Laporan Praktikum Bioinformatika.docx

LAPORAN PRAKTIKUM

DESAIN PRIMER PADA TEKNIK POLYMERASE CHAIN REACTION PADA GEN RESEPTOR GNRHR2 HOMO SAPIENS (HUMAN)

Oleh : Nur Aisyah Amini B2A018010

MATA KULIAH BIOLOGI SEL MOLEKULER PROGRAM STUDI S2 ILMU BIOLOGI FAKULTAS BIOLOGI UNIVERSITAS JENDERAL SOEDIRMAN 2018

BAB I. PENDAHULUAN GNRHR2 (Gonadotropin Releasing Hormone Receptor 2 (pseudogene)) merupakan gen reseptor yang dilepaskan oleh gonadotropin. Pada primata non-hominoid dan vertebrata non-mamalia, gonadotropin melepaskan gen reseptor hormon 2 (GnRHR2) mengkodekan tujuh-transmembran G-protein ditambah reseptor. Namun, pada manusia, kerangka bacaan yang sesuai berisi kodon stop prematur, yang telah disarankan untuk menyandikan residu selenocysteine, tetapi tidak ada bukti kuat untuk penggabungan selenocysteine (PMID: 12538601). Tampaknya transkripsi GnRHR2 manusia terjadi tetapi gen tidak mungkin menghasilkan protein multi-transmembran fungsional. Sebuah pseudogen non-transkripsi dari GnRHR2 terletak pada kromosom 14. Beberapa varian transkrip disambung alternatif telah ditemukan untuk gen ini. (NCBI, 2019) Dua hormon pelepas gonadotropin, GNRH (152760) dan GNRH2 (602352), telah diidentifikasi pada mamalia. Millar et al. dalam Carol (2009) mengkloning sebuah reseptor untuk GNRH2, ditunjuk GNRHR2, dari cDNA marmoset. GNRHR2 berbagi identitas urutan 41% dengan reseptor GNRH (GNRHR; 138850) dan, tidak seperti GNRHR, ia memiliki ujung terminal-C. Reseptor ini sangat selektif untuk GNRH2. GNRHR2 diekspresikan ke seluruh otak serta di beragam jaringan nonneural dan reproduksi. Faurholm et al dalan Carol (2009) mengkloning dan mengkarakterisasi GNRHR2 manusia. Protein GNRHR2 yang disimpulkan berbagi identitas urutan 90% dengan homolog marmoset dan identitas urutan 41% dengan GNRHR manusia. Transkrip GNRHR2 yang disambung diidentifikasi dalam hipofisis, pons, korteks frontal, otak kecil, kutub oksipital, tiroid, hipotalamus, plasenta, ginjal, prostat, dan testis. Namun, reseptor tidak memiliki kodon mulai metionin dan memiliki kodon in-frame stop pada ekson 2, menunjukkan bahwa gen tersebut mungkin tidak berfungsi. Tujuan studi ini adalah melakukan desain primer yang memenuhi kriteria optimal primer untuk igunakan pada teknik PCR pada gen GNRHR2 (Gonadotropin Releasing Hormone Receptor 2 (pseudogene).

Pada saat ini, desain primer dengan program open source di website lebih akurat dan efisien dibandingkan dengan desain primer konvensinal dengan penghitungan manual terdahulu, seperti program Primer3 Input (versi 4.1.) maupun Perl Primer. Faktor-faktor penting yang mempengaruhi kinerja primer pada PCR telah dimasukan sebagai parameter dalam program desain primer tersebut. Kriteria primer yang optimal adalah sebagai berikut : 1. Primer yang spesifik untuk fragmen DNA daerah target 2. Ukuran primer dalam kisaran 17-28 pb 3. Komposis basa nukleotida yaitu kandungan rata (G+C) 50-60% 4. Suhu melting (Tm) 55-80 °C 5. Suhu annealing (Tann) yaitu Tm - (2 –5 °C). 6. Struktur sekunder internal primer minimal untuk menghindari dimerisasi primer yaitu dG = -0,3 – 0 (analisis dimerisasi primer)

BAB II. METODA ANALISIS BIOINFORMATIKA Analisis bioinformatik pada gen GNRHR2 ini dilakukan dengan menggunakan beberapa program open source dari website sebagai berikut : 1. GeneBank Data base NCBI (National Centre for Biotechnology Information) a. Buka situs web : www.ncbi.nlm.nih.gov b. Pilih dalam kolom pencari : nucleotide c. Pilih dalam kolom pencari : NC_000001.11 (human mt DNA complete sequence) d. Penyuntingan daerah D-loop untuk situs polimorfisme T16189C dengan urutan nukleotida 1-241 e. Urutan nukleotida 1-241 (FASTA) sebagai input untuk desain primer dengan program Primer3 Input. 2) Desain Primer : Primer3 Input (versi 4.1). a. Buka situs web : www.google.com b. Pilih dalam kolom pencari : primer3 Input (version 4.1.), sehingga terbuka : www-genom.wi.mit.edu/cgi-bin/primer/primer3.cgi/ 9

