Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

DNA

5DNA fragments are bound to the filter in positions identical to those on the gel

6Hybridize filter with radioactively labeled probe.

The Human Genome Project

The Human Genome Project is a collaborative international, governmentand private-sponsored effort to map and sequence the entire human genome, some 3 billion base pairs distributed among the two sex chromosomes (X and Y) and 22 autosomes (chromosomes that are not sex chromosomes). Initial work identified and mapped at least 3000 genetic markers (genes or other recognizable loci on the DNA), evenly distributed throughout the chromosomes at roughly 100-kb intervals. At the same time, determination of the entire nucleotide sequence of the human genome began. The target date for completion is 2005. An ancillary part of the project is sequencing the genomes of other species (such as yeast, Drosophila melanogaster [the fruit fly], mice, and Arabidopsis thaliana [a plant]) to reveal comparative aspects of genetic and sequence organization (Table 13.1). Information about whole genome sequences of organisms has created a new branch of science called functional genomics. Functional genomics addresses global issues of gene expression, such as looking at all the genes that are activated during major metabolic shifts (as from growth under aerobic to growth under anaerobic conditions) or during embryogenesis and development of organisms. Functional genomics also provides new insights into evolutionary relationships between organisms.

The Human Genome Project is also vital to medicine. A number of human diseases have been traced to genetic defects, whose positions within the human genome have been identified. Among these are

cystic fibrosis gene

Duchenne muscular dystrophy gene* (at 2.4 megabases, the largest known gene in any organism)

Huntington’s disease gene

neurofibromatosis gene

neuroblastoma gene (a form of brain cancer)

amyotrophic lateral sclerosis gene (Lou Gehrig’s disease)

fragile X-linked mental retardation gene*

as well as genes associated with the development of diabetes, breast cancer, colon cancer, and affective disorders such as schizophrenia and bipolar affective disorder (manic depression).

Table 13.1

Completed Genome Nucleotide Sequences

1Genome size is given as millions of base pairs (mb). 2Marchantia is a bryophyte (a nonvascular green plant).

3The first complete sequence for the genome of a free-living organism.

Expression Vectors

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage . Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available.

R N A Expre s s i o n

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SP6 RNA polymerase. Large amounts of RNA product can be obtained in this manner; if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made.

P rotein Expression

Because cDNAs are DNA copies of mRNAs, cDNAs are uninterrupted copies of the exons of expressed genes. Because cDNAs lack introns, it is feasible to express these cDNA versions of eukaryotic genes in prokaryotic hosts that cannot process the complex primary transcripts of eukaryotic genes. To express a eukaryotic protein in E. coli, the eukaryotic cDNA must be cloned in an expression vector that contains regulatory signals for both transcription and translation. Accordingly, a promoter where RNA polymerase initiates transcription as well as a ribosome binding site to facilitate translation are engineered into the vector just upstream from the restriction site for inserting foreign DNA. The AUG initiation codon that specifies the first amino acid in the protein (the translation start site) is contributed by the insert (Figure 13.16).

Strong promoters have been constructed that drive the synthesis of foreign proteins to levels equal to 30% or more of total E. coli cellular protein. An example is the hybrid promoter, ptac, which was created by fusing part of the promoter for the E. coli genes encoding the enzymes of lactose metabolism (the lac promoter) with part of the promoter for the genes encoding the enzymes of tryptophan biosynthesis (the trp promoter) (Figure 13.17). In cells carrying ptac expression vectors, the ptac promoter is not induced to drive transcription of the foreign insert until the cells are exposed to inducers that lead to its activation. Analogs of lactose (a -galactoside) such as isopropyl- -thio- galactoside, or IPTG, are excellent inducers of ptac. Thus, expression of the foreign protein is easily controlled. (See Chapter 31 for detailed discussions of inducible gene expression.) The bacterial production of valuable eukaryotic proteins represents one of the most important uses of recombinant DNA technology. For example, human insulin for the clinical treatment of diabetes is now produced in bacteria.

