Phenylalanine hydroxylase (PAH) Gene Essay Example

Phenylalanine hydroxylase (PAH) Gene Paper Phenylalanine hydroxylase (PAH) Gene Background: We will write a custom essay sample on Phenylalanine hydroxylase (PAH) Gene specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on Phenylalanine hydroxylase (PAH) Gene specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on Phenylalanine hydroxylase (PAH) Gene specifically for you FOR ONLY $16.38 $13.9/page Hire Writer Phenylalanine hydroxylase (PAH) encodes the liver-secreted enzyme of the same name, a catalyst for the hydroxylation of tyrosine from phenylalanine, a rate-limiting step in the catabolism of the latter. This reaction only occurs in the presence of the cofactor tetrahydrobiopterin (BH4) as well as molecular oxygen and iron (1). Mutations in the PAH gene are generally caused by a change of an amino acid, for example, the change of arginine to tryptophan (2, 3). The numerous possible mutations in this gene result in a lack of enzyme activity. Thus, because of its main function, the deficiency in the activity of PAH causes a marked intolerance of the consumption of phenylalanine, an essential amino acid. This causes phenylketonuria (PKU), non-phenylketonuria hyperphenylalaninemia (non-PKU HPA), mild hyperphenylalaninemia (MHP), and other variant PKU (4, 5, 6). Defects in the PAH gene leads to the deficiency or the disruption of the production of the PAH enzyme; this is most commonly related to the resulting disorder, phenylketonuria. PKU is an autosomal, inborn, recessive disorder of phenylalanine metabolism (7). There are three common types of PKU. First, there is classical PKU, caused by the mutation of both alleles of the PAH gene in chromosome 12 which results in a severe deficiency or complete absence of the PAH enzyme, leading to toxic levels of unhydroxylated phenylalanine, typically over 10 times higher than normal concentrations (i.e. over 1000  µmol compared to the normal 100  µmol). Next, there is MHP, the mildest form of the PAH enzyme deficiency, with phenylalanine levels below 600  µmol but above normal. Thirdly, there is non-PKU HPA, caused by mutations in the PAH locus that hinder BH4 synthesis and regeneration. This relatively milder form of the disorder often results in heterozygous cases through a combination of mi ld and severe mutations (4, 7, 8). Severe classical PKU, if left untreated, is commonly known to result in the impedance of postnatal cognitive development causing mental retardation and in metabolic abnormalities causing increased phenylalanine in in the blood circulation and phenylpyruvic acid in the urine. PKU has also been known to cause skin abnormalities, organ damage, different kinds of posture peculiarities, pregnancy problems (maternal PKU), an odor describe as â€Å"mousy†, as well as other mental issues such as epilepsy, hyperactivity, and psychotic episodes (1,4,7,8). The most common negative effect associated with PKU, mental retardation, is caused by a neurotoxic effect of HPA. And while PKU is an inherited disorder, its negative effects could also be induced in the offspring of mothers with PKU, resulting not only in high fetus mortality rates but also in a high probability that the children are born with growth and mental retardations as well as malformations. This is known as PKU embryofetopath y or maternal PKU syndrome (8). Conversely, children born with non-PKU HPA and MHP have marked lower risks of being affect with the adverse effects of the disorder and can have normal development mentally and physically even with the absence of treatment (4,8). Despite the severe potential effects of classical PKU, newborn screening for high levels of phenylalanine has helped early diagnosis of the disorder, which is then followed by rapid treatment. Dietary restrictions of phenylalanine has been used for early treatment of PKU which, while not necessarily lead to complete normalization of IQ, was shown to be predictive of overall IQ with the complete lack of treatment in classical PKU patients leading to severe and irreversible cognitive retardation.(1,8) Thus, primary screening of neonates and children as well as awareness of the disorder for the parents are essential (3, 6). Results and Discussion: PAH chromosomal map position and nearby genes: The location of the PAH gene is at chromosome 12. Its long arm (q) is comprised of 13 exons with an approximate length of 90 kb. Figure 1 Chromosome 12 (9) Figure 1, above, is a representation of the entire chromosome 12 with both its short arm (p) and long arm (q) as it appears in the Ensembl website, albeit cropped to fit the page. This figure can be found by searching for the PAH gene and clicking on the â€Å"Location† link on the PAH listing. The website lists the location of the gene to be at â€Å"Chromosome 12: 103,232,104-103,311,381 reverse strand.