Bernard Allet

Mu insertion duplicates a 5 base pair sequence at the host inserted site

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.

Mapping of a cluster of genes for components of the transcriptional and translational machineries of Escherichia coli

A number of genes that code for components of translational machinery are closely linked to the genes for the RNA polymerase subunits β and β′ and are carried by a transducing phage, λrifd18. In order to map these genes, we have first investigated the physical structures of λrifd18 and the related, but independently isolated, transducing phage, λrifd12, using DNA heteroduplex analysis and digestion with various restriction enzymes. The order of the protein genes was determined by isolation of the DNA restriction fragments and examination of their ability to stimulate synthesis of proteins in vitro. From these experiments and from the results on the RNA gene mapping reported previously (Lund et al., 1976), the following order has been deduced: genes for 16 S rRNA, tRNA2Glu (gltT), 23 S rRNA, 5 S rRNA, translational elongation factor Tu (tufB), ribosomal proteins L1 and L11 (rplA and rplK, order not determined), ribosomal protein L7/L12 (rplL), RNA polymerase subunit β (rpoB), and RNA polymerase subunit β′ (rpoC). From heteroduplex analyses of the three independently isolated transducing phages, λrifd12, λrifd18, and 80rifr, we conclude that the gene order identified on λrifd18 is probably identical to that on the Escherichia coli chromosome, except that the identity of the tRNA gene in the rRNA transcription unit is ambiguous.

Structure analysis at the ends of the intervening DNA sequences in the chloroplast 23S ribosomal genes of C. reinhardii

All of the chloroplast 23S ribosomal genes of C. reinhardii are interrupted by a 0.87 kb sequence (Rochaix and Malnoë, 1978). We have sequenced the DNA across the two ends of this intervening element. In parallel, we have examined the nucleotide sequences in the corresponding part of the 23S ribosomal RNA. This allowed us to locate precisely the boundaries between the coding (that is, transcribed into mature 23S rRNA) and the noncoding DNA. The results show that the intervening sequence is flanked by two identical sets of 3 bp (5-CGT) oriented as direct repeats. In addition, a sequence of 5 bp (5-CGTGA) lies exactly next to one end and is found very close (16 bp) to the other end, in the coding part of the gene. These two sets are also oriented as direct repeats. Finally, sequences near one end of the intervening element are found with a few alterations near the other end, but in an inverted orientation. Possible interpretations of these results are discussed.

Partial correlation of the genetic and physical maps of bacteriophage Mu

Abstract λpMu phage particles that carry varying amounts of the Mu genome have been used to correlate the genetic and physical maps of phage Mu DNA. Two complementary techniques, restriction enzyme fragment analysis and electron microscope heteroduplex mapping, have been employed to identify the segments of Mu DNA present in the λpMu particles whose genetic complement had been determined by Mu amber mutant marker rescue. These analyses permitted us to assign a physical location of genes A to lys on one end (immunity end) of the Mu genome, and genes L to S on the other end (variable end). None of the hybrids described here included genes E to K which lie in the middle part of Mu DNA.

Synthesis of bacteriophage mu proteins in vitro

Abstract DNA extracted from purified Mu phage directs the synthesis of at least 12 unique proteins in a DNA-dependent protein-synthesizing system derived from Escherichia coli . A comparison of the proteins synthesized in vitro from various DNA templates, including Mu mutants, λpMu hybrid phages, or specific Mu fragments generated by digestion with restriction enzymes, has allowed us to assign several of the proteins to specific genes. Assuming that the products made in vitro are the same as those synthesized in vivo , our results lead to the following interpretations. (a) A gene in the immunity region codes a protein of 24,000 daltons which may be the phage repressor; (b) gene A product is a polypeptide of 70,000 daltons; a protein of 64,000 daltons in coded by gene M or N ; (c) gene B (or Bu ) product is 33,000 daltons and is synthesized in large quantities; (d) the product of gene R is 41,500 daltons; (e) two polypeptides of 53,000 and 35,000 daltons, respectively, may be coded by genes between E and L ; (f) a gene between B and C directs the sythesis of a protein of 14,000 daltons; and (g) a major protein is coded by DNA in the G-loop or β region.

Studies with a λ rif Transducing Phage Carrying the Genes for the β and β ′ Subunits of E. coli RNA Polymerase

INTRODUCTION Specialized transducing phages carrying specific bacterial genes or sets of genes have contributed greatly to our understanding of bacterial systems at both the genetic and the biochemical level. To facilitate the genetic analysis of Escherichia coli RNA polymerase (E.C. 2.7.7.6) and to provide a means for ultimately studying in vitro the regulation of the synthesis of the various polymerase subunits, λ transducing phages were isolated (Kirschbaum 1973a; Kirschbaum and Konrad 1973) that were initially shown to carry the gene for the β subunit (Kirschbaum 1973b). Subsequent genetic studies established that many of these transducing phages carried the structural gene for the β ′ subunit as well (Kirschbaum and Scaife 1974). This observation is consistent with the recent finding that the genes encoding the β and β ′ subunits are organized in the form of a single operon on the bacterial chromosome (Errington et al. 1974). The direct selection of phages carrying at least the β subunit gene ( rif ) was made possible by the isolation of a dominant rifampicin resistance mutation, rif d (Kirschbaum 1973a). This series of rif d transducing phages was generated by heat induction of an unusual λ lysogen in which λ C 1857 S 7 had been integrated into a site ( bfe ) on the bacterial chromosome that was very closely linked to rif (Figure 1) in a strain carrying the rif d mutation (Kirschbaum 1973a; Kirschbaum and Konrad 1973). This report is the result of several independent studies involving one such phage, λ c 1857 S 7d rif d 18 (hereafter referred to as λd rif d 18), and describes (a) the determination of the...