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Studying gene expression and function

The document discusses various methods for studying gene expression and function, including analyzing RNA transcripts. It describes techniques like northern hybridization, DNA-mRNA hybridization, S1 nuclease mapping, primer extension, and PCR that can be used to study transcripts and locate start/stop points. The document also covers methods for studying gene regulation, such as identifying protein binding sites through gel retardation assays and footprinting, and using deletion analysis to identify control sequences.

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Studying Gene Expression and Function Md. Murad Khan Lecturer Dept. of Microbiology Jagannath University
Introduction  All genes have to be expressed in order to function. Gene expression is the combined process of the transcription of a gene into mRNA, the processing of that mRNA, and its translation into protein (for protein-encoding genes).  Gene expression is the process by which the information encoded in a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.  To understand how a gene is expressed, the RNA transcript must be studied, in particularly:  the removal of introns,  the exact locations of the start and end points of transcription,  the signals that determine the start and finish of the transcription etc.
Introduction FIG: Some fundamentals of gene expression. mRNA = messenger RNA, tRNA = transfer RNA, rRNA = ribosomal RNA.
Studying the RNA Transcript of a Gene Transcript analysis by northern hybridization:  Northern hybridization, the RNA equivalent of Southern hybridization, is used to measure the length of a transcript.  An RNA extract is electrophoresed in an agarose gel, using a denaturing electrophoresis buffer (e.g., one containing formaldehyde) to ensure that the RNAs do not form inter- or intramolecular base pairs, as base pairing would affect the rate at which the molecules migrate through the gel.  After electrophoresis, the gel is blotted onto a nylon or nitrocellulose membrane, and hybridized with a labeled probe. If the probe is a cloned gene, the band that appears in the autoradiograph is the transcript of that gene.  The size of the transcript can be determined from its position within the gel, and if RNA from different tissues is run in different lanes of the gel, then the possibility that the gene is differentially expressed can be examined.
Studying the RNA Transcript of a Gene FIG: Northern hybridization. Three RNA extracts from different tissues have been electrophoresed in an agarose gel. The extracts are made up of many RNAs of different lengths so each gives a smear of RNA, but two distinct bands are seen, one for each of the abundant ribosomal RNAs. The sizes of these rRNAs are known (e.g. 4718 and 1874 nucleotides in mammals), so they can be used as internal size markers. The gel is transferred to a membrane, probed with a cloned gene, and the results visualized, for example by autoradiography if the probe has been radioactively labeled. Only lane 1 gives a band, showing that the cloned gene is expressed only in the tissue from which this RNA extract was obtained. Northern Hybridization
Studying the RNA Transcript of a Gene Transcript analysis by DNA-mRNA hybridization:  Nucleic acid hybridization occurs just as readily between complementary DNA and RNA strands as between single-stranded DNA molecules. If a hybrid is formed between a DNA strand, containing a gene, and its mRNA, then the boundaries between the double- and single-stranded regions will mark the start and end points of the mRNA.  Introns, which are present in the DNA but not in the mRNA, will “loop out” as additional single-stranded regions.  Now consider the result of treating the DNA–RNA hybrid with a single-strand specific nuclease such as S1. S1 nuclease degrades single-stranded DNA or RNA polynucleotides, including single-stranded regions at the ends of predominantly double stranded molecules, but has no effect on double-stranded DNA or on DNA– RNA hybrids.
Studying the RNA Transcript of a Gene FIG: A DNA-mRNA hybrid and the effect of treating this hybrid with a single-strand-specific nuclease such as S1.  S1 nuclease will therefore digest the non-hybridized single-stranded DNA regions at each end of the DNA–RNA hybrid, along with any looped-out introns. The single-stranded DNA fragments protected from S1 nuclease digestion can be recovered if the RNA strand is degraded by treatment with alkali. Limitation of the method:  Although the sizes of the protected DNA fragments could be measured by gel electrophoresis, this does not allow their order or relative positions in the DNA sequence to be determined.
Studying the RNA Transcript of a Gene Locating a transcription start point by S1 nuclease mapping:  A few subtle modifications to the previous technique allow the precise start and end points of the transcript and of any introns it contains to be mapped onto the DNA sequence. An example of the way in which S1 nuclease mapping is used to locate the start point of a transcript is shown in the figure.  Here, a Sau3A fragment that contains 100 bp of coding region, along with 300 bp of the leader sequence preceding the gene, has been cloned into an M13 vector and obtained as a single- stranded molecule. A sample of the RNA transcript is added and allowed to anneal to the DNA molecule. FIG: Locating a transcription start point by S1 nuclease mapping.
Studying the RNA Transcript of a Gene  The DNA molecule is still primarily single-stranded but now has a small region protected by the RNA transcript. All but this protected region is digested by S1 nuclease and the RNA is degraded with alkali, leaving a short single-stranded DNA fragment.  If these manipulations are examined closely it will become clear that the size of this single stranded fragment corresponds to the distance between the transcription start point and the right-hand Sau3A site. The size of the single-stranded fragment is therefore determined by gel electrophoresis and this information is used to locate the transcription start point on the DNA sequence. Exactly the same strategy could locate the end point of transcription and the junction points between introns and exons.
Studying the RNA Transcript of a Gene Transcript analysis by primer extension:  Primer extension—is less adaptable, because it can only identify the 5′ end of an RNA. It is, nonetheless, an important technique that is frequently used to check the results of S1 analyses.  Primer extension can only be used if at least part of the sequence of the transcript is known. This is because a short oligonucleotide primer must be annealed to the RNA at a known position, ideally within 100–200 nucleotides of the 5′ end of the transcript.  Once annealed, the primer is extended by reverse transcriptase . This is a cDNA synthesis reaction, but one that is very likely to proceed to completion as only a short segment of RNA has to be copied.
Studying the RNA Transcript of a Gene  The 3′ end of the newly synthesized strand of DNA will therefore correspond with the 5′ terminus of the transcript.  Locating the position of this terminus on the DNA sequence is achieved simply by determining the length of the single- stranded DNA molecule and correlating this information with the annealing position of the primer. FIG: Locating a transcription start point by primer extension.
