text:
Video: Live From Main Street Minneapolis
| Thu Jun. 12, 2008 2:43 PM EDT
FYI, if you ever find yourself wondering how independent progressive media in Minneapolis feel about playing host to the GOP convention this fall, wonder no more. They were pretty vocal about it earlier this week for Laura Flanders:
MoJo Troll Patrol encourages readers to sign in with Facebook, Twitter, Google, Yahoo, Disqus, or OpenID to comment. Please read our comment policy before posting.


text: Today's Lead: Soda Ban Goes Before State's Highest Court by David Howard King, Oct 18, 2013 Twitter The state's highest court will take up Mayor Michael Bloomberg's attempt to limit the size of sugary drinks. The policy that would have banned restaurants, theaters and food carts from selling sugary beverages in sizes larger than 16 ounces has been rejected by two other courts. The Appeals Court will take up the case next year, which means Bloomberg's successor will decide whether to continue to move the case forward. Joe Lhota, the Republican nominee for mayor, said he would end the appeal; Democratic nominee Bill de Blasio's camp issued a statement saying the candidate would "review the status" of the case if he is elected. The beverage industry has indicated that it is confident that the decision against Bloomberg's policy will be upheld. Other Stories We're Following Groups Propose Plan To Transform Pennsylvania Station (Crain's) New Details About Midtown East Rezoning Plan (Wall Street Journal) Moreland Commission Chair Took Donations From Panel's Contractor (Capital) In Scathing Ruling, Judge Denies Pro-Lhota PAC Bid (Daily News) De Blasio's Daughter Responds to Lhota Ad (Politicker) New Chief Envisions Performances on High Line (Wall Street Journal) Bike Share Users Unhappy About Full And Empty Docks: Survey (WNYC) Organization Tries To Make School Lunch A Mayoral Issue (Capital) Lawmakers Call For Education Chief's Resignation (Capital) Shadowy System Of Indigent Housing Unsafe And Unregulated, Report Says () Tweet Editor's Choice Rumored Scheme Brings Scrutiny to Bronx Political Machine How To Become A New York City Council Member Candidates Allege Wright Unfairly Influences Elections Alain Kaloyeros, Powerful Centerpiece of Buffalo Billion, Could Become Household Name Comments Comment(s) The comments section is provided as a free service to our readers. Gotham Gazette's editors reserve the right to delete any comments. Some reasons why comments might get deleted: inappropriate or offensive content, off-topic remarks or spam.

subset: commoncrawl


text:
Book your room at BeachWay Inn
Standard Room, 1 Queen Bed, Non SmokingMax Occupancy: 2 guests (up to 1 child, 1 infant)
Standard Room, 1 King Bed, Non SmokingMax Occupancy: 2 guests (up to 1 child, 1 infant)
Standard Room, 2 Double Beds, Non SmokingMax Occupancy: 4 guests (up to 3 children, 1 infant)
Standard Room, 2 Queen Beds, Non SmokingMax Occupancy: 4 guests (up to 3 children, 3 infants)
Get an overview of this hotelMotel in Arroyo Grande with free breakfast
A stay at BeachWay Inn places you in the heart of Arroyo Grande, convenient to Clark Center for the Performing Arts and Swinging Bridge.
This motel is within close proximity of Heritage House Museum and Gardens and The Great American Melodrama & Vaudeville.
Make yourself at home in one of the 15 guestrooms featuring refrigerators and flat-screen televisions. Complimentary wireless Internet access keeps you connected, and cable programming is available for your entertainment. Bathrooms have shower/tub combinations and complimentary toiletries. Conveniences include separate sitting areas and microwaves.
Make use of convenient amenities, which include complimentary wireless Internet access and tour/ticket assistance.
A complimentary continental breakfast is served daily.
The front desk is staffed during limited hours. Limited parking is available onsite.Information missing or incorrect? Tell us!
Hotel Amenities
Hotel Amenities
A complimentary breakfast is offered each morning. High-speed wireless Internet access is complimentary.
BeachWay Inn is a smoke-free property.
- Total number of rooms - 15
- Number of floors - 2
- Free breakfast
- Front desk (limited hours)
- Designated smoking areas (fines apply)
- Designated smoking areas
- Tours/ticket assistance
- Free WiFi
- Parking (limited spaces)
- Smoke-free property
- Number of buildings/towers - 1
Family Friendly Amenities
- Free Wi-Fi
- Refrigerator
- Microwave
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Parking (limited spaces)
Room Amenities
- Separate sitting area
- Refrigerator
- Microwave
- Private bathroom
- Free toiletries
- Shower/tub combination
- Cable TV service
- Flat-panel TV
- Free WiFi
- Room service
Where to Eat
Guests are offered a complimentary continental breakfast each morning.
Room service is available.
Hotel Policies
Check-in time starts at 3 PM
Minimum check-in age is 21
Check-in time ends at 10 PM
Check-out time is 11 AM
Payment types
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
Only service dogs are allowed
No rollaway/extra beds available
No cribs (infant beds) available
No elevators
Hotel Name
We should mention
Only registered guests are allowed in the guestrooms.


