Absence Of Inducer Research Articles

A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer

TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a ( recA −) only in the presence of a Pm inducer. In two tested E. coli recA + strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.

A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer.

TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a (recA(-)) only in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.

Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line.

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A gene is associated with an absence of p16 protein in human pituitary tumors. However, the effect of restoration of p16 protein expression in this tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system. Initial experiments showed that the p16/CDKN2A gene was not expressed, whereas a transcript for RB1 was detected as assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be homozygously deleted. The absence of p16/CDKN2A and presence of RB1, the down-stream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model system. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activated cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G1 arrest (80-82%). Western blot analysis showed concomitant expression of p16 protein in arrested cells and a shift in the phosphorylation status of pRB toward its hypophosphorylated form. To further confirm that expression of p16/CDKN2A mimicked its in vivo role, reversibility was assessed using alternate cycles in the presence and absence of inducer (isopropyl-1-thio-beta-D-galactopyranoside). Over three cycles the absence of induced expression of p16/CDKN2A resulted in release from G1 arrest. These results show that, in a pituitary cell line model, restoration of p16 expression is indeed sufficient to arrest cells in G1 and inhibit cell proliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.

Transfection of an Inducible p16/CDKN2A Construct Mediates Reversible Growth Inhibition and G1 Arrest in the AtT20 Pituitary Tumor Cell Line

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A gene is associated with an absence of p16 protein in human pituitary tumors. However, the effect of restoration of p16 protein expression in this tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system. Initial experiments showed that the p16/CDKN2A gene was not expressed, whereas a transcript for RB1 was detected as assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be homozygously deleted. The absence of p16/CDKN2A and presence of RB1, the downstream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model system. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activated cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G1 arrest (80–82%). Western blot analysis showed concomitant expression of p16 protein in arrested cells and a shift in the phosphorylation status of pRB toward its hypophosphorylated form. To further confirm that expression of p16/CDKN2A mimicked its in vivo role, reversibility was assessed using alternate cycles in the presence and absence of inducer (isopropyl-1-thio-β-d-galactopyranoside). Over three cycles the absence of induced expression of p16/CDKN2A resulted in release from G1 arrest. These results show that, in a pituitary cell line model, restoration of p16 expression is indeed sufficient to arrest cells in G1 and inhibit cell proliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.

Transcriptional activation of the chlorocatechol degradative genes of Ralstonia eutropha NH9.

Ralstonia eutropha (formerly Alcaligenes eutrophus) NH9 degrades 3-chlorobenzoate via the modified ortho-cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the cbnABCD operon encoding the degradative enzymes (including orfX of unknown function) and the divergently transcribed cbnR gene encoding the LysR-type transcriptional regulator of the cbn operon. The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes (tcbRCD orfXEF) of the 1,2, 4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51. Transcriptional fusion studies demonstrated that cbnR regulates the expression of cbnABCD positively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro-cis,cis-muconate (2-CM) and cis, cis-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol (catBCA) and chlorocatechol (clcABD) operons of Pseudomonas putida, in which only the intermediates of the regulated pathway, CCM for catBCA and 2-CM for clcABD, act as significant inducers. Specific binding of CbnR protein to the cbnA promoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position -20 to position -80 upstream of the cbnA transcriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position -50. Circular permutation gel shift assays demonstrated that CbnR bent the cbnA promoter region to an angle of 78 degrees and that this angle was relaxed to 54 degrees upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the catBCA and clcABD promoters may indicate a conserved transcriptional activation mechanism of ortho-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.

Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and of its cognate promoter.

Toluene-o-xylene monooxygenase is an enzymatic complex, encoded by the touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation in Pseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene-o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a touA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (P(ToMO)) was mapped, which showed the typical -24 TGGC, -12 TTGC sequences characteristic of sigma(54)-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the P. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of tou structural genes in the absence of inducers. Basal toluene-o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.