Studying the RNA Transcript of a Gene Transcript analysis by PCR:  Rapid amplification of cDNA ends (RACE) can be used to identify the 5′ and 3′ termini of RNA molecules and so, like S1 analysis, can be used to map the ends of transcripts. There are several variations to the RACE method. Here we will consider how the 5′ end of an RNA molecule can be mapped.  This procedure uses a primer that is specific for an internal region of the RNA molecule. The primer attaches to the RNA and directs the first, reverse transcriptase catalyzed, stage of the process, during which a single-stranded cDNA is made. As in the primer extension method, the 3′ end of the cDNA corresponds with the 5′ end of the RNA.
Studying the RNA Transcript of a Gene  Terminal deoxynucleotidyl transferase is now used to attach a series of A nucleotides to the 3′ end of the cDNA, forming the priming site for a second PCR primer, which is made up entirely of Ts and hence anneals to the poly(A) tail created by terminal transferase. Now the standard PCR begins, first converting the single-stranded cDNA into a double- stranded molecule, and then amplifying this molecule as the PCR proceeds. The PCR product is then sequenced to reveal the precise position of the start of the transcript. FIG: Identification of 5´ terminus by RACE.
Studying the Regulation of Gene Expression  Differentiation of cells involves wholesale changes in gene expression patterns, and the process of development from fertilized egg cell to adult requires coordination between different cells as well as time-dependent changes in gene expression.  A gene subject to regulation has one or more control sequences in its upstream region and that the gene is switched on and off by the attachment of regulatory proteins to these sequences.  A regulatory protein may repress gene expression, in which case the gene is switched off when the protein is bound to the control sequence, or alternatively the protein may have a positive or enhancing role, switching on or increasing expression of the target gene. FIG: Possible positions for control sequences in the region upstream of a gene.
Studying the Regulation of Gene Expression Identifying protein binding sites on a DNA molecule:  A control sequence is a region of DNA that can bind a regulatory protein. It should therefore be possible to identify control sequences upstream of a gene by searching the relevant region for protein binding sites. There are three different ways of doing this. (1) Gel retardation of DNA–protein complexes:  A gel retardation assay, also called electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis or gel shift assay or gel mobility shift assay or band shift assay, is a common electrophoresis technique used to study DNA-protein interactions.  A DNA fragment carrying a bound protein is identified by gel electrophoresis, as it has a lower mobility than the uncomplexed DNA molecule. The procedure is referred to as gel retardation. FIG: A bound protein decreases the mobility of a DNA fragment during gel electrophoresis.
Studying the Regulation of Gene Expression  The region of DNA upstream of the gene being studied is digested with a restriction endonuclease and then mixed with the regulatory protein or, if the protein has not yet been purified, with an unfractionated extract of nuclear protein (remember that gene regulation occurs in the nucleus).  The restriction fragment containing the control sequence forms a complex with the regulatory protein: all the other fragments remain as “naked” DNA. The location of the control sequence is then determined by finding the position on the restriction map of the fragment that is retarded during gel electrophoresis.  Limitation: A single control sequence may be less than 10 bp in size, so gel retardation is rarely able to pinpoint it exactly. FIG: Carrying out a gel retardation experiment.
Studying the Regulation of Gene Expression (2) Footprinting with DNase I:  The procedure generally called footprinting enables a control region to be positioned within a restriction fragment that has been identified by gel retardation.  Footprinting works on the basis that the interaction with a regulatory protein protects the DNA in the region of a control sequence from the degradative action of an endonuclease such as deoxyribonuclease (DNase) I. This phenomenon can be used to locate the protein binding site on the DNA molecule.  The DNA fragment being studied is first labeled at one end, and then complexed with the regulatory protein. Then DNase I is added, but the amount used is limited so that complete degradation of the DNA fragment does not occur. Instead the aim is to cut each molecule at just a single phosphodiester bond. FIG: A bound protein protects a region of a DNA molecule from degradation by a nuclease such as DNase I.
Studying the Regulation of Gene Expression  If the DNA fragment has no protein attached to it, the result of this treatment is a family of labeled fragments, differing in size by just one nucleotide each.  After removal of the bound protein and separation on a polyacrylamide gel, the family of labeled fragments appears as a ladder of bands. However, the bound protein protected certain phosphodiester bonds from being cut by DNase I, meaning that in this case the family of fragments is not complete, as the fragments resulting from cleavage within the control sequence are absent. Their absence shows up as a “footprint”, clearly seen in Figure shown here. FIG: DNase I footprinting.
Studying the Regulation of Gene Expression (3) Modification interference assays:  Gel retardation analysis and footprinting enable control sequences to be located, but do not give information about the interaction between the binding protein and the DNA molecule.  The more precise of these two techniques—footprinting—only reveals the region of DNA that is protected by the bound protein. Proteins are relatively large compared with a DNA double helix, and can protect several tens of base pairs when bound to a control sequence that is just a few base pairs in length. Footprinting therefore does not delineate the control region itself, only the region within which it is located. FIG: A bound protein can protect a region of DNA that is much longer than the control sequence.
Studying the Regulation of Gene Expression  The DNA fragments must first be labeled at one end. Then they are treated with a chemical that modifies specific nucleotides, an example being dimethyl sulphate, which attaches methyl groups to guanine nucleotides. This modification is carried out under limiting conditions so an average of just one nucleotide per DNA fragment is modified. Now the DNA is mixed with the protein extract. The key to the assay is that the binding protein will probably not attach to the DNA if one of the guanines within the control region is modified, because methylation of a nucleotide interferes with the specific chemical reaction that enables it to form an attachment with a protein. FIG: A modification interference assay.
Studying the Regulation of Gene Expression  If the DNA and protein mixture is examined by agarose gel electrophoresis two bands will be seen, one comprising the DNA–protein complex and one containing DNA with no bound protein. The band made up of unbound DNA is purified from the gel and treated with piperidine, a chemical which cleaves DNA molecules at methylated nucleotides. The products of piperidine treatment are now separated in a polyacrylamide gel and the labeled bands visualized.  The sizes of the bands that are seen indicate the position in the DNA fragment of guanines whose methylation prevented protein binding. These guanines lie within the control sequence. The modification assay can now be repeated with chemicals that target A, T, or C nucleotides to determine the precise position of the control sequence.
