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A M Fallon

Publications and source records attributed to A M Fallon.

At least 73 records · Page 4Linked to original sources
PubMed1990

Polybrene-mediated transfection of cultured lepidopteran cells: induction of a Drosophila heat shock promoter.

We have introduced hsp-cat plasmid DNA into Spodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 micrograms/ml) mixed with the polycation polybrene (100 micrograms/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from a Drosophila heat shock (hsp) gene. Expression of CAT activity in transfected Spodoptera cells was induced by a 2-h heat shock at 43 degrees C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure.

Animals↗
PubMed1990

Small heat shock proteins distinguish between two mosquito species and confirm identity of their cell lines.

We have adapted Aedes aegypti cells to Eagle's minimal medium containing nonessential amino acids, glutamine, and 5% fetal bovine serum. In this medium, cells maintained at 28 degrees C under a 5% CO2 atmosphere had a doubling time of 42 hr. Ninety-three percent of the cells in a typical population contained 6 full-size chromosomes and an apparent chromosome fragment. Ae. aegypti cells could be distinguished from Ae. albopictus cells by the sizes of the small heat shock proteins, whose synthesis was induced by a brief treatment at 41 degrees C. Analysis of the small heat shock proteins synthesized by dissected ovaries from Ae. aegypti and Ae. albopictus confirmed the species of origin of the 2 cell lines.

Aedes↗
PubMed1989

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells.

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.

Animals↗
PubMed1988

Identification of cDNAs corresponding to mosquito ribosomal protein genes.

Sequences encoding mosquito (Aedes albopictus) ribosomal proteins L8, L14 and L31 were identified from a cDNA library made from size-selected polyadenylated mRNA. Candidate cDNAs corresponding to moderately abundant mRNAs were screened by translation of hybrid-selected transcripts in wheat-germ lysates. Translation products were extracted with acetic acid and analyzed by electrophoresis in two dimensions in the presence of unlabeled ribosomal proteins. The identity of translation products that coelectrophoresed with purified ribosomal protein standards was supported by peptide mapping. The cDNAs corresponding to L8 (pL8) and L31 (pL31) hybridized to cytoplasmic mRNAs of 1.4 and 0.9 kb, respectively. In Southern blots of genomic DNA digested with BamHI, HindIII or EcoRI, the cDNA inserts from both pL8 and pL31 gave simple hybridization patterns suggestive of a low copy number for mosquito ribosomal protein genes.

Aedes↗
PubMed1987

Ribosome metabolism during the vitellogenic cycle of the mosquito, Aedes aegypti.

Ribosome accumulation and synthesis in the fat body of the mosquito Aedes aegypti increased by approx. 4-fold during 18 h after the blood meal, consistent with the pattern of total RNA accumulation during the synthetic phase of the vitellogenic cycle. By 24-30 h after the blood meal, the accumulated ribosomes began to be degraded, and the total RNA content in the fat body eventually returned to previtellogenic levels. A method has been developed for isolation of ribosomal subunits from fat body, and the ribosomal proteins have been shown to have properties similar to those from cultured Aedes albopictus cells by two-dimensional polyacrylamide gel electrophoresis. Proteins S1, S10, and A from the small ribosomal subunit are phosphorylated when fat body is incubated in Aedes saline in the presence of [32P]orthophosphate.

Adipose Tissue↗
PubMed1987

Pleiotropic changes in cycloheximide-resistant insect cell clones.

Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. From Aedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5 degrees C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show increased sensitivity to G-418, another antibiotic that interacts with the eukaryotic ribosome. However, there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%.

Aedes↗
PubMed1987

Properties of a ribonuclease from Aedes aegypti larvae.

1. The properties of a soluble ribonuclease from Aedes aegypti larvae have been compared with ribonuclease activity in adult female tissue. 2. In larval extracts ribonuclease activity was maximal at 40-45 degrees C whereas activity in tissue from adult females was highest at 50 degrees C. 3. Ribonuclease activity that was recovered in a 20-60% ammonium sulfate precipitate was further purified by batch elution from DEAE-Sephacel and from carboxymethylcellulose. 4. Ribonuclease activity in the partially purified fraction was sensitive to EDTA, stimulated by magnesium, had a pH optimum at 9.0 and a Mr of 45,000. 5. Agarose gels containing yeast RNA substrate were used to monitor partial purification of the larval ribonuclease.

