Ancient pests: the season of the Santorini Minoan volcanic eruption and a date from insect chitin.

Attributing a season and a date to the volcanic eruption of Santorini in the Aegean has become possible by using preserved remains of the bean weevil, Bruchus rufipes, pests of pulses, from the storage jars of the West House, in the Bronze Age settlement at Akrotiri. We have applied an improved pre-treatment methodology for dating the charred insects, and this provides a date of 1744-1538 BC. This date is within the range of others obtained from pulses from the same context and confirms the utility of chitin as a dating material. Based on the nature of the insect material and the life cycle of the species involved, we argue for a summer eruption, which took place after harvest, shortly after this material was transported into the West House storeroom.

Mid-Devensian climate and landscape in England: new data from Finningley, South Yorkshire.

While there is extensive evidence for the Late Devensian, less is known about Early and Middle Devensian (approx. 110-30 ka) climates and environments in the UK. The Greenland ice-core record suggests the UK should have endured multiple changes, but the terrestrial palaeo-record lacks sufficient detail for confirmation from sites in the British Isles. Data from deposits at Finningley, South Yorkshire, can help redress this. A channel with organic silts, dated 40 314-39 552 cal a BP, contained plant macrofossil and insect remains showing tundra with dwarf-shrub heath and bare ground. Soil moisture conditions varied from free draining to riparian, with ponds and wetter vegetated areas. The climate was probably low arctic with snow cover during the winter. Mutual climatic range (MCR), based on Coleoptera, shows the mean monthly winter temperatures of -22 to -2°C and summer ones of 8-14°C. Periglacial structures within the basal gravel deposits and beyond the glacial limits indicate cold-climate conditions, including permafrost. A compilation of MCR reconstructions for other Middle Devensian English sites shows that marine isotope stage 3-between 59 and 28 ka-experienced substantial variation in climate consistent with the Greenland ice-core record. The exact correlation is hampered by temporal resolution, but the Finningley site stadial at approximately 40 ka may correlate with the one of the Greenland stadials 7-11.

Gene copy number variation in schizophrenia.

Recent reports have highlighted the possibility that gene copy number variations play a role in the development of complex disorders and have suggested that some variations are very common in schizophrenic patients. We have carried out a comparative genomic hybridization screen using oligonucleotide probes of 891 candidate genes to look for very common copy number variance in schizophrenic patients. In addition we have developed a new approach for the detection and validation of putative copy number variation based upon established methods of allele quantification by DNA pooling and have used it to study 15 major candidates including dysbindin (DTNBP1), neuregulin (NRG1), RGS4 and DISC1. With the exception of positive control sequences, no copy number variations were found for any of the genes in any samples by the use of either technique. Our data for the genes studied are in line with the known existence and frequency of CNVs as reported by recent large scale studies and suggest that gene copy number variations are not more common in schizophrenics than controls, although large ethnic differences cannot be excluded.

Is there anybody in there? Entomological evidence from a boat burial at Øksnes in Vesterålen, northern Norway.

Although there are several well preserved Viking boat burials from Norway, until recently palaeoecological research on their context has often been limited. Research on fossil insect remains in particular can provide valuable forensic information even in the absence of an actual body. Here we present archaeoentomological information from a boat burial at Øksnes in Vesterålen, northeast Norway, an area where Norse and Sami traditions overlap. Excavated in 1934, organic preservation from the burial was limited to parts of the boat and a clump of bird feathers which were preserved in the Tromsø University Museum, and from which fossil insects were recovered. The insect assemblage from Øksnes includes the blowfly, Protophormia terraenovae (Rob.-Des.), which indicates exposure of the body and the probable timing of the burial. The high numbers of the human flea, Pulex irritans L. from among the feathers, suggests that these, probably from a pillow under the corpse, originated from within a domestic context. Deposition of flowers as part of the burial is discussed on the basis of the insect fauna. The absence of a body and any associated post burial decay fauna implies its exhumation and disposal elsewhere and this is discussed in the context of other exhumed medieval burials and Saga and other sources.

