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At least 1,243 records · Page 69Linked to original sources
PubMed1993

Formation of farnesal and 3-hydroxy-2,3-dihydrofarnesal from farnesol by protoplasts of Botryococcus braunii.

Farnesal and 3-hydroxy-2,3-dihydrofarnesal (3-hydroxy-3,7,11-trimethyl-6,10-dodecadiene-1-al) were formed from farnesol when the alcohol was incubated with the protoplast of Botryococcus braunii B race strain. This fact suggests the existence of farnesal hydratase in the alga. Feeding experiments showed that both farnesal and 3-hydroxy-2,3-dihydrofarnesal were efficiently incorporated into botryococcenes, triterpenoid hydrocarbons of the alga.

Carbon Radioisotopes↗
PubMed1993

Microamperometric estimation of photosynthesis inhibition in a single algal protoplast.

The single-cell level inhibition of photosynthesis by some chemicals was investigated from the light-irradiation induced response of intracellular oxygen reduction current at an ultramicroelectrode inserted into a single algal protoplast. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an electron-transport inhibitor, decreased a current peak appeared immediately after the light irradiation. The pI50 value (the negative decadic logarithm of the concentration needed for 50% inhibition) was determined from the change in response patterns and found to be 6.9 for DCMU. 2,4-Dichlorophenoxyacetic acid (2,4-D), an auxin herbicide, showed no effect on photosynthesis in the effective concentration range. 2,4-Dinitrophenol (DNP), a proton carrier, accelerated the oxygen production upon light irradiation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion.

The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.

Animals↗
PubMedDate not supplied

Ultrastructural nonisotopic mapping of nucleolar transcription sites in onion protoplasts.

The post- and preembedding ultrastructural localization of transcribing rRNA genes has been carried out in nucleoli of permeabilized onion growing root tip protoplasts by means of the nonisotopic bromouridine method. By means of both post- and preembedding approaches, major synthetic sites were identified with morphologically distinct subdomains of dense fibrillar components, with some signal also being associated with nucleolar fibrillar centers and vacuoles. Moreover, labeled medusoid fibrils within distinct domains seen in Lowicryl thin sections likely represent the morphological correlate of transcribing nucleolar genes.

Allium↗
PubMed1994

Uncoating of tobacco mosaic virus RNA in protoplasts.

The disassembly of tobacco mosaic virus particles during the establishment of infections in tobacco protoplasts was investigated by reverse transcription and PCR analysis of the parts of the viral RNA that remained encapsidated at various times after inoculation. Within the first 3 min, uncoating had proceeded from the 5' terminus of the viral RNA to a position at least 4635 nucleotides from the 5' terminus. Removal of coat protein subunits from the part of the virus particle near the origin of assembly sequence of the RNA occurred more slowly. The results also suggested that progeny virus particles began to be produced 35-40 min after inoculation.

Base Sequence↗
PubMed1994

Rice tungro bacilliform virus: transcription and translation in protoplasts.

Protoplasts from cell suspension cultures of Oryza sativa (monocot) and Orychophragmus violaceus (dicot) support transcription from the rice tungro bacilliform virus (RTBV) promoter and translation of the resulting mRNA despite the presence of a long leader sequence with strong secondary structure and 12 short open reading frames. Transcriptional elements located both upstream and downstream of the transcription initiation site are defined by deletion analysis and the functional TATA motif is determined. Expression of an open reading frame downstream of the entire leader is more efficient than that downstream of truncated derivatives. For optimal expression sequences in the 5' and 3' parts of the leader are required.

Base Sequence↗
PubMed2002

Positional effect of deletions on viability, especially on encapsidation, of Brome mosaic virus D-RNA in barley protoplasts.

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477-500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3'-proximal and the 5'-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.

3' Untranslated Regions↗
PubMed1983

Chromosome interactions and expression in fused Bacillus protoplasts.

