A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

A JAK-STAT pathway regulates wing vein formation in Drosophila.

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.

An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death.

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.

Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma.

The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.

Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.

Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Neural tube defects and abnormal brain development in F52-deficient mice.

F52 is a myristoylated, alanine-rich substrate for protein kinase C. We have generated F52-deficient mice by the gene targeting technique. These mutant mice manifest severe neural tube defects that are not associated with other complex malformations, a phenotype reminiscent of common human neural tube defects. The neural tube defects observed include both exencephaly and spina bifida, and the phenotype exhibits partial penetrance with about 60% of homozygous embryos developing neural tube defects. Exencephaly is the prominent type of defect and leads to high prenatal lethality. Neural tube defects are observed in a smaller percentage of heterozygous embryos (about 10%). Abnormal brain development and tail formation occur in homozygous mutants and are likely to be secondary to the neural tube defects. Disruption of F52 in mice therefore identifies a gene whose mutation results in isolated neural tube defects and may provide an animal model for common human neural tube defects.

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.