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The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-[Alpha]]purin-10(3H)-one ([M.sub.1]G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. [M.sub.1]G was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5[prime]-GGT([M.sub.1]G)TCCG-3[prime] into a gapped-duplex derivative of the vector. Unmodified and [M.sub.1]G-modified genomes containing either a cytosine or thy-mine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite [M.sub.1]G resulted in roughly equal numbers of [M.sub.1]G[approaches]A and [M.sub.1]G[approaches]T mutations with few [M.sub.1]G[approaches]C mutations. The total mutation frequency was [approximately equal to]1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite [M.sub.1]G allowed an estimate to be made of the ability of [M.sub.1]G to block replication. The (-)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when [M.sub.1]G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by [M.sub.1]G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of [M.sub.1]G and that [M.sub.1]G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that [M.sub.1]G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG[).sub.180] were grown for about 100 generations in muts, mutl, or muth strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG[).sub.180] insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recoginize three-base loops formed during replication and then generate long singlestranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.

Analysis of the extragenic suppressor mutation within the dnaA initiation gene that can inhibit a dnaX2016(Ts) DNA polymerization defect reveals that one mutation involves changing of Ala to Asp at codon 213, the second involving an Arg to Leu change at position 432 and the third involving a Thr to Lys change at the 435 codon. A study of the dominance in initiation and suppression activities of the mutant and wild-type alleles reveals that the wild-type allele exhibits a dominance in initiation over the Cs, Sx alleles that does not very with location. A chromosomal suppressor allele results in dominance of suppression while a plasmid-borne suppressor allele results in recessive suppression.

A spontaneous rifamycin resistance mutation in the RNA polymerase beta subunit rpoB gene blocks the thermosensitivity of the dnaR130 mutant of Escherichia coli in DNA replication initiation. One of the dnaR-inhibiting rpoB allele, represses dnaA46 or dnaA167 mutant thermosensitivity in replication initiation. A dnaA5 mutation eliminates thermoresistance of the dnaR mutant with the rpoB22 allele, while the dnaA46 mutation eliminates viability at high and low temperatures. Both dnaR and dnaA products activities influence the suppression of dnaR or dnaA defect suppression that is facilitated by rpoB.

Combining published data with results from an experimental analysis of the influence of flanking bases on T:G mispair repair during phage recombination using amber mutation, resulting in CAG sequences in bacteriophage lambda gene cI helps study mismatch repair of mutation in Escherichia coli gene lacI and lambda gene. The very short patch (VSP) repair system is the most capable repair system for the 5'CTAGG. Substitution of 3'G or 5C by any other base results in significant correction. Addition of usr+ plasmids complements the reduced VSP repair due to MutL or MutS deficiency for a T:G mispair in the 5'CTAGG 3'GGTACC context.

The ada-independent pathway for Escherichia coli aidB gene regulation involves two mutants abrB and abrD. The suppressor mutant abrD1 reduces aidB expression in the absence of sufficient aeration. This mutant is an allele of rpoS gene, as revealed by phenotypic and genetic analyses. This reduction is brought about by acetate at low pH values.

Cloning and analysis of the ftsW gene of Escherichia coli reveal that the ftsW gene product is a polypeptide and that the new mutant allele ftsW201 induces the temperature-dependent inhibition of cell division. Overexpression of the ftsW gene blocks cell proliferation. The cell division initiation protein FtsZ or FtsZ inhibitor has no effect on the inhibition of cell division by the ftsW201 allele. High level sensitivity of the ftsW201 mutant to FtsZ overproduction indicates the interaction between FtsZ and FtsW during early cell division stages.

Sequence analysis and complementation studies reveal the new human gene, activator of RNA decay (ard-1), that reverses the pleiotropic functions of deletion and temperature-sensitive mutations in the rne gene of Escherichia coli. The reversal facilitates the proper proliferation of rne-defective cells, stimulation of bulk mRNA decay, generation of site-specific RNase E-like cleavages and correction of abnormal shapes of cells. The ard-1 gene codes for a basic 13.3-kDa proline-rich peptide, which is homologous to eukaryotic proteins and Rne that mediate macromolecular transport and RNA linkage.