†(2) Though the website does not explicitly state where in chromosome 12 PAH is located, one can infer additional details from the provided images. For example, confusion can ensue from the fact that the indicated location in the image in the Ensembl website is on the long arm on q23.2, while previous sources have stated that it is located on q22-24.2. However, from the code in the location and the additional images, one can infer that these are the transcribed portions of the gene, two of which are illustrated in the site. Furthermore, one can see that the PAH gene is flanked by the genes insulin-like growth factor 1 (IGF1), or somatomedin C, and achaete-scute complex homolog 1 (ASCL1). To obtain the information, though, one needs to explore the interactive image (see Figure 2 below) and go to the individual pages of the neighbor genes. Figure 2 Detailed view of region near PAH (9) The NCBI website, however, while very extensive in details, and containing multiple transcripts pertaining to the PAH gene, can be somewhat confusing with regard to the Map Viewer. Going through the home page and directly searching for the desired gene results in a very large and confusing map, with the details of the gene and its neighboring gene beyond the page to right. For a beginner who is not quite sure what to look for, the NCBI Map Viewer can be very overwhelming. Focusing on the table and not the map, however, one can see that the PAH gene is located in Chromosome 12, in the long arm q22-q24.2; this information is under the heading â€Å"Cyto† (for cytogenic) and stated as â€Å"12q22-q24.2† (10). Again, this might not be immediately clear to a beginner. Furthermore, the different master map options (Morbid, Gene_cyto, etc.) individually show different arrangements of the symbols, not all of which seem to be genes. Thus, it is very hard to decipher which genes are actually near PAH, although zooming in on the â€Å"Genes on Sequence† and â€Å"Phenotype† maps do reveal the proximity of IGF1 and ASCL1. In all, for a beginner, the Ensembl website proved to be much easier to use to answer the first question. The intron/exon structure of the PAH gene: It was very difficult to find an illustration of the structure of the PAH gene in the NCBI website. However, the information page for the gene stated that the gene spans 90 kb with the entire sequence and its adjacent regions a total of 171 kb. Furthermore, it states that the gene contains 13 exons, which consequently means that it has 12 introns (number of introns is one less than the number of exons) (1). After some searching, however, beginning with clicking the available links for PAH in the Map Viewer table, the link â€Å"sv† led to a page with the title â€Å"Homo sapiens chromosome 12 genomic contig, GRCh37 reference primary assembly.† Searching for the gene gives the following (zoomed-in and cropped) structure:   Figure 3 Structure of PAH gene (11) Though not obvious from the first glance, later we will see that the bottom sequence actually represents the structure of the PAH, with the vertical green lines representing the 13 exons. After further searching, the following (rotated) PAH structure showing the 13 exons and 12 introns can be found in the Map Viewer under â€Å"ensRNA†:   Figure 4 Another illustration of the structure of PAH gene (11) Finding those, however, takes previous explicit knowledge and some work to track down the specific illustrations. In contrast, finding the number of exons and introns and an illustration of the structure of the PAH gene in the Ensembl website was very straightforward. The following illustration can be found in the same page as Figure 1: Figure 5 Ensembl illustration of PAH gene structure This strand, one of the transcripts available in the Ensembl page, clearly shows the 13 exons in a DNA sequence. Comparing this structure to Figures 3 and 4, the numbers and the arrangements of the exons and introns are exactly the same. However, relative to all the tedious searching needed to find the same answers in the NCBI website, the information needed for the question was instantly available from the Ensembl site, and the interface was very easy to understand. Common PAH mutations: Mutations in general can refer to abnormalities in function or structure of the concerned enzyme in the gene phenotype. As previously discussed, however, such as the causes of PKU and HPA, the human PAH gene has displayed allelic differences and pathogenic transformations throughout its structure. The common types of mutations and their occurrence according to a previous study are: missense mutations with 62% of the alleles, small or large deletions with 13%, splicing defects with 11%, silent polymorphisms with 6%, nonsense mutations with 5%, and insertions with 2% of the PAH alleles. (6) Table1 PAH mutation statistics Mutation Type: # of Mutation(s) Missense 336 Deletion 73 Splice 62 Silent 32 Nonsense 28 Insertion 10 Sil./Splice 3 Unknown 3 Total mutations: 547 Most reported Mutation (Association): p.R408W (214) Missense, as can be seen above, is the most common cause of mutation in the PAH gene, the molecular mechanism of this is the improper folding of the protein structure, causing aggregation or degradation. As mentioned earlier, the mutations of PAH are commonly caused by single changes in the amino acid. One of the missense mutations, for example, occurs in E1 nucleotide 1 with the change of ATG to GTG. However, there is also missense mutation in region E3 with sequence 187.000 in nucleotide 187; this is called ACC/CCC;CAC/AAC. The second most common type of mutation is deletion. An example of deletion mutation is in regions E2-12 with sequence 168.001 in nucleotide 168. This is called GAG/GAA;G/A and has been noted to have occurred in Palestinians Arabs. (2, 3, 12)   Other examples can be seen in Appendix (I). As mentioned earlier, there are three common variations of PKU: classical PKU, MHP, and non-PKU HPA. These variations which are basically different degrees of severity of the disorder are caused by the different kinds of mutations that cause varying PAH activity as well as allelic variations. The latter effect at