Electron Microscopic Visualization of Transcription

Miller, O. L.; Beatty, Barbara R.; Hamkalo, Barbara A.; Thomas, C. A.

doi:10.1101/sqb.1970.035.01.066

Cited by 77 publications

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“…DNP and RNP compaction. As visualized in chromatin spreads, nascent transcripts and the DNA templates to which they are attached are not naked RNA and DNA but rather nucleic acid-protein complexes, ribonucleoprotein (RNP) and deoxyribonucleoprotein (DNP) (46). In eucaryotes, there appears to be an inverse correlation between the level of transcriptional activity and the degree to which DNA is condensed by its associated proteins (43).…”

Section: Methodsmentioning

confidence: 99%

Transcription mapping of the Escherichia coli chromosome by electron microscopy

French

Miller

1989

J Bacteriol

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The distinctive double Christmas tree morphology of rRNA operons as visualized by electron microscopy makes them easy to recognize in chromatin spreads from Escherichia coli. On the basis of the pattern of nascent transcripts on nearby transcription units and the relative distances of the operons from one another and the replication origin, we are now able to specifically identify five of the seven rRNA operons in E. coli. The use of rRNA operons as markers of both position and distance has resulted in the morphological mapping of a significant portion of the E. coli chromosome; over 600 kilobase pairs in the 84-to 90-min and 72-min regions can now be recognized. Since individual rRNA operons could be identified, direct comparisons could be made of their transcriptional activities. As judged by the densities of RNA polymerases along the operons, rrnA, rrnB, rrnC, rrnD, and rnnE were all transcribed at similar levels, with one RNA polymerase every 85 base pairs.The ability to recognize individual operons and specific regions of the chromosome allows direct comparisons of various genetic parameters.Chromatin-spreading techniques provide the unique opportunity for direct visualization of in vivo transcriptional activity. In a chromatin spread, the contents of osmotically lysed cells are allowed to disperse at low ionic strength. Under these conditions, the bacterial chromosome unfolds and RNA polymerases with nascent transcripts are observed still attached to their DNA templates. A drawback of the technique, however, is that it is difficult to identify specific genes of interest among the many active transcription units that are visualized. The use of chromatin-spreading techniques to study the expression of specific bacterial genes has been limited, since the only chromosomal genes that have been identified to date are the rRNA operons (47,48). An alternative approach of visualizing specific genes on plasmids (23) is useful but does not allow analysis of the regulation of single-copy genes in their chromosomal environment.In chromatin spreads of Escherichia coli cells, rRNA operons are easily recognized by their dense packing with RNA polymerases and their distinctive double Christmas tree morphology (Fig. 1A). RNase III cleavage of nascent pre-rRNA transcripts between the 16S and 23S cistrons results in two gradients of increasing transcript length (30) (Fig. 1B). There are seven rRNA operons (22, 35, 50) distributed around the 4,700-kilobase-pair (kbp) E. coli chromosome (36). We have been able to specifically identify five of them on the basis of patterns of adjacent transcription units, their proximity to the replication origin, and their relative distances from one another. In well-spread preparations of chromatin, it is often possible to follow the bacterial chromosome for a considerable distance. Using the rRNA operons as markers of both position and distance, we have been able to map patterns of transcriptional activity over 600 kbp (-13%) of the E. coli chromosome. We have ultrastructurally identified 1...

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Section: Methodsmentioning

confidence: 99%

Transcription mapping of the Escherichia coli chromosome by electron microscopy

French

Miller

1989

J Bacteriol

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“…Chromatin-spreading techniques have previously allowed electron microscopic visualization of transcriptionally active but unidentified bacterial operons (1,2). Ultrastructural visualization of genetic activity allows regulation to be studied on a "per gene" basis, as opposed to the statistical approach inherent in the study of a population of cells.…”

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Electron microscopic visualization of trp operon expression in Salmonella typhimurium.

