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A Short ClC-2 mRNA Transcript Is Produced by Exon Skipping. Shijian Chu, Carol B. Murray, Minzhi M. Liu, Pamela L. Zeitlin; A Short ClC-2 mRNA Transcript Is Produced by Exon Skipping, Nucleic Acids Research , Volume 24, Issue 17, 1 September 1996, Pages 3453–3457, https://doi.org/10.1093/nar/24.17.3453. Download citation file: © 2017 Oxford University Press. ClC-2 is a voltage- and essay services, volume-regulated chloride channel expressed in many tissues. We have shown that ClC-2 in rat lung airways is significantly down-regulated after birth [ Murray, C.B. et al . (1995) Am. J. Respir. How To Critique A Thesis Paper? Cell Mol.

Biol ., 12, 597–604 ]. During PCR amplification from rat lung cDNA, a second transcript was identified which is 60 bp shorter than the full length sequence. Top 5 Essay? The peptide translated from this 60 bp sequence contains many positively charged amino acid residues. Rat genomic DNA sequencing showed that the 60 bp sequence is an intact exon. A 71% pyrimidine content and an AAG 3?-end splice site in the intron immediately upstream from the short, 60 bp sequence were identified which may account for the alternative splicing of the top 5 essay, following exon. Human genomic sequence analyses demonstrated similar intron-exon arrangement. A high CT content and research paper on classroom, an AAG 3? acceptor site were conserved in the intron corresponding to the rat upstream intron. Essay? The presence of the full length short form transcript was confirmed in rat kidney by RT-PCR, and the ratio of the long and research paper, the short form transcripts varied significantly according to the tissues examined, with the lowest long/short form ratio found in top 5 essay services, the lung among the tissues studied. Our data demonstrated that the alternatively spliced short form (ClC-2S) is transcribed in how to a thesis paper, many rat tissues, the ratio of the long/short form transcripts is lower in the lung compared with the services, brain, and the genomic organization in this area is conserved in rat and diagram, human. ClC-2 is a chloride channel belonging to a ClC family of chloride channels. More than 10 members of top 5 essay services, this family have been identified recently from a wide variety of species including yeast, fish and how to paper, mammals. The first member of the ClC chloride channel family, ClC-0, was identified by expression cloning from Torpedo marmorata electric organ ( 2 ). Other members of the family were later cloned from yeast Saccharomyces cerevisiae , and from a number of rat and human tissues ( 3–9 ). These channels can be tissue-specific or ubiquitous, and from epithelial or non-epithelial origins.

Among all the top 5 essay services, ClC chloride channels, the ClC-2 gene encodes a voltage- and volume-regulated chloride channel whose mRNA can be detected in many tissues in rat and human ( 8 , 10 ). The predicted protein product has 12 putative membrane-spanning regions in the N-terminal two-thirds of the sequence followed by a C-terminal hydrophilic domain proposed to muller thesis be cytosolic ( 8 ). Essay? Recently, two additional subforms of search engines, ClC-2, ClC-2G (ClC-2?) and services, ClC-2?, were identified from rabbit, the essay, former in the stomach and heart, and the latter in the heart ( 11 , 12 ). Both are PKA activated. Interestingly, the rat ClC-2 produces a chloride current in Xenopus oocytes only when it is essay services, under control of an upstream sequence from engines ClC-0 ( 8 ), whereas the rabbit forms were expressed without the services, attachment of the ClC-0 sequence. Although many chloride channels of the ClC family have been cloned and essay format, expressed in heterogenous systems, few studies have examined the regulation of their expression ( 3 ). Here we report the expression of a short form ClC-2 transcript (ClC-2S) by exon skipping, which is 60 bp shorter than the published sequence. The ratio of ClC-2/ClC-2S varies between the lung and other tissues. A high CT content and top 5, an AAG accepter site in the upstream intron are conserved in rat and human. Our results suggest that alternative splicing may constitute one of the critique a thesis paper, mechanisms that regulates ClC-2 expression and function in lung and other tissues. Adult rats and timed-pregnant dams were obtained from Harlan Sprague Dawley Inc. (Indianapolis, IN). Rats were euthanized by services intraperitoneal injection of sodium pentobarbital. Search Phd Thesis? Tissues from essay fetuses and adults were harvested, pooled (fetal tissues, according to their gestation period), flash-frozen in dry ice, and stored at ?70°C. Short On Transportation? The L2 cell line was derived from type II-like alveolar pneumocytes from normal adult female rat lung ( 13 , 14 ) and was obtained from American Type Culture Collection (ATCC, Rockville, MD).

It was maintained in top 5 services, monolayer culture in essay on transportation, Ham's F-12K without glutamine (Biofluids, Rockville, MD), containing 10% fetal calf serum, 2.5 µg/ml fungizone, 100 U/ml penicillin G and 100 U/ml streptomycin The cells were fed three times a week and split 1:10 when they reached 100% confluence. The human tracheal epithelial cell line 9/HTEo- ( 15 ) was obtained from top 5 essay services Dr D. Rikky Thesis? C. Essay Services? Gruenert and maintained in LHC-8 medium with glutamine (Biofluids), 10% fetal calf serum, 2.5 µg/ml fungizone, 100 U/ml penicillin G and 100 U/ml streptomycin. They were fed three times a week and split 1:5 when passed. Normal human bronchial primary cells, NHBE (Clonetics, San Diego, CA), were grown in serum free bronchial epithelial cell growth medium (BEGM, Clonetics). Total RNA was prepared from rat tissues or cultured cell monolayers using Trizol reagent (Gibco-BRL) following the manufacturer's protocol. The quality and quantity of the total RNA were examined by agarose gel electrophoresis and UV spectrophotometry. cDNA was prepared from 0.1 µg of total RNA with the cDNA Cycle Kit (Invitrogen, San Diego, CA), using the oligo dT primer. Format? For subsequent PCR amplification, 20% of the prepared cDNA was used in each 50 µl reaction as the template. PCR experiments were performed on a Perkin Elmer DNA Thermal Cycler 480.

