Biologia Molecular

2
Marcos na histria da Biologia Molecular e

Bioinformtica
1955 A sequncia da insulina bovina foi

a primeira protena a ser analisada
por F. Sanger
Biologia Computacional - 2010
1958 - Jack Kilby construiu o

primeiro circuito integrado
para a Texas Instruments

Bioinformtica
4
1981 - publicado o algoritmo Smith-Waterman algoritmo para o alinhamento de

sequncias.
1969 Foi criada a ARPANET para

estabelecer ligaes entre os
computadores de Stanford, UCSB,
Universidade de Utah e UCLA
1981 A IBM introduz no mercado o primeiro computador pessoal.

1983 lanado o Compact Disk (CD).
1970 - Howard Temin and David

Baltimore isolaram
independentemente a primeira
enzima de restrico
Biologia Computacional 2010
James Watson

Bioinformtica
1985 - publicado o algoritmo FASTP.

descrevem
David Baltimore
Kary Mullis
1985 - Kary Mullis e colaboradores

a reaco de PCR.
Howard Temin
Rosalind Franklin
Jack Kilby
F Sanger
INTRODUO
Francis Crick
1952-1953 - James Watson e Francis

Crick deduziram a estrutura da
molcula de DNA recorrendo ao
material ode difraco por raios X
obtido Rosalind Franklin
Section1: What is Life made of?
Cells
Fundamental working units of every living system.

Every organism is composed of one of two
radically different types of cells:
prokaryotic cells or
eukaryotic cells.
Prokaryotes and Eukaryotes are descended from the same primitive cell.
2 types of cells: Prokaryotes v.s.Eukaryotes
All extant prokaryotic and eukaryotic cells are the result of a total of 3.5
billion years of evolution.
Molcula comum a todas as clulas

vivas
DNA cido desoxiribonucleico molcula comum a todos os seres

vivos que existe no ncleo das
clulas eucariotas e no nucleide das
clulas procariotas.
Procariotas sem ncleo delimitado

por membrana
Eucariota ncleo delimitado por

membrana
Prokaryotes and Eukaryotes
Cells
Chemical composition-by weight

70% water
7% small molecules
salts
Lipids
amino acids
nucleotides
23% macromolecules
Proteins
Polysaccharides
lipids
biochemical (metabolic) pathways
translation of mRNA into proteins
According to the most recent evidence, there are three main branches to the tree of life.
Prokaryotes include Archaea (ancient ones) and bacteria.
Eukaryotes are kingdom Eukarya and includes plants, animals, fungi and certain algae.
Prokaryotes vs Eukaryotes
Prokaryotes v.s. Eukaryotes
Prokaryotes
Prokaryotes
Eukaryotes
Single cell
Single or multi cell
No nucleus
Nucleus
No organelles
Organelles
One piece of circular DNA Several pieces of linear

DNA - Chromosomes
No mRNA post
Exons/Introns splicing
transcriptional modification
Structural differences
Eubacterial (blue green algae)

and archaebacteria
only one type of membrane-plasma membrane forms
the boundary of the cell proper
The smallest cells known are

bacteria
Eukaryotes
Ecoli cell
3x106 protein molecules
1000-2000 polypeptide species.
plants, animals, Protista, and fungi

complex systems of internal
membranes forms
organelle and compartments
The volume of the cell is several

hundred times larger
Hela cell
5x109 protein molecules
5000-10,000 polypeptide species
Modelo da dupla hlice - Watson; Crick e

Franklin
Prokaryotic and Eukaryotic Cells

Chromosomal differences
Eukaryotes
Prokaryotes
The genome of E.coli contains
amount of t 4X106 base pairs
> 90% of DNA encode protein
Lacks a membrane-bound nucleus.
Circular DNA and supercoiled
domain
The genome of yeast cells contains

1.35x107 base pairs
A small fraction of the total DNA
encodes protein.
Many repeats of non-coding
sequences
All chromosomes are contained in
a membrane bound nucleus
Histones are unknown
DNA is divided between two or

more chromosomes
A set of five histones
DNA packaging and gene expression

regulation
Polinucletido Cadeia simples de DNA
Constituintes do DNA
Extremidades livres
DNA = cadeia de nucletidos