Experiments using DNA polymerase (Pol II) null mutant (polBDelta1) help analyze the participation of Pol II in the response of Escherichia coli to oxidative damage, adaptive mutation and recombination. Absence of Pol II in polBDelta1 increases the sensitivity of the mutant to H2O2. A comparative study of the sensitivities of PolBDelta1 and isogenic PolB(+) cells to peroxide demonstrate the role of Pol II in response to oxidation damage. Absence of PolB exhibits little influence on mutation during nonselective growth but increases the post plating mutation rate. PolBDelta1 mutation does not change the nature of homologous genetic recombination. Use of nalidixic acid increases the level of Pol II in E. coli K-12 C and B.

DNA sequencing of a 2,232-bp domain upstream of the pcm gene on the Escherichia coli chromosome indicates the presence of two open reading frames of 477 and 1524 bp that are directed in the same way as the pcm gene. Test of the gene product activity through the construction of a mutant with a kanamycin resistance element incorporated at a BstEII site in the center of the coding region reveals the poor survival of these cells in the immobile phase, high temperatures and in high-salt media.

Primer-extension analysis of 10Sa RNA from an Escherichia coli mutant that exhibits temperature-dependent RNase P activity reveals that the 10Sa precursor has a folded pre-tRNA-like structure in vivo and that RNase P cleavage of this structure yields the 5' terminal of the mature 10Sa RNA. The half-molecule structure includes an acceptor stem and a TCF stem loop. Mutations in the gene encoding 10Sa RNA decreases cell growth rate and a motility on semisolid agar.

Transcription-repair coupling, which causes the preferential repair of transcribed strands of mRNA encoding genes and suppressors of tRNA genes, and inadequate transcription of tRNA cause a decline in mutation frequency. Decline in mutation frequency in Escherichia coli cells is characterized by a quick and irreversible decline in the frequency of suppressor mutation and occurs only when cells are placed in a non-growth medium after mutagenic treatment.

Binding of cyclosporin A cyclophilin mutant of Escherichia coli F112W strain is homologous to human cyclophilin binding activity. A comparison of wild type E. coli cyclophilin and uncomplexed human cyclophilin reveals the difference in tryptophan 121 binding site of the two proteins, despite structural similarities at a pH of 6. 2.

Analyses of the X-ray crystal structures of a second-site revertant of a mutant of Escherichia coli dihydrofolate reductase confirm the nonadditive influence of F137S and D27S mutations on catalysis. An extended structural change that is propagated between these significantly distanced the regions through the helix alpha-B is evident for the double mutants.

The presence of the RecBCD protein, which ensures the viability of the Escherichia coli dam mutant, facilitates DNA repair. Recombination genes such as recQ+, recO+, recN+ and recF+ are important for the survival of the recBC sbcBC dam mutant. SOS gene subinduction in dam mutants is not inhibited by the mutations mutS, mutL and mutH.

The dnaE293(Ts) mutation's rendering of sensitivity to the bypass replication temperature despite the existence of active DNA Pol I implies that the Rip pathway elongation stage results due to DNA PolIII. The polymerization activity and the transition from 5' to 3' exonuclease activity of DNA Pol I suppress RecA activity in cSDR. Involvement of nick translation activity in this process cannot be ruled out.

High and low levels of overexpression of recF gene of Escherichia coli K-12 facilitates the analysis of the efficiency of the mutant alleles of recF gene in affecting partial activity. A high level is accomplished by incorporating a series of neutral mutations into recF, which deactivates a translational inhibiting factor responsible for hindering gene expression. Partial activity is observed in the three substitution mutations on the high-level overexpression plasmid. The rate of expression of recF is accelerated when a series of structurally neutral mutations cause modifications in the low-level overexpressing recF plasmid.

The effects of a chaperone, a product of the gene dnaK, on ribosome assembly in vivo, was examined to determine if the biogenesis of ribosomes in Escherichia coli results from self assembly of their different constituents, or entails the involvement of additional factors. The accumulation of ribosomal particles with various sedimentation constants such as 45S, 35S and 25S, and visual 30S and 50S ribosomal subunits, was monitored at nonpermissive temperature.

Ten unique mutations in the ilvA genes of Escherichia coli are isolated and identified. These mutations contain catalylically deficient, growth inhibiting forms of threonine deaminase, and their locations validate theories that threonine deaminase has an amino-terminal catalytic domain. Threonine deaminase regulates branched chain amino-acid synthesis.

The properties of amino acid residues that are required for gene expression activation by the TyrR protein were characterized using site-directed mutagenesis stimulation of either mtr or TyrR mutants with amino acid substitutions V-5 yields P (VP5), VF5, CST, CR7, DR9, RI10, RS10 and 16, but the capacity to prevent aroF was unchanged. Stimulation of mtr and TyrR, which are mediated by tyrosine or phenylalanine, are killed by tiny internal deletions of residues 10 to 19, 20 to 29 or 30 to 39. A reaction between trpR and TyrR proteins when tyrosine is present is implied since the suppression of TrpR, which is mediated by tyrosine, is dependent.