Before the Taq polymerase (Boehringer Mannheim, Indianapolis, IN) was added, it was mixed with TaqStart antibody according to the manufacturers protocol (Clontech, Palo Alto, CA). Primers used in RT-PCR are listed in services, Table 1 . Among all the short essay, primers, HRC5? and HRC3? were also used to evaluate the essay services, ratio of the two forms of the ClC-2 transcript. Action Research? Their sequences are identical to essay services human ClC-2 at search engines phd thesis, nt 2177–2195 and 2347–2369 respectively in the coding sequence ( 10 ). For evaluation of the ratio of the two forms, HRC5? was labeled with blue fluorescent dye FAM (Applied Biosystems, Inc., Foster City, CA) ( 16 ), and 25–31 cycles or 29–37 cycles of essay services, amplification were performed depending on the yield of the PCR products. In each cycle, the paper, reactions were denatured at 94°C for 1 min, annealed at 57°C for top 5 essay, 40 s and extended at 72°C for 40 s. Search Engines? PCR products were separated on a Model 373 ABI DNA Sequencer and quantified by GeneScan software (Applied Biosystems). In each PCR reaction, the quantity of the essay, fluorescence detected by the scanner was plotted on a log scale against the number of how to critique a thesis, cycles.

Only those points which were on a linear curve (before reaching the plateau area) were used for further analysis. The long/short form ratio of the ClC-2 products in each organ is the mean of three individual reactions each representing a different animal or cell culture. The one-way ANOVA followed by the Bonferroni were used to analyze these results. Cloning and sequencing of PCR fragments. PCR products were separated by agarose gel electrophoresis and the gel slices containing the bands of top 5, interest were excised. The DNA bands were extracted with the QiaQuick Gel Extraction Kit (Qiagen, Hilden, Germany).

The DNA fragments were then cloned into the pCR3 plasmid (Invitrogen). DNA sequencing was performed on the ABI DNA Sequencer Model 373. For direct sequencing of PCR fragments, the DNA in 50–100 µl of nurse maker, PCR reactions was concentrated by ethanol precipitation and resuspended in a 10 µl volume. The resuspended DNA was electrophoresed on a 3% NuSieve GTG agarose gel (FMC, Rockland, ME), electroeluted from the excised gel slice, and used as sequencing template. Essay? Agarose gel electrophoresis of PCR products of adult rat ClC-2 cDNA using HRC5? and HRC3? with 35 cycles. cDNA preparation and the amount used in each PCR reaction are described in the Material and Methods section. Outer lanes on both sides are molecular weight markers. Arrows indicate number of short essay, base pairs of the top 5, bands in the marker lanes. The sources for PCR products are brain ( 1 ), esophagus ( 2 ), heart ( 3 ), intestine ( 4 ), kidney ( 5 ), liver ( 6 ), lung ( 7 ), stomach ( 8 ) and testis ( 9 ) of rat, and action research on classroom, L2 cells ( 10 ). Top 5? The two major products are of 202 and essay format diagram, 142 bp.

Agarose gel electrophoresis of PCR products of essay services, adult rat ClC-2 cDNA using HRC5? and HRC3? with 35 cycles. cDNA preparation and the amount used in each PCR reaction are described in the Material and Methods section. Outer lanes on both sides are molecular weight markers. Nurse Resume Maker? Arrows indicate number of essay services, base pairs of the bands in the marker lanes. The sources for PCR products are brain ( 1 ), esophagus ( 2 ), heart ( 3 ), intestine ( 4 ), kidney ( 5 ), liver ( 6 ), lung ( 7 ), stomach ( 8 ) and testis ( 9 ) of rat, and L2 cells ( 10 ). The two major products are of 202 and 142 bp. Primers used in PCR experiments. Search Engines? Primers used in services, PCR experiments. A short form of ClC-2 transcript is produced in rat tissues. During PCR amplifications of the 3?-portion of essay, rat lung cDNA, in addition to the expected 202 nt fragment, a second smaller band was consistently amplified. The possibility of essay, non-specific amplification was less likely because the double-band pattern was observed using three different primer pairs (C2in5?/C2in3?, HRC5?/C2in3? and search engines phd thesis, HRC5?/HRC3?). PCR sequencing results localized the origin of the services, long and short PCR fragments to a segment of 160 bp between oligos HRC5? and HRC3? or A2214 to A2373 in the ClC-2 coding sequence ( 8 ) (GenBank accession no.

X64139). Although the format diagram, two-band pattern appeared in many different rat tissues, the ratio of these two bands varied reproducibly depending on the source of rat cDNA, suggesting a tissue-dependent expression pattern ( Fig. 1 ). Two methods, DNA sequencing and services, size determination of fluorescent-labeled PCR products, were used to investigate whether both PCR products were derived from search ClC-2. In the first approach, the amplified fragments from the adult rat lung were sequenced both after subcloning and directly from the top 5, PCR products. DNA sequencing data showed that the short fragment is identical to thesis the long fragment except for the absence of a 60 bp sequence ( Fig. 2 ). In the second approach, the long and short fragments were amplified from adult rat testes, trachea, intestine, kidney, liver, lung, brain, 19- and 21-day gestation fetal rat lungs and L2 cells with fluorescent labeled HRC5? and unlabeled HRC3?.

The exact size of each product in top 5 essay services, number of the thesis, bases was measured with fluorescent labeled molecular weight standards and top 5 essay, GeneScan software after separation on a sequencing gel. The long and short fragments (each with size confirmed) were present in every sample tested. As predicted from the DNA sequence, deletion of the 60 bp fragment would not cause any frame shift or termination but would produce a shorter form of ClC-2 protein by ?2 kDa (the predicted molecular weight of the essay, published rat ClC-2 protein is top 5, 99 kDa). Muller? The 60 bp fragment codes for a 20 amino acid residue peptide in the ClC-2 protein from L 747 to A 766 which is the most positively charged region in the protein. Essay Services? This region is located in the proposed C-terminal cytosolic domain, a region with little sequence homology to other members of the ClC chloride channel family. ( A ) Sequence alignment of the rat and human ClC-2 and the short form rat ClC-2 amplified by RT-PCR using the primers HRC5? and HRC3?. Amino acid sequences are shown beneath the cDNA sequences from which they are translated.

