Repressor forms of the enhancer-binding protein NrtC: some fail in coupling ATP hydrolysis to open complex formation by sigma 54-holoenzyme.

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between sigma54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by sigma70-holoenzyme. In this paper we characterize "repressor" mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma54-holoenzyme (in vitro activity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by sigma70-holoenzyme. The amino acid substitutions in NtrCrepressor proteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressor proteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrC(G219K) protein has "improved" DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressor proteins have impaired ATPase activity. We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activating transcription. These results demonstrate that NtrC(A216V) and NtrC(A220T) fail in a function of the central domain other than ATPase activity. Although they may fail in contact with sigma54-holoenzyme per se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.

Three novel plasmid R6K proteins act in concert to distort DNA within the alpha and beta origins of DNA replication.

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (alpha LIR and beta LIR), which are essential elements for the two distally located R6K alpha and beta DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of alpha LIR, whereas a gene coding for DDP3 is located at the 3' end of beta LIR. DDP1 and DDP2 are required for primary DNA distortion within alpha LIR or beta LIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within alpha LIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.

Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K.

The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein. Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype. We demonstrate here that these mutations prevent replication of a gamma origin core plasmid. To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants. We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.

Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain.

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.

Reversal of signal-mediated cellular retention by subunit assembly of human acetylcholinesterase.

The interrelationship between signal-mediated endoplasmic reticulum retention and control of subunit assembly in secreted complex proteins was examined in recombinant 293 cells expressing human acetylcholinesterase (HuAChE). This was achieved by analyzing the mutual effects of co-residing retention and dimerization signals on enzyme secretion by transfected cells. The function of putative signals within the COOH-terminal tetrapeptide CSDL of HuAChE was examined by site-directed mutagenesis. The CSDL tetrapeptide carries the free cysteine (Cys-580) involved in subunit assembly, yet it fails to function as a KDEL-type retention signal. This was demonstrated by mutations that increase similarity to the canonical retention signal (substitution of CSDL by KSDL) or those that deviate from it (substitution to CSAL). Cells expressing both types of mutants exhibited cell-associated HuAChE levels identical to that of wild type enzyme. Appendage of an engineered KDEL retention signal to a dimerization-impaired HuA-ChE subunit (the C580A mutant) resulted in intracellular retention of large amounts of fully active enzyme not prone to proteolytic degradation. On the other hand, attachment of KDEL to a native, dimerization-competent HuAChE polypeptide did not lead to intracellular retention and allowed efficient secretion of enzyme to the cell growth medium. Yet, appendage of KDEL to the native HuAChE led to some retardation in the transport of enzyme molecules through the Golgi apparatus, as manifested by increase in cellular population of endo H-resistant dimers, when compared with wild type enzyme. Taken together, these results indicate (alpha) that sub-unit dimerization mediated by the COOH-terminal cysteine of HuAChE can reverse the signal-mediated retention by masking recognition of KDEL by its cognate receptor and (b) that the native sequences of the acetylcholinesterase subunit polypeptide do not appear to function as a coupled retention/dimerization signal in the control of secretion of assembled enzyme molecules.

N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis.

The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related 'peripheral-site' functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.

Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding.

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme.

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Alpha and beta replication origins of plasmid R6K show similar distortions of the DNA helix in vivo.

Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.

Dual mechanism of repression at a distance in the lac operon.

The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants. Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site. The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression. This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold. Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements. During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously.

DNA dynamic flexibility and protein recognition: differential stimulation by bacterial histone-like protein HU.

The abundant E. coli "histone-like" protein HU is shown to be a differential effector of DNA recognition by three diverse control proteins. DNA recognition by lac repressor and catabolite activator protein is greatly stimulated, while specific aroH DNA recognition by trp repressor is inhibited. BaCl2, an agent previously shown to promote DNA bending, mimics the HU effect to give the same qualitative differential stimulation spectrum. The HU activation involves cooperativity, further suggesting that the various DNA bends and distortions induced during assembly of higher order HU:DNA structures are important for the HU stimulation. Thus, E. coli chromosomal DNA regulation is likely strongly influenced by HU protein that may promote a variety of alternative DNA structures that either facilitate or inhibit specific recognition by diverse control proteins.

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins.

The functional R6K alpha origin is composed of two DNA elements, one of 580 bp carrying the alpha origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previously identified as also required for gamma and beta origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for alpha origin activity. The function of the alpha origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the alpha origin depends on the R6K replication initiation protein pi. Within the 580 bp of the alpha origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the beta replicon. Deletion of the 96 bp or 98 bp results in inactivation of the alpha and the beta origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the alpha and the beta origins of R6K. DNA homology analysis performed on alpha, beta and gamma origin sequences, also reveals 10-23 bp sequences in the alpha and the beta origins that are related to the family of 22 bp direct repeats in the gamma origin which were shown previously to be binding sites for the pi protein.

Molecular aspects of genetic instability of an artificial 68 bp perfect palindrome in Escherichia coli.

An artificial 68 bp perfect palindrome carried on a plasmid (pAS807) is genetically unstable. An increase in the population of cells harbouring palindrome-deleted pAS807 derivatives (pAS807-V) is observed as the number of cell generations increases. The calculated frequency of palindrome excision events per cell generation and per plasmid replication round in Escherichia coli is 0.95 X 10(-4). Sequence analysis of eight independent isolates of palindrome-deleted molecules, reveals two symmetrical deletion types (three of type I and five of type II). The two types of pAS807-v molecules retain 19 bp of the original sequence of the 68 bp palindrome but differ in the content of the central 3 bp. The generation of the two deletion types is best explained by formation of intermediate cruciform structures. Following the fate of the palindrome in various bacterial mutants, we find that the excision events depend on functional polA1, polA(ex1), lig, texA343(recC343) and texA344(recB344) gene products. However, recB21 recC22 mutations do not affect palindrome excision.

Genetic instability of an artificial palindrom DNA sequence.

A short DNA palindrom, produced by head to head ligation of a 29 bp DNA fragment, was inserted into a 27,000 bp plasmid DNA element composed of two functional replicons (R6K, ColE1). Several plasmid types containing a single copy of this palindrom in different locations of insertion on the R6K sequence were obtained. The palindrom was engineered to possess a unique EcoRI recognition sequence at its axis of symmetry. The presence of this restriction site allowed to monitor the genetic stability of the artificial palindrom at their different insertion loci. Out of 5 different insertion locations, one (in pAS807) was found to lead to a significant destabilization of the palindrom. This insertion site lies within the replication control region of R6K. We have shown that the inserted palindrom in pAS8O7 does not affect the functionality of the R6K replication origins. Excission of the palindrom sequences from pAS8O7 was not accompanied by loss of the adjacent R6K DNA sequences. Different deletion derivatives of pAS807 were generated in-vitro in order to determine the driving unit of DNA sequences around the palindrom that are involved in its excision. The results imply that large DNA structure(s) around the palindrom are involved in its excission. Complete deletion of R6K sequences from either the left or the right side of the palindrom resulted in new configurations which stabilized the palindrom. A configuration of R6K DNA sequences exceeding 270 bp long sequence from both sides of the palindrom are necessary for the transition from a palindrom stable to palindrom unstable state. In addition evidence is presented to show that the excision process of palindrom sequences requires a functional polymerase I but not the gene product of recA.

New antibiotic substance produced by Salmonella typhimurium LT2.

Salmonella typhimurium LT2 excreted under certain conditions an antibiotic substance designated typhimuricin. It is suggested that the LT2 "cryptic" plasmid is involved in its production and in the immunity to it. Preliminary characterization of typhimuricin is presented.