An in vivo screening system to identify tumorigenic genes.

Screening for oncogenes has mostly been performed by in vitro transformation assays. However, some oncogenes might not exhibit their transforming activities in vitro unless putative essential factors from in vivo microenvironments are adequately supplied. Here, we have developed an in vivo screening system that evaluates the tumorigenicity of target genes. This system uses a retroviral high-efficiency gene transfer technique, a large collection of human cDNA clones corresponding to ~70% of human genes and a luciferase-expressing immortalized mouse mammary epithelial cell line (NMuMG-luc). From 845 genes that were highly expressed in human breast cancer cell lines, we focused on 205 genes encoding membrane proteins and/or kinases as that had the greater possibility of being oncogenes or drug targets. The 205 genes were divided into five subgroups, each containing 34-43 genes, and then introduced them into NMuMG-luc cells. These cells were subcutaneously injected into nude mice and monitored for tumor development by in vivo imaging. Tumors were observed in three subgroups. Using DNA microarray analyses and individual tumorigenic assays, we found that three genes, ADORA2B, PRKACB and LPAR3, were tumorigenic. ADORA2B and LPAR3 encode G-protein-coupled receptors and PRKACB encodes a protein kinase A catalytic subunit. Cells overexpressing ADORA2B, LPAR3 or PRKACB did not show transforming phenotypes in vitro, suggesting that transformation by these genes requires in vivo microenvironments. In addition, several clinical data sets, including one for breast cancer, showed that the expression of these genes correlated with lower overall survival rate.

Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency.

A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5' and 3' ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1 at the 5' and 3' sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1 enhances the integration or expression of the transgene, or both. Clearly, TJ1 is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed.

Short communication: emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO3-, NO2- or NH4+ -containing medium was investigated. 15N contents of gas sampled from gas-sealed pots, in which the plants were grown on 15N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC-MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO3--containing medium. N2O emission from clone 271 grown on NO3--containing medium was detected, but not from the wild-type plants. The N2O emission rate of clone 271 was 106 ng N2O mg-1 incorporated N week-1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild-type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2--containing medium and its emission rate increased with increasing NO2- levels in plants. We speculate that NO3- is reduced to NO2- and that a part of NO2- is metabolized to N2O in clone 271.

Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices.

To clarify the molecular structure of the integration sites of transgenes, we used particle bombardment to examine the DNA sequences of transgene loci. Three transgenic Arabidopsis lines gave a single Southern hybridization band with a selectable gene as the probe. Junction regions flanked by the transgenes were cloned by the inverse polymerase chain reaction method, and the characteristics of the DNA sequences of the 10 junction regions were investigated. All but two of these were AT-rich sequences bearing motifs characteristic of a scaffold/matrix-attachment region (S/MAR). Calculations showed that seven of them should have a propensity for curvature. An assay of in-vitro binding to tobacco nuclear matrices showed that all the junction regions bound to nuclear matrices and that the two input DNAs did not bind. The 12 chromosome/transgene (CT) junctions in these three transgene loci were investigated. Cleavage sites for topoisomerase I were found at 10 of the 12, near the junction point. The other two junctions had sites within 6bp of the junction point. The sequence near one terminal of the transgene in the transgene loci was compared with that near the other terminal. Short, direct repeats consisting of 4-6bp were present within 10bp of the junction points in the sequence. We speculate that the transgene introduced by particle bombardment is delivered on AT-rich S/MAR that has a propensity for curvature, and then a nucleotide near the short, direct repeat on the transgene is joined near the cleavage sites on the genome for topoisomerase I.

Preferential replication-dependent mutagenesis in the lagging DNA strand in Escherichia coli.

The mutation frequencies attributable to -1 frameshift or one-base substitution in the structural genes coding for resistance to chloramphenicol (Cm) and tetracycline (Tc) were followed over several cycles of DNA replication, and found to differ several-fold, depending on the orientation of the gene on the plasmid with respect to the direction of (unidirectional ColE1-type) replication. The mutation frequency was higher when the reporter gene was present in the plasmid in the same orientation as the direction of the origin, i.e., when the transcription template is the lagging daughter strand, than when the gene was inserted in the opposite orientation. This significant difference in reversion frequencies of genes with different polarities was demonstrated only for a brief period of cell growth (several cycles of replication) after induction of the dnaQ49 mutator, but was not observed when an increased number of replication cycles, was permitted, most probably due to fixation of the mutation into both strands. The mutated intermediate DNA which possesses a misaligned basepair in the Cm gene was demonstrated to be replicated into two progeny DNA molecules; one is the chloramphenicol-resistant (CmR) DNA synthesized from the template strand having the mutation and the other is the CmS DNA from the template strand without mutation. Our results suggest that replication-dependent mutagenesis may occur preferentially in the lagging strand.

Role of HU proteins in forming and constraining supercoils of chromosomal DNA in Escherichia coli.

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12-13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying both gyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.

Construction of a gyrA-gyrB-carrying plasmid overproducing functional DNA gyrase.