French¹,

Martin²,

Patterson³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Transcriptional activity of plasmids carrying wild-type and mutant trp operons was visualized in cell lysates of Salmonela typhimurium. Plasmid and transcription-unit sizes varied with the size of the cloned operon. Following 3-(3-indolyl)acrylic acid derepression, all operons of a particular type exhibited the same high level oftranscriptional activity.An estimated 11-14 transcripts must be initiated each minute to maintain the 190-base-pair spacing of RNA polymerases observed on the promoter-proximal half of the wild-type tip operon. A decline in RNA polymerase density was observed on promoter-distal portions of cloned tip operons, which may be attributable to premature transcription termination accompanying translation inhibition due to indolylacrylic acid's interference with normal tryptophanyl-tRNA synthetase activity.Chromatin-spreading techniques have previously allowed electron microscopic visualization of transcriptionally active but unidentified bacterial operons (1, 2). Ultrastructural visualization of genetic activity allows regulation to be studied on a "per gene" basis, as opposed to the statistical approach inherent in the study of a population of cells. Use of chromatin-spreading techniques to study the expression of specific bacterial genes has, however, been limited, since only those chromosomal genes with some distinct morphological characteristic, such as rRNA genes, could be identified.In chromosome spreads, plasmid DNA is easily distinguished from chromosomal DNA by its circularity and size. The use of multicopy plasmid cloning vectors in conjunction with chromatin-spreading techniques should allow genes of choice, identifiable by their occurrence on plasmids, to be studied. Additionally, the increase in copy number inherent in this method should enhance the probability of locating the gene of interest.We have successfully used this technique to visualize a specific operon, the tryptophan (trp) operon of Salmonella typhimurium. The wild-type and a variety of mutant trp operons have been established by in vivo cloning (3) on pBR322-derived recombinant plasmids in a host S. typhimurium strain lacking a chromosomal trp operon. We report here the visualization of wild-type trp operon expression and the effect that various mutations have on that expression. MATERIALS AND METHODSBacterial Strains. All plasmids were maintained in the same host, TB20. This strain, originally isolated by M. Demerec, carries a large deletion, A(trp-tonB-opp)101, that eliminates all trp sequences (4) and the two downstream loci tonB and opp (5).Media and Growth Conditions. Bacteria were grown to stationary phase overnight at 37°C in LB medium (6) containing ampicillin at 50 pug/ml. The overnight inoculum was diluted 100-fold into 50 ml of the same medium and incubated at 370C in a rotary shaker for 2.5-4 hr. To derepress the trp operon, 3-(3-indolyl)acrylic acid (IA) (Sigma) was added at a final concentration of 200 tkg/ml 15 min before the cells were harvested.Chromosome Spreading and Electron Microscopy. Ten...

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“…Cell walls from plants grown on completely inorganic medium were digested more effectively than those grown on agar supplemented with 1% glucose and 0.05% peptone. This suggests an increased synthesis of cell wall material in supplemented medium, an assumption that is supported by higher dry weights 6 .Optimal plasmolysis of Sphaerocarpos protoplasts was achieved in 0.4 M mannitol. Generally, the enzyme was dissolved directly in the plasmolyticum without addition of buffer.…”

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confidence: 73%

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confidence: 75%

“…Supported by extremely thin carbon films, even DNA preparations without appositional contrast could be observed by bright or dark field electron microscopy 4 ' 5 . Another way for visualizing genetic material was followed by MILLER et al 6 . Transcription complexes, consisting of DNA, RNA and protein, were sedimented onto the grids by centrifugation, and contrasted by using phosphotungstic acid (PTA) as a protein specific stain.…”

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Notizen: Enzymatic Isolation of Protoplasts from the Liverwort Sphaerocarpos donnellii Aust.

Schieder

Wenzel

1972

Zeitschrift Für Naturforschung B

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Fusion of free plant protoplasts opens possibilities to obtain new genetical and cytological results. Haploid material is superior to diploid for such experiments. Haploid protoplasts of Petunia 1 were isolated, but so far further development of these haploid protoplasts could not be achieved. Isolated moss protoplasts have been shown to be capable of regeneration 2 , and therefore would offer better material. Furthermore it is possible to easily commence here with axenic cultures. The composition of moss cell walls, however, differs strongly from those of higher plants 3~5 . Therefore only small quantities of protoplasts have been obtained by complicated mechanical methods 2 .This paper deals with the enzymatic degradation of cell walls and the isolation of protoplasts from normal and auxotrophic Sphaerocarpos donnellii Aust.Female plants of the wild strain were grown under sterile conditions on Benecke agar in petri dishes (temperature 18 + 1 °C, light intensity 2800 lux, 12 h per day). The plants were propagated vegetatively from thallus tips. Cell walls from plants grown on completely inorganic medium were digested more effectively than those grown on agar supplemented with 1% glucose and 0.05% peptone. This suggests an increased synthesis of cell wall material in supplemented medium, an assumption that is supported by higher dry weights 6 .Optimal plasmolysis of Sphaerocarpos protoplasts was achieved in 0.4 M mannitol. Generally, the enzyme was dissolved directly in the plasmolyticum without addition of buffer. pH was adjusted with 0.1 N HCl. The plants were not visibly damaged down to pH 4. The material was incubated in a shaking waterbath between 25 and 33 °C, higher temperatures caused denaturation of the cytoplasm. As long as the protoplasts were located in the thallus, the shaking frequency was 140 strokes/min, with an amplitude of 5.7 cm. The frequency was reduced to 65 strokes and amplitude to 2.4 cm, when the first protoplasts appeared.For digestion of the cell walls a number of enzymes were tested singly, in simultaneous and/or successive combinations. The glucosidases, hyaluronidase (EC 3.2

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Electron Microscopic Visualization of Transcription

Cited by 77 publications

References 0 publications

Transcription mapping of the Escherichia coli chromosome by electron microscopy

Transcription mapping of the Escherichia coli chromosome by electron microscopy

Electron microscopic visualization of trp operon expression in Salmonella typhimurium.

Notizen: Enzymatic Isolation of Protoplasts from the Liverwort Sphaerocarpos donnellii Aust.

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