c. Paste urutan nukleotida 1-241 (FASTA) pada kolom mispriming library d. Isi kolom new product size range : 1-241 e. Pilih kolom pilihan : pick primer f. Hasil desain primer : Primer3 output 3) Analisis dimerasasi primer : Perl Primer a. Pilih shortcut : Primer b. Paste pada kolom sequence forward primer : urutan left primer (Primer3 output) c. Paste pada kolom sequence reverse primer : urutan rigtt primer (Primer3 output) d. Pilih dalam kolom : calculate primer e. Hasil analisis dimerisasi primer ada dalam kolom Dimers

BAB III. HASIL DAN PEMBAHASAN 1. Analisa melalui situs NCBI untuk mendapatkan informasi mengenai gen GNRHR2 Homo sapiens (human) : a. Lokasi pada kromosom beserta orientasinya Location: 1q21.1 Chromosome 1 - NC_000001.11

Annotation release

Status

Assembly

Chr

Location

109

Current

GRCh38.p12 (GCF_000001405.38)

1

NC_000001.11 (145919013..145925 341)

105

previous assembly

GRCh37.p13 (GCF_000001405.25)

1

NC_000001.10 (145509820..145516 076, complement)

Gen GNRHR2 mempunyai 4 ekson. b. Panjang gen yang bersangkutan, jumlah ekson/intron NCBI Reference Sequence: NC_000001.11

Homo sapiens chromosome 1, GRCh38.p12 Primary Assembly FASTA Graphics LOCUS NC_000001

6329 bp

DNA

linear

CON 26-

MAR-2018 DEFINITION

Homo sapiens chromosome 1, GRCh38.p12 Primary Assembly.

ACCESSION

NC_000001 REGION: 145919013..145925341

VERSION

NC_000001.11

c. Transkrip (beserta alternatifnya) dan sekuen protein yang dihasilkan, serta pada kondisi apa masing2 alternatif tsb diekspresikan, serta organellenya. Pada primata non-hominoid dan vertebrata non-mamalia, gonadotropin melepaskan gen reseptor hormon 2 (GnRHR2) mengkodekan tujuh-transmembran Gprotein ditambah reseptor. Namun, pada manusia, kerangka bacaan yang sesuai berisi kodon stop prematur, yang telah disarankan untuk menyandikan residu selenocysteine, tetapi tidak ada bukti kuat untuk penggabungan selenocysteine (PMID: 12538601). Tampaknya transkripsi GnRHR2 manusia terjadi tetapi gen tidak mungkin menghasilkan protein multi-transmembran fungsional. Sebuah pseudogen non-transkripsi dari GnRHR2 terletak pada kromosom 14. Beberapa varian transkrip disambung alternatif telah ditemukan untuk gen ini Faurholm et al. (2001) mengkloning dan mengkarakterisasi GNRHR2 manusia. Protein GNRHR2 yang disimpulkan berbagi identitas urutan 90% dengan homolog

marmoset dan identitas urutan 41% dengan GNRHR manusia. Transkrip GNRHR2 yang disambung diidentifikasi dalam hipofisis, pons, korteks frontal, otak kecil, kutub oksipital, tiroid, hipotalamus, plasenta, ginjal, prostat, dan testis. Namun, reseptor tidak memiliki kodon mulai metionin dan memiliki kodon in-frame stop pada ekson 2, menunjukkan bahwa gen tersebut mungkin tidak berfungsi. 1. Sekuen mRNA NCBI Reference Sequence: NR_104034.1 LOCUS

NR_104034

786 bp

RNA

linear

PRI 12-MAY-2018

DEFINITION

Homo sapiens gonadotropin releasing hormone receptor 2

(pseudogene)(GNRHR2), transcript variant 3, non-coding RNA. ACCESSION

NR_104034

VERSION

NR_104034.1

KEYWORDS

RefSeq.

SOURCE

Homo sapiens (human)

ORGANISM

Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;

Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo. REFERENCE AUTHORS

1

(bases 1 to 786)

Saus E,Brunet A,Armengol L,Alonso P,Crespo JM, Fernandez-

Aranda F, Guitart M, Martin-Santos R, Menchon JM, Navines R, Soria V, Torrens M, Urretavizcaya M, Valles V, Gratacos M and Estivill X. TITLE

Comprehensive copy number variant (CNV) analysis of neuronal

pathways genes in psychiatric disorders identifies rare variants within patients JOURNAL PUBMED REMARK

J Psychiatr Res 44 (14), 971-978 (2010) 20398908 GeneRIF: Observational study of gene-disease association.

