SECUENCIACION Mol

8/8/2019 SECUENCIACION Mol

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TRABAJO DE BIOLOGIA MOLECULAR IIIFACULTAD DE CIENCIAS DE LA VIDA

INGENIERIA EN BIOTECNOLOGIA

Nombre: Diego Ruales

Biotecnologa 4toC28/09/10

SECUENCIA MUESTRA

1 gacttaattg gattgagcct tggtatggaa acctaccaag tgataacttt caaattcaga 61 gaaaccctgg aattaaaaat gggcaatcct

gagccaaatc ccgttttatg aaaacaaaca 121 aggatttcac aaagcgagaa taaataaagg ataggtgcag agactcaatg gaagctgttc

181 taagagatgg agttggctgc actttgttcg taaaggaatc cttccatcaa aactttccga 241 aaggatgaaa gataaaccta

tagacatatg tatacttatc gccaaattat tcaaaataat 301 taatgacgac tccaaccggt tctataattt ttttatatct atttttatga

aaaataaaag 361 aatttttttg aatcgattca aaataaaaat tgaaaaaaga attaaatatt cattgatcaa 421 atcattcact ccatcatagt

ctgataaatc tttttatttt tcagaattga ttaatcggat 481 aagaataaag atagagtccc attctacatg tcaatatcga caacaatgaa

atttatagta 541 agaggaaaat ccgtcgactt tagaaatcgt gagggttcaa gtccctctat ccccaaaaac 601 caggttgccg cctcaattat

ttatcttctg attttgttcg tgactcaaaa tgggttacgg 661 ttctcaatca ttctcacttt attatttcac aaaccgatcg gtgcggaaat

ttttttcatc 721 ataagccttg ggtgtgatat tgatatatat catacatgtc caaatgcaaa tgagcatctt 781 tgactaatgt attacaatta

ataatccatg tcattatttg tattatttgg actgtattga 841 aacttacaaa gttttctttt taaagataca agaaattcta ccagggccgg

gataatactt 901 tgtaatatct tttggttttt tttaattgac atagacccaa gtcctatatt aaaataaaat 961 gaggatggtg cgtcgtgaat

ggtcgggata gctcagctgg tagagcagag gactgaaaat 1021 c


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SECUNECIA NOCARDIA AOBENSIS PLASMIDO pYS1

1 tcgccatcga tcaggccgcc acgttctccg gtgtcgccta cctccagtcc accccgaaga

61 ccgtgttcgg tcagccggac gtcaaggaca ccaccgctga cggccggatc aagtgggaag

121 tccagcttgt ggcgggtttc aaggaccagt tcggtaaccc atcgcacgag gtcatcaagg

181 tcaacgtcgc ttcccacacc gatccgggcg agggcttgaa cccgtacacc cctgtccagc

241 tgatcaactt cgtcgtcggc gtcgtacctc cggaaatggg ccaggaccgc aacggcaacc

301 agaagatccg tggcggctcc acctggttcc gcgccgacga aatccgttcc accgcaacgc

361 cttccaacgg acgcaccaag acagcaaccg ccgaagccta agccgcacac gaagattcgt

421 ggcaagcccc tcccgctgag ggaggggtgc ggcagcacca acccaacgca ccaccggccc

481 gaatgcacgc gggccgactc ggtaaccacc tcgaactccc actgaccagg agaaacactg

541 tggacatcct gaccaccttc gaaatcctcg gcgccacctc gaccaccggc ctcgcctacc

601 tcggggctca atcgcgcggc tacggcctgc ggtactggca gatcaaggac cccgaactcc


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3781 gctggacaat cgaggcccgg ggaagcccga ccccaccacc ccgcgcacga aaaaggccgg

3841 ccgaatgctc gaccggcccg acgactcccc gcggctatgg ccgatccggc cagacctccg

3901 cccaggtctc tcgggccagc ctcgacgcgg accggttcgc agccgcggta acggtcagcc

3961 ccatcgcccg cgctacctcg gtctgcgtcg tcagcccgtt ccggagcgcg tatgccgcga

4021 gcagatgcgc cgcacgcttt gtgatccgct cggcgtgaga gaagtcctcc agcaacggaa

4081 tcagccactc ggcggccgac tgtccctctc gcacaggccc ttcctgtgag attctgcgag

4141 ctgtcttgac cagcaacgcc gctcgctctt cgacccattc tgccagctca ggcggggtct4201 cctgcggtgt catgtcgtat ctccttgcga ttcgagaaat cggtacctag agtgtagtcg

4261 tacagttttc catttctaga aactagaaat ccacctggaa ggaatccaag aaaatgcctc

4321 agatct

Shibayama,Y. ,Dabs,E.R. ,Yazawa,K. and Mikami,Y. Molecular an Cell Biology, University of the Witwatersrand, 1 Jan Smut Avenue,

Johannesburg, Gauteng 2050 South Africa (07-OCT-2009)

COMPARACION ENTRE LAS SECUENCIAS

Al comparar ambas secuencias genmicas la primera diferencia que salta a la vista que lasecuencia muestra es lineal mientras que la secuencia NOCARDIA AOBENSIS PLASMIDO

pYS1 es circular; la secuencia muestra solo es afectada por pocas metilaciones en sus sitios decorte por enzimas de restriccin por otro lado el plsmido en sus cortes por las enzimas derestricciones es afectado con mayor frecuencia por la metilacin; estas comparaciones tambintienen su base en que la secuencia muetra es de solo 1021pb mintras que el plasmido es de4326 pb. (NEBcutter 2010).


