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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.

There have been many reports that a metastable form of ice can exist in the atmosphere and that it transitions rapidly to stable, hexagonal ice at temperatures above about 200 K. Although this often-called cubic ice has also been created in laboratories over the years, we present here a method for the simple formation of this metastable phase in the laboratory, at one atmosphere, in relatively large volumes and at higher temperatures than previously reported. Evidence for this phase is found during the monitoring of optical transmission through bulk samples of quenched aqueous solutions. In our experiments, frozen samples were created by quenching 0.2 ml aqueous volumes in glass tubes to 195 K which are then warmed to and held at 267 K. Results show an unusual drop in optical transmission occurring in the first few minutes. Such a change is best explained by the transition of a metastable phase to hexagonal ice, rather than by any freeze concentration effects. In the minutes following nucleation and freezing of the sample, the average size of the poly-crystals forming the frozen matrix would be typically expected to increase due to recrystallization, causing lower side and back-scatter of the traversing light and so a subsequent increased optical transmission. However, the drop in transmission we see with samples nucleated at such a low temperature cannot be explained by recrystallisation but rather by a re-ordering of the ice, the grain boundaries, and the interstitial water.

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.

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.

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., Summary: Functional genomics was used to investigate trehalose synthesis genes after cold acclimation in Panagrolaimus sp. DAW1, an Antarctic nematode with the ability to survive intracellular freezing.

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.

The Antarctic nematodePanagrolaimus davidican survive intracellular freezing. Genetic studies indicate the culture strain (now designated asPanagrolaimussp. DAW1) is a different species to theP. davidiof field origin. This paper reports further attempts both to isolate DAW1 from Antarctic soils and to cultureP. davidiitself. Sequencing of the 18S rRNA gene of 151 individuals indicates that DAW1 is rare in the field, but characterising two new isolates shows that, nevertheless, it is present.Panagrolaimus davidiis common in the field, but cannot be cultured using the media tested here. These two species are difficult to distinguish morphologically, apart from the absence of males in DAW1 and its longer recurved tail. Whilst it is possible that DAW1 is an introduced species, the sites at Shackleton’s hut at Cape Royds are dominated byP. davidi, which is clearly an endemic species.

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.

Characterization of the major proteins in the crystalline styles of Perna canaliculus and Mytilus galloprovincialis was carried out to attempt to identify the molecular basis of differences in the physical structure of the styles and their biochemical significance. The bivalve P. canaliculus has a crystalline style that is typical of those with permanent hard-type styles, whereas the style of M. galloprovincialis is typical of bivalves with transient soft-type styles. The consistency of the styles is clearly related to their respective moisture content, and both style types contain a suite of high-molecular weight (>800 kDa and ∼95–170 kDa) glycolproteins and medium-molecular weight (40–50 kDa) protein duplexes. Total carbohydrates (as determined by the phenol-sulfuric acid method) make up a small proportion of the mass of the styles. It is suggested that these 40–50 kDa proteins play a role in the formation of the protein gel that establishes the structural integrity of the style. Their solubili...

Assays of chitinase, cellulase, and amylase activities in the crystalline styles of selected fast- and slow-growing families of the mussel Perna canaliculus were carried out on 2 occasions over 6 mo, during which time the shellfish grew in a normal mussel cultivation situation. The data did not support the hypothesis that differences in growth rate by these families could be related to differences in the expression of digestive enzymes in the styles, leading to improved nutritional efficiency by faster growing families. On the contrary, the styles of faster growing families in general exhibited lower rates of enzyme activity. Differences in enzyme activities between families appeared to be related to the size of the individuals rather than their genetic heritage. The crystalline styles of larger, more mature shellfish contained less moisture and less soluble protein, and the lower rates of enzyme activity in larger individuals may be indicative of a reallocation of style protein synthesis toward higher levels of insoluble structural proteins.

Morphological and genetic information was used to determine the species identity and relationships of four species of nematodes from the Victoria Land coast, Antarctica; and between laboratory and field populations ofPanagrolaimus davidiTimm. The 18S and D3 expansion segments of 28S rRNA were successfully amplified from the four species:Scottnema lindsayaeTimm,Plectus murrayiYeates,Eudorylaimusspp. andPanagrolaimus davidi. The four species from field populations mapped to three different nematode clades, indicating that if they are survivors in, rather than colonizers of, Antarctica they originate from several Gondwanan ancestors. The laboratory culture ofP. davidiis shown to be a different species to that of the field samples ofP. davidi, which were genetically identical to each other. There are several possible explanations for this observation, which require further investigation.