the locus of the gene determines the metabolic phenotype of the enzyme deficiency. In general, however, the mutations in the PAH gene are localized in a main part of the gene instead of being randomly distributed, as they occur either within or without the active site. What is interesting to note is that the PAH gene in intron 12 involves the single base change of guanine to adenine in the canonical 5-prime splice donor site where the first identified PKU mutation occurred. (3) Two out of the 6 links given by the Gene Gateway page were no longer working, one was solely dedicated to SNP, one was a link to a database that had links to other databases, and the last two were already explored thoroughly in previous parts of this assignment. The data presented in this section were mostly from the entire site dedicated to PAH gene mutations, the Phenylalanine Hydoxylase Locus Knowledgebase (5). This site, also a database, was arrived at after searching through the Locus Specific Mutation Databases which in turn arrived at from Human Genome Variation Society: Variation Databases and Related Sites. While the OMIM site did give some details about previous studies related to PAH gene mutations, they were more of a history of the mutations and examples of the studies. Finding the needed information was difficult because one needed to go through link after link and website after website, sometimes even arriving at the same website numerous times through different pathwa ys and still not obtaining any results. The PAHdb was by far, the only site that showed any data regarding the common mutations. Single nucleotide polymorphisms (SNPs) of the PAH gene: To date, 1220 SNPs for the PAH gene have been discovered, although GeneCards (2) states only 1097 from the NCBI website. In general, the SNPs involve the changing of a single base, as shown in Appendices I and II. Examples are the three found on exon 3, each of which has a single change of base, name cytocine, thiamine, and adeninine(13). Examples of these PAH gene SNPs are the rs63749677, rs63749676, rs63581460 and rs63499960; some of these are tabulated in Appendix (II). These SNPs are not randomly distributed as out of the 13 exons, they are seen in exons 1-7 and 12. Searching the NCBI website, however, resulted in 55 entries of SNPs with the following format: rs79931499 [Homo sapiens] CAATCCTTTGGGTGTATGGGTCGTAG[C/G]GAACTGAGAAGGGCCGAGGTATTGT 12 The above entry, an example of the results from the query in the NCBI SNP website, shows essential information about the SNP as well as options one can view. Compared to the other related links, which did not yield any useful information other than linking back to this site, the NCBI site dedicated purely to SNPs was simple and the information was easy to retrieve. Due to the very large number of SNPs, however, it would be difficult to evaluate all of them. Designing PCR primers: The given instructions and the program given in the website were rather straightforward, so the designing of the primer was the easiest part of the activity. The mRNA sequence was easily downloadable and the program was user-friendly (14). Being able to design primers this way was very fast and easy. The resulting primers are in Appendix (III). References: 1. [26/08/10]; Available from: http://www.ncbi.nlm.nih.gov/omim/612349 2. Hoeks M, den Heijer M, Janssen M. Adult issues in phenylketonuria. The Netherlands journal of medicine2009;67(1):2. 3. [21/09/09]; Available from: http://www.ensembl.org/index.html. 4. [26/08/10]; Available from: http://www.genecards.org/cgi-bin/carddisp.pl?gene=PAHsearch=pah#loc 5. [26/08/10]; Available from: http://www.pahdb.mcgill.ca. 6. Carter K, Byck S, Waters P, Richards B, Nowacki P, Laframboise R, et al. Mutation at the phenylalanine hydroxylase gene (PAH) and its use to document population genetic variation: the Quebec experience. European Journal of Human Genetics1998;6(1):61-70. 7.   [26/08/10]; Available from: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gndpart=phenylketonuria 8. [26/08/10]; Available from: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=genepart=pku 9. [26/08/10]; Available from: http://www.ensembl.org/Homo_sapiens/Location/View?db=core;g=ENSG00000171759;r=12:103232104-103311381;t=ENST00000307000 10. [26/08/10]; Available from: http://www.ncbi.nlm.nih.gov/projects/mapview/maps.cgi?taxid=9606chr=12MAPS=pheno,morbid,genec,decode,ensrna,ensgenes,rnaRn,rnaMm,rnaHs,rnaGga,rnaBt,gbdna,rna,ugHs,genes-rcmd=focusfill=80query=uid(136508683,136446655,12845117,12579049,8990832,717234,698472,11088097,11049717,6481463,570698,568170,34586070,16320694,13572526,34590012,128619463,415205)QSTR=pah 11. [26/08/10]; Available from: http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NT_029419.12v=65375409..65454686 12. *Robin A Williams, 2 Cyril DS Mamotte,2 *John R Burnett1,3. Phenylketonuria: An Inborn Error of Phenylalanine Metabolism 13.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚      [updated 21/09/09]; Available from: http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=5053 14.