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D L Wulff

Publications and source records attributed to D L Wulff.

13 recordsLinked to original sources
PubMed1991

The role of the OOP antisense RNA in coliphage lambda development.

We have made a derivative of bacteriophage lambda that makes no OOP antisense RNA. The mutant phage carries a point mutation that inactivates the OOP promoter, po. The phages lambda + and lambda po- have identical plaque morphologies, one-step growth curves, and frequencies of lysogenization of a sensitive host. OOP RNA synthesis is weakly repressed by the Escherichia coli LexA protein. Consonant with this inducibility of OOP RNA synthesis by ultraviolet light, we find a two-fold greater phage burst following ultraviolet induction of a lambda + than of a lambda po- prophage. In lambda + infections, OOP RNA causes two cleavage events in cll mRNA: one is in the 3'-end of the coding region, and the second is in the intercistronic region between the cll and O genes. The cll gene fragments are subject to additional hydrolytic events, and cll mRNA levels are several-fold lower in lambda + than in lambda po- infections late in the infection cycle. However, O mRNA levels are almost unaffected by the po- mutation.

Bacterial Proteins↗
PubMed1990

The cleavage specificity of RNase III.

We determined sites in lambda cII mRNA that are cleaved by RNase III in the presence of lambda OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cII mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the cII mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing.

Base Sequence↗
PubMed1990

RNase III-dependent hydrolysis of lambda cII-O gene mRNA mediated by lambda OOP antisense RNA.

The 77-nucleotide OOP antisense RNA of bacteriophage lambda complements lambda cII-O mRNA in a region that includes 55 nucleotides at the 3' end of the cII gene and 22 nucleotides in the intercistronic region between the cII and O genes. OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression by approximately 100-fold through an RNase III-dependent mechanism. Using primer extension analysis of cellular RNA isolated from an induced lambda lysogen that contains an OOP DNA plasmid, we have identified a cleavage site in cII-O mRNA within the region of complementarity with OOP RNA, at 13 nucleotides from the 3' end of that region. Ribonuclease protection experiments demonstrate that almost all cII-O mRNA in this overlap region is cleaved when OOP RNA is overproduced in RNase III+ cells but not in RNase III- cells. RNA fragments are detected that extend into the O gene from the cleavage sites, while the sister fragments that extend into the cII gene cannot be detected and must be eliminated by additional hydrolytic events. Differences in levels of uncleaved mRNA between RNase III+ and RNase III- cells are much less at several hundred nucleotides to either side of the target region. An alternate OOP RNA-dependent hydrolytic process occurs in RNase III- cells that results in cleavages in one of two regions, one close to the cleavage site observed in RNase III+ cells, and the second several nucleotides beyond the end of the complementary region between OOP RNA and cII-O mRNA. In this latter case, the fragments that extend into the cII gene are stable, while the sister O gene fragments are destroyed, in direct contrast to the RNase III-dependent process.

Bacteriophage lambda↗
PubMed1988

Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

The bacteriophage lambda transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth. We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein. A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization. The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host. An Escherichia coli HtpR- strain has been identified that greatly stabilizes these highly unstable cII mutants.

Amino Acid Sequence↗
PubMed1987

Mutations that affect the efficiency of translation of mRNA for the cII gene of coliphage lambda.

Starting with the lambda pRE-strain lambda ctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, lambda dya2 ctr1 cy3008 and lambda dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations in cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'. --The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII-mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A----G mutation four bases before the initial AUG codon, and cII3059, a GUU----GAU (Val2----Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed1987

Mutations that improve the pRE promoter of coliphage lambda.

The dya5 mutation, a C----T change at position -43 of the lambda pRE promoter, results in a twofold increase in pRE activity in vivo. Smaller increases in pRE activity are found for the dya2 mutation, a T----C change at position -1 of pRE, and the dya3 mutation, an A----G change at +5 of pRE. The mutant pRE promoters retain complete dependence on cII protein for activity. These observations argue, at least for pRE-like promoters, that promoter activities are influenced by nucleotide sequences at least eight nucleotides to the 5'-side of the conventional -35 region consensus sequence, and by nucleotide sequences near the start-site of transcription. Although Hawley and McClure (1983) found A-T pairs more frequently than G-TC pairs in the region of -40 to -45 of prokaryotic promoters, other mutations that change a G-TC pair to an A-T pair at positions -41, -44 and -45 of pRE do not result in increased promoter activity. We also found that a T----C change at positions -42 results in a mild decrease in promoter activity. These observations argue that Ts at positions -42 and -43 of pRE are required for maximum promoter activity, but do not support the hypothesis that As and Ts in the -40 and -45 region generally lead to higher promoter activities.

Bacteriophage lambda↗
PubMed1987

Cross-specificities between cII-like proteins and pRE-like promoters of lambdoid bacteriophages.