Studying the Regulation of Gene Expression Identifying control sequences by deletion analysis:  Gel retardation, footprinting, and modification interference assays are able to locate possible control sequences upstream of a gene, but they provide no information on the function of the individual sequences. Deletion analysis is a totally different approach that not only can locate control sequences (though only with the precision of gel retardation) but importantly also can indicate the function of each sequence.  The technique depends on the assumption that deletion of the control sequence will result in a change in the way in which expression of a cloned gene is regulated. For instance, deletion of a sequence that represses expression of a gene should result in that gene being expressed at a higher level. Similarly, tissue-specific control sequences can be identified as their deletion results in the target gene being expressed in tissues other than the correct one.
Studying the Regulation of Gene Expression FIG: The principle behind deletion analysis.
Studying the Regulation of Gene Expression  Reporter gene is a test gene that is fused to the upstream region of the cloned gene, replacing this gene. When cloned into the host organism the expression pattern of the reporter gene should exactly mimic that of the original gene, as the reporter gene is under the influence of exactly the same control sequences as the original gene.  The reporter gene must be chosen with care. The first criterion is that the reporter gene must code for a phenotype not already displayed by the host organism. The phenotype of the reporter gene must be relatively easy to detect after it has been cloned into the host, and ideally it should be possible to assay the phenotype quantitatively. These criteria have not proved difficult to meet and a variety of different reporter genes have been used in studies of gene regulation. FIG: A reporter gene.
Studying the Regulation of Gene Expression Carrying out a deletion analysis:  Deletions can be made in the upstream region of the construct by any one of several strategies, a simple example being shown in Figure here. The effect of the deletion is then assessed by cloning the deleted construct into the host organism and determining the pattern and extent of expression of the reporter gene. An increase in expression will imply that a repressing or silencing sequence has been removed, a decrease will indicate removal of an activator or enhancer, and a change in tissue specificity will pinpoint a tissue-responsive control sequence. FIG: Deletion analysis. A reporter gene has been attached to the upstream region of a seed-specific gene from a plant. Removal of the restriction fragment bounded by the sites R deletes the control sequence that mediates seed-specific gene expression, so that the reporter gene is now expressed in all tissues of the plant.
Identifying and Studying the Translation Product of a Cloned Gene HRT and HART can identify the translation product of a cloned gene:  Two related techniques, hybrid-release translation (HRT) and hybrid-arrest translation (HART), are used to identify the translation product encoded by a cloned gene. Both depend on the ability of purified mRNA to direct synthesis of proteins in cell-free translation systems. These are cell extracts, usually prepared from germinating wheat seeds or from rabbit reticulocyte cells (both of which are exceptionally active in protein synthesis) and containing ribosomes, tRNAs, and all the other molecules needed for protein synthesis.  The mRNA sample is added to the cell-free translation system, along with a mixture of the 20 amino acids found in proteins, one of which is labeled (often 35S-methionine is used). The mRNA molecules are translated into a mixture of radioactive proteins, which can be separated by gel electrophoresis and visualized by autoradiography. Each band represents a single protein coded by one of the mRNA molecules present in the sample.
Identifying and Studying the Translation Product of a Cloned Gene FIG: Cell-free translation. FIG: Hybrid-release translation. FIG: Hybrid-arrest translation.
Identifying and Studying the Translation Product of a Cloned Gene  Both HRT and HART work best when the gene being studied has been obtained as a cDNA clone. For HRT the cDNA is denatured, immobilized on a nitrocellulose or nylon membrane, and incubated with the mRNA sample. The specific mRNA counterpart of the cDNA hybridizes and remains attached to the membrane. After discarding the unbound molecules, the hybridized mRNA is recovered and translated in a cell-free system. This provides a pure sample of the protein coded by the cDNA.  Hybrid-arrest translation is slightly different in that the denatured cDNA is added directly to the mRNA sample. Hybridization again occurs between the cDNA and its mRNA counterpart, but in this case the unbound mRNA is not discarded. Instead the entire sample is translated in the cell-free system. The hybridized mRNA is unable to direct translation, so all the proteins except the one coded by the cloned gene are synthesized. The cloned gene’s translation product is therefore identified as the protein that is absent from the autoradiograph.
Identifying and Studying the Translation Product of a Cloned Gene Analysis of proteins by in vitro mutagenesis:  The best way of determining the relationship between the structure of a protein and its mode of structure is to induce a mutation in the gene coding for the protein and then to determine what effect the change in amino acid sequence has on the properties of the translation product. Under normal circumstances mutations occur randomly and a large number may have to be screened before one that gives useful information is found. A solution to this problem is provided by in vitro mutagenesis, a technique that enables a directed mutation to be made at a specific point in a cloned gene. FIG: A mutation may change the amino acid sequence of a protein, possibly affecting its properties.
Identifying and Studying the Translation Product of a Cloned Gene Different types of in vitro mutagenesis techniques:  An almost unlimited variety of DNA manipulations can be used to introduce mutations into cloned genes. The following are the simplest:  A restriction fragment can be deleted.  The gene can be opened at a unique restriction site, a few nucleotides removed with a double-strand-specific endonuclease such as Bal31, and the gene religated.  A short, double-stranded oligonucleotide can be inserted at a restriction site. The sequence of the oligonucleotide can be such that the additional stretch of amino acids inserted into the protein produces, for example, a new structure such as an a-helix, or destabilizes an existing structure.  Although potentially useful, these manipulations depend on the fortuitous occurrence of a restriction site at the area of interest in the cloned gene. Oligonucleotide-directed mutagenesis is a more versatile technique that can create a mutation at any point in the gene.
Identifying and Studying the Translation Product of a Cloned Gene FIG: Various in vitro mutagenesis techniques.