text:
The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology.
Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational "silencing" of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple "in-frame" stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli.
Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational "silencing" mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.
Bacterial pseudogenes are inactivated, presumably nonfunctional genes that can accumulate in the genomes of bacterial species, especially those undergoing processes such as niche selection or host specialization [1,2]. When a bacterial gene is under low selection pressure, it undergoes a period of frequent nucleotide substitutions because deleterious mutations are not efficiently purged. These mutations can cause the accumulation of in-frame stop codons, reading frame-shifts, or removal of traditional translational start codons or vital sections of the gene, giving rise to a pseudogene [3]. Mutations that destroy promoter or regulatory sequences can result in the "silencing" of transcription or translation or premature termination of protein synthesis [4].
The case of pseudogenes in Mycobacterium leprae, an obligate intracellular bacterium and etiologic agent of leprosy, is very dramatic. Its ~3.3 Mb genome consists of 1,614 open reading frames (ORFs) and 1,133 pseudogenes [5]. The pseudogenes represent 41% of the total genes http://genolist.pasteur.fr/Leproma/help/current.html webcite; the largest percentage found in any bacterial genome sequenced to date http://www.pseudogene.org/cgi-bin/db-gen.cgi?type=Prokaryote webcite.
The overall G+C content of M. leprae's genome is 57.8% [5]. This is 8% lower than that of its close relative M. tuberculosis [6], a feature usually described in species undergoing low selection pressure [7,8]. Interestingly, the G+C content of pseudogenes (56.5%) is lower than that of its ORFs (60.1%).
Pseudogenes are distributed throughout the M. leprae chromosome and are assigned to the majority of functional groups [5,9,10]. If these genes are indeed nonfunctional in M. leprae, they should no longer be required for survival in the specialized intracellular niche in which M. leprae resides. Therefore, the study of M. leprae pseudogenes is important to expand our understanding of the evolution of this obligate intracellular parasite and to establish the role that pseudogenes play in M. leprae's unique metabolism and parasitism.
Recently, pseudogene transcripts in M. leprae have been identified [11,12]. However, the extent of pseudogene transcription and the potential impact that these transcribed genes have on M. leprae have not been analyzed critically. The cost of expressing non-functional ORFs could be especially dramatic for M. leprae because the speed of pseudogene deletion appears to be slower than in other bacteria [13].
To address this, a study of the overall pseudogene transcriptional profile of M. leprae using a global M. leprae DNA microarray and reverse transcriptase-PCR analyses was conducted. The results demonstrated that a large number of M. leprae pseudogenes were transcribed during growth in the nude mouse foot pad, a model for lepromatous leprosy in man. Analyses of these transcribed pseudogenes using bioinformatics tools and in vitro methods demonstrated that potential mechanisms for transcription of these pseudogenes were associated with their residing within gene clusters or downstream of functional ORFs. In addition some of these genes contained functional promoters within their 5' upstream sequence. Since translation of this large number of pseudogenes could have a major impact on M. leprae's resources and energy consumption without apparent benefit to its survival and growth, mechanisms for translational "silencing" of these transcripts were also investigated using bioinformatics tools. Results demonstrated that the vast majority of these pseudogenes are "silenced". The "silencing" of these transcripts was found to be associated with: 1) the lack of a strong Shine-Dalgarno (SD), ribosomal binding site, in the 5'-UTR of the majority of these genes; 2) the lack of traditional translational start codons; 3) the presence of multiple in-frame stop codons; and 4) high Ka/Ks ratios indicating low functionality of putative protein products. These data indicated that the majority of pseudogenes were nonfunctional, inactivated genes. However, when one pseudogene containing a functional promoter, SD site, a traditional start codon, a very low Ka/Ks ratio, and encoding 3/4 of its M. tuberculosis ortholog was tested for protein production in E. coli, the predicted product was observed.
Transcriptome of M. leprae
Transcriptional analysis of M. leprae ORFs and pseudogenes using the M. leprae DNA microarray demonstrated that that 1,353 transcripts had a mean signal to noise ratio (SNR) cutoff value ≥ 2 (raw data are accessible through GEO Series accession # GSE17191 study at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17191 webcite). Several genes, positive on only 1 of 4 arrays were further analyzed and found positive using RT-PCR analysis (Additional File 1). Therefore the current transcriptome of nude mouse footpad-derived M. leprae consists of a total of 1,353 transcripts (Additional File 1). RT-PCR analysis was also used to validate the transcription of 20.5% of gene transcripts positive on the array. The transcriptome represents 49% of the total 2,747 genes surveyed and 867/1,353 (64%) transcripts were from ORFs or protein coding genes from a variety of functional gene categories (Additional File 1). Approximately 11% of these ORFs have previously been shown to produce proteins in armadillo-derived M. leprae, however no protein product was observed for any pseudogene in these studies (Additional File 1) [14,15]. The present study demonstrated that 486/1,353 (36%) of the gene transcripts detected were from pseudogenes, demonstrating that 43% of all pseudogenes found in the M. leprae genome were transcriptionally active (Additional file 1). This represents the largest number of transcriptionally-active pseudogenes reported to date.
Additional file 1. Transcriptional profile of M. leprae Thai-53 from the granulomatous foot pad tissue of nu/nu mouse using DNA microarray and RT-PCR analysis. The M. leprae gene transcripts detected using DNA microarray analysis experiments were performed using previously described protocols [37]. Microarrays were scanned using Bio-Rad VersArray Chip Reader and SpotFinder software to quantify fluorescence. Genes were positive for transcription if signal-to-noise ratios (SNR) were > 2-fold (raw data was deposited the public repository NCBI's Gene Expression Omnibus (GEO and are accessible through GEO Series accession number GSE17191 study at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17191 webcite. In addition, some gene transcripts were detected using RT-PCR and some of which have been previously published [11]. 1Mycobacterium leprae gene #: http://genolist.pasteur.fr/Leproma/ webcite. 2Mycobacterium tuberculosis gene #: http://genolist.pasteur.fr/Tuberculist/ webcite. 3Gene List: http://www.sanger.ac.uk/Projects/M_leprae/ webcite. 4M. leprae pseudogenes which have tested positive using for M. leprae gene transcripts using reverse transcriptase-PCR and cDNA from nude mouse footpad-derived M. leprae Thai-53. 5Data used with permission from Patrick Brennan [14,15].
Format: XLS Size: 294KB Download file
This file can be viewed with: Microsoft Excel Viewer
These transcribed pseudogenes were randomly distributed throughout the chromosome (Fig. 1; Additional File 1) and found in 26 of the 29 functional gene categories containing pseudogenes (Fig. 2). The largest number of transcribed pseudogenes 184/486 (38%) was found in the Functional Category V (Conserved Hypotheticals), which contains 776 genes and includes the largest number of pseudogenes in the M. leprae genome.
Figure 1. Distribution of transcribed pseudogenes within specific regions of the M. leprae chromosome. This graph represents the distribution of transcribed pseudogenes as a percent of the total number of pseudogenes within each specific region of the M. leprae chromosome (red) and the distribution of pseudogenes within each specific region of the M. leprae chromosome as a percent of the total number of pseudogenes within the genome (gray). http://www.sanger.ac.uk/Projects/M_leprae/ webcite.
Figure 2. Distribution of transcribed pseudogenes in functional gene categories of M. leprae. This graph represents the distribution of transcribed pseudogenes as a percent of the total number of pseudogenes within each functional gene category of M. leprae (red) and the distribution of pseudogenes within each functional gene category as a percent of the total number of pseudogenes within the genome (gray). http://www.sanger.ac.uk/Projects/M_leprae/ webcite.
Read-though transcription of pseudogenes
A plausible explanation for pseudogene transcription in M. leprae is read-through transcription as a result of their location within operons or downstream of transcribed ORFs. A total of 112 operons have been identified in the M. leprae TN strain (GeneChords-http://genomics10.bu.edu/cgi-bin/GeneChords/GeneChords.cgi webcite). However, their location within gene clusters has not been fully mapped. Results showed that 10% of transcribed pseudogenes were found within 20 gene clusters (Table 1) and an additional 10% were located downstream of transcribed ORFs (data not shown). These data suggested that 20% of all the pseudogenes could be potentially transcribed by read-through transcription.
Table 1. Transcribed M. leprae pseudogenes within gene clusters.
When M. leprae pseudogenes were experimentally analyzed for their presence within a polycistronic mRNA containing an upstream transcribed ORF, three of four gave the predicted RT-PCR fragment (Fig. 3) and sequencing of the PCR amplicons confirmed their predicted mRNA sequence (data not shown). Fig 3, Panel A depicts the chromosomal location of these pseudogenes, ORFs and primers used for PCR amplification; Panel B depicts results of agarose gel analysis of PCR products generated from M. leprae cDNA of these pseudogenes and their respective ORF cDNA. These data demonstrated the presence of a single mRNA transcript containing the predicted RT-PCR products from ML0831–ML0832-(pseudogene)-ML0833, ML1484c-ML1483c (pseudogene) and ML0180c-ML0179c (pseudogene). However, positioning of a pseudogene directly downstream from a transcribed ORF did not guarantee its transcription via a read-through mechanism since no read-through transcript of the predicted length was detected in the cDNA of ML0091c-ML0090c (Fig. 3B) even though individual gene transcripts were detected using microarray analysis (Additional File 1), indicating these genes were transcribed as independent genes.
Figure 3. Read-through transcription of M. leprae pseudogenes. This figure represents the results of RT-PCR analysis of transcriptional read-through between M. leprae pseudogenes and their upstream ORFs. Panel A shows mapped genomic regions where pseudogenes (ML0832, ML1483c, ML0179c, and ML0091c) and upstream ORFs are located http://genolist.pasteur.fr/Leproma/ webcite. Black arrows indicate direction of transcription. Small red arrows indicate forward and reverse primers for RT-PCR. Red line below primers and (bp) designate size of predicted PCR product if genes are expressed as a polycistronic mRNA. Panel B shows ethidium bromide-stained agarose gel analysis of these PCR amplicons: Lane 1, 100 bp DNA ladder (New England Biolabs); Lane 2, PCR amplicons from nu/nu footpad-derived M. leprae Thai-53 cDNA for each gene set; Lane 3, RT (-) control; Lane 4, 1 ng M. leprae DNA (positive control).
Identification of intrinsic stem loop structures
Intrinsic terminators between genes can stop transcript elongation and thus prevent read-through transcription. Hence the 3'UTR and coding regions of upstream transcribed ORFs of transcribed pseudogenes were analyzed for intrinsic stem loop terminator structures. The genomic ΔGcutoff for stem loop structures in the M. leprae TN genome was previously calculated to be -14.35 [16]. Therefore, only those ORFs which have stem loop structures downstream of the stop codon with ΔG values of < -14 were considered to contain potential intrinsic terminators. Using this criterion, only 27% of ORFs in the M. leprae genome contained intrinsic terminators in their 3'UTRs, demonstrating that the majority of M. leprae ORFs lack intrinsic terminators (Additional File 2). In addition, only 1.5% of transcribed ORFs upstream of transcriptionally active pseudogenes were found to contain stem loop structures with the potential to act as intrinsic terminators (Table 2). Interestingly, a strong putative intrinsic terminator (ΔG value = -21.84) was found within the 3'UTR of ML0091c, suggesting a potential mechanism for the lack of read-through transcription of the ML0090c pseudogene analyzed above. In contrast a strong intrinsic terminator was found within the coding sequence of ML0180c however, its presence did not stop read-through transcription of the downstream pseudogene ML0179c.
Additional file 2. Intrinsic terminators in M. leprae. This table gives predicted intrinsic terminators in the upstream region of M. leprae genes as a function of the genomic G+C content and palindrome-like structures with ΔG < -14 are considered as potential intrinsic terminators [16]. 1Mycobacterium leprae gene #: http://genolist.pasteur.fr/Leproma/ webcite. 2Algorithm Genome Scanner for Terminators (GeSTer) [16,38].
Format: XLS Size: 71KB Download file
This file can be viewed with: Microsoft Excel Viewer
Table 2. Prediction of intrinsic stem loop terminators in the 3'-UTR of transcribed ORFs located upstream of transcribed pseudogenes.
Pseudogene promoters
The presence of promoter-like sequences in the 5'UTR of transcribed M. leprae pseudogenes with translational start codons was investigated using "bend-it" DNA curvature analysis, alignment of promoter-like regions with that of mycobacterial homologs, and in vitro confirmation of promoter activity by cloning putative promoters into an E. coli promoter-less gfp expression-reporter vector. The presence of predicted promoter-like regions with strong upstream DNA static curvature between 9–16.8 deg/turn/peak were observed for 15/92 (16%) of these transcribed pseudogenes (Additional File 3). These promoters also aligned very well with that of other mycobacterial homologs (Table 3). Fig. 4 shows representative promoter-like structures for two of these pseudogenes in relationship to their initiation site, SD sequence, and translational start codon and aligned to that of homologous genes of other mycobacterial species. The function of 10 of these putative promoters was confirmed in E. coli promoter-gfp fusions by fluorescent microscopy (Table 3). Fig. 5 depicts a GFP positive clone for the promoter region of pyrR ML0531.
Additional file 3. M. leprae pseudogene promoter-like regions and truncation of transcribed pseudogenes compared to M. tuberculosis homologs. Using the M. leprae sequence data from 92 transcribed pseudogenes http://genolist.pasteur.fr/Leproma/ webcite, the presence of promoter-like regions and truncation of transcribed pseudogenes were analyzed and compared to M. tuberculosis homologs using "bend-it" DNA curvature analysis http://hydra.icgeb.trieste.it/dna/bend_it.html webcite. ML#: http://genolist.pasteur.fr/Leproma/ webcite. Rv#: http://genolist.pasteur.fr/TubercuList/ webcite. Start: Start codon as assigned to pseudogene http://genolist.pasteur.fr/Leproma/ webcite. ISp bp: Upstream intergenic space in bp as assigned http://genolist.pasteur.fr/Leproma/ webcite. HP deg/tn: Highest DNA curvature peak above threshold in degrees/helical turn found in the intergenic space as assigned by "bend-it" using DNase I values for bend parameters [41]. Pks: Number of static DNA curvature peaks with threshold > 5 degrees in the intergenic space as assigned by "bend-it" using DNase I values for bend parameters. # Stop: Number of stop codons present in the pseudogene frame. % Rv: The percentage of coding frame available before the first stop codon compared to the Mycobacterium tuberculosis ortholog as assigned in http://genolist.pasteur.fr/Leproma/ webcite.
Format: XLS Size: 89KB Download file
This file can be viewed with: Microsoft Excel Viewer
Figure 4. Promoter-like sequences upstream of transcribed M. leprae pseudogenes. This figure shows representative alignments of promoter-like sequences for M. leprae pseudogenes and their mycobacterial homologs. Panel A & B represent the pyrR (ML0531) and rpmB (ML1674) upstream promoter-like regions containing -35 and -10 regions and initiation site (I) in relationship to their ribosomal binding sites (RBS) and translational start codons (Start), respectively. * indicates identical nucleic acids in all strains. Mycobacterial species abbreviations http://www.ncbi.nlm.nih.gov/sites/entrez webcite: MAP-Mycobacterium avium, subsp paratuberculosis K-10. MAV-Mycobacterium avium 104. BCG-Mycobacterium bovis BCG Pasteur 1173P2. RV-Mycobacterium tuberculosis H37Rv. MSEG-Mycobacterium smegmatis MC2 155. ML-Mycobacterium leprae TN. MMCS-Mycobacterium MCS. MKMS-Mycobacterium KMS. MVAN-Mycobacterium vanbaalenii PYR-1
Figure 5. In vitro promoter activity in pyrR (ML0351) pseudogene. Panel A shows an image of GFP-fluorescent E. coli::pGlow-TOPO-TA/MLpyrR prom as a result of transformation of E. coli XL-1 Blue cells with the pGlow-TOPO-TA (promoterless and lacking a SD site) containing the upstream region of the M. leprae pyrR pseudogene; including the promoter, SD and start codon (Additional File 6, Fig. 3A) fused into the ATG of gfp. A clone containing the ampicillin-resistant phenotype and positive for the M. leprae pyrR promoter insert by PCR/DNA sequencing was analyzed by fluorescent microscopy using a Nikon Eclipse E400 fluorescent microscope using a GFP filter (excitation/emission maxima of 480 nm/560 nm). Panel B shows an image of E. coli::pGlow-TOPO-TA re-circularized vector (negative control for background fluorescence).
Table 3. Analysis of M. leprae pseudogene promoters in silico and in vitro promoter in E. coli analyses.
Translational start codons in transcribed pseudogenes
Potential mechanisms for translational "silencing" of pseudogenes transcribed in M. leprae were analyzed in silico using bioinformatics tools. Results demonstrated that 363/486 (75%) of transcribed pseudogenes lacked traditional translational start codons (AUG, GUG, UUG), greatly reducing the translation potential of transcribed pseudogenes into protein products (Additional File 4).
Additional file 4. Bioinformatics of 486 transcribed pseudogenes of M. leprae. Using M. leprae sequence data http://genolist.pasteur.fr/Leproma/ webcite, translational start codons (ATG, GTG, TTG) and translational stop codons (UGA, UAA, UAG) are listed as obtained using 5' 3' Frame 1 data from ExPasy Translate Tool http://us.expasy.org/tools/dna.html webcite for each transcribed pseudogene: 1 Mycobacterium leprae gene #: http://genolist.pasteur.fr/Leproma/ webcite. 2 Mycobacterium tuberculosis gene #: http://genolist.pasteur.fr/Tuberculist/ webcite. 3% Rv = % of the predicted full-length protein (in silico translated) when compared to the M. tuberculosis H37Rv homolog.
Format: XLS Size: 92KB Download file
This file can be viewed with: Microsoft Excel Viewer
Ribosome binding strength of the SD regions of transcribed pseudogenes
Our data indicated that although the SD sequences of transcribed pseudogenes were somewhat stronger than those of non-transcribed pseudogenes, the ribosome-binding capacity appeared significantly reduced when compared with functional genes (Fig. 6A). In addition, when the SD sequence conservation was estimated in the corresponding M. tuberculosis functional orthologs (n = 553), a larger SD degradation is found in M. leprae's non-transcribed pseudogenes relative to transcribed ones (Fig. 6B). However, it was noted that the peak in ribosomal binding efficiency varied along the upstream sequence of the pseudogene, flattening the obtained curve if only mean values were considered. Thus, individual measures of SD sequence conservation were obtained using Methods 1 and 2 described in Materials and Methods and indicated that 38% of transcribed pseudogenes had a putatively conserved SD region (Fig. 7). When the values of SD sequence conservation were compared among the different ORFs, transcribed pseudogenes showed intermediate values between non-transcribed pseudogenes and functional ORFs (Fig. 7; also see Additional File 5 for specific ribosome-binding values for all M. leprae genes and pseudogenes). Specifically, genes with degraded SD regions were more common among non-transcribed pseudogenes, whereas the opposite trend was found among ORFs with conserved SD regions (Additional File 5). The values of SD binding strength were always stronger for transcribed pseudogenes at all positions although they are not statistically significant. Thus, although transcribed pseudogenes have a reduced capacity for binding the 16S rRNA, a proportion of them appears to have conserved SD regions and might produce a protein product.
Additional file 5. Shine-Dalgarno signal strength of M. leprae genes. The translational potential for transcribed pseudogenes was determined by analysis of start codons (AUG, GUG, and UUG) at the 5' end of predicted pseudogenes using the M. leprae TN strain genome sequence 1http://genolist.pasteur.fr/Leproma/ webcite and regions upstream of these pseudogenes were examined for SD binding strength. The free energy values for the binding of the 3' end of the 16S rRNA with the region preceding each ORF at different positions (position zero indicates gene start). Lower free-energy values indicate higher binding strength. Estimates of SD sequence conservation for individual ORFs were obtained by quantifying the difference between the 2minimum and 3maximum free energy along the 50 nucleotides preceding the start of the gene 4(SD cons 1) and the difference between the free-energy value at position zero and the minimum value along the preceding 50 nucleotides 5(SD cons 2).
Format: XLS Size: 1.5MB Download file
This file can be viewed with: Microsoft Excel Viewer
Figure 6. Ribosome binding strength of M leprae genes. Graphs show the relative ribosomal binding strength of M. leprae genes. Panel A shows free energy values of the binding between the 3' sequence of the 16S rRNA and the region upstream of pseudogenes and ORFs. The position of the start codon of the genes is denoted by Pos (0). Lower values indicate stronger binding strength. Panel B shows the RBS ratios of M. leprae pseudogenes and their corresponding M. tuberculosis homologs.
Figure 7. Mean Shine-Dalgarno (SD) conservation for individual M. leprae genes. This graph shows the SD sequence conservation measured for individual M. leprae genes by calculating the difference between the minimum and maximum ribosome binding strength along the 30 bp upstream of gene start, SD (con 1), and by calculating the difference between the minimum ribosome binding strength along the 30 bp and the value at position zero, SD (con 2). Data indicate Means ± S.E for each gene category.