The distribution of nematodes at Cape Hallett and Gondwana station was found to be patchy but consistent, broad-scale habitat preferences were identified. These had been previously noted in McMurdo Dry Valleys soils and on Ross Island, indicating that habitat preferences are consistent across large scales and may play a role in determining range limits. Soil geochemistry, especially salinity, has a strong impact on distribution patterns. The distinct distribution patterns of the four species indicate different niches.Panagrolaimus davidiTimm is the only nematode that can survive within penguin rookeries, where salinity is high but bacterial food is plentiful.Scottnema lindsayaeTimm was found across the greatest range of habitats, including the driest sites and largest salinity range.Plectussp. was found in the wettest sites, with high organic content and low salinity.Eudorylaimus antarcticusSteiner (Yeates) was found at low densities and with other nematodes indicating a dependence on prey availability, an example of a biotic interaction structuring even these simplest ecosystems. Field-identifiable features could be used to identify probable nematode habitats, providing useful information for sampling and the selection of conservation areas.

An enzyme capable of hydrolysing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the Greenshell™ mussel Perna canaliculus was isolated and characterized. The enzyme was purified by sequential polyethylene glycol fractionation, anion exchange, hydrophobic interaction, gel filtration and hydroxyapatite chromatography. The enzyme was an acidic (pI ∼ 4.8), monomeric, 67 kDa, serine esterase with optimum activity at pH 8.0 and 25 °C. PTX2 and PTX1 were hydrolysed but the enzyme was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolysed short and medium chain length (C2-C10) 4-nitrophenyl-esters, okadaic acid C8-C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). MALDI-Tof MS/MS analysis showed that the enzyme had some homology with a juvenile hormone esterase from the Red Flour Beetle Tribolium castaneum, although BLAST searches of several data bases using de novo amino acid sequences failed to identify any homology with known proteins.

This is the first study to examine the tolerance of Antarctic springtails (Collembola) to ultraviolet radiation (UV). Survival of extended attenuated exposure to sunlight was examined for both individuals and aggregations of the speciesGomphiocephalus hodgsoniCarpenter over a 10 day period. Both individuals and aggregations demonstrated significantly higher survival and moult rates from control treatments kept in the dark to those exposed to UV. A photo-inhibitive element to moulting is indicated that may function to protect post-ecdysial springtails when their emergent cuticles are more sensitive to the external environment. DNA damage was measured in springtails directly exposed to sunlight for 5 h on a clear sunny day. Significant differences were found between treated animals and controls kept in the dark. There was some reduction of damage 12 and 24 h after exposure, when springtails had been placed in the dark to recover. This indicates the up-regulation of DNA repair mechanisms, with the 12 h treatment in particular showing no significant difference with controls. In addition to providing a first look at UV tolerance in these soil arthropods, these findings recommend employing strict protocols for collections of sample material for subsequent biological analysis in order to minimize the interactive effects of photo-damage.

Antarctic springtails are exemplars of extreme low temperature adaptation in terrestrial arthropods. This paper represents the first examination of such adaptation in the springtail, Gressittacantha terranova. Acclimatization state was measured in field-fresh samples over a 22-day period at the beginning of the austral summer. No evidence of temperature tracking was observed. Mean temperature of crystallization (T c) for all samples was −20.67 ± 0.32°C and the lowest T c recorded was −32.62°C. Ice affinity purification was used to collect antifreeze proteins (AFPs) from springtail homogenate. The purified ice fraction demonstrated both thermal hysteresis activity and recrystallisation inhibition. Growth-melt observations revealed that ice crystals grow normal to the c-axis (basal plane). Reverse-phased HPLC produce one clearly resolved peak (P1) and one compound peak (P2). Mass spectrometry identified the molecular mass of P1 as 8,599 Da. The P1 protein was also the most prominent in P2, although additional peptides of 6–7 KDa were also prominent. The main AFP of the Antarctic springtail, G. terranova has been isolated, although like other AFP-expressing arthropods, it shows evidence of expressing a family of AFPs.

