A relationship between DNA helix stability and recognition sites for RNA polymerase.

The RNA polymerase binding sites on the DNA of (i) the aroE-trkA-spc segment of the Escherichia coli genome, (ii) transposon Tn3, (iii) plasmid ColE1, and (iv) coliphage lambda were mapped by electron microscopy, with the use of the BAC technique; these maps were compared with the maps of the early-melting regions for the same genomes. The results indicate that in all these cases the binding sites for the E. coli RNA polymerase lie preferentially in the early melting regions of DNA. These data indicate that helix stability may be an important feature of the multipartite nature of the promoter structure.

Aminoglycoside-modifying enzymes of Staphylococcus aureus; expression in Escherichia coli.

Staphylococcus aureus plasmids PSH2, RN1956 and pWA1 code for an aminoglycoside phosphotransferase; plasmid pWA1 also encodes an aminoglycoside-aminocyclitol adenylyltransferase. S. aureus plasmid pWA2 confers resistance to erythromycin and sulfonamide. Using plasmid ColE1-ApR (RSF2124) as a vehicle, we have transferred the genes determining aminoglycoside phosphotransferase and aminoglycoside-aminocyclitol adenylyltransferase activities from S. aureus to Escherichia coli. The new plasmids obtained confer aminoglycoside-aminocyclitol resistant phenotypes to E. coli, similar to, and by the same mechanisms as "naturally" occurring plasmids. By contrast, the results obtained after cloning of plasmid pWA2 indicate that certain S. aureus antibiotic resistance determinants (e.g. for erythromycin (Em) and sulfonamide (Su) cannot be phenotypically expressed in E. coli. The DNA of the constructed hybrid plasmids has been analysed by agarose gel electrophoresis following digestion with restriction endonucleases, by ultracentrifugation in cesium chloride, by hybridization, and by electron microscopy. Each hybrid is a cointegrate replicon, composed of an entire S. aureus plasmid covalently joined to ColE1-ApR.

Bst71I: an isoschizomer of the type-IIS restriction enzyme, BbvI, recognizing the GCAGC(8/12) site.

A new type-IIS restriction enzyme, Bst71I, with the specificity 5'-GCAGC(N)8/3'-CGTCG(N)12 was isolated from Bacillus stearothermophilus (Promega No. 71). This enzyme is an isoschizomer of BbvI with somewhat improved characteristics for use by molecular biologists.

Identification of the gal3 insertion in Escherichia coli AS IS2.

The gal3 mutation in Escherichia coli, located in the operator-promoter region of the gal operon, is identified as an IS2 insertion in the polar orientation I relative to the direction of transcription. This mutation, which may be considered the earliest example of a polar mutation caused by an IS insertion, is shown by heteroduplex analysis of phage lambdagal3 to be located about 170 base pairs from the promoter-proximal end of the chlD-pgl deletion in lambdagal8. It appears indistinguishable in position, sequence and orientation from the IS2 insertion carried by lambdagal8-490. The endpoints of the bacterial DNA segments in lambdagal3 and lambdagal8 are physically mapped in relation to attL.

The site controlling the specificity of N action is outside the promoter-operator region: a triple hybrid phage lambda N21 imm434nin5.

A short interval of homology between imm lambda, imm434 and imm21 DNAs was identified near the leftward promoter-operator region. This homology, denoted Hs, was revealed by electron microscopic examination of lambda imm lambda/lambda imm21 and lambda imm434/lambda imm21 heteroduplexes, and permitted us to construct a special lambda hybrid (lambda hyB) which contains the N region of phage 21 and the adjacent imm region from phage 434. This triple hybrid, labmda N21 imm434nin5, was analysed by genetic, transcriptional and electronic micrographic techniques. Its leftward and rightward promoter-operator regions are of phage 434 specificity and are controlled by the 434 repressor. Surprisingly, the N21 gene of lambda hyB was found to be defective, perhaps to preserve the viability of the hybrid. Its leftward N-recognition system (nutL) is of phage 21 specificity since it responds only to the N21 function in complementation tests, as measured by antitermination of leftward transcription initiated at the pL promotor in the imm434 region. We conclude, therefore, that the pLoL region of 434 contains no information for the specificity of N antitermination. Both lambda imm21 and lambda hyB were found to be missing the tL1 terminator function (see also Salstrom and Szybalski, 1978b). In these phages, the tL2 terminator was found to be only 60% effective under N21 conditions, and therefore expression of their red-gam genes is sufficient to endow the lambda hyB and lambda N21- imm21nin5 phages with the Fec+ phenotype.

