Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers.

5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but is deficient in many cancers. Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map. Homozygous deletions of p16 and p15 are frequent malignant cell lines. However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown. We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR. The MTAP gene consists of eight exons and seven introns. Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions. Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL). A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4. Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB). These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes.

Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette.

We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.

Multiplex genotype determination at a large number of gene loci.

To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Etiolated cells of Chlamydomonas reinhardtii: choice material for characterization of mitochondrial membrane polypeptides.

We have investigated a light-conditional mutant of Chlamydomonas reinhardtii (J12) that is unable to synthesize chlorophyll in the dark with the aim of characterizing the mitochondrial membrane polypeptides of this alga. A crude membrane fraction derived from etiolated cells was analyzed by gel electrophoresis, immunoblot analysis, and pulse-labeling in the presence of specific protein synthesis inhibitors. This fraction contained both mitochondrial and etioplast membranes, and the latter contained appreciable amounts of subunits of the cytochrome b6f complex. The mitochondria-encoded subunit 1 of cytochrome-c oxidase called COX1 was identified, and its synthesis was detected in this membrane fraction. The redox-difference spectra of mitochondrial cytochromes were studied in whole cells and membrane fractions, in both respiratory-competent and -deficient strains. Mitochondrial membranes could be further purified after sucrose gradient centrifugation. The use of etiolated cells and their membrane extracts, in association with appropriate methodologies, opens ways to study the molecular genetics of mitochondria in C. reinhardtii and allows us to address the question of the cooperation established between the three genetic compartments of a plant cell.

Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis.

Hypoxia/reoxygenation is an important cause of tissue injury in a variety of organs and is classically considered to be a necrotic form of cell death. We examined the role of endonuclease activation, considered a characteristic feature of apoptosis, in hypoxia/reoxygenation injury. We demonstrate that subjecting rat renal proximal tubules to hypoxia/reoxygenation results in DNA strand breaks and DNA fragmentation (both by an in situ technique and by agarose gel electrophoresis), which precedes cell death. Hypoxia/reoxygenation resulted in an increase in DNA-degrading activity with an apparent molecular mass of 15 kDa on a substrate gel. This DNA-degrading activity was entirely calcium dependent and was blocked by the endonuclease inhibitor aurintricarboxylic acid. The protein extract from tubules subjected to hypoxia/reoxygenation cleaved intact nuclear DNA obtained from normal proximal tubules into small fragments, which further supports the presence of endonuclease activity. Despite unequivocal evidence of endonuclease activation, the morphologic features of apoptosis, including chromatin condensation, were not observed by light and electron microscopy. Endonuclease inhibitors, aurintricarboxylic acid and Evans blue, provided complete protection against DNA damage induced by hypoxia/reoxygenation but only partial protection against cell death. Taken together, our data provide strong evidence for a role of endonuclease activation as an early event, which is entirely responsible for the DNA damage and partially responsible for the cell death that occurs during hypoxia/reoxygenation injury. Our data also indicate that in hypoxia/reoxygenation injury endonuclease activation and DNA fragmentation occur without the morphological features of apoptosis.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.

The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Phosphorylation of ubiquitin-activating enzyme in cultured cells.

Ubiquitin-activating enzyme, E1, is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 exists as two isoforms in human cells which are separable by electrophoresis. These isoforms migrate with apparent molecular sizes of 110 kDa and 117 kDa in SDS/polyacrylamide gels. Immunoprecipitation of E1 from lysates of HeLa cells metabolically labeled with [32P]phosphate indicated the presence of a phosphorylated form of E1 which migrates at 117 kDa. Phospho amino acid analysis identified serine as the phosphorylated residue in E1. Phosphorylated E1 was also detected in normal and transformed cells from another human cell line. Phosphatase-catalyzed dephosphorylation of E1 in vitro did not eliminate the 117-kDa E1 isoform detected by Coomassie staining after SDS/polyacrylamide gel electrophoresis, thereby demonstrating that phosphorylation is not the sole structural feature differentiating the isoforms of E1. These observations suggest new hypotheses concerning mechanisms of metabolic regulation of the ubiquitin conjugation pathway.

Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

UNLABELLED Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but thatbokwas not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found thatbok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injuryin vitroand seizure-induced neuronal injuryin vivo Deletion ofbokalso increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death inbax-deficient neurons. Single-cell imaging demonstrated thatbok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bokdeficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death inbok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injuryin vitroandin vivo SIGNIFICANCE STATEMENT Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok controls neuronal Ca(2+)homeostasis and bioenergetics and, contrary to previous assumptions, exerts neuroprotective activitiesin vitroandin vivo Our results demonstrate that Bok cannot be placed unambiguously into the Bax-like Bcl-2 subfamily of pro-apoptotic proteins.

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhIRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown OS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Delta/as mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-1) could not be detected when any of the lasRI or rhIRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of OS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a CS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06