Structural Genomics of Minimal Organisms and Protein Fold Space

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of ‘unique’ protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated ‘hypothetical proteins’ inferred their molecular functions.     

Differential network expression during drug and stress response

MOTIVATION: The application of microarray chip technology has led to an explosion of data concerning the expression levels of the genes in an organism under a plethora of conditions. One of the major challenges of systems biology today is to devise generally applicable methods of interpreting this data in a way that will shed light on the complex relationships between multiple genes and their products. The importance of such information is clear, not only as an aid to areas of research like drug design, but also as a contribution to our understanding of the mechanisms behind an organism's ability to react to its environment. RESULTS: We detail one computational approach for using gene expression data to identify response networks in an organism. The method is based on the construction of biological networks given different sets of interaction information and the reduction of the said networks to important response sub-networks via the integration of the gene expression data. As an application, the expression data of known stress responders and DNA repair genes in Mycobacterium tuberculosis is used to construct a generic stress response sub-network. This is compared to similar networks constructed from data obtained from subjecting M.tuberculosis to various drugs; we are thus able to distinguish between generic stress response and specific drug response. We anticipate that this approach will be able to accelerate target identification and drug development for tuberculosis in the future. CONTACT: chris@lanl.gov SUPPLEMENTARY INFORMATION: Supplementary Figures 1 through 6 on drug response networks and differential network analyses on cerulenin, chlorpromazine, ethionamide, ofloxacin, thiolactomycin and triclosan. Supplementary Tables 1 to 3 on predicted protein interactions. http://www.santafe.edu/~chris/DifferentialNW.     

Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples

Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.     

Chromosome segregation: Aurora B gets Tousled

Aurora B kinases play important roles during mitosis in eukaryotic cells; new work in Caenorhabditis elegans has identified the Tousled kinase TLK-1 as a substrate activator of the model nematode's Aurora B kinase AIR-2 which acts to ensure proper chromosome segregation during cell division.     

The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato

The necessity to develop potato and tomato crops that possess durable resistance against the oomycete pathogen Phytophthora infestans is increasing as more virulent, crop-specialized and pesticide resistant strains of the pathogen are rapidly emerging. Here, we describe the positional cloning of the Solanum bulbocastanum-derived Rpi-blb2 gene, which even when present in a potato background confers broad-spectrum late blight resistance. The Rpi-blb2 locus was initially mapped in several tetraploid backcross populations, derived from highly resistant complex interspecific hybrids designated ABPT (an acronym of the four Solanum species involved:S. acaule, S. bulbocastanum, S. phureja and S. tuberosum), to the same region on chromosome 6 as the Mi-1 gene from tomato, which confers resistance to nematodes, aphids and white flies. Due to suppression of recombination in the tetraploid material, fine mapping was carried out in a diploid intraspecific S. bulbocastanum F1 population. Bacterial artificial chromosome (BAC) libraries, generated from a diploid ABPT-derived clone and from the resistant S. bulbocastanum parent clone, were screened with markers linked to resistance in order to generate a physical map of the Rpi-blb2 locus. Molecular analyses of both ABPT- and S. bulbocastanum-derived BAC clones spanning the Rpi-blb2 locus showed it to harbor at least 15 Mi-1 gene homologs (MiGHs). Of these, five were genetically determined to be candidates for Rpi-blb2. Complementation analyses showed that one ABPT- and one S. bulbocastanum-derived MiGH were able to complement the susceptible phenotype in both S. tuberosum and tomato. Sequence analyses of both genes showed them to be identical. The Rpi-blb2 protein shares 82% sequence identity to the Mi-1 protein. Significant expansion of the Rpi-blb2 locus compared to the Mi-1 locus indicates that intrachromosomal recombination or unequal crossing over has played an important role in the evolution of the Rpi-blb2 locus. The contrasting evolutionary dynamics of the Rpi-blb2/Mi-1 loci in the two related genomes may reflect the opposite evolutionary potentials of the interacting pathogens.     

Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.     

APOBEC3G/CEM15 (hA3G) mRNA levels associate inversely with human immunodeficiency virus viremia

APOBEC3G/CEM15 (hA3G) is a novel host factor that confers resistance to lentiviral infection under experimental conditions. Human immunodeficiency virus (HIV) type 1, however, produces viral infectivity factor (Vif) that targets hA3G for proteolysis, thereby escaping this defense system. To examine hA3G's contribution to the protection against HIV disease progression in humans, we quantified hA3G mRNA levels in peripheral blood mononuclear cells from 6 HIV-uninfected and 25 HIV-infected subjects; the latter group included 8 long-term nonprogressors (LTNPs) and 17 progressors. None of the HIV-infected subjects were receiving antiretroviral therapy. We found a striking inverse correlation between hA3G mRNA levels and HIV viral loads (P ≤ 0.00009) and a highly significant positive correlation between hA3G mRNA levels and CD4 cell counts (P ≤ 0.00012) in these patients. Furthermore, we discovered that the order of hA3G mRNA levels is LTNPs > HIV-uninfected subjects > progressors.     

A computational method to detect epistatic effects contributing to a quantitative trait

We develop a new computational method to detect epistatic effects that contribute to a complex quantitative trait. Rather than looking for epistatic effects that show statistical significance when considered in isolation, we search for a close approximation to the quantitative trait by a sum of epistatic effects. Our search algorithm consists of a sequence of random walks around the space of sums of epistatic effects. An important feature of our approach is that there is learning between random walks, i.e. the control mechanism that chooses steps in our random walks adapts to the experiences of earlier random walks. We test the effectiveness of our algorithms by applying them to synthetic datasets where the phenotype is a sum of epistatic effects plus normally distributed noise. Our test statistic is the rate of success that our methods achieve in identifying the underlying epistatic effects. We report on the effectiveness of our methods as we vary parameters that are intrinsic to the computation (length of random walks and degree of learning) as well as parameters that are extrinsic to the computation (number of markers, number of individuals, noise level, architecture of the epistatic effects).     

Changes in the extracellular proteome caused by the absence of the bldA gene product, a developmentally significant tRNA, reveal a new target for the pleiotropic regulator AdpA in Streptomyces coelicolor

The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.