Biomedical subjects
Publications and source records attributed to M Fiandt.
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.
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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.
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.
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.
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.
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.
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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.
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