Locations where introns are found are indicated by ‘*’. HRC5? and HRC3? sequences are double underlined. ( B ) Sequences of the rat intron upstream from the skipped exon and the corresponding intron from the human cell line 9/HTEo- (HTE). Two bases from the paper on classroom management, adjacent exons are attached at top 5 essay services, the ends of the engines, introns and marked with bold face. Possible branch point sequences are underlined. ( C ) Schematic drawing of the intron-exon organization in rat and 9/HTEo- (HTE) cells. Boxes indicate exons and straight lines indicate introns.

Lines above the exons and introns indicate splicing events. ( A ) Sequence alignment of the services, rat and human ClC-2 and rikky muller, the short form rat ClC-2 amplified by RT-PCR using the primers HRC5? and HRC3?. Amino acid sequences are shown beneath the cDNA sequences from top 5 essay services which they are translated. Locations where introns are found are indicated by ‘*’. HRC5? and HRC3? sequences are double underlined. ( B ) Sequences of the rat intron upstream from the skipped exon and the corresponding intron from the human cell line 9/HTEo- (HTE). Two bases from the adjacent exons are attached at the ends of the introns and marked with bold face. Possible branch point sequences are underlined. ( C ) Schematic drawing of the intron-exon organization in rat and 9/HTEo- (HTE) cells. Boxes indicate exons and straight lines indicate introns.

Lines above the exons and introns indicate splicing events. To confirm the presence of a full length ClC-2 transcript differing from the published sequence by how to the 60 bp deletion, nested RT-PCR was performed on cDNA from rat lung and kidney. The first amplification with an top 5 services outer primer pair (nt5?out and ct3?out) was followed by nested amplification with an inner primer pair (A5?in and ct3?in). A predominant band of ?3 kb was obtained from both rat lung and kidney. The PCR products were then cloned into vector plasmid pCR3 (Invitrogen) and two clones, pRK and pRL from rat kidney and lung respectively, were used for further analyses. DNA sequencing of the entire inserts in these two clones demonstrated that the nurse, majority of the sequence is identical to the published sequence except for top 5 essay services, two silent mutations (C 564 to A 564 , A 1422 to G 1422 ) and essay format, three missense mutations (C 830 to T 830 , T 932 to C 932 , G 1285 to A 1285 ) in pRL, and two missense mutations (G 1169 to T 1169 , A 2053 to G 2053 ), one single base pair deletion (C 475 ) and one 60 bp deletion (T 2239 to top 5 services G 2298 ) in pRK. Critique Paper? While the single base changes are likely the top 5 essay services, results of PCR error, the 60 bp deletion in pRK is identical to that found in the short form PCR product amplified by HRC5? and HRC3?, suggesting that the short form ClC-2 transcript (ClC-2S) is present in rat tissues with only the 60 bp sequence deletion, but no other alternative splicing. The short form ClC-2 transcript is produced by exon skipping. The genomic DNA structure in the relevant region was then investigated by cloning and sequencing.

A rat genomic clone containing part of the ClC-2 gene was selected from a rat genomic DNA library constructed in P1 phage ( 17 ). The subclones were prepared in plasmid pTZ19R (Pharmacia) and sequenced. The results revealed additional sequences at both ends of the 60 bp sequence characteristic of introns, which begin with GT at thesis, the 5? end and top 5 essay, end with AG at the 3? end, suggesting that the 60 bp sequence is an intact exon ( Fig. Search? 2 ). Top 5? This structure containing the exons and rikky muller, introns was also observed in a 0.6 kb PCR fragment amplified from adult rat lung cDNA using primers HRC5? and HRC3?, indicating that this 0.6 kb fragment might have been amplified from incompletely processed primary ClC-2 RNA or contaminating genomic DNA. Although no obvious CT tracts were found, a high CT content was observed in top 5 essay, the upstream intron (71%). Three possible branch point sequences (BPS), CTCTGAC from how to critique paper ?5 to ?11, CTATAAC from ?33 to ?39 and CAATGAC from ?134 to ?140 (in reference to the first base of the 60 bp exon), were observed in the upstream intron based on sequence similarity to the consensus YNCTGAC (where Y represents pyrimidine residues and N represents either purine or pyrimidine residues) ( 18 , 19 ), while no obvious BPS was seen in the downstream intron. In addition, A is the nucleotide preceding the 3? splice site AG in the upstream intron, which has been previously demonstrated to be less efficient in intron splicing ( 20 ).

Genomic DNA PCR analyses with the same primer pair in the human cell line 9/HTEo- revealed identical intron and exon sequences in a 0.7 kb PCR fragment from essay services NHBE cDNA, which may contain some pre-mRNA. The overall structure of the introns and the exons are similar to those of the rat ( Fig. 2 C). The exon sequences are identical to the published human ClC-2. In addition, a high CT content (69%), three possible branch point sequences and an AAG acceptor site were present in the upstream intron in these human cells ( Fig. Engines Phd Thesis? 2 B). Although the downstream intron of human and top 5 essay services, rat is also similar in paper on classroom, size, 112 and 134 bp respectively, none of them have a high CT content nor AAG 3? splice sites. Ratios of long/short form ClC-2 mRNA transcripts in rat tissues. Ratios of top 5 essay, long/short form ClC-2 mRNA transcripts in rat tissues. Adult rat lung has a decreased long/short form ratio of ClC-2 transcripts.

Initial agarose gel electrophoresis of the PCR products using HRC5? and HRC3? suggested that the brain had the highest ratio of long/short form transcript compared with other tissues, especially the lung and the L2 cells. To quantify the ratio of these long and short transcripts, RT-PCR was performed with a fluorescent-labeled oligonucleotide ( 16 ). As only one pair of primers was used in the PCR reactions and maker, two fragments were amplified, each fragment acted as the internal standard for the other. Services? PCR products were collected at short, different cycle numbers in top 5 services, each reaction, so that fractions during exponential amplification could be selected for further analysis. The PCR products were separated on sequencing gels and their fluorescence intensity was analyzed by GeneScan software. Examples of the RT-PCR plots are shown in Figure 3 . How To A Thesis? Adult rat brain was found to have the highest ratio of long/short form ClC-2 transcripts, while the lowest ratios were found in adult and top 5 services, fetal rat lungs (Table2). This difference between adult brain and all other tissues that we studied here is statistically significant ( P 0.05, by one way ANOVA, followed by muller the Bonferroni). Top 5 Essay? The lower long/short ratios in essay format, adult and fetal rat lungs are consistent with a similar ratio in the L2 cell line (type II-like rat alveolar pneumocytes) ( Table 2 ). Although the ratios of the two forms were estimated within each tissue and compared between tissues, this experiment did not intend to compare quantitatively the amount of each form alone between tissues.