Aedes↗
PubMed1986

Factors affecting polybrene-mediated transfection of cultured Aedes albopictus (mosquito) cells.

The polycation 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) is superior to calcium phosphate for the introduction of purified DNA into cultured Aedes albopictus (mosquito) cells. Adsorption of the polybrene-DNA complex to mosquito cells was essentially linear for 6 h. However, the rate of adsorption of DNA increased when the DNA-polybrene mixture was preincubated for several hours prior to addition to cells. A recombinant plasmid carrying an inducible chloramphenicol acetyltransferase gene under the control of a Drosophila heat shock protein (hsp) promoter was used to show that expression of transfected DNA was highest when cells were treated with a freshly prepared polybrene-DNA mixture. Optimal expression was observed in cells transfected with 4-13 micrograms of DNA per 10(6) cells; transfection with 24 micrograms of DNA resulted in reduced CAT expression. Variation in the polybrene-DNA ratio improved transfection with high levels of DNA. In mosquito cells, CAT expression was independent of DNA methylation.

Acetyltransferases↗
PubMed1986

Changes in ribonuclease activity during development of the mosquito, Aedes aegypti.

In the mosquito Aedes aegypti, quantitative and qualitative changes have been detected in ribonuclease activity during development. Ribonuclease activity is particularly high in extracts from larvae, relative to that in extracts from pupae or adults. Larval extract is enriched for a ribonuclease that is heat-labile, has an alkaline pH optimum, and is extremely sensitive to the divalent cation, manganese. Extract from adult females is enriched for a heat-stable component that has an acidic pH optimum and is more active at 56 than at 30 degrees C. Throughout the vitellogenic cycle, no major changes in ribonuclease activity were detected in fat body extracts.

Aedes↗
PubMed1985

Characterization of the ribosomal proteins from mosquito (Aedes albopictus) cells.

Proteins from the large and small subunits of Aedes albopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28-31 proteins ranging in molecular mass from 10 to 49 kDa. The large subunit contained 36-39 proteins that ranged in molecular mass from 11 to 53 kDa. The largest protein on the small subunit, S1, was the predominant phosphorylated ribosomal protein. Under long-term labelling conditions, L4 and L33 were also phosphorylated. Peptide mapping by partial proteolysis indicated that Ae. albopictus S1 may share partial amino acid homology with the phosphorylated ribosomal protein S6 from Drosophila melanogaster. Unlike Drosophila S6, however, Aedes S1 was not dephosphorylated during heat shock. Treatment of mosquito cells with the insect molting hormone 20-hydroxyecdysone did not affect phosphorylation of ribosomal proteins.

Aedes↗
PubMed1985

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells.

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.

Acetyltransferases↗
PubMed1982

Isolation and characterization of puromycin-resistant clones from cultured mosquito cells.

We have isolated from an established Aedes albopictus (mosquito) cell line clones which are resistant to the antibiotic puromycin. On the basis of growth and plating efficiency, clones Pur-8026 and Pur-8612 were five- and seven-fold more resistant, respectively, to puromycin than wild-type cells. In vitro protein synthesis was resistant to puromycin only in extracts prepared from Pur-8612 cells. Measurements of puromycin transport, cross-resistance to colchicine, and sensitivity to Tween-80 indicating that resistance in Pur-8026 cells was due to membrane alteration(s) affecting permeability to puromycin. This is the first description of puromycin resistant in insect cells and also the first report of puromycin resistance in an animal cell variant associated with an alteration at the level of protein synthesis.

Aedes↗
PubMed1982

Isolation and characterization of cycloheximide-resistant mosquito cell clones.

Following treatment of cultured mosquito cells (Aedes albopictus line of Singh) with the chemical mutagen ethyl methanesulfonate, we were able to isolate three cycloheximide-resistant clones. On the basis of growth kinetics, plating efficiency, and protein synthesis, these clones are 10- to 30-fold more resistant to cycloheximide than the parent cells. Cell-free lysates made from these cells retained 30-65% of their endogenous protein synthesizing ability in the presence of cycloheximide concentrations as high as 300 micrograms/ml. Protein synthesis in lysates from the parental cells, however, is reduced to about 10% of the control value (i.e., in the absence of drug) at 14 micrograms of cycloheximide/ml and was completely abolished at 75 micrograms/ml. These results indicate that cycloheximide resistance in these cells is likely due to an alteration in the protein synthetic machinery. This is the first description of cycloheximide-resistant insect cells, and the best example of cycloheximide resistance in cells originating from a higher eukaryotic organism.

