In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii).

Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rho-negative (Rho(-)) cells with that of SP cells by ginbuna transplantation experiments. Rho(-) cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho(-) cells was lower than that of SP cells, while Rho(-) cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho(-) cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts.

Epithelial cells challenged with a Rac-activating E. coli cytotoxin acquire features of professional phagocytes.

Activation of Rho, Rac and Cdc42 GTPases by an Escherichia coli cytotoxin (CNF1) has been reported to induce a phagocytic-like activity by epithelial cells in terms of a ruffle-driven capture and ingestion of large material. More recently, it has been reported that treatment with CNF1 induces superoxide anion release by these cells following a phagocytic stimulus. We herein show that in epithelial cells both transfection with the dominant form of Rac (RacV12) and treatment with the Rac-activating epidermal growth factor (EGF) may increase the secretion of superoxide anions on challenge with latex beads. Moreover, exposure to CNF1 induces a significant augmentation of acidic vesicles where the internalized particles were detectable. Our results indicate that (i) Rac is a pivotal GTPase for inducing in epithelial cells superoxide anion generation and (ii) the internalized material travels trough acidic compartments in CNF1-treated epithelial cells. Altogether this suggests a novel role for epithelial cells that, following Rac activation, might share with professional phagocytes the task of eliminating unwanted pathogens.

Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components.

Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system. The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB. Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination. The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination. The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.

Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Fluorescent modification of the cysteine 202 residue of Escherichia coli transcription termination factor rho.

The lone cysteine residue (Cys-202) of transcription termination factor rho has been modified with the sulfhydryl-specific dyes 5-iodoacetamidofluorescein and 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid. Labeling with both dyes is specific for the Cys-202 residue and is at least 90% complete. Rho protein is an RNA-dependent ATPase and exists as a hexamer of identical subunits in its activated (RNA-liganded) form. We find that chemical modification of rho at Cys-202 does not significantly change the properties of the protein; subunit assembly, RNA binding, and poly(rC)-activated ATP hydrolysis are all relatively unperturbed by the covalent attachment of these fluorescent moieties. On the other hand, the spectral, quenching, and anisotropy properties of the fluorescent groups are all significantly modified by attachment to the protein. No energy transfer is seen between fluorescein-labeled subunits within rho hexamers, indicating that the Cys-202 residues on these subunits are located at least 40 A apart. These fluorescently labeled rho molecules should represent useful probes to study the conformations and inter- and intrasubunit geometries of this termination factor at various stages of its interaction with nascent RNA transcripts.

Absence of a phosphorylated intermediate during ATP hydrolysis by Escherichia coli transcription termination protein rho.

We have established the suitability of adenosine 5'-O-(gamma-thio)triphosphate(ATP gamma S) as an analog of ATP for the nucleoside triphosphatase activity of Escherichia coli transcription termination protein rho (EC 3.6.1.3). Steady-state analysis gives a Vmax of 1.5 mumol min-1 mg-1, 9% of the value with MgATP as substrate, and indicates that ATP gamma S binds as tightly (based on Km and Ki versus ATP) to rho as does ATP. (gamma-S)[beta gamma-17O,gamma-17O,gamma-18O]ATP gamma S was used as substrate to produce chiral product inorganic [17O,18O]thiophosphate and determine the stereochemical course of the hydrolysis. The results of this determination, inversion at the thiophosphoryl phosphorus, indicate that the enzymatic hydrolysis of ATP by rho consists of a direct transfer of the phospho group to water without the existence of a phosphoenzyme or phospho-RNA intermediate.

rho factor-dependent transcription termination. Interference by a mutant rho.

The rho protein isolated from a strain of Escherichia coli with the rho1 (suA1) mutant allele is defective in interactions with RNA that are coupled to ATP hydrolysis. Here we show that the rho1 allele is partially dominant over wild-type and demonstrate that the mechanism of that dominance is due to an interference of wild-type rho factor function by the defective rho factor. The rho1 mutant protein can inhibit transcription termination and RNA-dependent ATPase activities of normal rho protein. Inhibition of the ATPase activity with excess RNA occurs by exchange of subunits to form hybrid hexamers in which the defective subunits apparently disrupt co-operative interactions essential for wild-type subunit function.

RNA sequence and secondary structure requirements for rho-dependent transcription termination.

The interaction of E. coli termination factor rho with the nascent RNA transcript appears to be a central feature of the rho-dependent transcription termination process. Based on in vitro studies of the rho-dependent termination of the transcript initiated at the PR promoter of bacteriophage lambda, and on earlier studies, Morgan, Bear and von Hippel (J. Biol. Chem. 258, 9565-9574, 1983) proposed a model defining the features of a potential binding site for rho protein on transcripts subject to rho-dependent termination. This model suggested that an effective rho binding site on a nascent RNA transcript should be: (i) greater than 70-80 nucleotide residues in length; (ii) essentially unencumbered with stable secondary structure; (iii) relatively sequence non-specific; and (iv) located within a few hundred nucleotide residues upstream of the potential rho-dependent terminus. In this paper we examine the sequences and secondary structures of several transcripts that exhibit rho-dependent termination to test this hypothesis further. Unstructured regions of approximately the expected size and location were found on all the transcripts examined. Though several short specific sequence elements were found to occur in a very similar arrangement on the lambda PR- and lambda PL-initiated transcripts of lambda phage, no such elements of sequence regularity were found on any of the other rho-dependent transcripts. The results of the sequence comparisons reported here strongly support the generality of the "unstructured binding site" hypothesis for rho-dependent termination.

Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase.

Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay. Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA. RNA molecules were preferentially released from enzyme molecules located within the termination region. Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor. Pretreatment of complexes with ribonuclease prevented dissociation of DNA. Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor. In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter. Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.

Internal promoters of the his operon in Salmonella typhimurium.

Two internal promoters in the his operon of Salmonella typhimurium have been precisely mapped genetically. The internal promoters are found in, or very close to, gene border regions in the his operon. The his operon was examined for the presence of additional internal promoters whose transcripts were sensitive to rho-mediated transcription termination and therefore had escaped detection. No new internal promoters were found. It is argued that the internal promoters described here are not likely to be fortuitous message start sites, but may play a physiologically important role in operon expression.

Transcriptional control of two gene subclusters in the tRNA operon of bacteriophage T4.

In bacteriophage T4 DNA, transcription units recognized in vitro by host RNA polymerase consist of promotor-proximal 'immediate early' (IE) genes and promotor-distal 'delayed early' (DE) genes separated from each other by rho-dependent transcription terminators. In vivo, the transition from IE to DE transcription requires phage-specific protein synthesis and can be prevented by chloramphenicol (CAM). Most of the information about IE/DE transition has been obtained by hybridizaton analyses of mixtures of RNA species synthesized simultaneously on several T4 transcription units (for review see ref. 3). A useful model for the study of T4 gene expression at the level of primary transcripts and individual gene products is provided by the T4 tRNA operon, a cluster of genes coding for eight T4-specific transfer RNAs and two stable RNAs (species 1 and 2) of unknown function (Fig. 1). The 10 genes of the tRNA operon are arranged in two subclusters (I and II) with a promotor located about 1 kilobase pair upstream. The primary transcripts and the final gene products of this region have been identified and isolated. Moreover, this genetic region was recently cloned and a part of it sequenced. We describe here the expression of T4 tRNA genes in vivo and in vitro in terms of the IE/DE concept and demonstrate that the two subclusters of the tRNA operon are subject to different modes of control.

Transcription in vitro of bacteriophage lambda 4S RNA: studies on termination and rho protein.

When bacteriophage lambdapga18 DNA is transcribed in a purified in vitro system by E. coli RNA polymerase (nucleoside triphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6), several major transcripts are synthesized. We have investigated transcriptional termination of one of these transcripts, the 4S, or "oop" RNA. Analysis by two-dimensional "fingerprinting" of T1 oligonucleotides reveals that transcription of the 4S RNA terminates at a specific site on the lambdapga18 DNA template, t-L with an efficiency of approximately 80%, i.e. 20% of transcripts are extended into larger RNAs. Addition of the E. coli protein rho to our transcription reactions has two effects: a) the efficiency of termination at the t-L site is increased to 100%; b) the number of 4S transcripts synthesized is increased by greater than 5-fold. Rho appears to stimulate 4S RNA synthesis by facilitating more rapid release of RNA polymerase from the t-L' termination site.