Our results suggest that ClC-2S is favored relative to top 5 essay ClC-2 in cells of action paper on classroom, lung-derivation compared with that of brain. Examples of RT-PCR plot from adult rat tissues and cells. Primers HRC5? and top 5 essay, HRC3? were used in the amplification. Number of cycles are labeled. Phd Thesis? Solid lines are short form products and dashed lines are long form products. Examples of RT-PCR plot from adult rat tissues and cells.

Primers HRC5? and top 5 essay, HRC3? were used in the amplification. How To Critique A Thesis Paper? Number of cycles are labeled. Solid lines are short form products and dashed lines are long form products. Rat ClC-2 transcript contains a truncated form produced by alternative splicing. A number of essay, ClC channels have been described among which ClC-2 is ubiquitously expressed in rat, rabbit and format, human tissues based on Northern hybridization ( 8 , 10 , 11 ). Top 5? ClC channel protein short forms, however, have been demonstrated only in a few cases. In rat ClC-K2, a 55 amino acid residue fragment in the second membrane spanning domain has been found truncated in a short form, ClC-K2S ( 3 ). In rabbit, a short form of ClC-2G has been reported with a 68 amino acid residue truncation at the very N-terminal end ( 12 ). In both cases, the authors speculated that different RNA processing might be the cause of the essay on transportation, truncation. ClC-2 is essay, different from ClC-K2 and ClC-2G in that there is a deleted portion within the critique, cytosolic domain, which is less conserved among ClC channel family members compared with other regions of the top 5, protein. Search Engines? This makes non-specific PCR amplification less likely.

The possibility of non-specific PCR amplification of other ClC channel genes is further eliminated by using three different primer pairs in our experiments, including using the full-length cDNA clone pRK as the services, PCR template, size determination of the PCR products by rikky muller GeneScan, and ultimately, cloning and top 5 services, sequencing of the two PCR fragments. Format? In addition to the observed exon skipping in the cytosolic domain, other ClC-2 transcripts in which exon-skipping occurs in services, other regions of the gene may also exist. Nurse? We have detected a full length ClC-2 cDNA from rat kidney by RT-PCR differing from the published rat brain sequence by only the 60 bp deletion, indicating the top 5, presence of the short form transcript ClC-2S without any other variations. The putative amino acid sequence of the deleted region is format diagram, highly positively charged, and services, the genomic structure is conserved in rat and human. Both of these may suggest a functional or structural importance of this alternative splicing and possible participation of the short form in the rat ClC-2 regulation or function. When the human cell lines 9/HTEo- and NHBE were analyzed by RT-PCR, ClC-2S was not detected. The conservation of the upstream intron in human and format, rat predicts that ClC-2S may be produced in human tissues in vivo . Alternative splicing generates diversity in chloride channel forms by several different mechanisms. Alternative RNA splicing is one of the most common strategies that cells use to essay services generate further diversity ( 21 ). The mechanisms involved in exon skipping are diverse and research paper on classroom management, not well understood. Small exon size itself may facilitate the aberrant splicing ( 22 ). However, a number of examples of exon skipping involve changes in some basic elements in their DNA sequences such as branch point sequences, polypyrimidine tracts and 3? splice sites in their upstream introns.

Mutations introduced at these points led to exon skipping, and return of these elements to their respective consensus sequences appeared to correct the skipping ( 23 , 24 ). Essay Services? While the structure of these conserved DNA elements is necessary for normal RNA splicing in diagram, many cases, only a few studies showed unusual amounts of pyrimidine residues in the introns upstream from deleted exons. In the case of CFTR (cystic fibrosis transmembrane conductance regulator) exon 9, an inverse relationship has been demonstrated between the length of a polythymidine tract in its intron 8 acceptor site and the proportion of top 5 services, exon 9 ? CFTR transcript, i.e., less exon 9 skipping was observed in cases with a longer polythymidine tract ( 25 ). High pyrimidine content is short on transportation, also evident in the rat ?-tropomyosin gene intron 6 (68%) where exon 7 is top 5 essay, skipped in skeletal muscle ( 26 ). The high pyrimidine content of the upstream intron in the ClC-2 gene may be one of the basic cis -acting elements which participate in the alternative splicing, however further evidence is needed to essay on transportation confirm its role. Another possible mechanism which could lead to exon skipping is the failure to recognize the 3?-splice site of the upstream intron. Competition between 3?-splice sites for recognition is largely dependent on their structure. Top 5 Services? AAG in the upstream intron 3?-splice site is unique to all of the ClC-2 introns sequenced to date (data not shown), others being either TAG or CAG, both preferred for recognition over AAG ( 20 ). The high pyrimidine content and the 3?-splice site sequence may constitute basic regulatory elements present in the rat genomic DNA. Since lung has a lower ratio of long form/short form transcripts than the brain, there might be other trans -acting regulatory factors with different abundance or activity in either lung or brain that interact with the upstream intron or splice sites and essay, either promote the essay services, exon skipping as seen in the lung or help retain the exon as seen in rikky muller thesis, the brain. ClC-2 and CFTR are both expressed in organs affected in cystic fibrosis. Top 5 Essay Services? CFTR is essay, a cAMP-regulated chloride channel ( 27 ) expressed in many secretory epithelia.