Nucletidos = acar + Fosfato +
base azotada
O acar deoxiribose - uma
pentose (5 tomos de carbono)
Fosfato
Deoxiribose
Bases azotadas = Pirimidinas e Purinas
Nucletidos e complementaridades
Adenosina
Ligaes entre as duas cadeias simples e

cadeias antiparalelas
Adenina = Timina
Guanina Citosina
Overview of organizations of life
Nucleus = library
Chromosomes = bookshelves
Genes = books
Almost every cell in an organism contains the same
libraries and the same sets of books.
Books represent all the information (DNA) that
every cell in the body needs so it can grow and
carry out its vaious functions.
Some Terminology
Genome: an organisms genetic material
Gene: a discrete units of hereditary information located on the

chromosomes and consisting of DNA.
Genotype: The genetic makeup of an organism
Phenotype: the physical expressed traits of an organism
Nucleic acid: Biological molecules(RNA and DNA) that allow organisms to

reproduce;
All Life depends on 3 critical molecules
More Terminology
The genome is an organisms complete set of DNA.
a bacteria contains about 600,000 DNA base pairs

human and mouse genomes have some 3 billion.
human genome has 24 distinct chromosomes.
Gene
RNAs
basic physical and functional units of heredity.

specific sequences of DNA bases that encode
instructions on how to make proteins.
Make up the cellular structure

large, complex molecules made up of smaller subunits
called amino acids.
RNA = cadeia de nucletidos

Nucletidos = acar + Fosfato +
base azotada
O acar ribose - uma pentose
(5 tomos de carbono)
Hold information on how cell works

Act to transfer short pieces of information to different parts of cell
Provide templates to synthesize into protein
Proteins
Form enzymes that send signals to other cells and regulate gene
activity
Form bodys major components (e.g. hair, skin, etc.)
A molcula de RNA
Constituintes do RNA
DNAs
Each chromosome contains many genes.
Proteins
Fosfato
Uracilo
Bases azotadas = Pirimidinas e Purinas
Pirimidinas
Cadeia simples
Ribose
4 nucletidos
diferentes
Adenina,Citosina,
Guanina e Uracilo
Purinas
As protenas so polmeros de aminocidos
Aminocidos - Grupos funcionais
26
Alanina
Ala
Arginina
Arg
Asparagina
Asn
cido Aspartico
Asp
Cistena
Cys
cido Glutamico
Glu
Glutamina
Gln
Glicina
Gly
Histidina
His
Isoleucina
Ile
Leucina
Leu
Lisina
Lys
Metionina
Met
Fenilalanina
Phe
Prolina
Pro
Serina
Ser
Treonina
Thr
Triptofano
Trp
Tirosina
Tyr
Valina
Val
Ao carbono alfa ligam-se:
H
NH2, grupo amina
COOH, grupo cido
R, radical, nico para cada aminocido
27
Tipos de aminocidos - Apolares
Tipos de aminocidos - Polares
28
29
Estruturas proteicas
Cadeia polipeptdica estrutura primria

Extremidade
amino
31
Extremidade
carboxilo
30
Montagem da protena final a partir de

diferentes subunidades
32
Principais funes das protenas

33
Tipo de protena
Funo
Exemplo
Enzima
Catlise biolgica
Enzimas activas em diferentes

processos biolgicos
Protenas de estrutura
Suporte
Fio de seda, fio de teia de aranha
Protenas de reserva
Reserva de aminocidos
Ovalbumina, casena, protenas

de reserva das sementes
Protenas de transporte
Transporte
Hemoglobina, transportadores
atravs de membranas
Receptores
Resposta a estmulos
Receptores das clulas nervosas
Hormonas
Regulao
Insulina
Protenas motoras
Movimento
Actina e miosina
Protenas de defesa
Proteco
Anticorpos
Sites de interesse
DNA, RNA, and the Flow of Information
34
Traduo - Sntese proteica
Replication
http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/nucleic_acids_1.html
Biochemistry
Transcription
http://www.biology.arizona.edu/biochemistry/biochemistry.html
Translation
Amino acids
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html
Electroforese
http://www.dnalc.org/ddnalc/resources/electrophoresis.html
DNA: The Code of Life
The structure and the four genomic letters code for all living organisms
Adenine, Guanine, Thymine, and Cytosine which pair A-T and C-G on
complimentary strands.
Overview of DNA to RNA to Protein
A gene is expressed in two steps