(HuGE Navigator) REFERENCE AUTHORS

2

(bases 1 to 786)

Grundker C, Fost C, Fister S, Nolte N, Gunthert AR and Emons

G. TITLE

Gonadotropin-releasing hormone type II antagonist induces

apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo JOURNAL PUBMED

Breast Cancer Res. 12 (4), R49 (2010) 20630060

REMARK

GeneRIF: Data indicate that GnRH-II antagonists seem to be

suitable drugs for an efficacious and less-toxic endocrine therapy for breast cancers,including triple-negative breast cancers. REFERENCE AUTHORS

3

(bases 1 to 786)

Yerges LM, Klei L, Cauley JA, Roeder K, Kammerer CM, Moffett

SP,Ensrud KE, Nestlerode CS, Marshall LM, Hoffman AR, Lewis C, Lang TF, Barrett-Connor E, Ferrell RE, Orwoll ES and Zmuda JM. CONSRTM

MrOS Research Group

TITLE

High-density association study of 383 candidategenes for

volumetric BMD at the femoral neck and lumbar spine among older men JOURNAL PUBMED

J. Bone Miner. Res. 24 (12), 2039-2049 (2009)

REMARK

19453261 GeneRIF: Observational study of gene-disease association.

(HuGE Navigator) REFERENCE

4

AUTHORS

(bases 1 to 786)

Gratacos M, Costas J, de Cid R, Bayes M, Gonzalez JR, Baca-

Garcia E, de Diego Y, Fernandez-Aranda F, Fernandez-Piqueras J, Guitart M,Martin-Santos R, Martorell L, Menchon JM, Roca M, Saiz-Ruiz J, Sanjuan J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X and Carracedo A. CONSRTM

Psychiatric Genetics Network Group

TITLE

Identification of new putative susceptibility genes for

several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment JOURNAL

Am. J. Med. Genet. B Neuropsychiatr. Genet. 150B (6), 808-816

(2009) PUBMED REMARK

19086053 GeneRIF: Observational study of gene-disease association.

(HuGE Navigator) REFERENCE

5

(bases 1 to 786)

AUTHORS

Stewart AJ, Katz AA, Millar RP and Morgan K.

TITLE

Retention and silencing of prepro-GnRH-II and type II GnRH

receptor genes in mammals JOURNAL PUBMED REFERENCE AUTHORS

Neuroendocrinology 90 (4), 416-432 (2009) 19657181 6

(bases 1 to 786)

van Biljon W, Wykes S, Scherer S, Krawetz SA and Hapgood J.

TITLE

Type II gonadotropin-releasing hormone receptor transcripts

in human sperm JOURNAL PUBMED REFERENCE

Biol. Reprod. 67 (6), 1741-1749 (2002) 12444048 7

(bases 1 to 786)

AUTHORS

Neill JD.

TITLE

GnRH and GnRH receptor genes in the human genome

JOURNAL

Endocrinology 143 (3), 737-743 (2002)

PUBMED REMARK REFERENCE

11861490 Review article 8

(bases 1 to 786)

AUTHORS

Faurholm B, Millar RP and Katz AA.

TITLE

The genes encoding the type II gonadotropin-releasing hormone

receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci JOURNAL PUBMED REFERENCE

Genomics 78 (1-2), 15-18 (2001) 11707068 9

(bases 1 to 786)

AUTHORS

Neill JD, Duck LW, Sellers JC and Musgrove LC.

TITLE

A gonadotropin-releasing hormone (GnRH) receptor specific for

GnRH II in primates JOURNAL PUBMED REFERENCE AUTHORS

Biochem. Biophys. Res. Commun. 282 (4), 1012-1018 (2001) 11352653 10 (bases 1 to 786) Millar R, Conklin D, Lofton-Day C, Hutchinson E, Troskie B,

Illing N, Sealfon SC and Hapgood J. TITLE

A novel human GnRH receptor homolog gene: abundant and wide

tissue distribution of the antisense transcript JOURNAL PUBMED COMMENT

J. Endocrinol. 162 (1), 117-126 (1999) 10396028 VALIDATED REFSEQ: This record has undergone validation or

preliminary review. The reference sequence was derived from DQ266410.1.Summary: In non-hominoid primates and nonmammalian vertebrates,the gonadotropin releasing hormone 2 receptor gene (GnRHR2) encodes a seven-transmembrane G-protein coupled receptor. However, in human, the corresponding reading frame contains a premature stop codon, which has been suggested to encode a selenocysteine residue,but there is no solid evidence for selenocysteine incorporation (PMID: 12538601). It appears that the human GnRHR2 transcription occurs

but the gene does not likely produce a functional multi-transmembrane protein. A non-transcribed pseudogene of GnRHR2 is located on chromosome 14. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Aug 2013]. Transcript Variant: This variant (3) lacks two exons but has an alternate 3' terminal exon, compared to variant 1. Publication Note:

This RefSeq record includes a subset of the

publications that are available for this gene. Please see the Gene record to access additional publications. ##Evidence-Data-START## Transcript exon combination :: AB239331.1, DQ266410.1 [ECO:0000332]RNAseq introns

