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1.  CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli 
Nucleic Acids Research  2014;43(1):530-543.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5′ handle (8 nt) and a 3′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5′ handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.
doi:10.1093/nar/gku1285
PMCID: PMC4288178  PMID: 25488810
2.  Insulin-like growth factor-1 induces regulatory T cell-mediated suppression of allergic contact dermatitis in mice 
Disease Models & Mechanisms  2014;7(8):977-985.
Allergic contact dermatitis (ACD) is triggered by an aberrant hyperinflammatory immune response to innocuous chemical compounds and ranks as the world’s most prevalent occupational skin condition. Although a variety of immune effector cells are activated during ACD, regulatory T (Treg) cells are crucial in controlling the resulting inflammation. Insulin-like growth factor-1 (IGF-1) regulates cell proliferation and differentiation and accelerates wound healing and regeneration in several organs including the skin. Recently IGF-1 has also been implicated in protection from autoimmune inflammation by expansion of Treg cells. Here, we demonstrate that ectopic expression of IGF-1 in mouse skin suppresses ACD in a Treg cell-specific manner, increasing the number of Foxp3+ Treg cells in the affected area and stimulating lymphocyte production of the anti-inflammatory cytokine interleukin 10. Similar therapeutic effects can be achieved with systemic or topical delivery of IGF-1, implicating this growth factor as a promising new therapeutic option for the treatment of ACD.
doi:10.1242/dmm.015362
PMCID: PMC4107326  PMID: 25056699
Insulin-like growth factor-1; Atopic dermatitis; Contact hypersensitivity; Regulatory T cells; Treg
3.  High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli 
RNA Biology  2013;10(5):716-725.
In Escherichia coli, the acquisition of new CRISPR spacers is strongly stimulated by a priming interaction between a spacer in CRISPR RNA and a protospacer in foreign DNA. Priming also leads to a pronounced bias in DNA strand from which new spacers are selected. Here, ca. 200,000 spacers acquired during E. coli type I-E CRISPR/Cas-driven plasmid elimination were analyzed. Analysis of positions of plasmid protospacers from which newly acquired spacers have been derived is inconsistent with spacer acquisition machinery sliding along the target DNA as the primary mechanism responsible for strand bias during primed spacer acquisition. Most protospacers that served as donors of newly acquired spacers during primed spacer acquisition had an AAG protospacer adjacent motif, PAM. Yet, the introduction of multiple AAG sequences in the target DNA had no effect on the choice of protospacers used for adaptation, which again is inconsistent with the sliding mechanism. Despite a strong preference for an AAG PAM during CRISPR adaptation, the AAG (and CTT) triplets do not appear to be avoided in known E. coli phages. Likewise, PAM sequences are not avoided in Streptococcus thermophilus phages, indicating that CRISPR/Cas systems may not have been a strong factor in shaping host-virus interactions.
doi:10.4161/rna.24325
PMCID: PMC3737330  PMID: 23619643
CRISPR adaptation; CRISPR/Cas systems; Escherichia coli; bacteriophage; high-throughput sequencing
4.  Pervasive generation of oppositely oriented spacers during CRISPR adaptation 
Nucleic Acids Research  2014;42(9):5907-5916.
During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that in Escherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.
doi:10.1093/nar/gku226
PMCID: PMC4027179  PMID: 24728991
5.  103Pd versus 125I ophthalmic plaque brachytherapy: preoperative comparative radiation dosimetry for 319 uveal melanomas 
Journal of Radiation Oncology  2014;3(4):409-416.
Objective
This study was conducted to compare the relative, clinical intraocular dose distribution for palladium-103 (103Pd) versus iodine-125 (125I) ophthalmic plaque radiation therapy.
Methods
Preoperative comparative radiation dosimetry was performed to evaluate 319 consecutive uveal melanomas treated between 2006 and 2012.
Results
There were 68 (21.3 %) anterior (iris and/or ciliary body) and 251 (78.7 %) choroidal melanomas examined in this study. According to AJCC staging, 7th edition, 146 (45.8 %) were T1, 126 (39.5 %) T2, 40 (12.5 %) T3, and 7 (2.2 %) T4. All were prescribed an equivalent tumor-apex dose. When compared to 125I, 103Pd was associated with a mean 41.9 % lower radiation dose to the opposite eye wall (p < 0.001), 12.7 % to the lens center (p < 0.001), 7.5 % to the optic disc (p = 0.008), and a 3.8 % decrease to the fovea (p = 0.034). However, subgroup analysis of smaller (T1-staged) tumors showed greater dose reductions to normal ocular structures compared to larger (T4-staged) tumors. Tumor and therefore plaque location also affected intraocular dose distribution. For example, palladium-103-related dose reductions to the fovea, optic nerve, and opposite eye wall were significantly greater for iris and ciliary body tumors compared to posterior choroidal melanomas (p < 0.001). After comparative dosimetry, 98.7 % (n = 315/319) were treated with 103Pd.
