Bioinformatics for beginners : genes, genomes, molecular evolution, databases and analytical tools

225 Pages·2014·52.76 MB·English

by  Supratim Choudhuri, Michael Kotewicz

BIOINFORMATICS
FOR BEGINNERS
BIOINFORMATICS
FOR BEGINNERS
Genes, Genomes, Molecular
Evolution, Databases
and Analytical Tools
Supratim Choudhuri
With contribution from Dr Michael Kotewicz
on the Optical Mapping of DNA
CenterforFoodSafetyandAppliedNutrition,FDA,
CollegePark,Maryland
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14 15 16 17 18 10 9 8 7 6 5 4 3 2 1
To my Family
Preface
As the title of the book suggests, this book is indeed Because this book is about bioinformatic analysis
for “beginners.” It is not intended for advanced stu- using web-based databases and tools, the emphasis is
dents of bioinformatics or practicing bioinformaticians. on sequence analysis. Global gene-expression profiling
This book has been written from the perspective of an hasnotbeenemphasizedotherthanashortdiscussion.
end-user who wants to use the freely available web- The makers of gene-expression analysis platforms pro-
based databases and tools for bioinformatic analysis. vide necessary software for analysis. Lastly, it is not
The audience of this book could include any scientist possible to show every type of analysis in a book with
or student who has a background in basic molecular a defined word count; nor is it possible to discuss all
biology but has not used web-based databases and thelinksandallthefunctionsassociatedwithadatabase
tools for sequence analysis, or has not done bioinfor- or analysis. Therefore, this book should serve as an
matic analysis on a regular basis. The total number of initial guide, and it is expected that the reader will
chapters is only nine. This is because related sections take it upon himself/herself to explore further using
have been combined into one chapter for coherence and the databases and tools. Terms such as program, tool,
understanding. These sections could have been easily algorithm, and web server have been used interchange-
split into separate stand-alone chapters to increase the ably throughout the book. These terms essentially mean
numberofchapters. thesamethinginthecontextofthisbook.However,the
More than a decade into the first human genome term web server could be used to mean both the hard-
sequencing, the use of bioinformatic analysis has been wareandthesoftware.
steadily increasing. There are more web-based freely Because the principal audience of the book is
available databases and analytical tools than ever supposed to be non-specialists, it was felt necessary to
before. Modern biology has pervaded even the social introduce the science and some core concepts of geno-
sciences. For example, sociologists and psychologists mics as well as some important genomic techniques
are now probing how the epigenomic effects of envi- before embarking on the bioinformatic analysis. By the
ronmental factors (including social factors) might same token, some fundamental aspects of molecular
shape the personality and behavior of the offspring evolution have been discussed in this book because the
postnatally. The National Center for Biotechnology goal of many applications of bioinformatics is to trace
Information has established an epigenomics database, the signatures of molecular evolution, as well as study
which will be immensely useful to scientists in the near the relatedness of taxa. In order to minimize the num-
future.Thus,bioinformaticshasbeenslowlybutsteadily ber of references in the text, reviews are cited wher-
pervading all branches of biology and beyond. In keep- ever possible.
ing with this, more and more bioinformatics books are
being written for experts, which do not necessarily cater Supratim Choudhuri
totheneedsofthenon-experts.
ix
Acknowledgment
Theauthorwouldliketoacknowledgetheinvaluable tools and databases and for making them freely
contributions of all scientists and engineers who available to facilitate bioinformatic analysis and
developed databases and online tools for analysis, and learning.
made them freely available. The author would also The author would like to thank Dr Steve Gendel for
like to acknowledge the contributions of the groups/ hiscarefulreadingoftheallergenicitypredictionsection
institutions/organizations for hosting and maintaining inChapter8,andprovidinghelpfulsuggestions.
these resources on web servers. A number of links The author would also like to thank manycolleagues
for freely available databases and web-based tools for for their encouragement, enthusiasm, and support for
analysis have been provided throughout the book. theproject.
Wherever possible, the latest relevant publications Last but not the least, the author is grateful to
(which usually include the previous publications as MrGrahamNisbetandMsCatherineMullaneofElsevier
well) describing these resources have been cited to for making this project a reality, helping to bring it to
acknowledge the contribution. The scientific com- successfulcompletion,andbeingavailablewheneverhelp
munity is truly grateful to the developers of these andadvicewereneeded.
