A new method of in situ hybridization

Manning, Jerry E.; Hershey, Neil D.; Broker, Thomas R.; Pellegrini, Maria; Mitchell, Herschel K.; Davidson, Norman

doi:10.1007/bf00333039

Cited by 114 publications

(44 citation statements)

References 17 publications

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“…The secondary antibodies were the IgGs raised in goats against rabbit immunoglobulins (Miles). These goat IgGs were covalently linked to polymethacrylate spheres (580 A in average diameter) (12). The spheres used in this communication were kindly provided by Norman Davidson and James Casey.…”

Section: Methodsmentioning

confidence: 99%

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

Chooi¹,

Leiby²

1981

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

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We describe a method for the localization of ribosomal proteins on electron microscopic spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates ofD. melanogaster egg chambers. The locations ofthe bound IgGs in the active transcripts are detected with goat antirabbit IgGs that have been labeled with electron-dense polymethacrylate spheres. By statistical analysis we can generate a confidence interval for the initial point of protein assembly for a particular protein. The first point of protein assembly for S14 is located near the 5' end of the pre-18S rRNA. In contrast, the first point of protein assembly for IA is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and LA are consistent with the 5' proximal and the 5' distal orientations of the pre-18S and the pre-28S rRNAs. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.

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Section: Methodsmentioning

confidence: 99%

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

Chooi¹,

Leiby²

1981

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

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“…Coupling of RNA and cytochrome c-biotin: The modification of these RNAs with cytochrome c-biotin is described elsewhere (10). In the case of the histone mRNA coupling, only 2 pg of mRNA were available; therefore, 20 lJg of E. coli 16S rRNA was added to the mRNA as carrier.…”

Section: Methodsmentioning

confidence: 99%

Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences

Pellegrini¹,

Holmes²,

Manning³

1977

Nucl Acids Res

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A method of enriching for long double-stranded segments of eukaryotic DNA carrying particular genes is described. A purified RNA coded for by the gene is covalently attached to biotin via the protein, cytochrome c. This modified RNA is hybridized to total nuclear, double-stranded DNA under conditions that allow the formation of R-loops. Avidin, which has a high affinity for biotin, is covalently attached to polymer spheres. The complexes of avidin-spheres with DNA:RNA-biotin R-loop hybrids band in CsCl at a much lower bouyant density than does free DNA. This density is a function of the length of DNA coupled per avidin-sphere. This method was used to prepare very long double-strands of DNA highly enriched in the coding sequences for the large rRNAs of D. melanogaster and L. donovani and the histone mRNAs of S. purpuratus. INTRODUCTIONThe isolation of long fragments of eukaryotic DNA containing specific

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“…The arrangement of ribosomal cistrons was studied by first enriching for double stranded DNA containing the rRNA genes from S. bullata DNA by application of the avidin-biotin method of gene enrichment (25). [24][25][26][27][28][29][30][31][32][33][34][35][36] hours after puparium formation (25,27). The DNA was further purified by centrifugation to equilibrium in a neutral cesium chloride buoyant density gradient.…”

Section: Introductionmentioning

confidence: 99%

Sequence arrangement of the rRNA genes of the dipteran Sarcophaga bullata

1981

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Velocity sedimentation studies of RNA of Sarcophaga bullata show that the major rRNA species have sedimentation values of 26S and 18S. Analysis of the rRNA under denaturing conditions indicates that there is a hidden break centrally located in the 26S rRNA species. Saturation hybridization studies using total genomic DNA and rRNA show that 0.08% of the nuclear DNA is occupied by rRNA coding sequences and that the average repetition frequency of these coding sequences is approximately 144. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA was determined by the examination of the structure of rRNA:DNA hybrids in the electron microscope. Long DNA strands contain several gene sets (18S + 26S) with one repeat unit containing the following sequences in the order given: (a) An 18S gene of length 2.12 kb, (b) an internal transcribed spacer of length 2.01 kb, which contains a short sequence that may code for a 5.8S rRNA, (c) A 26S gene of length 4.06 kb which, in 20% of the cases, contains an intron with an average length of 5.62 kb, and (d) an external spacer of average length 9.23 kb.

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A new method of in situ hybridization

Cited by 114 publications

References 17 publications

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences

Sequence arrangement of the rRNA genes of the dipteran Sarcophaga bullata

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