A plasmid carrying both gyrA and gyrB genes (pTS7) and a plasmid without the gyr gene (pTS4) were constructed. Introduction of pTS7 into the YK1100 (wild-type) cells resulted in an increase in the level of gyrA and gyrB mRNA by 5- to 6-fold over the level of the control transformant with pTS4. In the transformant cells carrying pTS7, the reporter plasmid pGP241, which is compatible with pTS plasmids, was significantly more highly negatively supercoiled than in the transformant with pTS4. The activity of DNA gyrase to supercoil the relaxed pGP241 DNA was 4-8 times higher with the S-30 extract from the transformant carrying pTS7 than with the extract from the transformant with pTS4.

Autoregulation of transcription of the hupA gene in Escherichia coli: evidence for steric hindrance of the functional promoter domains induced by HU.

The molecular mechanism of autoregulation of expression of the hupA gene in Escherichia coli was examined. The promoter of the gene contains a palindromic sequence with the potential to form a cruciform DNA structure in which the -35 sequence lies at the base of the stem and the -10 sequence forms a single-stranded loop. An artificial promoter lacking the palindrome, which was constructed by replacing a 10 nucleotide repeat for the predicted cruciform arm by a sequence in the opposite orientation, was not subject to HU-repression. DNA relaxation induced by deleting HU proteins and/or inhibiting DNA gyrase in cells results in increased expression from the hupA promoter. We propose that initiation of transcription of the hupA gene is negatively regulated by steric hindrance of the functional promoter domains for formation of the cruciform configuration, which is facilitated at least in part by negative supercoiling of the hupA promoter DNA region. The promoter region of the hupB gene also contains a palindromic sequence that can assume a cruciform configuration. Negative regulation of this gene by HU proteins may occur by a mechanism similar to that operating for the hupA gene.

IHF supresses the inhibitory effect of H-NS on HU function in the hin inversion system.

In the hin-mediated DNA inversion system, HU facilitates formation of the synaptic complex composed of two recombination sites spaced 996 bp apart and of the enhancer situated between them, by looping the DNA as to promote interaction of Hin invertase with the Fis enhancer factor [Johnson et al., Nature 329 (1987) 462-465]. The HU requirement for the in vivo hin-mediated inversion was demonstrated previously [Wada et al., Gene 76 (1989) 345-352; Hillyard et al., J. Bacteriol. 172 (1990) 5402-5407; Haykinson and Johnson, EMBO J. 12 (1993) 2503-2512] and in the current experiments. This HU action, however, required IHF when H-NS was present in the cell; i.e., the inversion reaction of the hin-invertible DNA fragment carried by the pKK1202R plasmid proceeded efficiently in host cells either deficient in H-NS or in the presence of both H-NS and IHF, but not in the cells depleted for IHF alone. The level of hin mRNA in mutant cells lacking HU or IHF, in which hin inversion did not occur, was normal or slightly increased. When IHF was absent, the stimulating effect of HU on in vitro DNA circle formation of a 125-bp hin fragment between hixL and the enhancer where Fis binds was inhibited by H-NS. The present study provides an example of a multi-component interaction between HU, H-NS and IHF on the hin DNA region, which contains three characteristic sites, a d(A/T)4 stretch and bent DNA site, and two putative IHF-binding sites.

Properties of DNA-binding of HU heterotypic and homotypic dimers from Escherichia coli.

The interactions of three forms of HU dimer from Escherichia coli with DNA were compared. The complexes formed between HU and short DNA fragments (35 to 132 bp), uncurved oligodeoxyribonucleotide (oligo) with and without 5-bp deoxyriboadenosine (dA) stretches and curved oligo with 5-bp dA stretches, were analyzed by the gel retardation technique. The binding of HU homodimers to the DNA was less efficient than that of HU heterodimer, and the HU-1 homodimer had lower DNA-binding capacity than the HU-2 homodimer. The equilibrium dissociation constant (KD) for DNA of the HU-1 homodimer was 3 times that of the HU heterodimer. The HU dimers had two- to fourfold higher affinity for DNA duplexes containing 5-bp dA stretches, particularly for curved DNA. The binding of HU heterodimer to the DNA was inhibited more by the curved DNA competitor than the uncurved DNA competitor without dA stretches.

Proton NMR study on a histone-like protein, HU alpha, from Escherichia coli and its complex with oligo DNAs.

It was confirmed that the flexible arm region of HU alpha forms an antiparallel beta-sheet and that all of the residues of phenylalanines, together with some of leucines and/or valines, form a hydrophobic core within the dimer of HU alpha. HU alpha protein alone is thermally labile and melts at 38 degrees C, but it becomes remarkably stabilized and melts at 59 degrees C in the presence of DNA. Several resonances from both HU alpha and DNA perturbed by their complex formation, notably those of His C-2 and C-4 protons, downfield shifted C alpha protons in the antiparallel beta-sheet, as well as Arg C delta and Lys C epsilon protons. The results indicated that a beta-sheet region of HU alpha binds to DNA, and also showed that rapid equilibrium occurs on the NMR time scale between bound and unbound states of HU alpha. A few intermolecular nuclear Overhauser effects (NOEs) were also observed between the protein and H1' protons of DNA in the complex, suggesting that HU alpha binds primarily to the minor groove of DNA.