Identifying and Studying the Translation Product of a Cloned Gene Using an oligonucleotide to create a point mutation in a cloned gene:  The gene to be mutated must be obtained in a single-stranded form and so is generally cloned into an M13 vector. The single-stranded DNA is purified and the region to be mutated identified by DNA sequencing. A short oligonucleotide is then synthesized, complementary to the relevant region, but containing the desired nucleotide alteration.  Despite this mismatch the oligonucleotide will anneal to the single-stranded DNA and act as a primer for complementary strand synthesis by a DNA polymerase. This strand synthesis reaction is continued until an entire new strand is made and the recombinant molecule is completely double-stranded.
Identifying and Studying the Translation Product of a Cloned Gene  After introduction, by transfection, into competent E. coli cells, DNA replication produces numerous copies of the recombinant DNA molecule. The semi- conservative nature of DNA replication means that half the double-stranded molecules that are produced are unmutated in both strands, whereas half are mutated in both strands. Similarly, half the resulting phage progeny carry copies of the unmutated molecule and half carry the mutation.  The phages produced by the transfected cells are plated onto solid agar so that plaques are produced. Half the plaques should contain the original recombinant molecule, and half the mutated version. Which are which is determined by plaque hybridization, using the oligonucleotide as the probe, and employing very strict conditions so that only the completely base-paired hybrid is stable.
Identifying and Studying the Translation Product of a Cloned Gene  Cells infected with M13 vectors do not lyse, but instead continue to divide. The mutated gene can therefore be expressed in the host E. coli cells, resulting in production of recombinant protein. The protein coded by the mutated gene can be purified from the recombinant cells and its properties studied. The effect of a single base pair mutation on the activity of the protein can therefore be assessed. FIG: One method for oligonucleotide-directed mutagenesis.
Identifying and Studying the Translation Product of a Cloned Gene Using PCR method to create a point mutation in a cloned gene:  The starting DNA molecule is amplified by two PCRs. In each of these, one primer is normal and forms a fully base-paired hybrid with the template DNA, but the second is mutagenic, as it contains a single base-pair mismatch corresponding to the mutation that we wish to introduce into the DNA sequence. This mutation is therefore present in the two PCR products, each of which represents one half of the starting DNA molecule.  The two PCR products are mixed together and a final PCR cycle carried out. In this cycle, complementary strands from the two products anneal to one another and are then extended by the polymerase, producing the full-length, mutated DNA molecule.
Identifying and Studying the Translation Product of a Cloned Gene  This technique, and related ones using PCR, is very quick and easy to carry out but a major problem is caused by the high error rate of the Taq polymerase used in PCR. This error rate makes it likely that not only the desired mutation, but also random ones, will be present at the end of the experiment. The PCR product therefore has to be cloned and the sequences of individual clones checked to find one that has just the desired mutation. FIG: One method for using PCR to create a directed mutation.
Studying Protein–Protein Interactions  Within living cells, few if any proteins act in total isolation. Instead, proteins interact with one another in biochemical pathways and in multiprotein complexes. Two important techniques, phage display and the yeast two hybrid system, enable these protein–protein interactions to be examined. Phage display:  This technique is called phage display because it involves the “display” of proteins on the surface of a bacteriophage, usually M13. This is achieved by cloning the gene for the protein in a special type of M13 vector, one that results in the cloned gene becoming fused with a gene for a phage coat protein.  After transfection of E. coli, this gene fusion directs synthesis of a hybrid protein, made up partly of the coat protein and partly of the product of the cloned gene. With luck this hybrid protein will be inserted into the phage coat so that the product of the cloned gene is now located on the surface of the phage particles.
Studying Protein–Protein Interactions  Normally this technique is carried out with a phage display library made up of many recombinant phages, each displaying a different protein. Large libraries can be prepared by cloning a mixture of cDNAs from a particular tissue or, less easily, by cloning genomic DNA fragments. The library consists of phages displaying a range of different proteins and is used to identify those that interact with a test protein. This test protein could be a pure protein or one that is itself displayed on a phage surface.  The protein is immobilized in the wells of a microtiter tray or on particles that can be used in an affinity chromatography column, and then mixed with the phage display library. Phages that are retained in the microtiter tray or within the column after a series of washes are ones that display proteins that interact with the immobilized test protein.
Studying Protein–Protein Interactions FIG: Phage display. (a) Display of proteins on the surface of a recombinant filamentous phage. (b) The gene fusion used to display a protein. (c) One way of detecting interactions between a test protein and a phage from within a display library. FIG: The yeast two hybrid system. (a) A pair of transcription factors that must interact in order for a yeast gene to be expressed. (b) Replacement of transcription factor 1 with the hybrid protein 1* abolishes gene expression as 1* cannot interact with transcription factor 2. (c) Replacement of transcription factor 2 with the hybrid protein 2* restores gene expression if the hybrid parts of 1* and 2* are able to interact.
Studying Protein–Protein Interactions The yeast two hybrid system:  The yeast two hybrid system is very different to phage display. This procedure is based on the discovery that gene expression in S. cerevisiae depends on interactions between pairs of transcription factors.  In the two hybrid system, a pair of transcription factors responsible for expression of a yeast gene is replaced by hybrid proteins, each one made partly of transcription factor and partly of test protein. The ability of this pair of hybrids to direct expression of the yeast target gene is then tested. To use the system, two yeast cloning experiments must be carried out.
Studying Protein–Protein Interactions  The first cloning experiment involves the gene whose protein product is being studied. This gene is ligated to the gene for one of the pair of transcription factors and the construct inserted into a yeast vector. The recombinant yeasts that are produced are not able to express the target gene, because this modified transcription factor cannot interact with its partner.  In the second cloning experiment, a hybrid version of the partner is made and cloned into the yeast cells. Restoration of expression of the target gene indicates that the two hybrid transcription factors can interact. The fusions are designed in such a way that this can only happen if the interactions occur between the test protein components of the hybrids, not between the transcription factor segments. Pairs of interacting test proteins are therefore identified. The second cloning experiment can involve a library of recombinants representing different proteins, so that one protein can be tested against many others.
THANKS TO ALL

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Studying gene expression and function

  • 1.
    Studying Gene Expression andFunction Md. Murad Khan Lecturer Dept. of Microbiology Jagannath University
  • 2.