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚      [21/09/09]; Available from: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi Appendices: Appendix (I) Examples 1. Systematic Name: c.1AG Region: E1 Reference (1st): Mutation Name: p.M1V Sequence: 0.000 JOHN SW, ROZEN R, LAFRAMBOISE R, LABERGE C, SCRIVER CR: Novel PKU mutation on haplotype 2 in French-Canadians. Am J Hum Genet 45:905-909, 1989 Other Name: ATG/GTG Length: 1 Nucleotide No.: 1 Rest. Site: -Xba I Mutation Type: Missense Syst. Name gDNA: Date Entered: 1997-01-31 CpG/Fs/Pm: No/No/No 2. Systematic Name: c.3GA Region: E1 EIKEN HG, KNAPPSKOG PM, APOLD J, SKJELKVÃ…LE L, BOMAN H: A de novo phenylketonuria mutation: ATG (Met) to ATA (Ile) in the start codon of the phenylalanine hydroxylase gene. Hum Mut 1:388-391, 1992 Mutation Name: p.M1I Sequence: 3.000 Other Name: ATG/ATA Length: 1 Nucleotide No.: 3 Rest. Site: -NspI Mutation Type: Missense Syst. Name gDNA: Date Entered: 1997-01-31 CpG/Fs/Pm: No/No/No 3. Systematic Name: c.117CG Region: E2 FORREST SM, DAHL HH, HOWELLS DW, DIANZANI I, COTTON RGH: Mutation detection in phenylketonuria by using chemical cleavage of mismatch: Importance of using probes from both normal and patient samples. Am J Hum Genet 49:175-183, 1991 Mutation Name: p.F39L Sequence: 117.000 Other Name: TTC/TTG Length: 1 Nucleotide No.: 117 Rest. Site: -MboII, +MaeIII Mutation Type: Missense Syst. Name gDNA: Erlandsen H, Pey AL, Gà ¡mez A, Pà ©rez B, Desviat LR, Aguado C, Koch R, Surendran S, Tyring S, Matalon R, Scriver CR, Ugarte M, Martà ­nez A, Stevens RC.: Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations. Date Entered: 1997-01-31 CpG/Fs/Pm: No/No/No Appendix (II) SNPs of the PAH gene Region Contig position mRNA pos dbSNP rs# cluster id Hetero- zygosity Function dbSNP allele Protein residue Codon pos Amino acid pos exon_12 26716405 1750 rs59326968 N.D. synonymous C Asn [N] 3 426 contig reference T Asn [N] 3 426 exon_7 26728783 1314 rs5030851 N.D. missense T Leu [L] 2 281 contig reference C Pro [P] 2 281 exon_6 26731200 1061 rs5030653 N.D. missense (22bp) [CIKPMLAN] 1 197 frame shift -/TGTATAAAACCCATGCTTGCTA 1 197 contig reference (22bp) [LYKTHACY] 1 197 26731262 1020 rs17852373 N.D. missense G Gly [G] 2 183 contig reference A Glu [E] 2 183 exon_3 26770856 671 rs5030842 N.D. missense C Pro [P] 1 67 contig reference T Ser [S] 1 67 contig reference A Ser [S] 3 36 exon_1 26793098 474 start codon 1 Appendix (III) Designed Primers Exon1 ENSE00001141448 CAGCTGGGGGTAAGGGGGGCGGATTATTCATATAATTGTTATACCAGACGGTCGCAGGCT TAGTCCAATTGCAGAGAACTCGCTTCCCAGGCTTCTGAGAGTCCCGGAAGTGCCTAAACC TGTCTAATCGACGGGGCTTGGGTGGCCCGTCGCTCCCTGGCTTCTTCCCTTTACCCAGGG CGGGCAGCGAAGTGGTGCCTCCTGCGTCCCCCACACCCTCCCTCAGCCCCTCCCCTCCGG CCCGTCCTGGGCAGGTGACCTGGAGCATCCGGCAGGCTGCCCTGGCCTCCTGCGTCAGGA CAACGCCCACGAGGGGCGTTACTGTGCGGAGATGCACCACGCAAGAGACACCCTTTGTAA CTCTCTTCTCCTCCCTAGTGCGAGGTTAAAACCTTCAGCCCCACGTGCTGTTTGCAAACC TGCCTGTACCTGAGGCCCTAAAAAGCCAGAGACCTCACTCCCGGGGAGCCAGCATGTCCA CTGCGGTCCTGGAAAACCCAGGCTTGGGCAGGAAACTCTCTGACTTTGGACAG PCR primer design: No mispriming library specified Using 1-based sequence positions OLIGO  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  start   Ã‚  len   Ã‚  Ã‚  tm   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  gc%   Ã‚  any     Ã‚  3  Ã‚  Ã‚  Ã‚  Ã‚     seq LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  369  Ã‚   20  Ã‚   59.83  Ã‚   55.00   6.00   2.00   Ã‚  TCCTCCCTAGTGCGAGGTTA RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚     522  Ã‚   20  Ã‚   59.98  Ã‚   55.00   3.00   2.00   Ã‚  CAGAGAGTTTCCTGCCCAAG SEQUENCE SIZE: 533 INCLUDED REGION SIZE: 533 PRODUCT SIZE: 154, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 3.00 1 CAGCTGGGGGTAAGGGGGGCGGATTATTCATATAATTGTTATACCAGACGGTCGCAGGCT 61 TAGTCCAATTGCAGAGAACTCGCTTCCCAGGCTTCTGAGAGTCCCGGAAGTGCCTAAACC 121 TGTCTAATCGACGGGGCTTGGGTGGCCCGTCGCTCCCTGGCTTCTTCCCTTTACCCAGGG 181 CGGGCAGCGAAGTGGTGCCTCCTGCGTCCCCCACACCCTCCCTCAGCCCCTCCCCTCCGG 241 CCCGTCCTGGGCAGGTGACCTGGAGCATCCGGCAGGCTGCCCTGGCCTCCTGCGTCAGGA 301 CAACGCCCACGAGGGGCGTTACTGTGCGGAGATGCACCACGCAAGAGACACCCTTTGTAA 361 CTCTCTTCTCCTCCCTAGTGCGAGGTTAAAACCTTCAGCCCCACGTGCTGTTTGCAAACC 421 TGCCTGTACCTGAGGCCCTAAAAAGCCAGAGACCTCACTCCCGGGGAGCCAGCATGTCCA 481 CTGCGGTCCTGGAAAACCCAGGCTTGGGCAGGAAACTCTCTGACTTTGGACAG KEYS (in order of precedence): left primer right primer ADDITIONAL OLIGOS start   Ã‚  len   Ã‚  Ã‚  tm   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  gc%   Ã‚  any     Ã‚  Ã‚  Ã‚  3  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     seq 1 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  339  Ã‚   20  Ã‚   59.77  Ã‚   50.00   Ã‚  3.00   Ã‚  1.00  Ã‚  Ã‚     ACGCAAGAGACACCCTTTGT RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   522  Ã‚   20  Ã‚   59.98  Ã‚   55.00   Ã‚  3.00   Ã‚  2.00   Ã‚  Ã‚  Ã‚  Ã‚  CAGAGAGTTTCCTGCCCAAG PRODUCT SIZE: 184, PAIR ANY COMPL: 6.00, PAIR 3 COMPL: 2.00 2 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   318  Ã‚   20  Ã‚   59.32  Ã‚   55.00   4.00   2.00 GTTACTGTGCGGAGATGCAC RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   522  Ã‚   20  Ã‚   59.98  Ã‚   55.00   3.00   2.00 CAGAGAGTTTCCTGCCCAAG PRODUCT SIZE: 205, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 2.00 3 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   157  Ã‚   20  Ã‚   60.07  Ã‚   55.00   2.00   0.00 CTGGCTTCTTCCCTTTACCC RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   337  Ã‚   20  Ã‚   59.32  Ã‚   55.00   4.00   3.00 GTGCATCTCCGCACAGTAAC PRODUCT SIZE: 181, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 1.00 4 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   156  Ã‚   20  Ã‚   60.07  Ã‚   55.00   3.00   0.00 CCTGGCTTCTTCCCTTTACC RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   337  Ã‚   20  Ã‚   59.32  Ã‚   55.00   4.00   3.00 GTGCATCTCCGCACAGTAAC PRODUCT SIZE: 182, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 2.00 Statistics con  Ã‚   too  Ã‚  Ã‚   in  Ã‚  Ã‚   in  