Biomedical subjects

J A Fuchs

Publications and source records attributed to J A Fuchs.

At least 55 records · Page 3Linked to original sources

PubMed1992

Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency.

Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841-845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded ln DNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA. As cellular uptake of DNA also correlated with the surface binding, these data support electrophoresis of surface bound DNA as the dominating mechanism for the DNA entry. Second, the variations of TE for different topological forms of DNA reflected their relative stability in the host cells. Third, since the loaded DNA could be either rapidly degraded by the host enzyme or expressed, they were unlikely coated with a layer of protective lipid membrane. Thus, PEF induced cellular uptake of DNA is unlikely by the endocytotic mechanisms as was reported previously for the liposomes (Chernomordik, L. V., A. V. Sokolov, and V. G. Budker. 1990.Biochim. Biophys. Acta. 1024:179-183).

Ampicillin Resistance↗

PubMed1992

Escherichia coli ribonucleotide reductase expression is cell cycle regulated.

The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.

Cell Cycle↗

PubMed1992

The Escherichia coli K-12 cyn operon is positively regulated by a member of the lysR family.

A regulatory gene, cynR, was found to be located next to the cyn operon but transcribed in the opposite direction. cynR encodes a positive regulatory protein that controls the cyn operon as well as its own synthesis. Positive regulation of the cyn operon requires cyanate and the cynR protein, but the negative autoregulation of the cynR gene appears to be independent of cyanate. The predicted amino acid sequence of the cynR protein derived from the DNA sequence was found to have significant homology to the predicted amino acid sequence of the lysR family of regulatory proteins.

Amino Acid Sequence↗

PubMed1991

Null mutation in the stringent starvation protein of Escherichia coli disrupts lytic development of bacteriophage P1.

As initial steps toward understanding the regulation and function of the stringent starvation protein (SSP) of Escherichia coli, we have isolated the ssp gene (encoding SSP), defined the operon in which ssp is found, and created insertion-deletion mutations of the ssp gene in recBC, sbc and recD strains by linear DNA transformation. During attempts to move the insertion-deletion structure to other strains by P1 transduction, we found that P1 was unable to form plaques on hosts lacking an intact ssp gene. The delta ssp mutation, however, did not affect transduction of the delta ssp strains and mutant strains were able to support lysogenic P1. When P1 lytic growth was induced, an increase in P1 DNA was detected without lysis or plaque formation. Examination of proteins synthesized in the delta ssp host during induction revealed the absence of P1 late gene products. Also, the apparent continued synthesis of early gene products during late time points was observed in the delta ssp host. The results reported here suggest that the defect in P1 lytic growth brought about by the absence of SSP occurs at the point at which bacteriophage P1 shifts from early to late gene expression. We also report the results of experiments on stable RNA synthesis following amino acid (aa) starvation induced by serine hydroxamate, and experiments on stable RNA synthesis following resupplementation of a limiting aa. These experiments show that SSP is not involved in stable RNA synthesis. Additionally, complementation studies have shown that ssp is identical to the previously described pog gene of E. coli.

Bacterial Proteins↗

PubMed1991

Nuclear magnetic resonance studies of recombinant Escherichia coli glutaredoxin. Sequence-specific assignments and secondary structure determination of the oxidized form.

Escherichia coli glutaredoxin (85 amino acid residues, Mr = 9100), the glutathione-dependent hydrogen donor for ribonucleotide reductase, was purified from an inducible lambda PL, expression system both with a natural isotope content and with uniform 15N labelling. This material was used for obtaining sequence-specific 1H magnetic resonance assignments and the identification of regular secondary structures in the oxidized form of the protein, which contains the redox-active disulfide Cys11-Pro-Tyr-Cys14. Oxidized glutaredoxin contains a four-stranded beta-sheet, with the peripheral strand 32-37 arranged parallel to the strand 2-7, which further combines with the two additional strands 61-64 and 67-69 in an antiparallel fashion. The protein further contains three helices extending approximately from residues 13-28, 45-54 and 72-84.

Amino Acid Sequence↗

PubMed1991

The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability.