In frame stop codons within transcribed pseudogenes
Deduced amino acid sequences for all transcribed pseudogenes were obtained using in silico translation tools. Results demonstrated that a total of 3,625 in-frame (5'3'frame 1) stop codons were found in 461/486 (95%) of the total number of transcribed pseudogenes and the remaining 25 pseudogenes did not contain stop codons (Additional File 4). The range of transcribed pseudogenes containing stop codons was from 1 to 40 stop codons/pseudogene) with 67% of these transcribed pseudogenes containing at least 5 in-frame stop codons (Fig. 8A). Evaluation of transcribed pseudogenes with translational start codons demonstrated that 95% of these genes contained in-frame stop codons with a range of 1–28/gene (Additional Table 4; Fig. 8B). When the number of stop codons per pseudogene was compared to pseudogene length in base pairs (bp), a significant correlation (p < 0.001) was found between gene length and the number of stop codons/pseudogene. For the majority of the expressed pseudogenes, the longer the gene, the greater the number of stop codons (Fig. 8C). Further analysis using the M. tuberculosis H37Rv homolog as an estimate of normal frame equivalency for 92 transcribed pseudogenes, demonstrated that deletion mutations have resulted in a reduced coding capacity between 97.7–0.46% of the predicted full-length protein (Additional File 3). This has resulted in an overall loss of 35.4% coding sequence in these pseudogenes. These deletions appeared to have contributed heavily to the presence of the large numbers of stop codons found in these genes and are a dominant force of pseudogene formation in this pathogen.
Figure 8. Translational stop codons within transcribed M. leprae pseudogenes. These graphs show M. leprae transcribed pseudogenes containing translational stop codons (UAA, UGA, or UAG). The number of in-frame (5'3' frame 1) stop codons/pseudogene was identified in silico by translation of the DNA sequence for each pseudogene http://genolist.pasteur.fr/Leproma/ webcite. using (ExPasy Translate Tool: http://us.expasy.org/tools/dna.html webcite): Panel A shows the % of transcribed pseudogenes containing the specified number of stop codons/pseudogene (e.g. 1–5, 6–10, etc), obtained by dividing the number of transcribed pseudogenes within each group by that of the total number of transcribed pseudogenes with stop codons; Panel B shows the % transcribed pseudogenes in each group containing translational start codons, obtained by dividing the number of transcribed pseudogenes within each group containing translational start codons by that of the total # pseudogenes with stop codons and translational started codons; Panel C, shows the # of stop codons/group as a function of gene length in base pairs (bp). The mean and standard deviation of the of the gene length (bp) from each group were compared to that of the other groups using GraphPad InStat software and all groups were significantly different (p < 0.001) from all other groups except for 21–25 vs 26–40.
Functionality predictions for transcribed pseudogenes
When M. leprae genes were analyzed for potential functionality by using homologs in M. tuberculosis H37Rv and Ka/Ks ratio analysis, results demonstrated that ORFs, annotated as functional, contained a mean Ka/Ks ratio of 0.31 ± 0.16 (Fig. 9A). Ninety percent of ORFs with Ka/Ks values > 0.5 were identified as hypothetical proteins or ribosomal proteins (data not shown). In comparison, of 216 pseudogenes analyzed (i.e. those with a functional homolog in M. tuberculosis for which unambiguous alignments could be obtained) the mean Ka/Ks ratio was 0.78 ± 0.35 (Fig. 9B). Thus, pseudogenes have undergone a dramatic shift in their Ka/Ks ratios, indicating they accumulate replacement substitutions at higher rates than functional genes. Given that Ka/Ks ratios of 1 cannot be achieved unless the M. tuberculosis ortholog used for comparison is also a pseudogene, the observed Ka/Ks values are extremely high and suggest that most pseudogenes evolve under lack of selection. In fact, only 48/216 (22%) of pseudogenes contained Ka/Ks ratios ≤ 0.5, indicating that they could potentially encode a functional protein. But even these cases could correspond to neutrally-evolving ORFs that have lost functionality only recently. However, of these 48 pseudogenes with low Ka/Ks ratios, transcripts were detected for only 23. When one of these pseudogenes, pyrR (Ka/Ks ratio = 0.09) containing a strong SD, a functional promoter, and a translational start codon (ATG) was analyzed for the ability to produce a translational product in E. coli, a protein product was observed (Fig. 10).
Figure 9. Functionality of M. leprae genes with a functional homolog in M. tuberculosis using Ka/Ks ratio analysis. This graph contains frequency histograms of ratios of nonsynomous (Ka) vs. synonymous (Ks) nucleic acid substitutions of M. leprae genes when compared to their homologous genes in M. tuberculosis. Panel A represents the frequency histogram for Ka/Ks ratios of M. leprae functional ORFs. Panel B represents the frequency histogram for Ka/Ks ratios of M. leprae pseudogenes that have a functional homolog in M. tuberculosis H37Rv.
Figure 10. Cloning and protein expression of the pyrR pseudogene in E. coli. Crude cell lysates of recombinant BL21-Star E. coli cells containing the M. leprae pyrR pseudogene (E. coli::pET200/D/TOPO/MLpyrR) were separated on 4–20% SDS-PAGE gradient gels, and the histidine epitope was detected by Western blotting using the Anti-Xpress™ antibody: Panel A, shows the M. leprae pyrR pseudogene sequence for PCR amplification, yellow bases depict primers for PCR, the numbered sequences are the coding region; Panel B, is an SDS-PAGE gel stained with commassie brilliant blue, containing proteins from the recombinant E. coli strains: Lane 1, Kaleidoscope Prestained Standards Invitrogen #161-0324; Lane 2, E. coli::pET200/D/lacZ; Lane 3, E. coli::pET200/D/lacZ, IPTG-induced 18 hr; Lane 4, E. coli::pET200/D-TOPO/MLpyrR; Lane 5, E. coli::pET200/D-TOPO/MLpyrR, IPTG-induced 18 hr. Panel C represents a Western blot for the histidine epitope on the recombinant E. coli::pET200/D-TOPO/MLpyrR using crude cell lysates from IPTG-induced 18 hr samples: Lane 1, E. coli::pET200/D/lacZ; Lane2, E. coli::pET200/D/TOPO/MLpyrR.
Thus, most pseudogenes, including those that are transcribed, appear to be under very low selection strength. It is also worth mentioning that 15/216 (7%) have Ka/Ks ratios > 1.4 (Fig. 8B), whereas these cases are basically absent in functional genes. These higher-than-expected non-synonymous substitutions are characteristic of proteins under positive selection, but only 4 of them are transcribed, and therefore, it is unlikely that these high ratios indicate accelerated protein evolution but simply sequence deterioration. These analyses could be considerably improved when the genome of the second strain of M. leprae BR4923 (just recently available) is analyzed, so that sequence evolutionary patterns could be compared between pseudogenes in both strains.
Pseudogenes are considered disabled copies of functional genes that were once active in the ancient genome and their identification has been relatively rare until the recent availability of a large number of fully sequenced and annotated genomes and the improvement in detection algorithms [17-19]. Analysis of these genomes demonstrated that pseudogenes are much more common than previously thought and that pseudogenes can represent a significant fraction of the genome [[5,18,19]; http://www.pseudogene.org/main.php webcite]. As a result, the coding potential of genomes has been shown to be substantially lower than originally predicted. For example, the human genome contains 16,326 pseudogenes and Escherichia coli K-12 genome, once thought to only possess a few pseudogenes, has been shown to harbor 134 inactivated genes. Mycobacterial species are no exception. M. tuberculosis H37Rv contains 278 inactivated genes http://www.pseudogene.org/cgi-bin/db-gen.cgi?type=Prokaryote webcite and the recently sequenced M. ulcerans genome has 727 pseudogenes (BuruList Web Server: http://genolist.pasteur.fr/BuruList/ webcite) [20]. The case of pseudogenes in M. leprae is very dramatic with over 1100 being documented [4,5,9]. This represents the largest number of any bacterial genome sequenced to date. These data strongly suggest that genome down-sizing through the accumulation of pseudogenes, as well as gene loss, has resulted in the very specialized requirements for M. leprae growth.
Although the precise mechanism resulting in the formation of this large number of pseudogenes in M. leprae is unclear, several possible mechanisms have been defined. It has been suggested that the loss of dnaQ-mediated proofreading activities of the DNA polymerase III and large-scale rearrangements and deletions arising from homologous recombination events may have contributed to this accumulation of pseudogenes [4,5]. The loss of sigma factors [21] and two-component systems [22] have also been proposed as possible mechanisms in M. leprae pseudogenization. The dynamics of this reductive process in M. leprae has recently been studied by reconstructing the gene content of the last common ancestor of M. leprae and its closest relative M. tuberculosis and comparing it with the present M. leprae genome [23]. Data from this study suggest that the loss of ancestral genes resulted in the loss of functional genes of M. leprae's ancestor and its divergence from M. tuberculosis and that pseudogenization events appear to be recent gradual evolutionary events in M. leprae's lineage (within the last 20 million years).
Pseudogene accumulation might promote adaptive microevolution resulting in transitioning from a free-living to a mutualistic lifestyle [1,2]; from multiple hosts to specific hosts and ultimately specific host cells. Therefore, pseudogenization of M. leprae's sigma factors [19] and stress response genes, resulting in limited response to environmental stress conditions [24], may have contributed at least in part to its adaptive evolution and to its extremely specialized niche within peripheral macrophages [24,25] and Schwann cells of peripheral nerves in humans [26].
In general, pseudogenes are considered to be 'junk' DNA sequences that are in the process of being removed from the genome. However, recently we and others have demonstrated the presence of a small number of pseudogene transcripts in M. leprae [11,12] and other bacterial species [27,28]. In addition, others have found that transcribed pseudogenes can be functional [29].
In the present study, further characterization of the overall pseudogene transcriptional profile of M. leprae in the nu/nu mouse foot pad granulomatous tissue by global DNA array and RT-PCR analyses demonstrated that not only does M. leprae possess the highest number of pseudogenes/genome it also possesses the highest rate of bacterial pseudogene transcription documented to date. There was no apparent bias for transcription of pseudogenes in M. leprae based on chromosomal location or functional gene category. Although the highest percentage of transcribed pseudogenes was found in functional category V (hypothetical proteins), this finding was not surprising as this category contains the largest percentage of pseudogenes in the genome [5]. Many pseudogenes belong to gene families that are large in close relatives such as M. tuberculosis but are simplified during the loss of redundancy that takes place after niche specialization [8]. Results of the present study demonstrated that a large number of these degenerated ORFs, which may no longer code for their appropriate functions, were expressed in M. leprae using transcriptional machinery, metabolic resources and energy without potential benefit to this organism. These direct and indirect costs have previously been suggested to select against the expression of pseudogenes in M. leprae by the erosion of sequences involved in transcription initiation [4]. Therefore, even though a large number of M. leprae pseudogenes are transcriptionally active, approximately 60% of M. leprae's pseudogenes are transcriptionally silent, presumably by this or similar mechanisms.
In silico analysis of transcribed pseudogenes suggested potential mechanisms for their transcription. Their positioning within gene clusters (operon-like organizations), or downstream of transcribed ORFs, along with the paucity of intrinsic terminators between functional ORFs and transcribed pseudogenes implies that several pseudogenes are transcribed via a read-through manner. These data support a previous study which demonstrated that ~74% of M. leprae ORFs lacked detectable intrinsic transcriptional terminators [16]. An exception to this was found in the present study when the transcriptional pair ML0180c-ML0179c (pseudogene), containing a strong terminator sequence (ΔG -38.4) within the ML0180c coding region, was found to be transcribed as a single gene transcript product. The question is why is the terminator not functional? Previous work by our group has shown that terminators do not function if they are inside coding regions. There could be various reasons for this, prominently the presence of ribosomes or formation of antitermination complexes. In this case, the terminator is inside the pseudogene coding region and factor(s) which prevent termination functions inside coding regions could come into play. Sequences upstream and downstream of terminators have also been shown to be important in some cases. These could be the reason(s) for its lack of functioning. Also it must be noted that ΔG is an important, but not the sole indicator of terminator efficiency. In fact, our work has also shown that most terminators in M. leprae have a ΔG lower than this value.
The present study also demonstrated that rho (ML1132) and ndk (ML1469c), a nucleoside diphosphate kinase associated with its activity [30], were among the 1353 genes expressed. However, to date nothing is known about rho-dependent transcript termination in M. leprae and therefore, the significance of this for pseudogene gene expression is unknown. In addition co-transcription of genes of unrelated function has been shown in intracellular species that have undergone massive genome reduction and low selection strength such as Buchnera, where after the elimination of DNA segments that included promoter regions, two unrelated genes ended up physically linked [31] and were shown by microarray analysis to be co-transcribed [32]. Thus, these imperfect regulatory mechanisms in which promoter-less ORFs or pseudogenes are unnecessarily expressed may not be uncommon in species undergoing low selection strength, such as those under episodes of genetic drift and small population sizes.
However, not all M. leprae pseudogenes appear to rely on read-through transcription as a mechanism of transcription. Putative promoters were identified in silico in the upstream region of M. leprae pseudogenes. When 10 of these were tested for promoter activity in a promoterless reporter E. coli system, all were positive. Therefore, while the selection against the expression of pseudogenes in M. leprae by the erosion of sequences involved in transcription initiation appears to be an effective transcriptional mechanism for "silencing" M. leprae pseudogenes, the presence of functional promoters contributes to pseudogene transcription in M. leprae.
Prokaryotic mRNAs generally contain within their 5'-UTRs an SD sequence that serves as a ribosome-binding site [33]. The loss of functional SD sequences results in the lack of efficient translational capability and therefore results in a reduction or loss of protein production. Recently it has been reported that the SD sequences of M. leprae pseudogenes are highly degraded or degenerate suggesting that translation is impaired in nonfunctional open reading frames (pseudogenes) in this pathogen and that this potentially reduces the metabolic investment on faulty proteins because, although pseudogenes can persist for long time periods in the genome, they would be effectively "silenced" [4]. The present study confirmed these results and further demonstrated that although they have lower ribosomal binding strength than ORFs, transcribed pseudogenes have higher ribosomal binding strength than non-transcribed pseudogenes. Therefore these data strongly suggest that some transcribed pseudogenes are actually translated in M. leprae. To test this hypothesis, the promoter, SD (strong ribosomal binding strength), start codon and partial coding region of the pyrR (ML0531) pseudogene was fused into the gfp gene in a promoterless reporter plasmid lacking a SD site and was transformed into E. coli. Results of this preliminary experiment suggested that the pyrR SD site initiated ribosomal binding and resulted in the translation of the pyrR-gfp fusion protein product yielding the green fluorescent phenotype. Thus, although the results of this study indicate that most pseudogenes have either no recognizable SD or weak SD sequences for binding to the anti-SD sequence of the 3' region of the 16S rRNA, some of the transcribed pseudogenes have intact ribosome-binding sequences of similar strength to the orthologs in M. tuberculosis.
In addition, the current study demonstrated that the majority of transcribed pseudogenes lack traditional prokaryotic translational start codons. It has been shown that alteration of start codons results in loss of translational efficiency [32,33]. Even though the lack of these sequences in the majority of M. leprae pseudogene transcripts appears to be an effective mechanism for translational "silencing", to date this has not yet been experimentally confirmed.
In-frame stop codons (elementary property that distinguishes a pseudogene from a functioning gene) were present in 95% of transcribed pseudogenes, whether or not they contained start codons. Therefore, if translation of transcribed pseudogenes initiates, a truncated protein product should result from the majority of M. leprae pseudogenes. In rare instances, the protein fragment is still functional as bad codons can also be bypassed or edited at the level of mRNA by recoding mechanisms. Recoding is the reprogramming of mRNA translation by localized alterations in the standard translational rules and recoding products can play critical cellular roles [34]. Typically three classes of recoding are known: 1) frameshift recoding; 2) bypass (hopping) recoding; and 3) codon redefinition involving site-specific recognition (usually but not limited to stop codon). Recoding is utilized in the expression of a minority of genes in probably all organisms and has been documented in M. avium, [Selenocysteine incorporation at the stop codon (UGA) to yield formate dehydrogenase http://recode.genetics.utah.edu/display.cfm#fdh_s_pro_mavi webcite]. To date recoding has not been documented in M. leprae or its close relative M. tuberculosis. However, if recoding does occur in M. leprae, it is unlikely that transcripts would be recoded to yield full length sequences when multiple stop codons occur in a single coding sequence. It is estimated that 80% of transcribed pseudogenes contain at least 3 stop codons within their sequence and 90% of these pseudogenes have < 50% of the predicted full-length protein when compared to the M. tuberculosis homolog due to deletion mutations. Therefore, it is predicted that if translated, these sequences will result in truncated proteins.
Using the non-synonymous to synonymous substitutions analysis as a measure of potential functionality of pseudogenes, we showed that only one third of these genes had similar Ka/Ks ratios to functional genes, regardless of whether they are transcribed or not. As explained above, this is an upper limit because part of the analyzed sequence evolution corresponds to the M. tuberculosis functional orthologs and because the pseudogenization process could be recent for some genes and therefore their Ka/Ks ratios would be close to normal. Therefore, although the number of pseudogenes for which unambiguous Ka/Ks ratios could be obtained was small, and at least one of these with a low Ka/Ks ratio was translated, these data suggest that most transcribed pseudogenes are in the process of degradation. However, this is an upper estimate because of the potential short time passed after pseudogenization and because part of the substitutions correspond to the functional homolog in M tuberculosis taken as reference. Additional support for these conclusions is that even though protein expression data has demonstrated the presence of > 300 proteins in protein extracts from armadillo-derived M. leprae, no pseudogene products were identified [14,15].
The data presented in this study strongly suggest that even though a large number of M. leprae's pseudogenes are transcriptionally active, translational "silencing" mechanisms ensure that valuable metabolites and energy are not wasted to produce proteins from the majority of these transcripts which have no apparent benefit for cellular survival or growth of M. leprae. However, it is unclear whether these pseudogene transcripts have an additional detrimental effect on M. leprae. Nevertheless, some pseudogene transcripts do appear to be capable of producing protein products. These genes and their potential translational products need to be studied more extensively to understand their full biological impact on M. leprae.
RNA purification and cDNA production
M. leprae was purified from the granulomatous hind footpad tissue of four individual nu/nu nude mice, six months post-infection as previously described [35]. M. leprae RNA was purified from individual bacterial preparations as previously described using TRIzol® (Invitrogen, Carlsbad, CA), FastPrep Blue RNA tubes and mechanical extraction using a FastPrep® 120 Instrument [36]. DNA was removed from these preparations using the Turbo DNA-free™ kit (Ambion, Austin, TX). DNA-free RNA aliquots were then stored at -80°C. This RNA was used for DNA microarray analysis or converted to cDNA for RT-PCR analysis using 1 μg RNA and the Advantage RT-for-PCR Kit (BD BioSciences, Clontech, Mountain View, CA) using random hexamers. Controls for DNA contamination consisted of 1 μg RNA incubated with the reverse transcription reagents, excluding the reverse transcriptase (RT-). Template cDNA was also made from BALB/c mouse spleen total RNA (BD Biosciences, Clontech).
M. leprae gene expression by microarray analysis
M. leprae whole genome DNA microarrays representing the 1,614 annotated ORFs and 1,133 identified pseudogenes, were obtained from the Leprosy Research Support and Maintenance of an Armadillo Colony Post-Genome Era, Part I: Leprosy Research Support Contract (NO1 AI-25469) at Colorado State University. Microarray experiments were performed using previously described protocols [37]. Microarrays were scanned using a Bio-Rad VersArray Chip Reader (Bio-Rad, Carlsbad, CA) and using SpotFinder Software (manufacturer) to quantify fluorescence. Genes were positive for transcription if the average mean signal to noise ratios (SNR) were > 2-fold for samples analyzed. Transcribed pseudogenes were then mapped to the M. leprae chromosome and functional gene categories which have transcribed pseudogenes were identified http://www.sanger.ac.uk/Projects/M_leprae/ webcite.
RT-PCR amplification
To validate ~20% of genes positive by microarray analysis, RT-PCR was performed using Thai-53 M. leprae cDNA, primers for PCR amplification [based on gene sequences from the M. leprae TN genome http://genolist.pasteur.fr/Leproma webcite/ using PrimerQuest http://www.idtdna.com webcite], and PCR analysis using recommended primer annealing temperature and 40 cycles of PCR. M. leprae DNA (1 ng) was used as a positive control. Reactions without reverse transcriptase (RT (-) reactions), buffer and mouse cDNA were used as negative controls for each assay. Amplicons were observed in ethidium bromide-stained 2% agarose gels using Gel Doc 2000 Gel Documentation System (Bio-Rad) and the amplicon sequence was confirmed using automated DNA sequencing.
Identification of transcriptional read-through mechanisms
To identify M. leprae pseudogenes potentially transcribed as a result of read-through transcription, the presence of these pseudogenes within gene clusters or directly downstream of transcribed ORFs was analyzed using Gene Cluster analysis (GeneChords-http://genomics10.bu.edu/cgi-bin/GeneChords/GeneChords.cgi webcite) and the M. leprae TN mapping data http://genolist.pasteur.fr/Leproma/ webcite. M. leprae cDNA was used as the template to amplify fragments representing the read-trough products of predicted size using PCR and forward primers within the upstream ORFs and reverse primers within the pseudogenes or in the genes downstream of the pseudogene (Fig. 3A). Products were analyzed for their predicted fragment sizes using agarose gel electrophoresis and the DNA sequence of the resultant PCR amplicons was confirmed by automated DNA sequencing.
Identification of stem loop structures indicative of intrinsic termination sequences
Since read-through transcription relies on the absence of transcript termination structures between the 3'-UTRs of transcribed ORFs and downstream genes or pseudogenes, the presence of stem loop structures, indicative of intrinsic terminators, was investigated in the annotated genome of M. leprae TN strain using the algorithm Genome Scanner for Terminators (GeSTer) [16,38]. The program accepts whole genome sequence information from GenBank (NCBI), and searches the sequences -20 to +270 with respect to the stop codon in the 3'-UTR of upstream ORFs for palindromic sequences, which could potentially form stem-loop structures when transcribed. It then sorts out the structures based on their ΔG. GeSTer further defines a genomic ΔGcutoff which is a function of the genomic G+C content of the bacterial species. Palindromic structures with ΔG value more negative than this cut-off are only considered as potential intrinsic terminators.
In silico identification of putative pseudogene promoters
Independent pseudogene transcription requires the presence of functional a promoter in the (5'UTR) of these genes. Promoters need DNA bend regions as the RNA polymerase complex initiates strand separation at the promoter -10 regions [39]. Putative pseudogene promoters were located by DNA curvature (bend) analysis [40,41] with the "bend-it" server http://hydra.icgeb.trieste.it/dna/index.php webcite using DNase I parameters [42] and the consensus bendability scale [42] with a 31 size sliding window and simple smoothing of plots. Upstream 200–300 nucleotide plots of intrinsic curvature, bendability, complexity and GC content troughs were used to locate promoter regions through coincidence of peaks and troughs. Intrinsic curvature peak heights of less than 5 degrees per helical turn were discarded [42]. The sigA promoter region 38 was used as a standard [43]. When these peaks and troughs coincided, the region was assigned a putative promoter site. To avoid pseudogenization effects on upstream regions it was necessary to use ClustalW alignments with upstream regions from normal mycobacterial genes of the Mtb complex, the MMAR-MUL and MAV-MAP complexes. If alignments could not be made, the ML data was discarded. ClustalW http://www.ebi.ac.uk/Tools/clustalw2/index.html webcite cross-species mycobacterial DNA alignments were used to assign putative promoter -10, -35 elements, initiation sites (I) and start codons to the predicted promoter-like regions and determine the distance (bp) of the promoter upstream from the start codon.
In vitro promoter analysis
To further characterize pseudogene promoters previously identified by "bend-it" DNA curvature and alignment analyses, approximately 150 bp fragments of upstream sequence of 10 pseudogenes containing putative promoter regions were amplified from M. leprae Thai-53 DNA using standard PCR, primers (Additional File 6) and cloned into the pGlow-TOPO-TA vector which contains a promoterless Cycle 3 GFP mutant reporter from the pGlow-TOPO-TA-Expression-Kit (Invitrogen). E. coli XL1-Blue Supercompetent cells were then transformed with these plasmids. Fluorescence was analyzed from ampicillin-resistant (100 μg/ml ampicillin in Luria Bertani agar) bacterial clones by mixing a portion of each colony with 10 μl sterile PBS and placing the bacterial suspension onto a glass slide. A cover slip was applied and slides were examined using a Nikon Eclipse E400 fluorescent microscope using a FITC/gfp filter (excitation/emissionmaxima of 480 nm/560 nm). Positive clones were considered those that contained fluorescence levels above cultures that contained only the pGlow-TOPO-TA re-circularized vector.
Additional file 6. M. leprae pseudogene promoter sequences for in vitro functional promoter analysis. This figure includes the 5'UTRs of M. leprae pseudogenes for cloning into the promoterless reporter vector pGlow-TOPO-TA promoterless reporter-gfp vector for identification of promoter activity. The legend designates which area corresponds to primers for PCR amplification the promoter region including -35 and -10- and initiation site (+1) as well as their position relative to the start codon for the pseudogene.
Format: DOCX Size: 17KB Download file
Prediction of pseudogene translational potential
The translational potential for transcribed pseudogenes was determined in silico by analysis of start codons (AUG, GUG, and UUG) at the 5' end of predicted pseudogenes using the M. leprae TN strain genome sequence http://genolist.pasteur.fr/Leproma/ webcite. Since the ability of a transcript to be translated appears to be dependent on conservation of SD sequences which bind the transcript to its complementary sequence in the 3'region of the 16S rRNA, upstream regions of these pseudogenes were examined for SD binding strength using previously described protocols [44]. These procedures calculate, based on base pair formation rules [45], the free energy values for the binding of the 3' end of the 16S rRNA with the region preceding each ORF at different positions (position zero indicates gene start). Lower free-energy values indicate higher binding strength. Estimates of SD sequence conservation for individual ORFs were obtained by quantifying the difference between the maximum and minimum free energy along the 50 nucleotides preceding the start of the gene (Method 1) and the difference between the free-energy value at position zero and the minimum value along the preceding 50 nucleotides (Method 2). Based on SD sequence conservation in M. tuberculosis, which has not undergone a pseudogenization process, values > 7 and 5 were taken as indicative of a conserved, functional SD region by the first and second measure, respectively.
Prediction of pseudogene functionality
The translational potential for transcribed pseudogenes was also investigated by estimating selection strength by calculating the rates of synonymous (Ks) and nonsynonomous (Ka) DNA substitutions (Ka/Ks ratios) in these sequences and compared to their corresponding functional homologs in M. tuberculosis H37Rv. Sequences of M. leprae pseudogenes and their corresponding M. tuberculosis H37Rv functional homologs were aligned using the program Pileup of the GCG Wisconsin package (Genetics Computer Group 1997). Only unambiguous alignments were considered. Rates of synonymous and non-synonymous substitutions were calculated using the Diverge command in GCG that applies Li's algorithm [46] with modifications [47]. The Ka/Ks ratio is indicative of selection strength: in functional genes, synonymous substitutions are more frequent than replacement substitutions. Nevertheless, the mean ratio was also calculated for M. leprae functional ORFs for comparison to pseudogenes. Typically in other bacteria if the Ka/Ks ratio is around 1, substitutions are equally frequent at all three codon positions and the ORF is likely to be a pseudogene. However, only 216 pseudogenes could be analyzed because of lack of homology with M. tuberculosis, alignment ambiguity, or because the start codon and/or reading frame could not be unequivocally identified.
A subset of transcriptionally active pseudogenes with translational start codons were further analyzed to determine the percentage of the predicted full-length protein of pseudogene when compared to the M. tuberculosis H37Rv homolog (% Rv) using the deduced translated protein sequences for M. leprae ExPasy Translate Tool http://us.expasy.org/tools/dna.html webcite and ClustalW alignment http://www.ebi.ac.uk/Tools/clustalw2/index.html webcite to the M. tuberculosis homolog.
Cloning and expression of the pyrR pseudogene protein
Primers designed to amplify the coding region corresponding to the entire 532 bp coding region of the pyrR pseudogene (including stop codons at codon 158 and 166 and beyond the end of the coding sequence) from Thai-53 DNA and containing CACC on the 5'terminus of the forward primer (Fig. 10A) were used with standard PCR and directionally cloned into the linearized, topoisomerase I-activated Champion™ pET200/D-TOPO vector (Invitrogen), containing an N-terminus 6× His-tag, an N-terminal tag of a β-galactosidase and T7lac promoter for high-level expression, and transformed into One Shot® BL21 Star™ (DE3) and Top10 for storage (Invitrogen) according to manufacturer's recommendations. A kanamycin-resistant (50 μg/ml in LB agar) clone BL21 Star™ was verified to contain the pyrR insert by PCR and subsequent automated DNA sequencing (E. coli::pET200/D-TOPO/MLpyrR). A culture was grown to an OD600 = 0.6 and induced for several hours with a final concentration of 1 mM isopropyl β-D thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO). Crude cell lysates from both non-induced and induced E. coli::pET200/D-TOPO/MLpyrR after 18 hr incubation at 37°C were separated using a SDS-PAGE (4% to 20% discontinuous gradient polyacrylamide gel, stained with coomassie brilliant blue (Bio-Rad), and compared against Kaleidoscope Pre-stained Standard markers (Bio-Rad). A Western blot using the Anti-Xpress™ antibody (Invitrogen) was used to verify the presence of the polyhistidine epitope on the recombinant protein product. Crude cell lysates from E. coli::pET200/D-TOPO/lacZ (Champion™ pET200/D-TOPO vector Kit), treated under the same conditions described above, were used as a control for this experiment however, this control lacked the histidine epitope tag.
Statistical analysis of data
Statistical analysis of data for this study was obtained from the comparison of means and standard deviations of raw data and performed using One-way Analysis of Variance (ANOVA) using Tukey-Kramer Multiple Comparisons Test GraphPad InStat software. All data with p < 0.05 were considered significant.
Authors' contributions
DLW: Project manager, experimental design, bioinformatics, manuscript writing and editing. TLP and ANM: Bioinformatics, cloning and promoter analysis, RNA purification, RT-PCR validation experiments and cloning and expression of PyrR protein. RS and AA*: M. leprae DNA microarray experiments and analysis, and assistance with writing and editing manuscript. VN and AM: Intrinsic terminationexperiments and assistance with writing and editing manuscript. AM: SD and Ka/Ks analyses and assistance with writing and editing manuscript. NM: Promoter "bend-it" and promoter alignment analysis and assistance with writing and editing manuscript. MM: Assistance with editing manuscript. TPG: Assistance with protein expression, experimental design and editing manuscript.
*Passed away before completion of the manuscript.
We wish to thank Mr. J.P. Pasqua, Dr. Maria T. Pena and Ms. Heidi Zhang for their excellent technical service to this project. This project was funded in part by: NIH-NIAID Contract # YI-AI-2646-01; HRSA/BPHC/Division of National Hansen's Disease Programs; CAPES, Coordenaçao de aperfeiçoamento de pessoal de nivel superior Fundação do Ministério da Educação, Brazil; and NIH NO1 AI-25469, N01 AI-40091, and AI-55298.
This manuscript recognizes the scientific contributions of Dr. Amol Amin, who tragically passed away before completion of this work.
Tong Z, Zhou D, Song Y, Zhang L, Pei D, Han Y, Pang X, Li M, Cui B, Wang J, Guo Z, Qi Z, Jin L, Zhai J, Du Z, Wang J, Wang X, Yu J, Wang J, Huang P, Yang H, Yang R: Pseudogene accumulation might promote the adaptive microevolution of Yersinia pestis.
J Med Microbiol 2005, 54(Pt 3):259-268. PubMed Abstract | Publisher Full Text
Toh H, Weiss BL, Perkin SA, Yamashita A, Oshima K, Hattori M, Aksoy S: Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host.
Genome Res 2006, 16(2):149-156. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Andersson JO, Andersson SG: Genome degradation is an ongoing process in Rickettsia.
Mol Biol Evol 1999, 16:1178-91. PubMed Abstract | Publisher Full Text
Mira A, Pushker R: The silencing of pseudogenes.
Mol Biol Evol 2005, 22(11):2135-38. PubMed Abstract | Publisher Full Text
Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, Honore N, Garnier T, Churcher C, Harris D, Mungall K, Basham D, Brown D, Chillingworth T, Connor R, Davies RM, Devlin K, Duthoy S, Feltwell T, Fraser A, Hamlin N, Holroyd S, Hornsby T, Jagels K, Lacroix C, Maclean J, Moule S, Murphy L, Oliver K, Quail MA, Rajandream MA, Rutherford KM, Rutter S, Seeger K, Simon S, Simmonds M, Skelton J, Squares R, Squares S, Stevens K, Taylor K, Whitehead S, Woodward JR, Barrell BG: Massive gene decay in the leprosy bacillus.
Nature 2001, 409:1007-11. PubMed Abstract | Publisher Full Text
Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature 1998, 393(6685):537-44.
Erratum in: Nature 1998, 396(6707):190PubMed Abstract | Publisher Full Text
Moran NA, Wernegreen JJ: Lifestyle evolution in symbiotic bacteria: insights from genomics.
Trends Ecol Evol 2000, 15(8):321-26. PubMed Abstract | Publisher Full Text
Rocha EP, Danchin A: Base composition bias might result from competition for metabolic resources.
Trends Genet 2002, 18(6):291-94. PubMed Abstract | Publisher Full Text
Eiglmeier K, Parkhill J, Honore N, Garnier T, Tekaia F, Telenti A, Klatser P, James KD, Thomson NR, Wheeler PR, Churcher C, Harris D, Mungall K, Barrell BG, Cole ST: The decaying genome of Mycobacterium leprae.
Lepr Rev 2001, 72:387-98. PubMed Abstract
Pushker R, Mira A, Rodríguez-Valera F: Comparative genomics of gene-family size in closely related bacteria.
Genome Biol 2004, 5(4):R27. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Williams DL, Torrero M, Wheeler PR, Truman RW, Yoder M, Morrison N, Bishai WR, Gillis TP: Biological implications of Mycobacterium leprae gene expression during infection.
J Mol Microbiol Biotechnol 2004, 8(1):58-72. PubMed Abstract | Publisher Full Text
Suzuki K, Nakata N, Bang PD, Ishii N, Makino M: High-level expression of pseudogenes in Mycobacterium leprae.
FEMS Microbiol Lett 2006, 259:208-14. PubMed Abstract | Publisher Full Text
Mira A, Pushker R, Rodriguez-Valera F: The Neolithic revolution of bacterial genomes.
Trends Microbiol 2006, 14(5):200-6. PubMed Abstract | Publisher Full Text
Marques MA, Espinosa BJ, Xavier da Silveira EK, Pessolani MC, Chapeaurouge A, Perales J, Dobos KM, Belisle JT, Spencer JS, Brennan PJ: Continued proteomic analysis of Mycobacterium leprae subcellular fractions.
Proteomics 2004, 4(10):2942-53. PubMed Abstract | Publisher Full Text
Marques MA, Neves-Ferreira AG, da Silveira EK, Valente RH, Chapeaurouge A, Perales J, da Silva Bernardes R, Dobos KM, Spencer JS, Brennan PJ, Pessolani MC: Deciphering the proteomic profile of Mycobacterium leprae cell envelope.
Proteomics 2008, 8(12):2477-91. PubMed Abstract | Publisher Full Text
Unniraman S, Prakash R, Nagaraja V: Alternate paradigm for intrinsic transcription termination in eubacteria.
J Biol Chem 2001, 276:41850-55. PubMed Abstract | Publisher Full Text
Lerat E, Ochman H: Psi-Phi: exploring the outer limits of bacterial pseudogenes.
Genome Res 2004, 14:2273-78. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lerat E, Ochman H: Recognizing the pseudogenes in bacterial genomes.
Nucleic Acids Res 2005, 33:3125-32. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Liu Y, Harrison PM, Kunin V, Gerstein M: Comprehensive analysis of pseudogenes in prokaryotes: widespread gene decay and failure of putative horizontally transferred genes.
Genome Biol 2004, 5(9):R64. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Stinear TP, Seemann T, Pidot S, Frigui W, Reysset G, Garnier T, Meurice G, Simon D, Bouchier C, Ma L, Tichit M, Porter JL, Ryan J, Johnson PD, Davies JK, Jenkin GA, Small PL, Jones LM, Tekaia F, Laval F, Daffé M, Parkhill J, Cole ST: Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer.
Genome Res 2007, 17(2):192-200. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Madan Babu M: Did the loss of sigma factors initiate pseudogene accumulation in M. leprae?
Trends Microbiol 2003, 11:59-61. PubMed Abstract | Publisher Full Text
Tyagi JS, Saini DK: Did the loss of two-component systems initiate pseudogene accumulation in Mycobacterium leprae?
Microbiology 2004, 150:4-7. PubMed Abstract | Publisher Full Text
Gomez-Valero L, Rocha E PC, Latorre A, Silva F: Reconstructing the ancestor of Mycobacterium leprae: The dynamics of gene loss and genome reduction.
Genome Res 2007, 17(8):1178-1185. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Williams DL, Pittman TL, Deshotel M, Oby-Robinson S, Smith I, Husson R: Molecular basis of the defective heat stress response in Mycobacterium leprae.
J Bacti 2007, 189:8818-27. Publisher Full Text
Krahenbuhl J, Adams LB: The role of the macrophage in resistance to the leprosy bacillus. In Macrophage-Pathogen Interactions. Edited by Zwilling BS, Eisenstein TK. Marcel Dekker, NY, NY; 1994:281-302.
Hagge DA, Oby Robinson S, Scollard D, McCormick G, Williams DL: A new model for studying the effects of Mycobacterium leprae on Schwann cell and neuron interactions.
J Infect Dis 2002, 186(9):1283-96.
Epub 2002 Oct 8PubMed Abstract | Publisher Full Text
Takahashi H, Watanabe H: A gonococcal homologue of meningococcal gamma-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent.
BMC Microbiol 2005, 5:56. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Davids W, Amiri H, Andersson SG: Small RNAs in Rickettsia: are they functional?
Trends Genet 2002, 18(7):331-334. PubMed Abstract | Publisher Full Text
Hirotsune S, Yoshida N, Chen A, Garrett L, Suglyama F, Takahashi S, Yagaml K-I, Wynshaw-Boris A, Yoshiki A: An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene.
Nature 2003, 423(6935):26-8. Publisher Full Text
Ingham C, Hunter IS, Smith MCM: Isolation and sequencing of the rho gene from Streptomyces lividans ZX7 and characterization of the RNA-dependent NTPase activity of the over expressed protein. [http://www.jbc.org/cgi/content/full/271/36/21803] webcite
Moran NA, Mira A: The process of genome shrinkage in the obligate symbiont Buchnera aphidicola.
Genome Biol 2001, 2(12):1-research0054. BioMed Central Full Text
Wilcox JL, Dunbar HE, Wolfinger RD, Moran NA: Consequences of reductive evolution for gene expression in an obligate endosymbiont.
Mol Microbiol 2003, 48(6):1491-1500. PubMed Abstract | Publisher Full Text
Hirose T, Sugiura M: Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence.
Nucleic Acids Res 2004, 32(11):3503-10. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Baranov PV, Gurvich OL, Fayet O, Prere MF, Miller WA, Gesteland RF, Atkins JF, Giddings MC: RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression.
Nucl Acids Res 2001, 29:264-267. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Truman RW, Krahenbuhl J: Viable M. leprae as a research reagent.
Int J Lepr Other Mycobact Dis 2001, 69(1):1-12. PubMed Abstract
Williams DL, Oby-Robinson S, Pittman TL, Scollard D: Purification of Mycobacterium leprae RNA for gene expression analysis from leprosy biopsy specimens.
BioTechniques 2003, 35:534.
536, 538, 540PubMed Abstract
Groathouse NA, Brown SE, Knudson DL, Brennan PJ, Slayden RA: Isothermal amplification and molecular typing of the obligate intracellular pathogen Mycobacterium leprae isolated from tissues of unknown origins.
J Clin Microbiol 2006, 44(4):1502-1508. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Unniraman S, Prakash R, Nagaraja V: Conserved economics of transcription termination in eubacteria.
Nucleic Acids Res 2002, 30(3):675-684. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rivetti C, Guthold M, Bustamente C: Wrapping of DNA around the E. coli RNA polymerase open-promoter complex.
EMBO Journal 1999, 18:4464-75. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Munteanu MG, Vlahovicek K, Parthasaraty S, Simon I, Pongor S: Rod models of DNA: sequence-dependent anisotropic elastic modeling of local bending phenomena.
Trends Biochem Sci 1998, 23(9):341-346. PubMed Abstract | Publisher Full Text
Goodsell DS, Dickerson RE: Bending and curvature calculations in B-DNA.
Nucleic Acids Res 1994, 22:5497-503. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Brukner I, Sánchez R, Suck D, Pongor S: Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides.
EMBO J 1995, 14:1812-8. PubMed Abstract | PubMed Central Full Text
Hu Y, Coates AR: Transcription of two sigma 70 homologue genes, sigA and sigB, in stationary-phase Mycobacterium tuberculosis.
J Bacteriol 1999, 181(2):469-76. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Osada Y, Saito R, Tomita M: Analysis of base-pairing potentials between 16S rRNA and 5'-UTR for translation initiation in various prokaryotes.
Bioinformatics 1999, 15:578-581. PubMed Abstract | Publisher Full Text
Turner DH, Sugimoto N, Jaeger JA, Longfellow CE, Freier SM, Kierzek R: Improved parameters for prediction of RNA structure.
Cold Spring Harb Symp Quant Biol 1987, 52:123-133. PubMed Abstract
Li WH: Unbiased estimation of the rates of synonymous and nonsynonymous substitution.
J Mol Evol 1993, 36:96-99. PubMed Abstract | Publisher Full Text
Pamilo P, Bianchi NO: Evolution of the Zfx and Zfy genes: rates and interdependence between the genes.
Mol Biol Evol 1993, 10(2):271-281. PubMed Abstract | Publisher Full Text


