A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

Ribosomal proteins from the yeast Saccharomyces cerevisiae were separated, on a preparative scale, by ion-exchange h.p.l.c. Proteins from the small and large ribosomal subunits were resolved, respectively, into 33 and 23 peaks, and most of the proteins present in these peaks were identified by using one- and two-dimensional gel electrophoresis. Several of the peaks appeared to contain a single protein uncontaminated by other species. Ribosomal proteins were also separated by using reverse-phase h.p.l.c. Analysis of the peaks resolved indicated that the order of elution for the proteins of both ribosomal subunits is, in certain cases, different for each of the two h.p.l.c. techniques used. Thus a combination of the two chromatographic methods employed here has the potential to facilitate the rapid and preparative separation of each of the proteins present in yeast ribosomes.     

Syntenic conservation between humans and cattle. I. Human chromosome 9

Bovine X hamster hybrid somatic cells have been used to investigate the syntenic relationship of nine loci in the bovine that have homologous loci on human chromosome 9. Six loci, ALDH1, ALDOB, C5, GGTB2, GSN, and ITIL, were assigned to the previously identified bovine syntenic group U18 represented by ACO1, whereas the other three loci, ABL, ASS, and GRP78, mapped to a new, previously unidentified autosomal syntenic group. Additionally, a secondary locus, ABLL, which cross-hybridized with the ABL probe, was mapped to bovine syntenic group U1 with the HSA 1 loci PGD and ENO1. The results predict that ACO1 will map proximal to ALDH1; GRP78 distal to ITIL and C5; GSN proximal to AK1, ABL, and ASS on HSA 9; GRP78 to MMU 2; and ITIL and GSN to MMU 4.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sj?gren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of approximately 9.5 and temperature of approximately 35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25-30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.     

The collaborative cross: a recombinant inbred mouse population for the systems genetic era

The mouse is the most extensively used mammalian model for biomedical and aging research, and an extensive catalogue of laboratory resources is available to support research using mice: classical inbred lines, genetically modified mice (knockouts, transgenics, and humanized mice), selectively bred lines, consomics, congenics, recombinant inbred panels, outbred and heterogeneous stocks, and an expanding set of wild-derived strains. However, these resources were not designed or intended to model the heterogeneous human population or for a systematic analysis of phenotypic effects due to random combinations of uniformly distributed natural variants. The Collaborative Cross (CC) is a large panel of recently established multiparental recombinant inbred mouse lines specifically designed to overcome the limitations of existing mouse genetic resources for analysis of phenotypes caused by combinatorial allele effects. The CC models the complexity of the human genome and supports analyses of common human diseases with complex etiologies originating through interactions between allele combinations and the environment. The CC is the only mammalian resource that has high and uniform genomewide genetic variation effectively randomized across a large, heterogeneous, and infinitely reproducible population. The CC supports data integration across environmental and biological perturbations and across space (different labs) and time.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Phenotypic variation resulting from a deficiency of epidermal growth factor receptor in mice is caused by extensive genetic heterogeneity that can be genetically and molecularly partitioned

The timing of lethality caused by homozygosity for a null allele of the epidermal growth factor receptor (Egfrtm1Mag) in mice is strongly dependent on genetic background. Initial attempts to genetically map background modifiers using Swiss-derived, outbred CD-1 mice were unsuccessful. To investigate the genetic architecture contributing to survival of Egfrtm1Mag homozygous embryos, the genetic variability segregating within the outbred population was partitioned by surveying viability of Egfrtm1Mag mutants using intercrosses between 129S6/SvEvTAC-Egfrtm1Mag and nine Swiss-derived, inbred strains: ALR/LtJ, ALS/LtJ, APN, APS, ICR/HaRos, NOD/LtJ, NON/LtJ, SJL/J, and SWR/J. The observations showed that these strains support varying levels of survival of Egfrtm1Mag homozygous embryos, suggesting that genetic heterogeneity within the CD-1 stock contributed to the original lack of Egfrtm1Mag modifier detection. Similar to the Swiss-derived intercrosses, nine congenic strains, derived from 129S6/SvEvTAC, AKR/J, APN, BALB/cJ, BTBR-T+ tf/tf, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ inbred backgrounds, also supported varying levels of survival of Egfrtm1Mag mutants. By intercrossing the congenic lines to create hybrid F1 embryos, different genetic backgrounds were found to have complementary modifiers. Analysis of the congenic lines argues against heterosis of outbred backgrounds contributing to Egfrtm1Mag phenotypic variability. A detailed analysis of the crosses suggests that modifiers function at three distinct stages of development. One class of modifiers supports survival of Egfrtm1Mag homozygous embryos to mid-gestation, another class supports development through the mid-gestation transition from yolk-sac to placental-derived nutrient sources, and a third class supports survival through later stages of gestation. Data from microarray analysis using RNA from wild-type and Egfrtm1Mag mutant placentas support the existence of extensive genetic heterogeneity and suggest that it can be molecularly partitioned. This method should be generally useful to partition heterogeneity contributing to other complex traits.