Enzim Restriksi dan Fungsinya
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Apa fungsi utama dari enzim restriksi pada sel bakteri?

  • Memotong DNA bakteri sendiri
  • Melindungi DNA dari DNA eksogen (correct)
  • Menghancurkan RNA
  • Meningkatkan ekspresi gen

    • Enzim restriksi dapat merusak DNA endogen bakteri karena DNA tersebut tidak dimetilasi.

    True

    Sebutkan dua jenis metilasi yang dapat terjadi pada DNA eukariotik!

    Metilasi pada gen dan metilasi pada jaringan spesifik.

    Enzim restriksi tipe II sebagian besar memerlukan __________ untuk berfungsi.

    <p>magnesium</p> Signup and view all the answers

    Jocokan jenis enzim restriksi dengan karakteristiknya:

    <p>Type I = Memerlukan ATP dan S-adenosyl-L-methionine Type II = Memotong di dalam atau dekat situs pengenalan Type III = Memotong di jarak pendek dari situs pengenalan Type IV = Menargetkan DNA yang dimodifikasi</p> Signup and view all the answers

    Mengapa bakteri tidak dapat menghancurkan DNA endogennya sendiri?

    <p>Karena DNA endogen dimetilasi</p> Signup and view all the answers

    HindII dan EcoRI adalah contoh dari enzim restriksi yang dinamai berdasarkan bakteri asalnya.

    <p>True</p> Signup and view all the answers

    Enzim restriksi jenis __________ adalah enzim dengan fungsi tunggal hanya untuk restriksi.

    <p>II</p> Signup and view all the answers

    Apa yang dimaksud dengan 'restriction site'?

    <p>Site spesifik yang dikenali oleh enzim restriksi</p> Signup and view all the answers

    Sticky ends dan blunt ends adalah dua jenis pemotongan DNAdari enzim restriksi.

    <p>True</p> Signup and view all the answers

    Apa fungsi dari enzim restriksi?

    <p>Melindungi sel dari DNA asing.</p> Signup and view all the answers

    Enzim restriksi memotong DNA di situs _____ yang dikenal.

    <p>pengakuan</p> Signup and view all the answers

    Cocokkan enzim restriksi dengan jenis potongannya:

    <p>EcoRI = Sticky ends AluI = Blunt ends HindIII = Sticky ends HaeIII = Blunt ends</p> Signup and view all the answers

    Apa yang terjadi jika ukuran urutan pengakuan lebih besar?

    <p>Frekuensi pemotongan menurun</p> Signup and view all the answers

    Isoschizomer memiliki situs pengakuan yang berbeda tetapi situs pemotongan yang sama.

    <p>True</p> Signup and view all the answers

    Sebutkan dua jenis potongan yang dihasilkan oleh enzim restriksi.

    <p>Sticky ends dan blunt ends.</p> Signup and view all the answers

    Proses untuk menentukan lokasi relatif dari berbagai situs pengakuan pada DNA disebut _____ mapping.

    <p>restriction</p> Signup and view all the answers

    Apa yang menjadi ciri utama plasmid yang digunakan sebagai vektor kloning?

    <p>Memiliki origin of replication dan marker seleksi.</p> Signup and view all the answers

    Plasmid dapat mereplikasi diri hanya setelah dimasukkan ke dalam sel eukariota.

    <p>False</p> Signup and view all the answers

    Sebutkan satu contoh marker seleksi yang biasa digunakan dalam plasmid.

    <p>ampr</p> Signup and view all the answers

    Plasmid harus memiliki _____ untuk dapat mereplikasi di dalam host.

    <p>origin of replication</p> Signup and view all the answers

    Cocokkan jenis bakteri dengan aplikasi kloningnya:

    <p>E. coli = Bakteri umum untuk kloning Bacillus sp. = Pembuatan enzim Pseudomonas sp. = Produksi antibiotik Streptomyces sp. = Sumber antibakteri</p> Signup and view all the answers

    Apa tujuan utama penggunaan host sel tertentu dalam eksperimen kloning?