Conclusion
Preoperative comparative radiation dosimetry was performed for a large cohort of patients with uveal melanoma. It influenced radionuclide selection, offered an opportunity for radiation sparing of critical vision-related intraocular structures, and typically increased radiation within the tumors.
doi:10.1007/s13566-014-0149-4
PMCID: PMC4241234  PMID: 25431638
Brachytherapy; Plaque; Palladium-103; Iodine-125; Choroidal melanoma
6.  Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition 
PLoS Genetics  2013;9(9):e1003742.
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.
Author Summary
CRISPR loci and their associated genes form a diverse set of adaptive immune systems that are widespread among prokaryotes. In these systems, the CRISPR-associated genes (cas) encode for proteins that capture fragments of invading DNA and integrate these sequences between repeat sequences of the host's CRISPR locus. This information is used upon re-infection to degrade invader genomes. Storing invader sequences in host genomes necessitates a mechanism to differentiate between invader sequences on invader genomes and invader sequences on the host genome. CRISPR-Cas of Staphylococcus epidermidis (Type III-A system) is inhibited when invader sequences are flanked by repeat sequences, and this prevents targeting of the CRISPR locus on the host genome. Here we demonstrate that Escherichia coli CRISPR-Cas (Type I-E system) is not inhibited by repeat sequences. Instead, this system is specifically activated by the presence of bona fide Protospacer Adjacent Motifs (PAMs) in the target. PAMs are conserved sequences adjoining invader sequences on the invader genome, and these sequences are never adjacent to invader sequences within host CRISPR loci. PAM recognition is not affected by base pairing potential of the target with the crRNA. As such, the Type I-E system lacks the ability to specifically recognize self DNA.
doi:10.1371/journal.pgen.1003742
PMCID: PMC3764190  PMID: 24039596
7.  CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 
Molecular Cell  2012;46(5):595-605.
Summary
The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR-interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a Protospacer Adjacent Motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R-loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.
doi:10.1016/j.molcel.2012.03.018
PMCID: PMC3372689  PMID: 22521689
8.  Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile 
PLoS Genetics  2013;9(5):e1003493.
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen.
Author Summary
The emergent human pathogen Clostridium difficile is a major cause of nosocomial diarrhea associated with antibiotic therapy. During the last few years, severe forms of C. difficile infections became more frequent due to the emergence of hypervirulent isolates. Despite intensive studies, many questions regarding the mechanisms controlling C. difficile virulence remain unanswered. We hypothesized that C. difficile, a member of an ancient group of bacteria, might widely use ancestral RNA–based mechanisms to control its gene expression for better adaptation to host conditions. Indeed, using next-generation sequencing technology, we identified a great number and a large diversity of potential RNA regulators in this pathogen. We obtained experimental evidence for regulatory roles of a particular class of regulatory RNAs responding to c-di-GMP, a universal bacterial signaling molecule regulating motility, biofilm formation, and virulence. We also detected abundant small RNA products of recently discovered adaptive prokaryotic immunity CRISPR-Cas systems that might be important for C. difficile survival in gut communities. Our findings suggest that small RNA molecules may play a major role in regulatory processes during C. difficile infection cycle and as such are promising targets of new therapeutic strategies.
doi:10.1371/journal.pgen.1003493
PMCID: PMC3649979  PMID: 23675309
9.  Human Involucrin Promoter Mediates Repression-Resistant and Compartment-Specific LEKTI Expression 
Human Gene Therapy  2011;23(1):83-90.
Abstract
Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector–mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral–derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter–transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type–related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice. We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization.
Di and colleagues describe a codon-optimized lentiviral vector platform capable of driving sustained expression of SPINK5, a gene encoding the lymphoepithelial Kazal type-related inhibitor (LEKTI) protein in human skin. This approach leads to durable and highly compartment-specific reconstitution of LEKTI in human skin grafted onto immunodeficient mice, and may prove effective for genetic skin disorders such as Netherton syndrome.
doi:10.1089/hum.2011.091
PMCID: PMC3260443  PMID: 21895535
10.  The antibacterial threaded-lasso peptide capistruin inhibits bacterial RNA polymerase 
Journal of molecular biology  2011;412(5):842-848.