In cystic fibrosis patients, mutant CFTR expression results in abnormal chloride permeability and significant respiratory and digestive tract symptoms. Our PCR results in rat gave lower ratios of long form/short form ClC-2 transcripts in the lung and intestine than in top 5 services, brain. These results, in addition to our previous observation that ClC-2 expression in rat lung is down-regulated after birth at both RNA and protein levels ( 1 ), may suggest that lower amounts of alternative chloride channels like ClC-2, or relatively lower amounts of certain forms, may contribute to disease pathogenesis in CF airways. This work was supported by R29 HL48274 to P. L. Zeitlin, Cystic Fibrosis Foundation Fellowship to S. Research On Classroom? Chu, and Bauernschmidt Fellowship from the essay, Hospital for the Consumptives of Maryland (Eudowood) to C. Essay Format Diagram? B. Services? Murray. Online ISSN 1362-4962 Print ISSN 0305-1048 Copyright © 2017 Oxford University Press. Oxford University Press is a department of the on classroom, University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Copyright 2017 Oxford University Press Privacy Policy Cookie Policy Legal Notices Site Map Accessibility Get Adobe Reader. This Feature Is Available To Subscribers Only.

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We hope that clears up any misconceptions you may have had. We invite you to scroll back to essay, the top and choose from one of our many resume libraries, and research on classroom start writing. cover letter for nursing. Should i include collegiate sports in my resume? And if so where? It depends how much professional experience you have. If you are a recent college grad, then it is acceptable to include on your resume. Good luck on top 5 services the job hunt!

Good luck on on transportation the job hunt! If the jobs are relevant to the ones you are applying for, then you can go as far back as you like. With regards to top 5 essay, your military experience, check out our military to how to a thesis, civilian resume guide: https://resumegenius.com/blog/go-shooting-guns-coffee-runs. Any of the templates in library 2 would be suitable for manufacturing careers. Services! Best of on transportation luck! I’ve worked in the same industry for the past 13 years. Multiple employers with jobs lasting two to three years each. The jobs have been similar, so the experience looks a bit repetitive. I need to find a template that highlights my experience without getting bogged down in the chronology, Any suggestions?

It provides ample space for top 5 your professional experience, while also highlighting your top qualifications. Search Engines! Good luck on top 5 the job hunt! hi resume genius.. i need template resume that suitable for trainer and short coach.. can u suggest to top 5 essay, me with template is suitable.. #128578; I had a job for 7 years and muller during that time I wore many hats, Executive Admin, Purchasing, Vendor Management, Project Coordination, etc. How would I write that on top 5 essay services my resume? Perhaps the short essay on transportation, Company name and top 5 essay services then all the related roles under that and essay the times I did those jobs? I was always the Executive Admin, but I did other jobs during that period. Yes, your suggestion is correct. Services! Start with the nurse resume maker, company name and included the related jobs with their own bullet points underneath. Good luck! Consider trying the ‘Job Hopper’ or the top 5 services, ‘Executive.’ They should able to fit all your jobs nicely.

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The Life of Galileo Context Essay. Restrictions on freedom will inevitably lead to conflict | Rosie Liberow As a young child, I always wanted to be bigger, older and rikky thesis, wiser. I . dreamed of being in high school, or being a perfect well dressed secretary working in an office. Yet they say time flies when you’re having fun, but it seems now that I yearn for those times again, those boundaries and limitations. Freedom is daunting; the world is big and dangerous and I often find myself wishing I was young again. For me, the liberty I do have.

Bertolt Brecht , Civil liberties , Conflict 1506 Words | 4 Pages. ESSAY PLAN CONFLICT - SHOULD I PURCHASE A 1940'S GENUINE MINK FUR COAT OR NOT? Opening para – browsing Ebay, personal . dilemma over ethics, state below my personal argue for and against purchase and wear of top 5, said coat FOR * animal is dead - (dead over 70 yrs), not slaughtered for me, mink farms don't exist any more, can't save them now * timeless and classic piece - should be enjoyed so wear it, take care of it, piece of history * cost effective purchase from ebay – have to go. American films , Animal rights , Conflict 1083 Words | 3 Pages. Essay About the Conflict in Syria. Essay about the Conflict in nurse Syria Foreign Affairs April 12th, 2012. Syria, a Middle Eastern country, is located on the east . Essay Services! coast of the Mediterranean Sea.

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Conflict , Family , Father 583 Words | 2 Pages. 800 Words Essay on Conflict for on transportation Students. division are called dissociative processes. Conflict , competition and mutual opposition are included in dissociative processes. Like . co-operation conflict is also regarded as a basic action. In this context A.W. Small says that conflict is the essay services fundamental form of essay format diagram, opposition oriented relationship and dissociative interaction. Karl Marx, George Simmel, Rulf Dahrendorf have analysed the top 5 essay services role of conflict in social structure. A.W.

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Conflict is a true test which . raises issues that otherwise wouldn’t be raised, issues which need to be dealt with in order for us to top 5 essay services grow as a person. As well as testing a person’s inner strength and understanding it can also expose their weaknesses and flaws. Trials and hardships test and shape us as we learn from our experiences of search engines phd thesis, dealing with them in our everyday lives, increasing our inner strength. American film actors , American television actors , Brad Pitt 507 Words | 2 Pages. ?“ Conflict exists in every scene and at every level: it is the essay services single driving force of the search play”. In light of this remark, . explore some of the ways Shakespeare presents conflict in Hamlet. In the course of your writing show how your appreciation of Middleton’s presentation of conflict in ‘The Revengers Tragedy’ has helped to top 5 services shape your ideas. (40 marks) Conflict is a driving force in William Shakespeare’s ‘Hamlet’ and maker, is presented to the audience in top 5 essay many different forms, ranging. Audience , Audience theory , Characters in Hamlet 1363 Words | 2 Pages. Intrastate Conflicts By Rania Maroun 500319319 Submitted To Dr. Abbas H. Gnamo Ted Rogers School of Business Management In partial . fulfillment for critique paper the requirements For POL 540 November 7, 2011 Ryerson University As a Lebanese citizen I have experienced firsthand the essay services tragic result which intrastate violence leaves on a nation and its people.

Intrastate conflicts have been the cause of the maker world’s many high profile displays of brutal and inhumane violent acts. In fact “most wars today. Ethiopia , Ethnic group , History of Somalia 2528 Words | 7 Pages. grow as a human being. The hardship of privations that conflict brings can in some individuals result in a growing capacity to stay strong, to . discover an inner resilience that the person may not have known about top 5 before the conflict started. Conflict can also bring about format a growth in essay moral strength in nurse the suffering that conflict creates the individual may come to a deeper understanding of right and wrong and grow in wisdom as a result. Conflict is a fight or battle, which happens among humans, causing.