text:
A still image of Aashika Bhatia as Meera in Meera
A still image of Aashika Bhatia as Meera in Meera in Meera TV Show
Courtesy: NDTV Imagine
Popular Search Terms: mira images aasika, aashika bhatia in meera, images of aashika bhatia in meera
Please Note: Images may have been watermarked to prevent other sites from hotlinking or scraping.
Uploaded by : tellybuzz
Join India Forums for free to comment on this story. Have an account already?
Use Quick Login to comment.
TV Show Meera
Celebrity : Aashika Bhatia
Forum Code
HTML Embed Code


text:
For a question abc, I would like to add multiple email accounts to it (notify abc of daily answers) so that some of friends can be alerted of possible updates to abc.
What is meta? ×
Meta Stack Exchange is where users like you discuss bugs, features, and support issues that affect the software powering all 150 Stack Exchange communities.
This isn't possible -- otherwise you could "sign up" a bunch of people for notifications and effectively annoy them with spam.
At the very least the emails would have to be confirmed before this could happen, making this impractical.
I suggest you encourage your friends to subscribe to tags, via email or RSS, instead -- http://blog.stackoverflow.com/2010/12/subscribe-to-tags-via-emai/


text:
Definitions for bello
This page provides all possible meanings and translations of the word bello
Bello is a town and municipality in Antioquia Department, Colombia and is a suburb of Medellín, the department capital. Bello is part of The Metropolitan Area of the Aburrá Valley in the department of Antioquia. It is bordered on the north by the municipality of San Pedro de los Milagros, on the east by the municipality of Copacabana, on the south by the municipality of Medellín and on the west by the municipalities of Medellín and San Jerónimo.
Images & Illustrations of bello
Translations for bello
From our Multilingual Translation Dictionary
Get even more translations for bello »
Find a translation for the bello definition in other languages:
Select another language:
Discuss these bello definitions with the community:
Word of the Day
Would you like us to send you a FREE new word definition delivered to your inbox daily?
Use the citation below to add this definition to your bibliography:
"bello." Definitions.net. STANDS4 LLC, 2015. Web. 25 Nov. 2015. <http://www.definitions.net/definition/bello>.


