Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context.

Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.

High circ-SEC31A expression predicts unfavorable prognoses in non-small cell lung cancer by regulating the miR-520a-5p/GOT-2 axis.

Dysregulation of circular RNAs (circRNAs) has recently been shown to play important regulatory roles in cancer development and progression, including non-small cell lung cancer (NSCLC). However, the roles of most circRNAs in NSCLC are still unknown. In this study, we found that hsa_circ_0001421 (circ-SEC31A) was upregulated in NSCLC tissues and cell lines. Increased circ-SEC31A expression in NSCLC was significantly correlated with malignant characteristics and served as an independent risk factor for the post-surgical overall survival of NSCLC patients. Reduced circ-SEC31A expression in NSCLC decreased tumor cell proliferation, migration, invasion, and malate-aspartate metabolism. Mechanistically, we demonstrated that silencing circ-SEC31A downregulated GOT-2 expression by relieving the sponging effect of miR-520a-5p, which resulted in significantly reduced malate-aspartate metabolism in NSCLC cells. Taken together, these results revealed the important role of circ-SEC31A in the proliferation, migration, invasion, and metabolic regulation of NSCLC cells, providing a new perspective on circRNAs in NSCLC progression.

Preventing BRCA1/ZBRK1 repressor complex binding to the GOT2 promoter results in accelerated aspartate biosynthesis and promotion of cell proliferation.

Breast cancer susceptibility gene 1 (BRCA1) has been implicated in modulating metabolism via transcriptional regulation. However, direct metabolic targets of BRCA1 and the underlying regulatory mechanisms are still unknown. Here, we identified several metabolic genes, including the gene which encodes glutamate-oxaloacetate transaminase 2 (GOT2), a key enzyme for aspartate biosynthesis, which are repressed by BRCA1. We report that BRCA1 forms a co-repressor complex with ZBRK1 that coordinately represses GOT2 expression via a ZBRK1 recognition element in the promoter of GOT2. Impairment of this complex results in upregulation of GOT2, which in turn increases aspartate and alpha ketoglutarate production, leading to rapid cell proliferation of breast cancer cells. Importantly, we found that GOT2 can serve as an independent prognostic factor for overall survival and disease-free survival of patients with breast cancer, especially triple-negative breast cancer. Interestingly, we also demonstrated that GOT2 overexpression sensitized breast cancer cells to methotrexate, suggesting a promising precision therapeutic strategy for breast cancer treatment. In summary, our findings reveal that BRCA1 modulates aspartate biosynthesis through transcriptional repression of GOT2, and provides a biological basis for treatment choices in breast cancer.

Cooperative STAT/NF-κB signaling regulates lymphoma metabolic reprogramming and aberrant GOT2 expression.

Knowledge of stromal factors that have a role in the transcriptional regulation of metabolic pathways aside from c-Myc is fundamental to improvements in lymphoma therapy. Using a MYC-inducible human B-cell line, we observed the cooperative activation of STAT3 and NF-κB by IL10 and CpG stimulation. We show that IL10 + CpG-mediated cell proliferation of MYClow cells depends on glutaminolysis. By 13C- and 15N-tracing of glutamine metabolism and metabolite rescue experiments, we demonstrate that GOT2 provides aspartate and nucleotides to cells with activated or aberrant Jak/STAT and NF-κB signaling. A model of GOT2 transcriptional regulation is proposed, in which the cooperative phosphorylation of STAT3 and direct joint binding of STAT3 and p65/NF-κB to the proximal GOT2 promoter are important. Furthermore, high aberrant GOT2 expression is prognostic in diffuse large B-cell lymphoma underscoring the current findings and importance of stromal factors in lymphoma biology.

Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner.

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

Serum aminotransferases in nonalcoholic fatty liver disease are a signature of liver metabolic perturbations at the amino acid and Krebs cycle level.

