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A 32 kilobase-pair fragment of intron 7 of the human dystrophin gene has been sequenced and analysed for the presence of repetitive elements and open reading frames. Two transposon-like human elements (THE-1 sequences), and three intervening, and related, long terminal repeat elements, together with three Alu sequences and a LINE sequence have been identified. These represent an unexpected clustering of highly-repetitive sequences within this single segment of intron DNA. Amplification of a region of chimpanzee genomic DNA by the polymerase chain reaction has provided evidence that at least one of the THE-1 sequences is present in the same position in the chimpanzee genome and the high homology between the human and chimpanzee sequences indicates that this element was fixed within the ancestral genome before the divergence of the two species. The possible role of repetitive, transposon-like sequences in natural mutagenesis of the dystrophin gene is discussed.

Clades that have undergone episodes of rapid cladogenesis are challenging from a phylogenetic point of view. They are generally characterised by short or missing internal branches in phylogenetic trees and by conflicting topologies among individual gene trees. This may be the case of the subfamily Trematominae, a group of marine teleosts of coastal Antarctic waters, which is considered to have passed through a period of rapid diversification. Despite much phylogenetic attention, the relationships among Trematominae species remain unclear. In contrast to previous studies that were mostly based on concatenated datasets of mitochondrial and/or single nuclear loci, we applied various single-locus and multilocus phylogenetic approaches to sequences from 11 loci (eight nuclear) and we also used several methods to assess the hypothesis of a radiation event in Trematominae evolution. Diversification rate analyses support the hypothesis of a period of rapid diversification during Trematominae history and only a few nodes in the hypothetical species tree were consistently resolved with various phylogenetic methods. We detected significant discrepancies among trees from individual genes of these species, most probably resulting from incomplete lineage sorting, suggesting that concatenation of loci is not the most appropriate way to investigate Trematominae species interrelationships. These data also provide information about the possible effects of historic climate changes on the diversification rate of this group of fish.

Alignment of the heparin-activated serpins indicates the presence of two binding sites for heparin: a small high-affinity site on the D-helix corresponding in size to the minimal pentasaccharide heparin, and a longer contiguous low-affinity site extending to the reactive center pole of the molecule. Studies of the complexing of antithrombin and its variants with heparin fractions and with reactive center loop peptides including intermolecular loop-sheet polymers all support a 3-fold mechanism for the heparin activation of antithrombin. Binding to the pentasaccharide site induces a conformational change as measured by circular dichroism. Accompanying this, the reactive center becomes more accessible to proteolytic cleavage and there is a 100-fold increase in the kass for factor Xa but only a 10-fold increase for thrombin, to 6.4 x 10(4) M-1 s-1. To obtain a 100-fold increase in the kass for thrombin requires in addition a 4:1 molar ratio of disaccharide to neutralize the charge on the extended low-affinity site. Full activation requires longer heparin chains in order to stabilize the ternary complex between antithrombin and thrombin. Thus, addition of low-affinity but high molecular weight heparin in conjunction with pentasaccharide gives an overall kass of 2.7 x 10(6) M-1 s-1, close to that of maximal heparin activation.

Anhydrobiosis, or extreme desiccation tolerance, is one of the strategies that allows terrestrial Antarctic organisms to survive in a harsh environment. A new study in BMC Genomics analyses gene expression in an Antarctic nematode during desiccation, and sheds new light on this phenomenon.

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

Levels of recrystallization have been measured by two distinct techniques; a splat cooling assay and a new device, an optical recrystallometer, which measures the change in light transmittance through a frozen sample. Both techniques indicate the presence of recrystallization inhibitors in a grass extract and in other samples. The advantages of each method of measuring recrystallization are discussed.