text:
2000 Nissan Altima • 144,856 miles
Car turns over but has no injector pulse. I can make it run by spraying fuel into the intake tube. After a min it gains injector pulse and runs fine. If I shut of for a real short period it will restart fine but if I leave it off for a few minutes it will loose injector pulse again. Does anyone have any ideas? I have only two codes while running a tps and a engine coolant sensor. I fixed the coolant sensor and ohm the tps and it showed to be fine. Please help me thanks.
November 3, 2012.
November 3, 2012.
This could be a wiring problem or a bad Control Module but it would require a considerable amount of testing to confirm something like that or locate the wiring issue.
Nov 3, 2012.
Nov 3, 2012.
My car has 157k on it and the coolant light came on today flashing i looked at the resivoir and it was empty filled it up and then later on that day it was empty again i looked at the ...
1 answer • 1996 Saturn SC2 • 157,000 miles
Dear SIR. Now I have some question to ask you. 1 How can i change (step)water coolant? 2 How much 1 lite oil per kilometre? 3 How can i maintain moonroof? Thanks so much
1 answer • 2006 Honda Accord • 40,000 miles
1992 Ford Tempo mileage: 65,000. My daughter has a 1992 Ford Tempo that she has been driving for about 4 years. She is very conscientious in regularly changing oil, and routinely having...
1 answer • 1992 Ford Tempo • 65,000 miles
This is about "Red" antifreeze, it's harmful attributes to engine gaskets and corrosion to all parts of the cooling and heating systems. I noticed that comments have been made on ...
1 answer • 2000 Chevrolet S-10 • 71,000 miles