Aedes↗
PubMed1979

Regulation of ribosomal protein synthesis in Escherichia coli by selective mRNA inactivation.

In an Escherichia coli strain lysogenic for lambda spc2 transducing phage, an extra copy of ribosomal protein (r-protein) genes in the spc and alpha operons are carried on the phage chromosome. Expression of genes in the spc operon in this merodiploid strain was compared with that in a control "haploid" strain carrying lambda trkA phage. It was found that the synthesis rate of spc mRNA, relative to other reference mRNA in the merodiploid strain, is about 2-fold higher than that in the control strain; yet, no dosage effect was observed in the synthesis rate of r-proteins in the spc or alpha operon. The spc mRNA was found to be more rapidly degraded in the merodiploid strain than in the control strain, and its steady-state amount, relative to reference mRNA, was only slightly higher in the merodiploid strain than in the control strain. Thus, E. coli cells have the ability to regulate the rate of r-protein synthesis regardless of the rate of transcription of r-protein genes, presumably by inactivation of the mRNA followed by its degradation. A model is proposed which involves selective inactivation of r-protein mRNA by a feedback mechanism. The model can explain coordinated synthesis of r-proteins and other observations related to selective expression of certain alleles in diploid strains.

Bacterial Proteins↗
PubMed1979

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.

Alleles↗
PubMed1978

Some rRNA operons in E. coli have tRNA genes at their distal ends.

We have previously isolated seven rRNA operons on plasmids or lambda transducing phages and identified various tRNAs encoded by these operons. Each of the seven operons has one of two different spacer tRNA gene arrangements between the genes for 16S and 23S rRNA: either tRNAGlu2 or both tRNAIle1 and tRNAAla1B genes. In addition, various tRNA genes are located at or near the distal ends of rRNA operons. In particular, genes for tRNATrp and tRNAAsp1 are located at the distal end of rrnC at 83 min on the E. coli chromosome. Experiments with various hybrid plasmids, some of which lack the rRNA promoter, have now demonstrated that this promoter is necessary for expression of the distal tRNA genes. Rifampicin run-out experiments have also provided evidence that the tRNATrp gene is located farther from its promoter than the spacer tRNA gene or the 5S RNA gene. These results confirm the localization of genes for tRNATrp and tRNAAsp1 at the distal end of rrnC and strongly suggest that they are co-transcribed with the genes for 16S, tRNAGlu2, 23S and 5S RNA. Other such distal tRNAs have been identified, and it is suggested that they too are part of rRNA operons.

Chromosome Mapping↗
PubMed1977

Identification and organization of ribosomal protein genes of Escherichia coli carried by lambdafus2 transducing phage.

We describe the isolation of lambdafus2, which carries bacterial DNA from the str-spc region of the Escherichia coli chromosome. Genes for 27 ribosomal proteins were found on the genome of this transducing phage by identifying the ribosomal proteins whose synthesis was stimulated after infection of UV-irradiated bacteria. These were genes for S3, S4, S5, S7, S8, S10, S11, S12, S13, S14, S17, S19, L2, L3, L4, L5, L6, L14, L15, L16, L17, L18, L22 L23, L24, L29, and L30. Subsets of these genes were identified on the genomes of the phages lambdaspc1, lambdaspc2-delta 9, and lambdaspc2-delta16, all of which carry parts of the bacterial DNA present on lambdafus2. From the known structures of these phage genomes, it has been possible to determine the relative order of many of these genes on the lambdafus2 genome and thus on the E. coli chromosome. Our evidence also suggests that the genes for S3, S17, S19, L2, L4, L16, L22, L23, and L29 are part of a single transcription unit. These results, along with the observations described in the previous and accompanying papers, indicate that the ribosomal protein genes on lambdafus2 are organized into at least four transcription units.

Coliphages↗