:: single sample supports all

introns SAMEA2151119, SAMEA2151741 [ECO:0000348] ##Evidence-Data-END## ##RefSeq-Attributes-START## unitary pseudogene :: PMID: 11352653; inferred protein-coding ortholog GeneID: 574163 ##RefSeq-Attributes-END## PRIMARY COMP

REFSEQ_SPAN 1-786

PRIMARY_IDENTIFIER PRIMARY_SPAN

FEATURES source

DQ266410.1

1-786

Location/Qualifiers 1..786 /organism="Homo sapiens" /mol_type="transcribed RNA" /db_xref="taxon:9606" /chromosome="1" /map="1q21.1"

gene

1..786 /gene="GNRHR2" /gene_synonym="GnRH-II-R" /note="gonadotropin releasing hormone receptor2 (pseudogene)" /pseudo /db_xref="GeneID:114814" /db_xref="HGNC:HGNC:16341" /db_xref="MIM:612875"

misc_RNA

1..786

/gene="GNRHR2" /gene_synonym="GnRH-II-R" /product="gonadotropin releasing hormone receptor 2 (pseudogene), transcript variant 3" /pseudo /db_xref="GeneID:114814" /db_xref="HGNC:HGNC:16341" /db_xref="MIM:612875" exon

1..230 /gene="GNRHR2" /gene_synonym="GnRH-II-R" /inference="alignment:Splign:2.1.0" /pseudo

exon

231..451 /gene="GNRHR2" /gene_synonym="GnRH-II-R" /inference="alignment:Splign:2.1.0" /pseudo

exon

452..786 /gene="GNRHR2" /gene_synonym="GnRH-II-R" /inference="alignment:Splign:2.1.0" /pseudo

ORIGIN 1 atcttcgttc cctgcagaac cttgacagtt gaacaagtga cctcctccag aacagatgga 61 gagtctccag aagcagaggc tttagtgaac gaaattcgca ataatcagct ccagatcctg 121 aaaaggaggg cgaagaatca gtggccaaag ctaaccgctt catacccaca cttcatcctc 181 ctcagtttct ctccaggcca ccatgtctgc aggcaacggc accccttggg atgccacctg 241 gaatatcact gttcaatggc tggctgtgga catcgcatgt cggacactga tgttcctgaa 301 actaatggcc acgtattctg cagctttcct gcctgtggtc attggattgg accgccaggc 361 agcagtactc aacccgcttg gatcccgttc aggtgtaagg aaacttctgg gggcagcctg

421 gggacttagt ttcctgcttg ccttccccca gggatctaga actagaaata ccatttgacc 481 cagccatccc attactgggt atatacccaa aggactataa atcatgctgc tataaagaca 541 catgcacaca tgtttattgc ggcactattc acaacagcaa agacttggaa ccaacccaaa 601 tgtctaataa tgatagactg gattaagaaa atgtggcaca tatacaccat ggaatactat 661 gcagccgtaa aaaatgatga gttcatgtcc tttgtaggga catggatgaa attggaaatc 721 atcattctca gtaaactatt gcaaggacaa aaaaccaaac accgcatgtt ctcactcata 781 gatggg //

(2) Sekuen protein UniProtKB/Swiss-Prot: Q96P88.4 LOCUS

GNRR2_HUMAN

292 aa

linear

PRI 07-NOV-2018

DEFINITION

PUTATIVE PSEUDOGENE: RecName: Full=Putative gonadotropin-

releasing hormone II receptor; Short=GnRH II receptor; Short=GnRH-II-R; AltName: Full=Type II GnRH receptor. ACCESSION

Q96P88

VERSION

Q96P88.4

DBSOURCE

UniProtKB: locus GNRR2_HUMAN, accession Q96P88; class: standard. extra accessions:E7EN27,Q8TCX8 created: Feb 12, 2003. sequence updated: Oct 3, 2012. annotation updated: Nov 7, 2018. xrefs: AF403014.1, AAL27000.1, AY081843.1, AAL89821.1,

AL160282.18 xrefs (non-sequence databases): UniGene:Hs.356873, UniGene:Hs.711567, ProteinModelPortal:Q96P88, STRING:9606.ENSP00000354569, DrugBank:DB01406, DrugBank:DB00644, DrugBank:DB00666, PhosphoSitePlus:Q96P88, DMDM:408360337, PaxDb:Q96P88, PRIDE:Q96P88, ProteomicsDB:77648, DisGeNET:114814,

GeneCards:GNRHR2, HGNC:16341, MIM 612875, neXtProt:NX_Q96P88, eggNOG:KOG3656, eggNOG:ENOG410XRW9, HOVERGEN:HBG107957, InParanoid:Q96P88, TreeFam:TF106499, Reactome:R-HSA-375281, Reactome:R-HSA-416476, PRO:PR:Q96P88, Proteomes:UP000005640, CleanEx:HS_GNRHR2, GO:0005887, GO:0005886, GO:0004968, GO:0032870, GO:0007186, InterPro:IPR000276, InterPro:IPR017452, InterPro:IPR027983, InterPro:IPR001658, PANTHER:PTHR24241:SF116, Pfam:PF00001, PRINTS:PR00529, PRINTS:PR00237, PROSITE:PS50262 KEYWORDS

Cell membrane; Complete proteome; Disulfide bond; G-protein

coupled receptor; Membrane; Phosphoprotein; Receptor; Reference proteome; Transducer; Transmembrane; Transmembrane helix. SOURCE ORGANISM

Homo sapiens (human) Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;

Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo. REFERENCE

1

(residues 1 to 292)

AUTHORS

Faurholm,B., Millar,R.P. and Katz,A.A.