Australia , Ethics , Happiness 410 Words | 2 Pages. ?‘ Conflict can both divide and unite the people who encounter it.’ Conflict is an inherent part of the human condition; causing . Services! polarization of populations depending on paper, one’s beliefs, however conflict can also unite by the action of a pacifist movement from either party. Conflict has gradation of severity, varying from personal disputes, to the potential of services, another world war, caused by format diagram, the relentless terrorism. Martin Luther King Jr, is a prime example, when a population unites due to persuasion. African American , Islam , Martin Luther King, Jr. 963 Words | 2 Pages. The epic of The Odyssey by Homer is the second oldest surviving Greek text. Top 5! The story contains many conflicts such as man v god, man v . Nurse! himself, and man v society. Top 5 Essay! This paper will explore some of the important conflicts in this classic tale.

Man v god is the most important conflict in the story. Towards the beginning of the story Odysseus goes to war against the Trojans and wins. What he doesn’t know is resume, that the great God Poseidon favored Odysseus and essay, his army and helped them win the Trojan. Homer , Marriage , Odysseus 1023 Words | 3 Pages. is different lies at engines phd thesis, the root of conflict Score: 45/50 Statement of explanation The exploration of this context prompt, fear . of what is different lies at the root of conflict , has been chosen to be written in the form of a persuasive essay . Much like the text response, this essay only presents the one point of essay, view, that agreeably the fear of the different than being different is what drives conflicts . However, unlike a conventional text response, this essay does not strictly revolve around. Salem witch trials , The Crucible 1649 Words | 3 Pages. With reference to a conflict over the use of a local resource that you have studied, discuss the extent to engines phd thesis which all interest groups involved . Services! can be satisfied with its outcomes. The Bingley relief road, stretching from Cottingley Bar to Crossflatts, was completed in rikky thesis 2004 at a cost of ?47.9 million. It was built with the aim of reducing congestion in the centre of essay services, Bingley, through improving journey times and reliability; for over 20 years, around 36,000 vehicles a day travelling through Bingley brought.

Bingley , City of Bradford , Lane 1497 Words | 2 Pages. Internal conflict is used to explore the nurse idea of how humans prioritize their justice in following their morals to their loyalty using contrast. . An example of this is in Montana 1948 by Larry Watson. The novel shows a young boy David, who experiences a series of events and watches his father Wes’ internal conflict of services, arresting his brother Frank as well as David’s conflicts within himself of format, choosing to grow up and telling his parents about top 5 services his uncle’s murder. They both struggle to choose whether to. Choice , Conflict , Crime 991 Words | 3 Pages.

Conflict is a main theme in war poetry as will be shown throughout this assessment. In ‘The Man He Killed’ By Thomas Hardy the speaker is a . young soldier who has killed an enemy in the Boer War and is experiencing guilt and search phd thesis, regret about his actions, as further on in the poem he considers him as a friend had they met under different circumstances ‘You shoot a fellow down You’d treat if met where any bar is’. The theme of the poem is about the man that the top 5 essay services young soldier has killed. The poem is spoken. Alfred, Lord Tennyson , Charge of the Light Brigade , Crimean War 1190 Words | 3 Pages. ?Sacrifice is often necessary to resolve conflict The power of research, sacrifice should never be underestimated. Where have the capacity to give up a . possession or what is valuable to us for the sake of other’s considerations, tensions and essay, conflict can usually be resolved. As a naive and rikky muller, confused teenager, I left the country and all that I had ever known. The idyllic setting of Surrey Hills, where the autumn leaves created a dusty orange wave on the concrete pavement was replaced by a bluestone cottage.

Ku Klux Klan , Martin Luther King, Jr. , Morality 1671 Words | 3 Pages. ?My conflict is top 5 services, about my terrible roommate that I had my freshman year at Navarro College back when I was still living back in Texas. Her name . is Brittany and when we first met during freshman orientation, we got along fine. Sure we came from completely different cultures and resume maker, backgrounds but we chatted and although we didn’t find much in common she was a nice girl. I didn’t want to assume living next to her would be alright but no red flags cropped up so for the first few weeks it was smooth sailing. Ass , Conflict , I Decided 1057 Words | 3 Pages. The duality of the human soul in the guise of good and evil is the main preoccupation in the adventure novel 'Lord of the essay Flies'. Throughout the novel, the . conflict is dramatized by the contradicting views between Ralph and Jack, who represent civilisation and savagery.

The differing views are expressed by each boy's strong attitudes towards authority. Ralph uses leadership to establish rules and to enforce the British moral codes that the muller boys were raised up to use. However, Jack demands the complete. English-language films , Evil , God 1303 Words | 4 Pages.

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Pick the 4 Best UC Personal Insight Questions for YOU! If you’re applying to any of the University of top 5 services, California schools, you need to write four short essays. To start, read through all eight of the Personal Insight Questions you have to choose from. (Find specific ideas and strategies for each of the 8 new Personal Insight Questions at the bottom of this post!) The goal is to how to critique, write four short essays that as a whole will provide the top 5 services, UC admissions deciders with a picture of what makes you unique and special—and help set you apart from the competition. read more… UC Essay Prompt 8: What Sets You Apart? University of paper, California Essay Prompts for Fall 2017. (Ideas for Answering Personal Insight Question No. 8) UC Essay Prompt 8: What is the one thing that you think sets you apart from other candidates applying to the University of top 5 essay services, California? Out of the eight new University of essay format diagram, California essay prompts for Fall 2017, I think that most students should consider writing about Prompt 8 as one of their four required mini-essays.

More than any of the other questions, this prompt targets most directly what makes you YOU, and asks to understand something about top 5, your most fundamental qualities, characteristics or values. It is also the most open-ended of the bunch. read more… NEW University of California Essay Prompts for Fall 2017! First New University of short essay on transportation, California Essay Prompts. for their College Application in 10 Years! The University of California just listed brand new college application essay prompts—for the first time in a decade! The two essays had to be a total of no more than 1,000 words. The UC is now calling its new essay prompts, “Personal insight questions,” and students must choose four out of essay services, eight to answer.

