I. Sequence Organization of Drosophila melanogaster 5S rRNA and 4S RNA Genes. II. In Vitro Studies on Replication of Plasmid DNAs

Year: 1979 DOI: 10.7907/d94z-c749
I. The sequence organizations of Drosophila melanogaster 4S RNA (tRNA) and 5S rRN A genes have been investigated. Segments of Drosophila DNA containing these genes have been propagated in recombinant plasmids using Escherichia coli as a host and Co1E1 as a vector.

Electron microscope partial denaturation mapping, mapping by ferritin labeling and restriction enzyme-gel electrophoresis analysis all indicate that the Drosophila DNA inserts of the 5S rRNA gene containing plasmids consist of tandem repeats of 5S genes and spacer regions. The repeat length is approximately 380 nucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer of length 260 ntp. Little heterogeneity in the lengths of the repeats has been detected.

A tRNA gene containing clone has been characterized by electron microscopic methods and by restriction endonuclease mapping. Four tRNA genes were detected on a 9.34 kb fragment of DNA. Three of these genes appear to be identical and different from the fourth. No evidence was found for extensive sequence homology in the sequences surrounding the genes. In situ hybridization with cRNA transcribed from the plasmid showed localization at region 42A of chromosome 2R.

II. An improved system for in vitro replication of Escherichia coli plasmid DNAs has been developed and characterized. Endogenous DNA is removed from the extracts, making replication dependent upon exogenous DNA even when plasmid containing cells are used as a source of extracts. Replication in this system requires E. coli DNA polymerases I and III, RNA polymerase, DNA gyrase, and the products of at least five genes required for E. coli replication (dnaB, dnaC, dnaG, dnaP, dnaZ).

This system has been used to study the replication properties of the ampicillin resistant plasmid RSF1030. We have found that, like Co1E1, RSF1030 replicates unidirectionally from a unique origin. We have also investigated the replication of pFH118, a high copy number mutant derived from Co1E1. The mutation in pFH118 is due to the insertion of 20-30 base pairs into the coding sequence for a 100 nucleotide RNA that is transcribed during DNA replication. Results of reversion studies suggest that this RN A plays a role in determining plasmid copy number. Furthermore, the RN As transcribed from Co1E1 and RSF1030 have significant sequence homology, although the plasmids were isolated independently and have been thought to have no sequence homology.