Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria

Mitochondria are tailored to meet the metabolic and signaling needs of each cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations to produce a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. The protein expression data were largely concordant with large-scale surveys of RNA abundance and both measures indicate tissue-specific differences in organelle composition. RNA expression profiles across tissues revealed networks of mitochondrial genes that share functional and regulatory mechanisms. We also determined a larger "neighborhood" of genes whose expression is closely correlated to the mitochondrial genes. The combined analysis identifies specific genes of biological interest, such as candidates for mtDNA repair enzymes, offers new insights into the biogenesis and ancestry of mammalian mitochondria, and provides a framework for understanding the organelle's contribution to human disease.     

Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities

The selective adsorption of supercoiled plasmid, open-circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes. Genomic DNA and open-circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid. As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid. The advantage of gyrolite adsorbents is their lower unit price and ability to selectively adsorb DNA impurities without binding supercoiled plasmid under certain conditions. The effects of ionic strength, temperature, chelating agent, divalent cation, and lyotropic salts on adsorption of highly purified plasmid are studied to understand the forces that bind DNA to gyrolite, a structure with hydrophilic and hydrophobic characteristics. The results indicate that DNA binding is governed by hydrogen bonding, electrostatic bridging with divalent cations, shielding of electrostatic repulsion, hydrophobic adsorption, and disruption of integral surface water layer on gyrolite. On the basis of results from a range of Hofmeister series salts, strongly hydrated anions may enhance DNA adsorption by promoting hydrophobic interactions between DNA and gyrolite. Conversely, the very weakly hydrated chaotrope I(-) may enhance adsorption by strongly associating with hydrophobic siloxanes of gyrolite, thereby disrupting an integral water layer, which competes for hydrogen bonding sites.     

Systematic detection of errors in genetic linkage data.

Construction of dense genetic linkage maps is hampered, in practice, by the occurrence of laboratory typing errors. Even relatively low error rates cause substantial map expansion and interfere with the determination of correct genetic order. Here, we describe a systematic method for overcoming these difficulties, based on incorporating the possibility of error into the usual likelihood model for linkage analysis. Using this approach, it is possible to construct genetic maps allowing for error and to identify the typings most likely to be in error. The method has been implemented for F2 intercrosses between two inbred strains, a situation relevant to the construction of genetic maps in experimental organisms. Tests involving both simulated and real data are presented, showing that the method detects the vast majority of errors.     

Identification of polymorphic simple sequence repeats in the genome of the zebrafish.

The zebrafish has drawn a great deal of attention as a developmental system because it offers the ability to combine excellent embryology and genetics. Here, we report that simple sequence repeats are abundant in the zebrafish genome and are highly polymorphic between two outbred lines, making them useful markers for the construction of a genetic map of this organism.     

Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan

Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMSF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMSF over half its surface, only the neurons on the treated half extended neurites. The active factor in CMSF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80 degrees C; it was stable to heating to 60 degrees C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with [35S]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparin sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparin sulfate proteoglycan and other molecules. CMSF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMSF.     

Reversible S-nitrosation and inhibition of HIV-1 protease.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.     

BB rat diabetes susceptibility and body weight regulation genes colocalize on Chromosome 2

The genetic etiology of Type 1 (insulin-dependent) diabetes mellitus is complicated by the apparent presence of several diabetes susceptibility genetic regions. Type 1 diabetes in the inbred BioBreeding (BB) rat closely resembles the human disorder and was previously shown to involve two genes: the lymphopenia (lyp) region on Chromosome (Chr) 4 and RT1 ^u in the major histocompatibility complex (MHC) on Chr 20. In addition, a segregation analysis of an F₂ intercross between the diabetes-prone congenic BB DR ^lyp/lyp,u/u and F344^{+/+, lv/lv} rats indicated that at least one more genetic factor was responsible for Type 1 diabetes. In this study, we generated F₂N₂ progeny in a cross between non-diabetic F₂(DR ^lyp/lyp,u/u × F344) ^lyp/lyp,u/u and diabetic DR ^lyp/lyp,u/u rats. In a subsequent total genome scan, a third factor was mapped to the 21.3-cM region on Chr 2 between D2Mit14 and D2Mit15 (peak LOD score 4.7 with 67% penetrance). Interestingly, the homozygosity of the BB allele (b/b) for the Chr 2 region was significantly associated with a greater weight reduction after fasting than the homozygosity of the F344 allele (f/f, p < 0.008). In conclusion, the development of Type 1 diabetes in the congenic DR ^lyp/lyp rat is controlled by at least three genes: lymphopenia, MHC, and a third factor that may play a role in metabolism and body weight regulation. Received: December 1998 / Accepted: 10 May 1999     

Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1’s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.     

A high-resolution linkage-disequilibrium map of the human major histocompatibility complex and first generation of tag single-nucleotide polymorphisms

Autoimmune, inflammatory, and infectious diseases present a major burden to human health and are frequently associated with loci in the human major histocompatibility complex (MHC). Here, we report a high-resolution (1.9 kb) linkage-disequilibrium (LD) map of a 4.46-Mb fragment containing the MHC in U.S. pedigrees with northern and western European ancestry collected by the Centre d'Etude du Polymorphisme Humain (CEPH) and the first generation of haplotype tag single-nucleotide polymorphisms (tagSNPs) that provide up to a fivefold increase in genotyping efficiency for all future MHC-linked disease-association studies. The data confirm previously identified recombination hotspots in the class II region and allow the prediction of numerous novel hotspots in the class I and class III regions. The region of longest LD maps outside the classic MHC to the extended class I region spanning the MHC-linked olfactory-receptor gene cluster. The extended haplotype homozygosity analysis for recent positive selection shows that all 14 outlying haplotype variants map to a single extended haplotype, which most commonly bears HLA-DRB1*1501. The SNP data, haplotype blocks, and tagSNPs analysis reported here have been entered into a multidimensional Web-based database (GLOVAR), where they can be accessed and viewed in the context of relevant genome annotation. This LD map allowed us to give coordinates for the extremely variable LD structure underlying the MHC.