Identifying and Analysing mRNA

 The levels and expression patterns of mRNA dictate many cellular processes and therefore there is much interest in the ability to analyse and determine levels of a particular mRNA. Technologies such as real-time or quantitative PCR , microarray and now RNA sequencing (RNA-Seq) are employed to perform high-throughput analysis. A number of other informative techniques have been developed that allow the fine structure of a particular mRNA to be analysed, and the relative amounts of an RNA quantified by non-PCR-based methods. This is important, not only for gene regulation studies, but may also be used as a marker for certain clinical disorders. Traditionally, the Northern blot has been used for detection of particular RNA transcripts by blotting extracted mRNA and immobilising it on a nylon membrane. Subsequent hybridisation with labelled gene probes allows precise determination of the size and abundance of a transcript. However, much use has been made of a number of nucleases that digest only single-stranded nucleic acids and not double-stranded molecules. In particular, the ribonuclease protection assay (RPA) has allowed much information to be gained regarding the nature of mRNA transcripts ( Figure 1). In the RPA, single-stranded mRNA is hybridised in solution to a labelled single-stranded RNA probe that is in excess. The hybridised part of the complex becomes protected, whereas the non-hybridised part of the probe made from RNA is digested with RNase A and RNase T1. The protected fragment may then be analysed on a high-resolution polyacrylamide gel. This method may give valuable information regarding the mRNA in terms of the precise structure of the transcript (transcription start site, intron/exon junctions, etc.). It is also quantitative and requires less RNA than a Northern blot. A related technique, S1 nuclease mapping , is similar, although the non-hybridised part of a DNA probe, rather than an RNA probe, is digested, this time with the enzyme S1 nuclease.

Fig1. Steps involved in the ribonuclease protection assay (RPA). PAGE, polyacrylamide gel electrophoresis.

The PCR has also had an impact on the analysis of RNA via the development of a technique known as reverse transcriptase-PCR ( RT-PCR). Here, the RNA is isolated and a first-strand cDNA synthesis undertaken with reverse transcriptase; the cDNA is then used in a conventional PCR. Under certain circumstances, a number of thermostable DNA polymerases have reverse transcriptase activity, which obviates the need to separate the two reactions and allows the RT-PCR to be carried out in one tube. One of the main benefits of RT-PCR is the ability to identify rare or low levels of mRNA transcripts with great sensitivity. This is especially useful when detecting, for example, viral gene expression and furthermore provides the means of differentiating between latent and active virus (Figure 2).

Fig2. Representation of the detection of active viruses using RT-PCR.

In many cases, the analysis of tissue-specific gene expression is required, and again the PCR has been adapted to provide a solution. This technique, termed differential display, is also an RT-PCR-based system requiring that isolated mRNA be first converted into cDNA. In a subsequent step, one of the PCR primers, designed to anneal to a general mRNA element such as the poly(A) tail in eukaryotic cells, is used in conjunction with a combi nation of arbitrary 6–7 bp primers that bind to the 5′ end of the transcripts. Consequently, this results in the generation of multiple PCR products with reproducible patterns (Figure 3). Comparative analysis by gel electrophoresis of PCR products generated from different cell types therefore allows the identification and isolation of those transcripts that are differentially expressed. As with many PCR-based techniques, the time to identify such genes is dramatically reduced to a few days compared to several weeks that are required to construct and screen cDNA libraries using traditional approaches.

Fig3. Analysis of gene expression using differential display PCR.

Analysing Genes In Situ

Gross chromosomal changes are often detectable by microscopic examination of the chromosomes within a karyotype. Single or restricted numbers of base substitutions, deletions, rearrangements or insertions are far less easily detectable, but may induce similarly profound effects on normal cellular biochemistry. In situ hybridisation makes it possible to determine the chromosomal location of a particular gene fragment or gene mutation. This is carried out by preparing a radiolabelled DNA or RNA probe and applying this to a tissue or chromosomal preparation fixed to a microscope slide. Any probe that does not hybridise to complementary sequences is washed off and an image of the distribution or location of the bound probe is viewed by autoradiography (Figure 4). Using tissue or cells fixed to slides it is also possible to carry out in situ PCR and qPCR. This is a highly sensitive technique, where PCR is carried out directly on the tissue slide with the standard PCR reagents. Specially adapted thermal cycling machines are required to hold the slide preparations and allow the PCR to proceed. This allows the localisation and identification of, for example, single copies of intra cellular viruses and, in the case of qPCR, the determination of initial concentrations of nucleic acid.

