M. Fiandt

Deletions and insertions in the immunity region of coliphage lambda: revised measurement of the promoter-startpoint distance.

Abstract Plaque-forming λc I − mutants which carry internal deletions in the immunity region, including genes c I and rex , were isolated. Among seven independent isolates, only three demonstrably different deletions were obtained. The largest deletion (KH54 or KH115), which eliminates almost all of the DNA between promoters p L and p R , spans the distance from 74.1 to 78.4 %λ, as measured in relation to the left (0 %λ) and right (100 %λ) termini of the mature λ DNA molecule. There were four representatives of a second (74.7–77.7%λ) and one of a third (75.2–78.1%λ) deletion. In addition, a mutant carrying a 1400-nucleotide pair-long insertion in gene c I was isolated. The c I- rex deletions served as markers for more accurate measurements of the physical positions of the endpoints of several bio insertions, a dy plasmid, and the endonucleolytic cleavage sites for the restriction nucleases by electron micrography of DNA heteroduplexes. This led to a reevaluation of our earlier estimate of the interval length between the p L promoter mutation sex 1 and the s L startpoint for the major leftward operon of λ. In agreement with the independent results of others, we find that the p L to s L distance is within the limits of 24–62 nucleotide pairs ( s L - sex 1) or 11 to 45 nucleotide pairs ( s L - Hin II cut in o L ).

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.

Precise measurement of the b2 deletion in coliphage lambda

Abstract The length of the b2 deletion is 12.0 ± 0.4%λ, as determined by comparison of the electron micrographically measured lengths of the b2+-containing AvaI fragment 3 from phage λ DNA and the corresponding fragment 3′ from λb2. This is equal to approx. 5700 base pairs, when the λ length is calculated as 48 000 base pairs, based on the current sequence data for phage φX174 and the φX174-RFII/λ DNA length ratio.

N-Independent Leftward Transcription in Coliphage Lambda: Deletions, Insertions and New Promoters Bypassing Termination Functions

SummaryLambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of λN+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.

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).

Physical mapping of the trp endpoint in the N-tL segment of phage λtrpE-A

Abstract In the transducing phage λ trp E-A, the region between the att site and gene N is replaced by the trp operon. The right terminus of the trp 60-3 substitution is within the imm 21 region just to the left of gene N at 71.8 %λ units, measured from the left terminus of the mature λpapa genome. These measurements are consistent with the notion that it is the deletion of the t L terminator which permits the p L -promoted transcription to extend from the λ genome into the trp operon, even when synthesis of the N product is blocked by chloramphenicol. The t L site must therefore be located within an area between the 71.1 %λ ( imm 21) and 71.8 %λ ( trp 60-3) boundaries.

Physical mapping of coliphage λatt2

Abstract The transducing phage lines of the type λ att 2 , which arise by abnormal excision of an integrated prophage, carry bacterial DNA from both the left and right of the prophage including the two prophage attachment sites, att L and att R. The excision sites, X L and X R , have been mapped with respect to the attachment sites by DNA heteroduplex analysis of a particular λ att 2 . The X L - att L segment, containing no known E. coli genes, is about 1300 base-pairs long (2.78 ± 0.13 %λ units). The att R - X R segment, including the bio operons and the uvrB locus, is about 7800 base-pairs long (16.8 ± 0.5 %λ units). X L and X R appear to be favored sites for the generation of λ att 2 .

Construction and properties of A ColE1::Tn3-cosλ 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.

λimmλ·434: A phage with a hybrid immunity region

Abstract The genomes of coliphages λ and λimm434 are identical with the exception of the immunity region, which codes for the respective repressors and corresponding operators. Although previous genetic studies failed to detect any recombinations that might have occurred within the immunity regions of these two phages, electron-micrographic heteroduplex mapping demonstrated a short interval of homology between λ and λimm434 DNA located in this otherwise heterologous region. We report the isolation of a recombinant which resulted from a cross-over event between λ and λimm434 within the immunity region. This phage, denoted λimmλ·434, carries the leftward promoter-operator as well as gene rex of λ and the rightward promoter-operator as well as gene tof (cro) of phage 434. Since the recombination occurred within the cI gene, phage λimmλ·434 must carry a hybrid repressor gene.

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.