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PadminiNarayanan-Intro-2018.pptx
1. •
Introduction,
Medical Application and
Recent Advances
in
Molecular Biology and Bioinformatics
Refresher Course in Molecular Biology and Bioinformatics, 2018
Padmini Narayanan, Ph.D.
pnarayan@bcm.edu
2. Learning Objectives
• Why do you need to attend this series? What can
you expect from this course? How can you translate
this knowledge in to better patient care?
• To provide a solid introduction to molecular biology
with an emphasis on techniques and bioinformatic
tools that are currently used to investigate the
molecular mechanisms and its application to
diagnose and treat diseases.
• To introduce and give context to all the following
lectures of this course
3. From: Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical
sciences
Brief Bioinform. 2016;19(2):286-302. doi:10.1093/bib/bbw114
7. • Diseases are inherently complex with multitude of
interactions on different levels – genomic-environmental-
transcriptome-etc.
• Network analysis approach offers sophisticated means for
multidimensional omnics data integration.
• Its integrative informatics – pulling and curating
information from various “-omnics” – identifying
functional subnetworks in complex disease phenotypes –
prioritizing candidate genes (GWAS)
• Exploring biochemical networks for identifying drug
combinations and off target effects
• PPI networks – revel novel pathways – molecular
underpinning of diseases
Translational Bioinformatics – Clinical
Significance
8. • Using this approach – new methods/ models can be
developed
• Better understanding of different aspects of the disease
progression
• Generate new diagnostic tools – biomarkers – prognostic
and drug targets
• Valid hypothesis generation , targeted therapy –
personalized medicine
Translational Bioinformatics –
Clinical Significance
9. RESOURCES
• The Cancer Genomic Atlas (TCGA)
• Therapeutically Applkicable Research to generate Effective
Treatments (TARGET)
• Cancer Target and Driver Discovery Network (CTD2)
• NCI Genomic Data commons (GDC)
11. Replication
Gene and Gene
regulatory units
Transcription
Translation
Posttranslational modifications
Splicing
Transport, Stability
Structure,
Transport,
Stability
Interactions
Activity
Downstream signaling
Structural support
https://www.dnalc.org/resources/animations/
12. Exceptions to Central Dogma of
Molecular Biology
RNA dependent
RNA synthesis
DNA RNA Protein
RNA Interference
Reverse transcription
GENOTYPE PHENOTYPE
13. Increasing genome repertoire and
functional diversity
Genome
-20-25,000 genes
Transcriotome
~100,000 transcripts
Proteome
<1,000,000
proteins
Alternative
promoters Alternative
splicing
rnRNAediting
Post-translational
modifications
14. A genome is an organism’s complete set of
•DNA, including all of its genes
Each genome contains all of the information
•needed to build and maintain that organism
In humans, a copy of the entire genome has
•more than 3 billion DNA base pairs and is
•contained in all cells that have a nucleus
DNA structure and Genomes
Aug 10th 2018 Dr. Yasminka Jakubek
DNA- Structure and Genomes
15. Human Genome, 3x109bp packaged
into chromosomes
<3 % is expressed as
proteins!!
~80% is transcribed
What does the transcripts
do??
remaining 98–99% (non-
coding regions) holds
structural and functional
relevance
Regulatory
function
http://www.sciencemag.org/news/2012/09/human-
genome-much-more-just-genes
16. DNA Replication
~160 proteins are involved in replication
Numerous genetic diseases result from mutations in these
proteins or from errors in DNA replication or repair
e.g. recQ Helicases: Premature Aging, cancer predisposition
(Werner’s syndrome)
DNA replication and PCR methods
Aug 17th 2018 - Dr. Aparna Krishnavajhala
17. PCR: in vitro DNA Replication
PCR Applications
Molecular Biology and Genetics
Research
ClinicalApplications
Forensic Sciences
RT-PCR: Reverse Transcription PCR
Real time quantitative-PCR (qPCR)
Nested PCR
PCR Arrays
Multiplex PCR
1st cycle 2nd cycle 30th cycle
2
2
=4 copies 2
3
=8 copies 2
31
=2 Billion copies
18. DNA Sequencing
Very important tool for biologists
Sequence of genes
Positioning of genes
Sequences of regulatory regions
Applications:
HealthCare
Diagnostics
Forensics (DNA fingerprinting)
Agriculture
Research
Next generation DNA sequencing
methods
Aug 24th 2018 Dr. Devon Marie Fitzgerald
19. • Sanger Sequencing – base by base sequencing of a locus – 1kb
per run
• DNA-microarrays – hybridization of the DNA sample with a set of
pre-defined oligonucleotide probes distributed across the entire
genome or enriched around regions of interest
• Next-generation sequencing (NGS) - fragmentation of the
genomic DNA into pieces that are subsequently sequenced and
aligned to a reference sequence. Allows detection of novel changes
• Whole Exome Sequencing (WES)– specific region – sequencing of all
variants in the coding regions – reveals protein affecting modifications
• Whole Genome Sequencing (WGS) of an individual – identify all rare
coding and non-coding variations.
