An Escherichia coli recBCsbcBrecF host permits the deletion-resistant propagation of plasmid clones containing the 5'-terminal palindrome of minute virus of mice

The deletion events that have plagued attempts to maintain molecular clones with long palindromic DNA sequences in Escherichia coli have been shown to be less frequent in recBCsbcB hosts [Collins et al., Gene 19 (1982) 139-146]. This study sought to determine if such hosts would permit the stable propagation of plasmid clones carrying the deletion-generating, 206 nucleotide (nt) long, imperfect palindrome derived from the 5' terminus of the genome of minute virus of mice (MVM), an autonomous parvovirus [Astell et al., Nucleic Acids Res. 11 (1983) 999-1018]. To this end these hybrid plasmids were used to transform several different mutant recBCsbcB hosts, followed by the isolation and restriction mapping of plasmid DNA from selected transformants. Characterization of plasmid DNA isolated from a recBCsbcBrecF host indicated deletion-resistant propagation of the intact species. Sequence analysis of unamplified and chloramphenicol (Cm)-amplified plasmid DNA obtained from these clones confirmed the integrity of the palindromic region of the viral DNA insert.     

An IS4 transposition causes a 13-bp duplication of phage lambda DNA and results in the constitutive expression of the cI and cro gene products

We have examined the nature of the additional DNA present in lambda hyp- mutants (Eisen et al., 1982). This DNA is an IS4 element in orientation I, in the y region of bacteriophage lambda at nucleotide position 39,139 (see Moore et al., 1979). Our assignment is based on (i) the similarity in size derived from the PstI, AvaI, and HindII restriction pattern and (ii) the DNA sequence of both the left and right lambda-IS4 DNA junctions in phage lambda hyp15rev4. The IS4 integration event resulted in the duplication of 13 bp of lambda DNA in contrast to the 11- and 12-bp duplications previously observed at the sites of IS4 integrations elsewhere (Klaer et al., 1981).     

The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase alpha in vitro was analyzed using circular, single-stranded M13 or phi X174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3'-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by alpha-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.     

Characterization of two types of rRNA gene repeat units from the crustacean Artemia

We have previously described that Artemia rRNA genes are organized with a basic repeat unit of 16.5 kb [Cruces et al., Biochem. Biophys. Res. Commun. 98 (1981) 404-409]. Here we describe the organization of the DNA coding for rRNA of a different population of this crustacean that has a repeat unit of 12.2 kb. Both types of repeat units have been cloned and the organization of the external spacers studied by restriction analysis. Both external spacers contain repeated sequences, but they are not homologous to each other. Sequences from the external spacer of the 16.5 kb repeat are also found elsewhere in the genome, within sequences not related to rRNA genes.     

Escherichia coli molecular genetic map (1500 kbp): update II

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter-selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.     

Analysis of cosmids using linearization by phage lambda terminase

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage λ terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested' with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage λ (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.     

The nucleotide sequence of the polypeptide IX gene of human adenovirus type 3

The nucleotide sequence of the DNA segment encompassing the polypeptide IX gene of class B human adeno-virus serotype 3 (Ad3) has been determined using cloned restriction fragments. There is only a single, open translational reading frame capable of specifying a protein of 138 amino acids, comparable to the M_r 12000–13000 of protein IX detected in virions (Wadell, 1980). The corresponding region of a closely related class B virus, Ad7, is virtually identical (Dijkema et al., 1981), but the comparable segments of class C viruses Ad2 or Ad5 are much less homologous (Aleström et al., 1980; Maat et al., 1980). There are 150 single bp changes and 19 deletion-insertions, at least one frameshift, together affecting 210 nucleotides within the 455 bp comparison positions of the protein-coding regions of Ad2 (423 bp) and Ad3 (417 bp). Each of the 19 deletion-insertions involves an integral multiple of 3 bp in phase with the open translation frame. There is no “TATA” promoter box in Ad3 DNA at the position comparable to that of Ad2. The deduced protein sequences near the amino-terminus are extensively conserved between the two classes of viruses, but the carboxy-terminal portion and the nucleotide sequences flanking the gene are much more diverged. In both classes, these N- and C-terminal regions of the inferred proteins are linked by an alanine-rich chain, an arrangement suggestive of two functional domains.