Transcription: RNA synthesis
2) Translation: Protein synthesis
1)
Como traduzida a informao gentica - Cdigo
Cell Information: Instruction book of Life
Gentico
39
DNA, RNA, and Proteins

are examples of strings
written in either the fourletter nucleotide of DNA
and RNA (A C G T/U)
or the twenty-letter amino
acid of proteins. Each
amino acid is coded by 3
nucleotides called codon.
(Leu, Arg, Met, etc.)
Cdigo de trs letras tripleto em

que cada tripleto codifica ara um
nico aminocido.
64 combinaes possveis
(combinaes de 4 bases 3 a 3).
20 aminocidos essenciais.
O mesmo aminocido pode ser

codificado mais de um codo.
Trs codes para parar a traduo

codes stop.
O Cdigo Gentico UNIVERSAL
Grelhas de leitura
Redundncia no cdigo gentico: codes e aminocidos
1 Grelha de Leitura
3 Grelha de Leitura
40
2 Grelha de Leitura
Metionina e Triptfano possuem um nico codo.
O cdigo gentico redundante e degenerado.
Arginina, Leucina e Serina so os aminocidos com mais codes.
A sequncia de nucletidos de uma

molcula de mRNA lida na direco 5
para 3, tripleto a tripleto (
A mesma sequncia de RNA pode
especificar trs sequncias diferentes de
amino cidos, dependendo da grelha de
leitura.
Na prtica apenas uma das grelhas
contm a mensagem correcta.
10
Revisiting the Central Dogma
DNA the Genetics Makeup
In going from DNA to proteins, there is

an intermediate step where mRNA is
made from DNA, which then makes
protein
This known as The Central Dogma
Why the intermediate step?
DNA is kept in the nucleus, while
protein sythesis happens in the
cytoplasm, with the help of
ribosomes
What are genes?
-physical and functional traits that are passed on

from one generation to the next.
Genes were discovered by Gregor Mendel in the
1860s while he was experimenting with the pea
plant. He asked the question:
Genes are inherited and are

expressed
genotype (genetic makeup)

phenotype (physical expression)
On the left, is the eyes

phenotypes of green and
black eye genes.
MUtAsHONS
The DNA can be thought of as a sequence of the

nucleotides: C,A,G, or T.
What happens to genes when the DNA sequence is
mutated?
11
The Good, the Bad, and the Silent
Mutations can serve the organism in three ways:
The Good : A mutation can cause a trait that enhances the organisms function:
Genes are Organized into Chromosomes
Mutation in the sickle cell gene provides resistance to malaria.
The Bad :
A mutation can cause a trait that is harmful, sometimes fatal to the

organism:
Huntingtons disease, a symptom of a gene mutation, is a degenerative

disease of the nervous system.
What are chromosomes?

It is a threadlike structure found in the nucleus of the cell
which is made from a long strand of DNA. Different
organisms have a different number of chromosomes in their
cells.
Thomas Morgan(1920s) - Evidence that genes are located on
chromosomes with a certain order was discovered by genetic
experiments performed with flies.
The Silent:
A mutation can simply cause no difference in the function of the

organism.
th
Campbell, Biology, 5 edition, p. 255
Portrait of Morgan
http://www.nobel.se/medicine/laureates/1933/morgan-bio.html
Chromosomes
Genes Make Proteins
Organism
Number of base pair
number of Chromosomes
---------------------------------------------------------------------------------------------------------
genome-> genes ->protein(forms cellular structural & life functional)>pathways & physiology
Prokayotic
Escherichia coli (bacterium)
4x106
Eukaryotic
1.35x107
17
Drosophila melanogaster(insect)
1.65x108
Homo sapiens(human)
2.9x109
23
Zea mays(corn)
5.0x109
10
Saccharomyces cerevisiae
(yeast)
12
Proteins: Workhorses of the Cell
20 different amino acids
Human Genome Composition
different chemical properties cause the protein chains to fold up

into specific three-dimensional structures that define their
particular functions in the cell.
Proteins do all essential work for the cell
build cellular structures

digest nutrients
execute metabolic functions
Mediate information flow within a cell and among cellular
communities.
Proteins work together with other proteins or nucleic acids as
"molecular machines"
structures that fit together and function in highly
specific, lock-and-key ways.
Definition of a Gene
Regulatory regions: up to 50 kb upstream of +1 site
Exons:
protein coding and untranslated regions (UTR)