The importance and identification of regulatory polymorphisms and their mechanisms of action.

The search for the genetic variations underlying all human phenotypes is in its infancy but must be one of the long term goals of the scientific community. There is evidence that most, if not all human phenotypes, including illnesses are influenced by the genetic makeup of the individual. There are an estimated 11 million human genetic polymorphisms with a minor allele frequency >1% and possibly many times that number of rare sequence variants. The proportion of these sequence variants which have any functional effect is unknown but it is likely that the majority of those which influence illness lie outside of the amino acid coding regions of genes, and affect the regulation of gene expression--these are called rSNPs. Recent research suggests that about 50% of genes have one or more common rSNPs associated with them and probably most if not all genes have an rSNP within the human population. In the long term, determining which polymorphisms are potentially functional must be done bio-informatically using algorithms based upon experimental data. However, at the current time, the limited data that has been obtained does not allow the creation of such an algorithm. In vitro studies suggest that a large proportion of rSNPs lie within the core and proximal promoter regions of genes but it is not clear how the majority of these influence transcription, as they do not appear to be within any known transcription factor binding sites. However, promoter regions possess a number of sequence-dependent characteristics which make them distinct from the rest of the genome, namely stability, curvature and flexibility. Subtle changes to these features may underlie the mechanisms by which many polymorphisms exert their function.

Gene copy number variation in schizophrenia.

The possibility that gene copy number variations play a role in the development of complex disorders is a topic of considerable interest. Recent reports have highlighted the large number of such variations that exist and that their occurrence varies considerably between populations. A recent report has suggested that copy number variations in four genes (GRIK3, EFNA5, AKAP5 and CACNG2) may be associated with schizophrenia. One problem with this area of study is the validation of high throughput methods such as comparative genomic hybridisation, as the latter inevitably generates false positives. We have used two contrasting methodologies to determine the validity of the findings reported above which if true would have major implications for the pathogenesis of schizophrenia. Samples from a UK population were tested using a method of allele quantification by DNA pooling and samples from Belgium and northern Sweden were tested using Multiplex Amplicon Quantification (MAQ). Both methods were used to test DNA samples used in the original investigation. No copy number variations were found for any of the genes in any samples. Our data suggests that more reliable methods need to be used to validate the existence of CNVs before full scale association studies are carried out.

Endosomal location of dopamine receptors in neuronal cell cytoplasm.

Five subtypes of dopamine receptor exist in two subfamilies: two D(1)-like (D(1) and D(5)) and three D(2)-like (D(2), D(3) and D(4)). We produced novel monoclonal antibodies against all three D(2)-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D(3) receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.

Strong bias in the location of functional promoter polymorphisms.

A considerable proportion of heritable human phenotypic variation is thought to result from altered gene expression. Unfortunately, it is currently impossible to use bioinformatic analysis to discriminate between DNA sequence variants that are likely to influence gene expression and those that are not. In an attempt to define some of the characteristics of promoter polymorphisms with functional effects on gene expression, we examined 674 haplotypes representing 247 unique gene promoters using a standardized reporter gene assay system. Sequence variants that altered gene expression by 1.5-fold or more were strongly biased toward a location in the core and proximal promoter regions, 50% being within the first 100 bases 5' to the transcription start site. No bias was seen in the allele frequencies of functional and nonfunctional sequence variants. Only 33% of the functional variants were found in known consensus transcription factor binding sequences or motifs, which suggests that either there are many unknown transcription factor binding motifs or other, unknown mechanisms are involved. The genes with functional polymorphisms that are reported here for the first time include AGTRL2, CAT, CHRNA5, CTSG, CYP2D6, DLD, ERCC1, GABRA1, GABRP, HNRPH3, HIP1, IGKV1-9, KCNJ15, KCNK6, KLK1, MSMB, MYOC, NPY2R, NOTCH4, ORM2, PEDF, PTPRCAP, ST16 (IL24), SULT1A1, and TSHR.