Technologies and methods to identify genetic
variants - Simple Nucleotide Variants (SNVs) &
Structural variants (SVs)
20. From: Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical
sciences
Brief Bioinform. 2016;19(2):286-302. doi:10.1093/bib/bbw114
21. Questions
Is DNA the only genetic material?
Is DNA sequence/gene alone
responsible for heritable traits?
Can heritable changes take place
without changes in DNA Sequence?
22. • Mitotically or meiotically
heritable changes in gene
expression that don’t
involve a change in DNA
sequence.
• Macromolecules that bind
and functionally affect the
metabolism of the DNA
• Dependent on
cell/tissue type!
What is epigenetics?
1. DNA methylation:
CpG islands:
• transcription starting sites
(UTR-areas): hypo-
/hypermethylation
• imprinting (X-inactivation):
hypermethylation
Repetitive sequences:
• Transposable elements:
hypermethylation
2. Histone modifications:
• Acetylation: HAT/HDAC-
enzymes
• Methylation: H3K4me3:
near the transcription start
sites
• Phosphorylation,
Ubiquitylation,
Sumoylation
24. Epigenomics
• The Encyclopedia of DNA Elements
(ENCODE) Program that screens the entire
genome and map every detectable
functional unit in the database.
• identify signature patterns, such as DNA
methylation, histone modification and
transcription factors, suppressors that bind
the specific region.
• 88% of trait associated variants detected
with GWAS fall in non-coding regions,
ENCODE will tremendously impact their
assessment and phenotype-related
interpretation
25. DNA Repair, Genomic Instability
o DNA is constantly mutating Reactive oxygen species
Replication errors
Radiation
Mutagen
Virus
o Mutation in a DNA repair gene can cripple the
repair process (Werner, Rothmund Thomson)
o Repair mechanisms restore DNA to its original
state
•Application:
Disease pathology (Cancer, accelerated aging etc)
DNA repair, recombination and genomic instability
Sept 7th 2018 Dr. Devon Marie Fitzgerald
26. • Manipulating DNA molecule – study effect of genes –
develop novel medicine
• Recent advances – rather than studying the DNA taken
out of the genome –able to modify genes directly in
their endogenous context – any organism
• Elucidate their effect in relevant environment –
functional organization of the genome at the system
levels – identify causal genetic variations.
Genome editing
A new era in molecular biology
Just CRISPR it!!!
27. Targeted Genome editing and
CRISPR/Cas9
A new era in molecular biology
Genome engineering
with Cas9 nuclease
Origin: Bacterial immune system
Guide RNA
Cas9 (CRISPR associated endonuclease)
Make precise modifications in
target DNA (HDR)
Generate knock-outs (NHEJ)
Activate or repress target
genes (promoter binding)
Genome wide screens
(disease/pathway associated
novel genes)
Targeted genomic editing CRISPR
Sep 14th 2018Dr. Hyun Hwan
28. CRISPR applications and Animal Models
CRISPR application and Animal Models
Sep 21nd 2018 Dr. Sirena Soriano
Hsu. P, et al. Cell 2014 June5, 157(6).
Genetic and epigenetic control of
cells with genomic engineering –
Broad rage of application.
Application of genomic eng - causal genetic
mutation – Rapid identification of epigenetic
variants associated with altered biological
functions or disease phenotype – effectively
recapitulated in animal and
29. Gene Expression:
Transcription, Alternative splicing, RNA Seq
Regulation of gene expression is the critical link between
the genome and cellular morphology/physiology
• Transcription
• Transcriptional Factors
• Transcriptional Regulation
Sep 28th 2018 Dr. Tao Ling
90% of human genes are
alternatively spliced!