    Introduction  All geneshave to be expressed in order to function. Gene expression is the combined process of the transcription of a gene into mRNA, the processing of that mRNA, and its translation into protein (for protein-encoding genes).  Gene expression is the process by which the information encoded in a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.  To understand how a gene is expressed, the RNA transcript must be studied, in particularly:  the removal of introns,  the exact locations of the start and end points of transcription,  the signals that determine the start and finish of the transcription etc.
  • 3.
    Introduction FIG: Some fundamentalsof gene expression. mRNA = messenger RNA, tRNA = transfer RNA, rRNA = ribosomal RNA.
  • 4.
    Studying the RNATranscript of a Gene Transcript analysis by northern hybridization:  Northern hybridization, the RNA equivalent of Southern hybridization, is used to measure the length of a transcript.  An RNA extract is electrophoresed in an agarose gel, using a denaturing electrophoresis buffer (e.g., one containing formaldehyde) to ensure that the RNAs do not form inter- or intramolecular base pairs, as base pairing would affect the rate at which the molecules migrate through the gel.  After electrophoresis, the gel is blotted onto a nylon or nitrocellulose membrane, and hybridized with a labeled probe. If the probe is a cloned gene, the band that appears in the autoradiograph is the transcript of that gene.  The size of the transcript can be determined from its position within the gel, and if RNA from different tissues is run in different lanes of the gel, then the possibility that the gene is differentially expressed can be examined.
  • 5.
    Studying the RNATranscript of a Gene FIG: Northern hybridization. Three RNA extracts from different tissues have been electrophoresed in an agarose gel. The extracts are made up of many RNAs of different lengths so each gives a smear of RNA, but two distinct bands are seen, one for each of the abundant ribosomal RNAs. The sizes of these rRNAs are known (e.g. 4718 and 1874 nucleotides in mammals), so they can be used as internal size markers. The gel is transferred to a membrane, probed with a cloned gene, and the results visualized, for example by autoradiography if the probe has been radioactively labeled. Only lane 1 gives a band, showing that the cloned gene is expressed only in the tissue from which this RNA extract was obtained. Northern Hybridization
  • 6.
    Studying the RNATranscript of a Gene Transcript analysis by DNA-mRNA hybridization:  Nucleic acid hybridization occurs just as readily between complementary DNA and RNA strands as between single-stranded DNA molecules. If a hybrid is formed between a DNA strand, containing a gene, and its mRNA, then the boundaries between the double- and single-stranded regions will mark the start and end points of the mRNA.  Introns, which are present in the DNA but not in the mRNA, will “loop out” as additional single-stranded regions.  Now consider the result of treating the DNA–RNA hybrid with a single-strand specific nuclease such as S1. S1 nuclease degrades single-stranded DNA or RNA polynucleotides, including single-stranded regions at the ends of predominantly double stranded molecules, but has no effect on double-stranded DNA or on DNA– RNA hybrids.
  • 7.
    Studying the RNATranscript of a Gene FIG: A DNA-mRNA hybrid and the effect of treating this hybrid with a single-strand-specific nuclease such as S1.  S1 nuclease will therefore digest the non-hybridized single-stranded DNA regions at each end of the DNA–RNA hybrid, along with any looped-out introns. The single-stranded DNA fragments protected from S1 nuclease digestion can be recovered if the RNA strand is degraded by treatment with alkali. Limitation of the method:  Although the sizes of the protected DNA fragments could be measured by gel electrophoresis, this does not allow their order or relative positions in the DNA sequence to be determined.
  • 8.
    Studying the RNATranscript of a Gene Locating a transcription start point by S1 nuclease mapping:  A few subtle modifications to the previous technique allow the precise start and end points of the transcript and of any introns it contains to be mapped onto the DNA sequence. An example of the way in which S1 nuclease mapping is used to locate the start point of a transcript is shown in the figure.  Here, a Sau3A fragment that contains 100 bp of coding region, along with 300 bp of the leader sequence preceding the gene, has been cloned into an M13 vector and obtained as a single- stranded molecule. A sample of the RNA transcript is added and allowed to anneal to the DNA molecule. FIG: Locating a transcription start point by S1 nuclease mapping.
  • 9.
    Studying the RNATranscript of a Gene  The DNA molecule is still primarily single-stranded but now has a small region protected by the RNA transcript. All but this protected region is digested by S1 nuclease and the RNA is degraded with alkali, leaving a short single-stranded DNA fragment.  If these manipulations are examined closely it will become clear that the size of this single stranded fragment corresponds to the distance between the transcription start point and the right-hand Sau3A site. The size of the single-stranded fragment is therefore determined by gel electrophoresis and this information is used to locate the transcription start point on the DNA sequence. Exactly the same strategy could locate the end point of transcription and the junction points between introns and exons.
  • 10.
    Studying the RNATranscript of a Gene Transcript analysis by primer extension:  Primer extension—is less adaptable, because it can only identify the 5′ end of an RNA. It is, nonetheless, an important technique that is frequently used to check the results of S1 analyses.  Primer extension can only be used if at least part of the sequence of the transcript is known. This is because a short oligonucleotide primer must be annealed to the RNA at a known position, ideally within 100–200 nucleotides of the 5′ end of the transcript.  Once annealed, the primer is extended by reverse transcriptase . This is a cDNA synthesis reaction, but one that is very likely to proceed to completion as only a short segment of RNA has to be copied.
  • 11.
    Studying the RNATranscript of a Gene  The 3′ end of the newly synthesized strand of DNA will therefore correspond with the 5′ terminus of the transcript.  Locating the position of this terminus on the DNA sequence is achieved simply by determining the length of the single- stranded DNA molecule and correlating this information with the annealing position of the primer. FIG: Locating a transcription start point by primer extension.
  • 12.
    Studying the RNATranscript of a Gene Transcript analysis by PCR:  Rapid amplification of cDNA ends (RACE) can be used to identify the 5′ and 3′ termini of RNA molecules and so, like S1 analysis, can be used to map the ends of transcripts. There are several variations to the RACE method. Here we will consider how the 5′ end of an RNA molecule can be mapped.  This procedure uses a primer that is specific for an internal region of the RNA molecule. The primer attaches to the RNA and directs the first, reverse transcriptase catalyzed, stage of the process, during which a single-stranded cDNA is made. As in the primer extension method, the 3′ end of the cDNA corresponds with the 5′ end of the RNA.