xi
C H A P T E R
1
Fundamentals of Genes and Genomes(cid:1)
O U T L I N E
1.1 Biological Macromolecules, Genomics, 1.9.1 Configuration and Chirality of Amino Acids 15
and Bioinformatics 2 1.9.2 Ionic Character of AminoAcids 16
1.9.3 Relationship between Protein Function
1.2 DNAasthe Universal GeneticMaterial 2
and the Location of Amino Acids in
1.3 DNADouble Helix 2 the PolypeptideChain 16
1.3.1 StructuralUnitsof DNA 2 1.9.4 Linkage between Amino Acids—The
1.3.2 Linkage between Nucleotides 3 Peptide Bond 17
1.3.3 Base-Pairing Rules, Double Helix, 1.9.5 Four Levels of ProteinStructure 17
and Triple Helix 4 1.9.6 Acidic and Basic Proteins 17
1.3.4 Single-Stranded DNA 4 1.9.7 Nonstandard AminoAcids in
1.3.5 Base Sequenceand the Genetic Code 5 PolypeptideChains 18
1.4 Conformationsof DNA 5 1.10 Genome Structureand Organization 18
1.10.1 TheStructure of aRepresentative
1.5 Typical Eukaryotic Gene Structure 5
Genome—The Human Genome 19
1.5.1 Transcribed Region 7
1.10.2 Functional SequenceElementsinthe
1.5.1.1 Intron-SplicingSignals 7
Genome 21
1.5.1.2 EffectofIntronPhaseon
1.10.2.1 Promoters 21
AlternativeSplicing 9
1.10.2.2 Enhancers 21
1.5.1.3 EvolutionofIntrons 10
1.10.2.3 LocusControlRegions 21
1.5.2 50-FlankingRegion of TranscribedGenes 11
1.10.2.4 Insulators 22
1.5.3 30-FlankingRegion of TranscribedGenes 11
1.10.3 Epigenetic Modifications of the GenomeCan
1.6 Mutations in the DNA Sequence 12 Editthe Language Written inthe DNA
Sequence and Add an Extra Layer of
1.7 Some Features ofRNA 12
Complexityin Genome Expression 22
1.7.1 Instability of mRNA 12
1.10.3.1 HistoneCode 23
1.7.2 50- and 30-Untranslated Regions of mRNA 12
1.10.3.2 TheDynamicsofEpigenetic
1.7.3 Secondary Structures in RNA 13
Changes 24
1.8 CodingVersus Noncoding RNA 14 1.10.4 Lessons Learned from the Second Phase
1.8.1 Small Noncoding RNA,Long Noncoding of the ENCODE Project about the DNA
RNA, Competing Endogenous RNA, Elements inthe Human Genome and
and Circular RNA 14 itsEpigenetic Modifications 24
1.9 Protein Structure andFunction 15 References 25
(cid:1)Theopinionsexpressedinthischapteraretheauthor’sownandtheydonotnecessarilyreflecttheopinionsoftheFDA,theDHHS,
ortheFederalGovernment.
1
BioinformaticsforBeginners. 2014PublishedbyElsevierInc.
2
1. FUNDAMENTALSOFGENESANDGENOMES
1.1 BIOLOGICAL MACROMOLECULES, copies the single-stranded RNA genome into a single-
GENOMICS, AND BIOINFORMATICS strandedDNA,whichthenproducesadouble-stranded
viral DNA genome. The double-stranded viral DNA
Genetic information is stored in the cell in the form genomeisreferredtoastheprovirus,whichgetsincor-
of biological macromolecules, such as nucleic acids porated into the host genome from where it keeps pro-
and proteins. The genetic information not only drives ducing more retrovirus particles with single-stranded
the functioning of the whole organism, but also drives RNAgenomes.
the evolutionary engine. Thus, an understanding of the
molecular basis of life is fundamental to understanding
how genetic information shapes life and drives its 1.3 DNA DOUBLE HELIX
evolution. The following discussion captures some
fundamental aspects of the structure and function of The structure of the DNA double helix and its
genesandgenomeswithspecialnotes(inboxes)onthe building blocks are described in all biology textbooks.
applicationsofthisinformation. Here,someotheraspectsarealsohighlighted,including
the information in Box 1.1. DNA is a double-stranded
right-handed helix; the two strands are complementary
1.2 DNA AS THE UNIVERSAL becauseofcomplementarybasepairing,andantiparallel
GENETIC MATERIAL because the two strands have opposite 50230 orientation
(Figure1.1A).ThediameterofthehelicalDNAmolecule
is 20A˚ (52nm). The helical conformation of DNA
With some exceptions, deoxyribonucleic acid (DNA)
istheuniversalgeneticmaterial.Insomeviruses,termed creates the alternate major groove and minor groove
(Figure1.1B).