30. Gene Expression: Non-Coding RNA and
Personalized Medicine
• A non-coding RNA (ncRNA) is a functional RNA
molecule that is not translated into a protein.
• Non-coding RNAs: Junk or Critical Regulators in
Health and Disease?
o Prader–Willi syndrome (snoRNA-HBII52)
o Alzheimer's disease (lncRNA-BACE-AS)
o Cancer (miR200)
RNAi therapeutics – potent –specific mechanism –
targeting gene expression – siRNA bound o RNA induced
silencing complex(RISC) mediated target mRNA
degradation using endonucleases (Arg).
Pathway present in all mammalian cells.
First-in-Humans Trial of an RNA Interference
Therapeutic Targeting VEGF and KSP in Cancer
Patients with Liver Involvement Cancer Discovery
April 2013 3; 406
31. Oct – 5th 2018 – Dr.Narayan Sastri Palla
Gene expression – Non-coding RNA and
personalized medicine. Breaking the genetic
enigma
32. Translation and Protein Function
Prediction and detection
mRNA to Protein
Single nucleotide change can alter codon and possibly aminoacid
Change in amino acid sequence causes changes in
– 3-D structure of protein
– Defective protein folding
– Protein function
Cystic fibrosis
– Misfolded CFTR protein
– Protein retained in ER
Huntington disease
– Trinucleotide repeats
– Multiple CAG repeats
Antibiotics often target bacterial
•Translation
33. From: Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical
sciences
Brief Bioinform. 2016;19(2):286-302. doi:10.1093/bib/bbw114
34. Protein Detection
• Extraction of proteins
• Separation of proteins
• Identification of proteins
• Quantification of Proteins
• Western Blot
• Immunohistochemistry/Immunofluorescence
• ELISA/EIA
• Immunoprecipitation
• Mass-Spectrometry
• X-Ray Chrystallography
36. Immunohistochemistry
Avidin-biotin complex detection
Signal amplification is
not possible
Suitable for high
abundant ptn.
Moderate signal
amplification – commonly
used.
Substrate usually Horse Radish peroxidase (HRP) or
alkaline phosphatase (AP) or the primary or secondary ab
will be conjugated with fluorophore.
37. Immuno-colocalization of GP130 surface receptor
with Lipid rafts in Pediatric AML blasts
260
125
70
38
15 Caveolin-1
Kasumi
HS5
THP-1
K562
K61
MLLM-13
NB4
A16468-surface_unstim-surface.fcs
FITC-A
Count 10
0
10
1
10
2
10
3
10
4
0
10
19
29
38
GP130 Surface staining
observed using flowcytometry
Translation, function prediction and detection of
proteins
Oct 12th 2018 Dr. Nikhil Jain
38. Protein Modification:
Localization and activity
Enzymes, Receptors, Cytokines, Hormones etc.
Translation, Transport and activity
Metabolic disorders
Types of protein modification
Molecular Biology Techniques
Activity Assay
Identification of Post-translational modification
Protein Modification – Localization and activity
Oct 19th 2018 Dr. Poonam Sarkar
39. Cell cycle and chromosomal
Instability
Historical context
Preservation and propagation of gene
associated diseases
Molecular biology techniques used to
these disorders
Molecular biology application in treatment
Introduction to relevant Bio-informatic tools
Oct 26th 2018 Dr. Renuka Ramankutty Menon
40. • Based on isolation of DNA
sequence of interest to obtain
multiple copies invitro
• Insertion of genetic information
in to replicating vehicles
• Plasmids, viral vectors, BAC etc.
• Different techniques of cloning
and vectors tailored to the final
application.
• Lecture – Structure of plasmids,
cloning sites, restriction
enzymes- cloning softwares and
clone a gene in a vector.
Molecular Cloning and its
application
Nov 2nd 2018 Dr. Paramahamsa Maturu
41. http://www.genome.gov/20019523
• A genome-wide association study is an approach that involves
rapidly scanning markers across the complete sets of DNA, or
genomes, of many people to find genetic variations associated
with a particular disease.
• Once new genetic associations are identified, researchers can
use the information to develop better strategies to detect,
treat and prevent the disease. Such studies are particularly
useful in finding genetic variations that contribute to common,
complex diseases, such as asthma, cancer, diabetes, heart
disease and mental illnesses.