Two groups of Bacillus subtilis DNA sequences, which substantiate the mutations of various Escherichia coli mutants that are lacking in sidorophore by imperfect enterobactin biosynthesis enzymes, were separated. Only an entD mutation was substantiated by the first, while different combinations of ent B, ent E and ent C and ent A mutations were substantiated by the second. The two genes were seen in the nucleotide sequence of the B. subtilis DNA existing in plasmid pENTA22, a clone in which B subtilis ent D homologue is expressed. B. subtilis genes and E. coli ent D genes can be exchanged.

Wild-type human glutathione reductase (hGR) and inactive mutants with single or double mutations at residues 446 and 447 were isolated and characterized. The mutant G446E/F447P-hGR behaved like a poorly folded monomeric protein. The results suggest that the sequence around G446 can control dimerization as well as domain folding. The role of interface residues as a folding signal and the prospect to develop dimerization inhibitors of hGR as antiparasitic drugs are discussed.

Experimental analyses of the tolC mutant derivatives of the deep rough (rfa) mutants show that the cumulative effects of rfa and tolC mutations on the outer membrane hydrophobic barrier depend on their responsivity to the hydrophobic agents in Escherichia coli K-12 strains. The cumulative effects imply that a mutual process does not mediate the cumulative effects of the mutations in changing the outer membrane permeability. An efflux system develops in E. coli when the hydrophobic agents slowly enter the low-permeability outer membrane, which prevents the influence of noxious hydrophobic agents.

Molecular dynamics calculations restrained by NMR-derived distances and torsion angles were used to demonstrate the structure of the formamidopyrimidine (AFB-[alpha]-FAPY) adduct which interconverts between [alpha] and [beta] anomers. The structural distortions and the less favorable stacking interactions induced by the AFB-[alpha]-FAPY adduct provide insight into its lower stability as compared to the AFB-[beta]-FAPY adduct in duplex DNA.

A unique mutation is described that has resulted in an increase in sugar efflux. The results have shown that the primary defect induced by the mutation has involved the decrease in affinity of [H.sup.+].

The article studies the crystal structures of lactose permease from Escherichia coli (LacY) that exhibits two six-helix bundles with 2-fold pseudosymmetry separated by a large hydrophilic cavity. Findings reveal that the primary defect induced by the mutation might include a decrease in affinity for [H.super.+].

A mutational analysis performed for UvrD1 by considering the crystal structure of a DNA-bound Escherichia coli UvrD-ADP?Mg[F.sub.3] transition state mimetic is described. The results showed that the strength of the Ku-binding function of UvrD1 which lies within its C-terminal 270 amino acid segment could be influenced by deleting the 90 amino acid C-terminal domain, diminished DNA unwinding, without affecting ATP hydrolysis or binding to the DNA helicase substrate.

Combination of high-resolution Fourier transform ion cyclotron resonance mass spectrometry and top-down fragmentation techniques were used to analyze the mechanistic details of hydrogen peroxide reduction by Escherichia coli bacterioferritin comigratory protein (BCP). The mutant BCP enzyme adopted a different and novel mechanistic pathway and reacted with peroxide to form a sulfenic acid on Cys-45 and is also a substrate for reduction by thioredoxin.

Four individual site-directed mutations are constructed for each of the residues of Asparagine 153 and Aspartate 155 in Escherichia coli understand their role in the active site biotin synthase. The results indicated that the protein residues, Asparagine 153 and Aspartate 155 that form hydrogen bonds to S-adenosyl-L-methionine (AdoMet) and dethiobiotin (DTB) are significant for retaining intermediates during the catalytic cycle and for targeting the reactivity of the 5?-deoxyadenosyl radical.

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. 15 (Vib), is analyzed. The refolding processes of the mutants are followed by measuring changes in Trp fluorescence and the high similarity of time courses has suggested cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

The strains [P.sub.phoB]::[P.sub.tac] and [P.sub.pstS]::[P.sub.tac] from Pho regulon in Escherichia coli are used to characterize phenotypes resulting from various [DELTA]phoU mutations in plasmids. The experimental results suggested that PhoU plays a significant role in controlling the abundance of the Pst[SCAB.sub.2] transporter through the PhoBR two-component signaling pathway and its activity.