Functional Analysis of the Zn2Cys6Transcription Factors Oaf1p and Pip2p: DIFFERENT ROLES IN FATTY ACID INDUCTION OF β-OXIDATION INSACCHAROMYCES CEREVISIAE

Fatty acid induction of the peroxisomal beta-oxidation machinery in Saccharomyces cerevisiae involves transcriptional control of genes regulated by the oleate response element (ORE). Glucose as the preferred carbon source antagonizes this effect. Induction is dependent on the Zn(2)Cys(6) family members Oaf1p and Pip2p, which bind to this element as a heterodimer. We show here by ectopically expressing both components and LexA fusion derivatives that this transcription factor complex is only active in the presence of oleate. In contrast to Pip2p, Oaf1p is responsive to oleate activation in the absence of the other component of the heterodimer. Therefore, it is the exclusive receptor of the oleate signal. Pip2p is active also under noninducing conditions but is effectively inhibited when complexed with Oaf1p in the absence of inducer. It contributes to the trans-activation properties of the Oaf1p-Pip2p heterodimer and is required for efficient binding of Oaf1p to OREs in vivo. Repression of ORE-dependent transcription by glucose occurs via both Oaf1p and Pip2p. By dissecting functional domains of both proteins, we identified a region required for regulated activity of the C-terminal activation domain. These findings allow us to postulate a model for carbon source-regulated transcription of peroxisomal protein genes.

Vaccinia virus WR gene A5L is required for morphogenesis of mature virions.

The vaccinia virus WR A5L open reading frame (corresponding to open reading frame A4L in vaccinia virus Copenhagen) encodes an immunodominant late protein found in the core of the vaccinia virion. To investigate the role of this protein in vaccinia virus replication, we have constructed a recombinant virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major vaccinia virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion.

Protein binding in vivo to OP2 promoter of the Pseudomonas putida TOL plasmid.

The transcription of OP2 encoding enzymes for m-toluate catabolism on the Pseudomonas putida TOL plasmid is activated by basal-level XylS protein in the presence of m-toluate or by overproduced XylS protein in the absence of m-toluate. In this study, in vivo dimethyl sulfate (DMS) footprinting was performed to understand the mechanism of transcriptional regulation of OP2 promoter by XylS. In the presence of overproduced XylS without m-toluate, several protected nucleotides were observed, indicating the binding of RNA polymerase to DNA. However, the protection was canceled upon addition of m-toluate. These results suggest that RNA polymerase is retained by XylS on the OP2 promoter in the absence of inducer, and is released by m-toluate binding to XylS, concomitant with transcription.

INTERACTION OF TRIBUTYLTIN WITH 3,3',4,4',5- PENTACHLOROBIPHENYL-INDUCED ETHOXYRESORUFIN O -DEETHYLASE ACTIVITY IN RAT HEPATOMA CELLS

Interaction of tributyltin ( TBT) with 3,3',4,4',5-pentachlorobiphenyl (PCB-126)-induced ethoxyresorufin O -deethylase ( EROD) activity was examined in vitro using H4IIE rat hepatoma cells. H4IIE cells were exposed to TBT and PCB-126, individually or in combination, at different concentrations. TBT was cytotoxic at concentrations greater than 98 n M . PCB-126 was not cytotoxic in the concentration range of 49 to 3140 p M . At concentrations greater than 49 n M , PCB-126 enhanced the cytotoxicity of TBT in the 24-98 n M range. In the absence of inducers of EROD activity, TBT significantly inhibited constitutive EROD activity in H4IIE cells in a concentration-dependent manner. EROD activity in H4IIE cells was significantly increased by exposure to PCB-126 alone. This effect was potentiated by coexposure to low, noncytotoxic concentrations of TBT. The induction of cytochrome P-4501A (CYP1A) activity in the presence of both an inducer (PCB-126) and low concentrations of an inhibitor (TBT) indicates that TBT does not interfere with the Ah receptor binding, but acts at the transcriptional level. Potentiation of EROD activity and cytotoxicity as a consequence of coexposure to PCB-126 and TBT is of considerable toxicological significance, given their coaccumulation in a variety of marine organisms.