Genome-wide association study
42. • Introduction to genome-wide association studies
(GWAS), clinical relevance of GWAS of complex
disease
• Introduction to relevant databases to identify
haplotype blocks and performing genetic and
epigenetic profiling or susceptibility loci
• Experimental techniques to determine underlying
mechanisms of susceptibility loci
• Walkthrough of a leukemia-specific post-GWAS
analysis using HaploReg and RegulomeDB
•
GWAS- Population Genetics
46. •OMIM
Online Mendelian Inheritance in Man®
Compendium of genes & genetic phenotypes
Contain information on all known Mendelian
•disorders (~15,000 genes)
Relationship between phenotype & genotype
Initiated 1960s by Dr. Victor A. McKusick
McKusick-Nathans Institute of Genetic
•Medicine, JHU
www.omim.org
48. •
Home About Statistics • Downloads Y Help Y External Links Terms of Use Contact Us MIMmatch NEW a Select Language I •
I insulin Search
Advanced Search - I Search History I Display Options • I Retrieve Corresponding: Gene Map Clinical Synopsis
Search: 'insulin'
Results: 1 - 10 of 1,056 I Show 1 GO I Download As • I 1 2 3 4 5 6 7 8 9 10 Next Last
* 147670. INSULIN RECEPTOR; INSR
Crogenetic location: 19p132, Genomic coordinates (GRC1137): 19:7,112 765-7251,010
I'vatching terms: insulin
2 * 176730. INSULIN; INS
ENS-IGF2 SPLICED READ-THROUGH TRANSCRIPTS, INCLUDED
Crogenetic location 1105.5, Genomic coordinates (GRCIL37). 11:2,181,008-2,182,438
Gene Tests, Links
Gene Tests, ICD+, Links
Home About Statistics • Downloads Y Help Y External Links Terms of Use • Contact Us MI Mmatch NEW al Select Language I V
I insulin Search
Advanced Search • I Search History I Display Options •
•*176730
•INSULIN; INS
•Alternative titles; symbols
PROINSULIN
•Other entities represented in this entry:
•INS-IGF2 SPLICED READ-THROUGH TRANSCRIPTS, INCLUDED; INSIGF, INCLUDED
•HGNC Approved Gene Symbol: INS
•Cytogenetic location: 11p15.5Genomic coordinates(GRCh37): 11:2,181,008-2,182,438or..New
Gene-Phenotype Relationships
•-:3+• Table of Contents for *176730
•Title
•Gene-Phenotype Relationships
•Text
•Description
•Gene Structure
•Mapping
•Gene Function
•Biochemical Features
•Molecular Genetics
•Animal Model
•History
•Allelic Variants
•Table View
•See Also
•References
•Contributors
•Creation Date
•Edit History
•External Links for Entry:
•Genome
DNA
Protein
Gene Info
...Clinical Resources
Variation
Animal Models
•Location Phenotype Phenotype
•MINI
number
125S52
•613370
•3.10.5.5
•Hypexproinsulinenta
•Maturity-onset diabetes of the voting, type 10
•Phenotype
mapping key
•606176
•616214
•Diabetes mellitus, insulin-dependent, 2
•Diabetes mellitus, permanent neonatal
•3
•3
•3
•3
•T W Y T
49. •Gene
http://www.ncbi.nlm.nih.gov/gene
•F ; 7 2 :
•NCBI Resources 1:vi How To NI
•Gene •Gene No.
•Advanced •Help
•Gene
•Gene integrates information from a wide range of species. A record may include nomenclature, Reference
Sequences (RefSeqs), maps, pathways, variations, phenotypes, and links to genome-, phenotype-, and locus-specific
resources worldwide.
•Using Gene Gene Tools Other Resources
•Gene Quick Start Submit GeneRIFs 1-10moloGene
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50. •1 •of 64 Next > Last »
•P
a
g
e
•Aliases
•Location
•Chromosome 1, •ER-alpha, Esr
Esrl •estrogen receptor 1 [Rattus
•NCBI Resources 17.1 How To 1,71 •Sign in to NCBI
•Gene •V
•Help
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Alternatively
spliced Annotated
genes Non-coding
Protein-coding
Pseudogene
•Results: 1 to 20 of 1271
•0 See also 13 discontinued or replaced items.