  • 13.
    Studying the RNATranscript of a Gene  Terminal deoxynucleotidyl transferase is now used to attach a series of A nucleotides to the 3′ end of the cDNA, forming the priming site for a second PCR primer, which is made up entirely of Ts and hence anneals to the poly(A) tail created by terminal transferase. Now the standard PCR begins, first converting the single-stranded cDNA into a double- stranded molecule, and then amplifying this molecule as the PCR proceeds. The PCR product is then sequenced to reveal the precise position of the start of the transcript. FIG: Identification of 5´ terminus by RACE.
  • 14.
    Studying the Regulationof Gene Expression  Differentiation of cells involves wholesale changes in gene expression patterns, and the process of development from fertilized egg cell to adult requires coordination between different cells as well as time-dependent changes in gene expression.  A gene subject to regulation has one or more control sequences in its upstream region and that the gene is switched on and off by the attachment of regulatory proteins to these sequences.  A regulatory protein may repress gene expression, in which case the gene is switched off when the protein is bound to the control sequence, or alternatively the protein may have a positive or enhancing role, switching on or increasing expression of the target gene. FIG: Possible positions for control sequences in the region upstream of a gene.
  • 15.
    Studying the Regulationof Gene Expression Identifying protein binding sites on a DNA molecule:  A control sequence is a region of DNA that can bind a regulatory protein. It should therefore be possible to identify control sequences upstream of a gene by searching the relevant region for protein binding sites. There are three different ways of doing this. (1) Gel retardation of DNA–protein complexes:  A gel retardation assay, also called electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis or gel shift assay or gel mobility shift assay or band shift assay, is a common electrophoresis technique used to study DNA-protein interactions.  A DNA fragment carrying a bound protein is identified by gel electrophoresis, as it has a lower mobility than the uncomplexed DNA molecule. The procedure is referred to as gel retardation. FIG: A bound protein decreases the mobility of a DNA fragment during gel electrophoresis.
  • 16.
    Studying the Regulationof Gene Expression  The region of DNA upstream of the gene being studied is digested with a restriction endonuclease and then mixed with the regulatory protein or, if the protein has not yet been purified, with an unfractionated extract of nuclear protein (remember that gene regulation occurs in the nucleus).  The restriction fragment containing the control sequence forms a complex with the regulatory protein: all the other fragments remain as “naked” DNA. The location of the control sequence is then determined by finding the position on the restriction map of the fragment that is retarded during gel electrophoresis.  Limitation: A single control sequence may be less than 10 bp in size, so gel retardation is rarely able to pinpoint it exactly. FIG: Carrying out a gel retardation experiment.
  • 17.
    Studying the Regulationof Gene Expression (2) Footprinting with DNase I:  The procedure generally called footprinting enables a control region to be positioned within a restriction fragment that has been identified by gel retardation.  Footprinting works on the basis that the interaction with a regulatory protein protects the DNA in the region of a control sequence from the degradative action of an endonuclease such as deoxyribonuclease (DNase) I. This phenomenon can be used to locate the protein binding site on the DNA molecule.  The DNA fragment being studied is first labeled at one end, and then complexed with the regulatory protein. Then DNase I is added, but the amount used is limited so that complete degradation of the DNA fragment does not occur. Instead the aim is to cut each molecule at just a single phosphodiester bond. FIG: A bound protein protects a region of a DNA molecule from degradation by a nuclease such as DNase I.
  • 18.
    Studying the Regulationof Gene Expression  If the DNA fragment has no protein attached to it, the result of this treatment is a family of labeled fragments, differing in size by just one nucleotide each.  After removal of the bound protein and separation on a polyacrylamide gel, the family of labeled fragments appears as a ladder of bands. However, the bound protein protected certain phosphodiester bonds from being cut by DNase I, meaning that in this case the family of fragments is not complete, as the fragments resulting from cleavage within the control sequence are absent. Their absence shows up as a “footprint”, clearly seen in Figure shown here. FIG: DNase I footprinting.
  • 19.
    Studying the Regulationof Gene Expression (3) Modification interference assays:  Gel retardation analysis and footprinting enable control sequences to be located, but do not give information about the interaction between the binding protein and the DNA molecule.  The more precise of these two techniques—footprinting—only reveals the region of DNA that is protected by the bound protein. Proteins are relatively large compared with a DNA double helix, and can protect several tens of base pairs when bound to a control sequence that is just a few base pairs in length. Footprinting therefore does not delineate the control region itself, only the region within which it is located. FIG: A bound protein can protect a region of DNA that is much longer than the control sequence.
  • 20.
    Studying the Regulationof Gene Expression  The DNA fragments must first be labeled at one end. Then they are treated with a chemical that modifies specific nucleotides, an example being dimethyl sulphate, which attaches methyl groups to guanine nucleotides. This modification is carried out under limiting conditions so an average of just one nucleotide per DNA fragment is modified. Now the DNA is mixed with the protein extract. The key to the assay is that the binding protein will probably not attach to the DNA if one of the guanines within the control region is modified, because methylation of a nucleotide interferes with the specific chemical reaction that enables it to form an attachment with a protein. FIG: A modification interference assay.
  • 21.
    Studying the Regulationof Gene Expression  If the DNA and protein mixture is examined by agarose gel electrophoresis two bands will be seen, one comprising the DNA–protein complex and one containing DNA with no bound protein. The band made up of unbound DNA is purified from the gel and treated with piperidine, a chemical which cleaves DNA molecules at methylated nucleotides. The products of piperidine treatment are now separated in a polyacrylamide gel and the labeled bands visualized.  The sizes of the bands that are seen indicate the position in the DNA fragment of guanines whose methylation prevented protein binding. These guanines lie within the control sequence. The modification assay can now be repeated with chemicals that target A, T, or C nucleotides to determine the precise position of the control sequence.
  • 22.