Contribution of the Pmra promoter to expression of genes in the Escherichia coli mra cluster of cell envelope biosynthesis and cell division genes.

Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802-5811, 1997). Disruption of this promoter (Pmra) on the chromosome and its replacement by the lac promoter (Pmra::Plac) led to isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from Pmra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which Pmra::Plac promoter expression was repressed or fully induced. It was confirmed that the Pmra promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced Pmra::Plac cells. This was correlated with an accumulation of the nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP-N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when Pmra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the Pmra promoter.

PRD--a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria.

Several operon-specific transcriptional regulators, including antiterminators and activators, contain a duplicated conserved domain, the PTS regulation domain (PRD). These duplicated domains modify the activity of the transcriptional regulators both positively and negatively. PRD-containing regulators are very common in Gram-positive bacteria. In contrast, antiterminators controlling beta-glucoside utilization are the only functionally characterized members of this family from gram-negative bacteria. PRD-containing regulators are controlled by PTS-dependent phosphorylation with different consequences: (i) In the absence of inducer, the phosphorylated EIIB component of the sugar permease donates its phosphate to a PRD, thereby inactivating the regulator. In the presence of the substrate, the regulator is dephosphorylated, and the phosphate is transferred to the sugar, resulting in induction of the operon. (ii) In gram-positive bacteria, a novel mechanism of carbon catabolite repression mediated by PRD-containing regulators has been demonstrated. In the absence of PTS substrates, the HPr protein is phosphorylated by enzyme I at His-15. This form of HPr can, in turn, phosphorylate PRD-containing regulators and stimulate their activity. In the presence of rapidly metabolizable carbon sources, ATP-dependent phosphorylation of HPr at Ser-46 by HPr kinase inhibits phosphorylation by enzyme I, and PRD-containing regulators cannot, therefore, be stimulated and are inactive. All regulators of this family contain two copies of PRD, which are functionally specialized in either induction or catabolite repression.

Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability

E. coli recombinant expression systems that utilize lac operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression. Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system. We show here that the pET system, in which phage T7 RNA polymerase gene is expressed via lac operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase. Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well. Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH 6.0. Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells were passaged repeatedly in non-selection medium. These findings both provide a method to enhance the stability of lac-based recombinant expression systems, and suggest that derepression of the lac operon in the absence of inducer may be part of a general cellular response to nutrient limitation.

Production of D-amino acids from d, L-5-substituted hydantoins by an Agrobacterium tumefaciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity

Conversion of D,L-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine by Agrobacterium tumefaciens RU-OR involved a racemase, an hydantoinase and an unusual D-selective N-carbamylamino acid amidohydrolase which was active at alkaline pH and was not inhibited by N-carbamyl-L-amino acids. Enzyme activity was induced by growth in media containing 2-thiouracil. A mutant strain (RU-ORL5) was isolated, which expressed both the hydantoinase and N-carbamylamino acid amidohydrolase enzymes in the absence of inducer. © Rapid Science Ltd. 1998

Repression of the A8L gene, encoding the early transcription factor 82-kilodalton subunit, inhibits morphogenesis of vaccinia virions.

The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and A8L genes, respectively. A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particles. The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle. To address these and other questions, we constructed and characterized a conditionally lethal recombinant vaccinia virus in which the A8L gene is stringently repressed by the Escherichia coli lac operator system. The amount of 82-kDa protein synthesized could be regulated by the amount of inducer: from undetectable to higher than normal levels. Virus replication, as determined by plaque formation or virus yield upon synchronous infection, was dependent on inducer. Nevertheless, de novo synthesis of the 82-kDa subunit was not required for viral early, intermediate, and late gene expression or DNA replication. Overexpression of the A8L gene alone, produced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect. Laser confocal fluorescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated in the absence of inducer, indicating that the A8L protein has a role in morphogenesis of the core and subsequent events.