    <p>Untuk mengekspresikan informasi genetik.</p> Signup and view all the answers

    Chimeric plasmids adalah plasmid yang hanya berasal dari satu organisme.

    <p>False</p> Signup and view all the answers

    Apa yang dimaksud dengan 'multiple cloning site' (MCS) dalam plasmid?

    <p>Tempat di mana DNA asing dapat dimasukkan tanpa mengganggu fungsi plasmid.</p> Signup and view all the answers

    Apa fungsi dari gen resistensi antibiotik pada pDNA?

    <p>Memungkinkan seleksi sel yang mengandung plasmid</p> Signup and view all the answers

    Plasmid konjugatif tidak memerlukan bantuan plasmid lain untuk melakukan konjugasi.

    <p>False</p> Signup and view all the answers

    Sebutkan salah satu keuntungan dari plasmid pUC.

    <p>Memiliki situs kloning ganda (polylinker) dengan beberapa situs pemotongan yang unik.</p> Signup and view all the answers

    Ukuran maksimum fragmen DNA yang dapat dimasukkan ke dalam plasmid adalah _____ kb.

    <p>5</p> Signup and view all the answers

    Cocokkan jenis plasmid dengan karakteristiknya:

    <p>Plasmid konjugatif = Memerlukan bantuan dari plasmid lain untuk konjugasi Plasmid non-konjugatif = Memiliki daerah tra dan mob pAT153 = Derivatif dari pBR322 dengan peningkatan jumlah salinan pUC = Memiliki situs kloning ganda (polylinker)</p> Signup and view all the answers

    Apa keuntungan utama dari penggunaan E. coli sebagai vektor dalam rekayasa genetik?

    <p>E. coli mudah untuk dimodifikasi dan diisolasi.</p> Signup and view all the answers

    S. cerevisiae merupakan mikroba prokariotik yang paling umum digunakan dalam rekayasa genetik.

    <p>True</p> Signup and view all the answers

    Sebutkan dua jenis vektor yang digunakan untuk E. coli.

    <p>Plasmid dan Bakteriofag</p> Signup and view all the answers

    Plasmid adalah bahan genetik berbentuk ______ yang ditemukan di bakteri.

    <p>sirkular</p> Signup and view all the answers

    Cocokkan jenis vektor dengan karakteristiknya:

    <p>Plasmid = DNA ekstrakromosom sirkular Bakteriofag = Vektor dengan kemampuan infeksi sel Vektor hibrida = Kombinasi plasmid dan bakteriofag Kromosom buatan = Struktur DNA besar untuk eukariot</p> Signup and view all the answers

    Apa yang dimaksud dengan 'selectable marker' dalam vektor?

    <p>Tanda untuk mengidentifikasi sel yang berhasil ditransformasi.</p> Signup and view all the answers

    Situs pengenalan unik pada DNA vektor diperlukan untuk memudahkan penyisipan DNA rekombinan.

    <p>True</p> Signup and view all the answers

    Apa keuntungan menggunakan C. reinhardtii dalam rekayasa genetik?

    <p>Menggabungkan keuntungan mikroba dengan struktur sel tanaman.</p> Signup and view all the answers

    Apa yang menjadi ciri utama dari promoter konstitutif?

    <p>Mengatur ekspresi gen pada semua jaringan</p> Signup and view all the answers

    Promoter induktif dapat diaktifkan oleh faktor biotik dan abiotik.

    <p>True</p> Signup and view all the answers

    Sebutkan satu contoh dari promoter induktif!

    <p>Sistem heat-shock pada Drosophila</p> Signup and view all the answers

    Promoter __________ digunakan untuk mengatur ekspresi gen hanya pada jaringan tertentu.

    <p>khusus jaringan</p> Signup and view all the answers

    Apa kriteria penting untuk promoter yang teratur secara kimia?

    <p>Berasal dari organisme dengan evolusi jauh dari target</p> Signup and view all the answers

    Promoter sintetis dirancang untuk hanya aktif dalam spesies tertentu.

    <p>True</p> Signup and view all the answers

    Apa tujuan pengembangan promoter konstitutif dalam rekayasa genetik?

    <p>Untuk mengproduksi protein yang diperlukan selama semua tahap perkembangan tanaman.</p> Signup and view all the answers

    Cocokkan jenis promoter dengan karakteristiknya:

    <p>Promoter konstitutif = Ekspresi di hampir semua jaringan Promoter induktif = Diaktifkan oleh faktor lingkungan Promoter spesifik jaringan = Ekspresi terbatas pada jaringan tertentu Promoter sintetis = Dibuat secara buatan untuk tujuan tertentu</p> Signup and view all the answers

    Promoter yang dapat diatur dengan suhu tinggi disebut __________.

    <p>promoter heat-shock</p> Signup and view all the answers

    Study Notes

    Cloning and Recombinant DNA

    • Cloning and recombinant DNA are techniques used to transfer DNA fragments between organisms.
    • A plasmid, a circular double-stranded DNA molecule, is a vehicle for cloning. Plasmids are often used to transfer antibiotic resistance genes between bacteria.
    • Restriction enzymes cut DNA at specific recognition nucleotide sequences, called restriction sites. They're often found in bacteria, acting as a defense mechanism against foreign DNA.
    • Restriction enzymes can create blunt ends or sticky ends when cutting DNA. Sticky ends have complementary overhangs, facilitating easier ligation.
    • DNA ligase seals the sugar-phosphate backbone of DNA fragments. It's crucial for joining DNA fragments together during recombinant DNA technology.
    • Cloning vectors are plasmids or bacteriophages modified to carry foreign DNA. They need an origin of replication, a selectable marker (e.g., antibiotic resistance gene), and multiple cloning sites.
    • Types of restriction enzymes are categorized by their composition, enzyme co-factor requirements. and the nature and position of their DNA cleavage sites.
    • Isoschizomers have similar recognition sites but different restriction sites.
    • Isocaudomers have different recognition sites but similar restriction sites.

    Basic Steps in Gene Cloning

    • Isolate DNA and fragment it into desired sizes.
    • Insert the DNA fragments into a carrier molecule (vector).
    • Introduce the recombinant DNA into a host cell (e.g., bacteria).
    • Select host cells containing the desired DNA fragment.

    Contents

    • Cloning principles
    • Restriction enzymes
    • Host cells
    • Vectors
    • Promoters
    • Transformant selection
    • Mobile DNA
    • Vector criteria and types

    Restriction Enzymes

    • Restriction enzymes are molecular scissors that cut DNA at specific sites.
    • They act as a protection mechanism against foreign DNA.
    • Bacterial endogenous DNA is protected by methylation.
    • Recognition sites are short sequences of nucleotides (4-8 base pairs long).
    • Many recognition sequences are palindromic.

    How Bacteria Prevent Restriction Enzymes

    • Bacteria methylate their own DNA to prevent cleavage by restriction enzymes.

    Types of Restriction Enzymes

    • There are four general groups of restriction enzymes.
      • Type I, II, III, and IV.

    Characteristics of Restriction Enzymes

    • Type I enzymes cleave DNA at sites far from the recognition sequence. They require ATP and S-Adenosyl-L-methionine.
    • Type II enzymes cleave DNA within or close to the recognition sequence. They often require magnesium.
    • Type III enzymes cleave DNA at specific distances from the recognition sequence. They need ATP.
    • Type IV enzymes cleave modified DNA such as methylated, hydroxymethylated, and glucosyl-hydroxymethylated DNA.

    Nomenclature of Restriction Enzymes

    • Restriction enzymes are named after the bacteria from which they are purified.
    • The name includes the genus, species, and strain.

    Restriction Site

    • A restriction site is the specific nucleotide sequence recognized by a restriction enzyme.
    • Recognition sequences vary in length and many are palindromic (read the same forward and backward on complementary strands).

    Palindromic Sequence

    • A palindromic sequence reads the same forward and backward on complementary DNA strands.
    • The size of the recognition sequence determines how often a restriction enzyme will cut.

    Function and Mechanism of Restriction Enzymes

    • Restriction enzymes protect cells by restricting the function of infecting DNA.
    • They recognize specific sequences and cut DNA, creating blunt ends or sticky ends.

    Sticky Ends and Blunt Ends

    • Sticky ends are DNA fragments with complementary overhangs.
    • Blunt ends are DNA fragments with no overhangs.

    Restriction mapping

    • A technique to determine the relative locations of different recognition sites of different enzymes in a DNA molecule—cutting with various combinations of restriction enzymes and determining the sizes of fragments.

    DNA Ligase

    • A molecular glue that joins DNA fragments together.
    • Seals the sugar-phosphate backbone of DNA.
    • Efficiently seals sticky ends but can also seal blunt ends, though with lower efficiency.
    • T4 DNA ligase is commonly used.

    Plasmids

    • Naturally occurring, extrachromosomal, circular, double-stranded DNA molecules.
    • Important for transferring genetic material (e.g., antibiotic resistance) between bacteria.
    • Can be modified to carry new genes, which makes them useful cloning vectors.

    Cloning Vectors

    • Plasmids are commonly used cloning vectors.
    • Cloning vectors require an origin of replication.
    • A selectable marker.
    • Multiple cloning sites.

    Chimeric Plasmids

    • Combinations of different segments.

    Host Cells

    • Ideal host cells are easy to handle, propagate, have diverse strains, and accept a variety of vectors.
    • Prokaryotes (bacteria) are commonly used in many cloning protocols.
    • Eukaryotes are used when the goal is gene expression.
    • Bacteria (e.g., E. coli)
    • Fungi (e.g., Yeast)
    • Plants
    • Animals

    Prokaryotic Hosts

    • E. coli is commonly used for cloning and is well-characterized. But it has limitations on fragment size for effective cloning.
    • other bacteria can also be used as hosts.

    Eukaryotic Hosts

    • Microbes (e.g., yeasts) and more complex organisms (e.g., plants, animals) with specific advantages for particular genetic engineering purposes.

    Artificial Chromosomes

    • Artificial chromosomes (e.g., YACs and BACs) are used to clone large DNA fragments.
    • BACs are more stable than YACs, which helps in cloning large segments of DNA.

    Promoter

    • Promoter is a specific DNA sequence that dictates where, when, and how much transcription occurs.
    • Promoters may be constituitive—always active—or inducible—activated by specific factors.
    • Promoters may be tissue-specific, acting only in certain cells or during specific development stages.

    Types of Promoters

    • Constitutive promoters
    • Inducible promoters
    • Tissue-specific/developmental-stage specific promoters
    • Synthetic promoters

    Common Reporter Genes

    • Antibiotic-resistance genes
    • LacZ gene of E. coli
    • GUS reporter gene
    • GFP (Green Fluorescent Protein) gene (often isolated from jellyfish)

    Bacterial Transformation

    • Uptake of foreign DNA by bacteria.
    • Selection process often uses antibiotic resistance, since transformed cells are more likely to thrive in an antibiotic-containing medium.

    Reporter Gene

    • Genes that encode easily detectable proteins.
    • Allows scientists to follow gene expression in a specific pathway of cells.

    LacZ Gene

    • Used as a reporter gene in molecular biology.
    • Its product, beta-galactosidase, catalyzes the breakdown of certain substrates, which can be used to identify bacterial cells with the appropriate DNA.

    GUS Reporter Gene

    • Encodes β-glucuronidase (GUS).
    • Compatible with Agrobacterium-mediated transformation.
    • Commonly used to analyze promoter activity in plants, because the presence of GUS enzyme is easily tested by visualization.

    Fluorescent Protein

    • Proteins that fluoresce.
    • GFP is a common fluorescent protein used in research.
    • Used to analyze promoter activity.

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    Description

    Uji pengetahuan ini menguji pemahaman Anda tentang enzim restriksi pada sel bakteri. Anda akan menjawab pertanyaan mengenai fungsi, jenis, dan karakteristik enzim restriksi dalam pemotongan DNA. Pelajari lebih lanjut tentang struktur dan mekanisme kerja enzim ini.

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