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   no  Ã‚  Ã‚   tm  Ã‚  Ã‚   tm   high   high  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   high sid   many  Ã‚   tar   excl  Ã‚   bad  Ã‚  Ã‚   GC  Ã‚   too  Ã‚   too  Ã‚   any  Ã‚  Ã‚   3   poly  Ã‚   end ered  Ã‚  Ã‚   Ns  Ã‚   get  Ã‚   reg  Ã‚   GC% clamp  Ã‚   low   high compl compl  Ã‚  Ã‚  Ã‚   X   stab  Ã‚  Ã‚   ok Left  Ã‚  Ã‚   3637  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚   162  Ã‚  Ã‚  Ã‚   0  Ã‚   419   2558  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   2  Ã‚  Ã‚   22  Ã‚  Ã‚   73  Ã‚   401 Right  Ã‚   3701  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚   130  Ã‚  Ã‚  Ã‚   0  Ã‚   321   2817  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   2  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚   78  Ã‚   353 Pair Stats: considered 140, unacceptable product size 129, high end compl 3, ok 8 primer3 release 1.1.4 KEYS (in order of precedence): left primer right primer ADDITIONAL OLIGOS start   Ã‚  len   Ã‚  Ã‚  tm   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  gc%   Ã‚  any     Ã‚  3  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     seq 1 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   19  Ã‚   20  Ã‚   60.21  Ã‚   50.00   5.00   2.00   Ã‚  Ã‚  Ã‚  GCAGTGCCCTCCAGAAAATA RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   265  Ã‚   20  Ã‚   58.12  Ã‚   40.00   3.00   0.00   Ã‚  TCAAAGATGACCCCAAAAGA PRODUCT SIZE: 247, PAIR ANY COMPL: 2.00, PAIR 3 COMPL: 0.00 2 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   19  Ã‚   20  Ã‚   60.21  Ã‚   50.00   5.00   2.00  Ã‚     GCAGTGCCCTCCAGAAAATA RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   260  Ã‚   22  Ã‚   60.05  Ã‚   40.91   4.00   0.00   Ã‚  GATGACCCCAAAAGATTTACCA PRODUCT SIZE: 242, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 1.00 3 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   45  Ã‚   20  Ã‚   60.39  Ã‚   50.00   6.00   1.00  Ã‚     AGCCATGGACAGAATGTGGT RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   265  Ã‚   20  Ã‚   58.12  Ã‚   40.00   3.00   0.00   Ã‚  TCAAAGATGACCCCAAAAGA PRODUCT SIZE: 221, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 1.00 4 LEFT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   19  Ã‚   20  Ã‚   60.21  Ã‚   50.00   5.00   2.00   Ã‚  Ã‚  Ã‚  GCAGTGCCCTCCAGAAAATA RIGHT PRIMER  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   258  Ã‚   20  Ã‚   57.92  Ã‚   40.00   4.00   0.00   Ã‚  TGACCCCAAAAGATTTACCA PRODUCT SIZE: 240, PAIR ANY COMPL: 4.00, PAIR 3 COMPL: 1.00 Statistics con  Ã‚   too  Ã‚  Ã‚   in  Ã‚  Ã‚   in  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   no  Ã‚  Ã‚   tm  Ã‚  Ã‚   tm   high   high  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   high sid   many  Ã‚   tar   excl  Ã‚   bad  Ã‚  Ã‚   GC  Ã‚   too  Ã‚   too  Ã‚   any  Ã‚  Ã‚   3   poly  Ã‚   end ered  Ã‚  Ã‚   Ns  Ã‚   get  Ã‚   reg  Ã‚   GC% clamp  Ã‚   low   high compl compl  Ã‚  Ã‚  Ã‚   X   stab  Ã‚  Ã‚   ok Left     Ã‚  7708  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚   791  Ã‚  Ã‚  Ã‚   0   4562  Ã‚   600  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚   14  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚   52   1689 Right  Ã‚   7734  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   0   1269  Ã‚  Ã‚  Ã‚   0   4609  Ã‚   311  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚  Ã‚   6  Ã‚  Ã‚  Ã‚   0  Ã‚  Ã‚   44   1495 Pair Stats: considered 2222, unacceptable product size 2195, high end compl 6, ok 21 primer3 release 1.1.4

Gold Rush essays

Gold Rush essays The California Gold Rush was one of the most important events in the 19th century. Without it America would not be as populated and economically strong as it is today. The California Gold Rush brought many foreigners into the United States and triggered migration to the Western Coast. The California Gold Rush taught people that failure was okay and made people take risks. The California Gold Rush was the Las Vegas of the 19th century. The California Gold Rush blew the United States economy sky high and gave America a very useful and attractive resource. The California Gold Rush started at John Sutters Mill in the beginning of 1848. The first gold, discovered in January, 1848. Now if Mrs. Wimmer California Gold Rush possesses a piece which has been found earlier than mine Mr. Marshall can tell, as it was probably received from him. I think Mr. Marshall could have hardly known himself, which was exactly the first little piece, among the whole. As soon as the first piece was discovered the word spread and many within the area joined. Within time many people came from many different places within and outside the United States. People came to California to escape poverty, become rich, to cover their debts, and to try to go into early retirement. Many Argonauts were left by their spouses for going to California and were criticized by many for not helping their family. From 1848 to 1852 Californias population grew from 14,000 to 223,856 . The spread of Gold Fever was extremely rapid due to the fact that the United States had just won the Mexican War. The press (media) also had a big part in the California Gold Rush because it made people believe that every single gold digger that came to California would become a millionaire in no time and with very little effort. The news spread outside of the United States and soon immigrants started coming from countries such as China, Chili, M...