Gene expression of the DNA repair enzyme, photolyase (E.C. 4.1.99.3) was examined in the gonads, eggs, embryos and larval stages of the Antarctic sea urchin, Sterechinus neumayeri. Partial sequencing of the gene revealed two highly conserved regions, including a 300 bp region representing the binding site for the cofactor flavin adenine dinucleotide. The second 1200 bp region, likely representing a second light-harvesting cofactor binding site, was identified in a second sea urchin species, Strongylocentrotus frascicanus. Probes for photolyase were developed from the shorter sequence, and expression in sea urchin developmental tissue and stages, and in response to in situ exposure to ultraviolet radiation was quantified using PCR and RT-qPCR, with concentrations of photolyase normalized to actin concentrations. Photolyase was expressed in all tissues and developmental stages examined. In controlled field-based experiments in McMurdo Sound, Antarctica, we found evidence of both constitutive expression of photolyase and induction in response to in situ exposure of embryos to UV-R. Induction of photolyase was observed in response to greater ambient UV-R (such as shallower water depths or sea ice-free regions).

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.

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.

The non-Antarctic Notothenioidei families, Bovichtidae, Pseudaphritidae and Eleginopsidae, diverged early from the main notothenioid lineage. They are important in clarifying the early evolutionary processes that triggered notothenioid evolution in the Antarctic. The early-diverged group represents 8% of all notothenioid species and never established themselves on the Antarctic shelf. Most attention has been paid to the Antarctic notothenioids and their limited physiological tolerance to climate change and increased temperatures. In this review, we discuss key life history traits that are characteristic of the non-Antarctic early-diverged notothenioid taxa as well as the genetic resources and population differentiation information available for this group. We emphasise the population fitness and dynamics of these species and indicate how resource management and conservation of the group can be strengthened through an integrative approach. Both Antarctic waters and the non-Antarctic regions face rapid temperature rises combined with strong anthropogenic exploitation. While it is expected that early-diverged notothenioid species may have physiological advantages over high Antarctic species, it is difficult to predict how climate changes might alter the geographic range, behaviour, phenology and ultimately genetic variability of these species. It is possible, however, that their high degree of endemism and dependence on local environmental specificities to complete their life cycles might enhance their vulnerability.

Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5 degrees C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at -1 degrees C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at -4 degrees C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).

SUMMARY The relative importance of freezing tolerance and cryoprotective dehydration in the Antarctic nematode Panagrolaimus davidi has been investigated. If nucleation of the medium is initiated at a high subzero temperature (-1°C), the nematodes do not freeze but dehydrate. This effect occurs in deionised water, indicating that the loss of water is driven by the difference in vapour pressure of ice and supercooled water at the same temperature. If the nematodes are held above their nucleation temperature for a sufficient time, or are cooled slowly, enough water is lost to prevent freezing (cryoprotective dehydration). However, if the medium is nucleated at lower temperatures or if the sample is cooled at a faster cooling rate, the nematodes freeze and can survive intracellular ice formation. P. davidi thus has a variety of mechanisms that ensure its survival in its harsh terrestrial Antarctic habitat.

As part of our studies to examine the molecular basis of cold-adaptation, we have determined the kinetic properties, thermal stability and deduced amino acid sequence of the enzyme lactate dehydrogenase (LDH) from an Antarctic zoarcid fish, Lycodichthys dearborni. Unlike Antarctic notothenioid fish which are endemic to the Southern Ocean, zoarcid fish are cosmopolitan and have a substantially longer evolutionary history as a sub-order. The A4-LDH isoform was isolated and purified from the white muscle of L. dearborni. The kinetic parameters K m PYR and k cat were determined at temperatures from 0 to 25°C. K m PYR was substantially higher at low temperatures than those from Antarctic and temperate notothenioid fish, whereas k cat at these temperatures was essentially the same as those of the other fish LDH in this study. The sequence of L. dearborni A4-LDH was determined from cDNA derived from white muscle RNA and found to be similar to, but distinct from, the A4-LDH sequences of Antarctic notothenioid fish. Molecular modelling based on the structure of the A4-LDH from Pagothenia borchgrevinki suggested that three conservative amino acid changes within the core of the protein that are not directly part of the active site but which might nonetheless influence the active site, may be important in cold-adaptation in L. dearborni A4-LDH, and that several other changes on the surface of the protein might also play a role in cold-adaptation.