text:
Tronox completes Exxaro acquisitionComment on this story
Tronox Limited' an Australian holding company' announced on Friday that it has completed its acquisition of Exxaro Resources Limited's mineral sands business.
This means that Tronox is now the world's largest fully integrated producer of titanium ore and titanium dioxide.
"This combination empowers Tronox with unique and game-changing competitive advantages' creating a stronger and more balanced company poised to deliver even greater shareholder value'" said Tom Casey' Chairman and CEO of Tronox Limited. "We now have the ability to sell into a lucrative titanium feedstock market while assuring our titanium dioxide customers that we have the supply to deliver quality products at reasonable prices."
Under the terms of the transaction' Exxaro received approximately 38.5% of the voting securities of Tronox Limited. Tronox's operations include South Africa's KZN Sands and Namakwa Sands as well as 100% of its former joint venture with Exxaro at Tiwest in Western Australia. The company employs approximately 3'500 workers in 16 locations worldwide.
Tronox will list its Class A Shares on the New York Stock Exchange on June 18' 2012 under the ticker symbol TROX. - I-Net Bridge
Comment Guidelines
- Please read our comment guidelines.
- Login and register, if you haven' t already.
- Write your comment in the block below and click (Post As)
- Has a comment offended you? Hover your mouse over the comment and wait until a small triangle appears on the right-hand side. Click triangle () and select "Flag as inappropriate". Our moderators will take action if need be.
- Verified email addresses: All users on Independent Media news sites are now required to have a verified email address before being allowed to comment on articles. You are only required to verify your email address once to have full access to commenting on articles. For more information please read our comment guidelines


text:
This Thanksgiving, we're thankful for all these awesome celebs
The one thing that makes a guy lose interest
This 7-year-old boy learned all about the power of helping others
The first Captain America: Civil War trailer is here!
How cute are these DIY turkey pops?
Ditch the dress: How to shimmer and shine in bold bottoms
This is *exactly* how you should do your makeup this Thanksgiving
Believe it or not, you *can* stay fit this Thanksgiving
#ThatAwkwardMomentWhen... Tell us your awkward story for a chance to be in the mag!
GL's Exclusive Look at Liv and Maddie's "Ask-Her-More-A-Rooney"
More Friends = More Fun
GL Tweets !
31 MINUTES AGO If you're a #Marvel fan, you absolutely *have* to watch the #CaptainAmericaCivilWar trailer: https://t.co/YYy5dXDbCB pic.twitter.com/x7eNiJT1DR
1 HOURS AGO You *can* make an awesome pie. Here's how: https://t.co/StusTik6aM pic.twitter.com/o86zR2CsqK
2 HOURS AGO #GIVEAWAY! Win a pretty #DIY pillow from @American_Girl and @TweetSimplicity here: https://t.co/fiZXRecHzF pic.twitter.com/Mbjhal4pZr
sponsored links
Check these out, too...
Trend: High-low hem
Sweet and High-Low Sheer Coral Dress, $37, lulus.com
Trend: Wild skirts
Wild skirts on the lower half of a dress are a great way to
add visual interest and texture to your dance outfit. Fringe, ruffles, layers,
and tulle are all fun and funky or you can go for feathers like the dress
Feather Dress, $90, delias.com
Trend: Pale metallics
Sparkle and shine will never go out of style when it comes
to picking out a dress for a big dance. Go this-season with sequins, glitter
and metallic prints in neutral hues. Shine on!
Trend: Pretty pastels
Pastels are taking over this season, and color us obsessed.
This dress has a beautiful lace pattern, but it's the color that has us head
over heels. Strut your stuff in a selection of precious pastels from lilac to
peach or this gorgeous soft blue.
Darling Lace Amelia Dress, $135, asos.com
POSTED IN fashion, trend, spring, dresses, weekend fun, dance
WIN IT! Can *you* solve the mystery?
Dive into the weird, wonderful world of Curiosity House: The Shrunken Head! CLICK HERE for your chance to win it—and to explore Dumfrey's Dime Museum of Freaks, Oddities and Wonders.