SWOT Analysis Essay Example | Topics and Well Written Essays - 2000 words

SWOT Analysis - Essay Example Valley National Bank (VNB), as one of the leading regional banks, offers better opportunities for professionals in banking for building their career. In the context of opportunities available in Valley National Bank, there are diverse job opportunities such as Benefit Specialists, Branch Sales Manager, Consumer Loans Quality Analyst, Loan Review Manager, Secondary Marketing Manager and Senior Attorney. Valley National Bank presently employs over 56 staff members in a variety of positions. The bank also provides salaries and package benefits to permanent employees. It can also be observed that Valley National Bank provides certain permanent and wide ranging employment positions. Valley National Bank is considered as one of the recognized leaders in financial services due to its outstanding performances, enhanced customer services and diversified products. The bank offers wide ranging products having an asset of over US$16 billion and 211 branches along with a diverse set of customers. There are different benefits offered by the bank in the sector of health and welfare. Moreover, Valley National Bank provides high priority on customer offering services through commercial and wealth management (CareerBuilder, LLC, â€Å"Overview†). ... An association with the bank in a wide range of functionalities and positions would help an individual to growth his/her future by a considerable extent (CareerBuilder LLC, â€Å"Overview†) With these considerations, the paper intends to prepare a SWOT analysis of the organization and comprehend the prevailing employment opportunities for an individual within the premises. SWOT ANALYSIS OF VALLEY NATIONAL BANK Valley National Bank is an association of banking that has been chartered in 1927, within the laws incorporated in the United States. It is headquartered in New Jersey. The bank is operating in around 198 full service offices all over central and northern along with central part of New Jersey and city of New York in Brooklyn, Manhattan and Queens. The bank facilitates a variety of commercial, retail and wealth management services. Other services that are offered by the bank are internet banking, telephones, automatic teller machines facilities of safe and night deposits, letter of credit, foreign exchange and foreign banking account maintenance. The bank provides various affluent services to the customers for 24 hours in a day and for 7 days in a week (Form 10-K, â€Å"Business†) With regard to strengths of the organization i.e. Valley National Bank, it can be stated that the staff and managers in the organization are skillful and the operational activities are handled according to the guidelines of the organization. The managerial skills that are persuaded by management facilitate in maintaining efficiency in the activities performed in the bank. Moreover, it can be observed that the employees who are employed in the bank possess a great learning experience as valuable

Human motivation Research Paper Example | Topics and Well Written Essays - 4000 words

Human motivation - Research Paper Example The behavioral scientists have mainly confirmed that raised productivity is a task of the level of human wellbeing. This notion surpasses awareness being focused on instruments only since at the core of the company and its complete structure, the employees enabled with proficiency, knowledge, mind-sets and intellect is the most important their nature in factor grouping of capital generation and creation procedure. Companies, regardless of their nature, always target at attaining their commercial objectives, or else, the endurance of such venture will be more of a delusion and not a reality such extremely significant. The achievement of any company is mostly calculated by the level of its productivity. Even though, this can be considered autonomous on the mind-set as well as self-esteem of the personnel in form of their level of job dedication. No corporation can consequently afford to overlook any of the factors that may contribute to the improving of the dedication levels of its emp loyees, which is motivation. During every productive activity, the fundamental components and factors incorporate property, funds, labor, and the capitalist (Bruce, 2002, p. 56). The laborers and the capitalists are human and as such extremely significant within any productive venture. They make use of the other aspects for the understanding for the objective of the venture. It can then be adduced that human beings have a very significant position within any system and especially in organizations.... Companies, regardless of their nature, always target at attaining their commercial objectives, or else, the endurance of such venture will be more of a delusion and not a reality. The achievement of any company is mostly calculated by the level of its productivity. Even though, this can be considered autonomous on the mind-set as well as self-esteem of the personnel in form of their level of job dedication. No corporation can consequently afford to overlook any of the factors that may contribute to the improving of the dedication levels of its employees, which is motivation. During every productive activity, the fundamental components and factors incorporate property, funds, labor, and the capitalist (Bruce, 2002, p. 56). The laborers and the capitalists are human and as such extremely significant within any productive venture. They make use of the other aspects for the understanding for the objective of the venture. It can then be adduced that human beings have a very significant po sition within any system and especially in organizations. Therefore, they should be considered with a high concern so that they can chip in successfully and competently throughout productive activities. Effect of Motivational Theories A simple meaning of motivation is what makes individuals put actual effort as well as energy into what they perform. One may anticipate the motivational theories to be the ideal place to search for a normally agreed upon meaning. However, the field of motivation is described by a large quantity of various theoretical frameworks and models that make it tricky, if not impractical, to recognize similarities and differences

Stop gambling Essay Example for Free