1 to 178 exons per gene (mean 8.8)
8 bp to 17 kb per exon (mean 145 bp)
Introns:
splice acceptor and donor sites, junk DNA

average 1 kb 50 kb per intron
Gene size:
Largest 2.4 Mb (Dystrophin). Mean 27 kb.
Genes eucariotas
13
Central Dogma Revisited

Splicing
Transcription
DNA
Terminology for Splicing
hnRNA
Nucleus
mRNA
Spliceosome
Translation
protein
Ribosome in Cytoplasm
Base Pairing Rule: A and T or U is held together by 2

hydrogen bonds and G and C is held together by 3
hydrogen bonds.
Note: Some mRNA stays as RNA (ie tRNA,rRNA).
Exon: A portion of the gene that appears in both the

primary and the mature mRNA transcripts.
Intron: A portion of the gene that is transcribed but
excised prior to translation.
Spliceosome: A organelle that carries out the splicing
reactions whereby the pre-mRNA is converted to a
mature mRNA.
Splicing (Eukaryotes)
Unprocessed RNA is
composed of Introns and
Extrons. Introns are removed
before the rest is expressed
and converted to protein.
Sometimes alternate splicings
can create different valid
proteins.
A typical Eukaryotic gene has
4-20 introns. Locating them
by analytical means is not
easy.
HOW CAN WE ANALYZE DNA?
14
Sequenciao
Sequenciao - determinar a ordem dos

nucletidos que constituem uma molcula de DNA ou
dos A.A. que constituem um pptido.
A ordem dos nucletidos determina a ordem dos A.A
e consequentemente a estrutura da protena e a sua
funo.
Alteraes na sequncia de DNA podem levar a alteraes
na protena codificada por esse DNA.
Os Mtodos Clssicos
Maxam & Gilbert: Mtodo qumico (1977) modificao

qumica e posterior quebra da molcula de DNA
http://www.springerprotocols.com/Abstract/doi/10.1385/0-89603-064-4:333
Sanger: Mtodo biossinttico (1977) - terminao da

sntese da cadeia de DNA
Ambos os mtodos geram fragmentos marcados de diferentes
tamanhos que so posteriormente separados por electroforese
Alteraes pontuais
Gene A
from
per son 1
Codon result s in
particular Amino
Acid (AA) sequ ence
GCA
Ala
AGA
Arg
GAT
Asp
AAT
Asn
TGT
Cys
Gene A
from
per son 2
Codon chan ge
makes no differen ce
in AA
(redu ndant code)
Codon chan ge
result s in different
AA sequ ence
GCG
AGA
GAT
AAT
TGT
Ala
Arg
Asp
Asn
Cys
GCA
Ala
AAA
Lys
GAT
Asp
AAT
Asn
TGT
Cys
Gene A
from
per son 3
Ex. Anemia Falciforme e Fibrose qustica
Electroforese A chave
DNA fragments having a difference
of one nucleotide can be separated
on gel electrophoresis
32P
ATCGATCGAT
32P
ATCGATCGA
32P
ATCGATCG
32P
ATCGATC
32P
ATCGAT
32P
ATCGA
32P
ATCG
32P
ATC
32P
AT
32P
A
But these bands cant tell us
the identity of the terminal
nucleotides
Polyacrylamide Gel Electrophoresis
G
C
T
A
If those band with the same
terminal nucleotide can be
grouped, then it is possible
to read the whole sequence
15
O mtodo de Sanger
Para o mtodo de Sanger
Usa molculas anlogas aos

nucletidos do DNA mas sem o
grupo hidroxilo na posio 3 -
Vrias cpias de DNA em cadeia simples - template DNA
Um primer adequado para dar incio replicao
DNA polimerase para sintetizar novas molculas de DNA

