The construction and replication properties of hybrid plasmids composed of the r-determinant of R100.1 and the plasmids pCRI or pSC201

Summary We have cloned the entire r-determinant of the antibiotic resistance plasmid R100.1 on the plasmid vectors pCR1 and pSC201. We find that the hybrid plasmids segregate from cultures in which replication of the vector is blocked. This suggests that the r-det is not capable of autonomous replication.     

Phytochrome control of its own accumulation and leaf expansion in tentoxin- and norflurazon-treated mung bean seedlings

Primary leaves of 4-day-old, dark-grown mung bean [ Vigna radiata (L.) Wilczek cv. Berken] seedlings were exposed to 24 h of white light (200 μmol m⁻² s⁻¹) which was terminated by a 15 min, phytochrome-saturating red or far-red light exposure. Phytochrome content (in vivo and in vitro) and leaf area were monitored during the subsequent dark period. Red light treatments resulted in lower phytochrome content and greater leaf expansion than did far-red treatments. Phytochrome accumulation and leaf expansion were less in norflurazon- (no carotenoids and very low Chl) than in tentoxin- (very low Chl) treated leaves. After 3 days of darkness, leaf expansion was about 25% greater and phytochrome content was about 50% less in red- than in far-red-treated leaves of all treatments. These effects generally took longer to develop in norflurazon- than in tentoxin-treated tissues. Norflurazon-treated tissues exposed to long white light periods apparently do not as accurately reflect phytochrome-controlled photomorphogenic events of green tissues as do tentoxin-treated tissues of mung bean seedlings.     

On the applicability of adaptive bioprocess state estimators

This article presents an industrial case study, examining the application of a novel adaptive biomass estimator to an industrial microfungi production process. It is our intention that this contribution should focus upon the implementation issues of the algorithm, in preference to a rigorous theoretical development. The novel algorithm adopted is developed from Adaptive Inferential Estimation studies of Guilandoust and co-workers. The technique utilizes input-output process measurements obtained at different frequencies, thereby providing more frequent estimates of biomass concentration than are otherwise available from off-line laboratory analyses. The algorithm is particularly suited to the biotechnology industry, as it is capable of utilizing irregular assay measurements with varying delays.Although this article demonstrates the encouraging industrial implications of the adaptive algorithm, like all adaptive techniques currently developed, it is restricted by the inability to perform robust on-line system identification. The ultimate selection of a "suboptimal" "fixed parameter" algorithm for on-line implementation, is therefore directly attributable to these inadequacies. Aspects of data acquisition, data pretreatment, and data quality are critical for real process applications, and while some practical approaches are adopted here, many important implementation problems remain unresolved. (c) 1993 John Wiley & Sons, Inc.     

The influence of tryptophan on mobility of residues in the trp repressor of Escherichia coli

The relative mobility of residues in the trp repressor of Escherichia coli has been examined in the absence and presence of the corepressor L-tryptophan by one- and two-dimensional 1H NMR. A comparison of relative intensities of cross peaks in NOESY and COSY spectra allowed a rigid Tyr and a mobile Tyr residue, three mobile Ser residues and three mobile Lys residues to be detected. The two Tyr residues were assigned by selective nitration with tetranitromethane. The singly nitrated molecule (on Tyr7) binds the trp operator with an affinity close to that of the unmodified repressor. Measurements of the intraring cross-relaxation rate constant as a function of temperature for Tyr7 shows the presence of considerable internal motion on the subnanosecond time scale in the flexible N-terminal arm. The order parameter, S2, characterising the motion is 0.35, which increases to about 0.5 in the presence of Trp. Trp decreases both the amplitude of the motion and the rate of the motion. At least three of the six Ser residues of the trp repressor have greater mobility than expected for a rigid body, and two of the Ser residues are sensitive to the presence of Trp. The more mobile Ser residues are probably those on the N-terminal arm and the C-terminal sequence. These results complement the single-crystal X-ray diffraction studies for which the electron density of the first ten and last three amino acid residues is weak. The solution data are consistent with proposals that the flexible N-terminal arm of the trp repressor makes important contacts with the DNA.     

Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase

Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be blocked by preincubation with ouabagenin, a rapidly reversible aglycone derivative of ouabain. The reduction in [3H]ouabain binding is due to a decrease in the number of binding sites rather than an alteration of the affinity of the enzyme for ouabain. Differential labeling at pH 8.2 with 1.0 mM 5,5'-dithiobis-(2-nitrobenzoic acid), preincubated with or without 5 microM ouabagenin, followed by tryptic digestion and reverse-phase high performance liquid chromatography of the generated soluble peptides reveals a single peptide labeled by the sulfhydryl probe that is protected by ouabagenin. From these results it is concluded that there is a single sulfhydryl group, essential for ouabain binding, presumably located in the ouabain binding site of lamb kidney (Na,K)-ATPase.     

Two sides of a cellular coin: CD4(+)CD3- cells regulate memory responses and lymph-node organization

We propose that CD4(+)CD3(-) cells have two functions: a well-established role in organizing lymphoid tissue during development, and a newly discovered role in supporting T-cell help for B cells both during affinity maturation in germinal centres and for memory antibody responses. As CD4(+)CD3(-) cells express the HIV co-receptors CD4 and CXC-chemokine receptor 4, we think that infection of these cells by HIV, and their subsequent destruction by the host immune system, could help to explain the loss of memory antibody responses and the destruction of lymphoid architecture that occur during disease progression to AIDS.     

A hidden-state Markov model for cell population deconvolution.

Microarrays measure gene expression typically from a mixture of cell populations during different stages of a biological process. However, the specific effects of the distinct or pure populations on measured gene expression are difficult or impossible to determine. The ability to deconvolve measured gene expression into the contributions from pure populations is critical to maximizing the potential of microarray analysis for investigating complex biological processes. In this paper, we describe a novel approach called the multinomial hidden Markov model (MHMM) that produces: (i) a maximum a posteriori estimate of the fraction represented by each pure population and (ii) gene expression values for each pure population. Our method uses an unsupervised, probabilistic approach for handling missing data points and clusters genes based on expression in pure populations. MHMM, used with several yeast datasets, identified statistically significant temporal dynamics. This method, unlike the linear decomposition models used previously for deconvolution, can extract information from different types of data, does not require a priori identification of pure gene expression, exploits the temporal nature of time series data, and is less affected by missing data.