Fig4. General scheme for in situ hybridisation.

An alternative labelling strategy used in karyotyping and gene localisation is fluorescence in situ hybridisation ( FISH). This method (sometimes also termed chromosome painting) is based on in situ hybridisation, but different gene probes are labelled with different fluorophores, each specific for a particular chromosome. The advantage of this method is that separate gene regions may be identified and com parisons made within the same chromosome preparation. The technique is also of great interest in genome mapping for ordering DNA probes along a chromosomal segment.

Analysing Protein–DNA Interactions

To determine potential transcriptional regulatory sequences, genomic DNA fragments may be cloned into specially devised promoter probe vectors. These contain sites for insertion of foreign DNA that lie upstream of a reporter gene. A number of reporter genes are currently used, including the lacZ gene encoding β-galactosidase, the cat gene encoding chloramphenicol acetyl transferase ( CAT) and the lux gene, which produces luciferase and is determined in a bioluminescent assay. Fragments of DNA potentially containing a promoter region are cloned into the vector and the constructs transfected into eukaryotic cells. Any expression of the reporter gene will be driven by the foreign DNA, which must therefore contain promoter sequences ( Figure 5). These plasmids and other reporter genes, such as those using green fluorescent protein ( GFP) or the firefly luciferase gene, allow quantification of gene transcription in response to transcriptional activators.

Fig5. Assay for promoters using the reporter gene for chloramphenicol acetyl transferase (CAT).

The binding of a regulatory protein or transcription factor to a specific DNA site results in a complex that may be analysed by the technique termed electrophoretic mobility shift assay ( EMSA) or gel retardation assay. It may also be adapted to study protein RNA interactions. In gel electrophoresis, the migration of a DNA fragment bound to a protein of a relatively large mass will be retarded in comparison to the DNA fragment alone. For gel retardation to be useful, the region containing the promoter DNA element must be digested or mapped with a restriction endonuclease before it is complexed with the protein. The location of the promoter may then be defined by finding the position on the restriction map of the fragment that binds to the regulatory protein and therefore retards it during electrophoresis. One potential problem with gel retardation is finding the precise nucleotide binding region of the protein, since this depends on the accuracy and detail of the restriction map and the convenience of the restriction sites. However, it is a useful first step in determining the interaction of a regulatory protein with a DNA binding site. Large-scale methods now employ massively parallel DNA sequencing after initial immunoprecipitation of the chromatin with an antibody. This method, termed ChIP-Seq ( chromatin immunoprecipitation sequencing), has enabled the genome-wide identification of transcription factor binding sites in DNA.

Most methods used in identifying regulatory sites in DNA rely on the fact that the interaction of a DNA-binding protein with a regulatory DNA sequence will protect that DNA sequence from degradation by an enzyme such as DNase I in a footprinting assay. In its basic form, the DNA regulatory sequence is first labelled at one end and then mixed with the DNA-binding protein (Figure 6). DNase I is added under conditions favouring a partial digestion. This limited digestion ensures that a number of fragments are produced where the DNA is not protected by the DNA-binding protein; the region protected by the DNA-binding protein will remain undigested. All the fragments are then separated on a high-resolution polyacrylamide gel alongside a control digestion where no DNA-binding protein is present. The resulting gel will contain a ladder of bands representing the partially digested fragments. Where DNA has been protected, no bands appear; this region or hole is termed the DNA footprint. The position of the protein-binding sequence within the DNA may be elucidated from the size of the fragments either side of the footprint region.

Fig6. Steps involved in DNA footprinting.

Footprinting is a more precise method of locating a DNA–protein interaction than gel retardation; however, it is unable to give any information as to the precise interaction or the contribution of individual nucleotides. Identifi cation of regulatory regions in DNA may alternatively be accomplished on a genome-wide scale by sequencing using techniques such as DNase-Seq. Owing to the large scale, this approach requires bioinformatic analysis in order to provide genome-wide footprints.

