Comparative analyses of multi-species sequences from targeted genomic regions

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.     

Failure to find holes in the T-cell repertoire

The ability of an animal to respond to a given antigenic peptide depends on its major histocompatibility complex (MHC) type. Some peptides are not immunogenic when combined with a particular form of the MHC-encoded molecule. This non-responsiveness is regulated by immune response (Ir) genes and is thought to arise by one of two distinct mechanisms. Either the MHC-encoded molecules physically fail to interact with the antigen, preventing the activation of T cells with appropriate receptors, or they limit the expressed repertoire of T cell clones so that no T cells are available to be activated by existing complexes of MHC-encoded molecules and antigen. Experimental evidence has been generated to support both mechanisms. However, the relative importance of each has not been clearly established. In this study we started with a peptide that was immunogenic in B10 mice; it was thus known to be able to interact with the MHC molecule, and T cells existed which could recognise the peptide-MHC complex. Based on previous experiments, we then changed only those parts of the peptide that we thought interacted with the T-cell receptor. All the new analogues created were still immunogenic, confirming that the amino-acid substitutions that we had made did not prevent productive interactions with the MHC-encoded molecule. No limitations ('holes') in the T-cell repertoire were found. The experiments demonstrate the vast potential of the T-cell population to recognize many different analogues, each in a unique way, and suggest that constraints on the diversity of the T-cell repertoire may not be a major explanation for Ir gene defects.     

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

Warming experiments underpredict plant phenological responses to climate change

Warming experiments are increasingly relied on to estimate plant responses to global climate change. For experiments to provide meaningful predictions of future responses, they should reflect the empirical record of responses to temperature variability and recent warming, including advances in the timing of flowering and leafing. We compared phenology (the timing of recurring life history events) in observational studies and warming experiments spanning four continents and 1,634 plant species using a common measure of temperature sensitivity (change in days per degree Celsius). We show that warming experiments underpredict advances in the timing of flowering and leafing by 8.5-fold and 4.0-fold, respectively, compared with long-term observations. For species that were common to both study types, the experimental results did not match the observational data in sign or magnitude. The observational data also showed that species that flower earliest in the spring have the highest temperature sensitivities, but this trend was not reflected in the experimental data. These significant mismatches seem to be unrelated to the study length or to the degree of manipulated warming in experiments. The discrepancy between experiments and observations, however, could arise from complex interactions among multiple drivers in the observational data, or it could arise from remediable artefacts in the experiments that result in lower irradiance and drier soils, thus dampening the phenological responses to manipulated warming. Our results introduce uncertainty into ecosystem models that are informed solely by experiments and suggest that responses to climate change that are predicted using such models should be re-evaluated.     

A Met-to-Val mutation in the skeletal muscle Na+ channel alpha-subunit in hyperkalaemic periodic paralysis.

HYPERKALAEMIC periodic paralysis (HYPP) is an autosomal dominant disease that results in episodic electrical inexcitability and paralysis of skeletal muscle. Electrophysiological data indicate that tetrodotoxin-sensitive sodium channels from muscle cells of HYPP-affected individuals show abnormal inactivation. Genetic analysis of nine HYPP families has shown tight linkage between the adult skeletal muscle sodium channel alpha-subunit gene on chromosome 17q and the disease (lod score, z = 24; recombination frequency 0 = 0), strongly suggesting that mutations of the alpha-subunit gene cause HYPP. We sequenced the alpha-subunit coding region isolated from muscle biopsies from affected (familial HYPP) and control individuals by cross-species polymerase chain reaction-mediated complementary DNA cloning. We have identified an A----G substitution in the patient's messenger RNA that causes a Met----Val change in a highly conserved region of the alpha-subunit, predicted to be in a transmembrane domain. This same change was found in a sporadic case of HYPP as a new mutation. We have therefore discovered a voltage-gated channel mutation responsible for a human genetic disease.     

Hypothalamic K(ATP) channels control hepatic glucose production

Obesity is the driving force behind the worldwide increase in the prevalence of type 2 diabetes mellitus. Hyperglycaemia is a hallmark of diabetes and is largely due to increased hepatic gluconeogenesis. The medial hypothalamus is a major integrator of nutritional and hormonal signals, which play pivotal roles not only in the regulation of energy balance but also in the modulation of liver glucose output. Bidirectional changes in hypothalamic insulin signalling therefore result in parallel changes in both energy balance and glucose metabolism. Here we show that activation of ATP-sensitive potassium (K(ATP)) channels in the mediobasal hypothalamus is sufficient to lower blood glucose levels through inhibition of hepatic gluconeogenesis. Finally, the infusion of a K(ATP) blocker within the mediobasal hypothalamus, or the surgical resection of the hepatic branch of the vagus nerve, negates the effects of central insulin and halves the effects of systemic insulin on hepatic glucose production. Consistent with these results, mice lacking the SUR1 subunit of the K(ATP) channel are resistant to the inhibitory action of insulin on gluconeogenesis. These findings suggest that activation of hypothalamic K(ATP) channels normally restrains hepatic gluconeogenesis, and that any alteration within this central nervous system/liver circuit can contribute to diabetic hyperglycaemia.     

Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory cells

Biochemical and biophysical studies on Aplysia sensory neurons indicate that inhibitory responses to the molluscan peptide FMRFamide are mediated by lipoxygenase metabolites of arachidonic acid. These compounds are a new class of second messengers in neurons.     

Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization

Cardiac failure has a principal underlying aetiology of ischaemic damage arising from vascular insufficiency. Molecules that regulate collateral growth in the ischaemic heart also regulate coronary vasculature formation during embryogenesis. Here we identify thymosin beta4 (Tbeta4) as essential for all aspects of coronary vessel development in mice, and demonstrate that Tbeta4 stimulates significant outgrowth from quiescent adult epicardial explants, restoring pluripotency and triggering differentiation of fibroblasts, smooth muscle cells and endothelial cells. Tbeta4 knockdown in the heart is accompanied by significant reduction in the pro-angiogenic cleavage product N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Although injection of AcSDKP was unable to rescue Tbeta4 mutant hearts, it significantly enhanced endothelial cell differentiation from adult epicardially derived precursor cells. This study identifies Tbeta4 and AcSDKP as potent stimulators of coronary vasculogenesis and angiogenesis, and reveals Tbeta4-induced adult epicardial cells as a viable source of vascular progenitors for continued renewal of regressed vessels at low basal level or sustained neovascularization following cardiac injury.