    Studying the Regulationof Gene Expression Identifying control sequences by deletion analysis:  Gel retardation, footprinting, and modification interference assays are able to locate possible control sequences upstream of a gene, but they provide no information on the function of the individual sequences. Deletion analysis is a totally different approach that not only can locate control sequences (though only with the precision of gel retardation) but importantly also can indicate the function of each sequence.  The technique depends on the assumption that deletion of the control sequence will result in a change in the way in which expression of a cloned gene is regulated. For instance, deletion of a sequence that represses expression of a gene should result in that gene being expressed at a higher level. Similarly, tissue-specific control sequences can be identified as their deletion results in the target gene being expressed in tissues other than the correct one.
  • 23.
    Studying the Regulationof Gene Expression FIG: The principle behind deletion analysis.
  • 24.
    Studying the Regulationof Gene Expression  Reporter gene is a test gene that is fused to the upstream region of the cloned gene, replacing this gene. When cloned into the host organism the expression pattern of the reporter gene should exactly mimic that of the original gene, as the reporter gene is under the influence of exactly the same control sequences as the original gene.  The reporter gene must be chosen with care. The first criterion is that the reporter gene must code for a phenotype not already displayed by the host organism. The phenotype of the reporter gene must be relatively easy to detect after it has been cloned into the host, and ideally it should be possible to assay the phenotype quantitatively. These criteria have not proved difficult to meet and a variety of different reporter genes have been used in studies of gene regulation. FIG: A reporter gene.
  • 25.
    Studying the Regulationof Gene Expression Carrying out a deletion analysis:  Deletions can be made in the upstream region of the construct by any one of several strategies, a simple example being shown in Figure here. The effect of the deletion is then assessed by cloning the deleted construct into the host organism and determining the pattern and extent of expression of the reporter gene. An increase in expression will imply that a repressing or silencing sequence has been removed, a decrease will indicate removal of an activator or enhancer, and a change in tissue specificity will pinpoint a tissue-responsive control sequence. FIG: Deletion analysis. A reporter gene has been attached to the upstream region of a seed-specific gene from a plant. Removal of the restriction fragment bounded by the sites R deletes the control sequence that mediates seed-specific gene expression, so that the reporter gene is now expressed in all tissues of the plant.
  • 26.
    Identifying and Studyingthe Translation Product of a Cloned Gene HRT and HART can identify the translation product of a cloned gene:  Two related techniques, hybrid-release translation (HRT) and hybrid-arrest translation (HART), are used to identify the translation product encoded by a cloned gene. Both depend on the ability of purified mRNA to direct synthesis of proteins in cell-free translation systems. These are cell extracts, usually prepared from germinating wheat seeds or from rabbit reticulocyte cells (both of which are exceptionally active in protein synthesis) and containing ribosomes, tRNAs, and all the other molecules needed for protein synthesis.  The mRNA sample is added to the cell-free translation system, along with a mixture of the 20 amino acids found in proteins, one of which is labeled (often 35S-methionine is used). The mRNA molecules are translated into a mixture of radioactive proteins, which can be separated by gel electrophoresis and visualized by autoradiography. Each band represents a single protein coded by one of the mRNA molecules present in the sample.
  • 27.
    Identifying and Studyingthe Translation Product of a Cloned Gene FIG: Cell-free translation. FIG: Hybrid-release translation. FIG: Hybrid-arrest translation.
  • 28.
    Identifying and Studyingthe Translation Product of a Cloned Gene  Both HRT and HART work best when the gene being studied has been obtained as a cDNA clone. For HRT the cDNA is denatured, immobilized on a nitrocellulose or nylon membrane, and incubated with the mRNA sample. The specific mRNA counterpart of the cDNA hybridizes and remains attached to the membrane. After discarding the unbound molecules, the hybridized mRNA is recovered and translated in a cell-free system. This provides a pure sample of the protein coded by the cDNA.  Hybrid-arrest translation is slightly different in that the denatured cDNA is added directly to the mRNA sample. Hybridization again occurs between the cDNA and its mRNA counterpart, but in this case the unbound mRNA is not discarded. Instead the entire sample is translated in the cell-free system. The hybridized mRNA is unable to direct translation, so all the proteins except the one coded by the cloned gene are synthesized. The cloned gene’s translation product is therefore identified as the protein that is absent from the autoradiograph.
  • 29.
    Identifying and Studyingthe Translation Product of a Cloned Gene Analysis of proteins by in vitro mutagenesis:  The best way of determining the relationship between the structure of a protein and its mode of structure is to induce a mutation in the gene coding for the protein and then to determine what effect the change in amino acid sequence has on the properties of the translation product. Under normal circumstances mutations occur randomly and a large number may have to be screened before one that gives useful information is found. A solution to this problem is provided by in vitro mutagenesis, a technique that enables a directed mutation to be made at a specific point in a cloned gene. FIG: A mutation may change the amino acid sequence of a protein, possibly affecting its properties.
  • 30.
    Identifying and Studyingthe Translation Product of a Cloned Gene Different types of in vitro mutagenesis techniques:  An almost unlimited variety of DNA manipulations can be used to introduce mutations into cloned genes. The following are the simplest:  A restriction fragment can be deleted.  The gene can be opened at a unique restriction site, a few nucleotides removed with a double-strand-specific endonuclease such as Bal31, and the gene religated.  A short, double-stranded oligonucleotide can be inserted at a restriction site. The sequence of the oligonucleotide can be such that the additional stretch of amino acids inserted into the protein produces, for example, a new structure such as an a-helix, or destabilizes an existing structure.  Although potentially useful, these manipulations depend on the fortuitous occurrence of a restriction site at the area of interest in the cloned gene. Oligonucleotide-directed mutagenesis is a more versatile technique that can create a mutation at any point in the gene.
  • 31.
    Identifying and Studyingthe Translation Product of a Cloned Gene FIG: Various in vitro mutagenesis techniques.
  • 32.