Construction and properties of a ColE1::Tn3-cos lambda plasmid for determining RNA polymerase binding sites on ColE1 and Tn3.

To determine the location of the RNA polymerase binding sites on the ColE1 plasmid and Tn3 transposon, a special hybrid ColE1::Tn3-cos lambda molecule was constructed which contains the left arm of phage lambda DNA and the right lambda terminal fragment. This permits orienting ColE1 molecules, since the RNA polymerase binding pattern of these two lambda fragments are known to be distinct. ColE1 DNA contains seven binding sites and Tn3 binds three RNA polymerases, with some of the latter probably involved in the expression of the transposition of functions of this transposon. The relationship of these sites to the positions and orientations of known promoters, transcripts, genes and functions is discussed.

Packaging of plasmid DNA containing the cohesive ends of coliphage lambda.

High yields of ColE1::Tn3-cos lambda plasmid genomes packaged in phage lambda virions (2.10(9) per ml) are produced by thermal induction of E. coli W3350 (lambda cI1857S7) lysogens carrying the plasmid DNA. The plasmid DNA is packaged in the linear form, with the right m' terminus of lambda being associated with the lambda tail.

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.

N-independent leftward transcription in coliphage lambda: deletions, insertions and new promoters bypassing termination functions.

Lambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of lambda N+ nutL- (Fec-) strains. In addition to true nutL+ reversions, three classes of second-site mutations were identified: (1) ninL deletions that remove a region containing either tL1 or both tL1 and tL2 termination signals, or only a small region (defining the rut site) just upstream from tL1, (2) new constitutive promoters that map just upstream from the tL2 termination site and which are created either by point mutations (hip) or by short insertion sequences (isp), (3) small internal deletions in gene cro. The positions and individual effects of these mutations, some of which only partially abolish termination function, provide evidence for a complex multipartite structure of the termination signals.

Formation of macromolecular complexes and other effects of DNA treatment with diethyl and dimethyl sulfate: physico-chemical and electron microscopic studies.

In vitro exposures of isolated DNA to one of the two carcinogenic and mutagenic chemicals, diethylsulfate or dimethylsulfate, induces several kinds of physicochemical and morphological alterations. These changes are detectable by a variety of independent techniques. A fraction of DNA treated briefly with either of these two chemicals moves during velocity centrifugation experiments less rapidly than the bulk of control DNA and more rapidly through gels during electrophoresis. This apparent decrease in size is paralleled by the formation of large DNA aggregates with mobilities indicating molecular weights several times that of the untreated, control DNA. The presence of a basic protein in the incubation mixture increases the rate of formation of such complexes. the tendency of the alkylated DNA to bind to both biological and non-biological materials is reflected in the increased attachment of DNA to columns built with methyl-esterified serum albumin and in its quantitative retention on nitrocellulose filters. DNA exposed to dimethylsulfate decreases its density in CsCl gradients. A mixture of two or more DNAs of different densities exposed to this chemical produces an u.v.-absorbing band which is found in such gradients at an intermediate density. If the alkylation reaction is carried out in the presence of a protein, a portion of DNA bands at a density intermediate between the density of DNA and that of the protein, even in the presence of an ionic detergent in the gradient. Under the electron microscope the alkylated DNA shows multiple single-strand breaks and peeling-off whiskers of denatured DNA. Aggregates of DNA molecules become visible upon further incubation of DNA with the alkylating agent. We suggest that the DNA-DNA and DNA- protein complexes play an important role in the process of carcinogenesis and mutagenesis.

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA.

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).

Insertion sequence IS2 near the gene for prophage lambda excision.

In this study we characterize a variant of the lambdacI857S7 prophage, designated lambdabi2cI857S7, which carries a DNA insertion. The insertion sequence is IS2, and it resides in the antipolar orientation II just upstream from the gene for prophage excision (xis) at 61.6%lambda. This bi2 insertion mutant could prove valuable for studies on possible recombination functions of IS2 DNA and of its effect on the lambda integration and excision functions.