DNA Cloning: The Basics:- Expression of Cloned Genes Produces Large Quantities of Protein

Frequently it is the product of the cloned gene, rather than the gene itself, that is of primary interest—particularly when the protein has commercial, therapeutic, or research value. With an increased understanding of the fundamentals of DNA, RNA, and protein metabolism and their regulation in E. coli, investigators can now manipulate cells to express cloned genes in order to study their protein products.

Most eukaryotic genes lack the DNA sequence ele ments—such as promoters, sequences that instruct RNA polymerase where to bind—required for their expression in E. coli cells, so bacterial regulatory sequences for transcription and translation must be inserted at ap propriate positions relative to the eukaryotic gene in the vector DNA. (Promoters, regulatory sequences, and other aspects of the regulation of gene expression are discussed in Chapter 28.) In some cases cloned genes are so efficiently expressed that their protein product represents 10% or more of the cellular protein; they are said to be overexpressed. At these concentrations some foreign proteins can kill an E. coli cell, so gene expression must be limited to the few hours before the planned harvest of the cells.

Cloning vectors with the transcription and transla tion signals needed for the regulated expression of a cloned gene are often called expression vectors. The rate of expression of the cloned gene is controlled by replacing the gene’s own promoter and regulatory sequences with more efficient and convenient versions supplied by the vector. Generally, a well-characterized promoter and its regulatory elements are positioned near several unique restriction sites for cloning, so that genes inserted at the restriction sites will be expressed from the regulated promoter element (Fig. 9–11). Some of these vectors incorporate other features, such as a bacterial ribosome binding site to enhance translation of the mRNA derived from the gene, or a transcription termination sequence.

Genes can similarly be cloned and expressed in eukaryotic cells, with various species of yeast as the usual hosts. A eukaryotic host can sometimes promote post translational modifications (changes in protein structure made after synthesis on the ribosomes) that might be required for the function of a cloned eukaryotic protein.

FIGURE 9–10 Probe to detect the gene for a protein of known amino acid sequence. Because more than one DNA sequence can code for any given amino acid sequence, the genetic code is said to be “de generate.” (As described in Chapter 27, an amino acid is coded for by a set of three nucleotides called a codon. Most amino acids have two or more codons; see Fig. 27–7.) Thus the correct DNA sequence for a known amino acid sequence cannot be known in advance. The probe is designed to be complementary to a region of the gene with minimal degeneracy, that is, a region with the fewest possible codons for the amino acids—two codons at most in the example shown here. Oligonucleotides are synthesized with selectively randomized sequences, so that they contain either of the two possible nucleotides at each position of potential degeneracy (shaded in pink). The oligonucleotide shown here represents a mixture of eight different sequences: one of the eight will complement the gene perfectly, and all eight will match at least 17 of the 20 positions.

FIGURE 9–11 DNA sequences in a typical E. coli expression vector. The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end encoding the amino terminus proximal to the promoter. The promoter allows efficient transcription of the inserted gene, and the transcription termination sequence sometimes improves the amount and stability of the mRNA produced. The operator (O) permits regulation by means of a repressor that binds to it. The ribosome binding site pro vides sequence signals needed for efficient translation of the mRNA derived from the gene. The selectable marker allows the selection of cells containing the recombinant DNA.

اخر الاخبار

اخبار العتبة العباسية المقدسة

المجمع العلمي ينظّم سفرة دينية لطلبة مشاريعه القرآنية في كربلاء

عبر مجلّة (تراث الجنوب) المحكّمة.. قسم شؤون المعارف يوثق إرث مدن جنوب العراق

في ذكرى تأسيسه.. متحف الكفيل يستعرض مسيرته في حفظ التراث الإنساني والإسلامي

قسم الشؤون الفكرية وجامعة ميسان يبحثان تعزيز التعاون العلمي المشترك

المزيد

الآخبار الصحية

السجائر الإلكترونية والإقلاع عن التدخين.. نتائج متباينة تثير الجدل العلمي

مصر تحقق إنجازاً دولياً جديداً في منظمة الصحة العالمية

دراسة: السباحة فعّالة أكثر من الجري في الحفاظ على صحة القلب

عامل خطر في بداية الحمل يؤخر تطور الكلام والمهارات الحركية لدى الرضع

المزيد

الآخبار التكنلوجية

أحلامك ليست عشوائية.. إليك ما يفعله دماغك أثناء نومك

روسيا.. العلماء يضاعفون الاستقرار الحراري للألواح الشمسية ثلاث مرات

علماء يكشفون سر سرعة الدلافين في السباحة

سلمي أو دموي.. هكذا تنتقل السلطة في مجتمع الجرذان

المزيد

مواضيع ذات صلة

From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information

DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins

DNA Cloning: The Basics:- Specific DNA Sequences Are Detectable by Hybridization

DNA Cloning: The Basics:- Cloning Vectors Allow Amplification of Inserted DNA Segments

DNA Cloning: The Basics:- Restriction Endonucleases and DNA Ligase Yield Recombinant DNA

Other Functions of Nucleotides:- Adenine Nucleotides Are Components of Many Enzymes Cofactors

Other Functions of Nucleotides:- Nucleotides Carry Chemical Energy in Cells