Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

Normal levels of immunocompetence in possums (Trichosurus vulpecula) exposed to different laboratory housing conditions post capture

Specific and non-specific immunological tests were used to monitor aspects of the immune response in captive possums. The tests included total and differential white blood cell counts, lymphocyte transformation assay, and enzyme linked immunosorbant assay. The level of free cortisol present in possum plasma samples was evaluated as an endocrine marker for stress. Four different housing conditions were used to test whether stress could be managed or avoided in captive animals. Animals were caged individually or as groups in pens. Bacille Calmette-Gurein (BCG) and tetanus toxoid immunization was used to evoke primary cell mediated and antibody responses in test animals. The results indicated that there was no significant difference in immunological responses or endocrine parameters in animals held under any of the housing conditions. The results infer that wild possums adapt quickly post-capture to novel housing conditions and produce representative patterns of immunity when held in housing conditions and fed ad libitum.     

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction.     

Cyanogenic Eucalyptus nobilis is polymorphic for both prunasin and specific beta-glucosidases

Cyanogenesis (i.e. the evolution of HCN from damaged plant tissue) requires the presence of two biochemical pathways, one controlling synthesis of the cyanogenic glycoside and the other controlling the production of a specific degradative beta-glucosidase. The sole cyanogenic glycoside in Eucalyptus nobilis was identified as prunasin (D-mandelonitrile beta-D-glucoside) using HPLC and GC-MS. Seedlings from three populations of E. nobilis were grown under controlled conditions and 38% were found to be acyanogenic, a proportion far greater than reported for any other cyanogenic eucalypt. A detailed study of the acyanogenic progeny from a single open-pollinated parent found that 23% lacked a cyanogenic beta-glucosidase, 32% lacked prunasin and 9% lacked both. Of the remaining seedlings initially identified as acyanogenics, 27% contained either trace amounts of beta-glucosidase or prunasin, while 9% contained trace amounts of both. Results support the hypothesis that the two components necessary for cyanogenesis are inherited independently. Trace amounts are likely to result from the presence of non-specific beta-glucosidases or the glycosylation of the cyanohydrin intermediate by non-specific UDP glycosyl transferases.     

Increasing cell biomass in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

Background  

Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.     

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies,in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A_2A receptor (A_2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A_2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A_2AR controls. The A_2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.