•Name/Gene ID Description
•Results by taxon
• Top Organisms
[Tree]Homo sapiens
(644) Mus musculus
(300) Rattus norvegicus
(294)
•Gene
•A
•Table of contents
•Summary
•Genomic context
•Genomic regions, transcripts, and products
•Bibliography
•Variation
•Pathways from BioSystems
•Interactions
•General gene information
•Markers, Homology, Gene Ontology
•General protein information
•NCB! Reference Sequences
(RefSeq) Related sequences
•Additional links
•Related information
•Unactive previous page of results
•Esrl estrogen receptor 1 [ Rattus norvegicus (Norway rat) ]
•Gene ID 24890, updated on 25-Jul-2015
•Esr1 provided by RGD
•estrogen receptor 1 provided by RGD
•RGD:2581
•Ensembl:ENSRNOG00000019358; Vega: OTTRNOG00000001366
•protein coding
•PROVISIONAL
•Rattus norvegicus
•Eukaryota: Metazoa; Chordata; Craniata; Vertebrata: Euteleostomi; Mammalia; Eutheria; Euarchontoglires;
•Glires; Rodentia; Sciurognathi; Muroidea; Muridae, Murinae; Rattus
•Esr; ER-alpha: RNESTROR
•acts as a transcriptional activator when bound to estrogen; may play a role in myocardial regulation IRGD,
•Feb 2006]
•Annotation category suggests misassembly
•Summary
•Official Symbol
Official Full
Name Primary
source See
related Gene
type Ref Seq
status Organism
Lineage
• Also
known as
Summary
•Annotation
51. •Summary
Genomic context O0
Genomic regions, transcripts, and products 00
•111.7, Bibliography O0
•Variation
•Pathways from BioSystems
•NM 012689.1 } NP 036821.1 estrogen receptor
•See identical proteins and their annotated locations for
NP_036821.1Status: PROVISIONAL
• Source sequence(s) Y00102
UniProtKafSwiss-Prot P06211
• Related ENSRNORX1000026350, OTTRNOP00000001225,
ENSRNOT00000028350, OTTRNOT00000002098
•Conserved Domains 44) summary
•cd06949
•Location:315 —*552
•cd07171
•Location:185 — 266
•pfarnl 2743
•Location:557 —* 600
• pfarn 021,59 Oest_recep, Oestrogen receptor
Location:42 — 186
•Genomic regions, transcripts, and products
•Bibliography
•Variation
•Pathways from BioSystems
•Interactions
•General gene information
• Markers, Homology, Gene Ontology
General protein information
•NCBI Reference Sequences (RefSeq)
Related sequences
•BioAssay by Target (Summary)
•BioProjects
•BioSystems
•Conserved Domains
•Full text in PMC
•Full text in PMC nucleotide
•Gene neighbors
•Genome
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•Map Viewer
•Nucleotide
•Probe
•Protein
·ubChenn Compound
Pubehem Substance
·ubMed
·ubrded (GeneRIF)
PubMed(nucleatide/PMC)
RefSeq Proteins
•RefSeq RNAs
•SNP
•NR_LBD_ER: Ligand binding domain of Estrogen receptor,
which are activated by the hormone 17beta-estradicil (estrogen)
•NR_DBD_ER; DNA-binding domain of estrogen receptors (ER)
is composed of two G4-type zinc fingers
•ESR1_C; Oestrogen-type nuclear receptor final C-terminal
•NCBI Reference Sequences (RefSeq)
•Ref Seqs maintained independently of Annotated Genomes
•These reference sequences exist independently of genonne builds. Explain
•mRNA and Protein(s)
52. •
• •
•
•Gene Card
•www.genecards.org
GeneCardsSuite GeneCards MalaCards LiteMap Discovery PathCards GeneAnalyIics GeneALaCart VarElect GenesLikeMeGeneLac
•GeneCards
®
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•Free for academic non-profit institutions_ Other users need a Commercial license ;MTV On
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Pius
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•Home User Guide Analysis Tools- News And Views About My Genes Log In Sign Up
•GeneCardse: The Human Gene Database
•GeneCards is a searchable, integrative database that provides
comprehensive, user-friendly information on all annotated and predicted
human genes. It automatically integrates gene-centric data from >100 web
sources, including genomic, transcriptomic, proteomic, genetic, clinical
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•GeneALaCart •OGenesLikeMe
•Jump to section for this gene:
•Aliases
53. •
15) GeneCardsSultf GeneCards MalaCardsLifeMap Discovery PathCards GeneAna!rim GeneALaCart liarElect GenesLikeMe GeneLoc
GeneCards®
Free Ix academic non-profit institution& Other users need a Commercial license WEIZMANN
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A ati
MILUPORG Proteins & Enzymes
Jump io Aliases Compounds Disorders Domains Expression Function Ge nom ics Localization Orthologs Research Products for AR Gene
section Paralogs Pathways Products
Proteins Publications Sources =3 Transcripts Variants Antibodies Proteins More...