adicionando novos nucletidos extremidade 3
Um conjunto de nucleotidos normais
Uma pequena poro de dideoxinucletidos marcados

radioactivamente ou com corantes fluorescentes
didexinucletidos
Mtodo de Sanger actualizado
Originalmente eram feitas quatro reaces em separado cada

uma com um dideoxinucletido diferente.
Actualmente todos os intervenientes (DNA, primers, ddNTPs

fluorescentes) so colocados simultaneamente.
A electroforese em gel de poliacrilamida foi substituda por

electroforese capilar.Os produtos so lidos com um laser.
16
Leitura das sequenciaes
Diagrama de electroforese
http://multimedia.mcb.harvard.edu/BSM_start.html
Dados no tratados cromatograma
Programas que se podem usar
Chromas (http://www.technelysium.com.au/chromas.html)
Finch (http://www.geospiza.com/Products/finchtv.shtml)
BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html)
Limite da sequenciao (mtodo de Sanger) 600-700 nucletidos
Sequncia de m qualidade
Sequenciao de genomas mtodo tradicional

DNA
Shake or restriction enzymes
DNA fragments
Vector
Circular genome
(bacterium, plasmid)
Known
location
(restriction
site)
17
Different Types of Vectors
Shotgun Sequencing
genomic segment
VECTOR
Size of insert (bp)
Plasmid
2,000 - 10,000
Cosmid
40,000
BAC (Bacterial Artificial

Chromosome)
70,000 - 300,000
YAC (Yeast Artificial

Chromosome)
> 300,000
Not used much
recently
Ligao dos fragmentos
cut many times at

random (Shotgun)
~500 bp
~500 bp
Get one or two

reads from each
segment
Fragment Assembly
Desafio computacional: a ligao de pequenos fragmento

individuais (reads) numa s sequncia genmica (superstring)
reads
At ao fim da dcada de 1990 a ligao dos fragmentos do

genoma humano gerados por shotgun era considerado um problema
sem resoluo
Cover region with ~7-fold redundancy

Overlap reads and extend to reconstruct the
original genomic region
18
Challenges in Fragment Assembly
Repeats: A major problem for fragment assembly

> 50% of human genome are repeats:
- over 1 million Alu repeats (about 300 bp)
- about 200,000 LINE repeats (1000 bp and longer)
Repeat
Low-Complexity DNA (e.g. ATATATATACATA)
Microsatellite repeats
Repeat
Repeat
Green and blue fragments are interchangeable when

assembling repetitive DNA
(a1ak)N where k ~ 3-6

(e.g. CAGCAGTAGCAGCACCAG)
Transposons/retrotransposons
SINE
Short Interspersed Nuclear Elements
(e.g., Alu: ~300 bp long, 106 copies)
LINE
LTR retroposons
Long Interspersed Nuclear Elements

~500 - 5,000 bp long, 200,000 copies
each end
Gene Families
Segmental duplications
Long Terminal Repeats (~700 bp) at

genes duplicate & then diverge
~very long, very similar copies
Overlap-Layout-Consensus
Assemblers:
Repeat Types
ARACHNE, PHRAP, CAP, TIGR, CELERA
Repeties so grandes desafios
Do two aligned fragments really overlap, or are they

from two copies of a repeat?
Overlap: find potentially overlapping reads
Layout: merge reads into contigs and

contigs into supercontigs
Consensus: derive the DNA sequence and

correct read errors
Solution: repeat masking hide the repeats!!!
Masking results in high rate of misassembly (up to 20%)
Misassembly means alot more work at the finishing step
..ACGATTACAATAGGTT..
19
Assemble the genome

1.
Sequence different reads
2.
Merge Reads into Contigs
3. Link Contigs into Supercontigs

Normal density
Too dense:
Overcollapsed?
?????
Inconsistent links:
Overcollapsed?
Unambiguous
3.
Link Contigs into Supercontigs (contd)
Find all links between unique contigs
3.
Link Contigs into Supercontigs (contd)
Fill gaps in supercontigs with paths of

overcollapsed contigs
Connect contigs incrementally, if 2 links
20
Sequncia consenso
Pirosequenciao
https://www.roche-applied-science.com/publications/multimedia/genome_sequencer/presentation/wbt.htm
Cobertura do genoma > 7x

