麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.     

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSO_A type, as well as with the conversion signal of the Escherichia coli phage X174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

On-line state recognition in a yeast fed-batch culture using error vectors

The physiological states with respect to cell growth and ethanol production in a yeast fed-batch culture expressed in linguistic form could be recognized on-line by fuzzy inferencing based on error vectors. The error vector was newly defined here in a macroscopic elemental balance equation. The physiological states for cell growth and ethanol production were characterized by error vectors using many experimental data from fed-batch cultures. Fuzzy membership functions were constructed from the frequency distributions of the error vectors and state recognition was performed by fuzzy inferencing. In particular, an unusual physiological state for a yeast cultivation, in which aerobic ethanol production was accompanied by very low cell growth, could be recognized accurately. According to the results of the state recognition, an energy parameter, the P/O ratio in the metabolic reaction model was adaptively estimated, and the cell growth was successfully evaluated with the estimated P/O. (c) 1995 John Wiley & Sons, Inc.     

Traveling Waves and the Processing of Weakly Tuned Inputs in a Cortical Network Module

Recent studies have shown that local cortical feedback can havean important effect on the response of neurons in primary visualcortex to the orientation of visual stimuli. In this work, westudy the role of the cortical feedback in shaping thespatiotemporal patterns of activity in cortex. Two questionsare addressed: one, what are the limitations on the ability ofcortical neurons to lock their activity to rotatingoriented stimuli within a single receptive field? Two, can thelocal architecture of visual cortex lead to the generation ofspontaneous traveling pulses of activity? We study theseissues analytically by a population-dynamic model of ahypercolumn in visual cortex. The order parameter thatdescribes the macroscopic behavior of the network is thetime-dependent population vector of the network. We firststudy the network dynamics under the influence of a weakly tunedinput that slowly rotates within the receptive field. We showthat if the cortical interactions have strong spatialmodulation, the network generates a sharply tuned activityprofile that propagates across the hypercolumn in a path thatis completely locked to the stimulus rotation. The resultantrotating population vector maintains a constant angular lagrelative to the stimulus, the magnitude of which grows with thestimulus rotation frequency. Beyond a critical frequency thepopulation vector does not lock to the stimulus but executes aquasi-periodic motion with an average frequency that is smallerthan that of the stimulus. In the second part we consider thestable intrinsic state of the cortex under the influence of isotropic stimulation. We show that if the local inhibitoryfeedback is sufficiently strong, the network does not settleinto a stationary state but develops spontaneous travelingpulses of activity. Unlike recent models of wave propagation incortical networks, the connectivity pattern in our model isspatially symmetric, hence the direction of propagation ofthese waves is arbitrary. The interaction of these waves withan external-oriented stimulus is studied. It is shown that thesystem can lock to a weakly tuned rotating stimulus if thestimulus frequency is close to the frequency of the intrinsic wave.     

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34⁺ cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34⁺ cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.     

Physical characterization of a glucose-dehydrogenase-bearing plasmid from ketoacid-producingErwinia herbicola

Erwinia herbicola (ATCC 21998), a facultative anaerobe, has two plasmids: pVQ1 and pVQ2. Curing with mitomycin C indicated that pVQ2 was cryptic but pVQ1, a 7.4-kb plasmid, bears a 4.3SacI fragment which strongly hybridized to the C-terminal region of the glucose dehydrogenase gene ofAcinetobacter calcoaceticus. A restriction map of plasmid pVQ1 is presented.The authors are with the Department of Biotechnology, Regional Research Laboratory, Canal Road, Jammu Tawi-180 001, India;     

Rapid micropreps and minipreps of Ti plasmids and binary vectors fromAgrobacterium tumefaciens

A simple and versatile procedure has been developed for the isolation of both large helper/Ti plasmids and binary vectors fromAgrobacterium tumefaciens. Using a slightly modified alkaline lysis protocol, intact plasmid can be recovered from cultures grown in standard micro-centrifuge tubes or culture tubes in sufficient yield and purity to allow for restriction analysis on ethidium bromide stained gels of the >200 kb Ti plasmid DNA. Contamination by chromosomal DNA is minimal and there is thus no need for isopycnic gradient purification. This same procedure can be combined with a high temperature treatment (37°C) and antibiotic selection to generate preparations containing binary vector DNA that are virtually free of interfering Ti plasmid DNA. Restriction patterns produced from these binary vector DNA preparations are unambiguous and therefore preliminary screening by Southern hybridization can be eliminated.