Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

Functional and physiological consequences of genetic variation at phosphoglucose isomerase: Heat shock protein expression is related to enzyme genotype in a montane beetle

Allele frequency variation at the phosphoglucose isomerase (PGI) locus in Californian populations of the beetle Chrysomela aeneicollis suggests that PGI may be undergoing natural selection. We quantified (i) apparent Michaelis-Menten constant (Km) of fructose 6-phosphate at different temperatures and (ii) thermal stability for three common PGI genotypes (1–1, 1–4, and 4–4). We also measured air temperature (Ta) and beetle body temperature (Tb) in three montane drainages in the Sierra Nevada, California. Finally, we measured 70-kDa heat shock protein (Hsp70) expression in field-collected and laboratory-acclimated beetles. We found that PGI allele 1 predominated in the northernmost drainage, Rock Creek (RC), which was also significantly cooler than the southernmost drainage, Big Pine Creek (BPC), where PGI allele 4 predominated. Allele frequencies and air temperatures were intermediate in the middle drainage, Bishop Creek (BC). Differences among genotypes in Km (1–1 > 1–4 > 4–4) and thermal stability (4–4 > 1–4 > 1–1) followed a pattern consistent with temperature adaptation. In nature, Tb was closely related to Ta. Hsp70 expression in adult beetles decreased with elevation and differed among drainages (BPC > BC > RC). After laboratory acclimation (8 days, 20°C day, 4°C night) and heat shock (4 h, 28–36°C), Hsp70 expression was greater for RC than BPC beetles. In RC, field-collected beetles homozygous for PGI 1–1 had higher Hsp70 levels than heterozygotes or a 4–4 homozygote. These results reveal functional and physiological differences among PGI genotypes, which suggest that montane populations of this beetle are locally adapted to temperature.

Saccharomyces Genome Database provides tools to survey gene expression and functional analysis data

Upon the completion of the Saccharomyces cerevisiae genomic sequence in 1996 [Goffeau,A. et al. (1997) Nature, 387, 5], several creative and ambitious projects have been initiated to explore the functions of gene products or gene expression on a genome-wide scale. To help researchers take advantage of these projects, the Saccharomyces Genome Database (SGD) has created two new tools, Function Junction and Expression Connection. Together, the tools form a central resource for querying multiple large-scale analysis projects for data about individual genes. Function Junction provides information from diverse projects that shed light on the role a gene product plays in the cell, while Expression Connection delivers information produced by the ever-increasing number of microarray projects. WWW access to SGD is available at genome-www.stanford.edu/Saccharomyces/.

Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+ chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Functional genomics: Probing plant gene function and expression with transposons

Transposable elements provide a convenient and flexible means to disrupt plant genes, so allowing their function to be assessed. By engineering transposons to carry reporter genes and regulatory signals, the expression of target genes can be monitored and to some extent manipulated. Two strategies for using transposons to assess gene function are outlined here: First, the PCR can be used to identify plants that carry insertions into specific genes from among pools of heavily mutagenized individuals (site-selected transposon mutagenesis). This method requires that high copy transposons be used and that a relatively large number of reactions be performed to identify insertions into genes of interest. Second, a large library of plants, each carrying a unique insertion, can be generated. Each insertion site then can be amplified and sequenced systematically. These two methods have been demonstrated in maize, Arabidopsis, and other plant species, and the relative merits of each are discussed in the context of plant genome research.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: Similarity of functional domains to subtilisin-like endoproteases.

A 69-kDa proteinase (P69), a member of the pathogenesis-related proteins, is induced and accumulates in tomato (Lycopersicon esculentum) plants as a consequence of pathogen attack. We have used the polymerase chain reaction to identify and clone a cDNA from tomato plants that represent the pathogenesis-related P69 proteinase. The nucleotide sequence analysis revealed that P69 is synthesized in a preproenzyme form, a 745-amino acid polypeptide with a 22-amino acid signal peptide, a 92-amino acid propolypeptide, and a 631-amino acid mature polypeptide. Within the mature region the most salient feature was the presence of domains homologous to the subtilisin serine protease family. The amino acid sequences surrounding Asp-146, His-203, and Ser-532 of P69 are closely related to the catalytic sites (catalytic triad) of the subtilisin-like proteases. Northern blot analysis revealed that the 2.4-kb P69 mRNA accumulates abundantly in leaves and stem tissues from viroid-infected plants, whereas the mRNA levels in tissues from healthy plants were undetectable. Our results indicate that P69, a secreted calcium-activated endopeptidase, is a plant pathogenesis-related subtilisin-like proteinase that may collaborate with other defensive proteins in a general mechanism of active defense against attacking pathogens.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis--i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilinage differentiation/maturation through the erythroid or granulocytic lineage. In the initial HPC differentiation stages, the RB gene is gradually induced at the mRNA and protein level in both erythroid and granulopoietic cultures. In late HPC differentiation and then precursor maturation, RB gene expression is sustained in the erythroid lineage, whereas it is sharply downmodulated in the granulocytic series. Functional studies were performed by treatment of HPC differentiation culture with phosphorothioate antisense oligomer targeting Rb mRNA; coherent with the expression pattern, oligomer treatment of late HPCs causes a dose-dependent and selective inhibition of erythroid colony formation. These observations suggest that the RB gene plays an erythroid- and stage-specific functional role in normal adult hematopoiesis, particularly at the level of late erythroid HPCs.

CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression.

Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3-fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions.