lacZ Reporter Gene Research Articles

The transcription factor GCN4 regulates PHM8 and alters triacylglycerol metabolism in Saccharomyces cerevisiae.

PHM8 is a very important enzyme in nonpolar lipid metabolism because of its role in triacylglycerol (TAG) biosynthesis under phosphate stress conditions. It is positively regulated by the PHO4 transcription factor under low phosphate conditions; however, its regulation has not been explored under normal physiological conditions. General control nonderepressible (GCN4), a basic leucine-zipper transcription factor activates the transcription of amino acids, purine biosynthesis genes and many stress response genes under various stress conditions. In this study, we demonstrate that the level of TAG is regulated by the transcription factor GCN4. GCN4 directly binds to its consensus recognition sequence (TGACTC) in the PHM8 promoter and controls its expression. The analysis of cells expressing the P PHM8 -lacZ reporter gene showed that mutations (TGACTC-GGGCCC) in the GCN4-binding sequence caused a significant increase in β-galactosidase activity. Mutation in the GCN4 binding sequence causes an increase in PHM8 expression, lysophosphatidic acid phosphatase activity and TAG level. PHM8, in conjunction with DGA1, a mono- and diacylglycerol transferase, controls the level of TAG. These results revealed that GCN4 negatively regulates PHM8 and that deletion of GCN4 causes de-repression of PHM8, which is responsible for the increased TAG content in gcn4∆ cells.

Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.

Modeling Metazoan Apoptotic Pathways in Yeast.

This chapter describes techniques for characterizing metazoan apoptotic pathways using Saccharomyces cerevisiae. Active forms of the major apoptotic effectors-caspases, Bax and Bak-are all lethal to yeast. Using this lethality as a readout of caspase/Bax/Bak activity, proteins and small molecules that directly or indirectly regulate the activity of these effectors can be investigated in yeast, and apoptotic inhibitors can be identified using functional yeast-based screens. Caspase activity can also be monitored in yeast by cleavage-dependent liberation of a transcription factor from the plasma membrane, enabling it to activate the lacZ reporter gene. This system can be used to define the sequences that can be efficiently cleaved by particular caspases.

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.

N- and C-terminal non-conserved residues contribute to transactivation by a sea anemone (Nematostella vectensis) NF-κB transcription factor

NF-κB is an evolutionarily conserved eukaryotic transcription factor that plays a role in many important developmental and immune-related processes by activating target gene expression. The goal of these experiments was to define the sequences required for a sea anemone NF-κB's intrinsic transactivation activity by using mutant proteins with serial deletions of the N- and C-terminal sequences. Deletion mutants were constructed that were missing the C-terminal 15, 32 or 47 amino acids (aa) or the N-terminal 17, 27 or 47 aa of the 440 aa NF-κB protein from the starlet sea anemone, Nematostella vectensis (Nv), a simple model organism in the phylum Cnidaria. These Nv-NF-κB mutants were expressed as GAL4 fusion proteins in yeast, and their transactivation activities were assessed by LacZ reporter gene assays. The deletion of 47 aa from either the N terminus or the C terminus of NF-κB completely inactivates the transactivation function of Nv-NF-κB. In addition, we identified proline-258 in the center of the protein as a key residue for the transactivation function of Nv-NF-κB. Taken together, these results demonstrate that non-conserved N- and C-terminal residues are both required for the transcriptional activating function of the sea anemone NF-κB protein, suggesting that it has a novel functional domain structure among known NF-κB proteins.

Generation of an 870kb deletion encompassing the Skt/Etl4 locus by combination of inter- and intra-chromosomal recombination.

BackgroundEtl4lacZ (Enhancer trap locus 4) and SktGt (Sickle tail) are lacZ reporter gene integrations into the same locus on mouse chromosome 2 targeting a gene that is expressed in the notochord of early embryos and in multiple epithelia during later development. Both insertions caused recessive mutations that resulted exclusively in mild defects in the caudal vertebral column. Since notochord-derived signals are essential for formation of the vertebral column the phenotypes suggested that the lacZ insertions interfered with some notochord-dependent aspect of vertebral development. As both insertions occurred in introns it was unclear whether they represent hypomorphic alleles or abolish gene function. Here, we have generated a definitive null allele of the Skt/Etl4 gene and analysed homozygous mutants.ResultsWe have introduced loxP sites into three positions of the gene based on additional upstream exons that we identified, and deleted approximately 870 kb of the locus by a combination of inter- and intra-chromosomal Cre-mediated recombinations in the female germ line of mice. This deletion removes about 90 % of the coding region and results in the loss of the SKT/ETL4 protein. Similar to the Etl4lacZ and SktGt alleles our deletion mutants are viable and fertile and show only mild defects in caudal vertebrae due to abnormal intervertebral disc development, although with higher penetrance. No other tissue with Skt/Etl4 expression that we analysed showed obvious defects.ConclusionThe complete loss of Skt/Etl4 function affects only development of caudal notochord derivatives and is compensated for in its other expression domains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0302-0) contains supplementary material, which is available to authorized users.

A lacZ Reporter-Based Strategy for Rapid Expression Analysis and Target Validation of Mycobacterium tuberculosis Latent Infection Genes.

We report a novel lacZ fusion vector and demonstrate its utility for expression analysis of genes associated with Mycobacterium tuberculosis latent infection. The vector contains E. coli (oriE) and mycobacterial (oriM) origins of replication, a kanamycin resistance gene (Km(r)) as selection marker, and a lacZ reporter gene in fusion with MCS for cloning of upstream regulatory sequence of the desired genes. β-galactosidase activity of the vector was standardized for expression analysis under latent mycobacterial conditions using Phsp60, a constitutive mycobacterial promoter, utilizing Mycobacterium smegmatis as model organism. Validation of the vector was done by cloning and expression analysis of PhspX (alpha crystalline) and Picl (isocitrate lyase), promoters from two of the genes shown to be involved in M. tuberculosis persistence. Both genes showed appreciable levels of β-galactosidase expression under hypoxia-induced persistent conditions in comparison to their actively replicating state. Expression analysis of a set of hypothetical genes was also done, of which Rv0628c showed increased expression under persistent conditions. The reported fusion vector and the strategy can be effectively used for short listing and validation of drug targets deduced from various non-conclusive approaches such as bioinformatics and microarray analysis against latent/persistent form of mycobacterial infection.

Abstract 14205: A Role of Inositol 1,4,5-trisphosphate Receptors to Inhibit the Progression of Pulmonary Arterial Hypertension

Backgroud: Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel in response to extracellular stimulation. There are three subtypes of IP3R (IP3R1, 2 and 3) and IP3R2 may be predominantly expressed in the pulmonary arterial smooth muscle cells (PASMCs). Objectives: To elucidate the role of IP3R2 in the progression of pulmonary arterial hypertension (PAH). Methods: The detailed expression pattern of IP3R2 in pulmonary vasculature during development and adulthood was investigated in lung sections of transgenic mice in which a LacZ reporter gene was knocked into the IP3R2 locus. In order to elucidate whether IP3R2 play a role in progression of PAH, we utilized chronic hypoxia (10% O2 for 6 weeks) -induced PAH mouse models. The severity of PAH was evaluated by transthoracic echocardiography, the weight of right ventricle, and the thickness of tunica media of pulmonary arteries, and compared between wild type (WT) and IP3R2 knock out (KO) mice. Proliferation and apoptosis in PASMCs were also assessed in both mice by immuno-staining with anti-phospho-histone H3 (PHH3) antibody and TUNEL assay, respectively. Further, in vitro, capacitative Ca2+ entry was assessed by Ca2+ imaging in primarily cultured PASMCs from WT and IP3R2 KO lungs by loading with fluorescent Ca2+ probe (Fluo-4AM). Results: Strong expression of the IP3R2 was detected in pulmonary arteries, predominantly in medial wall, from embryonic periods to the adulthood. IP3R2 KO mice showed higher RV pressure than those in WT mice by echocardiography. RV hypertrophy and medial wall thickness of PASMCs were more severely progressed in KO. There was no significant increase of PHH3 positive cells, while there was significant decrease of TUNEL positive cells in KO PASMCs compared with WT, suggesting that apoptosis was inhibited in KO PASMCs. Ca2+ imaging revealed that store-operated Ca2+ entry (SOCE) was enhanced in KO compared with WT. Taken together, loss of IP3R2 function leads to the acceleration of the progression of chronic hypoxia-induced PAH, with depressed apoptosis and enhanced SOCE in PASMCs. Conclusions: Although IP3R2 functions in increase of cytosolic Ca2+, it may play an inhibitory role in pathophysiology of PAH by influencing to the apoptosis and SOCE in PASMCs.

Vector promoters used in Klebsiella pneumoniae.

Much effort has been devoted to the metabolic engineering of Klebsiella pneumoniae; however, our knowledge of the actual expression level of promoters used in K. pneumoniae is limited. In this study, the expression levels of three promoters were compared systematically by using the lacZ reporter gene with different carbon sources in K. pneumoniae. The results showed that, although promoters PT5 and Ptac designed for Escherichia coli were functional, PT5 appeared more efficient and the induction/repression ratio of Ptac was decreased extremely in K. pneumoniae. The basal level of Ptac for lacZ expression reached 396.5U/mg, which was 9.5-fold higher compared with PT5 in LB medium, indicating Ptac can be used as an efficient "constitutive" promoter as well as an efficient induced promoter in K. pneumoniae. In different carbon sources medium, a newly constructed endogenous constitutive Pbud proved to be a stable and weak promoter. On the basis of our data, a set of Pbud and Ptac promoters could meet the broad range (about 1,000 orders of magnitude) of gene expression needed for engineered K. pneumoniae in glycerol-based medium.

Prediction and identification of an acid-inducible promoter from Lactococcus lactis ssp. cremoris MG1363

Using a promoter online prediction system, the gene sequence of GroEL protein from Lactococcus lactis ssp. cremoris MG1363 was analysed and a putative promoter, designated as gro, was further studied. Under acidic conditions, gro showed preliminary promoter function by driving the expression of lacZ reporter gene in Escherichia coli DH5α. In addition, gro drove the gene expression of GroEL and green fluorescent protein. In this paper, an acid-inducible promoter was successfully identified. This promoter has a wide prospect in the field of biotechnology.

CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic Geobacillus kaustophilus and Geobacillus thermodenitrificans.

The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to use citrate or C4-dicarboxylates like fumarate or succinate as the substrates for growth. The genomes of the sequenced Geobacillus strains (nine strains) each encoded a two-component system of the CitA family. The sensor kinase of G. thermodenitrificans (termed CitAGt) was able to replace CitA of Escherichia coli (CitAEc) in a heterologous complementation assay restoring expression of the CitAEc-dependent citC-lacZ reporter gene and anaerobic growth on citrate. Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed DcuSGk) was able to replace DcuSEc of E. coli. It responded in the heterologous expression system to C4-dicarboxylates and to citrate, suggesting that DcuSGk is, like DcuSEc, a C4-dicarboxylate sensor with a side-activity for citrate. DcuSGk, unlike the homologous DctS from Bacillus subtilis, required no binding protein for function in the complementation assay. Thus, the thermophilic G. thermodenitrificans and G. kaustophilus contain citrate and C4-dicarboxylate sensor kinases of the CitA and DcuS type, respectively, and retain function and substrate specificity under mesophilic growth conditions in E. coli.

Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

Cells from the hematopoietic lineage are only present transiently during healing in a mouse model of non-severe burn injury.

IntroductionThe aim of our study is to identify the contribution of hematopoietic-derived cells to burn-wound healing in a non-severe injury. There are many conflicting reports of the contribution of bone marrow-derived cells to wound healing and whether these are hematopoietic or mesenchymal in origin. The role of hematopoietic lineage cells is investigated in this study in the context of the response to burn injury.MethodsTransgenic mice expressing the LacZ reporter gene in all cells of the hematopoietic lineage underwent a non-severe full-thickness burn injury (8 % of total body surface area). Wounds were assessed for LacZ-positive cells at days 7, 14, and 28 post-injury by using whole-mount staining. Cells were also cultured from the wounds at each time point and analysed for expression of fibroblast and myofibroblast markers.ResultsAt day 7, positive cells were identified in the wounds representing the inflammatory response. Some dermal cells were also identified at this early stage. At day 14, positive cells were also identified and were cultured from the wound tissue samples. However, by day 28, no positive cells could be detected or cultured from the healed wound tissue. Isolated LacZ-positive cells did not express collagen 1 or α-smooth muscle actin proteins, indicating that they had not differentiated into dermal fibroblast-type cells.ConclusionsIn this model of burn injury, hematopoietic lineage cells were present in the healing wound only transiently and did not appear to contribute to the long-term scar population. This is in contrast with reports demonstrating that fibrocytes contribute a long-term sustained population in scar tissue. This work demonstrates that in a non-severe burn injury model there is a sustained transient contribution of hematopoietic cells to the healed wound. Further characterisation of the types and extent of wounding required to establish a long-term hematopoietic response will be important in determining future cell-based therapies.

Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

The Escherichia colifadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ–proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensisfabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensisfabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of ShewanellafabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0172-2) contains supplementary material, which is available to authorized users.

Abstract 362: Ccn1 Induction and Its Role in the Neointima Formation-induced by Carotid Artery Ligation in Mice

Aim: Atherosclerosis, characterized by neointima formation, occurs preferentially at areas of disturbed flow. Extracellular matrix protein CCN1 is upregulated at the site of atherosclerotic lesions in humans and in animal models. Our aim was to examine the expression of CCN1-regulated by disturbed flow and its role in neointima formation. Methods and Results: Ccn1+/lacZ mice, in which a lacZ reporter gene was inserted to be driven by Ccn1-promoter, were subjected to complete ligation of the left common carotid artery. Progressive reduction in luminal diameters of the ligated artery were observed using B mode ultrasound compared with the right common carotid artery. Ccn1 expression, assessed by whole-mount X-gal staining, was detected in the intima and the media of carotid arteries 3 days after ligation, and the staining intensified between 1-4 weeks in the neointima after ligation. Specifically, Ccn1 expression was observed in endothelial cells, smooth muscle cells, and macrophages in the neointima. To dissect the role of CCN1 in the development of arterial lesions, knock-in mice carrying the integrin α6β1-binding-deficient mutant allele Ccn1-dm were tested in the carotid artery ligation model. Compared with wild-type mice, Ccn1dm/dm mice displayed significantly less neointima after ligation with improved endothelial function, less endothelial apoptosis, and lower oxidative stress, suggesting that the arterial expression of CCN1 induces endothelial dysfunction through its receptor integrin α6β1. Conclusions: CCN1 is a critical pathophysiological regulator mediating neointima formation induced by disturbed flow-generated after carotid artery ligation. CCN1 and its receptor integrin α6β1 represent potential therapeutic targets for atherosclerosis.

317. HH67.2, a Promising Novel Adeno-Associated Virus (AAV) Vector, Shows Improved Muscle Specificity and Infectivity Compared To AAV9

Currently, AAV9 serotype is considered a robust heart and muscle gene delivery vehicle widely used in preclinical studies and in a clinical trial for spinal muscle atrophy (SMA). However, the systemic delivery of AAV9 with large vector doses leads to undesirable effects such as ectopic gene expression in other tissues. These shortcomings may be overcome by using certain strategies such as the development of novel AAV vectors with muscle-specific tropism. Previously, our lab identified a novel muscle specific AAV vector, HH67, by DNA shuffling and in vivo selection. Similar to other evolved tissue-specific vectors, the HH67 vector showed reduced liver tropism, but its transduction efficiency in heart and muscle tissues was still lower than that of AAV9. We set out to introduce site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of AAV capsids which has been shown to significantly increase gene transfer efficiency for certain AAV serotypes. In this study, we investigated whether two capsid mutations in HH67, Y445F and Y731F (named HH67.2), would enhance gene delivery to the muscle while maintaining low non-specific transgene expression. We found that the HH67.2 mutant had a significant increase in transgene delivery to the muscle compared to AAV9 after systemic delivery in C57BL/6 mice via tail-vein injection. In the muscle, expression of the LacZ reporter gene (2.5 × 10^5±65413 units/μg HH67.2 vs 7.2 × 10^4±8211 units/μg AAV9; p=0.0049) and the DNA genome copy number (4.50±2.07 VG/cell HH67.2 vs 1.15±0.197 VG/cell AAV9; p=0.0118) both increased 3.5-fold compared to AAV9. Furthermore, liver and other non-specific organs showed up to an 11-fold decrease in HH67.2 vector copy number (11.8±2.5 VG/cell HH67.2 liver vs 126.8±39.6 VG/cell AAV9 liver; p<0.0001). However, HH67.2 and AAV9 had no significant difference in gene expression in the heart. LacZ expression results were confirmed by vector genome copy number via quantitative PCR and X-Gal staining. Finally, we also introduced the same Y-F mutations in three similarly shuffled and in vivo selected AAV capsids such as M41, H43 and H48, but the mutations decreased infectivities of all these capsids. Our results suggest that HH67.2 is a novel AAV vector with enhanced muscle specificity and infectivity compared to AAV9 and could be a promising vehicle to deliver therapeutic genes to skeletal muscle or heart.**This work was part of Carrie Martin and Christina Mayer's research project for the Honors Program at the UNC Eshelman School of Pharmacy.

603. Novel DNA-Shuffled AAV Capsids Show Robust and Unique Gene Delivery and Expression Profiles in the CNS Via Intrathecal Injection

Aim: Adeno-associated virus (AAV) has great potential for treatment of central nervous system (CNS) diseases. Currently, a few AAV serotypes including AAV9 effectively deliver genes in the CNS. We aimed to screen more AAV serotypes for expression in CNS and compare them to AAV9.Material and methods: Using an existing library containing shuffled capsid genes of AAV serotypes 1 through 9, we characterized a few novel capsids for high expression in the CNS in vivo. Three novel serotypes, AAVH43, AAVH48, and AAVHH67 containing the reporter gene lacZ driven by a CMV promoter were injected in C57BL/6 mice and AAV9 was included as a positive control group (n=4 per group). Prior to vector injection, 25% mannitol solution 200μl was given by tail vein to deduce intracranial pressure. Twenty minutes later, 10μl of virus vectors (1×1011 vg) were slowly injected into the cerebrospinal fluid (CSF) at the intervertenral space between lumbar vertebrae L3 and L4 by a 33-gauge needle connected to a 50μl Hamilton syringe with PE10 tubing. Two weeks later, tissue samples were harvested for lacZ staining and immunostaining.Results: LacZ gene delivered by AAVH43, AAVH48, and AAVHH67 vectors expressed efficiently and similarly to AAV9 in the spinal cord by intrathecal injection. The entire spinal cord from the lumbar up to cervical region showed gene expression. The infected cells were mainly distributed in the posterior funiculus and gray matter. In addition, all 4 vectors infected dorsal root ganglia neurons very efficiently. Immunostaining showed AAVH43, AAVH48 and AAVHH67 vectors with a CMV promoter infected mainly neurons but not glial cells, similar to AAV9. In addition, AAVH43 and AAVH48 had some common characteristics and infected cerebellar cortex much more efficiently than AAV9. Those neurons showed distinctive morphology as Purkinje cells. Further confirmation with cell-type specific antibodies is needed.Conclusion: The AAVH43, AAVH48 and AAVHH67 vectors have similar ability to infect the spinal cord and dorsal root ganglia, equivalent to AAV9, when delivered by intrathecal injection. Moreover, the cerebellar cortex is more susceptible to AAVH43 and AAVH48 infection. With further investigation, the three novel AAV serotypes could be suitable alternative candidates for CNS gene delivery and therapy.

Eukaryote-like Ser/Thr protein kinase PrkA modulates sporulation via regulating the transcriptional factor σ(K) in Bacillus subtilis.

Protein kinase A (PrkA), also known as AMP-activated protein kinase, functions as a serine/threonine protein kinase (STPK), has been shown to be involved in a variety of important biologic processes, including pathogenesis of many important diseases in mammals. However, the biological functions of PrkA are less known in prokaryote cells. Here, we explored the function of PrkA as well as its underlying molecular mechanisms using the model bacterium Bacillus subtilis168. When PrkA is inhibited by 9-β-D-arabinofuranosyladenine (ara-A) in the wild type strain or deleted in the ΔprkA mutant strain, we observed sporulation defects in B. subtilis 168, suggesting that PrkA functions as a sporulation-related protein. Transcriptional analysis using the lacZ reporter gene demonstrated that deletion of prkA significantly reduced the expression of the transcriptional factor σK and its downstream genes. Complementation of sigK gene in prkA knockout mutant partially rescued the phenotype of ΔprkA, further supporting the hypothesis that the decreased σK expression should be one of the reasons for the sporulation defect resulting from prkA disruption. Finally, our data confirmed that Hpr (ScoC) negatively controlled the expression of transcriptional factor σK, and thus PrkA accelerated sporulation and the expression of σK by suppression of Hpr (ScoC). Taken together, our study discovered a novel function of the eukaryotic-like STPK PrkA in spore development as well as its underlying molecular mechanism in B. subtilis.

Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR.

OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an “injury response” control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.