The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions.

AIM: To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE. METHODS: ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays. RESULTS: 78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells. CONCLUSIONS: While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region.

We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.

Stress-induced activation of the sigma B transcription factor of Bacillus subtilis.

The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals. On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions. Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response. After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression. Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth. Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells. However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus.

Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon.

Transcription factor sigma B of Bacillus subtilis is active during the stationary growth phase, but its physiological role remains unknown. Understanding the function and regulation of genes controlled by sigma B (csb genes) should provide important clues to sigma B function in stationary-phase cells. To this end, we used a genetic approach to identify six new csb genes. This strategy relies on two elements: (i) random transcriptional fusions between the Escherichia coli lacZ gene and genes on the B. subtilis chromosome, generated in vivo with transposon Tn917lacZ, and (ii) a plate transformation technique to introduce a null sigB mutation into the fusion-bearing recipients directly on indicator plates. This strategy allowed the comparison of fusion expression in strains that were isogenic save for the presence or absence of a functional sigma B protein. Beginning with 1,400 active fusions, we identified 11 that were wholly or partly controlled by sigma B. These fusions mapped to six different loci that exhibit substantial contrasts in their patterns of expression in the logarithmic and stationary growth phases, suggesting that they participate in diverse cellular functions. However, for all six loci, the sigma B-dependent component of their expression was manifest largely in the stationary phase. The high frequency of six independent csb loci detected in a random collection of 1,400 fusions screened, the fact that four of the six new loci were defined by a single fusion, and the absence of the previously identified ctc and csbA genes in the present collection strongly suggest that sigma B controls a large stationary-phase regulon.

Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase and is controlled by stationary-phase transcription factor sigma B.

Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon, but in no case has the physiological function of any gene in this regulon been identified. Here we show that transcription of gtaB is partly dependent on sigma B in vivo and that gtaB encodes UDP-glucose pyrophosphorylase. The gtaB reading frame was initially identified by a sigma B-dependent Tn917lacZ fusion, csb42. We cloned the region surrounding the csb42 insertion, identified the reading frame containing the transposon, and found that this frame encoded a predicted 292-residue product that shared 45% identical residues with the UDP-glucose pyrophosphorylase of Acetobacter xylinum. The identified reading frame appeared to lie in a monocistronic transcriptional unit. Primer extension and promoter activity experiments identified tandem promoters, one sigma B dependent and the other sigma B independent, immediately upstream from the proposed coding region. A sequence resembling a factor-independent terminator closely followed the coding region. By polymerase chain reaction amplification of a B. subtilis genomic library carried in yeast artificial chromosomes, we located the UDP-glucose pyrophosphorylase coding region near gtaB, mutations in which confer phage resistance due to decreased glycosylation of cell wall teichoic acids. Restriction mapping showed that the coding region overlapped the known location of gtaB. Sequence analysis of a strain carrying the gtaB290 allele found an alteration that would change the proposed initiation codon from AUG to AUA, and an insertion-deletion mutation in this frame conferred phage resistance indistinguishable from that elicited by the gtaB290 mutation. We conclude that gtaB encodes UDP-glucose pyrophosphorylase and is partly controlled by sigma B. Because this enzyme is important for thermotolerance and osmotolerance in stationary-phase Escherichia coli cells, our results suggest that some genes controlled by sigma B may play a role in stationary-phase survival of B. subtilis.

Activation of Bacillus subtilis transcription factor sigma B by a regulatory pathway responsive to stationary-phase signals.

Alternative transcription factor sigma B of Bacillus subtilis controls a stationary-phase regulon induced under growth conditions that do not favor sporulation. Little is known about the metabolic signals and protein factors regulating the activity of sigma B. The operon containing the sigma B structural gene has the gene order orfV-orfW-sigB-rsbX, and operon expression is autoregulated positively by sigma B and negatively by the rsbX product (rsbX = regulator of sigma B). To establish the roles of the orfV and orfW products, orfV and orfW null and missense mutations were constructed and tested for their effects on expression of the sigma B-dependent genes ctc and csbA. These mutations were tested in two contexts: in the first, the sigB operon was under control of its wild-type, sigma B-dependent promoter, and in the second, the sigB operon promoter was replaced by the inducible Pspac promoter. The principal findings are that (i) the orfV (now called rsbV) product is a positive regulator of sigma B-dependent gene expression; (ii) the orfW (now called rsbW) product is a negative regultor of such expression; (iii) sigma B is inactive during logarithmic growth unless the rsbW product is absent; (iv) the rsbX, rsbV, and rsbW products have a hierarchical order of action; and (v) both the rsbV and rsbW products appear to regulate sigma B activity posttranslationally. There are likely to be at least two routes by which information can enter the system to regulate sigma B: via the rsbX product, and via the rsbV and rsbW products.

Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor sigma B of Bacillus subtilis.

We describe a general, in vivo method for identifying Bacillus subtilis genes controlled by specific, nonessential regulatory factors. We establish the use of this approach by identifying, isolating, and characterizing a gene dependent on sigma B, an alternate transcription factor which is found early in stationary phase but which is not essential for sporulation. The method relies on two features: (i) a plate transformation technique to introduce a null mutation into the regulatory gene of interest and (ii) random transcriptional fusions to a reporter gene to monitor gene expression in the presence and absence of a functional regulatory product. We applied this genetic approach to isolate genes comprising the sigma B regulon. We screened a random Tn917lacZ library for fusions that required an intact sigma B structural gene (sigB) for greatest expression, converting the library strains from wild-type sigB+ to sigB delta::cat directly on plates selective for chloramphenicol resistance. We isolated one such fusion, csbA::Tn917lacZ (csb for controlled by sigma B), which mapped between hisA and degSU on the B. subtilis chromosome. We cloned the region surrounding the insertion, identified the csbA reading frame containing the transposon, and found that this frame encoded a predicted 76-residue product which was extremely hydrophobic and highly basic. Primer extension and promoter activity experiments identified a sigma B-dependent promoter 83 bp upstream of the csbA coding sequence. A weaker, tandem, sigma A-like promoter was likewise identified 28 bp upstream of csbA. The csbA fusion was maximally expressed during early stationary phase in cells grown in Luria broth containing 5% glucose and 0.2% glutamine. This timing of expression and medium dependence were very similar to those for ctc, the only other recognized gene dependent on sigma B.

In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes.

The promoter region preceding the hutUH operon in Klebsiella aerogenes contains two oppositely oriented, overlapping promoters. In the absence of catabolite gene activator protein-cyclic AMP (CAP-cAMP), transcription proceeds primarily from the backward-oriented promoter (Pc), whose function has not yet been determined, and only very weakly from the forward hutUH promoter, hutUp. In the presence of CAP-cAMP, Pc is repressed and transcription from hutUp is favored. Two protein components required for this in vitro transcription system, RNA polymerase (RNAP) and CAP, were purified from K. aerogenes and were shown to be functionally interchangeable with the corresponding proteins from Escherichia coli, suggesting that E. coli RNAP could be used to study some aspects of hut transcription. We showed that a gradual activation of hutUp (by increasing concentrations of CAP, cAMP, or glycerol) resulted in a parallel repression of Pc, arguing in favor of a direct competition between the two promoters. The presence of a DNA sequence resembling the consensus for CAP-binding sites and centered at nucleotide -82 (relative to hutUp) initially suggested that a primary role of CAP was to repress Pc, thereby indirectly activating hutUp. However, the relatively slow formation of open complexes at Pc, even in the absence of CAP-cAMP, showed that Pc is a weak promoter and likely to be a poor competitor for RNAP. The observed dominance of Pc over hutUp suggested that the latter is an even weaker promoter. Thus, repression of Pc would not be sufficient to cause the observed increase in hutUp activity, and the CAP-cAMP complex must play a direct role in the activation of hutUp.

Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria.

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.

Gene encoding the alpha core subunit of Bacillus subtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S11, and L17.

We describe the genetic and transcriptional organization of the promoter-distal portion of the Bacillus subtilis alpha operon. By DNA sequence analysis of the region surrounding rpoA, the gene for the alpha core subunit of RNA polymerase, we identified six open reading frames by the similarity of their products to their counterparts in the Escherichia coli transcriptional and translational apparatus. Gene order in this region, given by gene product, was IF1-B-S13-S11-alpha-L17. Gene order in E. coli is similar but not identical: SecY-B-S13-S11-S4-alpha-L17. The B. subtilis alpha region differed most strikingly from E. coli in the presence of IF1 and the absence of ribosomal protein S4, which is the translational regulator of the E. coli alpha operon. In place of the gene for S4, B. subtilis had a 177-base-pair intercistronic region containing two possible promoter sequences. However, experiments with S1 mapping of in vivo transcripts, gene disruptions in the alpha region, and a single-copy transcriptional fusion vector all suggested that these possible promoters were largely inactive during logarithmic growth, that the major promoter for the alpha operon lay upstream from the region cloned, and that the genes in the IF1 to L17 interval were cotranscribed. Thus, the transcriptional organization of the region resembles that of E. coli, wherein the alpha operon is transcribed primarily from the upstream spc promoter, but the absence of the S4 gene suggests that the translational regulation of the region may differ more fundamentally.

Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis.

