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P Youderian

Publications and source records attributed to P Youderian.

At least 19 recordsLinked to original sources
PubMed1995

Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.

Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues. Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt). Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro. Whereas three of these eight mutant repressors are unstable in E. coli, all are made at similar steady-state levels in Salmonella typhimurium. Three of the eight mutant holorepressors are lethal when overproduced in S. typhimurium, because they confer an induced auxotrophy. Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA. These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work.

Amino Acid Sequence↗
PubMed1994

A site-specific endonuclease derived from a mutant Trp repressor with altered DNA-binding specificity.

Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second "recognition" alpha-helix of the helix-turn-helix DNA-binding motif. Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors. Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot. One mutant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type repressor. A site-specific nuclease was derived from Lys79 repressor by constructing a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct. This nuclease has an altered specificity of DNA binding in vitro. When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency.

Bacterial Proteins↗
PubMed1992

Mutant lambda repressors with increased operator affinities reveal new, specific protein-DNA contacts.

The binding specificities of four mutant lambda cI repressor proteins with increased affinities for operator DNA were examined. Two mutant repressors (Glu34----Lys and Glu83----Lys) have the same specificity of binding as wild-type repressor, whereas two (Gly48----Ser and Gly48----Asn) have new binding specificities. The Gly48----Asn mutant repressor recognizes lambda operators with changes at base pair 3 with a different order of affinity than wild-type repressor, suggesting that the side chain of Asn48 makes additional specific DNA contacts at or near this base pair. When paired with a change that disrupts the specific interaction of the amino-terminal arm of lambda repressor with DNA (Lys4----Gln), one change that increases the affinity of repressor (Gly48----Ser) suppresses the binding defect of the Lys4----Gln repressor, resulting in a double mutant repressor with a new binding specificity different than that of both its parents and of wild type. These results lend strong support to the model of direct recognition of the lambda operator by lambda repressor proposed from the crystal structure of the repressor/operator complex.

Bacteriophage lambda↗
PubMed1992

Somatic cell mapping of the human cyclophilin B gene (PPIB) to chromosome 15.

The polymerase chain reaction (PCR) technique was used to generate a unique probe complementary to the hydrophobic 5' end of the human cyclophilin B gene. This unique probe was hybridized to DNAs from human x hamster hybrid somatic cell lines retaining different combinations of human chromosomes. The gene was assigned to chromosome 15.

Amino Acid Isomerases↗
PubMed1991

Mutant tryptophan aporepressors with altered specificities of corepressor recognition.

The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.

Apoproteins↗
PubMed1991

Automated kinetic assay of beta-galactosidase activity.

An automated kinetic assay for beta-galactosidase activity in Escherichia coli was developed to permit the measurement of many independent samples simultaneously. Bacteria are grown, lysed from without (by adsorption of a high multiplicity of bacteriophage T4) and assayed in microtiter plates with 96 wells. Absorbance data are collected and analyzed by computer. The growth and lysis procedure, apparatus and software used in this assay can be used for other spectrophotometric enzyme assays.

Automation↗
PubMed1989

Phage lambda Cro protein and cI repressor use two different patterns of specific protein-DNA interactions to achieve sequence specificity in vivo.

By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.

Amino Acids↗
PubMed1988

Mutant Trp repressors with new DNA-binding specificities.

Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of the Escherichia coli trpR (tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes. Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensus trp operator. Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities. These new specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair. The Trp repressor does not use the first two amino acids of its "recognition alpha-helix," Ile79 and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites.

Alanine↗
PubMed1988

Mutations that improve the binding of yeast FLP recombinase to its substrate.

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

Codon↗
PubMed1988

Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages.

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.

Chromosomes, Bacterial↗
PubMed1988

Phase variation in Salmonella: analysis of Hin recombinase and hix recombination site interaction in vivo.

The bacteriophage P22-based challenge phase selection was used to characterize the binding of Salmonella Hin recombinase to the wild-type hixL and hixR recombination sites, as well as to mutant and synthetic hix sequences in vivo. Hin recombinase binds to the hixL or hixR recombination sites and represses transcription from an upstream promoter in the challenge phage system. Hin-mediated repression results from Hin associating into multimers either prior to binding or during the binding process at the hix operator sites (cooperativity). The ability of Hin multimers to repress transcription is eliminated when the hix 13-bp half-sites are rotated to opposite sides of the DNA helix by inserting 4 bp between them. Insertion of 1 bp between half-sites reduces overall repression. Hin also binds one of the hixL half-sites to repress transcription, but only when high levels of Hin protein are present in the cell. Mutations have been identified in the hix sites that impair Hin binding. Five of the 26 bp in the hix sites are critical; sites with base-pair substitutions at these five positions show greatly reduced binding. Three additional base pairs make minor contributions to binding. These results are consistent with the results of binding studies between Hin and the hix sites in vitro.

Base Sequence↗
PubMed1987

Spontaneous mutations occur near dam recognition sites in a dam- Escherichia coli host.

The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.

Chromosome Deletion↗
PubMed1987

DNA specificity determinants of Escherichia coli tryptophan repressor binding.

We have analyzed the sequence-specific interaction between the Escherichia coli tryptophan (Trp) repressor and its operator using challenge phage vectors. These phages, derivatives of Salmonella phage P22 that have substitutions of synthetic, symmetric trp operators for the P22 mnt operator, provide a genetic assay for DNA binding in vivo. Phages carrying operators that retain the determinants of Trp repressor binding efficiently lysogenize cells producing repressor; in contrast, phages with operators missing critical determinants kill such hosts. The binding determinants revealed by this assay corroborate a simple docking model for the Trp repressor-operator interaction postulated from the repressor crystal structure, and account for both the specificity of repressor binding and the ability of Trp repressor to recognize multiple, tandem DNA sites.

Bacterial Proteins↗
PubMed1986

Structural and regulatory divergence among site-specific recombination genes of lambdoid phage.

The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini.

Amino Acid Sequence↗
PubMed1986

General selection for specific DNA-binding activities.

We present a general strategy for the selection of bacterial clones that express DNA-binding activities corresponding to particular DNA recognition sites. The selection uses a "challenge phage" vector, P22 Kn9 arc-amH1605, into which is substituted a synthetic DNA-binding site for a site that controls transcription of the P22 antirepressor (ant) gene. Constitutive synthesis of antirepressor channels a challenge phage into lytic development and efficiently kills an infected host, unless the substituted site is bound by a specific protein; in this case, the challenge phage prefers lysogenic development, and the host survives and acquires an antibiotic-resistance phenotype. Infections with challenge phages carrying the E. coli Lac operator, phage lambda OL1 operator, or synthetic, "idealized" E. coli Trp and Tn10 Tet operators select clones that express each of the corresponding binding activities. The use of challenge phage vectors may be extended to select clones that express eukaryotic DNA-binding activities.

Base Sequence↗
PubMed1985

The bacteriophage P22 arc and mnt repressors. Overproduction, purification, and properties.

The arc and mnt genes of bacteriophage P22 encode small repressor proteins. We have cloned these genes onto plasmids that overproduce Arc and Mnt to greater than 1% of the soluble cellular protein. Both proteins were purified to greater than 95% homogeneity, and N-terminal sequences and amino acid compositions were determined. These data, in combination with previously determined gene sequences, establish the complete protein sequences for Arc (53 residues) and Mnt (82 residues). Both proteins have melting temperatures between 45 and 55 degrees C and can be renatured to a fully active species. Arc is a dimer in solution and Mnt is a tetramer.

Amino Acid Sequence↗