Three proteins have been identified in the milk of the common brush tail possum. Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include beta-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas beta-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100-110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpecula beta-lactoglobulin, along with two other macropod beta-lactoglobulins, forms a subclass of beta-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents.

Isolated and established in culture from the Antarctic in 1988, the nematode Panagrolaimus davidi has proven to be an ideal model for the study of adaptation to the cold. Not only is it the best-documented example of an organism surviving intracellular freezing but it is also able to undergo cryoprotective dehydration. As part of an ongoing effort to develop a molecular understanding of this remarkable organism, we have assembled both a transcriptome and a set of genomic scaffolds. We provide an overview of the transcriptome and a survey of genes involved in temperature stress. We also explore, in silico, the possibility that P. davidi will be susceptible to an environmental RNAi response, important for further functional studies.

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family.

A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence.

With the transition of Antarctica from seasonally cold to a permanently frozen state came a very significant transition in the surrounding waters. During the seasonal cold period the formation of sea ice and reduction of water temperature created a set of problems for marine organisms. Seasonal changes that could previously be avoided by behavioural adaptations became permanent and had a profound effect on the marine fauna and flora.

Population genetics patterns of marine fish in general and of Southern Ocean fish in particular range from virtual panmixia over ocean-wide scale to deeply fragmented populations. However the causes underlying these different patterns are not properly understood. In this paper, we tested the hypotheses that population connectivity is positively related to a combination of life history traits, namely duration of pelagic larval period and the tendency towards pelagic life style in the adulthood. To do so, we analysed the variability of six microsatellite and one mitochondrial marker (cytochrome b) in three Southern Ocean fish species (Trematomus newnesi, Trematomus hansoni and Trematomus bernacchii). They share a recent common ancestor but notably differ in their duration of pelagic larval period as well as pelagic versus benthic lifestyle. We sampled over a range of more than 5000 km for all three species and used a number of population genetics tools to investigate past and contemporary levels of connectivity. All species experienced population fluctuations, but coalescent simulations suggested that contemporary populations are in migration-drift equilibrium. Although global FST values were rather low, a significant population structure separated the High-Antarctic from the Peninsular regions in all species. The level of genetic differentiation was much lower in the pelagic versus benthic species. Present data suggest that past and present genetic structuring in the Southern Ocean are indeed related with the ecological traits of Antarctic fish, however the relative importance of individual factors remains unclear.

A major secreted aspartic proteinase from Candida albicans has been crystallized in the presence of inhibitors to prevent autodegradation. With pepstatin a cleaved form of the enzyme was nevertheless found in the crystals whereas with the inhibitor A70450 the enzyme remained intact. The crystals containing pepstatin were not suitable for X-ray data collection while the crystals containing A70450 grew by vapour diffusion as tetragonal bipyramids, space group P4(3)2(1)2 (or P4(1)2(1)2), a = b = 76.2 A, c = 126.1 A, with one molecule in the asymmetric unit and they diffract to beyond 2.2 A.

The serpins are a widely distributed group of serine proteinase inhibitors found in plants, birds, mammals and viruses. Despite the great evolutionary divergence of these organisms, their serpins art highly conserved, both in sequence and structurally. Amino acid sequences were aligned by a combination of automatic algorithms and by consideration of conserved structural elements in those serpins for which crystal structures exist. The program HOMED was used which allowed the alignment of amino acids to be simultaneously converted into the equivalently aligned nucleotide sequences. The aligned amino acids were used as the basis for superposition of the four known three-dimensional structures for which coordinates are available and compared with an optimal three-dimensional superposition in order to estimate the reliability of the sequence alignment. Phylogenetic relationships implied by these nucleotide sequence alignments were determined by the method of maximum parsimony. The proposed gene tree suggested that as much diversity existed between the plant serpin and mammalian serpins as was present among mammalian serpins and provided further evidence that the architecture of serpin molecules is highly constrained.