Aspartic acid 26 in Escherichia coli thioredoxin is located at the bottom of a hydrophobic cavity, near the redox-active disulfide of the active site. Asp 26 is embedded in the protein except for part of the surface of one carboxyl oxygen. The high degree of evolutionary conversion of Asp 26 suggests that it plays a critical role in thioredoxin function. We have determined the pKa of Asp 26 by a novel electrophoretic method based on the relative electrophoretic mobilities of wild-type thioredoxin and of D26A thioredoxin (with Asp 26 replaced by alanine). The pKa of Asp 26 determined by this technique is 7.5, more than 3 units above the pKa of a solvated carboxyl side chain. The titration of Asp 26 is thermodynamically linked to the stability of thioredoxin. As expected if thioredoxin stability depends on the ionization state of Asp 26, delta Go WT, the free energy of the cooperative denaturation reaction of wild-type thioredoxin by guanidine hydrochloride, varies with pH in a sigmoidal fashion in the vicinity of pH 7.5. Over the same pH range, the free energy for D26A folding, delta Go D26A, is pH independent and D26A is highly stabilized compared to wild type. From the thermodynamic cycle describing the linkage of Asp 26 titration to thioredoxin stability, the difference in free energy between wild-type thioredoxin with protonated Asp 26 and wild-type thioredoxin with deprotonated Asp 26, delta delta Go (COOH----COO-), is calculated to be 4.9 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1991

Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa.

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.

Aspartic Acid↗

PubMed1991

Escherichia coli glutaredoxin: cloning and overexpression, thermodynamic stability of the oxidized and reduced forms, and report of an N-terminal extended species.

Escherichia coli glutaredoxin (MW 9700) catalyzes intracellular redox reactions utilizing a disulfide/dithiol enzymatic mechanism involving the active-site residues -Cys-Pro-Tyr-Cys-. It is functionally related to the thioredoxin family and is expected to share similar three-dimensional structure [Eklund, H., Cambillau, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We constructed an overexpression system in which production of glutaredoxin is controlled by temperature-sensitive expression of the phage T7 promoter. In addition to glutaredoxin, a second gene product is observed; this species, which we call glutaredoxin N, is glutaredoxin extended by the sequence Met-Arg-Arg-Glu-Ile- at the N terminus. We have begun characterization of the structure and stability of the oxidized and reduced forms of glutaredoxin (grx-S2 and grx-(SH)2, respectively). Secondary structure calculated from CD data agrees with that predicted from the three-dimensional model of Eklund et al. The cooperative denaturation reactions of oxidized and reduced glutaredoxin were measured in temperature-induced and guanidine hydrochloride induced unfolding experiments. Surprisingly, oxidized and reduced glutaredoxins are very similar in stability. In heat-induced denaturation, monitored by CD, Tm is 55 and 57 degrees C for oxidized and reduced, respectively. In GuHCl denaturation, monitored by fluorescence, the midpoint denaturant concentrations are 2 M for both oxidized and reduced. It follows that the redox potentials of the disulfide bond are similar in unfolded and folded glutaredoxin. This is unexpected because in E. coli thioredoxin the oxidized form is far more stable than the reduced [Kelley, R.F., Shalongo, W., Jagannadham, M.V., & Stellwagen, E. (1987) Biochemistry 26, 1406-1411] and the redox potential of folded thioredoxin is significantly more negative than that of unfolded thioredoxin [Lin, T.-Y., & Kim, P. (1989) Biochemistry 28, 5282-5287].

Amino Acid Sequence↗

PubMed1991

Kinetics of electron transfer from thioredoxin reductase to thioredoxin.

The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry. The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument. The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics. The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins. A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization. Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration. Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.

Electron Transport↗

PubMed1990

Characterization of Escherichia coli thioredoxins with altered active site residues.