•SummariesAR or Gene o
•Entrez Gene Summary forAR Gene Cf:
•The androgen receptor gene is more than 90 kb long and codes for a protein that has 3 major functional domains:
the N-terminal domain, DNA-binding domain, and androgen-binding domain. The protein functions as a steroid-
hormone activated transcription factor. Upon binding the hormone ligand, the receptor dissociates from accessory
proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes.
This gene contains 2 polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts
in the N-terminal transactivation domain of its protein. Expansion of the polyglutamine tract causes spinal bulbar
muscular atrophy (Kennedy disease). Mutations in this gene are also associated with complete androgen
insensitivity (CAIS). Two alternatively spliced variants encoding distinct isoforms have been described. [provided
by RefSeq, Jul 2008]
•GeneCardsSummary forAR Gene
•AR (Androgen Receptor) is a Protein Coding gene. Diseases associated with AR include prostate cancer
and hypospadias 1, x-linked. Among its related pathways are Pathways in cancer and Akt Signaling. GO
annotations related to this gene include sequence-specific DNA binding transcription factor activity and
chromatin binding. An important para/og of this gene is NR3C1.
•UniProtKBjSwiss-Prot for AR Gene ANDR_HUMAN,P10275
•Steroid hormone receptors are ligand-activated transcription factors that regulate eukaryotic gene expression and
affect cellular proliferation and differentiation in target tissues. Transcription factor activity is modulated by bound
coactivator and corepressor proteins. Transcription activation is down-regulated by NROB2. Activated, but not
phasphorylated, by HIPK3 and ZIPIVIDAPK3.
•Tocris Summary for AR Gene C
54. •
•pJurnp to
section
•Research Products WAR Gene
•Antibodies Proteins More
•Aliases Compounds Disorders Domains Expression Function Genomics Localization Orthologs
•Paralogs Pathways Products Proteins Publications Sources Summaries Transcripts Variants
Localization for All Gene
Addgene plasmids for AR
RD R&D Systems cDNA Clones for AR
(Androgen R 3CA )
Subcellular locations from UniProtKB/Swiss-Prot for AR Gene
Nucleus. Cytoplasm. Note—Predominantly cytoplasmic in unligated form but translocates to the
nucleus upon ligand-binding. Can also translocate to the nucleus in unligated form in the presence
of GNB2L1. P13275-ANDR HUMAN Cell Line Products
I—a:—"—ff... an atarr 1-1—, (n
Junin to
section
Aliases Compounds Disorders Domains Expression Function Genomics Localization Orthologs
Paralogs Pathways Products Proteins Publications Sources Summaries Transcripts Variants
•Expression toeAft Gene
mRNA expression in normal human tissues for AR Gene
•Microarray
•BrnGPS < Intensity >5'1
•0 1 1 0 1 0 0
•I
•RNAseqGTExSAGE (Serial Analrisuf Gene Expression)
•ClaIxFPKM)14 inumlna Body MapCLAP TAG. GCACCTTCAG
• 1000 0 1 10 100 1000 0 1 10 100 1000
•I I I I I I I
•B o n e
M a r r o w
W h o l e B l o o d
W h i t e B l o o d
C e l l s L y m p h
N o d e
T h y m u s
B r a i n C o r t e x
C e r e b e l l u m
•Swine
•Spinal Cord
third Nerve
•Heart
•Merry
•Smooth
Muscle
Skeletal
Muscle Small
Intestine
Colon
Adipocyte
Kidney Liver
•Major
Tissues
Research Products tor MI Gene
•Antibodies Proteins More...