Analogous systems for expression of foreign genes in eukaryotic cells include vectors carrying promoter elements derived from mammalian viruses, such as simian virus 40 (SV40), the Epstein – Barr virus, and the human cytomegalovirus (CMV). A system for high-level expression of foreign genes uses insect cells infected with the baculovirus expression vector. Baculoviruses infect lepidopteran insects (butterflies and moths). In engineered baculovirus vectors, the foreign gene is cloned downstream of the promoter for polyhedrin, a major viral-encoded structural protein, and the recombinant vector is incorporated

13.2 ● DNA Libraries 413

Linearize

RNA transcription by

SP6 RNA polymerase

SP6 RNA polymerase

● Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SP6 are useful for making RNA transcripts in vitro. SP6 RNA polymerase works efficiently in vitro and recognizes its specific promoter with high specificity. These vectors typically have a polylinker adjacent to the SP6 promoter. Successive rounds of transcription initiated by SP6 RNA polymerase at its promoter lead to the production of multiple RNA copies of any DNA inserted at the polylinker. Before transcription is initiated, the circular expression vector is linearized by a single cleavage at or near the end of the insert so that transcription terminates at a fixed point.

Bacterial promoter and ribosomebinding site

Bacterial plasmid

mRNA

N

● A ptac protein expression vector contains the hybrid promoter ptac derived from fusion of the lac and trp promoters. Expression from ptac is more than 10 times greater than expression from either the lac or trp promoter alone. Isopropyl- -D-thiogalacto- side, or IPTG, induces expression from ptac as well as lac.

Recovery of eukaryotic protein product

● A typical expression-cloning vector. Eukaryotic coding sequences are inserted at the restriction site just downstream from a promoter region where RNA polymerase binds and initiates transcription. Transcription proceeds through a region encoding a bacterial ribosome-binding site and into the cloned insert. The presence of the bacterial ribosome-binding site in the RNA transcript ensures that the RNA can be translated into protein by the ribosomes of the host bacteria.

Biology of the Gene, 4th edition. Copyright 1987 by James D. Watson. Reprinted by permission of Benjamin/Cummings Publishing Co., Inc.)

into insect cells grown in culture. Expression from the polyhedrin promoter can lead to accumulation of the foreign gene product to levels as high as 500 mg/L.

Screening cDNA Expression Libraries with Antibodies

Antibodies that specifically cross-react with a particular protein of interest are often available. If so, these antibodies can be used to screen a cDNA expression library to identify and isolate cDNA clones encoding the protein. The cDNA library is introduced into host bacteria, which are plated out and grown overnight, as in the colony hybridization scheme previously described. DNAbinding nylon membranes are placed on the plates to obtain a replica of the bacterial colonies. The nylon membrane is then incubated under conditions that induce protein synthesis from the cloned cDNA inserts, and the cells are treated to release the synthesized protein. The synthesized protein binds tightly to the nylon membrane, which can then be incubated with the specific antibody. Binding of the antibody to its target protein product reveals the position of any cDNA clones expressing the protein, and these clones can be recovered from the original plate. Like other libraries, expression libraries can be screened with oligonucleotide probes, too.

Fusion Protein Expression

Some expression vectors carry cDNA inserts cloned directly into the coding sequence of a vector-borne protein-coding gene (Figure 13.18). Translation of the recombinant sequence leads to synthesis of a hybrid protein or fusion protein. The N-terminal region of the fused protein represents amino acid sequences encoded in the vector, whereas the remainder of the protein is encoded by the foreign insert. Keep in mind that the triplet codon sequence within the cloned insert must be in phase with codons contributed by the vector sequences to make the right protein. The N-terminal protein sequence contributed by the vector can be chosen to suit purposes. Furthermore, adding an N-terminal sig-

Ptac

13.2 ● DNA Libraries 415

FIGURE 13.18 ● A typical expression vector for the synthesis of a hybrid protein. The cloning site is located at the end of the coding region for the protein -galactosidase. Insertion of foreign DNAs at this site fuses the foreign sequence to the -galactosidase coding region (the lacZ gene). IPTG induces the transcription of the lacZ gene from its promoter plac, causing expression of the fusion protein.