And they are each supposed to be under 350 words. (So, total under 1,400 words.) I love that there are more prompts to choose from, with themes ranging from leadership, creativity, service and essay on transportation, personal individuality to more serious ones, such as sharing personal challenges or barriers to success. I’ve written separate posts for each of the 8 new prompts, and you can find them below. Just click the blue link to top 5 services, get specific ideas and strategies for each Personal Insight Question. After reading through these new prompts, I believe it will be important to short essay on transportation, choose four that show a nice variety as well as balance to showcase who you are.

In essence, these mini-essays will serve as your one personal statement, in that the goal is to top 5 services, highlight your most core qualities and values–and help you stand out format diagram, from the top 5, crowd. These new prompts are great news since they allow more flexibility in how you write about yourself. I also believe they should also be easier to craft than writing two longer essays. If you are just starting the admissions process, I would just take the nurse maker, time to read through them and start thinking about which ones you might like to write about. Unlike the old prompts, each of these new University of California essay prompts includes a series of additional questions, which can help you brainstorm ideas for your essays and also help you understand exactly what the UCs want to learn from you. That is top 5 essay super helpful, so read them closely! The UC also provided a new worksheet–Personal Insight Questions: Guide for Freshman Applicants–to help students brainstorm and nurse, craft their answers to these new prompts.

This is an invaluable tool, and I urge everyone to use it! One piece of advice: If you are a student who has faced significant challenges or obstacles so far in essay your life or personal background, I would strongly urge you to write about them. Numbers 4 and especially 5 would be the most obvious ones to help you write about those issues. I also believe those two prompts will produce the most poignant and meaningful mini-essays. (I would suggest all students explore if they could include Number 4 or 5 as part of their four essays for the same reason.) I also think UC Essay Prompt 8 is search engines phd thesis a terrific prompt, since it asks you to top 5, write about something unique about yourself. Resume! What a perfect opportunity to top 5 essay, stand out! The main danger of some of the other prompts is that you simply answer them and short essay on transportation, they end up being dull or boring to top 5 essay services, read. Even though these will be shorter essays, they still need to format, be engaging and meaningful! As with all essays, I would advise you to always try to think of specific examples to support any main point you make.

Even though these are shorter pieces, look for your real-life moments and top 5 essay services, experiences to help you illustrate your points, especially when talking about your leadership, creativity, talents, skills, favorite subjects or volunteer activities. And ditto for rikky muller thesis sharing personal struggles. One bit of essay services, sad news about search, these new prompts is that this will make it much more difficult, if not impossible, for students to recycle their essays from top 5 essay, other applications, especially The Common Application. Students also will need to research on classroom, come up with four rather than two strong topic ideas. The key is to essay services, look at resume maker these new University of California essay prompts as opportunities to showcase yourself through a variety of top 5 services, lens so the on classroom, admissions deciders can get an essay services accurate picture of your individuality. Locations of UC campuses. Here are the new University of California essay prompts and on transportation, admissions instructions from the UC web site for incoming freshman (I will address the top 5 services, new University of search, California transfer essay prompts in an upcoming post):

Freshman: Personal insight questions. What do you want UC to know about top 5 essay, you? Here’s your chance to tell us in your own words. You will have 8 questions to choose from. You must respond to only 4 of the format, 8 questions. Each response is limited to a maximum of essay, 350 words. Which questions you choose to how to critique a thesis, answer is top 5 services entirely up to search engines phd thesis, you: But you should select questions that are most relevant to your experience and that best reflect your individual circumstances.

All questions are equal: All are given equal consideration in the application review process, which means there is top 5 no advantage or disadvantage to choosing certain questions over others. There is no right or wrong way to essay diagram, answer these questions: It’s about getting to know your personality, background, interests and achievements in your own unique voice. Remember, the top 5 essay, personal questions are just that — personal . Which means you should use our guidance for each question just as a suggestion in case you need help. The important thing is expressing who are you, what matters to you and what you want to search engines phd thesis, share with UC. Things to consider: A leadership role can mean more than just a title. It can mean being a mentor to others, acting as the person in charge of a specific task, or a taking lead role in organizing an event or project. Top 5 Essay Services! Think about your accomplishments and what you learned from the essay on transportation, experience. What were your responsibilities? Did you lead a team? How did your experience change your perspective on essay services, leading others?

Did you help to critique paper, resolve an important dispute at your school, church in your community or an organization? And your leadership role doesn’t necessarily have to be limited to top 5 services, school activities. For example, do you help out or take care of your family? Things to consider: What does creativity mean to you? Do you have a creative skill that is action paper on classroom important to services, you? What have you been able to do with that skill? If you used creativity to solve a problem, what was your solution?

What are the steps you took to solve the problem? How does your creativity influence your decisions inside or outside the classroom? Does your creativity relate to your major or a future career? Things to consider: If there’s a talent or skill that you’re proud of, this is the time to share it. You don’t necessarily have to diagram, be recognized or have received awards for your talent (although if you did and you want to top 5 services, talk about, feel free to do so). Why is this talent or skill meaningful to you? Does the talent come naturally or have you worked hard to develop this skill or talent? Does your talent or skill allow you opportunities in essay or outside the classroom? If so, what are they and how do they fit into your schedule? Things to consider: An educational opportunity can be anything that has added value to your educational experience and better prepared you for college.

For example, participation in an honors or academic enrichment program, or enrollment in top 5 essay services an academy that’s geared toward an occupation or a major, or taking advanced courses that interest you — just to muller thesis, name a few. If you choose to write about educational barriers you’ve faced, how did you overcome or strived to top 5 essay services, overcome them? What personal characteristics or skills did you call on to overcome this challenge? How did overcoming this barrier help shape who are you today? Things to consider: A challenge could be personal, or something you have faced in your community or school. Why was the challenge significant to you? This is a good opportunity to talk about essay on transportation, any obstacles you’ve faced and what you’ve learned from the experience. Top 5 Essay! Did you have support from someone else or did you handle it alone? If you’re currently working your way through a challenge, what are you doing now, and does that affect different aspects of research on classroom management, your life? For example, ask yourself, “How has my life changed at home, at essay services my school, with my friends, or with my family?” Things to essay format, consider: Discuss how your interest in the subject developed and describe any experience you have had inside and outside the top 5 essay, classroom — such as volunteer work, summer programs, participation in student organizations and/or activities — and what you have gained from your involvement.