Heterodiploid bacteria can be regenerated from fused mixed protoplasts. In both B. megaterium and B. subtilis, early selective effects during regeneration may strongly affect the phenotype of the products. Among the products are diploid prototrophs, whose stability is still in doubt. It is not known whether the prototrophy results from complementation or from recombination during the selection. In the case of B. subtilis, regeneration in a permissive, uncrowded and non-selective environment leads to production of rather large yields of heterodiploids, either biparental or recombinant. These seem to be only partially stable, but while remaining diploid they give rise to a wide variety of genetic recombinants. In general rather few prototrophs are found when selection is applied to the bacterial diploids. The phenotypic properties of B. subtilis diploids reveal incomplete expression of the demonstrable gene inventory. Biparental diploids appear commonly to show the phenotype of only one of the contained parent types, as if one chromosome remains unexpressed. For diploid recombinants data are insufficient to reveal the status of the chromosomes. Non-expression is believed to be due to structural complexity of particular chromosomes, or their parts. Recombination occurring within bacterial heterodiploid clones appears to satisfy many of the expectations for genetic recombination in eukaryotes--including that of being in part at least classically reciprocal, with intraclonal reciprocals in equivalent numbers. While short map intervals show reduced recombination, large numbers of recombinations (one to two thirds) involve exchanges at or near the terminus and origin of bidirectional replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1992

Multiple ocs-like elements required for efficient transcription of the mannopine synthase gene of T-DNA in maize protoplasts.

Regulatory elements controlling transcriptional activity of the mannopine synthase 2' promoter (mas 2') were defined by analysis of deletion mutants in transient expression assays in maize protoplasts. Deletion of the region between -305 and -290 containing sequence similarity to the octopine synthase (ocs) promoter element reduced activity by 67% compared to wild type activity. Less than 1% of the activity remained in 5' deletions downstream of -153. Inclusion of various heterologous enhancer-like sequences immediately upstream of position -325 increased activity by up to 7.5-fold. Insertion of the -325 to -275 sequence alone, or in combination with heterologous enhancer-like elements, restored activity of some of the 5'-deletion mutants. Restoration of activity was not obtained with mutants deleted past position -127. Our results suggest that a single class of nuclear proteins from maize interact with high affinity at elements designated mas b (-306 to -275; mas 1' element), d (-127 to -108), and e (-82 to -39; mas 2' element) as well as the 20 bp element from the ocs promoter. Although the binding site at mas d only appears to accommodate a single protein, this element has the potential to make a weak, but positive, contribution to the activity of the mas 2' promoter. The binding of nuclear proteins could not be demonstrated at mas a and c, both of which showed limited homology to the ocs element. Mutational evidence suggested that mas a and c may also contribute to mas 2' transcription.

Agrobacterium tumefaciens↗
PubMed1991

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts.

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3' deleted by cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5'----3' as well as 3'----5' RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

Bacillus thuringiensis↗
PubMed1993

Effects of promoter, intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts.

Various chimaeric promoter regions coupled to the uidA beta-glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5' transcribed region decreased expression strength.

Enhancer Elements, Genetic↗
PubMed1991

Control of transient expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barley aleurone layers.

Gibberellic acid (GA3) and abscisic acid (ABA) control the transcription of alpha-amylase genes in barley aleurone cells. This control is likely to be exerted through cis-acting hormone-responsive elements in the promoter region of the gene. In order to further define these elements, we have developed procedures for obtaining transient expression of chimaeric genes in protoplasts prepared from mature barley aleurone layers. Constructs with heterologous constitutive promoters and with heterologous and homologous GA3- and ABA-regulated promoters were expressed specifically by these cells. This system would appear to offer great potential in gene regulation studies especially for hormonally regulated homologous genes. Functional analysis of a barley alpha-amylase gene has been performed using this system. A 2050 bp fragment from a high-pI alpha-amylase gene was fused to a reporter gene (GUS) and control of its expression was examined. Deletion analysis of this promoter fragment showed that major GA- and ABA-responsive elements occurred between 174 and 41 bp upstream from the transcription initiation site.

Abscisic Acid↗
PubMed1993

Differential expression of U5snRNA gene variants in maize (Zea mays) protoplasts.

The small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 participate in the removal of introns from pre-messenger RNAs in the nucleus. Three genes encoding U5snRNAs, the RNA moiety of U5snRNPs, have been isolated from maize. As in other plant UsnRNA gene families the three maize U5snRNA genes exhibit sequence variation. Two of the gene variants (MzU5.1 and MzU5.2) are clearly expressed after transfection into maize leaf protoplasts while the third gene variant (MzU5.3) is expressed at very low levels. These different levels of expression cannot be directly correlated with sequence changes in the highly conserved Upstream Sequence Element (USE) required for expression of Arabidopsis UsnRNA genes nor with differential stability of the U5snRNA transcripts. Further sequence elements may therefore have a role in regulating maize UsnRNA gene expression.

Base Sequence↗
PubMed1994

Factors affecting the re-formation of vacuoles in evacuolated protoplasts and the expression of the two vacuolar proton pumps.