BACKGROUND: Extensive epidemiologic studies have shown that cardiovascular disease and the metabolic syndrome (MetS) are associated with serum concentrations of liver enzymes; however, fundamental characteristics of this relation are currently unknown. OBJECTIVE: We aimed to explore the role of liver aminotransferases in nonalcoholic fatty liver disease (NAFLD) and MetS. DESIGN: Liver gene- and protein-expression changes of aminotransferases, including their corresponding isoforms, were evaluated in a case-control study of patients with NAFLD (n = 42), which was proven through a biopsy (control subjects: n = 10). We also carried out a serum targeted metabolite profiling to the glycolysis, gluconeogenesis, and Krebs cycle (n = 48) and an exploration by the next-generation sequencing of aminotransferase genes (n = 96). An in vitro study to provide a biological explanation of changes in the transcriptional level and enzymatic activity of aminotransferases was included. RESULTS: Fatty liver was associated with a deregulated liver expression of aminotransferases, which was unrelated to the disease severity. Metabolite profiling showed that serum aminotransferase concentrations are a signature of liver metabolic perturbations, particularly at the amino acid metabolism and Krebs cycle level. A significant and positive association between systolic hypertension and liver expression levels of glutamic-oxaloacetic transaminase 2 (GOT2) messenger RNA (Spearman R = 0.42, P = 0.03) was observed. The rs6993 located in the 3' untranslated region of the GOT2 locus was significantly associated with features of the MetS, including arterial hypertension [P = 0.028; OR: 2.285 (95% CI: 1.024, 5.09); adjusted by NAFLD severity] and plasma lipid concentrations. CONCLUSIONS: In the context of an abnormal hepatic triglyceride accumulation, circulating aminotransferases rise as a consequence of the need for increased reactions of transamination to cope with the liver metabolic derangement that is associated with greater gluconeogenesis and insulin resistance. Hence, to maintain homeostasis, the liver upregulates these enzymes, leading to changes in the amounts of amino acids released into the circulation.

DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China.

We investigated the effectiveness of the standard 648 bp mitochondrial COI barcode region in discriminating among Satyrine species from China. A total of 214 COI sequences were obtained from 90 species, including 34 species that have never been barcoded. Analyses of genetic divergence show that the mean interspecific genetic divergence is about 16-fold higher than within species, and little overlap occurs between them. Neighbour-joining (NJ) analyses showed that 48 of the 50 species with two or more individuals, including two cases with deep intraspecific divergence (>3%), are monophyletic. Furthermore, when our sequences are combined with the conspecific sequences sampled from distantly geographic regions, the "barcoding gap" still exists, and all related species are recovered to be monophyletic in NJ analysis. Our study demonstrates that COI barcoding is effective in discriminating among the satyrine species of China, and provides a reference library for their future molecular identification.

Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.

Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes.

Expression and purification of a functional recombinant aspartate aminotransferase (AST) from Escherichia coli.

Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and α-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at -20ºC. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.

Molecular modeling and functional confirmation of a predicted fatty acid binding site of mitochondrial aspartate aminotransferase.

Molecular interactions are necessary for proteins to perform their functions. The identification of a putative plasma membrane fatty acid transporter as mitochondrial aspartate aminotransferase (mAsp-AT) indicated that the protein must have a fatty acid binding site. Molecular modeling suggests that such a site exists in the form of a 500-Å(3) hydrophobic cleft on the surface of the molecule and identifies specific amino acid residues that are likely to be important for binding. The modeling and comparison with the cytosolic isoform indicated that two residues (Arg201 and Ala219) were likely to be important to the structure and function of the binding site. These residues were mutated to determine if they were essential to that function. Expression constructs with wild-type or mutated cDNAs were produced for bacteria and eukaryotic cells. Proteins expressed in Escherichia coli were tested for oleate binding affinity, which was decreased in the mutant proteins. 3T3 fibroblasts were transfected with expression constructs for both normal and mutated forms. Plasma membrane expression was documented by indirect immunofluorescence before [(3)H]oleic acid uptake kinetics were assayed. The V(max) for uptake was significantly increased by overexpression of the wild-type protein but changed little after transfection with mutated proteins, despite their presence on the plasma membrane. The hydrophobic cleft in mAsp-AT can serve as a fatty acid binding site. Specific residues are essential for normal fatty acid binding, without which fatty acid uptake is compromised. These results confirm the function of this protein as a fatty acid binding protein.

Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV.

Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

Effects of sulfur dioxide on hypoxic pulmonary vascular structural remodeling.

Hypoxic pulmonary hypertension is a pathophysiological process important in the development of various cardiopulmonary diseases. Recently, we found that sulfur dioxide could be produced endogenously by pulmonary vessels, and that it showed vascular regulatory capabilities. In this paper, we examined the role of sulfur dioxide in hypoxic pulmonary vascular structural remodeling (HPVSR). A total of 48 Wistar rats were divided into six groups. Rats in the hypoxic group, hypoxic+sulfur dioxide group, and hypoxic+hydroxamate group were left under hypoxic conditions, whereas the control group, control+sulfur dioxide group, and control+hydroxamate group rats were left in room air. For each group, we measured the pulmonary arterial pressure, sulfur dioxide content in plasma and lung tissue, glutamate oxaloacetate transaminase 1 and 2 mRNAs, micro- and ultra-structural changes in pulmonary arteries, proliferation of pulmonary smooth muscle cells, vascular collagen metabolism, pulmonary endothelial cell inflammatory response, and pulmonary vascular endothelin-1 production in the rats. In hypoxic rats, the content of sulfur dioxide in plasma and lung tissue decreased significantly in comparison with those in the control groups, and significant pulmonary hypertension, pulmonary vascular structural remodeling, and increased vascular inflammatory response were also observed in hypoxic rats. Sulfur dioxide donor significantly downregulated Raf-1, mitogen-activated protein kinase kinase-1 (MEK-1) and p-ERK/ERK, and inhibited pulmonary vascular smooth muscle cell proliferation, collagen remodeling and pulmonary vascular endothelial cell nuclear factor-kappaB (NF-kappaB), and intercellular adhesion molecule 1 (ICAM-1) expressions. It also prevented pulmonary hypertension and pulmonary vascular structural remodeling in association with the upregulated sulfur dioxide/glutamate oxaloacetate transaminase pathway. Hydroxamate, however, advanced pulmonary hypertension, pulmonary vascular structural remodeling, and inflammatory response of the pulmonary artery in association with a downregulated sulfur dioxide/glutamate oxaloacetate transaminase pathway. The results suggested that sulfur dioxide markedly inhibited Raf-1, MEK-1, and the phosphorylation of extracellular signal-regulated kinase (ERK), and then inhibited pulmonary arterial smooth muscle cell (PASMC) proliferation induced by hypoxia. The downregulated sulfur dioxide/glutamate oxaloacetate transaminase pathway may be involved in the mechanisms responsible for pulmonary hypertension and pulmonary vascular structural remodeling.

Downregulation of Gnas, Got2 and Snord32a following tenofovir exposure of primary osteoclasts.

Clinical observations have implicated the antiretroviral drug tenofovir with bone density loss during the management of HIV infection. The goal of this study was to investigate the in vitro effects of tenofovir exposure of primary osteoclasts in order to gain insights into the potential mechanisms for the drug-induced bone density loss. We hypothesized that tenofovir may alter the expression of key genes involved in osteoclast function. To test this, primary osteoclasts were exposed to physiologically relevant concentrations of the prodrug tenofovir disoproxil fumarate (TDF), then intensive microarray analysis was done to compare tenofovir-treated versus untreated cells. Specific downregulation of Gnas, Got2 and Snord32a were observed in the TDF-treated cells. The functions of these genes help to explain the basis for tenofovir-associated bone density loss. Our studies represent the first analysis of the effects of tenofovir on osteoclast gene expression and help to explain the basis of tenofovir-associated bone density loss in HIV-infected individuals.

[Marker characters of variation induced in Triticum aestivum L. by nicotinic acid].

