Abstract
A transmission electron microscope method for gene mapping by in situ hybridization to Drosophila polytene chromosomes has been developed. As electron-opaque labels, we use colloidal gold spheres having a diameter of 25 nm. The spheres are coated with a layer of protein to which Escherichia coli single-stranded DNA is photochemically crosslinked. Poly(dT) tails are added to the 3' OH ends of these DNA strands, and poly(dA) tails are added to the 3' OH ends of a fragmented cloned Drosophila DNA. These probe--dA strands are hybridized in situ to polytene chromosome squashes. Gold spheres are linked to the hybridized probe--dA strands by A.T base pairing. The sphere positions relative to the chromosome bands can be observed by transmission electron microscopy. The method shows low background and high resolution.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Artavanis-Tsakonas S., Schedl P., Tschudi C., Pirrotta V., Steward R., Gehring W. J. The 5S genes of Drosophila melanogaster. Cell. 1977 Dec;12(4):1057–1067. doi: 10.1016/0092-8674(77)90169-6. [DOI] [PubMed] [Google Scholar]
- Bender W., Davidson N. Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules. Cell. 1976 Apr;7(4):595–607. doi: 10.1016/0092-8674(76)90210-5. [DOI] [PubMed] [Google Scholar]
- Gerhard D. S., Kawasaki E. S., Bancroft F. C., Szabo P. Localization of a unique gene by direct hybridization in situ. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3755–3759. doi: 10.1073/pnas.78.6.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hershey N. D., Conrad S. E., Sodja A., Yen P. H., Cohen M., Jr, Davidson N., Iigen C., Carbon J. The sequence arrangement of Drosophila melanogaster 5s DNA cloned in recombinant plasmids. Cell. 1977 Jul;11(3):585–598. doi: 10.1016/0092-8674(77)90076-9. [DOI] [PubMed] [Google Scholar]
- Horisberger M., Rosset J. Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem. 1977 Apr;25(4):295–305. doi: 10.1177/25.4.323352. [DOI] [PubMed] [Google Scholar]
- Procunier J. D., Tartof K. D. Genetic analysis of the 5S RNA genes in Drosophila melanogaster. Genetics. 1975 Nov;81(3):515–523. doi: 10.1093/genetics/81.3.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robins D. M., Ripley S., Henderson A. S., Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. doi: 10.1016/0092-8674(81)90267-1. [DOI] [PubMed] [Google Scholar]
- Roychoudhury R., Jay E., Wu R. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 1976 Jan;3(1):101–116. doi: 10.1093/nar/3.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tereba A., Lai M. M., Murti K. G. Chromosome 1 contains the endogenous RAV-0 retrovirus sequences in chicken cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6486–6490. doi: 10.1073/pnas.76.12.6486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss B., Richardson C. C. End-group labeling of nucleic acids by enzymatic phosphorylation. Cold Spring Harb Symp Quant Biol. 1966;31:471–478. doi: 10.1101/sqb.1966.031.01.061. [DOI] [PubMed] [Google Scholar]
- Wimber D. E., Steffensen D. M. Localization of 5S RNA genes on Drosophila chromosomes by RNA-DNA hybridization. Science. 1970 Nov 6;170(3958):639–641. doi: 10.1126/science.170.3958.639. [DOI] [PubMed] [Google Scholar]