Juxtaposed regions of extensive and minimal linkage disequilibrium in human Xq25 and Xq28

Linkage disequilibrium (LD), or the non-random association of alleles, is poorly understood in the human genome. Population genetic theory suggests that LD is determined by the age of the markers, population history, recombination rate, selection and genetic drift. Despite the uncertainties in determining the relative contributions of these factors, some groups have argued that LD is a simple function of distance between markers. Disease-gene mapping studies and a simulation study gave differing predictions on the degree of LD in isolated and general populations. In view of the discrepancies between theory and experimental observations, we constructed a high-density SNP map of the Xq25-Xq28 region and analysed the male genotypes and haplotypes across this region for LD in three populations. The populations included an outbred European sample (CEPH males) and isolated population samples from Finland and Sardinia. We found two extended regions of strong LD bracketed by regions with no evidence for LD in all three samples. Haplotype analysis showed a paucity of haplotypes in regions of strong LD. Our results suggest that, in this region of the X chromosome, LD is not a monotonic function of the distance between markers, but is more a property of the particular location in the human genome.     

Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects

Astrocytomas are the leading cause of brain cancer in humans. Because these tumours are highly infiltrative, current treatments that rely on targeting the tumour mass are often ineffective. A mouse model for astrocytoma would be a powerful tool for dissecting tumour progression and testing therapeutics. Mouse models of astrocytoma have been designed to express oncogenic proteins in astrocytes, but have had limited success due to low tumour penetrance or limited tumour progression. We present here a mouse model of astrocytomas involving mutation of two tumour-suppressor genes, Nf1 and Trp53. Humans with mutations in NF1 develop neurofibromatosis type I (NF1) and have increased risk of optic gliomas, astrocytomas and glioblastomas. The TP53 tumour suppressor is often mutated in a subset of astrocytomas that develop at a young age and progress slowly to glioblastoma (termed secondary glioblastomas, in contrast to primary glioblastomas that develop rapidly de novo). This mouse model shows a range of astrocytoma stages, from low-grade astrocytoma to glioblastoma multiforme, and may accurately model human secondary glioblastoma involving TP53 loss. This is the first reported mouse model of astrocytoma initiated by loss of tumour suppressors, rather than overexpression of transgenic oncogenes.     

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.     

Construction of a genetic toggle switch in Escherichia coli

It has been proposed' that gene-regulatory circuits with virtually any desired property can be constructed from networks of simple regulatory elements. These properties, which include multistability and oscillations, have been found in specialized gene circuits such as the bacteriophage lambda switch and the Cyanobacteria circadian oscillator. However, these behaviours have not been demonstrated in networks of non-specialized regulatory components. Here we present the construction of a genetic toggle switch-a synthetic, bistable gene-regulatory network-in Escherichia coli and provide a simple theory that predicts the conditions necessary for bistability. The toggle is constructed from any two repressible promoters arranged in a mutually inhibitory network. It is flipped between stable states using transient chemical or thermal induction and exhibits a nearly ideal switching threshold. As a practical device, the toggle switch forms a synthetic, addressable cellular memory unit and has implications for biotechnology, biocomputing and gene therapy.     

A clonogenic common myeloid progenitor that gives rise to all myeloid lineages

Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.     

The S receptor kinase determines self-incompatibility in Brassica stigma

The self-incompatibility possessed by Brassica is an intraspecific reproductive barrier by which the stigma rejects self-pollen but accepts non-self-pollen for fertilization. The molecular/biochemical bases of recognition and rejection have been intensively studied. Self-incompatibility in Brassica is sporophytically controlled by the polymorphic S locus. Two tightly linked polymorphic genes at the S locus, S receptor kinase gene (SRK) and S locus glycoprotein gene (SLG), are specifically expressed in the papillar cells of the stigma, and analyses of self-compatible lines of Brassica have suggested that together they control stigma function in self-incompatibility interactions. Here we show, by transforming self-incompatible plants of Brassica rapa with an SRK28 and an SLG28 transgene separately, that expression of SRK28 alone, but not SLG28 alone, conferred the ability to reject self (S28)-pollen on the transgenic plants. We also show that the ability of SRK28 to reject S28 pollen was enhanced by SLG28. We conclude that SRK alone determines S haplotype specificity of the stigma, and that SLG acts to promote a full manifestation of the self-incompatibility response.     

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.     

The Ras-MAPK pathway is important for olfaction in Caenorhabditis elegans

The Ras-MAPK (mitogen-activated protein kinase) signal transduction pathway is well known to control cellular proliferation and differentiation in response to extracellular signals, but its other functions are less understood. In Caenorhabditis elegans this pathway regulates several developmental events, such as vulval induction and progression of meiosis, but its function in the nervous system is unknown. Here we report that the Ras-MAPK pathway is involved in olfaction in this organism. Mutational inactivation and hyperactivation of this pathway impairs efficiency of chemotaxis to a set of odorants. Experiments in which let-60 ras was expressed using a heat-shock promoter and a cell-specific promoter show that a normal activity of LET-60 Ras is required in mature olfactory neurons. Application of the odorant isoamylalcohol to wild-type animals leads to the activation of MAP kinase in olfactory neurons within 10 seconds. This induction is dependent on the function of the nucleotide-gated channel TAX-2/TAX-4 and the voltage-activated calcium channel subunit UNC-2. These results suggest a dynamic regulatory role for the Ras-MAPK pathway in perception and transmission of sensory signals in olfactory neurons.