TITLE

The genes encoding the type II gonadotropin-releasing hormone

receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci JOURNAL PUBMED REMARK REFERENCE

Genomics 78 (1-2), 15-18 (2001) 11707068 NUCLEOTIDE SEQUENCE [GENOMIC DNA]. 2

(residues 1 to 292)

AUTHORS

Neill,J.D.

TITLE

GnRH and GnRH receptor genes in the human genome

JOURNAL

Endocrinology 143 (3), 737-743 (2002)

PUBMED REMARK

11861490 NUCLEOTIDE SEQUENCE [GENOMIC DNA], AND TISSUE SPECIFICITY.;

TISSUE=Testis REFERENCE AUTHORS

3

(residues 1 to 292)

Gregory,S.G., Barlow,K.F., McLay,K.E., Kaul,R.,

Swarbreck,D.,Dunham,A., Scott,C.E., Howe,K.L., Woodfine,K., Spencer,C.C.,Jones,M.C., Gillson,C., Searle,S., Zhou,Y., Kokocinski,F., McDonald,L., Evans,R., Phillips,K., Atkinson,A., Cooper,R., Jones,C., Hall,R.E., Andrews,T.D., Lloyd,C., Ainscough,R., Almeida,J.P.,

Ambrose,K.D., Anderson,F., Andrew,R.W., Ashwell,R.I., Aubin,K., Babbage,A.K., Bagguley,C.L., Bailey,J., Beasley,H., Bethel,G., Bird,C.P., Bray-Allen,S., Brown,J.Y., Brown,A.J., Buckley,D., Burton,J., Bye,J., Carder,C., Chapman,J.C., Clark,S.Y., Clarke,G., Clee,C., Cobley,V., Collier,R.E., Corby,N., Coville,G.J., Davies,J., Deadman,R., Dunn,M., Earthrowl,M., Ellington,A.G., Errington,H., Frankish,A., Frankland,J., French,L., Garner,P., Garnett,J., Gay,L., Ghori,M.R., Gibson,R., Gilby,L.M., Gillett,W., Glithero,R.J., Grafham,D.V., Griffiths,C., Griffiths-Jones,S., Grocock,R., Hammond,S., Harrison,E.S., Hart,E., Haugen,E., Heath,P.D., Holmes,S., Holt,K., Howden,P.J., Hunt,A.R., Hunt,S.E., Hunter,G., Isherwood,J., James,R., Johnson,C., Johnson,D., Joy,A., Kay,M., Kershaw,J.K., Kibukawa,M., Kimberley,A.M., King,A., Knights,A.J., Lad,H., Laird,G., Lawlor,S., Leongamornlert,D.A., Lloyd,D.M., Loveland,J., Lovell,J., Lush,M.J., Lyne,R., Martin,S., Mashreghi-Mohammadi,M., Matthews,L., Matthews,N.S., McLaren,S., Milne,S., Mistry,S., Moore,M.J., Nickerson,T., O'Dell,C.N., Oliver,K., Palmeiri,A., Palmer,S.A., Parker,A., Patel,D., Pearce,A.V., Peck,A.I., Pelan,S., Phelps,K., Phillimore,B.J., Plumb,R., Rajan,J., Raymond,C., Rouse,G., Saenphimmachak,C., Sehra,H.K., Sheridan,E., Shownkeen,R., Sims,S., Skuce,C.D., Smith,M., Steward,C., Subramanian,S., Sycamore,N., Tracey,A., Tromans,A., Van Helmond,Z., Wall,M., Wallis,J.M., White,S., Whitehead,S.L., Wilkinson,J.E., Willey,D.L., Williams,H., Wilming,L., Wray,P.W., Wu,Z., Coulson,A., Vaudin,M., Sulston,J.E.,Durbin,R., Hubbard,T., Wooster,R., Dunham,I., Carter,N.P.,

McVean,G., Ross,M.T., Harrow,J., Olson,M.V., Beck,S., Rogers,J.,

Bentley,D.R., Banerjee,R., Bryant,S.P., Burford,D.C.,

Burrill,W.D., Clegg,S.M., Dhami,P., Dovey,O., Faulkner,L.M., Gribble,S.M., Langford,C.F., Pandian,R.D., Porter,K.M. and Prigmore,E. TITLE

