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            I-Labeled DNA·RNA Hybrids in Cytological Preparations

Altenburg, Lewis C.; Getz, Michael J.; Crain, William R.; Saunders, Grady F.; Shaw, Margery W.

doi:10.1073/pnas.70.5.1536

Cited by 6 publications

(2 citation statements)

References 20 publications

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“…125I was incorporated into RNA by a combination of the methods of Getz et al (1972) and Altenberg et al (1973). Greater than 99% of the incorporated label was rendered acid soluble by digestion with pancreatic RNase and a specific activity of 1.5 X 105 cpm/ug was obtained.…”

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confidence: 99%

Transcription of isolated mouse liver chromatin

Bacheler

Smith

1976

Biochemistry

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Analysis of RNA transcription from isolated mouse liver chromatin has been undertaken by means of RNA-excess hybridizations with small amounts of radioactive DNA. This analysis indicates that mouse liver chromatin is a restricted template for the in vitro synthesis of RNA complements to repetitive DNA, but more RNA species are synthesized than are found in the RNA isolated from mouse liver nuclei. Extraction with 0.5 M NaC1 destroys the template restriction of isolated chromatin. RNA synthesized in vitro from DNA or chromatin templates by Escherichia coli RNA polymerase, as well as in vivo mouse liver nuclear RNA, were each hybridized to 125I-labeled DNA of high, intermediate, or low reiteration frequency. Chromatin-primed and nuclear RNA saturate a smaller portion of each DNA fraction than does DNA-primed RNA. However, chromatin-primed RNA saturates more high and low reiteration frequency DNA than does nuclear RNA. Simultaneous hybridization of nuclear-and chromatin-primed RNA with 125I-labeled DNA indicates that chromatin-primed RNA contains all of the sequences present in nuclear RNA. Extraction of chromatin with 0.5 MNaC1 leads to removal of histone F1, as well as a wide variety of non-histone proteins. When used as a template for in vitro RNA synthesis, such salt-extracted chromatin produced RNAs that hybridize as large a portion of each DNA fraction as does DNA-primed RNA.

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confidence: 99%

Transcription of isolated mouse liver chromatin

Bacheler

Smith

1976

Biochemistry

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“…Henning (1973) has discussed the problem of background in LM in situ hybridization experiments and suggested that a final treatment with a dilute solution of TCA may effectively reduce the background. Altenburg et al (1973) treated some of their slides with a solution of trypsin after hybridization and RNase treatment in a bid to reduce background due to binding to protein of the radioactive iodine they used.…”

Section: Discussion O F C E R T a I N T E C H N I C A L Stepmentioning

confidence: 99%

Molecular hybridization of RNA and DNA in situ: visualization at the electron microscope level

Jacob

Moar

Gillies

et al. 1976

Journal of Microscopy

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SUMMARY Electron microscopic visualization of molecular hybrids formed in situ is feasible at the present time. It can be accomplished by two alternative approaches. In one, the in situ hybridization is carried out on ultrathin sections of target embedded in glycol methacrylate. In the other, whole cells are used for hybridization and they are subsequently prepared for electron microscopy. The choice of the method to be adopted depends on the type of target tissue. When there is a choice, the second approach seems preferable. Some of the important technical steps in the hybridization procedure, such as DNA denaturation in ultrathin sections, have been discussed and attention has been drawn to practical problems that may arise during the preparatory steps. Our light microscope experiments demonstrate that preparations made after glutaraldehyde fixation have a lower hybridization efficiency than those fixed with 3: 1 methanol‐acetic acid. Attempts are therefore being made to explore the possibility of using methanol‐acetic acid for electron microscope in situ hybridization. First results of straightforward fixation show that the preservation of nuclear structure may be fairly satisfactory for the purpose. However, the cumulative effects of subsequent treatments in the procedure still remain to be examined. For electron microscope autoradiographs (EM ARG) of hybridized preparations, the most suitable emulsion at present appears to be Ilford L4. Various factors conducive to optimum resolution consistent with maximum efficiency in this emulsion have been pointed out. Practical problems that may arise in autoradio graphs of hybridized preparations such as background and variation of grain density in adjacent sections have also been considered.

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