A search for markers of epigenetic variability in spring common wheat form Genotroph-1, obtained after the treatment of seeds and growing plants of spring common wheat cultivar Kazakhstanskaya-126 with nicotinic acid of innate origin (0.1-0.01% water solutions), and in a winter wheat line Gostianum-88 along with a common wheat cultivar Alem obtained on the basis of Genotroph-1 was conducted under laboratory conditions. The possibility of using isoenzymes as markers for studying variation induced by natural nicotinic acid with high frequency was demonstrated. Enzyme glutamate oxaloacetate transaminase-2, as a marker by which modified plants and lines obtained on the basis of these plants differ from the original cultivar, was identified.

Fatty acid binding protein facilitates sarcolemmal fatty acid transport but not mitochondrial oxidation in rat and human skeletal muscle.

The transport of long-chain fatty acids (LCFAs) across mitochondrial membranes is regulated by carnitine palmitoyltransferase I (CPTI) activity. However, it appears that additional fatty acid transport proteins, such as fatty acid translocase (FAT)/CD36, influence not only LCFA transport across the plasma membrane, but also LCFA transport into mitochondria. Plasma membrane-associated fatty acid binding protein (FABPpm) is also known to be involved in sacrolemmal LCFA transport, and it is also present on the mitochondria. At this location, it has been identified as mitochondrial aspartate amino transferase (mAspAT), despite being structurally identical to FABPpm. Whether this protein is also involved in mitochondrial LCFA transport and oxidation remains unknown. Therefore, we have examined the ability of FABPpm/mAspAT to alter mitochondrial fatty acid oxidation. Muscle contraction increased (P < 0.05) the mitochondrial FAT/CD36 content in rat (+22%) and human skeletal muscle (+33%). By contrast, muscle contraction did not alter the content of mitochondrial FABPpm/mAspAT protein in either rat or human muscles. Electrotransfecting rat soleus muscles, in vivo, with FABPpm cDNA increased FABPpm protein in whole muscle (+150%; P < 0.05), at the plasma membrane (+117%; P < 0.05) and in mitochondria (+80%; P < 0.05). In these FABPpm-transfected muscles, palmitate transport into giant vesicles was increased by +73% (P < 0.05), and fatty acid oxidation in intact muscle was increased by +18% (P < 0.05). By contrast, despite the marked increase in mitochondrial FABPpm/mAspAT protein content (+80%), the rate of mitochondrial palmitate oxidation was not altered (P > 0.05). However, electrotransfection increased mAspAT activity by +70% (P < 0.05), and the mitochondrial FABPpm/mAspAT protein content was significantly correlated with mAspAT activity (r = 0.75). It is concluded that FABPpm has two distinct functions depending on its subcellular location: (a) it contributes to increasing sarcolemmal LCFA transport while not contributing directly to LCFA transport into mitochondria; and (b) its primary role at the mitochondria level is to transport reducing equivalents into the matrix.

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China.

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.

The promoter for C4-type mitochondrial aspartate aminotransferase does not direct bundle sheath-specific expression in transgenic rice plants.

For NAD-malic enzyme (NAD-ME)-type C4 photosynthesis, two types of aspartate aminotransferase (AAT) are involved. We examined the expression pattern of the Panicum miliaceum mitochondrial Aat gene (PmAat) and P. miliaceum cytosolic Aat gene (PcAat) in transgenic rice plants, which were specifically expressed in bundle sheath cells (BSCs) and mesophyll cells (MCs), respectively. Expression of a beta-glucuronidase (GUS) reporter gene under the control of the PcAat promoter was regulated in an organ-preferential and light-dependent manner in the transgenic rice plants. However, the PmAat promoter drove the GUS expression in all organs we tested without light dependency, and this non-preferential expression pattern was also observed in transgenic rice with introduction of the intact PmAat gene. The expression patterns of the rice counterpart Aat genes to PmAat or PcAat showed that the rice mitochondrial Aat (RmAat1) gene was expressed in all organs tested in a light-independent manner, while expression of the rice cytosolic Aat (RcAat1) gene showed an organ-preferential and light-dependent pattern. Taking these results together, we can generalize that the regulatory system of BSC-specific or light-dependent expression of mitochondrial Aat is not shared between P. miliaceum (C4) and rice (C3) and that the expression of the C4 genes introduced into rice mimics that of their counterpart genes in rice.