Stop gambling Essay Today, I’m going to talk about gambling. Nowadays, gambling becomes a entertainment in our life. Some people go there just for fun, but most people go there for money, they think they can make money by gambling. As we known, only very few people can make money from gambling, the winner always not belong to gamblers, so the problem comes out. Most people with gambling problems say they lost control over how much time and money they spend on gambling. Meanwhile, they ignored other responsibilities. They knew they had problems, but only gambling seemed important. Many people who gamble overly have mixed feelings about gambling. They know they are causing problems for the people they love. They may become anxious and unhappy, and often hate themselves. They feel they can’t give up on all the time, money and emotion they have put into gambling. They can’t accept that they will never win the money back what they have lost. Some people still believe their system will pay off, their luck will change or they are due to win. Others believe continue to gamble is the only way to get the money back. Eventually they will lose their money, family, even life. Also, it will make some serious consequences, like suicide, crime, and destructive behaviors. Other people promise to quit, but it is not easy. If they quit now, they will feel like a loser. They will have to face all the problems that gambling has caused. But, only to stop gambling can help them to start a new life. So here are some solutions to help people to quit the gambling. 1 Make a decision on a given day when you wake up that you will not gamble. Decide that for this one day you will not gamble. If you have to do it an hour at a time, it is okay. Sometimes you may have to do it in a minute at a time. it is okay, as you promise yourself in a given moment that you will not gamble. Schedule your day in a very structured way so you do not have a lot of free time. This can really help a lot, as you will have no time to gamble. 2. Find something at that time to replace your gambling. One of the way to quit gambling is to be able to find healthy replacements that will substitute for your gambling activities. Exercise, go shopping, go out with friends, or do some cooking. You can also watch a movie, or listen to some soft music or do some reading and so on. Anything, and I mean anything not go back to gamble. Your goal is to stop gambling, or begin to reduce gambling frequency. 3. Remember the feeling when you leave the casino or lost a lot of money on a bet. Think about how bad it feels when you lose a lot of money. Remember that feeling, and allow yourself to feel that feeling when you are gambling again. Sometimes these feelings can exactly imitate how you feel after a gambling. This can really stop you from going out and placing a bet. So my conclusion is gambling as a entertainment is ok for people, but if you addict to it, eventually you will get hurt. So before gambling, please ask yourself, how you going to be responsible of yourself, your family, and your future.

Hackers- Who Are They? And What Do They Do? Essay -- essays research pa

Computer Hackers Outline Thesis : Contrary to popular belief, not only do you not have to be smart to be a hacker, you do not even need to know much about computers. I. Hacking today A. More like exploration in the 60's and 70's B. Cannot call themselves hackers II. Different Types of hackers A. A hacker defined B. A cracker defined C. A phreaker defined III. You do not have to be intelligent A. Not like you have seen on television B. Programs used are highly accessible on the Internet C. Some start out with trial and error VIII. Online Legalities A. Unauthorized access to, or use of a computer B. Alteration or destruction of data without authorization C. Theft of computer services D. Computer fraud (using a computer to commit other crimes) E. Denial of access to a computer F. Unauthorized possession of passwords __________________________________ Hackers Many of the people in our society today often think that computer hackers are very smart individuals, who have special talents and abilities enabling them to crack passwords, enter mainframes, send viruses, and crash computer systems. Contrary to popular belief, not only do you not have to be smart to be a hacker, you do not even need to know much about computers. Modern day hacking usually ends in destruction of other peoples property, or changing important private information. Also, as stated by a 42-year-old security agent in San Francisco, â€Å" ‘Hacking in the 1960s and 1970s was a little more like gentlemanly exploring than it was taking things from people† ’(Weise). These days hackers cannot call themselves hackers without the threat of being called a â€Å"hacker wanna-be† or a â€Å"script kiddie.† A â€Å"hacker wanna-be† is the same thing as script kiddie, but is another slang term for a person who uses guess-work and programs that he did not write (Weise). Although, most people think of a hacker as someone who breaks into computers and sends viruses, there are several different types of hackers, broken up into three different groups. The first type of hacker is the hacker himself. The defination of a hacker is : â€Å"[hack$dr (hà £k=dr)] One who enjoys the intellectual challenge of creatively overcoming or circumventing limitations† (Dict... ...ere are actually very strict guidelines on the Internet of what you can and cannot do.The Book of Online Law states that it is illegal for you to have "unauthorized access to, or use of a computer, alteration or destruction of data without authorization, theft of computer services, computer fraud (using a computer to commit other crimes), denial of an others access to a computer " (Smedinghoff). Hacking is starting a program that grants you access to things you normally would not be able to do, or access. You do not have to be smart to be a hacker. Anyone can get hacker tools and programs just by looking for them, downloading them, and then putting them to use. Modern day hacking is not how you see it on television. It is a simple process which any one can do using one of many programs that are available online. There are several different types of hackers. They each depend on what type of hacking is being done. There is one last thing for you to be aware of, now that you know how easy it is to be a hacker. You have to be careful of all the many Internet legalities. You can get in serious trouble and not know you were doing anything wrong until it is too late.

