Chloramphenicol Acetyltransferase Activity Research Articles

Experimental modification in thermal stability of oligomers by alanine substitution and site saturation mutagenesis of interfacial residues

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.

Pioglitazone improves skeletal muscle functions in reserpine-induced fibromyalgia rat model

Background Fibromyalgia (FM) is characterized by musculoskeletal pain, fatigue, sleep and memory disturbance. There is no definitive cure yet for FM-related health problems. Peroxisome proliferator-activated receptor’s (PPAR’s) activation is associated with insulin sensitisation and improved glucose metabolism. PPAR-γ was reported to alleviate FM allodynia. Limited data are discussing its effect on motor disorders. Objective To investigate the potential effect of PPAR-γ agonists (pioglitazone, as one member of thiazolidinediones (TZD)) on motor dysfunction in reserpine-induced FM in a rat model. Materials and methods Thirty-six male Wistar rats were divided into negative control (n = 9) and reserpine-induced FM (n = 27) groups. The latter was subdivided into three equal subgroups (n = 9), positive control (untreated FM model), pioglitazone-treated and GW9662-treated. We evaluated muscle functions and activity of chloramphenicol acetyltransferase, superoxide dismutase, malondialdehyde, and serum levels of interleukin-8 and monocyte chemoattractant protein-1. Results Pioglitazone significantly relieved fatigue, improved muscle performance, reduced inflammatory cytokines and enhanced antioxidant’s activity, while GW9662, a known PPAR-γ antagonist, aggravated the FM manifestations in the rat model. Conclusion PPAR-γ agonists show a promising role against FM-associated motor dysfunctions.

Antibiotic-Resistant and Non-Resistant Bacteria Display Similar Susceptibility to Dielectric Barrier Discharge Plasma.

Here, we examined whether antibiotic-resistant and non-resistant bacteria show a differential susceptibility to plasma treatment. Escherichia coli DH5α were transformed with pPRO-EX-HT-CAT, which encodes an ampicillin resistance gene and chloramphenicol acetyltransferase (CAT) gene, and then treated with a dielectric barrier discharge (DBD) plasma torch. Plasma treatment reduced the viable cell count of E. coli after transformation/selection and further cultured in ampicillin-containing and ampicillin-free medium. However, there was no significant difference in viable cell count between the transformed and untransformed E. coli after 1 min- and 2 min-plasma treatment. Furthermore, the enzyme-linked immunosorbent assay (ELISA) and acetyltransferase activity assay showed that the CAT activity was reduced after plasma treatment in both transformed and selected E. coli grown in ampicillin-containing or ampicillin-free medium. Loss of lipopolysaccharide and DNA damage caused by plasma treatment were confirmed by a Limulus test and polymerase chain reaction, respectively. Taken together, these findings suggest the plasma acts to degrade components of the bacteria and is therefore unlikely to display a differential affect against antibiotic-resistant and non-resistant bacteria. Therefore, the plasma method may be useful in eliminating bacteria that are recalcitrant to conventional antibiotic therapy.

Designing seryl-tRNA synthetase for improved serylation of selenocysteine tRNAs.

Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNASec , which is subsequently converted by selenocysteine synthase to Sec-tRNASec . Escherichia coli SerRS serylates tRNASec poorly; this may hinder efficient production of designer selenoproteins invivo. Guided by structural modelling and selection for chloramphenicol acetyltransferase activity, we evolved three SerRS variants capable of improved Ser-tRNASec synthesis. They display 10-, 8-, and 4-fold increased kcat /KM values compared to wild-type SerRS using synthetic tRNASec species as substrates. The enzyme variants also facilitate invivo read-through of a UAG codon in the position of the critical serine146 of chloramphenicol acetyltransferase. These results indicate that the naturally evolved SerRS is capable of further evolution for increased recognition of a specific tRNA isoacceptor.

MicroRNA-200c/141 upregulates SerpinB2 to promote breast cancer cell metastasis and reduce patient survival.

The microRNA-200 (miR-200) family is associated with tumor metastasis and poor patient prognosis. We found that miR-200c/141 cluster overexpression upregulated SerpinB2 in the MDA-MB-231 triple-negative (TN) breast cancer cell line. We observed transcription factor (c-Jun, c-Fos, and FosB) upregulation, nuclear localization of c-Jun, and increased SerpinB2 promoter-directed chloramphenicol acetyltransferase activity in miR-200c/141 cluster-overexpressing cells relative to controls. Additionally, miR-124a and miR-26b, which directly target SepinB2, were downregulated compared to controls. In mouse xenograft models, miR-200c/141 cluster overexpression promoted lymph node and lung metastasis, and siRNA-mediated SerpinB2 knockdown decreased lung metastasis, suggesting that SerpinB2 mediates miR-200c/141-induced lung metastasis. We also explored the clinical significance of SerpinB2 protein status through analysis of primary breast tumor samples and The Cancer Genome Atlas (TCGA) data. High SerpinB2 levels were associated with reduced survival and increased lymph node metastasis in breast cancer patients. SerpinB2 was overexpressed in the TN breast cancer subtype as compared to the luminal subtype. The present study demonstrates that SerpinB2 promotes miR-200c/141 cluster overexpression-induced breast cancer cell metastasis, and SerpinB2 overexpression correlates with increased metastatic potential and unfavorable outcomes in breast cancer patients. SerpinB2 may be a useful biomarker for assessing metastasis risk in breast cancer patients.