A high proportion of polymorphisms in the promoters of brain expressed genes influences transcriptional activity.

There is increasing interest in the possibility that polymorphisms affecting gene expression are responsible for a significant proportion of heritable human phenotypic variation, including human disease. We have sought to determine if polymorphisms in the promoters of brain expressed genes are commonly functional. We screened for polymorphism 56 genes previously reported to be differentially expressed in the brains of schizophrenics [Y. Hakak, J.R. Walker, C. Li, W.H. Wong, K.L. Davis, J.D. Buxbaum, V. Haroutunian, A.A. Fienberg, Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. 98 (2001) 4746-4751.]. We found 60 variants distributed across 31 of the genes. A total of 77 haplotypes representing 28 different putative promoters were analyzed in a reporter gene assay in two cell lines. Of a total of 54 sequence variants represented in the haplotypes, 12 (or around 22%) were functional according to a highly conservative definition. These were found in the promoters of eight genes: NPY, PCSK1, NEFL, KIAA0513, LMO4, HSPA1B, TF and MDH1. We therefore estimate that around 20-25% of promoter polymorphisms in brain expressed genes are functional, and this is likely to be an underestimate. Our data therefore provide for the first time empirical evidence that promoter element polymorphisms, at least in brain expressed genes, should be afforded a high priority for molecular genetic studies.

Low gene expression conferred by association of an allele of the 5-HT2C receptor gene with antipsychotic-induced weight gain.

OBJECTIVE: Association has been reported between the C allele of a -759C/T polymorphism in the promoter of the 5-HT2C receptor gene (HTR2C) and antipsychotic-induced weight gain, suggesting that polymorphic HTR2C expression influences this phenotype. The authors tested this polymorphism, and other promoter variants, for effects on HTR2C transcription. METHOD: Six HTR2C promoter haplotypes constructed from four polymorphisms were cloned into a luciferase reporter gene plasmid. Their transcriptional activities were then compared in two human cell lines. RESULTS: All haplotypes containing the -759C allele showed less transcriptional activity than haplotypes containing the -759T allele. The A allele of a -997G/A polymorphism was also associated with reduced expression. CONCLUSIONS: These findings suggest that the -759C allele is functional and results in relative underexpression of HTR2C. Reduced expression of HTR2C mRNA may underlie vulnerability to weight gain following antipsychotic treatment.

Association between dopamine D3 receptor gene polymorphisms and schizophrenia in an isolate population.

There are several lines of evidence implicating the dopamine D3 receptor in the pathophysiology of schizophrenia. The Ser9Gly polymorphism of the dopamine D3 receptor gene (DRD3) has been the most extensively investigated DRD3 variant in connection with the disease but results have been inconclusive. Recent reports indicate that the Ser9Gly polymorphism is in linkage disequilibrium with other markers, but association studies between DRD3 haplotypes and schizophrenia have had mixed results. Genetic heterogeneity may be one of the causes of contradicting results. In order to clarify the role of DRD3 alterations in the aetiology of disease, we have investigated three D3 genetic variants (Ser9Gly, -205-G/A, -7685-G/C) in a sample of patients with schizophrenia or schizoaffective disorder (N=118) and controls (N=162) recruited from a human isolate from Navarra (Northern Spain) of Basque origin. Although no association was found between the Ser9Gly or the -205-A/G polymorphisms and disease, an excess of allele -7685-C was observed in patients (p=0.002 after correction for multiple analyses). Haplotype analysis shows the three markers to be in strong linkage disequilibrium (p<0.0001) and strongly associated with disease (p<1x 10(-5)). These results may suggest that these polymorphisms exert a combined or synergistic effect on susceptibility to schizophrenia, or are in linkage with an unknown causative factor. However, further replication in independent samples is required.