•— mouse, rat tor AK
•Predesigned siRNA for gene silencing in
human,mouse,rat for AR
• I I Santa Cruz Biot echnology (SCBT)
s iRNA f or A R S ee all 4 - A R s iRNA
(h)
•AR sh RNA (h) Lentiviral Particles
•Clone Products
• I OriGene clones in human,mouse for
AR
•OriGene ORF clones in mouse,rat for
AR Custom cloning services - gene
synthesis, suhcloning, mutagenesis,.
variant library, vector shuttling
•GenScript: cDNA clones in your preferred
vector
•NM_001011645 Nk1_000044
•0Vector BioLabs ready-to-use
•adenovirusiAAV for human, mouse, rat
•Addgene plasmids for AR
• RD R&D Systems cDNA Clones for AR
(Androgen RiNR3C4)
•Sino Biological Human cDNA Clone for
AR
•ACA AAA., ACCI.CC,APJ—AAA—I.
55. •NCBI
•http://www.ncbi.nlm.nih.gov/
•e .
•NCBI Resources 1...7.1 How To 1:.71
N ucleot ide
Nucleotide horn° sapiens p531
•Save search Limits Advanced
•Display Settings: CI Summary, 20 per page, Sorted by Default order
•0 Found 16050 nucleotide sequences. Nucleotide (15791) EST (224) GSS ()
Results: Ito 20 of 15791 Pagel F-1 of 790I
0 Homo sapiens mRNA for P53, complete cds
12,451 bp linear mRNA
Accession! AB082923.1 GI: 23491728
GenBank FASTA Graphics Related Sequences
Homo sapiens p53 (p53) gene, axon 7 and partial cds
2. 110 bp linear DNA
Accession: JF923572.1 GI: 349734069
GenBank FASTA Graphics Related Sequences
56. •NCB! Resouires 1:7..) How To 1:7.)
Nucleotide [Nucleotide
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•Display Settings: CI GenBank Send: CI
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Homo sapiens m RNA for P53, complete cds
GenBank: A13082923.1
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Basic Features
(3 Default features
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LOCUS AP082023 2451 bp mRNA linear PRI 01-APR-2003
DEFINITION Demo sapiens mRNA for P55, complete cds.
ACCESSION AP082925
VERSION AP082923.1 GI:23491728
KEYWORDS
SOURCE Goma sapiens (human)
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Euarchontoliresr. Primates; Saplorrhinir.
Catarrhini; Dominidae; Homo.
REFERENCE 1
AUTHORS Azuma,K., Shichijo,S., Maeda,Y., Nakatsura,T., Nonaka,Y., Fujii,T.,
Koike,K. and Itoh,K.
TITLE Mutated p55 gene encodes a nonmutated epitope recognized by
EILA-2+4601-restricted and tumor cell-reactive CTLs at tumor site
JOURNAL Cancer Res. 65 {4), 854-858 {2005)
PUSHED 12591757
REFERENCE 2 {bases 1 to 2451)
AUTDORS Shichijo,S. and Itoh,K.
TITLE Direct Submission
JOURNAL Submitted (26-MAR-2002) Shigeki Shichijo, Kurume Univ. School of
Med., Dep. Immunol..r. 67-Asahi-machi, Kurume, Fukuoka 830-0011,
Japan {E-mail:shichijoOmed.kurume-u.ac.jp, Tel:81-542-31-7551,
Fax:81-942-11-7699)
FEATURES Location/ Qualifiers
scsnrnp 1..249.1
Analyze this sequence R
Pick Primers
Highlight Sequence Featur
Sequence
Articles about the TP53
Nemo-like kinase is critica
in response [Cell De
Prp.rtrintir: uniHe of TPS: WAW
57. •NCBI Primer Blast
•http://www.ncbi.nlm.nih.gov/tools/primer-blast/
•Primer-BLAST •firictirro
•;61/Primer-BLAST: Finding primers specific to your PCR template (using Primer3 and BLAST).More... Tips for finding specific primers
•Reset page Save search parameters Retrieve recent results
•PCRTemplate
•Enter accession, gi, or FASTA sequence (A refseq record is preferred)Clear
•Or, upload FASTA file •(Choose File j no fib selected
•M
i
n •Opt •M. Max Tm difference
•Intron length range
•Intron inclusion
•(Tm)
•Exoniintron selection
•Exon junction span
Exon junction match
•A refseq mRNA sequence as PCR template Input Is required for options In the section
• No preference1-41V.)