New PAH gene promoter KLF1 and 3′-region C/EBPalpha motifs influence transcription in vitro

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50% in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60% of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli

BackgroundThe occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. MethodsTo understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV–vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. ResultsWe observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37μM. ConclusionsIt was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. General significanceUnderstanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.

On-chip enzymatic assay for chloramphenicol acetyltransferase using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Herein, we report a chloramphenicol (CAP) acetyltransferase (CAT) activity assay based on self-assembled monolayers on gold as an alternative to conventional CAT reporter gene assay systems, which sometimes require toxic materials and complicated steps that limit their use. A CAP derivative presented on a monolayer was converted to the acetylated CAP by CAT in the presence of acetyl-CoA. The conversion was directly monitored by observing the molecular weight changes in CAP using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CAT activity was determined under various reaction conditions by changing reaction times, CAT and acetyl-CoA concentrations. As a practical application, we identified gene expression in bacteria that were transformed with pCAT plasmid DNA. Our strategy can provide a simple and rapid assay that eliminates some commonly used but potentially detrimental steps in enzymatic assays, such as radioactive labeling and complicated separation and purification of analytes prior to detection.

Post-transcriptional regulation by distal Shine-Dalgarno sequences in the grpE-dnaK intergenic region of Streptococcus mutans.

A unique 373 bp region (igr66) between grpE and dnaK of Streptococcus mutans lacks a promoter but is required for optimal production of DnaK. Northern blotting using probes specific to hrcA, igr66 or dnaK revealed multiple transcripts produced from the dnaK operon and 5'-RACE mapped 5' termini of multiple dnaK transcripts within igr66. One product mapped to a predicted 5'-SL (stem-loop) and two others mapped just 5' to Shine-Dalgarno (SD)-like sequences located immediately upstream to dnaK and to a predicted SL 120 bp upstream of the dnaK start codon (3'-SL). A collection of cat reporter-gene strains containing mutant derivatives of igr66 were engineered. Chloramphenicol acetyltransferase (CAT) activity varied greatly between strains, but there were no correlative changes in cat mRNA levels. Interestingly, mutations introduced into the SD-like sequences 5' to the 3'-SL resulted in an 83-98% decrease in CAT activity. Markerless point mutations introduced upstream of dnaK in the SD-like sequences impaired growth at elevated temperatures and resulted in up to a 40% decrease in DnaK protein after heat shock. Collectively, these results indicate processing within igr66 enhances translation in a temperature dependent manner via non-canonical ribosome binding sites positioned >120 bp upstream of dnaK.

Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus.

BackgroundExotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear.ResultsWe compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants.ConclusionsWith the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0294-5) contains supplementary material, which is available to authorized users.

Versatile synthesis of probes for high-throughput enzyme activity screening

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination. FigureThe Nimzyme high-throughput enzyme activity assay allows for the detection of enzyme activity in cell lysate. Fluorous interactions between a specialized substrate probe and a nanostructure-initiator mass spectrometry surface allow for in situ cleanup and the subsequent collection of unambiguous mass spectra. One of the main hurdles that prevents the widespread adoption of this technology is the need to chemically synthesize the required probes. Here, we present a simplified route to derive Nimzyme probes from a wide variety of biologically interesting substrates. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-013-6888-z) contains supplementary material, which is available to authorized users.

Interplay between EGR1 and SP1 is critical for 13-cis retinoic acid-mediated transcriptional repression of angiotensin type 1A receptor.

Recently, we have demonstrated that 13-cis retinoic acid (13cRA) downregulates rat angiotensin type 1A receptor (Agtr1a) gene transcription through a MAP kinase (ERK1/2)-dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA-mediated Agtr1a downregulation. Rat Agtr1a chloramphenicol acetyltransferase (CAT) promoter construct containing a sequence -2541 and -1836 bp upstream of the start site demonstrated reduced CAT activity; this region possesses a specificity protein 1 (SP1) consensus sequence (5'-TGGGGCGGGGCGGGG-3'). Mobility shift analysis using untreated nuclear extracts in the presence of mithramycin A suggests that the trans-acting factor binding to this cis-acting element is SP1. 13cRA significantly reduced specific binding without any change in SP1 protein expression. Studies showed that 13cRA treatment maximally phosphorylates ERK1/2 within 5-10 min, which translocates to the nucleus, activating early growth response protein 1 (Egr1) mRNA expression at 20 min followed by de novo protein synthesis, leading to an EGR1/SP1 interaction. siRNA silencing of Egr1 restored Agtr1a mRNA and protein expression in 13cRA-treated cells, and Sp1 silencing results in complete loss of Agtr1a expression. Our study suggests that 13cRA-mediated activation of ERK1/2, through EGR1, is capable of disrupting SP1, the requisite trans-activator for Agtr1a expression, providing a novel paradigm in Agtr1a gene transcription.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.

