Isolation of Lambda Transducing Phage with the bio Genes Inserted between Lambda Genes P and Q

Plaque-forming, biotin-transducing phages were constructed with the bio genes inserted between lambda genes P and Q. These phages were isolated for the eventual aim of fusing the lambda Q gene to the bio operon. The following steps were used to construct these phages: A defective temperature-sensitive lysogen was constructed with the bio genes adjacent to and to the left of lambda genes beta NcI857OPQSRA. Heat-resistant survivors were screened for deletions with endpoints in the bio operon and to the right of lambda P and to the left of lambda A. Five of approximately 1,600 heat-resistant survivors had these properties. Two had the gene order bioAB .... lambda QSRA. When these two strains were lysogenized with lambda cI857b221 and heat induced, the desired transducing phages were obtained. We characterized these phages and studied one in detail. Two-thirds of the plaque-forming transducing phages isolated carried the entire bioB gene and only part of the bioA gene, and one-third carried the entire bioA and bioB genes. The phages isolated lost the bio genes upon propagation, indicating that they contain a partial duplication of phage genes. The duplication was shown not to involve the entire lambda Q gene in one of these phages, lambda bioq1b221. A recombinant of this phage, lambda Nam7am53c17b221, failed to form plaques under biotin-derepression conditions. We conclude that if the lambda Q gene was fused to the bio operon in this phage, not enough lambda Q gene product was made to allow phage propagation.     

Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts

A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R-qut-t'R1 module. Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression. This facilitates cloning with lambda red- gam- vectors in recA- hosts.     

Organization and structure of the Qa genes of the major histocompatibility complex of the C3H mouse: implications for Qa function and class I evolution.

We have determined the structure and organization of the entire Qa family of class I genes from the major histocompatibility complex of the C3H mouse. Restriction maps of overlapping lambda and cosmid clones reveal that there are only five Qak genes: Q1k, Q2k, Q4k, Q10k and a Q5/9 hybrid, presumably generated by unequal homologous recombination. The resulting deletion of Q6-Q9 is consistent with the Qa-2null phenotype of this mouse strain. We have sequenced the Qak genes, and predict that each may encode a class I molecule with a structure comparable with that proposed for the transplantation antigens. Furthermore, these Qa products should be able to bind peptides and interact with appropriate T-cell receptors. Interestingly, in comparing Qak and H-2k sequences, we find limited evidence of interlocus gene conversion between Qa and H-2 loci, suggesting that the Qa genes are not likely to serve as a reservoir of genetic information for the generation of H-2 diversity within this haplotype.     

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.     

Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains.

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a lambda(max) of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color-controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.     

Sequences required for antitermination by phage 82 Q protein

The gene Q antiterminator proteins of phages lambda and 82 modify RNA polymerase at sites (named qut) that are close to, and apparently inseparable from the promoters themselves. Modification occurs while RNA polymerase has paused close to the start site, at nucleotide 16 for lambda, and nucleotides 15 and 25 for phage 82. We present a deletion analysis of the phage 82 qut site that identifies sequences required for pausing and shows that these sequences also are required for efficient Q function in vivo and in vitro. We show (1) that deletions as close as +5 to the RNA start site retain some ability to be modified by Q82, suggesting that part of the qut site is in the non-transcribed region of the promoter; (2) that NusA protein is required for activity of Q82 on certain qut82 site deletions, whereas it only modestly stimulates antitermination from the native qut82 site; and (3) that qut82 is active only on RNA polymerase that initiates at the qut-associated promoter, and not on RNA polymerase that initiates upstream and passes through an otherwise active qut82 site.     

Genetic Analysis of the System that Reduces Biotin-d-Sulfoxide in Escherichia coli

Four genes of Escherichia coli whose products are needed to reduce biotin-d-sulfoxide to biotin have been mapped: bisA next to chlA, bisB next to chlE, bisC linked to xyl, and bisD next to chlG. A defective lambda transducing phage, lambdadbis5, which carries all the bacterial genes between the lambda attachment site and chlE, was isolated and shown to have lost the phage genes from int through Q.