Béla Sain

The number of rRNA genes in Escherichia coli

The three different ribosomal RNA species of Escherichia colt’ (5 S, 16 S and 23 S) originate from a single 30 S precursor RNA. The DNA coding for this 30 S rRNA is called an rRNA gene set (rm). It has long been known that these genes are redundant in bacteria, their number has been estimated on the basis of saturation hybridization experiments as being 510 (for a review see [ 1 ] ). The most reliable measurements suggested the existence of six rm genes [2]. Plasmids and transducing phages helped to map unambiguously five of these, which are now designated rrnA (85 min), rrnB (88 min), rrnC (83 min), rmD (71 min) and rmE (89 min) [3]. In this paper we present evidence strongly suggesting that the actual number of rRNA gene sets is seven in E. coli K12.

The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434

A new plasmid vector, pNS1, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. It is a derivative of pBR327, and it includes a regulatory region from the lambdoid phage 434. The expression of the TcR gene of pNS1 is under the control of the ORpR operator-promoter of phage 434, which is regulated by the repressor gene cI. The cloning sites of pNS1 (StuI, NdeI, HpaI, HindIII, AsuII and EcoRI) are situated within cI; hence insertion of foreign DNA into these sites causes depressed expression of the TcR gene from pR thus conferring the TcR phenotype on the harboring Escherichia coli strain. The use of pNS1 is facilitated by the presence of another selectable marker, ApR, its small size, and its known nucleotide sequence; no special host strain is required.

Cloning of an E. coli ribosomal RNA gene and its promoter region from λrifd18

The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results: (1) We failed to clone an EcoRI fragment with the rRNA promoter region in plasmid RSF2124. (2) A smaller EcoRI-BamHI fragment with the rRNA promoter was also unclonable by itself, but one recombinant was found containing this fragment together with another large (7 Mdaltons) fragment, derived from phage lambda. The presence of this large fragment proved to be essential. The identity of these DNA fragments in the recombinant clone was confirmed by redigestion with several restriction enzymes, hybridization with rRNA, and in vitro transcription experiments, which showed preferential rRNA transcription. (3) A BamHI fragment encompassing the entire rRNA gene was easily cloned. Such stable clones carried a doubled number of rRNA genes. In vitro transcription using the recombinant plasmid resulted in 70% rRNA transcription. These recombinant clones allow the easy purification of the relevant DNA fragments for further investigation including sequencing.

Restriction endonuclease analysis of the transducing bacteriophage lambda rif d18

The λ rif d18 bacteriophage carries essential parts of the E. coli genome which can not be mapped by conventional methods. The phage DNA was analysed with four restriction endonucleases (endo R. BamHI, Sall, HpaI and EcoRI) and a physical map was constructed.

Homology between rRNA of Escherichia coli and mitochondrial DNA of maize

Chloroplast and mitochondria of higher plants possess their own DNA coding for organelle-specific RNAs and proteins. The similarity between the translation systems of chloroplast and mitochondria and those of bacteria may serve as a proof of the evolutionary relationship between these semiautonomous cell organelles and procaryotic organisms [l-3], Recently, compa~son of 16 S rRNA sequences of Escherichia coZi and maize chloroplast has revealed a 76% homology between the two rRNAs [4]. A similar result was obtained from sequencing data of 16 S rRNA of E~g~e~~ ~acili~ chloroplast [S]. We demonstrate here the homology between rRNA of E. coli and mitochondrial DNA of maize based on Southern hybridization experiments.

Physical mapping of bacteriophage lambda DNA with restriction endonuclease HpaI

The restriction endonuclease from Haemophilus parainfluenzae, endoR.HpaI cleaves lambdacI857s7 DNA into 14 fragments. The sizes of these fragments were determined and a physical map was constructed. The ordering of the fragments was carried out using different deletion and substitution mutants of lambda phage, double cleavages with another restriction enzyme, endoR.BamHI, and partial protection of individual HpaI recognition sites by the antibiotics distamycin A and actinomycin D. HpaI produces fragments from the left arm of the lambda DNA genome, which may help in investigating the structure and function of this part of the phage.

Cloning of the promoters of an Escherichia coli rRNA gene New experimental system to study the regulation of rRNA transcription

The promoters of the rrnB gene of Escherichia coli have been cloned on a multicopy, pBR322-derived plasmid by deleting most of the structural part of rrnB and fusing the terminators of the gene immediately to the promoters. Several further deletions were constructed to vary the promoter-terminator distance, destroy or damage selectively any of the promoters or terminators, and vary the distance between the two pairs of P1 P2 and P3 P4 promoters. All these transcription signals were shown to function on the plasmids in vitro and in vivo. The truncated in vivo transcription products initiated at the P1 and P2 promoters of the recombinant plasmids were found to be stable, and the accumulated transcripts could be easily distinguished from the chromosome-coded rRNA. This provides a convenient experimental system to study the regulation of rRNA biosynthesis.

Bacteriophage purification by gel chromatography.

Abstract We have developed an inexpensive procedure for bacteriophage purification suitable for small- and medium-scale preparations (up to one liter of lysate). The method consists of precipitation with polyethylene glycol 6000 and gel chromatography on a Bio-Gel A-5m column. The purity of the phage preparation is comparable to that obtained by CsCl step gradient ultracentrifugation.

MITOCHONDRIAL DNA OF MAIZE

ABSTRACT Mitochondrial DNA of higher plants differs in many characteristics from yeast and mammalian ones. The large size of plant mitochondrial DNAs predicts that additional information is coded by them in addition to the known mitochondrial functions. The cytoplasmic male sterility is such an additional trait in maize. In the present paper we characterize the mitochondrial genome of maize by comparative restriction endonuclease analysis. Colony banks of mtDNAs of sterile cmsS, cmsT and normal maize lines were constructed in pBR322 plasmid vector. Mitochondrial plasmid-like molecules of cmsS maize line were cloned into PstI-site of pBR322 by the homopolymer extension method. By using recombinant plasmid pCK5D1 containing 3.4MD mitochondrial plasmid and Southern hybridization method episomal nature of mitochondrial plasmid-like molecules was established. Homologous sequences were found by hybridizing the mtDNA of maize with rRNA of E.coli. These results suggest that the mitochondrial systems of maize may serve as a good tool for clarifying of the evolutional origine of eucaryotic transposable elements and the plant mitochondrial genome.

RESTRICTION ENDONUCLEASES AS TOOLS IN PHYSICAL MAPPING AND RESTRUCTURING OF BACTERIAL DNA

ABSTRACT 1. The restriction endonuclease of Bacillus sphaericus had been purified to homogeneity. The enzyme offers several advantages both as a practical tool in biochemical research, and as a model system in studying sequence-specific DNA-protein interactions. It is stable, easy to purify and is produced in higher amounts than any other restriction endonuclease so far reported. Its corresponding methylase is also known. The enzyme cleaves single-stranded DNA as well heteroduplexes with methylated complementary strand. 2. The use of restriction endonucleases as tools is illustrated by a study of bacterial ribosomal RNA genes. The number of E.coli rRNA genes had been determined by analyzing the fragmentation pattern of E.coli DNA resulting from BamHI, and Sall digestion. The detailed physical map of a transducing bacteriophage carrying an rRNA gene had been established. Restriction endonuclease-generated fragments corresponding to the total rRNA gene or its promoter region had been cloned with the plasmid vector pBR 313. A more detailed map of the cloned fragment was prepared. Sequencing of the promoter is in progress.