Rapid kinetic analysis of transcription elongation by Escherichia coli RNA polymerase.

Nucleotide incorporation during transcription by RNA polymerase is accompanied by pyrophosphate formation. Rapid release of pyrophosphate from the elongation complex at a rate consistent with productive transcription elongation occurs only in the presence of the correct next nucleotide for incorporation into the transcript.

Cell culture monitoring by impedance mapping using a multielectrode scanning impedance spectroscopy system (CellMap).

We report on the impedance mapping of in vitro cellular morphology by electrical impedance spectroscopy, using microelectrodes. A micro multielectrode system was designed, fabricated, assembled, tested and demonstrated for the monitoring of anchorage-dependent cell behavior and morphology. This system allowed continuous, label-free, quantitative monitoring and visualization of cell adhesion, spreading, proliferation and detachment due to cell cycle processes as well as cell-drug interaction, with spatio-temporal resolution. OvCa429 ovarian cancer cells were monitored in vitro over a period of 70 hours by inoculating the cell suspension directly on the multielectrode device. The phase angle of impedance was observed to develop a distinctive shape as a result of cell attachment and proliferation. The shape of the phase angle curve reverted back to the pre-attachment shape upon detachment of cells from the substrate, caused by the addition of trypsin to the cell culture medium. The impedance data of the cell culture were then successfully modeled as a multi-parametric equivalent circuit. The model incorporated both interfacial and cell-layer impedance parameters. Upon addition of trypsin, the cell-layer parameters showed a marked decline and were eventually eliminated from the multi-parametric model, confirming the correlation of the model to the electrode-cell-electrolyte system. These experiments demonstrate the applicability of the impedance mapping technique in visualizing and quantifying physiological changes in the cell layer due to cellular processes as well as the effect of external chemical stimulus on cells (cell-drug interaction).

Role of the Pseudomonas aeruginosa las and rhl quorum-sensing systems in rhlI regulation.

In Pseudomonas aeruginosa the LasR-LasI and RhlR-RhlI quorum-sensing (QS) systems control expression of numerous virulence genes in a population density-dependent fashion. In this study, we investigated regulation of the autoinducer synthase gene rhlI, which is responsible for C(4)-HSL signal production. Primer extension analysis was used to map the rhlI transcriptional start site and an upstream regulatory region was identified. Expression studies revealed that (i) this regulatory region is important for rhlI expression and (ii) although the rhl QS system will induce rhlI, las is the dominant regulator. Furthermore, we found that control of rhlI in Escherichia coli is markedly different than in P. aeruginosa.

Evaluation of Hbr (MITE) markers for assessment of genetic relationships among maize ( Zea mays L.) inbred lines.

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker ( Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (

Use of epitope tags for routine analysis of transgene expression.

Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3' nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.

The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation.

The checkpoint kinase Chk1 is an important mediator of cell cycle arrest following DNA damage. The 1.7 A resolution crystal structures of the human Chk1 kinase domain and its binary complex with an ATP analog has revealed an identical open kinase conformation. The secondary structure and side chain interactions stabilize the activation loop of Chk1 and enable kinase activity without phosphorylation of the catalytic domain. Molecular modeling of the interaction of a Cdc25C peptide with Chk1 has uncovered several conserved residues that are important for substrate selectivity. In addition, we found that the less conserved C-terminal region negatively impacts Chk1 kinase activity.

Mammographically detected ductal carcinoma in situ treated with conservative surgery with or without radiation therapy: patterns of failure and 10-year results.

OBJECTIVE: The authors reviewed their institution's experience treating mammographically detected ductal carcinoma in situ (DCIS) of the breast with breast-conserving therapy (BCT) to determine 10-year rates of local control and survival, patterns of failure, and factors associated with outcome. SUMMARY BACKGROUND DATA: From January 1980 to December 1993, 177 breasts in 172 patients were treated with BCT for mammographically detected DCIS of the breast at William Beaumont Hospital, Royal Oak, Michigan. METHODS: All patients underwent an excisional biopsy, and 65% were reexcised. Thirty-one breasts (18%) were treated with excision alone, whereas 146 breasts (82%) received postoperative radiation therapy (RT). All patients undergoing RT received whole-breast irradiation to a median dose of 50.0 Gy. One hundred thirty-six (93%) received a boost to the tumor bed for a median total dose of 60.4 Gy. Median follow-up was 5.9 years for the lumpectomy alone group and 7.2 years for the lumpectomy + RT group. RESULTS: In the entire population, 15 patients had an ipsilateral breast recurrence. The 5- and 10-year actuarial rates of ipsilateral breast recurrence were 7.8% and 7.8% for lumpectomy alone and 8.0% and 9.2% for lumpectomy + RT, respectively. Eleven of the 15 recurrences developed within or immediately adjacent to the lumpectomy cavity and were designated as true recurrences or marginal misses (TMM). Four recurred elsewhere in the breast. Eleven of the 15 recurrences were invasive, whereas 4 were pure DCIS. Only one patient died of disease, yielding 5- and 10-year actuarial cause-specific survival rates of 100% and 99.2%, respectively. Eleven patients were diagnosed with subsequent contralateral breast cancer, yielding 5- and 10-year actuarial rates of 5.1% and 8.3%, respectively. Clinical, pathologic, and treatment-related factors were analyzed for an association with ipsilateral breast failure or TR/MM. No factors were significantly associated with ipsilateral breast failure. In the entire population, the omission of RT and younger age at diagnosis were significantly associated with TR/MM. Patients younger than 45 years at diagnosis had a significantly higher rate of TR/MM in both the lumpectomy + RT and lumpectomy alone groups. None of the 37 patients who received a postexcisional mammogram had an ipsilateral breast failure versus 15 in the patients who did not receive a postexcisional mammogram. CONCLUSIONS: Patients diagnosed with mammographically detected DCIS of the breast appear to have excellent 100-year rates of local control and overall survival when treated with BCT. These results suggest that the use of RT reduces the risk of local recurrence and that patients diagnosed at a younger age have a higher rate of local recurrence with or without the use of postoperative RT.