We have investigated the activation of transcription from the pRE promoters of phages lambda, 21 and P22 by the lambda and 21 cII proteins and the P22 c1 (cII-like) protein, using an in vivo system in which cII protein from a derepressed prophage activates transcription from a pRE DNA fragment on a multicopy plasmid. We find that each protein is highly specific for its own cognate pRE promoter, although measureable cross-reactions are observed. The primary recognition sequence for cII protein on lambda pRE is a pair of TTGC repeat sequences in the sequence 5'-TTGCN6TTGC-3' at the -35 region of the promoter. This same sequence is found in 21 pRE, while P22 pRE has the sequence 5'-TTGCN6TTGT-3', which is the same as that of lambda ctr1, a pRE+ variant of lambda. lambda ctr1 pRE is half as active as lambda + pRE when assayed with either the lambda cII or the P22 c1 proteins. Therefore, the single base change in the P22 repeat sequence cannot explain why the P22 c1 protein is much more active with P22 pRE than lambda pRE. The dya5 mutation, a G----A change at position -43 of pRE, makes pRE a stronger promoter when assayed with either the lambda or 21 cII proteins or the P22 c1 protein. We conclude that efficient activation of a cII-dependent promoter by a cII protein requires sequence information in addition to the TTGC repeat sequences. We do not know the characteristics of the proteins which are responsible for the specificity of each protein for its own cognate promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed1987

OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression through an RNase III-dependent mechanism.

OOP RNA is a major short (77 bases) transcript that is made from bacteriophage lambda DNA both in vivo and in vitro. OOP RNA is synthesized in the opposite direction to mRNA for the lambda cII gene, and the final 55 bp of the OOP region overlaps the 3' end of the cII gene. We find that a multicopy plasmid containing an OOP DNA fragment inhibits cII expression from a derepressed prophage by approximately 100-fold, using an in vivo assay in which cII protein activates galactokinase synthesis from a cII-dependent promoter on a multicopy plasmid. A large inhibitory effect is also observed when the po promoter for OOP RNA is replaced by the strong lambda pL promoter, but not when po is deleted. Plasmids that provide a large excess of "anti-OOP" RNA (RNA that is complementary to OOP RNA) make OOP RNA a less effective inhibitor of cII expression. Inhibition by the OOP DNA plasmid is not observed in an Escherichia coli strain deficient in RNase III. We propose that the 3' end of cII mRNA and OOP RNA form a double-stranded complex that is a substrate for the host enzyme RNase III, resulting in degradation of cII mRNA. Deletion studies on the OOP DNA plasmid indicate that no specific sequence between the promoter and terminator stem structure is required for the inhibitory effect. Lambda cII expression from an induced prophage is increased twofold in the presence of a large excess of anti-OOP RNA. This experiment, in which the prophage is the sole source of OOP RNA, suggests a physiological role for OOP RNA in regulating cII-gene expression.

Bacteriophage lambda↗
PubMed1979

Mutations reducing the activity of c17, a promoter of phage lambda formed by a tandem duplication.

We report the isolation of four independently selected mutations (scs) in the c17 promoter of phage lambda that reduce or eliminate the promoter activity. The c17 promoter is not normally present in lambda, and has been shown to be generated by a tandem duplication which creates a "Pribnow Box," a heptamer sequence implicated in promoter activity. This sequence is located upstream from the site of transcription initiation and is present, with some variation, in all promoters whose sequences have been determined. Analysis of the c17 duplications carrying the scs mutations reveals that three of these mutants carry single base-pair changes in the most highly conserved base pairs of the Pribnow Box and that the other mutation is a reversion to the wild type sequence in this region (i.e., a loss of the duplicated base pairs).

Base Sequence↗
PubMed1978

The relationship between function and DNA sequence in an intercistronic regulatory region in phage lambda.

rho factor-mediated transcription termination at the tr1 terminator site of bacteriophage lambda is examined. Mutations affecting the termination event are characterised. These mutations define features of the site which seem to be important to terminator function. In addition, other related transcriptional and translational regulatory elements are defined within the region surrounding the termination site. The potential molecular interactions and structural overlaps of these control signals apparently couple the regulation of the decision between lytic and lysogenic growth patterns by phage lambda.

Base Sequence↗
PubMed1976

Lambda cin-1, a new mutation which enhances lysogenization by bacteriophage lambda, and the genetic structure of the lambda cy region.

Seven lambda cy mutants have been mapped within a small region located approximately halfway between the rightward boundary of the imm434 region and the lambda cII gene. The seven mutants lie at four sites separated by a total distance of about 12 nucleotide pairs, as estimated from recombination frequencies. Six of the seven mutants lie on the right side of the cy fine structure map, spanning a total distance of about 3-5 nucleotide pairs. Lying approximately 11-21 nucleotide pairs to the left of the leftmost cy mutant is a newly described mutation called cin-1, for c independent. The cin-1 mutation allows some lysogenization when coupled with any cy, cII or cIII mutant, but not when coupled with a defective cI gene. The cin-1 mutation, like cy mutants, has a cis-dominant action upon the cI gene in mixed infections. The observation that gammaimm434 cin-1-cy2001 lysogenizes efficiently, but not gammaimm434 cin-1 cy2001 cII68 nor any other gammaimm434 cin-1 cy derivative, is interpreted to mean that all of the cy mutants on the right side of the cy fine structure map inactivate a binding site for cII/cIII function, but that cy2001, the single mutant on the left side of the cy fine structure map, does not inactivate that binding site.

Chromosome Mapping↗
PubMed1976

Mutations masking the lambda cin-1 mutation.

A lambda cnc mutation masks the phenotype of the lambda cin-1 mutation. Eleven cnc mutations have been found. All appear to be identical and all map at a site just to the right of the cin-1 site. It is probable that cin-1 and cnc affect adjacent, or almost adjacent nucleotide pairs. Neither the cin-1 nor the cnc mutation involves large additions or deletions of genetic material.

Chromosome Mapping↗