The DNA sequence and biological annotation of human

chromosome 1 JOURNAL PUBMED

Nature 441 (7091), 315-321 (2006)

REMARK

16710414 NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Erratum:[Nature. 2006 Oct 26;443(7114):1013. Banerjee, R

[added]; Bryant, SP [added]; Burford, DC [added]; Burrill, WDH [added]; Clegg, SM [added]; Dhami, P [added]; Dovey, O [added]; Faulkner, LM [added]; Gribble, SM [added]; Langford, CF [added]; Pandian, RD [added]; Porter, KM [added]; Prigmore, E] REFERENCE AUTHORS

4

(residues 1 to 292)

Morgan,K., Conklin,D., Pawson,A.J., Sellar,R., Ott,T.R. and

Millar,R.P. TITLE

A transcriptionally active human type II gonadotropin-

releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q.12 JOURNAL PUBMED REMARK REFERENCE

Endocrinology 144 (2), 423-436 (2003) 12538601 FUNCTION. 5

(residues 1 to 292)

AUTHORS

Stewart,A.J., Katz,A.A., Millar,R.P. and Morgan,K.

TITLE

Retention and silencing of prepro-GnRH-II and type II GnRH

receptor genes in mammals JOURNAL PUBMED REMARK COMMENT

Neuroendocrinology 90 (4), 416-432 (2009) 19657181 FUNCTION. On Oct 5, 2012 this sequence version replaced Q96P88.3. [FUNCTION] Putative receptor for gonadotropin releasing

hormone II (GnRH II) which is most probably non-functional. {ECO:0000269|PubMed:12538601, ECO:0000269|PubMed:19657181}.

[SUBCELLULAR LOCATION] Cell membrane; Multi-pass membrane protein. [TISSUE SPECIFICITY] Expressed in many tissues. {ECO:0000269|PubMed:11861490}. [PTM] Phosphorylated on the C-terminal cytoplasmic tail. {ECO:0000305}. [SIMILARITY] Belongs to the G-protein coupled receptor 1 family. {ECO:0000255|PROSITE-ProRule:PRU00521}. [CAUTION] Could be the product of a pseudogene. A frameshift introducing a premature stop codon in exon 1 most probably results in the inactivation of that transcribed gene in human. However, alternative splicing may allow the production of a truncated but possibly functional protein containing 5 transmembrane domains which is shown here. {ECO:0000305}. [SEQUENCE CAUTION] Sequence=AAL27000.1; Type=Erroneous gene model prediction; Evidence={ECO:0000305}. FEATURES source

Location/Qualifiers 1..292 /organism="Homo sapiens" /db_xref="taxon:9606"

gene

1..292 /gene="GNRHR2"

Protein

1..292 /product="Putative gonadotropin-releasing hormone II

receptor" /note="GnRH II receptor; GnRH-II-R; Type II GnRH receptor" /UniProtKB_evidence="Uncertain" Region

1..292 /region_name="Mature chain" /experiment="experimental evidence, no additional

details recorded" /note="Putative gonadotropin-releasing hormone II receptor. /FTId=PRO_0000069497." Region

1..28 /region_name="Topological domain" /experiment="experimental evidence, no additional

details recorded"

/note="Extracellular. {ECO:0000255}." Region

7..233 /region_name="7tm_1" /note="7 transmembrane receptor (rhodopsin family);

pfam00001" /db_xref="CDD:278431" Bond

bond(26,101) /bond_type="disulfide" /experiment="experimental evidence, no additional

details

recorded" /note="{ECO:0000255|PROSITE-ProRule:PRU00521}."

Region

29..49 /region_name="Transmembrane region" /experiment="experimental evidence, no additional

details recorded" /note="Helical; Name=1. {ECO:0000255}." Region

50..67 /region_name="Topological domain" /experiment="experimental evidence, no additional

details recorded" /note="Cytoplasmic. {ECO:0000255}." Region

68..88 /region_name="Transmembrane region" /experiment="experimental evidence, no additional

details recorded" /note="Helical; Name=2. {ECO:0000255}." Region

89..115 /region_name="Topological domain" /experiment="experimental evidence, no additional

details

recorded" /note="Extracellular. {ECO:0000255}."

Region

96 /region_name="Conflict" /experiment="experimental evidence, no additional

details recorded" /note="V -> D (in Ref. 2; AAL89821). {ECO:0000305}." Region

116..136 /region_name="Transmembrane region"

/experiment="experimental evidence, no additional details recorded" /note="Helical; Name=3. {ECO:0000255}." Region

137..177 /region_name="Topological domain" /experiment="experimental evidence, no additional

details recorded" /note="Cytoplasmic. {ECO:0000255}." Region

178..198 /region_name="Transmembrane region" /experiment="experimental evidence, no additional

details

recorded" /note="Helical; Name=4. {ECO:0000255}."