Escherichia coli thioredoxin is a small disulfide-containing redox protein with the active site sequence Cys-Gly-Pro-Cys-Lys. Mutations were made in this region of the thioredoxin gene and the mutant proteins expressed in E. coli strains lacking thioredoxin. Mutant proteins with a 17-membered or 11-membered disulfide ring were inactive in vivo. However, purified thioredoxin with the active site sequence Cys-Gly-Arg-Pro-Cys-Lys is still able to serve as a substrate for thioredoxin reductase and a reducing agent in the ribonucleotide reductase reaction, although with greatly reduced catalytic efficiency. A smaller disulfide ring, with the active site sequence Cys-Ala-Cys, does not turn over at a sufficient rate to be an effective reducing agent. Strain in the small ring favors the formation of intermolecular disulfide bonds. Alteration of the invariant proline to a serine has little effect on redox activity. The function of this residue may be in maintaining the stability of the active site region rather than participation in redox activity or protein-protein interactions. Mutation of the positively charged lysine in the active site to a glutamate residue raises the Km values with interacting enzymes. Although it has been proposed that the positive residue at position 36 is conserved to maintain the thiolate anion on Cys-32 (Kallis & Holmgren, 1985), the presence of the negative charge at this position does not alter the pH dependence of activity or fluorescence behavior. The lysine is most likely conserved to facilitate thioredoxin-protein interactions.

Amino Acid Sequence↗

PubMed1990

The cyanase operon and cyanate metabolism.

Cyanase is an inducible enzyme in E. coli that catalyzes bicarbonate-dependent decomposition of cyanate. It is encoded as part of an operon we have named the cyn operon, which includes three genes in the following order: cynT (cyanate permease), cynS (cyanase), and cynX (protein of unknown function). The direction of transcription is opposite to that of the lac operon, and the 3'-end of the cyn operon overlaps the 3'-end of the lac operon by 98 nucleotides. The gene cynR (regulatory protein) is located upstream from the cyn operon, and its transcription is opposite that of the cyn operon. The genes of the cyn operon and the cynR gene have been cloned, sequenced and over-expressed. Cyanate at concentrations of about 1 mM is toxic to strains of E. coli lacking the cyanase gene, but strains in which the inducible gene for cyanase is present can grow on cyanate as the sole source of nitrogen at concentrations as high as 20 mM. The presence of cyanase itself is not sufficient to overcome cyanate toxicity--the permease must also be present. Strains lacking the cyanase gene, but having a functional permease gene, are extremely sensitive to cyanate. Uptake of cyanate involves the product of the permease gene in an energy-dependent process. It appears that the cyn operon has evolved to function in detoxification/decomposition of cyanate arising from both intra- and extracellular sources.

Aminohydrolases↗

PubMed1990

Regulation of the operon encoding ribonucleotide reductase: role of the negative sites in nrd repression.

Expression of the nrd genes was previously shown to be controlled by both positive and negative regulation (C. K. Tuggle and J. A. Fuchs, EMBO J. 5:1077-1085, 1986). Two regions, one located 5' and one located 3' of the nrd promoter (nrdP), were identified as negative regulatory sites since deletion of these sequences increased nrd expression. These regions of DNA have sequence similarities, and a looping mechanism was proposed to explain the requirement for two distinct sites in nrd repression. To investigate the role of these sequences in regulating nrd, a gel electrophoresis assay was used to detect the proteins that bind to the nrd regulatory sites. A protein that bound to restriction fragments containing the negative regulatory sites but not to other DNA fragments was identified in cell extracts and was partially purified. DNase I footprinting experiments showed that the binding protein protects the 5' negative site previously identified in vivo. The 3' negative site also identified in vivo was not required in vitro for high-affinity protein binding to the 5' site, but lower-affinity binding to this site could be detected. Specific binding to the 5' site was found to be elevated approximately 10-fold in crude extracts from thymine-starved cells as compared with that in extracts from unstarved cells. This higher activity was also evident in purified preparations, suggesting that thymine starvation increases the expression of the negative regulatory protein. The finding that a purified protein preparation binds both negative regulatory sites indicates that this preparation contains the nrd repressor protein or proteins. Insertion of 37 base pairs (3.5 helix turns) of DNA at a HpaII site or 35 base pairs (3.3 turns) at a MnlI site between the 5' regulatory sites and nrdP abolished the increase in nrd expression resulting from thymine starvation in vivo, but negative regulation appeared to be less affected than when either negative site was deleted. Insertion of DNA in these constructs was shown not to affect repressor binding in vitro, indicating either that a simple model of DNA looping to bring equivalent operator sites into physical proximity does not explain repression at nrd or that the distance between sites is sufficient that helical turns are of little importance.

