp. DAW1 is undergoing an intracellular freezing event. The onset of freezing correlated with a high production of genes involved in cuticle formation and subsequently, after 24 h in a frozen state, protease production. In addition to the mapping of RNA sequencing, we have focused on a select set of genes arising both from the expression profiles, as well as implicated from other cold tolerance studies, to undertake qPCR. Among the most abundantly represented transcripts in the RNA mapping is the zinc-metalloenzyme, neprilysin, which also shows a particularly strong upregulated signal through qPCR once the nematodes have frozen.

Relative efficacy of nicotinamide treatment of a mouse model of infantile Niemann-Pick C1 disease.

Nicotinamide delivered in drinking water at about 2 g/kg/day significantly prolonged survival and showed a suggestive improvement on memory in the Npc1 nih / Npc1 nih mouse model of infantile NPC1 disease. It is likely that this role is due to its function as a histone deacetylase (HDAC) inhibitor although another HDAC inhibitor, valproic acid, was without effect. Nicotinamide could also work by preventing/reversing oxidative stress.

Establishing RNAi in a Non-Model Organism: The Antarctic Nematode Panagrolaimus sp. DAW1.

The Antarctic nematode Panagrolaimus sp. DAW1 is one of the only organisms known to survive extensive intracellular freezing throughout its tissues. Although the physiological mechanisms of this extreme adaptation are partly understood, the molecular mechanisms remain largely unknown. RNAi is a method that allows the examination of gene function in a direct, targeted manner, by knocking out specific mRNAs and revealing the effects on the phenotype. In this study we have explored the viability of RNAi in Panagrolaimus sp. DAW1. In the first trial, nematodes were fed E. coli expressing Panagrolaimus sp. DAW1 dsRNA of the embryonic lethal genes rps-2 and dhc, and the blister gene duox. Pd-rps-2(RNAi)-treated nematodes showed a significant decrease in larval hatching. However, qPCR showed no significant decrease in the amount of rps-2 mRNA in Pd-rps-2(RNAi)-treated animals. Several soaking protocols for dsRNA uptake were investigated using the fluorescent dye FITC. Desiccation-enhanced soaking showed the strongest uptake of FITC and resulted in a significant and consistent decrease of mRNA levels of two of the four tested genes (rps-2 and tps-2a), suggesting effective uptake of dsRNA-containing solution by the nematode. These findings suggest that RNAi by desiccation-enhanced soaking is viable in Panagrolaimus sp. DAW1 and provide the first functional genomic approach to investigate freezing tolerance in this non-model organism. RNAi, in conjunction with qPCR, can be used to screen for candidate genes involved in intracellular freezing tolerance in Panagrolaimus sp. DAW1.

Ice-shell purification of ice-binding proteins.

Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable.