Perjalanan Ilmu Biologi Molekuler

Washington State University [email protected]

9/15/2016

An Introduction to Molecular Biology


Washington State University

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Perjalanan Ilmu Biologi Molekuler •

Mikroskop ditemukan tahun 1665

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Robert Hooke (1635-1703) organisma terdiri dari sel

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Matthias Schleiden (18041881) dan Theodor Schwann (1810-1882) melanjutkan studi tentang sel

• Robert Hooke

• Matthias Schleiden Washington State University

• Theodor Schwann [email protected]

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Transformasi di Bakteria • Eksperimen dilakukan oleh Frederick Griffith in 1928 • Mengamati perubahan Streptococcus pneumoniae – Dari virulent (S), koloni tepi rata, berkapsul  avirulent (R) koloni tepi bergerigi tanpa kapsul

• S bakteri yang dipanasi dapat mentransformasi R bakteri menjadi S bakteri

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1800 - 1870

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1865 Gregor Mendel menemukan postulat dasar hereditas – Individu organism mempunyai 2 alternatif hereditas yang diwariskan (dominan vs resesif)

Mendel: The Father of Genetics

1869 Johann Friedrich Miescher menemukan DNA yang diberi nama “nuclein”. Johann Miescher


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1880 - 1900 • 1881 Edward Zacharias menunjukkan chromosome terdiri dari nuclein. • 1899 Richard Altmann mengubah nama “nuclein” menjadi asam nukleat. • 1900, struktur kimia 20 amino acids dapat diidentifikasi



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1900-1911 1902 - Emil Hermann Fischer pemenang Nobel prize Menunjukkan asam amino saling berikatan membentuk protein •

– Postulated: protein properties are defined by amino acid composition and arrangement, which we nowadays know as fact •

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1911 – Thomas Hunt Morgan menemukan gen dalam kromosom adalah unit terkecil hereditas 1911 Pheobus Aaron Theodore Lerene menemukan RNA


Emil Fischer

Thomas Hunt Morgan

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1940 - 1950

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1941 – George Beadle dan Edward Tatum menemukan bahwa gen membuat protein George Beadle

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1950 – Edwin Chargaff menemukan bahwa – Cytosine berpasangan dengan Guanine – Adenine berpasangan dengan Thymine



Edward Tatum

Edwin Chargaff

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1950 - 1952

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1950s – Mahlon Bush Hoagland mengisolasi tRNA pertama kali

Mahlon Hoagland

1952 – Alfred Hershey dan Martha Chase menyebutkan bahwa gen adalah DNA

Hershey Chase Experiment Washington State University

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Gen terdiri dari mRNA Hershey & Chase menginvestigasi bacteriophage, virus particle – Virus ini tidak mempunyai aktifitas metabolisme sendiri – Ketika virus menginfeksi host, maka host akan mulai mensintesis protein virus – Gen visrus akan bereplikasi dan melakukan asembling dengan proteinnya membentuk partikul virus baru. Ada virus yang mengandung DNA gen, ada yang RNA gen

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Procedur Transformasi Virus

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1952 - 1960

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1952-1953 James D. Watson dan Francis H. C. Crick menunjukkan bahwa struktur DNA adalah double helix

James Watson and Francis Crick

1956 George Emil Palade menunjukkan bahwa ribosom organel tempat pembuatan protein yang ada di sitoplasma George Emil Palade



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1970 •

1970 Howard Temin dan David Baltimore mampu mengisolasi enzim restriksi yang pertama kali.

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DNA dapat dipotong menjadi fragment di bagian tertentu yang disebut dengan “site-specific restriction enzymes”; – Fragment DNA ini dapat digabungkan kembali dan dimasukkan ke dalam bakteri inang (gene cloning or recombinant DNA technology)


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1970- 1977

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1977 Phillip Sharp dan Richard Roberts menunjukkan bahwa premRNA proses pemotongan intron dan menggabungan exon.