Leishmania donovani is the only cause of visceral leishmaniasis in East Africa; previous descriptions of L. infantum and "L. archibaldi" from this region are a consequence of convergent evolution in the isoenzyme data.

Isoenzyme-based studies have identified 3 taxa/species/'phylogenetic complexes' as agents of visceral leishmaniasis in Sudan: L. donovani, L. infantum and "L. archibaldi". However, these observations remain controversial. A new chitinase gene phylogeny was constructed in which stocks of all 3 putative species isolated in Sudan formed a monophyletic clade. In order to construct a more robust classification of the L. donovani complex, a panel of 16 microsatellite markers was used to describe 39 stocks of these 3 species. All "L. donovani complex" stocks from Sudan were again found to form a single monophyletic clade. L. donovani ss stocks from India and Kenya were found to form 2 region-specific clades. The partial sequence of the glutamate oxaloacetate transaminase (GOT) gene of 17 L. donovani complex stocks was obtained. A single nucleotide polymorphism in the GOT gene appeared to underlie the isoenzyme classification. It was concluded that isoenzyme-based identification is unsafe for stocks isolated in L. donovani endemic areas and identified as L. infantum. It was also concluded that the name L. archibaldi is invalid and that only a single visceralizing species, Leishmania donovani, is found in East Africa.

Fine-scale genetic structure in a free-living ungulate population.

The fine-scale genetic structure of wild animal populations has rarely been analysed, yet is potentially important as a confounding factor in quantitative genetic and allelic association studies, as well as having implications for population dynamics, inbreeding and kin selection. In this study, we examined the extent to which the three spatial subunits, or hefts, of the Village Bay population of Soay sheep (Ovis aries) on St Kilda, Scotland, are genetically structured using data from 20 microsatellite and protein loci. Allele frequencies differed significantly among three hefts in all the study years we considered (1987-2000 inclusive). Small but significantly positive F(ST) and negative F(IS) values were observed in most years, indicating that the hefts are genetically differentiated, and that within each heft there is more observed heterozygosity than would be expected if each were an isolated breeding population. Males showed less fidelity to their natal heft, and as a consequence higher levels of relatedness within hefts were observed among females than among males. There was a significant negative relationship between geographical proximity and relatedness in pairwise comparisons involving females, and on average pairs of females located within 50 m of each other were related at the equivalent level of second cousins. Structure is therefore largely driven by incomplete postnatal dispersal by females. Mating appears to be random with respect to the spatial-genetic substructure of the hefts, and therefore genetic structure does not contribute to the overall rate of inbreeding in the population. However, genetic substructure can lead to allelic associations and generate environmental effects within lineages that have the potential to confound heritability analyses and allelic association studies.

A genetic algorithm for the identification of conformationally invariant regions in protein molecules.

Understanding macromolecular function often relies on the comparison of different structural models of a molecule. In such a comparative analysis, the identification of the part of the molecule that is conformationally invariant with respect to a set of conformers is a critical step, as the corresponding subset of atoms constitutes the reference for subsequent analysis for example by least-squares superposition. A method is presented that categorizes atoms in a molecule as either conformationally invariant or flexible by automatic analysis of an ensemble of conformers (e.g. crystal structures from different crystal forms or molecules related by non-crystallographic symmetry). Different levels of coordinate precision, both for different models and for individual atoms, are taken explicitly into account via a modified form of Cruickshank's DPI [Cruickshank (1999), Acta Cryst. D55, 583-601] and are propagated into error-scaled difference distance matrices [Schneider (2000), Acta Cryst. D56, 715-721]. All pairwise error-scaled difference distance matrices are then analysed simultaneously using a genetic algorithm. The algorithm has been tested on several well known examples and has been found to converge rapidly to reasonable results using a standard set of parameters. In addition to the description of the algorithm, a criterion is suggested for testing the identity of two three-dimensional models within experimental error without any explicit superposition.