Figure 1.5.4 in Ausubel, F. M., et al.,1987. Current Protocols in Molecular Biology. New York: John Wiley & Sons.)

416 Chapter 13 ● Recombinant DNA: Cloning and Creation of Chimeric Genes

Cl

Br

OH H

X-gal

● The structure of 5- bromo-4-chloro-3-indolyl- -D-galactopyra- noside, or X-gal.

Specific interactions between proteins (so-called protein – protein interactions) lie at the heart of many essential biological processes. Stanley Fields, Cheng-Ting Chien, and their collaborators have invented a method to identify specific protein – protein interactions in vivo through expression of a reporter gene whose transcription is dependent on a functional transcriptional activator, the GAL4 protein. The GAL4 protein consists of two domains: a DNA-binding (or DB) domain and a transcriptional activation (or TA) domain. Even if expressed as separate proteins, these two domains will still work, provided they can be brought together. The method depends on two separate plasmids encoding two hybrid proteins, one consisting of the GAL4 DB domain fused to protein X, and the other consisting of the GAL4 TA domain fused to protein Y (figure, part a). If proteins X and Y interact in a specific protein–protein interaction, the GAL4 DB and TA domains are brought together so that transcription of a reporter gene driven by the GAL4 promoter can take place (figure, part b). Protein X, fused to the GAL4-DNA binding domain (DB), serves as the “bait” to fish for the protein Y “target” and its fused GAL4 TA domain. This method can be used to screen cells for protein “targets” that interact specifically with a particular “bait” protein. To do so, cDNAs encoding proteins from the cells of interest are inserted into the TA-containing plasmid to create fusions of the cDNA coding sequences with the GAL4 TA domain coding sequences, so a fusion protein library is expressed. Identification of a target of the “bait” protein by this method also yields directly a cDNA version of the gene encoding the “target” protein.

(a)

TA

X Y

DB

lacZ Reporter Gene

(b)

TA

X

Y

DB

lacZ Reporter Gene

● Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle.

2 duplex

DNA molecules

amount of total genomic DNA, a particular sequence can be produced in quantity in a few hours.

PCR amplification is an effective cloning strategy if sequence information for the design of appropriate primers is available. Because DNA from a single cell can be used as a template, the technique has enormous potential for the clinical diagnosis of infectious diseases and genetic abnormalities. With PCR techniques, DNA from a single hair or sperm can be analyzed to identify particular individuals in criminal cases without ambiguity. RT-PCR, a variation on the basic PCR method, is useful when the nucleic acid to be amplified is an RNA (such as mRNA). Reverse transcriptase (RT) is used to synthesize a cDNA strand complementary to the RNA, and this cDNA serves as the template for further cycles of PCR.

In Vitro Mutagenesis

The advent of recombinant DNA technology has made it possible to clone genes, manipulate them in vitro, and express them in a variety of cell types under various conditions. The function of any protein is ultimately dependent on its amino acid sequence, which in turn can be traced to the nucleotide sequence of its gene. The introduction of purposeful changes in the nucleotide sequence of a cloned gene represents an ideal way to make specific structural changes in a protein. The effects of these changes on the protein’s function can then be studied. Such changes constitute mutations introduced in vitro into the gene. In vitro mutagenesis makes it possible to alter the nucleotide sequence of a cloned gene systematically, as opposed to the chance occurrence of mutations in natural genes.