SUMMARY To determine if an Antarctic species repairs DNA at rates equivalent to warmer water equivalents, we examined repair of UV-damaged DNA in echinoid embryos and larvae. DNA repair by photoreactivation was compared in three species Sterechinus neumayeri (Antarctica), Evechinus chloroticus (New Zealand) and Diadema setosum (Tropical Australia) spanning a latitudinal gradient from polar (77.86°S) to tropical (19.25°S) environments. We compared rates of photoreactivation as a function of ambient and experimental temperature in all three species, and rates of photoreactivation as a function of embryonic developmental stage in Sterechinus. DNA damage was quantified from cyclobutane pyrimidine dimer (CPD) concentrations and rates of abnormal embryonic development. This study established that in the three species and in three developmental stages of Sterechinus, photoreactivation was the primary means of removing CPDs, was effective in repairing all CPDs in less than 24 h, and promoted significantly higher rates of normal development in UV-exposed embryos. CPD photorepair rate constant (k) in echinoid embryos ranged from 0.33 to 1.25 h-1, equating to a time to 50% repair of between 0.6 and 2.1 h and time to 90%repair between 3.6 and 13.6 h. We observed that experimental temperature influenced photoreactivation rate. In Diadema plutei, the photoreactivation rate constant increased from k=0.58 h-1to 1.25 h-1, with a Q10=2.15 between 22°C and 32°C. When compared among the three species across experimental temperatures (-1.9 to 32°C), photoreactivation rates vary with a Q10=1.39. Photoreactivation rates were examined in three developmental stages of Sterechinus embryos, and while not significantly different, repair rates tended to be higher in the younger blastula and gastrula stages compared with later stage embryos. We concluded that photoreactivation is active in the Antarctic Sterechinus, but at a significantly slower (non-temperature compensated) rate. The low level of temperature compensation in photoreactivation may be one explanation for the relatively high sensitivity of Antarctic embryos to UV-R in comparison with non-Antarctic equivalents.

The construction of a simple ice nucleation spectrometer is described. It uses 10 microliter droplets loaded into glass capillary tubes which are then inserted into an aluminium holder. Each holder takes six capillary tubes surrounding a central thermocouple. Four holders are placed into a cooling block, cooled by fluid from a programmable refrigerated circulator, and the thermocouples interfaced to a computer to record temperatures. Freezing of each sample is detected by an exotherm on the temperature recording, with 24 samples recorded per run. The spectrometer was tested using deionized water, an extract from a New Zealand alpine cockroach and an extract of lawn grass. The cockroach extract is estimated to contain about 10(3) more nucleators, active at -5 degrees C, than the grass extract.

Dear Editors, The urease ELISA assay is based on a pH change which occurs when urease-conjugated anti- body reacts with a urea substrate-releasing am- monia (Chandler et al., 1982). The distinctive yel- low to purple colour change in the bromocresol purple indicator means that the assay can be read either by eye or by means of an ELISA plate reader. Recently Lo et al. (1988) demonstrated that the urease ELISA was prone to false positive reactions which occurred when ammonia pro- duced in one well was transferred in aerosols to adjacent wells during the incubation time required for the reaction to develop. This finding, together with some inconsistent results with putative H-Y antisera in our laboratory, prompted us to carry out a detailed investigation of the assay system as it pertained to H-Y serology. The H-Y antigen is a widely conserved minor histocompatibility antigen that characterises the heterogametic sex, i.e., males in mammals, and females in birds and fishes (Nakamura et al., 1987). T cell immune respon- siveness to the H-Y antigen has been well docu- mented (Simpson, 1982), but since the first report of a polyclonal antibody to this antigen (Goldberg

It is an article of faith among biochemists and molecular biologists that precious enzymes must be stored on ice. The usual reason given is that, at temperatures around freezing, enzyme activity is minimized and protein stability maximized. There is considerable evidence supporting this, but is it true for all enzymes? What about enzymes from organisms that spend part or all of their lives at temperatures around freezing? How do they manage to maintain normal enzymatic function at low temperatures? Can we learn something from cold-adapted proteins that would allow us better to understand how proteins function?

Background: Infections caused by Candida albicans, a common fungal pathogen of humans, are increasing in incidence, necessitating development of new therapeutic drugs. Secreted aspartic proteinase (SAP) activity is considered an important virulence factor in these infections and might offer a suitable target for drug design. Amongst the various SAP isozymes, the SAP2 gene product is the major form expressed in a number of C. albicans strains. Results The three-dimensional structures of SAP2 complexed with the tight-binding inhibitor A70450 (a synthetic hexapeptide analogue) and with the general aspartic proteinase inhibitor pepstatin A (a microbial natural product) have been determined to 2.1 a and 3.0 a resolution, respectively. Although the protein structure retains the main features of a typical aspartic proteinase, it also shows some significant differences, due mainly to several sequence insertions and deletions (as revealed by homology modelling), that alter the shape of the binding cleft. There is also considerable variation in the C-terminal structural domain. Conclusion The differences in side chains, and in the conformations adopted by the two inhibitors, particularly at their P4, P3 and P′2 positions (using standard notation for protease-inhibitor residues), allows the A70450 structure to complement, more accurately, that of the substrate-binding site of SAP2. Some differences in the binding clefts of other SAP isoenzymes may be deduced from the SAP2 structure.