Phillip Sharp



Richard Roberts

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1986 - 1995

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1986 Leroy Hood: mengembangkan sekuensing

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1986 Human Genome projek dimulai

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1990 Human Genome dipublikasi

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1995 Publikasi susunan DNA di kromosom 3, 11, 12, dan 22 (mapping genes)

Leroy Hood


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1995-1996

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1995 John Craig Venter: mensekuen genom bakteri yang pertama kali

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1995 Automated fluorescent sequencing instruments and robotic operations

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1996 Sekuensing eukaryotic genomeyeast-pertama kali



John Craig Venter

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1997 - 1999

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1997 E. Coli dapat disekuensing

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1998 PerkinsElmer, Inc.. Developed 96-capillary sequencer

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1998 Complete sekuen dari genom Caenorhabditis elegans

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1999 Sekuensung kromosom no 22 manusia


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2000-2001

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2000 sekuensing genom Drosophila melanogaster

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2001 International Human Genome Sequencing: draft pertama sekuen genom manusia dipublikasi



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2003 – sekarang • April 2003 Genom manusia selesai dilakukan, Genom tikus mulai dilakukan. • April 2004 genom rat dilakukan.


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1869 Johann Friedrich Miescher menemukan DNA yang diberi nama “nuclein”. Johann Miescher

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1950 – Edwin Chargaff menemukan bahwa – Cytosine berpasangan dengan Guanine – Adenine berpasangan dengan Thymine

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1952-1953 James D. Watson dan Francis H. C. Crick menunjukkan bahwa struktur DNA adalah double helix




James Watson and Francis Crick [email protected]

DNA •

Struktur DNA double helix sugar molecule – phosphate group – and a base (A,C,G,T)

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DNA selalu dibaca dari ujung 5’ ke ujung 3’ untuk transkripsi dan replikasi 5’ ATTTAGGCC 3’ 3’ TAAATCCGG 5’

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Informasi Sel

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DNA, RNA, dan Protein hanya ditulis dengan 4 huruf saja (A C G T/U)

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20 jenis asam amino hanya dikode oleh 3 nukleotida saja, yang disebut codon (Leu, Arg, Met, etc.)


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Lecture PowerPoint to accompany

Molecular Biology Fourth Edition

Robert F. Weaver Chapter 2 The Molecular Nature of Genes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.


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The Chemical Nature of Polynucleotides • The component parts of DNA – Nitrogenous bases: • Adenine (A) • Cytosine (C) • Guanine (G) • Thymine (T)

– Phosphoric acid – Deoxyribose sugar


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RNA • RNA component parts – Nitrogenous bases • Like DNA except Uracil (U) replaces Thymine – Phosphoric acid – Ribose sugar • Nucleosides lack the phosphoric acid


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Purines and Pyrimidines • Adenine and guanine are related structurally to the parent molecule purine • Cytosine, thymine and uracil resemble pyrimidine


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DNA Linkage • Nucleotides are nucleosides with a phosphate group attached through a phosphodiester bond • Nucleotides may contain one, two, or even three phosphate groups linked in a chain


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A Trinucleotide The example trinucleotide has polarity – Top of molecule has a free 5’-phosphate group = 5’ end – Bottom has a free 3’hydroxyl group = 3’ end

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Summary • DNA and RNA are chain-lie molecules composed of subunits called nucleotides • Nucleotides contain a base linked to the 1’position of a sugar and a phosphate group • Phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’-hydroxyl groups by phosphodiester bonds


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DNA Structure The Double Helix • Rosalind Franklin’s x-ray data suggested that DNA had a helical shape • The data also indicated a regular, repeating structure • DNA was believed to require an irregular sequence • Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned to the interior

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DNA Helix • Structure compared to a twisted ladder – Curving sides of the ladder represent the sugarphosphate backbone – Ladder rungs are the base pairs – There are about 10 base pairs per turn

• Arrows indicate that the two strands are antiparallel


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Summary • The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside • The bases pair in a specific way: – Adenine (A) with thymine (T) – Guanine (G) with cytosine (C)


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Genes Made of RNA Hershey & Chase investigated bacteriophage, virus particle by itself, a package of genes – This has no metabolic activity of its own – When virus infects a host cell, the cell begins to make viral proteins – Viral genes are replicated and newly made genes with viral protein assemble into virus particles

Some viruses contain DNA genes, but some viruses have RNA genes, either double- or singlestranded Washington State University