One efficient technique for in vitro mutagenesis is PCR-based mutagenesis. Mutant primers are added to a PCR reaction in which the gene (or segment of a gene) is undergoing amplification. The mutant primers are primers whose sequence has been specifically altered to introduce a directed change at a particular place in the nucleotide sequence of the gene being amplified (Figure 13.22). Mutant versions of the gene can then be cloned and expressed to determine any effects of the mutation on the function of the gene product.

13.4 ● Recombinant DNA Technology:

An Exciting Scientific Frontier

The strategies and methodologies described in this chapter are but an overview of the repertoire of experimental approaches that have been devised by molecular biologists in order to manipulate DNA and the information inherent in it. The enormous success of recombinant DNA technology means that the molecular biologist’s task in searching genomes for genes is now akin to that of a lexicographer compiling a dictionary, a dictionary in which the “letters,” i.e., the nucleotide sequences, spell out not words, but genes and what they mean. Molecular biologists have no index or alphabetic arrangement to serve as a guide through the vast volume of information in a genome; nevertheless, this information and its organization are rapidly being disclosed by the imaginative efforts and diligence of these scientists and their growing arsenal of analytical schemes.

Recombinant DNA technology now verges on the ability to engineer at will the genetic constitution of organisms for desired ends. The commercial production of therapeutic biomolecules in microbial cultures is already established (for example, the production of human insulin in quantity in E. coli cells). Agricultural crops with desired attributes, such as enhanced resistance to her-

Gene in plasmid with mutation target site X

'

Thermal denaturation; anneal mutagenic primers which also introduce a unique restriction site

Taq DNA polymerase; many cycles of PCR

Many copies of plasmid with desired site-specific mutation

Transform E.coli cells. Screen single colonies for plasmids with unique restriction site (≡ mutant gene)

● One method of PCR-based site-directed mutagenesis. Template DNA strands are separated by increased temperature, and the single strands are amplified by PCR using mutagenic primers (represented as bent arrows) whose sequences introduce a single base substitution at site X (and its complementary base X ; thus the desired amino acid change in the protein encoded by the gene). Ideally, the mutagenic primers also introduce a unique restriction site into the plasmid that was not present before. Following many cycles of PCR, the DNA product can be used to transform E. coli cells. Single colonies of the transformed cells can be picked. The plasmid DNA within each colony can be isolated and screened for the presence of the mutation by screening for the presence of the unique restriction site by restriction endonuclease cleavage. For example, the nucleotide sequence GGATCT within a gene codes for amino acid residues Gly-Ser. Using mutagenic primers of nucleotide sequence AGATCT (and its complement AGATCT) changes the amino acid sequence from Gly-Ser to Arg-Ser and creates a BglII restriction site (see Table 11.5). Gene expression of the isolated mutant plasmid in

E. coli allows recovery and analysis of the mutant protein.

The gene defective in cystic fibrosis codes for CFTR (cystic fibrosis transmembrane conductance regulator), a membrane protein that pumps Cl out of cells. If this Cl pump is defective, Cl ions remain in cells, which then take up water from the surrounding mucus by osmosis. The mucus thickens and accumulates in various organs, including the lungs, where its presence favors infections such as pneumonia. Left untreated, children with cystic fibrosis seldom survive past the age of 5 years.

ADA SCID (adenosine deaminase – defective severe combined immunodeficiency) is a fatal genetic disorder caused by defects in the gene that encodes adenosine deaminase (ADA).

The consequence of ADA deficiency is accumulation of adenosine and 2 -deoxyadenosine, substances toxic to lymphocytes, important cells in the immune response. 2 -Deoxyadenosine is particularly toxic because its presence leads to accumulation of its nucleotide form, dATP, an essential substrate in DNA synthesis. Elevated levels of dATP actually block DNA replication and cell division by inhibiting synthesis of the other deoxynucleoside 5 -triphosphates (see Chapter 27). Accumulation of dATP also leads to selective depletion of cellular ATP, robbing cells of energy. Children with ADA SCID fail to develop normal immune responses and are susceptible to fatal infections, unless kept in protective isolation.