The re-formation of vacuoles in miniprotoplasts (evacuolated mesophyll protoplasts) of tobacco was investigated under different conditions. When a constant osmolarity was maintained, increasing the concentration of NaCl in the medium enhanced the regeneration of vacuoles compared to the control (0.5 M mannitol used as osmoticum). An enhanced growth rate of miniprotoplasts could also be observed under low-osmolarity conditions, by substitution of NaCl for KCl or NaNO3, or with different effectors (glycinebetaine and methyljasmonate). Using the polymerase chain reaction, one cDNA fragment of the B-subunit of the vacuolar ATPase and two fragments of the tonoplast-bound pyrophosphatase (PPase) of tobacco were cloned. Southern blot analyses indicates that for both proteins more than one gene is present in tobacco. During the regeneration of vacuoles the transcript level of the PPase increased earlier than that of the B-subunit of the vacuolar ATPase under all conditions tested (0.5 M mannitol, 0.3 M mannitol, and 0.25 M NaCl, respectively). Under salt-stress conditions (0.25 M NaCl used as osmoticum), the expression level of both proton pumps is enhanced compared to the control. This increase is not specifically due to salt stress but generally to an increased growth rate of the vacuole, since under low-osmolarity conditions the expression of the vacuolar pumps is enhanced, too.

Adenosine Triphosphatases↗
PubMed1995

Calcium-sensitivity of the plasmalemmal delayed rectifier potassium current suggests that calcium influx in pulvinar protoplasts from Mimosa pudica L. can be revealed by hyperpolarization.

Isolated protoplasts from pulvinar motor cells of Mimosa pudica were studied using conventional whole-cell patch clamp techniques. With internal solutions weakly buffered for Ca2+ (0.2 mM EGTA), a rundown of the outward delayed rectifier K+ current was induced by hyperpolarizing the holding potential, and this effect was strongly promoted by high external Ca2+ concentrations. This rundown could be reversed by coming back to less hyperpolarized holding potentials or by lowering the external [Ca2+]. Such rundown was absent when pipette internal solutions strongly buffered (10 mM EGTA) for Ca2+ were used. Ionomycin induced rundown of the K+ current with internal solutions containing 0.2 mM but not 10 mM EGTA. The hyperpolarization-associated rundown was reversibly blocked by Gd3+ and La3+.

Bepridil↗
PubMed1992

Elimination of by-products in 11 beta-hydroxylation of substance S using Curvularia lunata clones regenerated from NTG-treated protoplasts.

Stable mutants showing improved 11-hydroxylation of Substance S were isolated, following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and regeneration of uninucleate protoplasts of the appropriate fungal strains. This procedure was especially suitable for obtaining more directed 11 beta-hydroxylation of Substance S with Curvularia lunata IM 2901. Apart from producing cortisol (11 beta-hydroxy-S), the parent strain formed several by-products that significantly lowered the yield of the desired 11 beta-hydroxyderivative. Isolated mutants of this microorganism carried out directed 11 beta-hydroxylation with only a small amount of one of the by-products, which resulted in a much higher yield of cortisol.

Biotransformation↗
PubMed1990

Molecular changes in protoplast-derived rice plants.

To determine whether regeneration of rice plants from protoplast culture induces DNA polymorphisms, progeny plants from direct regenerants of such cultures were examined for restriction fragment length polymorphisms (RFLP analysis). Significantly increased levels of DNA polymorphism were found compared with those in non-tissue culture control plants. Analysis with gene sequences representative of different functional domains, revealed that such polymorphisms are apparently widespread and not associated with any particular region. Analysis by comparative digestion with both methylation-sensitive and insensitive restriction enzymes revealed that methylation changes cannot be regarded as a major factor in the induction of these DNA polymorphisms.

DNA↗
PubMed1991

Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplasts.

Deletions were made in the cauliflower mosaic virus polyadenylation sequence which was cloned downstream of the beta-glucuronidase gene (gus). The populations of mRNAs generated in tobacco mesophyll protoplasts by transient expression with the various constructs were analysed using a polymerase chain reaction procedure. When no deletion was present in the sequence, the mRNA appeared to be polyadenylated at two major polyadenylation sites. A deletion upstream from the AATAAA sequence made the population of polyadenylated mRNAs very heterogenous at their 3' ends. A deletion downstream of the AATAAA sequence had no effect on the choice of the site. Alternative polyadenylation sites were used when the native polyadenylation site was deleted. These results are discussed in relation to data obtained with other polyadenylation sequences from both plants and animals.

Base Sequence↗