A sequncia consenso derivada do alinhamento dos diferentes fragmentos
necessrio um nmero suficiente de leituras para garantir um consenso
estatticamente significativo
Deste modo so corrigidos os erros de leitura
TAGATTACACAGATTACTGA TTGATGGCGTAA CTA

TAGATTACACAGATTACTGACTTGATGGCGTAAACTA
TAG TTACACAGATTATTGACTTCATGGCGTAA CTA
TAGATTACACAGATTACTGACTTGATGGCGTAA CTA
TAGATTACACAGATTACTGACTTGATGGGGTAA CTA
O DNA quebrado em fragmentos de ~100 pb que so posteriormente desnaturados

(ssDNA).
ssDNA ligam-se a esferas microscpicas individualizadas.
Em cada esfera corre uma reaco de PCR de modo a que fique coberta de ~10
milhes de cpias do mesmo fragmento.
Cada esfera colocada individualmente num poo e cada poo recebe um cocktail de
reagentes:
TAGATTACACAGATTACTGACTTGATGGCGTAA CTA
Sequenciadores de ltima gerao

Two Short Read Technologies
DNA polimerase para adicionar deoxinucletidos ssDNA

APS - adenosine phosphosulfate
ATP sulfurylase enzima que produz ATP a partir de adenosine phosphosulfate (APS) e
pirosfato (PPi).
luciferina
luciferase enzima que cataliza a converso da luciferina em oxiluciferina com libertao de
luz.
Comparao Sanger vs. Next Gen
Solexa technology / Illumina GA

http://www.illumina.com/company/technology.ilmn
ABI SOLID
HeliScope single molecule sequencer technology
http://www.helicosbio.com/Technology/TrueSingleMole
culeSequencing/tSMStradeHowItWorks/tabid/162/Def
ault.aspx
21
A importncia da Bioinformtica
O Futuro
22

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2

Marcos na histria da Biologia Molecular e

1955 A sequncia da insulina bovina foi

Biologia Computacional - 2010

1958 - Jack Kilby construiu o

Marcos na histria da Biologia Molecular e

1981 - publicado o algoritmo Smith-Waterman algoritmo para o alinhamento de

1969 Foi criada a ARPANET para

1981 A IBM introduz no mercado o primeiro computador pessoal.

1970 - Howard Temin and David

Biologia Computacional 2010

Biologia Computacional 2010

Marcos na histria da Biologia Molecular e

1985 - publicado o algoritmo FASTP.

1985 - Kary Mullis e colaboradores

1952-1953 - James Watson e Francis

Biologia Computacional 2010

Section1: What is Life made of?

Fundamental working units of every living system.

Biologia Computacional 2010

2 types of cells: Prokaryotes v.s.Eukaryotes

Biologia Computacional 2010

Molcula comum a todas as clulas

Biologia Computacional 2010

DNA cido desoxiribonucleico molcula comum a todos os seres

Procariotas sem ncleo delimitado

Eucariota ncleo delimitado por

Biologia Computacional 2010

Prokaryotes and Eukaryotes

Chemical composition-by weight

Biologia Computacional 2010

Biologia Computacional 2010

Prokaryotes v.s. Eukaryotes

Single or multi cell

One piece of circular DNA Several pieces of linear

Eubacterial (blue green algae)

the boundary of the cell proper

The smallest cells known are

plants, animals, Protista, and fungi

organelle and compartments

The volume of the cell is several

5x109 protein molecules

5000-10,000 polypeptide species

Biologia Computacional 2010

Modelo da dupla hlice - Watson; Crick e

Prokaryotic and Eukaryotic Cells

> 90% of DNA encode protein

Lacks a membrane-bound nucleus.

Circular DNA and supercoiled

The genome of yeast cells contains

Histones are unknown

DNA is divided between two or

A set of five histones

DNA packaging and gene expression

Biologia Computacional 2010

Biologia Computacional 2010

Polinucletido Cadeia simples de DNA

DNA = cadeia de nucletidos

Bases azotadas = Pirimidinas e Purinas

Biologia Computacional 2010

Biologia Computacional 2010

Ligaes entre as duas cadeias simples e

Overview of organizations of life

Biologia Computacional 2010