Has your interest in the subject influenced you in choosing a major and/or career? Have you been able to rikky muller thesis, pursue coursework at a higher level in this subject (honors, AP, IB, college or university work)? Things to consider: Think of community as a term that can encompass a group, team or a place – like your high school, hometown, or home. You can define community as you see fit, just make sure you talk about your role in that community. Was there a problem that you wanted to fix in your community? Why were you inspired to act? What did you learn from essay, your effort? How did your actions benefit others, the wider community or both? Did you work alone or with others to initiate change in how to critique paper your community? Things to consider: Don’t be afraid to brag a little.

Even if you don’t think you’re unique, you are — remember, there’s only one of you in the world. From your point of view, what do you feel makes you belong on one of UC’s campuses? When looking at your life, what does a stranger need to understand in order to know you? What have you not shared with us that will highlight a skill, talent, challenge, or opportunity that you think will help us know you better? We’re not necessarily looking for top 5 what makes you unique compared to others, but what makes you, YOU. Here are some additional details about these new University of California essay prompts that you might find helpful or interesting (This was released from the admissions department today.): The new questions also provide students with better direction and focus on topics that are important to campuses. Each new question aligns to one or more of the 14 comprehensive review criteria (nine criteria for transfer students) that campuses consider in their admissions decisions. Essay! “We hope this new format will not only provide us with additional insight into top 5 essay services applicants, but also allow students to better choose the questions that speak to them most directly,” stated a UC admissions director. Last Minute Help for UC College App Essay Writers!

The University of California announced NEW essay prompts for 2016-17. Read about how to answer them HERE. It’s Not Too Late to Write Your College App Essays. Before the Nov. 30 Deadline. Yes, you have waited until the last minute. But don’t despair! There’s still time to management, pound out services, two awesome essays! If you’re working on your two college application essays for the University of California freshman application, I’ve put together a short list of my most helpful posts. Read through them, watch the videos, and get crankin!!

Prompt 1: Describe the world you come from engines, — for example, your family, community or school — and tell us how your world has shaped your dreams and top 5 essay, aspirations. read more… If you are applying to the University of California schools, you have until the end of this month (this Sunday, Nov. 30!). As busy seniors, some of you might have waited to write your two college application essays over Thanksgiving, when you have some days off and can catch up. The key, however, is to not let this last minute deadline dash ruin your Thanksgiving. So yes, you are really cutting it close. If you don’t have a plan, it could hang over your head the entire holiday weekend. To not let these essays ruin one of the best times of the search engines, year–when you are supposed to be feasting with your family, watching football games and focusing on all you have to top 5 essay services, be grateful for–take a few minutes to map out a plan.

These essays don’t have to take days and days to write. If you can latch on to some strong topic ideas, and then pound out a rough draft, you could crank them out in a matter of hours. read more… UC College Application Essay Instant Boot Camp. UPDATE: as of engines phd thesis, March 23, 2016 The University of California announced NEW essay prompts for essay 2016-17. Read about how to answer them HERE. This post is now outdated. The information is no longer relevant!! If you are working on your two college application essays for search engines phd thesis the University of California freshman application, I’ve put together a short list of my most helpful posts. Prompt 1: Describe the world you come from top 5 essay, — for example, your family, community or school — and tell us how your world has shaped your dreams and aspirations.

Sample College App Essays for “Describe the World You Come From” UPDATE: as of March 23, 2016 The University of California announced NEW essay prompts for action paper on classroom 2016-17. Read about how to essay services, answer them HERE. This post is now outdated. How To Critique A Thesis! The information is no longer relevant!! Looking for your World to answer the essay, University of how to critique a thesis, California Prompt 1? A high school English teacher contacted me this week asking if I had any sample essays for the University of California college application Prompt 1. She was using my guides and Essay Hell blog posts to help teach her students how to write their college application essays. read more… How to Write the UC Transfer Essay: Why This Major? CHANGED its prompts.

for transfer students. Read Strategies for the New University of California. Transfer Essays for top 5 essay services the updated information on the new prompts. ON NEW UC TRANSFER ESSAYS! (OUTDATED!!) Why You Chose Your Major: A Love Story. If you want to transfer into action any of the University of California schools (UCLA, Berkeley, UCI, UC Davis, UC Santa Cruz, etc.), you need to write two college application essays. One is the same prompt that all students are required to write—which basically asks for top 5 essay a personal statement style essay. It’s known as Prompt 2, and I wrote “Personal Quality, Talent, Achievement…” as a guide on how to rikky muller thesis, write this essay in essay services a narrative style. Now I want to offer some ideas on how to, how to answer the second prompt required for transfer students:

Transfer Student Prompt 1: What is your intended major? Discuss how your interest in the subject developed and services, describe any experience you have had in the field — such as volunteer work, internships and nurse resume maker, employment, participation in student organizations and activities — and what you have gained from your involvement. read more… Don’t Let UC Deadline Ruin Your Turkey Day! If you are applying to the University of California schools, you have until the end of this month. As busy seniors, some of you might have waited to essay, write your essays over Thanksgiving, when you have some days off and essay format diagram, can catch up. Yes, it would be better if you already had them in, but there is top 5 still time. The key, however, is to not let this last minute deadline dash ruin your Thanksgiving. Critique Paper! The holiday comes late this year–Nov.

27–so it’s closer to the Nov. 30th deadline than ever. So yes, you are really cutting it close. If you don’t have a plan, it could hang over your head the entire holiday weekend. read more… A Peek into the Many Worlds of Prompt 1 of the UC Essays. UPDATE: The University of California announced NEW essay prompts for 2016-17.

Read about essay services, how to answer them HERE . The following content in phd thesis this post is no longer relevant and top 5 services, is outdated! About three years ago, I wrote a post to essay on transportation, try to help students applying to services, the University of diagram, California find topics to answer Prompt 1 for their college application essay: Describe the services, World You Come From . Since I shared my advice in my Describe the World You Come From post , I have received more than a 100 comments from students. Most have specific questions, mainly trying to see if their idea of a “world” would make a great essay. Since then, I tried to short, answer most of essay services, their questions. This year, I am so swamped with tutoring students, however, that I’m not able to answer all the questions right now.

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