RNA viruses, RNA is the genetic material. The term
ribovirus is used for viruses with single- and double-
stranded RNA genomes, including retroviruses, which
1.3.1 Structural Units of DNA
areRNA-basedforaportionoftheirlifecycle.1
AmongtheRNAviruses,retrovirusesarewellknown; DNA is composed of structural units called nucleo-
they include the notorious AIDS virus. Retroviruses tides (deoxyribonucleotides). Each nucleotide is com-
are unique because in their life cycle they have both posed of a pentose sugar (20-deoxy-D-ribose); one of
RNA and DNA versions of their genome. A complete the four nitrogenous bases—adenine (A), thymine (T),
retroviruscontainsanRNAgenome.TheRNAgenome guanine(G),orcytosine(C);andaphosphate.Thepentose
encodes some protein products that are necessary for sugar has five carbon atoms and they are numbered 10
converting the single-stranded RNA genome into a (1-prime)through50 (5-prime).Thebaseisattachedtothe
double-stranded DNAgenomeandthenitssubsequent 10carbonatomofthesugar,andthephosphateisattached
integration into the host genome. One such protein to the 50 carbon atom (Figure 1.1A). The sugar and base
product of the retroviral genome is the reverse form a nucleoside, whereas nucleoside plus phosphate
transcriptase(RT)enzyme.Uponentryintothecell,the makes a nucleotide. Hence, nucleoside5sugar1base,
reverse transcriptase is produced from the viral RNA whereasnucleotide5sugar1base1phosphate.Table1.1
genomeusingthehostcellularmachinery.TheRTthen shows the naming of nucleosides and nucleotides.
BOX1.1
1. ThemajorgroovesinDNAcanbindproteins.This nature.Thecomplementaryandantiparallelnature
isanimportantpropertyofDNAstructurebecause ofdouble-strandednucleicacidsisanimportant
themajorgroovesintheupstreamregulatoryregions propertytorememberwhiledesigning
ofagenebindtranscription-regulatoryproteins. syntheticoligonucleotidesforhybridization
Forexample,forZn-fingertranscriptionfactors, (probesorprimers).
eachZnfingerrecognizesandbindstoaspecific 3. Byconvention,nucleicacid(DNAorRNA)
trinucleotidesequenceinthemajorgrooveofDNA.2 sequenceiswritten50-30 fromlefttoright,suchas
2. Anydouble-strandednucleicacid(whetherDNA 50-ATGTAAGCAC-30.Ifthe50-30 designationisnot
doublestrand,DNA(cid:3)RNAhybriddoublestrand, mentioned,itisassumedthatthesequencehasbeen
orRNA(cid:3)RNAdoublestrand)isantiparallelin writtenina50-30 direction,followingconvention.
BIOINFORMATICSFORBEGINNERS
3
1.3. DNADOUBLEHELIX
FIGURE 1.1 DNAstructure.(A)TwonucleotidesoftheDNAdoublehelix,showingtheirantiparallelorientation,twoH-bondsbetween
A andT andthree H-bondsbetweenG and C; (B) theDNAdoublehelixshowingthe majorandminor groovesas wellasthe diameterof
the molecule; (C) the convention of classifying the two sides of the phosphodiester bond and the products generated from their cleavage;
(D)thefrontside(Watson(cid:3)Crickedge)andthebackside(Hoogsteenedge)ofapurine;(E)howHoogsteenH-bondingaidsintheformation
ofthetriplehelix(seeSection1.3.3);(F)theantiandthesynconformationsofbasesaroundtheN-glycosidicbond.
Each nucleotide in DNA (as well as in RNA) has one
TABLE1.1 NamingofNucleosidesandNucleotides
replaceable hydrogen, which is what makes the DNA
Nucleoside Nucleotide (andRNA)acidic.