Reversible male sterility: a novel system for the production of hybrid corn.

Hybrid corn seed is traditionally produced using either mechanical/hand detasseling or cytoplasmic male sterility, or a combination of both. In recent years, the development of transgenic systems to produce hybrid seed in several crops has attracted much attention. Here we describe a transgenic mechanism for production of hybrid corn, reversible male sterility (RMS), in which the action of the cytotoxic gene used to introduce male sterility is suppressed by the application of a chemical to the plant. Reversion of the sterility allows the RMS parent to be self-fertilized, a step which overcomes the need to remove fertile sib plants prior to making the hybrid cross. The key enabling technology in RMS is the use of a plant gene promoter which is specifically induced by chemical application. We have exemplified RMS in transgenic corn plants and believe that it provides specific benefits in the production of hybrid corn seed.

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment.

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 x B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by the bar or pat genes, was more efficient than selection on kanamycin, mediated by the nptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants--transgene silencing, as well as poor transgene transmission to progeny was observed in some plant lines in which the parent plants had expressed the transgene.

Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific expression of the TMV coat protein in transgenic tobacco plants affects the level of coat protein-mediated virus protection.

Transgenic tobacco plants were produced that express a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) under the control of the promoter from a ribulose bisphosphate carboxylase small subunit (rbcS) gene. Plant lines expressing comparable levels of CP from the rbcS and cauliflower mosaic virus 35S promoters were compared for resistance to TMV. In whole plant assays the 35S:CP constructs gave higher resistance than the rbcS:CP constructs. On the other hand, leaf mesophyll protoplasts isolated from both plant lines were equally resistant to infection by TMV. This indicated that the difference in resistance between the lines in the whole plant assay reflects differences at the level of short- and/or long-distance spread of TMV. Therefore, we propose that the difference in tissue-specific expression between the 35S and rbcS promoters accounts for greater resistance in the plant lines that express the 35S:CP chimeric genes.

Effect of protein aggregation state on coat protein-mediated protection against tobacco mosaic virus using a transient protoplast assay.

To address the mechanism(s) of protection against tobacco mosaic virus (TMV) infection conferred by expression of the TMV capsid protein (CP) gene in transgenic tobacco plants, a transient protection assay has been developed. Introduction of either purified viral CP or virus inactivated by ultraviolet irradiation into tobacco protoplasts induced a transient protection to challenge virus introduced concomitantly or shortly thereafter. The transient protection was characterized and the effects of different aggregation states of TMV CP were tested in the transient assay system. Tobacco mosaic virus CP preparations composed largely of helical, virus-like, aggregates conferred a less transient protection against TMV and greater protection against a distantly related virus than did preparations composed primarily of smaller aggregates.

Resistance to TMV in transgenic plants results from interference with an early event in infection.

Constitutive expression of the tobacco mosaic virus (TMV) coat protein (CP) gene in transgenic tobacco plants results in inhibition of disease symptom development following inoculation with TMV. Evidence is presented here that this protection is also observed in leaf mesophyll protoplasts isolated from these plants. Protoplasts were resistant to infection by TMV at concentrations of 10 microgram/ml to 1 mg/ml when introduced by either electroporation or polyethylene glycol-mediated inoculation. There was little protection against infection by TMV RNA and the protection was lost as the concentration of TMV RNA in the inoculum increased. When virus was incubated briefly at pH 8.0 prior to inoculation, protection broke down in a manner similar to that observed following RNA inoculation. Analogous results were obtained in experiments with whole plants. Because virus treated in this manner has presumably lost little or no CP, these results suggest that expression of the TMV CP gene in transgenic plant cells prevents TMV from uncoating. A model is presented for the mechanism of this blockage which relates these results to early events in TMV infection.

Direct visualization of RecA protein binding to and unwinding duplex DNA following the D-loop cycle.

The RecA protein of Escherichia coli will promote the plectonemic joining of a linear single-stranded DNA molecule with a homologous supertwisted double-stranded (ds) DNA molecule. As shown by others, this reaction is characterized by a single cycle of joint formation and dissociation, termed the D-loop cycle. The released DNA products appear by electron microscopy to be topologically identical to the reactant DNAs, yet a second cycle of joining is not observed. This implies that either the RecA protein-single-stranded DNA filament or the dsDNA must be altered during the pairing reaction such that further joint formation is inhibited. Shibata et al. (Shibata, T., DasGupta, C., Cunningham, R. P., Williams, J. K. G., Osber, L., and Radding, C. M. (1982) J. Biol. Chem. 256, 7565-7572) proposed that the dsDNA was inactivated due to the binding of RecA protein following the D-loop cycle, but were unable to describe the structure of the putative RecA-protein-dsDNA complex. Here we have extended those studies to show that if fresh dsDNA is added to a reaction mixture following completion of the D-loop cycle, joint formation is stimulated, but only with the freshly added dsDNA. Following completion of the D-loop cycle, a labile RecA protein-dsDNA complex, in which the dsDNA is partially unwound can be preserved by glutaraldehyde fixation and visualized by electron microscopy. This result provides direct evidence that the block to a second cycle of joining is due to the presence of RecA protein remaining bound to the released dsDNA.

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange.

The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.