SUMMARY
Capistruin, a ribosomally synthesized post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is unknown. Capistruin is a threaded-lasso peptide (lariat peptide), comprising an N-terminal 9-amino-acid ring followed by a 10-amino-acid C-terminal tail that is threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capustruin, like MccJ25, inhibits wild-type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25 due to a mutation in an RNAP subunit gene exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.
doi:10.1016/j.jmb.2011.02.060
PMCID: PMC3143284  PMID: 21396375
lariat peptides; capistruin; microcin J25 (MccJ25); RNA polymerase; RNA polymerase inhibitor
11.  Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli 
Molecular microbiology  2010;77(6):1367-1379.
CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently “silent” E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.
doi:10.1111/j.1365-2958.2010.07265.x
PMCID: PMC2939963  PMID: 20624226
12.  The Physiological Period Length of the Human Circadian Clock In Vivo Is Directly Proportional to Period in Human Fibroblasts 
PLoS ONE  2010;5(10):e13376.
Background
Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior.
Methodology/Principal Findings
In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer.
Conclusions/Significance
We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.
doi:10.1371/journal.pone.0013376
PMCID: PMC2958564  PMID: 21042402
13.  Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases▿  
Journal of Bacteriology  2009;191(20):6273-6280.
Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the α-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNAAsp by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.
doi:10.1128/JB.00829-09
PMCID: PMC2753047  PMID: 19684138
14.  Maturation of the Translation Inhibitor Microcin C▿  
Journal of Bacteriology  2009;191(7):2380-2387.
Microcin C (McC), an inhibitor of the growth of enteric bacteria, consists of a heptapeptide with a modified AMP residue attached to the backbone of the C-terminal aspartate through an N-acyl phosphamidate bond. Here we identify maturation intermediates produced by cells lacking individual mcc McC biosynthesis genes. We show that the products of the mccD and mccE genes are required for attachment of a 3-aminopropyl group to the phosphate of McC and that this group increases the potency of inhibition of the McC target, aspartyl-tRNA synthetase.
doi:10.1128/JB.00999-08
PMCID: PMC2655510  PMID: 19168611
15.  Analysis of Promoter Targets for Escherichia coli Transcription Elongation Factor GreA In Vivo and In Vitro▿ †  
Journal of Bacteriology  2007;189(24):8772-8785.
Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.
doi:10.1128/JB.00911-07
PMCID: PMC2168603  PMID: 17766423
16.  Transcription regulation of the EcoRV restriction–modification system 
Nucleic Acids Research  2005;33(21):6942-6951.
When a plasmid containing restriction–modification (R–M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R–M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R–M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription.
doi:10.1093/nar/gki998
PMCID: PMC1310966  PMID: 16332697
17.  Structure-Activity Analysis of Microcin J25: Distinct Parts of the Threaded Lasso Molecule Are Responsible for Interaction with Bacterial RNA Polymerase 
Journal of Bacteriology  2005;187(11):3859-3863.
Peptide microcin J25 (MccJ25) inhibits bacterial RNA polymerase. We show that thermolysin-cleaved MccJ25 and MccJ25 lacking amino acids 13 to 17 also inhibit transcription. Our data and structural analysis of intact and thermolysin-digested MccJ25 suggest that distinct regions of MccJ25 are involved in transcription inhibition and cell entry.
doi:10.1128/JB.187.11.3859-3863.2005
PMCID: PMC1112051  PMID: 15901712
18.  Expression and Functional Analysis of Uch-L3 during Mouse Development 
Molecular and Cellular Biology  2000;20(7):2498-2504.
Mice homozygous for the s1Acrg deletion at the Ednrb locus arrest at embryonic day 8.5. To determine the molecular basis of this defect, we initiated positional cloning of the s1Acrg minimal region. The mouse Uch-L3 (ubiquitin C-terminal hydrolase L3) gene was mapped within the s1Acrg minimal region. Because Uch-L3 transcripts were present in embryonic structures relevant to the s1Acrg phenotype, we created a targeted mutation in Uch-L3 to address its role during development and its possible contribution to the s1Acrg phenotype. Mice homozygous for the mutation Uch-L3Δ3-7 were viable, with no obvious developmental or histological abnormalities. Although high levels of Uch-L3 RNA were detected in testes and thymus, Uch-L3Δ3-7 homozygotes were fertile, and no defect in intrathymic T-cell differentiation was detected. We conclude that the s1Acrg phenotype is either complex and multigenic or due to the loss of another gene within the region. We propose that Uch-L3 may be functionally redundant with its homologue Uch-L1.
PMCID: PMC85452  PMID: 10713173

Results 1-18 (18)