Literature as a Medium Which Conveys the Culture of the Target Language Essay

The purpose of this paper is to familiarize foreign language instructors with the effectiveness of using literature as a mean of bridging the cultural gap between students and the target language. It will also discuss how teaching literature can help students become more effective communicators in the target language and at the same time making them appreciate other cultures different from their own. This may lead to say that, one of the major functions of teaching literature in a foreign language classroom is to serve as a medium to transmit the culture of the target language of the people who speak the language in which it is written. Therefore, literary texts can offer to the students a colorful world that reflects the culture of the language community. As readers; students get engaged within these texts, they come to understand how the characters in such literary works experience life and how they speak and behave in different settings. However , the study of literature can be considered as a method in which students can dive into the cultural experience of the language throughout the imagery and metaphors mentioned in a given text, as Gray points out: â€Å" Students learn to see a world through another’s eye,observing human values , different kind of living and discovering that others live very different societies. they will understand and become broadly aware of the social,political,historical,cultural events happening in a given society. . The role of literature in language teaching Literature, when defined as a subject of study, is an activity that involves and uses language (O’Sullivan, 1991). Ganakumaran et al (2003) stated that it is also referred to as the big â€Å"L† that focuses on the literariness of a text. It is an example of language in use and a context for language use. Literature is seen as a medium in which students’ appreciation of different system language organizat ion can be developed. Therfore, studying literature, on the whole, may not be considered as a reaction but rather as an interaction between the reader , the writer and their cultures. From an other parameter ,the teaching of literature in foreign language classrooms is essential and can be used as a perfect instrument to stimulate and speed up the teaching and learning process Carter and Long (1991). In other words, literature contributes in helping the students to explore knowledge ,promote their literacy development and fosters their language proficiency. Also, literature may provide effective, attitudinal and experiential factors which will motivate students to read McKay (1982). As a matter of fact, Obeidat (1997) said that literature has much to offer than language would normally do, since it has greater freedom and since it acknowledges no linguistic barriers which could refrain our ability to use language. Therefore, it is undeniable that literature helps to improve students’ command of language and the ability to use it freely, not solely as a linguistic, but as a cultural, intellectual, social and psychological medium of expression. In terms of using literature to teach language, Brumfit and Carter (1986) asserted that literary texts not only can grip the reader’s imagination more strongly but they can also be promoted for the examination of language at work. Littlewood (2005) also observed that although such texts were predominantly valuable for developing reading skills, it could be exploited for purposes such as explaining grammar teaching and indicating various types of language usage. Since literary texts contain language intended for native speakers, literature stands as a model for language learners to become familiar with different forms and conventions (Collie and Slater, 1991). Containing real examples of grammatical structures and vocabulary items, the literary texts raise learners’ awareness of the range of the target language and advance their competence in all language skills (Povey, 1967). Second, using literature in language teaching has the advantage of providing cultural information about the target language. Literary texts increase foreign language learners’ insight into the country and the people whose language is being learnt (Collie and Slater, 1991), which fosters learners’ ability to interpret discourse in different social and cultural target language contexts (Savvidou, 2004). . Literature as an instruments that stimulates the knowledge of the culture Teaching literature stimulates the imagination of the students and develops their critical abilities by getting them involved in interpreting the different symbols and meanings found in the text. They become more productive and adventurous when they perceive the richness and diversity of the language. Therefore, it is vital if educators create a harmonious literary atmosphere ,they may enable their students to immerse themselves in the story by turning the classroom into a cultural island where students interact with the text, share different viewpoints about the settings, the style, the language. Researchers have recognized that the benefits of using literature in three core areas: First , literature is beneficial to language development( John and Louis, 1987; Morgan, 1998; Myonghee, 2004;Sage,1987); second, literature enhances the knowledge of ulture and society which is too complicated to be captured by any single piece of expository writing; Third, literature fosters critical thinking by offering readers multiple perspectives especially in books with issues such as immigration, cultural differences, social upheavals (Edmondson 1995/6). John Lye (2003) believed that literature captures the complexities of the human situation, illustrating (dramatically, imaginatively, compellin gly) the grounds and the meanings of values, and the nuances of our experiences. It also allows us imaginatively to inhabit the lives, the experiences, the meanings of others. Lye(2003) added that literature challenges the sentimentalization of values, and those distortions of values which serve the ends of the powerful, the opportunistic, and those who want only to serve their own desires ,from an other parameter, literature explores and reflects conflicts in the culture itself, conflicts in the values and ideas of the culture, the conflict of social groups (ethnic, racial, class, gender, religious and so forth), in addition to that ,it offers a sense of identity and community, by establishing and founding narratives which capture the ‘essence’ of what typifies the culture or community, or by dramatizing those particular stories and images which give the culture a sense of common meaning and purpose. 4.  Applying literature and culture in language teaching classroom When it comes to learning a new language there can be no substitute for actually living in a country where this language is spoken. Besides, teaching literature does not mean ,limit students to take a passive role but to stimulate students to be engaged proactively within the target language. However, foreign language teachers can provide literary texts best by turning their literature classes into a community where students can engage themselves actively with the culture and the language they are studying and where the emphasis in class could be on ‘cultural experience rather than cultural awareness. As Duff and Maley point out, â€Å"literature offers universal themes which are relevant to students’ own experience,it is also a mirror that reflects and heightens each learner’s perception of the social world. Thus, literary texts are opened to multiple interpretations and interactions†. Students cannot remain passive if they are to interpret and understand what they are reading. They are placed â€Å"in an active interactional role in working with and making sense of the target language guided by the teacher, they have to actively participate and interact with each other. Literature and culture in foreign language teaching can provide linguistic and artistic elements and perspectives to students.  Through literature as well as culture, students could have the opportunity to establish a connection with the target language and realize that their own personal, social,cultural and historical contexts have been influenced by a foreign language and a foreign culture and how, in a symbiotic process, this other culture has been nourished. Culture, on the other hand, offers an interdisciplinary field that includes artistic discourses, social conventions, and reflexive impacts. It opens the door for students to increase their knowledge of the target culture as they can contemplate and critically comment on people’s way of life, values, attitudes, and beliefs, and regard how these elements can have an impact in linguistic categories and forms. Indeed,language found a soulmate in literature, and they are inserted in culture.