text:
$7.99Embroidery shop owner Marcy Singer is about to have the rug pulled out from under her....Marcy can't wait to see the new exhibit at the Tallulah Falls museum on antique tapestries and textiles, including beautiful kilim rugs. But her enthusiasm quickly turns to terror when, the day after the exhibition opens, she discovers a dead body behind her store, the Seven-Year Stitch, wrapped up in a most
$7.99When murder strikes the small town of Tallulah Falls, embroidery shop owner Marcy Singer isn't afraid of getting down to the knitty-gritty to clear her name.... For most small-business owners in Tallulah Falls, the upcoming Renaissance Faire is a wonderful way to promote their specialty shops. For Marcy's nemesis, Nellie, and her sister, Clara, it's an opportunity to finally put Marcy and her shop
$7.99Embroidery shop owner Marcy Singer is about to find out that show biz and sew biz don't mix! Marcy's mom Beverly is the costume designer for a lavish, Bollywood-style production...and she suggests Tallulah Falls as a great place to shoot part of the film. Everyone at the embroidery shop, and around town, is in a flutter that a glamorous movie production is taking place in their midst. But when the
$7.99Embroidery shop owner Marcy Singer gets hung up on a tapestry that may lead to sunken treasure and be the motive for murder.... When Marcy's friend Reggie, Tallulah Falls' local librarian, asks her to teach an embroidery class as therapy for domestic abuse victims, she gladly agrees. One of the women wants to flee from her abusive husband but is afraid to leave her elderly father-in-law behind.
$7.99Although she's taken an interest in Irish embroidery, Marcy Singer can't help but abandon her needlecraft when handsome local brewer Todd finds himself accused of murder. Both Todd and his friend Blake's fingerprints are on the murder weapon, and neither is talking about what happened. Marcy is determined to stitch together some luck from more than a few four-leaf clovers and prove that the
$7.99Cupid's arrow is making its way to Tallulah Falls, Oregon, bringing lots of business to Marcy Singer's embroidery specialty shop-like the bride who wants her mother's dress embellished with jewels. But someone wants to squash all the romance-and in this case they'll go as far as murder...
$7.99Marcy Singer owns The Seven Year Stitch, an embroidery specialty shop in the small town of Tallulah Falls, Oregon. Trouble strikes when an elderly woman brings an antique piece of embroidery into the shop-and promptly dies of unnatural causes. Now Marcy has to stitch together clues to catch a crafty killer.
$7.99First in a new mystery series that will have readers stitching-and itching for more When Marcy Singer opens an embroidery specialty shop in quaint Tallulah Falls, Oregon, she throws a soiree and a Stitch-In. Soon, Marcy's sign- up sheet for embroidery classes fills up and everyone in town seems willing to raise a glass-or a needle-to support the newly-opened Seven Year Stitch. Then Marcy finds
$2.99A Family Divided by Color is a heartfelt story about Abby Brenner who faces impossible struggles. She became a mother at fifteen. She was on welfare, lived in a run-down house, while raising two sons, Peter and Alex. Peter is the son of a white man that loves money over his son. Alex is the son of a black man, who has no desire to be a part of his life. Abby struggles to keep sanity in her life as
$7.99In the latest mystery from the bestselling author of Wicked Stitch, the future of Marcy Singer's embroidery shop is dangling by a thread…Marcy's shop, the Seven-Year Stitch, is one year old this October so it's time to party in little Tallulah Falls, Oregon. Aside from the Halloween decorations and festive stitching kits and patterns, Marcy's got all sorts of celebratory swag, including Seven-Year
Down Low Diva (5 Star Publications Presents)Take a small town girl, and add Delicious Divas Escort Service to her mix...and you will get a Down Low Diva. Christina lets her hidden fantasies and skills take her on a vacation from the long time love of her life to a secret society where her every dream can come true. But she didn t fulfill her sexual desires of endless nights of unlimited fun with sex toys and tantalizing fruits for money.


text:
- Filed Under
Brandi Varney, Newark, vs. Robert Varney, Mooresville, N.C.
Annette L. Halfhill, Sunbury, vs. Dennis L. Halfhill, Sunbury.
Jason S. Lawrence, Heath, vs. Jennifer D. Lawrence, Newark.
Amy Chapins, Johnstown, vs. Richard Chapins, Pataskala.
Denise Davis, Newark, vs. John Davis, Wooster.
Anthony C. Rings, Buckeye Lake, vs. Keisha R. Rings, Blacklick.
Lori L. Amos, Nashport, vs. Donald J. Amos, Nashport.
Melissa S. McMullen, Newark, and Dusty T. McMullen, Newark.
Debra A. Wilson, Reynoldsburg, vs. James L. Wilson III, Columbus.
Compiled from staff reports.
Janet Worth, Newark, vs. Douglas Weaver, Newark.
Tina M. Queen, Newark, vs. Dale S. Queen, Newark.
Marlene M. Isaac, Granville, vs. Gregory T. Isaac, Grove City.
Christine Walters, Pataskala, vs. Heath Walters, Pataskala.
Jessica Ortiz, Newark, vs. Christopher Ortiz, Newark.
Mary E. Nelson, Newark, and Robert L. Nelson, Heath.
Jennifer M. Ferguson, Newark, vs. James M. Ferguson, Newark.
Robert L. Roderick, Newark, vs. Sharon L. Roderick, Versailles.
Ruth E. Legg, Pataskala, vs. Patrick S. Legg, Columbus.
George W. Edmonson Jr., Centerburg, vs. Kelly M. Edmonson, Mount Vernon.
Diane Neighbarger, Newark, vs. John Neighbarger, Newark.
Leslie A. Wright, Newark, vs. Gary E. Wright, Newark.
Deborah Benner, Mount Vernon, vs. Wayne Benner, Newark.
Sally Warner, Newark, vs. Thad Warner, Newark.
Amber R. Armstrong-Johnson, Newark, vs. William R. Johnson Jr., Newark.
Heath C. Schotts, Newark, vs. Heather L. Schotts, Columbus.
Christie L. Hammond, Newark, vs. Kevin J. Hammond, Glenford.
Kimberly J. Carter, Newark, vs. Curtis E. Carter, Newark.


text:
Chavez declares 3-day mourning for refinery blast victims
This affects all of us. It's very sad, very painful." Venezuelan President Hugo ChavezVenezuelan President Hugo Chavez has declared three days of mourning following the lethal explosion in the country's largest refinery, which claimed the lives of dozens of people. "This affects all of us. It's very sad, very painful," Chavez said by phone on state television. The Venezuelan president called for a thorough investigation into the cause of the tragic incident at the Amuay plant, located on the Paraguana Peninsula in the northwestern Falcon State. Chavez also pledged to provide the required assistance for those affected in the incident.
According to Vice President Elias Jaua, who has traveled to the area, 39 people, including a 10-year-old child and 18 National Guard troops, were killed and over 80 people were wounded in the Saturday explosion.The blast also inflicted damage to hundreds of homes and shops nearby. However, the emergency officials say the worst is over and the fire is now under control. Energy Minister Rafael Ramirez said the explosion was sparked by a gas leak, the cause of which was not known. Venezuela is South America's biggest oil producing nation and the Amuay refinery, one of the biggest in the world, produces 645,000 barrels of crude a day. ASH/HSN/MA


text:
Recent Posts
Tagsabroad a cappella athletics blog campus culture Community dining environment fall farmers market fashion Feb Break Febs finals first-years food fun global liberal arts Housing ice cream for breakfast independent projects Independent Research International International Students Organization internships ISO J-Term Languages MCMP music professors registration Senior Seminar snow Snow Bowl speakers Student Organizations students study abroad Theater Vermont weather winter winter carnival workshops
Tags » independent projects
Science and the Liberal Arts
As a chemistry major, I'm often asked about the opportunities at Middlebury for the natural sciences. "Middlebury is known for its language and environmental studies programs," some will say with the underlying question being "Middlebury isn't necessarily known for their … Continue reading
Senior Project-Studio Art
Since the fall I have been working hard preparing for my Senior show, in May, which will be culmination of my Studio Art career here at Middlebury. Its been quite the experience. I spent a lot of fall semester with … Continue reading
Tags: independent projects, murals, studio art
Anticipating J-term
Christmas was an amazing time this year. I spent tons of time with my family talking, eating, laughing, watching movies, and just hanging out. With less than a week before we return for J-term the anticipation is in the air. … Continue reading
Tags: independent projects, J-Term, studio time, workshops


text:
Siemens to Reduce Costs and Sell Assets
BERLIN—Siemens AG said it aimed to cut €6 billion, or about $7.5 billion, in costs by 2014 and would sell several assets in a push to improve returns for shareholders. The industrial company posted a 27% decline in fiscal-year net.
Munich-based Siemens on Thursday also announced the €680 million acquisition of LMS International, a Belgian supplier of simulation software for testing transport and energy equipment.


text:
Product Rating
1 customer reviews
Beautiful Dress!
By Yuliya C. from Walpole, MA on 9/29/2014
Size: TRUE
This is a very beautiful dress! We Love it! It looks great in person as well! I was hesitant to order it since it was written that it was "100% Polyester" - but it is actually not! The black with flowers Chiffon skirt is Polyester, BUT the pink top and lining of the skirt is %100 Cotton and very nice and thin! My daughter wanted to sleep in it when we received it! :) Thanks!
More Reviews on this item


text:
Warner Bros.(LONDON) -- Original Gremlins star Zach Galligan says that the success of a Jurassic World, a movie that is set decades after the original, has had an influence on the making of Gremlins 3.
Galligan told an audience at London's Prince Charles Cinema on Wednesday, during a special Q&A, that he thinks the franchise won't start from scratch, thus there's a "chance" he'll likely be in the next one, which will come out more than 25 years after Gremlins 2.
"It's not going to be a reboot," he told the audience in London, who had come out for a special screening of Gremlins, followed by questions for Galligan, according to the BBC. "It will not be a remake in any way, shape, or form. [Director] Chris Columbus has come out and said that the first film is very near and dear to his heart and as long as he is alive it will never be remade."
Galligan expanded on his statements.
"So the only thing they will be, and apparently are going to be doing, is something along the lines of like Jurassic World, where it will be 30 years later," he continued.
As of now, Jurassic World, which starred Chris Pratt, has made north of $1.6 billion worldwide, reports Box Office Mojo, and the movie picked up where the original Jurassic Park franchise left off.
There will be new stars but Galligan added that "some of the characters from the first one" would be in it, but that "I swear on my life, I don't know if I'm in it or not, but I think there is probably a decent chance since I'm one of the few people left standing from the first movie."
Reports of a new Gremlins movie started back in early 2014. In April, Deadline reported that Goosebumps writer Carl Ellsworth would be involved in the script.
Copyright © 2015, ABC Radio. All rights reserved.


text:
I buy larger quantities and use inert gas for storage. I used to tig and mig weld so I have regulators and such. I bought a small gas cylinder from my welding gas supplier. It costs very little to fill it, so I blanket my chems when opened and periodically. I also use this on the wifes wine bottles
By the way, I should have started this thread in the correct location.