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Physical Chemistry of Nucleic Acids DNA and RNA molecules can appear in several different structural variants – Changes in relative humidity will cause variation in DNA molecular structure – The twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA was identified in 1979


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A Variety of DNA Structures • High humidity DNA is called the B-form • Lower humidity from cellular conditions to about 75% and DNA takes on the A-form – Plane of base pairs in A-form is no longer perpendicular to • When wound in a leftthe helical axis handed helix, DNA is – A-form seen when hybridize termed Z-DNA one DNA with one RNA strand in solution Washington State University

• One gene requires ZDNA for activation 2-36

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Variation in DNA between Organisms • Ratios of G to C and A to T are fixed in any specific organism • The total percentage of G + C varies over a range to 22 to 73% • Such differences are reflected in differences in physical properties Washington State University

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DNA Melting

• With heating, noncovalent forces holding DNA strands together weaken and break • When the forces break, the two strands come apart in denaturation or melting • Temperature at which DNA strands are ½ denatured is the melting temperature or Tm • GC content of DNA has a significant effect on Tm with higher GC content meaning higher Tm Washington State University

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DNA Denaturation • In addition to heat, DNA can be denatured by: – Organic solvents – High pH – Low salt concentration

• GC content also affects DNA density – Direct, linear relationship – Due to larger molar volume of an A-T base pair than a G-C base pair 2-39


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Summary • GC content of a natural DNA can vary from less than 25% to almost 75% • GC content has a strong effect on physical properties that increase linearly with GC content – Melting temperature, the temperature at which the two strands are half-dissociated or denatured – Density – Low ionic strength, high pH and organic solvents also promote DNA denaturation


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DNA Renaturation • After two DNA strands separate, under proper conditions the strands can come back together • Process is called annealing or renaturation • Three most important factors: – Temperature – best at about 25 C below Tm – DNA Concentration – within limits higher concentration better likelihood that 2 complementary will find each other – Renaturation Time – as increase time, more annealing will occur Washington State University

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Polynucleotide Chain Hybridization Hybridization is a process of putting together a combination of two different nucleic acids – Strands could be 1 DNA and 1 RNA – Also could be 2 DNA with complementary or nearly complementary sequences


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DNA Sizes DNA size is expressed in 3 different ways: – Number of base pairs – Molecular weight – 660 is molecular weight of 1 base pair – Length – 33.2 Å per helical turn of 10.4 base pairs

Measure DNA size either using electron microscopy or gel electrophoresis


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DNAs of Various Sizes and Shapes

• Phage DNA is typically circular • Some DNA will be linear • Supercoiled DNA coils or wraps around itself like a twisted rubber band Washington State University

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Summary • Natural DNAs come in sizes ranging from several kilobases to thousands of megabases • The size of a small DNA can be estimated by electron microscopy • This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled


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Relationship between DNA Size and Genetic Capacity How does one know how many genes are in a particular piece of DNA? – Can’t determine from DNA size alone – Factors include: • How DNA is devoted to genes? • What is the space between genes?

– Can estimate the upper limit of number genes a piece of DNA can hold


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DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions • Gene encodes protein • Protein is abut 40,000 D

– How many amino acids does this represent? • • • •

Average mass of an amino acid is about 110 D Average protein – 40,000 / 110 = 364 amino acids Each amino acid = 3 DNA base pairs 364 amino acids requires 1092 base pairs


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DNA Genetic Capacity How large is an average piece of DNA? – E. coli chromosome • 4.6 x 106 bp • ~4200 proteins

– Phage l (infects E. coli) • 4.85 x 104 bp • ~44 proteins

– Phage x174 (one of smallest) • 5375 bp • ~5 proteins Washington State University

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DNA Content and the C-Value Paradox • C-value is the DNA content per haploid cell • Might expect that more complex organisms need more genes than simple organisms • For the mouse or human compared to yeast this is correct • Yet the frog has 7 times more per cell than humans


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C-Value Paradox • The observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradox • Most likely explanation for the paradox is that DNA that does not code for genes is present when the less complex organism has more DNA


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Summary • There is a rough correlation between DNA content and number of genes in a cell or virus • This correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widely • C-value paradox is probably explained not by extra genes, but by extra noncoding DNA in some organisms


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Perjalanan Ilmu Biologi Molekuler

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