Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml.

Long-term potentiation activates the GAP-43 promoter: Selective participation of hippocampal mossy cells

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing dl-aminophosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, β-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of β-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

The 3′-untranslated region of CaMKIIα is a cis-acting signal for the localization and translation of mRNA in dendrites

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The α subunit of Ca2+-calmodulin-dependent protein kinase II (CaMKIIα) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKIIα provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKIIα promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3′-untranslated region of the CaMKIIα gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKIIα gene, only the lacZ transcripts bearing the 3′-untranslated region are localized to dendrites. The β-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa

Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response. Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen Pseudomonas aeruginosa, the number and types of genes controlled by quorum sensing have not been studied systematically. We have constructed a library of random insertions in the chromosome of a P. aeruginosa acyl-HSL synthesis mutant by using a transposon containing a promoterless lacZ. This library was screened for acyl-HSL induction of lacZ. Thirty-nine quorum sensing-regulated genes were identified. The genes were organized into classes depending on the pattern of regulation. About half of the genes appear to be in seven operons, some seem organized in large patches on the genome. Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites. Many of the genes identified showed a high level of induction by acyl-HSL signaling.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in Escherichia coli. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5′-diphosphate 3′-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene, hisG, was examined by using a transcriptional lacZ fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of hisG-lacZ mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the hisG-lacZ fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024–7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

Mammalian Trithorax and Polycomb-group homologues are antagonistic regulators of homeotic development

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of Trx. Bmi-1 was first identified as a collaborator in c-myc-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs. Here, we note the axial-skeletal transformations and altered Hox expression patterns of Mll-deficient and Bmi-1-deficient mice were normalized when both Mll and Bmi-1 were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll-deficient compared with Bmi-1-deficient mice demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8-lacZ reporter construct. Reexpression of MLL was able to overcome repression, rescuing expression of Hoxc8-lacZ in Mll-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target Hox gene.

Phenotypic alterations in insulin-deficient mutant mice

Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing β cells and glucagon-positive α cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcription-coupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of δ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.

Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli

Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial LacZ reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent β-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of β-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.

Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′ genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.

A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a “patch” near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and Pi signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6Kγ attP “genome targeting” suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.