The physiological roles of the gene subset defined by early-blocked sporulation mutations (spo0) and their second-site suppressor alleles (rvtA11 and crsA47) remain cryptic for both vegetative and sporulating Bacillus subtilis cells. To test the hypothesis that spo0 gene products affect global regulation, we assayed the levels of carbon- and nitrogen-sensitive enzymes in wild-type and spo0 strains grown in a defined minimal medium containing various carbon and nitrogen sources. All the spo0 mutations (except spo0J) affected both histidase and arabinose isomerase levels in an unexpected way: levels of both carbon-sensitive enzymes were two- to six-fold higher in spo0 strains compared to wild type, when cells were grown on the derepressing carbon sources arabinose or maltose. There was no difference in enzyme levels with glucose-grown cells, nor was there a significant difference in levels of the carbon-independent enzymes glutamine synthetase and glucose-6-phosphate dehydrogenase. This effect was not due to a slower growth rate for the spo0 mutants on the poor carbon and nitrogen sources used. The levels of carbon-sensitive enzymes were not simply correlated with sporulation ability in genetically suppressed spo0 mutants, but the rvtA and crsA suppressors each had such marked effects on wild-type growth and enzyme levels that these results were difficult to interpret. We conclude that directly or indirectly the spo0 mutations, although blocking the sporulation process, increase levels of carbon-sensitive enzymes, possibly at the level of gene expression.

Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster.

We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a lambda gt11 expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding S11) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.

Regulation of hutUH operon expression by the catabolite gene activator protein-cyclic AMP complex in Klebsiella aerogenes.

RNA polymerase transcribed the hutUH operon of Klebsiella aerogenes if the catabolite gene activator protein (CAP) and cyclic AMP (cAMP) were present or if the DNA template was derived from a promoter mutant in which hutUH expression was independent of the need for positive effectors. In the absence of CAP or cAMP, not only was hutUH transcription absent, but transcription in the opposite direction (toward hutC) was initiated at a site (pC) ca. 70 base pairs from the site (pUH) of hutUH mRNA initiation. When the pC promoter was cloned in front of a promoterless galK gene, active expression of galK was observed. Thus, the pC promoter is active in vivo as well as in vitro. Transcription from pUH and pC may be mutually exclusive, with the major effect of CAP and cAMP being to prevent transcription from pC, thus relieving the antagonistic effect on transcription from pUH. This "double-negative" control by CAP-cAMP is supported by two observations: (i) CAP-cAMP was unable to activate transcription from pUH if RNA polymerase had been previously bound to pC and (ii) a mutation that allowed transcription from pUH in the absence of positive effectors simultaneously eliminated the activity of pC. An alternative model, in which CAP-cAMP is required for pUH expression and RNA polymerase binding at pC serves to modulate this control in some unknown way, is also considered. The physiological role of the transcript from pC other than regulation of pUH is unknown.

A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA.

The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.

Genetic and physical maps of Klebsiella aerogenes genes for histidine utilization (hut).

Deletion derivatives of the hut-containing plasmid pCB101 were tested against point mutants defective in individual genes of the histidine utilization (hut) operons using a complementation/recombination assay. Location of the genes of the right operon, hutU and hutH, was confirmed by direct assay of the gene products, urocanase and histidase; location of the repressor gene was identified by measuring the ability of the plasmid-carried genes to repress the formation of histidase from a chromosomal location. The analysis of eight deletion plasmids unambiguously confirms the map order of the hut genes as hutI-G-C-U-H, and demonstrates that, in Klebsiella aerogenes, the hutU and hutH genes are transcribed from their own promoter. In addition, the genetic map of hut can be aligned with the restriction map of the hut DNA in plasmid pCB101. One of the deletion plasmids studied apparently encodes a defective histidase subunit that is trans-dominant to active histidase. Another deletion, which completely removes the left operon, hutIG, allows high level expression of the hutUH operon and thus overproduction of a toxic intermediate.

Growth, enzyme levels, and some metabolic properties of an Escherichia coli mutant grown on L-threonine as the sole carbon source.

A mutant of Escherichia coli (designated E. coli SBD-76) that utilizes L-threonine as the sole carbon source was isolated. In contrast with levels in extracts of wild-type cells, the levels of threonine dehydrogenase in extracts of this mutant were 100-fold higher than levels of threonine aldolase or degradative threonine dehydratase. Catabolite repression of threonine dehydrogenase was manifested in wild-type, but not SBD-76, cells. For purposes of isolating enzymes, large quantities of SBD-76 cells with the elevated threonine dehydrogenase level could be grown in a fermentor in modified Fraser medium containing 1% glycerol, rather than in the 0.2% L-threonine minimal medium used to isolate the mutant. SBD-76 cells grown on L-threonine excreted glycine and aminoacetone into the medium, and extracts of the mutant strain catalyzed a quantitative conversion of L-threonine to glycine and aminoacetone.

L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12.

L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.