Base Sequence↗

PubMed1990

Health risk factors among the Amish: results of a survey.

The Amish are a unique societal subgroup deliberately setting themselves apart from the modern world through religious beliefs and practices. Few reports and no systematic studies have been conducted on Amish health behaviors. A representative sample of 400 Amish adults residing in Holmes County, Ohio, were interviewed using the Behavioral Risk Factor Survey (BRFS). For comparison, a representative sample of 773 non-Amish adults responded to the same survey through telephone interviews. Prevalence of self-reported hypertension was lower among Amish than non-Amish, obesity was greater among Amish than non-Amish women but similar among men, and stress responses were more prevalent among Amish than non-Amish. While some differences between groups could be influenced by response biases, many are supported by less systematic observations of Old Order Amish societies. Patterns of health behavior reflect characteristics of Amish culture and may be responsible for certain favorable mortality rates among the Amish population. Need for cultural sensitivity is evident in health promotion program design and implementation.

Adolescent↗

PubMed1989

Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii.

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.

Amino Acid Sequence↗

PubMed1989

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120.

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli. Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.

Amino Acid Sequence↗

PubMed1989

Identification and characterization of a cyanate permease in Escherichia coli K-12.

Escherichia coli contains an inducible enzyme, cyanase, that catalyzes the decomposition of cyanate into ammonia and bicarbonate. The gene encoding cyanase, cynS, was cloned and found to be on a DNA fragment that contained the lac operon. Characterization of a plasmid encoding cyanase indicated that a 26-kilodalton (kDa) protein of unknown function was also induced by cyanate (Y-C. Sung, D. Parsell, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 169:2639-2642, 1987). The gene encoding the 26-kDa protein was located between cynS and its promoter, indicating the existence of a cyn operon. The 26-kDa protein was identified as a cyanate permease that transports exogenous cyanate by active transport. E. coli was shown to contain a cyanate transport system that is energy dependent and saturable by cyanate.

Aminohydrolases↗

PubMed1989

In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus.

The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322. Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase. A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB. The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination. The resulting strain had no detectable glutathione synthetase activity. The gshB allele containing the kan insertion was used to map gshB on the E. coli chromosome by P1 transduction. The results indicated that gshB is located at 63.4 min, between metK and speC. The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E. coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987).

Chromosome Mapping↗

PubMed1988

Characterization of the cyn operon in Escherichia coli K12.

Escherichia coli can overcome the toxicity of environmental cyanate by hydrolysis of cyanate to ammonia and bicarbonate. This reaction is catalyzed by the enzyme cyanase, encoded by the cynS gene. The nucleotide sequence of cynS has been reported (Sung, Y.-c., Anderson, P. M., and Fuchs, J. A. (1987) J. Bacteriol. 169, 5224-5230). The nucleotide sequence of the complete cyn operon has now been determined. The cyn operon is approximately 2600 base pairs and includes cynT, cynS, and cynX, which encode cyanate permease, cyanase, and a protein of unknown function, respectively. Two cyanate-inducible transcripts of 1500 and 2500 nucleotides, respectively, were detected by Northern blot analysis. S1 nuclease mapping experiments indicated that two different cyn mRNAs have a common 5'-end and two different 3'-ends. One 3'-end was located within the coding region of cynX, whereas the other 3'-end includes the entire DNA sequence of cynX. The longer transcript contained 98 nucleotides complementary to lac mRNA produced by the predominant lac transcription termination sequence. Termination vectors were used to show that both 3'-ends were generated by sequences that caused transcriptional termination in vivo. Expression vectors were used to demonstrate that a protein corresponding to the expected size was synthesized from the DNA fragment containing the open reading frame designated cynX. The predicted amino acid sequence of cynX indicates that it is a very hydrophobic protein. The level of cynX expression was significantly less than that of cynT or cynS expression.

Amino Acid Sequence↗