Functional analysis of polymorphisms in the promoter regions of genes on 22q11.

Segmental aneusomy, which includes chromosome 22 deletion syndrome (del(22)(q11.2q11.2)), has been associated with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face (CAF) syndrome, cat-eye syndrome (CES), der(22) syndrome, and duplication of the del(22)(q11.2q11.2) syndrome's typically deleted region. Adults with del(22)(q11.2q11.2) may develop psychiatric illnesses, including schizophrenia, schizoaffective disorder, and bipolar disorder, suggesting that lower gene dosage leads to a predisposition to these illnesses. In a bid to identify important regulatory polymorphisms (SNPs) that may emulate changes in gene dosage of the genes within the common deletion, we have analyzed the promoter region of 47 genes (44 of which encode a protein with known function) encoding proteins in and around 22q11 for sequence variants. A total of 33 of the promoters contained polymorphisms. Of those, 25 were cloned into a reporter gene vector, pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671), using a cotransfected CMV-SPAP plasmid as an internal control. Five genes (PRODH, DGCR14, GSTT2, SERPIND1, and a gene tentatively called DKFZP434P211) showed activity differences between haplotypes of greater than 1.5-fold. Of those, PRODH, which encodes proline dehydrogenase, has previously been highlighted in relation to schizophrenia, and the functional promoter polymorphism reported here may be involved in pathogenic mechanisms.

The human hyaluronan synthase genes: genomic structures, proximal promoters and polymorphic microsatellite markers.

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.

Promoter polymorphisms in glutathione-S-transferase genes affect transcription.

The glutathione-S-transferases are a group of enzymes that play a major role in detoxification and defence against toxic, carcinogenic and other compounds. We analysed the proximal promoters of 14 genes encoding glutathione-S-transferase for polymorphism. Ten of the promoters contained sequence variants, nine of which we were able to clone into a reporter gene vector, pGL3. The relative ability of each haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671) using a cotransfected CMV-SEAP plasmid as a control. Four genes (GSTA1, GSTA2, GSTM4 and GSTT2) showed activity differences greater than 1.5-fold between haplotypes, and a fifth gene (MGST1) showed a 1.4-fold difference. The promoter sequence variants in these genes may therefore play a role in human variation, susceptibility to diseases and the effects of drugs.

Support for RGS4 as a susceptibility gene for schizophrenia.

BACKGROUND: The gene encoding the regulator of G-protein signaling 4 has recently been associated with susceptibility to schizophrenia. This finding is particularly interesting, because it was replicated within the same study and also because there are functional, positional, and expression data to support the regulator of G-protein signaling 4 as a schizophrenia candidate gene. Although the original report was highly suggestive, a limitation was that the study was conducted on rather small samples. METHODS: We have examined a large case (n = 709) control (n = 710) sample for association between schizophrenia using four markers investigated in the earlier study, denoted single nucleotide polymorphisms 1, 4, 7, and 18. RESULTS: We were able to replicate the associations with single nucleotide polymorphisms 4 and 18 that had previously been reported individually and have also identified significant association with haplotypes constructed from single nucleotide polymorphisms 1 and 4. CONCLUSIONS: Our data give modest support for the hypothesis that the regulator of G-protein signaling 4 is a susceptibility gene for schizophrenia.

Morphine, cocaine and antidepressant induced motivational activity and midbrain dopaminergic neurotransmission.