    Identifying and Studyingthe Translation Product of a Cloned Gene Using an oligonucleotide to create a point mutation in a cloned gene:  The gene to be mutated must be obtained in a single-stranded form and so is generally cloned into an M13 vector. The single-stranded DNA is purified and the region to be mutated identified by DNA sequencing. A short oligonucleotide is then synthesized, complementary to the relevant region, but containing the desired nucleotide alteration.  Despite this mismatch the oligonucleotide will anneal to the single-stranded DNA and act as a primer for complementary strand synthesis by a DNA polymerase. This strand synthesis reaction is continued until an entire new strand is made and the recombinant molecule is completely double-stranded.
  • 33.
    Identifying and Studyingthe Translation Product of a Cloned Gene  After introduction, by transfection, into competent E. coli cells, DNA replication produces numerous copies of the recombinant DNA molecule. The semi- conservative nature of DNA replication means that half the double-stranded molecules that are produced are unmutated in both strands, whereas half are mutated in both strands. Similarly, half the resulting phage progeny carry copies of the unmutated molecule and half carry the mutation.  The phages produced by the transfected cells are plated onto solid agar so that plaques are produced. Half the plaques should contain the original recombinant molecule, and half the mutated version. Which are which is determined by plaque hybridization, using the oligonucleotide as the probe, and employing very strict conditions so that only the completely base-paired hybrid is stable.
  • 34.
    Identifying and Studyingthe Translation Product of a Cloned Gene  Cells infected with M13 vectors do not lyse, but instead continue to divide. The mutated gene can therefore be expressed in the host E. coli cells, resulting in production of recombinant protein. The protein coded by the mutated gene can be purified from the recombinant cells and its properties studied. The effect of a single base pair mutation on the activity of the protein can therefore be assessed. FIG: One method for oligonucleotide-directed mutagenesis.
  • 35.
    Identifying and Studyingthe Translation Product of a Cloned Gene Using PCR method to create a point mutation in a cloned gene:  The starting DNA molecule is amplified by two PCRs. In each of these, one primer is normal and forms a fully base-paired hybrid with the template DNA, but the second is mutagenic, as it contains a single base-pair mismatch corresponding to the mutation that we wish to introduce into the DNA sequence. This mutation is therefore present in the two PCR products, each of which represents one half of the starting DNA molecule.  The two PCR products are mixed together and a final PCR cycle carried out. In this cycle, complementary strands from the two products anneal to one another and are then extended by the polymerase, producing the full-length, mutated DNA molecule.
  • 36.
    Identifying and Studyingthe Translation Product of a Cloned Gene  This technique, and related ones using PCR, is very quick and easy to carry out but a major problem is caused by the high error rate of the Taq polymerase used in PCR. This error rate makes it likely that not only the desired mutation, but also random ones, will be present at the end of the experiment. The PCR product therefore has to be cloned and the sequences of individual clones checked to find one that has just the desired mutation. FIG: One method for using PCR to create a directed mutation.
  • 37.
    Studying Protein–Protein Interactions Within living cells, few if any proteins act in total isolation. Instead, proteins interact with one another in biochemical pathways and in multiprotein complexes. Two important techniques, phage display and the yeast two hybrid system, enable these protein–protein interactions to be examined. Phage display:  This technique is called phage display because it involves the “display” of proteins on the surface of a bacteriophage, usually M13. This is achieved by cloning the gene for the protein in a special type of M13 vector, one that results in the cloned gene becoming fused with a gene for a phage coat protein.  After transfection of E. coli, this gene fusion directs synthesis of a hybrid protein, made up partly of the coat protein and partly of the product of the cloned gene. With luck this hybrid protein will be inserted into the phage coat so that the product of the cloned gene is now located on the surface of the phage particles.
  • 38.
    Studying Protein–Protein Interactions Normally this technique is carried out with a phage display library made up of many recombinant phages, each displaying a different protein. Large libraries can be prepared by cloning a mixture of cDNAs from a particular tissue or, less easily, by cloning genomic DNA fragments. The library consists of phages displaying a range of different proteins and is used to identify those that interact with a test protein. This test protein could be a pure protein or one that is itself displayed on a phage surface.  The protein is immobilized in the wells of a microtiter tray or on particles that can be used in an affinity chromatography column, and then mixed with the phage display library. Phages that are retained in the microtiter tray or within the column after a series of washes are ones that display proteins that interact with the immobilized test protein.
  • 39.
    Studying Protein–Protein Interactions FIG:Phage display. (a) Display of proteins on the surface of a recombinant filamentous phage. (b) The gene fusion used to display a protein. (c) One way of detecting interactions between a test protein and a phage from within a display library. FIG: The yeast two hybrid system. (a) A pair of transcription factors that must interact in order for a yeast gene to be expressed. (b) Replacement of transcription factor 1 with the hybrid protein 1* abolishes gene expression as 1* cannot interact with transcription factor 2. (c) Replacement of transcription factor 2 with the hybrid protein 2* restores gene expression if the hybrid parts of 1* and 2* are able to interact.
  • 40.
    Studying Protein–Protein Interactions Theyeast two hybrid system:  The yeast two hybrid system is very different to phage display. This procedure is based on the discovery that gene expression in S. cerevisiae depends on interactions between pairs of transcription factors.  In the two hybrid system, a pair of transcription factors responsible for expression of a yeast gene is replaced by hybrid proteins, each one made partly of transcription factor and partly of test protein. The ability of this pair of hybrids to direct expression of the yeast target gene is then tested. To use the system, two yeast cloning experiments must be carried out.
  • 41.
    Studying Protein–Protein Interactions The first cloning experiment involves the gene whose protein product is being studied. This gene is ligated to the gene for one of the pair of transcription factors and the construct inserted into a yeast vector. The recombinant yeasts that are produced are not able to express the target gene, because this modified transcription factor cannot interact with its partner.  In the second cloning experiment, a hybrid version of the partner is made and cloned into the yeast cells. Restoration of expression of the target gene indicates that the two hybrid transcription factors can interact. The fusions are designed in such a way that this can only happen if the interactions occur between the test protein components of the hybrids, not between the transcription factor segments. Pairs of interacting test proteins are therefore identified. The second cloning experiment can involve a library of recombinants representing different proteins, so that one protein can be tested against many others.
  • 42.