Base (base1sugar) (base1sugar1phosphate)
Adenine Deoxyadenosine DeoxyadenylicacidOR 1.3.2 Linkage between Nucleotides
(sugar5deoxyribose) deoxyadenosinemonophosphate
The nucleotides are joined by 50(cid:3)30 phosphodiester
Guanine Deoxyguanosine DeoxyguanylicacidOR
(sugar5deoxyribose) deoxyguanosinemonophosphate linkage; that is, the 50-phosphate of a nucleotide is
linked to the 30-OH of the preceding nucleotide by a
Cytosine Deoxycytidine DeoxycytidylicacidOR
phosphodiester linkage. In a linear DNA molecule, the
(sugar5deoxyribose) deoxycytidinemonophosphate
50-end has a free phosphate and the 30-end has a free
Thymine Deoxythymidine DeoxythymidylicacidOR
OH group (Figure 1.1A). Each phosphodiester bond
(sugar5deoxyribose) deoxythymidinemonophosphate
has two sides: a 30-side that is linked to the 30-end of
Uracil Uridine(inRNA) UridylicacidORuridine the preceding nucleotide, and a 50-side that is linked to
(inRNA) (sugar5ribose) monophosphate
50-end of the following nucleotide. The 30-side is called
BIOINFORMATICSFORBEGINNERS
4
1. FUNDAMENTALSOFGENESANDGENOMES
the A side by convention and its cleavage generates of the DNA double helix, whereas the Watson(cid:3)Crick
a 50-PO4 product. The 50-side is called the B side by edge is internal. In normal base pairing in DNA and
convention and its cleavage generates a 30-PO product RNA (Watson(cid:3)Crick base pairing), the Watson(cid:3)Crick
4
(Figure1.1C). edge (i.e. the front) of the two complementary bases
is involved. However, the Hoogsteen edge provides an
additional hydrogen bonding site. Therefore, the AaT
and GaC base pairs in the normal double helix can
1.3.3 Base-Pairing Rules, Double Helix,
and Triple Helix form additional hydrogen bonds (Hoogsteen hydro-
gen bonds) to give rise to a triple helix involving the
In the double-stranded DNA, A pairs with T by two Hoogsteen edge of the purines, i.e. N7 of A and G
hydrogenbondsandGpairswithCbythree hydrogen for the third strand (Figure 1.1E). Hoogsteen hydrogen
bonds (Figures 1.1A and 1.1B); thus GC-rich regions bonds can also form in RNA. In nucleic acids, the
of DNA have more hydrogen bonds and consequently presence of a stretch of homopurine allows a stretch
are more resistant to thermal denaturation. Each of homopyrimidine to hybridize through Hoogsteen
nucleotide pair (AaT and GaC) has a molecular hydrogen bonding to form a section of DNA triple
weight of approximately 660Da (sodium salt; 610 helix. The homopyrimidine-containing third strand is
without sodium). In the helical double-stranded DNA oriented parallel to the oligopurine strand (Figure 1.1E),
molecule, the sugar(cid:3)phosphate backbone lies outside whereas the homopurine-containing third strand is oriented
and the bases are inside. Base pairs are stacked and antiparallel to the oligopurine strand (see Box 1.2).3(cid:3)5
horizontal; hence they are perpendicular to the axis For bases, two conformational variations are possi-
of DNA. Because of the stacked nature of the base ble. The bond joining the 10-carbon of the deoxyribose
pairs in DNA, spatially flat molecules can intercalate sugar to the base is the N-glycosidic bond. Rotation
between them. Of the four bases, A and G are purines about this base-to-sugar glycosidic bond gives rise to
whereas T and C are pyrimidines. In double-stranded syn and anti conformations. The anti conformation is
DNA,apurinepairswithapyrimidine(AwithTandG the most common one (Figure 1.1F); however, the syn
with C). Therefore, total amount of purine should conformation can trigger the formation of triple helix
equal total amount of pyrimidine; in other words, the (Figure 1.1E)and also play a role in transversion muta-
purine/pyrimidine ratio should be 1.0 or close to 1.0. tion (see Molecular basis of mutation, Section 2.3.1 in
This purine(cid:3)pyrimidine equivalence in double-stranded Chapter2).
DNAiscalledChargaff’srule.
In the bases, the side with the N1 position of
1.3.4 Single-Stranded DNA
the heterocyclic ring is the “front,” also called the
Watson(cid:3)Crick edge (Figure 1.1D); the opposite side is Many DNA viruses have single-stranded DNA (for
the “back,” also called the Hoogsteen edge. Purines example, ϕX-174, parvoviruses). RNA viruses have
have an imidazole ring, which forms the “back”; so in RNA as the genetic material, and the RNA genome can
purines, the N7 position of the imidazole ring is part besingleordoublestranded.Single-strandedDNAdoes
of the Hoogsteen edge (Figure 1.1D). The Hoogsteen not have base equivalence and hence does not follow
edge of the bases is located towards the edge (outside) Chargaff’sbaseequivalencerule.
BOX1.2
1. EachphosphatehasthreereplaceableH1; 3. Thepurine(cid:3)pyrimidineequivalencecanbe
phosphodiester-bondformationbetweentwo utilizedtodetermineifaDNAmoleculefroman
nucleotidesleavesonereplaceableH1.These unknownsourceisdoublestrandedorsingle
replaceableH1maketheDNA(andRNA)acidic stranded.Inadouble-strandedDNAmolecule,
(Figures1.1and1.3). thepurine/pyrimidineratioshouldbe1.0(orclose
2. Theintercalationpropertyofspatiallyflatmolecules to1.0);incontrast,inasingle-strandedDNA
isutilizedtovisualizeDNA(andRNA)inagelusing moleculethisequivalenceislacking.
flataromaticmoleculesthatfluoresceunderUV, 4. ThedifferentialthermalstabilityofAT-richversus
suchasethidiumbromideandacridineorange. GC-richregionsindouble-strandednucleicacids
Theintercalationofthesemoleculescanalsocause istakenintoconsiderationwhiledesigning
frameshiftmutationduringDNAreplication. oligonucleotidesforhybridizationfordifferent
BIOINFORMATICSFORBEGINNERS