Region

199..216 /region_name="Topological domain" /experiment="experimental evidence, no additional

details recorded" /note="Extracellular. {ECO:0000255}." Region

217..237 /region_name="Transmembrane region" /experiment="experimental evidence, no additional

details recorded" /note="Helical; Name=5. {ECO:0000255}." Region

238..292 /region_name="Topological domain" /experiment="experimental evidence, no additional

details

recorded" /note="Cytoplasmic. {ECO:0000255}."

ORIGIN 1 msagngtpwd atwnitvqwl avdiacrtlm flklmatysa aflpvvigld rqaavlnplg 61 srsgvrkllg aawglsflla fpqlflfhtv hcagpvpftq cvtkgsfkaq wqettynlft 121 fcclfllplt amaicysriv lsvsrpqtrk gshapagefa lprsfdncpr vrlralrlal 181 lilltfilcw tpyyllgmwy wfsptmltev ppslshilfl lgllnapldp llygaftlgc 241 rrghqelsid sskegsgrml qeeihafrql evqktvtsrr agetkgisit si //

3) Format Fasta Sequen Homo sapiens gonadotropin releasing hormone receptor 2 (pseudogene) (GNRHR2), transcript variant 3, non-coding RNA NCBI Reference Sequence: NR_104034.1 GenBank Graphics >NR_104034.1 Homo sapiens gonadotropin releasing hormone receptor 2 (pseudogene) (GNRHR2), transcript variant 3, non-coding RNA ATCTTCGTTCCCTGCAGAACCTTGACAGTTGAACAAGTGACCTCCTCCAGAACAGATGGAGAGTCTCCAG AAGCAGAGGCTTTAGTGAACGAAATTCGCAATAATCAGCTCCAGATCCTGAAAAGGAGGGCGAAGAATCA GTGGCCAAAGCTAACCGCTTCATACCCACACTTCATCCTCCTCAGTTTCTCTCCAGGCCACCATGTCTGC AGGCAACGGCACCCCTTGGGATGCCACCTGGAATATCACTGTTCAATGGCTGGCTGTGGACATCGCATGT CGGACACTGATGTTCCTGAAACTAATGGCCACGTATTCTGCAGCTTTCCTGCCTGTGGTCATTGGATTGG ACCGCCAGGCAGCAGTACTCAACCCGCTTGGATCCCGTTCAGGTGTAAGGAAACTTCTGGGGGCAGCCTG GGGACTTAGTTTCCTGCTTGCCTTCCCCCAGGGATCTAGAACTAGAAATACCATTTGACCCAGCCATCCC ATTACTGGGTATATACCCAAAGGACTATAAATCATGCTGCTATAAAGACACATGCACACATGTTTATTGC GGCACTATTCACAACAGCAAAGACTTGGAACCAACCCAAATGTCTAATAATGATAGACTGGATTAAGAAA ATGTGGCACATATACACCATGGAATACTATGCAGCCGTAAAAAATGATGAGTTCATGTCCTTTGTAGGGA CATGGATGAAATTGGAAATCATCATTCTCAGTAAACTATTGCAAGGACAAAAAACCAAACACCGCATGTT CTCACTCATAGATGGG

4) Hasil Blasting gen Homo sapiens gonadotropin releasing hormone receptor 2 (pseudogene) (GNRHR2), transcript variant 3, non-coding RNA

5) Hasil pengeditan 10 sequen menggunakan bioedit

6) Hasil urutan primer dari program Primer3 versi 4.0 nukleotida 1-781 sebagai berikut : Primer3 Output

No mispriming library specified Using 1-based sequence positions OLIGO start len tm LEFT PRIMER 13 20 60.15 RIGHT PRIMER 210 20 60.41 SEQUENCE SIZE: 360 INCLUDED REGION SIZE: 360

gc% 50.00 50.00

any 3.00 2.00

3' seq 3.00 GAAAAGGAGGGCGAAGAATC 0.00 GGGAAAAGAAAGGGAGACCA

PRODUCT SIZE: 198, PAIR ANY COMPL: 2.00, PAIR 3' COMPL: 0.00 1 GCTCCAGATCCTGAAAAGGAGGGCGAAGAATCAGTGGCCAAAGCTAACCGCTTCATACCC >>>>>>>>>>>>>>>>>>>> 61 ACACTTCATCCTGTGAGGTAAATGCTATGGCCAAGGCACAGGGCGTGTGGAGGAATGAGT 121 TGGGTTTTTGAAGTTGATTAGATTGGAGCCTCCTATGTCTACTTCTTTCTACACAGACAC 181 AGTAACAATCTGGTCTCCCTTTCTTTTCCCCACAAGGGGATGATATTTCTACCACCTCAC <<<<<<<<<<<<<<<<<<<< 241 TCATATTTTTAGCTGAAATGATTTTCCTGGTATGTCTGTTATTTTGTGCAGTCCCCAAGA 301 CCAGAGACCAATGTCCGGAGAGAAGAGGGTGAGACAGAGAGGAAGCTCACCTCCTTGTAG KEYS (in order of precedence): >>>>>> left primer <<<<<< right primer

