Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene

Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1–5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.     

Molecular detection and identification of phytoplasmas associated with Catharanthus roseus,Pinus eldarica,and Petunia hybrida in southeastern Iran's urban green spaces

Given the potential for urban green spaces to provide fresh and healthy environments for humans, exploring the issues that threaten plants in these places is crucial. Phytoplasma-related symptoms were encountered on some plants in urban green spaces in the province of Kerman, southeastern Iran, between 2017 and 2019. Affected periwinkles and petunias exhibited phytoplasma disease symptoms, including virescence, phyllody, and witches'-broom. However, ball or disc-like shoot proliferation symptoms were noticed on the trunks and branches of pine trees. PCR was performed with phytoplasma-detecting universal primers, targetting and amplifying the 16S rRNA gene, and determining whether phytoplasmas are implicated in the symptomatic plants. The infection of the symptomatic plants was confirmed using nested-PCR amplification of expected DNA sizes for phytoplasmas. No product, however, was amplified from sampled symptomless plants. The sequencing of nested-PCR products was performed to obtain sequences encasing the standard F2nR2 fragments. The resulted sequences were submitted to iPhyClassifier, the universal phytoplasma classification platform, for the taxonomic assignment of the found phytoplasmas compared with previously identified ‘Candidatus Phytoplasma’ species, groups, and subgroups. The results revealed that phytoplasma strains related to the species ‘Ca. P. trifolii’ (16SrVI-A subgroup) infect periwinkles and pines. However, strains from the species ‘Ca. P. aurantifolia’ (16SrII-D subgroup) and ‘Ca. P. phoenicium’ (16SrIX-C subgroup) were found in petunias and periwinkles, respectively. To the best of our knowledge, phytoplasmas from the 16SrVI-A and 16SrII-D subgroups are the first reported to infect these plants in Kerman province, while a related strain from the subgroup 16SrIX-C is the first recorded to infect periwinkles in Iran and the second in the world.     

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.     

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.