Exon at 5' shift Exonat S' sIde
•7'4
•Minimalnumberofbasesthatmustannealtoexons attheTor3'sideoftheJunction
Primerpairmustbeseparatedbyatleastoneintrononthe=respondinggenomIcDNA0,
•MinMax
•1000 101300130
•at a n •a a LIZ a Al
•AB082923.1
•Primer Parameters
•Use my own forward primer
(5'->3' on plus strand)
•Use my own reverse primer (5'-
>3' on minus strand)
•PCR product size
•# of primers to return •1
0
•C
l
e
a
r
•C
l
e
a
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•*low- Amplicon size 50-150bp for qPCR
•Primer melting temperatures 57.0 60.0 63.0 3
•Range
•From To
•Clem
•Forward primer
Reverse primer
•Mln Max
•713 1060
58. •
•Primer pair 3
• Sequence (5'->31 Template strand Length Start Stop Tm GC% Self complementarity Self 3' complementarity
• Forward primer AAAGTCTAGAGCCACCGTCC Plus 20 65 84 59.10 55.00 6.00 0.00
• Reverse primer GCAGTCTGGCTGCCAATCC Minus 19 186 168 61.41 63.16 7.00 3.00
• Product length 122
• Exon Junction 174/175 (reverse primer) on template NM 001276696.1
•Products on Intended target
•>NM 001126114.2 Homo sapiens tumor protein p53 (TP53), transcript variant 3, mRNA
•product length = 122
•Forward primer 1 AAAGTCTAGAGCCACCGTCC 20
•Template 65
•Reverse primer 1 GCAGTCTGGCTGCCAATCC 19
•Template 186 169
•Products on potentially unintended templates
•>NM 001125118.1 Home sapiens tumor protein p53 (TP53), transcript variant 8, mRNA
•product length = 122
•Forward primer 1 AAAGTCTAGAGCCACCGTCC 20
•8 4
•Template 65 94
•Reverse primer 1 GCAGTCTGGCTGCCAATCC 19
•Template 186 169
•>NM_001126113.2 Homo sapiens tumor protein p53 (TP53), transcript variant 4, mRNA
•product length = 122
•Forward primer 1 AAAGTCTAGAGCCACCGTCC 20
•Template 65 94
•Reverse primer 1 GCAGTCTGGCTGCCAATCC 19
•Template 196 169
59. •
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61. •vvvvw.hprd.org
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0 4 . B r o w s e
I oiw a s /
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•nformatics
•Comparison of Protein-Protein Interaction !Databases, published in MC Ei
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62. Human Protein
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.▪."••-••
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the proteins that have both 8H2 and SH3 domains. Similarly, if more than one field is filled in, it will be treated as an 'AND' c
•information go to the FAQ
Protein Name E53
Accession Number RefSeq V
HPRD Identifier
Gene Symbol
Chromosome Locus
Molecular Class See List
See List
PTMs
Cellular Component See List
See List
Domain Name
Motif See List
Expression See List
Length of Protein Sequence From : to : in amino acids
Molecular Weight From : to : in kDa
Diseases
seam Clear_
63. •are at HPRD » Query » p53
ILT:plocularClass Transcription factor
•p53
•'Molecular Function Transcription factor ac
•Regulation of nucleol
•Biological Process nucleoside, nucleotide
•acid metabolism ; Apc
•GO TO: Isc
•PThs&SUBSTRATES
•INTERACTIONS ETERNAL LINKS1
If ALTERNATE NAMES DISEASES
SEQUENCE
SUMMARY
•I Protein Sequence 393AA NP 0011195341
•HEEPQSDPSI
T
hIPLSSSVPSQ
RICSDSDGLAP
EVRVCACPGR
GSRAHSSHLK
•EPPLSWFS DLWKLLPENN
RTYQGSYGFR LGFLHSGTAK
PUIIRVEGN LRVEYLDDRN
DRRTEEENLR KKGEPHHELP
SKRGINTSRE ECKLMFRTEGP
•-VLSPLPSQAM
SVICTYSPAL
TFRESVVVPY
PGSTRRALPN
DSD
•DDLMLSPDDI
NEMFCQLART
EPPEVGSDCT
NTSSSPQPPY
•EQWFTEDPGP DEAPRMPEAA PPVAPAPAAP
TPAAPAPAPS CP1NLWVDST PPPGTRVRAM AIYKQSQHHT
EVVRRCPHHE TIHYNYMOINS SCMGGMNRRP ILTIITLEDS
SGNLLGRNSF RPLDGEYFTL QIRGRERFEM FRELNEALEL
ZDAQAGREPG
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