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APLICACIONES

Cuatro nocardioformas bacterianos fueron aisladas de fuentes clnicas respiratorias (W7467,W7811, W9013 y W8061T) en los Estados Unidos. El examen macroscpico mostr hifas areasescasas e hifas de sustrato de color beige y rojo. Mostraron marcadores taxonmicos que eran

compatibles con la clasificacin de Nocardia: es decir, cido, meso diaminopimlico, arabinosa ygalactosa como azcares de diagnstico, el difosfatidilglicerol fosfolpidos, fosfatidiletanolamina,fosfatidilinositol y mannosides fosfatidilinositol, un menaquinona con una cadena lateral omega-cclica isopreno MK- 8 (H4cycl.), un perfil de cidos grasos no ramificados compuesto de grasassaturadas y cidos grasos monoinsaturados con una cantidad considerable de cidotuberculosterico, y los cidos miclicos que abarca desde 52 hasta 62 tomos de carbono contres principales cidos miclicos que fueron mono-y poli insaturados que muestra una longitudde cadena C54, C56 y C58. Secuencias de genes y caracteres fenotpicos mostraron queestaban ms estrechamente relacionados con aobensis Nocardia


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ENZIMATaqII

ABSTRACTThe TspDTI restriction endonuclease, which shows a novel recognition specicity 5-ATGAA(N11/9)-3, was isolated from Thermus sp. DT. TspDTI appears to be a `twin' of restrictionendonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI wasisolated from the same location as TspDTI, it recognizes a related sequence 5-ACGGA (N11/9)-


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3 and has conserved cleavage positions. Both enzymes resemble two other class- IISendonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences ofTspGWI tryptic peptides exhibit 88.9100% similarity to the TaqII sequence. All four enzymeswere puried to homogeneity; their polypeptide sizes (114.5122 kDa) make them the largestclass-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of

class-IIS restriction endonucleases of a common origin is herein proposed.

Abstract

Background: Restriction-modification systems are a diverse class of enzymes. They areclassified into four major types: I, II, III and IV. We have previously proposed the existence of aThermus sp. Enzyme family, which belongs to type II restriction endonucleases (REases),however, it features also some characteristics of types I and III. Members include relatedthermophilic endonucleases: TspGWI, TaqII,TspDTI, and Tth111II.

Results: Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme

that

recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, andmutagenised the tspgwi gene and investigated the properties of its product, the bifunctionalTspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, acloning method was devised, based on amino acid sequencing of internal proteolytic fragments.The deduced amino acid sequence of the enzyme shares significant sequence similarity withanother representative of the Thermus sp. family TaqII. Interestingly, these enzymes recognisesimilar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, butdiffer in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an


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allosteric activator for cleavage. Both the restriction endonuclease (REase) andmethyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolatedfrom Thermus sp.) are dependent on the presence of divalent cations.

Conclusion: TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like

domains; particularly noticeable is the central HsdM-like module comprising a helical domain anda highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containingDPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E) XK nucleasedomain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependenttranslocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI areautonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appearsto be a streamlined 'half' of a Type I enzyme.

Real-time PCR analysesThe tissue samples, dissected from the infected hamsters, were homogenized in 500 ml of Trizolreagent and total RNAs were extracted in accordance with the instructions of the manufacturer.First strand cDNA was synthesized using toal RNA and random primer. For real time RT-PCR,total RNA was reverse-transcribed, and then it was PCR-amplified by ABI Prism 7900HT (AppliedBiosystems, USA) using SYBR Premix Ex TaqII (Takara, Japan). Specific HPRT (hypoxanthine-guanine phosphoribosyltransferase) primers were used as an internal control. The data wereanalyzed with Sequence Detection Systems ver 1.7a software (Applied Biosystems). Theexpression levels of the target genes were calculated using the threshold cycle time (Ct), the firstcycle number at which emitted fluorescence exceeds 106the standard deviation (SD) of base-lineemission as measured in the cycles of PCR. Standard curve was generated using known cDNA

concentration (10-fold dilution from 10 ng, 1 pg/reaction). Normalized results were expressed asthe ratio pg RNA of NiV N gene to pg RNA of the HPRT gene.


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Tipo II

Los sistemas de restriccin de tipo II normalmente contienen enzimas individuales de restriccin y

modificacin de las codificadas por genes separados. La enzimas de restriccin de tipo II suelen

reconocen secuencias especficas de ADN y se unir en las posiciones constantes en o cerca de esa

secuencia para producir 5-fosfato y 3-hidroxilo. Por lo general, requieren iones Mg2 + como cofactor,

aunque algunos tienen requisitos ms exigentes. Las metiltransferasas suelen reconocer la misma

secuencia, aunque algunos son ms promiscuos. Tres tipos de metiltransferasas de ADN se han

encontrado como parte del tipo II que forman los sistemas de R-M o C5-metilcitosina, N4-N6 metilcitosina

o metiladenina. Roberts et al. 2005 Nucl. Acids Res. 31: 1805-1812 (REBASE ref 7998).

Bibliografa

Roberts et al. 2005 Nucl. Acids Res. 31: 1805-1812 (REBASE ref 7998).

http://rebase.neb.com/rebase/rebtypes.html

Shibayama,Y. ,Dabs,E.R. ,Yazawa,K. and Mikami,Y. Molecular an Cell Biology, University of theWitwatersrand, 1 Jan Smut Avenue, Johannesburg, Gauteng 2050 South Africa (07-OCT-2009)http://www.ncbi.nlm.nih.gov/pubmed

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