ADDITIONAL OLIGOS

start

len

tm

gc%

any

3'

seq

1 LEFT PRIMER 13 20 60.15 50.00 3.00 3.00 GAAAAGGAGGGCGAAGAATC RIGHT PRIMER 212 20 60.41 50.00 1.00 1.00 TGGGGAAAAGAAAGGGAGAC PRODUCT SIZE: 200, PAIR ANY COMPL: 2.00, PAIR 3' COMPL: 1.00 2 LEFT PRIMER 13 20 60.15 50.00 3.00 3.00 GAAAAGGAGGGCGAAGAATC RIGHT PRIMER 213 20 60.41 50.00 1.00 0.00 GTGGGGAAAAGAAAGGGAGA PRODUCT SIZE: 201, PAIR ANY COMPL: 2.00, PAIR 3' COMPL: 2.00 3 LEFT PRIMER 25 20 59.82 50.00 6.00 2.00 GAAGAATCAGTGGCCAAAGC RIGHT PRIMER 213 20 60.41 50.00 1.00 0.00 GTGGGGAAAAGAAAGGGAGA PRODUCT SIZE: 189, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00 4 LEFT PRIMER 25 20 59.82 50.00 6.00 2.00 GAAGAATCAGTGGCCAAAGC RIGHT PRIMER 210 20 60.41 50.00 2.00 0.00 GGGAAAAGAAAGGGAGACCA PRODUCT SIZE: 186, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00 Statistics con sid ered Left 2289 Right 2374

too many Ns 0 0

in tar get 0 0

in excl reg 0 0

no bad GC GC% clamp 6 0 6 0

tm too low 961 1016

tm high high too any 3' high compl compl 671 0 2 692 0 1

poly X 0 0

high end stab 44 37

ok 605 622

Pair Stats: considered 195, unacceptable product size 176, ok 19 primer3 release 1.1.4 (primer3_results.cgi release 0.4.0)

7) Analisis menggunakan Pearl Primer Hasil analisis Dimerisasi Primer dengan PERL PRIMER dari urutan Primer yang diperoleh dengan Program3 (Primer3 Output) adalah : 1. Most stable 3' extensible primer-dimers (at 37°C), if any 2. More stable non-extensible primer-dimers (at 37°C), if any a. Forward vs. Forward: -0.08 kcal/mol 5' GAAAAGGAGGGCGAAGAATC 3' ........||........ 3' CTAAGAAGCGGGAGGAAAAG 5' b. Forward vs. Reverse: -0.24 kcal/mol 5' GAAAAGGAGGGCGAAGAATC 3' .||........|| 3' ACCAGAGGGAAAGAAAAGGG 5'

c. Reverse vs. Reverse: -1.92 kcal/mol

5' GGGAAAAGAAAGGGAGACCA 3' ||...............|| 3' ACCAGAGGGAAAGAAAAGGG 5'

Berdasarkan atas hasil desain primer diperoleh urutan primer yang masih sesuai dengan kriteria primer yang optimal dengan kriteria sebagai berikut : 1. Primer yang spesifik untuk daerah 1..292 gen GNRHR2 2. Ukuran primer 20 pb masih dalam kisaran 17-28 pb 3. Kandungan rata (G+C) 50 % masih dalam kisaran 50-60% 4. Suhu melting (Tm) 60,15 °C dan 60,41 °C masih dalam kisaran 55-80 °C 5. Struktur sekunder internal primer berupa nilai dG primer forward- forward (-0,08 kcal/mol), forward - reverse (-0,24 kcal/mol), reverse –reverse (-1,92 kcal/mol) masih dalam kisaran dG = -0,3 – 0 kcal/mol (analisis dimerisasi primer) BAB IV. SIMPULAN Pasangan primer yang diperoleh dari desain primer gonadotropin releasing hormone receptor 2 (pseudogene) (GNRHR2), transcript variant 3, non-coding RNA dari data GeneBank pada NCBI, khususnya pada urutan nukleotida 1-241 adalah : FORWAD PRIMER : GAAAAGGAGGGCGAAGAATC REVERSE PRIMER : GGGAAAAGAAAGGGAGACCA PCR Product Size: 198 pb

DAFTAR PUSTAKA Carol A. Bocchini: 6/24/2009 http://omim.org/entry/612875 . Desaulniers, A. 2017. Expression and Role of Gonadotropin-Releasing Hormone 2 and Its Receptor in Mammals. University of Nebraska – Lincoln Wahyono, D.J. 2018. Buku Panduan Praktikum Genetika Molekuler. Fakultas Biologi Universitas Jenderal Soedirman, Purwokerto. Wahyono, D.J. 2018. Buku Panduan Praktikum Bioinformatika. Fakultas Biologi Universitas Jenderal Soedirman, Purwokerto..