Characterization of Two Metagenome-Derived Esterases That Reactivate Chloramphenicol by Counteracting Chloramphenicol Acetyltransferase

Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

Specificity protein, Sp1-mediated increased expression of Prdx6 as a curcumin-induced antioxidant defense in lens epithelial cells against oxidative stress

Peroxiredoxin 6 (Prdx6) is a pleiotropic oxidative stress-response protein that defends cells against reactive oxygen species (ROS)-induced damage. Curcumin, a naturally occurring agent, has diversified beneficial roles including cytoprotection. Using human lens epithelial cells (hLECs) and Prdx6-deficient cells, we show the evidence that curcumin protects cells by upregulating Prdx6 transcription via invoking specificity protein 1 (Sp1) activity against proapoptotic stimuli. Curcumin enhanced Sp1 and Prdx6 mRNA and protein expression in a concentration-dependent manner, as evidenced by western and real-time PCR analyses, and thereby negatively regulated ROS-mediated apoptosis by blunting ROS expression and lipid peroxidation. Bioinformatic analysis and DNA–protein binding assays disclosed three active Sp1 sites (−19/27, −61/69 and −82/89) in Prdx6 promoter. Co-transfection experiments with Sp1 and Prdx6 promoter–chloramphenicol acetyltransferase (CAT) constructs showed that CAT activity was dramatically increased in LECs or Sp1-deficient cells (SL2). Curcumin treatment of LECs enhanced Sp1 binding to its sites, consistent with curcumin-dependent stimulation of Prdx6 promoter with Sp1 sites and cytoprotection. Notably, disruption of Sp1 sites by point mutagenesis abolished curcumin transactivation of Prdx6. Also, curcumin failed to activate Prdx6 expression in the presence of Sp1 inhibitors, demonstrating that curcumin-mediated increased expression of Prdx6 was dependent on Sp1 activity. Collectively, the study may provide a foundation for developing transcription-based inductive therapy to reinforce endogenous antioxidant defense by using dietary supplements.

Pax6 Controls the Expression of Critical Genes Involved in Pancreatic α Cell Differentiation and Function*

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased β and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

The construction and characterization of the bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts of Marek's disease viruses

BackgroundMarek's disease virus (MDV) has a bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts. By sequencing for the promoters from 8 different strains (CVI988, 814, GA, JM, Md5, G2, RB1B and 648A), it is found, comparing with the other 7 MDV strains, CVI988 has a 5-bp (from -628 to -632) deletion in this region, which caused a Sp1 site destroyed. In order to analysis the activity of the promoter, the complete bi-directional promoters from GA and CVI988 were, respectively, cloned into pCAT-Basic vector in both directions for the recombinants pPGA(pp38)-CAT, pPGA(1.8 kb)-CAT, pPCVI(pp38)-CAT and pPCVI(1.8 kb)-CAT. The complete promoter of GA was divided into two single-direction promoters from the replication of MDV genomic DNA, and cloned into pCAT-Basic for pdPGA(pp38)-CAT and pdPGA(1.8 kb)-CAT as well. The above 6 recombinants were then transfected into chicken embryo fibroblasts (CEFs) infected with MDV, and the activity of chloramphenicol acetyltransferase (CAT) was measured from the lysed CEFs 48 h post transfection.ResultsThe results showed the activity of the divided promoters was decreased on both directions. In 1.8-kb mRNA direction, it is nearly down to 2.4% (19/781) of the whole promoter, while it keeps 65% (34/52) activity in pp38 direction. The deletion of Sp1 site in CVI988 causes the 20% activity decreased, and has little influence in pp38 direction.ConclusionThe present study confirmed their result, and the promoter for the 1.8-kb mRNA transcripts is a much stronger promoter than that in the orientation for pp38.

Transcriptional activation of insulin-like growth factor binding protein 6 by 17β-estradiol in SaOS-2 cells

Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micronM) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5-CCTTCA CCTG-3] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.

Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell-Free Protein Synthesis

Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.

Identification of a Novel Prophage-Like Gene Cluster Actively Expressed in Both Virulent and Avirulent Strains of Leptospira interrogans Serovar Lai

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.