A molecular model for transmembrane segments 1 and 2 from the fungal proton pumping ATPase has been developed, and this structure is predicted to form a helical hairpin loop structure in the membrane. This region was selected because it is highly conformationally active and is believed to be an important site of action for clinically important therapeutics in related animal cell enzymes. The hairpin loop is predicted to form an asymmetric tightly packed structure that is stabilized by an N-cap between D140 and V142, by hydrogen bonding between residues in the turn region and the helices, and by pi-pi interactions between closely apposed aromatic residues. A short four-residue S-shaped turn is stabilized by hydrogen bonding but is predicted to be conformationally heterogeneous. The principal effect of mutations within the hairpin head region is to destabilize the local close packing of side groups which disrupts the pattern of hydrogen bonding in and around the turn region. Depending on the mutation, this causes either a localized or a more global distortion of the primary structure in the hairpin region. These altered structures may explain the effects of mutations in transmembrane segments 1 and 2 on ATP hydrolysis, sensitivity to vanadate, and electrogenic proton transport. The conformational sensitivity of the hairpin structure around the S-turn may also account for the effects of SCH28080 and possibly ouabain in blocking ATPase function in related animal cell enzymes. Finally, the model of transmembrane segments 1 and 2 serves as a template to position transmembrane segments 3 and 8. This model provides a new view of the H(+)-ATPase that promotes novel structure/function experimentation and could serve as the basis for a more detailed model of the membrane sector of this enzyme.

Background Circum-Antarctic waters harbour a rare example of a marine species flock – the Notothenioid fish, most species of which are restricted to the continental shelf. It remains an open question as to how they survived Pleistocene climatic fluctuations characterised by repeated advances of continental glaciers as far as the shelf break that probably resulted in a loss of habitat for benthic organisms. Pelagic ecosystems, on the other hand, might have flourished during glacial maxima due to the northward expansion of Antarctic polar waters. In order to better understand the role of ecological traits in Quaternary climatic fluctuations, we performed demographic analyses of populations of four fish species from the tribe Trematominae, including both fully benthic and pelagic species using the mitochondrial cytochrome b gene and an intron from the nuclear S7 gene. Results Nuclear and cytoplasmic markers showed differences in the rate and time of population expansions as well as the likely population structure. Neutrality tests suggest that such discordance comes from different coalescence dynamics of each marker, rather than from selective pressure. Demographic analyses based on intraspecific DNA diversity suggest a recent population expansion in both benthic species, dated by the cyt b locus to the last glacial cycle, whereas the population structure of pelagic feeders either did not deviate from a constant-size model or indicated that the onset of the major population expansion of these species by far predated those of the benthic species. Similar patterns were apparent even when comparing previously published data on other Southern Ocean organisms, but we observed considerable heterogeneity within both groups with regard to the onset of major demographic events and rates. Conclusion Our data suggest benthic and pelagic species reacted differently to the Pleistocene ice-sheet expansions that probably significantly reduced the suitable habitat for benthic species. However, the asynchronous timing of major demographic events observed in different species within both "ecological guilds", imply that the species examined here may have different population and evolutionary histories, and that more species should be analysed in order to more precisely assess the role of life history in the response of organisms to climatic changes.