Positive motivational properties of opioids, stimulants and serotonin selective reuptake inhibitors have been reported following place preference conditioning. The possibility that these effects are associated with changes in dopamine concentration in the nucleus accumbens or striatum was investigated. Male Wistar rats were place conditioned in a three compartment model to vehicle or drug (morphine 2.5 mg/kg, cocaine 5 mg/kg, sertraline 5 mg/kg or paroxetine 15 mg/kg) alternately for 8 days using a 30 min pre-treatment time. Control animals received saline only. Nucleus accumbens and striatal tissue were dissected 72 h after final drug dose, and the concentration of dopamine and its metabolites determined using high performance liquid chromatography (HPLC). Striatal dopamine D1-like receptor density was also determined through radioligand binding. Significant place preference (P<0.05) was observed with morphine, cocaine and sertraline. Morphine treated subjects showed a significant decrease (P<0.05) in striatal dopamine concentration, whilst cocaine and sertraline treatment resulted in a significant increase in striatal dopamine levels. Nucleus accumbens concentrations of dopamine, and striatal dopamine D1-like receptor density remained unchanged. The changes in striatal dopamine concentrations are consistent with withdrawal from opioid and stimulant compounds, and suggest that place preference conditioning may, in part, result from negative motivational or aversive effects.

A high proportion of chromosome 21 promoter polymorphisms influence transcriptional activity.

We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes.

Forensic archaeoentomology--An insect fauna from a burial in York Minster.

An insect fauna associated with the medieval burial of Archbishop Greenfield, interred in December 1315 in a lead coffin within a stone sarcophagus beneath the floor of York Minster, is examined and compared with the limited entomological data from other medieval burials. The implications of the archaeoentomological data are discussed. The fauna is dominated by the so-called coffin beetle Rhizophagus parallelocollis and the generalised staphylinid predator Quedius mesomelinus, together with a number of subterranean fungal feeders. The beetle assemblage is probably immediately post burial, and the lead coffin in the case of Greenfield had not been able to shield the body from decay.

Will we ever find the genes for addiction?

AIM: To assess the likelihood of finding genes which predispose to addiction and to present this information in a form accessible to the general readership of Addiction. METHODS: Review of the evidence that genetic factors play a significant role in the process of addiction and the proximity of the identification of these factors. RESULTS: The search for the genetic susceptibility variants for many complex illnesses has been ongoing for decades, with increased pace in the last 20 years. However, until very recently only a small number of such variants have been found. Recent studies have used several thousand samples in genome-wide association studies and the latest genotyping technology and have reported a growing number of successes, but have highlighted the need for even larger samples and new statistical methods or new experimental approaches to identify fully the genes involved in the disease process. The phenotype for addiction to drugs is not well defined, and the heritability of addiction to drugs of abuse is far from clear and may be small compared to that of many other complex disorders. The absolute requirement for the administration of drugs before addiction can occur, and other environmental factors known to have a major effect, makes the selection of both probands and controls challenging for genetic studies. Many candidate genes put forward so far as susceptibility genes may be unrelated to the underlying process referred to as addiction but, rather, are related to the propensity to take drugs in the first place. CONCLUSIONS: It is the underlying biological process which changes to an alternative state following addiction, which is the target of investigation, and it is not clear that even genome-wide association studies with sample sizes a magnitude greater than those reported so far would identify the genes involved which have the largest effect. Ultimately, modern neurobiological approaches may identify this process and the genes involved, and even at this stage identifying the susceptibility variants will require both biological as well as genetic analysis.

Fossil insect evidence for the end of the Western Settlement in Norse Greenland.

The fate of Norse farming settlements in southwest Greenland has often been seen as one of the great mysteries of North Atlantic colonization and expansion. Preservation of organic remains in the permafrost of the area of the Western Settlement, inland from the modern capital Nuuk, allowed very detailed study of the phases of occupation. Samples were taken from house floors and middens during the process of archaeological excavations and from insect remains were abstracted and identified in the laboratory. In this study, we present a new paleoecological approach principally examining the fossil fly faunas from house floors. The results of our study provide contrasting detailed pictures of the demise of two neighboring farms, Gården under Sandet and Nipaatsoq, one where abandonment appears as part of a normal process of site selection and desertion, and the other where the end was more traumatic. The level of detail, which was obtained by analysis of the dipterous (true fly) remains, exceeds all previous work and provides insights otherwise unobtainable.