Designed Disulfide between N-terminal Domains of Lactose Repressor Disrupts Allosteric Linkage

Substitution of Cys for Val at position 52 of the lac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site. This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lac repressor-operator complex (Schumacher, M. A., Choi, K. Y., Zalkin, H., and Brennan, R. G. (1994) Science 266, 763-770; Lewis, M., Chang, G., Horton, N.C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized. In the reduced form, V52C bound operator DNA with slightly increased affinity. Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with approximately 6-fold higher affinity than wild-type protein. Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor. In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding. In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mM. Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding.

2-chloromuconate and ClcR-mediated activation of the clcABD operon: in vitro transcriptional and DNase I footprint analyses.

In Pseudomonas putida, the plasmid-borne clcABD operon encodes enzymes involved in 3-chlorocatechol degradation. Previous studies have demonstrated that these enzymes are induced when P. putida is grown in the presence of 3-chlorobenzoate, which is converted to 3-chlorocatechol, and that ClcR, a LysR-type regulator, is required for this induction. The clcABD operon is believed to have evolved from the chromosomal catBCA operon, which encodes enzymes that utilize catechol and is regulated by CatR. The inducer for the catBCA operon is an intermediate of the catechol pathway, cis,cis-muconate. In this study, we demonstrate by the use of in vitro transcription assays and lacZ transcription fusions in vivo that the analogous intermediate of the 3-chlorocatechol pathway, 2-chloromuconate, is the inducer of the clcABD operon. The DNase I footprints of ClcR with and without 2-chloromuconate were also determined. An extended region of the promoter from -79 to -25 was occupied in the absence of inducer, but the -35 region was unprotected. When 2-chloromuconate was added to the binding assays, the footprint contracted approximately 4 bp at the proximal end of the promoter, and the -35 region was contacted. It is interesting to note that CatR actually extends its footprint 14 bp on the catBCA promoter in response to its inducer. Although CatR and ClcR change their nucleotide protection patterns in different manners when exposed to their respective inducers, their final footprints resemble each other. Therefore, it is possible that their transcriptional activation mechanisms may be evolutionarily conserved.

Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms.

Regulation of the Escherichia coli tna operon: nascent leader peptide control at the tnaC stop codon.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination at Rho-dependent termination sites in the leader region of the operon. Tryptophan induction is dependent on translation of a short leader peptide coding region, tnaC, that contains a single, crucial tryptophan codon. Recent studies suggest that during induction, the TnaC leader peptide acts in cis on the translating ribosome to inhibit its release at the tnaC stop codon. In the present study we use a tnaC-UGA-'lacZ construct lacking the tnaC-tnaA spacer region to analyze the effect of TnaC synthesis on the behavior of the ribosome that translates tnaC. The tnaC-UGA-'lacZ construct is not expressed significantly in the presence or absence of inducer. However, it is expressed in the presence of UGA suppressors, or when the structural gene for polypeptide release factor 3 is disrupted, or when wild-type tRNATrP is overproduced. In each situation, tnaC-UGA-'lacZ expression is reduced appreciably by the presence of inducing levels of tryptophan. Replacing the tnaC UGA stop codon with a sense codon allows considerable expression, which is also reduced, although to a lesser extent, by the addition of tryptophan. Inhibition by tryptophan is not observed when Trp codon 12 of tnaC is changed to a Leu codon. Overexpression of tnaC in trans from a multicopy plasmid prevents inhibition of expression by tryptophan. These results support the hypothesis that the TnaC leader peptide acts in cis to alter the behavior of the translating ribosome.

Construction by gene targeting in human cells of a "conditional' CDC2 mutant that rereplicates its DNA.

We describe a novel gene targeting strategy for the genetic analysis of essential genes in mammalian cells and its use to study the role of the cell cycle control gene CDC2 in human cells. A cell line (HT2-19) was generated in which endogenous CDC2 gene expression and cell viability depend on the presence of an inducer in the growth medium. In the absence of inducer, HT2-19 cells undergo extensive DNA rereplication and apoptosis. Rereplication is indicative of a role for human CDC2 in a control mechanism, previously undetected in mammalian cells, that prevents premature entry into S-phase.