efore Darwin, most biologists adheredto a platonic model of nature. Thisimplied that the biological realm con-sisted of a finite set of essentially immutablenatural forms that, like inorganic formssuch as atoms or crystals, are an intrinsicpart of the eternal order of the world. Justas, today, we account for the form of atomsand crystals by a set of physical laws or‘constructional rules’, so pre-darwinianbiologists sought to account for the originof biological forms in terms of a set ofgenerative physical laws often referred to asthe ‘laws of form’.For many biologists today, platonicbiology is an anachronism irretrievably laidto rest, and the idea that biological formsmight be intrinsic features of nature generat-ed by physical laws is treated with increduli-ty. However, recent advances in proteinchemistry suggest that at least one set ofbiological forms — the basic protein folds —is determined by physical laws similar tothose giving rise to crystals and atoms. Theygive every appearance of being invariantplatonic forms of precisely the type that thepre-darwinian biologists were seeking. Protein folds, the basic constructionalunits of proteins, each consist of a foldedchain of between 80 and 200 amino acids.Some proteins consist of a single fold, butmost are a combination of two or more.During the 1970s, as the three-dimensionalstructure of an increasing number of foldswas determined, it became apparent thatthe folds could be classified into a finitenumber of distinct structural families con-taining a number of closely related forms.The fact that protein folds could be classifiedin this manner provided the first line ofevidence that the folds might be naturalforms.Further evidence that the folds doindeed represent a finite set of naturalforms is provided by detailed structuralstudies carried out over the past two decadeswhich have revealed that the structure ofthe folds can be accounted for by whatamounts to a set of ‘constructional rules’governing the way that the various sec-ondary structural motifs, such as a-helicesand b-sheets, can be combined and packedinto compact three-dimensional structures.One is inevitably reminded of the atom-building rules governing the assembly ofsubatomic particles into the 92 atoms of theperiodic table. Consideration of these ‘constructionallaws’ suggests that the total number ofpermissible folds is bound to be restrictedto a very small number — about 4,000,according to one estimate. Confirmationthat this is probably so is provided by a differ-ent type of estimate, based on the discoveryrate of new folds. Using this method,Cyrus Chothia of Britain’s Medical ResearchCouncil estimated that the total number offolds utilized by living organisms may not bemore than 1,000. Subsequent estimates havegiven figures of between 500 and 1,000.Whatever the final figure, the fact thatthe total number of folds represents a tinystable fraction of all possible polypeptideconformations, determined by the laws ofphysics, reinforces the notion that the folds,like atoms, represent a finite set of built-innatural forms.The robustness of the folds offers anoth-er clue. The fact that the folds can retaintheir native conformations in the face ofmultiple different sorts of short-term defor-mations caused by the molecular turbulenceof the cell, and in the face of extensive, long-term evolutionary changes in their amino-acid sequences, is precisely what would beexpected if they are natural forms, specifiedby physical law. Again, the fact that thesame fold can be specified by many differ-ent, apparently unrelated amino-acidsequences, suggesting multiple separate dis-coveries during the course of evolution, isfurther evidence that the folds are intrinsicfeatures of the order of nature. Finally, thefact that in many cases the same fold isadapted to very different biochemical func-tions is precisely what would be expected ifprotein functions are secondary adaptationsof a set of primary, immutable, naturalforms. If forms as complex as the protein foldsare intrinsic features of nature, might someof the higher architecture of life also be deter-mined by physical law? The robustness ofcertain cytoplasmic forms, for example thespindle apparatus and the cell form of ciliateprotozoans such as

The involvement of new protein and messenger ribonucleic acid synthesis in long-term potentiation was studied in the anaesthetized rat dentate gyrus using several inhibitors of protein synthesis (anisomycin, emetine, cycloheximide and puromycin) and an inhibitor of messenger ribonucleic acid synthesis (actinomycin D). When injected for 1 h just prior to tetanization, the four inhibitors of protein synthesis produced a mild reduction of long-term potentiation of the excitatory postsynaptic potential measured 10 min after tetanization. Anisomycin produced a significantly faster decay of long-term potentiation, while the other inhibitors had more moderate effects. Actinomycin D failed to affect long-term potentiation. In a second experiment, the time-dependency of the anisomycin effect was examined. Anisomycin injected immediately after tetanization promoted decay of long-term potentiation, but when injected after a 15-min delay, the drug had no effect. Inhibition of protein synthesis for 4 h prior to tetanization did not have any more effect on long-term potentiation than inhibition for 1 h. In no experiment was long-term potentiation of the population spike affected by drug manipulation. These results suggest that for long-term potentiation of the excitatory postsynaptic potential to be maintained for at least 3 h proteins must be synthesized from already existing messenger ribonucleic acid, and that this synthesis is mostly completed within 15 min after tetanization.

Haemoglobin is a major protein induced during post-gastrula development of Artemia. Three haemoglobins are formed from the association of two different subunits, and they are expressed differentially during post-gastrula development. These haemoglobins are unusual in that they are of high molecular weight (Mr 260,000) and are composed of two globin chains (Mr 130,000) each of which is a polymer of eight covalently linked myoglobin-like domains [1]. Each polypeptide is encoded by a single large mRNA which may have originated as a result of successive tandem duplication and fusion of a single ancestral globin gene [2].

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.