Histone-like proteins are required for cell growth and constraint of supercoils in DNA.

The gene hns of Escherichia coli K-12, which encodes the histone-like protein H-NS, was inactivated by insertion of a DNA (gene hph) encoding hygromycin phosphotransferase. The growth rates of two mutants lacking either one or the other of the histone-like proteins, HU and IHF, were not affected by introduction of the hns mutation. However, cells depleted of HU, IHF and H-NS simultaneously, could not be constructed by P1 phage-mediated transduction. These results, together with our previous finding that cells deficient in both HU and IHF are viable at 30-37 degrees C [Kano and Imamoto, Gene 89 (1990) 133-137] showed that E. coli cells deficient in any two of these three histone-like proteins are viable at 30-37 degrees C, and suggested that simultaneous deficiency of all three of the proteins is lethal. There were no detectable differences in the levels of superhelicity of the reporter plasmids isolated from cells deficient in either IHF or H-NS, or from wild-type cells, but about 15% decrease in negative superhelicity was detected for the reporter plasmid isolated from cells lacking HU and lacking both HU and H-NS. However, the cryptic bgl operon, whose expression was reported to be regulated through topological changes of cellular DNA, could not be activated in cells depleted of HU or IHF. The bgl operon was expressed in cells depleted of both HU and H-NS as well as in cells depleted of H-NS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.

Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.

Chimeric HU-IHF proteins that alter DNA-binding ability.

Chimeric proteins between Escherichia coli histone-like HU and IHF were constructed by genetic engineering, in which part of the arm region was replaced by the corresponding region of IHF alpha (designated as HupANhimA) or IHF beta (HupANhimD); alternatively, an alpha-helix 2-beta 1 region was replaced by the corresponding region of IHF alpha (HupAXhimA) or IHF beta (HupAXhimD) (symbols N and X indicate NotI and XhoI junctions). These proteins were synthesized in a hupA-hupB double-deletion mutant. HupANhimA exhibited marked reduction in nonspecific DNA binding in vitro, and a drastic loss of HU activity in replicative transposition of Mu phage in vivo. HupANhimD also showed a significant reduction in the ability for DNA binding, though this protein supported Mu phage development. In contrast, the other two chimeric HU proteins showed only slight changes in nonspecific DNA-binding ability: they retained activities for transposition of Mu phage in vivo. These observations confirm that the flexible arm of HU-2, a domain proposed for DNA binding [Tanaka et al., Nature 310 (1984) 376-381; Goshima et al., Gene 96 (1990) 141-145], plays an important role in the physiological function of this protein. The results indicate that a unique conformation of the arm structure of HU protein, particularly the N-terminal half of a two-strand antiparallel beta-ribbon of the structure, is important for the DNA-binding ability of this protein.

Construction and characterization of an Escherichia coli mutant with a deletion of the metZ gene encoding tRNA (f1Met).

The Escherichia coli metZ gene encoding tRNA (f1Met) was replaced by the chloramphenicol-resistance-encoding gene. The resulting mutant exhibited slightly lower growth rates as compared to the wild type at 37 degrees C or 42 degrees C, but grew apparently slower than the latter at 30 degrees C, indicating a slight cold sensitivity of growth. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or trpA'::lac' Z fusion gene, in the metZ deletion mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB' ::lac'Z fusion gene, whose start codons are GUG, were also synthesised in the deletion mutant. These results provide evidence that tRNA (f1Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA (f1Met), a minor N-formyl methionine-specific tRNA, in the tRNA (f1Met)-depleted cells.

HU-1 mutants of Escherichia coli deficient in DNA binding.

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.

Characterization of HU-like protein from Bifidobacterium longum.

A HU-like protein (HBl) of Bifidobacterium longum was purified and characterized. HBl is heat-stable and acid-resistant, and has a molecular weight of about 9.1 kDa as estimated by its mobility on electrophoresis. HBl is intermediate in basicity (pI 9.8) between the HU-1 and HU-2 proteins of Escherichia coli, and is dissociated from a calf thymus DNA-cellulose column at 300-400 mM NaCl. Its amino acid composition shows many similarities with that of E coli HU. The NH2-terminal amino acid sequence of HBl also shows significant similarities to the consensus sequence deduced from the sequences of eleven HU-like proteins from prokaryotic sources. Chemical crosslinking analysis indicated that the HBl protein predominantly forms a homotypic dimer.

Participation of hup gene product in replicative transposition of Mu phage in Escherichia coli.

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that mutator phage Mucts62 was unable to replicate in a hupA hupB double mutant, although it could replicate in hupA or hupB single mutant as efficiently as in the wild-type strain. Mucts62 was able to lysogenize the double mutant at 30 degrees C; cell killing occurred when the lysogen was incubated at 42 degrees C, but did not result in phage production. High-frequency non-replicative integration of Mu into host genomic DNA soon after infection could not be detected in the hupAB double mutant. These results provide the evidence that HU protein is essential for replicative transposition of Mu phage in E. coli, and also participates in high-frequency conservative integration.