Structure-based mutational analysis is used for probing the architecture of the glutamine binding pocket in Escherichia coli glutaminyl-tRNA synthetase (GlnRS). The studies have shown that the relative absence of direct hydrogen bonds to glutamine is compensated partially by additional binding energy derived from the enhanced stability of this circular network.

The overinduction of the yjb operon and that of other genes involved in extracellular polysaccharide (EPS) production are shown to be an adaptive response to osmotic stress in an rpoS-deficient background and that these overinduced genes are of special value for an rpoS-deficient strain. The results have shown that alternative stress response strategies have played a role in the absence of RpoS or when its activity is reduced, allowing the cells to survive and proliferate even without its general stress protection.

The Fe(III) complex-bound full-length Escherichia coli (Ec) DOS proteins mutated at Met95, Arg97, and Phe113 in the putative distal side is used to examine their molecular mechanism, catalytic activities and spectral characteristics in the absence and presence of the external axial ligands, cyanide and imidazole. The results revealed that Met95 and Phe113 located at close proximity to the ligand access channel of the Fe(III) complex influence the catalytic enhancement caused by ligand binding, heme coordination structure, and ligand binding kinetics.

The site-directed homoserine o-succinyltransferase (MetA) mutants are used to identify two amino acid residues responsible for the sensitivity of [MetA.sub.E.coli] to both and acids. The improved growth in Escherichia coli at higher temperatures is attributed to MetA stabilization and they provide new insight to design E. coli strains that grow at higher temperatures, thus helping in reducing the cost of cooling bioprocesses.

An evolved Escherichia coli mutant deficient in three terminal oxidases is used to demonstrate the fermentation of glucose to D-lactic acid under aerobic growth conditions. The results have shown the ability of the E. coli strains to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.

Pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes that encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase, respectively is reported. Out of the four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux, pyruvate formation rate, and pyruvate yield.

The specific role of Arg97 is established by generating Arg97Ala, Arg97Glu and Arg97Ile mutant Escherichia coli (Ec DOS) PAS structure proteins and examining the binding to [O.sub.2], CO and cyanide, as well as redox potentials. The binding behavior of cyanide to the Fe(III) complexes of the Arg mutant proteins is similar to that of [O.sub.2], which is evident from the [K.sub.d] values, suggesting electrostatic interactions between cyanide and Arg97.

Escherichia coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster was generated to study the role of the O antigen in persistence and colonization in the animal host. The data results suggested that the O polysaccharide side chain of Escherichia coli O157:H7 lipopolysaccharide (LPS) is a required factor for efficient bovine intestinal colonization.

The glutamic acid residue of transmembrane helix III in subunit I (E107L) mutant cytochrome bd from Escherichia coli is studied by using electrometry and absorption spectroscopy with a microsecond time resolution to elucidate the role of E107 in translocation of protons within the protein molecule. The studies have shown that E107 is either the second group or a key residue of a proton delivery pathway leading from the cytoplasm toward the second group.

Ser72 at the active site of the Escherichia coli dUTPase is mutated to an alanine and the properties of the mutant are examined. The studies have shown that the Escherichia coli dUTPase has not catalyzed the hydrolysis of the [alpha],[beta]-imido-dUTP.Mg, indicating that the analogue has provided the hydrogen in the bond to the serine [beta]-OH.

A method for breeding phage resistance and the genetic nature of the resultant Escherichia coli mutant is described. The results showed that the two strains, D198 and JM109 have comparable growth rates and produce the same of recombinant LacZ activity.

The quantitation, reconstitution, and the cross-linking analysis of the [F.sub.O] complex isolated from wildtype mutant were performed to study the c ring of the mutant strain and its stoichiometry. The data obtained revealed that the stoichiometry of the subunit c rings remains constant even after the reduction of the synthesis of subunit c.

The glutamine at position 263 in Escherichia coli NrfA was mutated to understand the roles of the active site calcium ion and coordinating glutamine residue in nitrite reduction to ammonia by cytochrome c nitrite reductase. The findings provided insight into important function of unusual Q263-calcium ion pair to increase substrate affinity through its role in stabilization of a network of hydrogen bonded water molecules in the active site heme distal ligand.

The characteristics of ygaVP genes of Escherichia coli that are formed by fusion of tributylin (TBT) luxAB are examined. The results show that a TBT-activated promoter is located upstream of ygaVP and is constitutive in a ygaVP mutant, indicating that YgaV is an autoregulated TBT-inducible repressor.