In addition to the detection of DNA sequences that contribute to the regulation of gene expression, an ingenious way of detecting the protein transcription factors has been developed. This is termed the yeast two-hybrid system. Transcription factors have two domains, one for DNA binding and the other to allow binding to further proteins ( activation domain ). These occur as part of the same molecule in natural transcription factors, for example TFIID. However, they may also be formed from two separate domains. Thus a recombinant molecule is formed, encoding the protein under study as a fusion with the DNA-binding domain. The fusion construct cannot, however, activate transcription. Genes from a cDNA library are expressed as a fusion with the activator domain; these also cannot initiate transcription. However, when the two fractions are mixed together, transcription is initiated if the domains are complementary ( Figure 7). This is indicated by the transcription of a reporter gene such as the cat gene. The technique is not just confined to transcription factors and may be applied to any protein system where interaction occurs.

Fig7. Yeast two-hybrid system (interaction trapping technique). Transcription factors have two domains, one for DNA binding (A) and the other to allow binding to further proteins (B). Thus a recombinant molecule is formed from a protein (C) as a fusion with the DNA-binding domain. It cannot, however, activate transcription alone. Genes from a cDNA library (D) are expressed as a fusion with the activator domain (B) but also cannot initiate transcription alone. When the two fractions are mixed together, transcription is initiated if the domains are complementary and expression of a reporter gene takes place.

Transgenics and Gene Targeting

In many cases it is desirable to analyse the effect of certain genes and proteins in an organism rather than in the laboratory. Furthermore, the production of pharmaceutical products and therapeutic proteins is also desirable in a whole organism. This also has important consequences for the biotechnology industry (Table 1). The introduction of foreign genes into germ-line cells and the production of an altered organism is termed transgenics. There are two broad strategies for transgenesis. The first is direct transgenesis in mammals, whereby recombinant DNA is injected directly into the male pronucleus of a recently fertilised egg. This is then raised in a foster mother animal resulting in an offspring that is all transgenic. Selective transgenesis is where the recombinant DNA is transferred into embryo stem (ES) cells. The cells are then cultured in the laboratory and those expressing the desired protein selected and incorporated into the inner cell mass of an early embryo. The resulting transgenic animal is raised in a foster mother, but in this case the transgenic animal is a mosaic or chimeric since only a small proportion of the cells will be expressing the protein. The initial problem with both approaches is the random nature of the integration of the recombinant DNA into the genome of the egg or embryo stem cells. This may produce proteins in cells where it is not required or disrupt genes necessary for correct growth and development.

Table1. Use of transgenic mice for investigation of selected human disorders

A refinement of this approach is gene targeting which involves the production of an altered gene in an intact cell, a form of in vivo mutagenesis as opposed to in vitro mutagenesis. The gene is inserted into the genome of, for example, an ES cell by specialised viral-based vectors. The insertion is non-random, however, since homologous sequences exist on the vector to the gene and on the gene to be targeted. Thus, homologous recombination may introduce a new genetic property to the cell, or inactivate an already existing one, termed gene knockout . Perhaps the most important aspect of these techniques is that they allow animal models of human diseases to be created. This is useful since the physiological and biochemical consequences of a disease are often complex and difficult to study, impeding the development of diagnostic and therapeutic strategies.

Modulating Gene Expression by RNAi

There are a number of ways of experimentally changing the expression of genes. Traditionally, methods have focussed on altering the levels of mRNA by manipulation of promoter sequences or levels of accessory proteins involved in control of expression. In addition, post-mRNA production methods have also been employed, such as antisense RNA, where a nucleic acid sequence complementary to an expressed mRNA is delivered into the cell. This antisense sequence binds to the mRNA and prevents its translation. A development of this theme and a process that is found in a variety of normal cellular processes is termed RNA interference (RNAi). This process of RNAi is now a well-established and powerful technique that has been widely applied to identify the function of genes and the resulting proteins either in cells grown in culture or even in vivo using model organisms. Essentially the technique may be used to decrease the expression of a gene termed gene knockdown and differs from the method of gene knockout, where the expression of a gene is abolished.

One important feature of the method is the design and synthesis of the dsRNA ( double-strand RNA) to the gene of interest. This is introduced into the appropriate cell line after which the RNAi pathway in the cell is activated. A potential problem in the dsRNA design process is the so-called off-target effect , where the expression of multiple genes is inadvertently reduced, as well as the gene under study. This can be addressed in part by employing a number of computational methods that assist in the design of the dsRNA, thus minimising off-target effects. Indeed, improvements of the technique have also included the synthesis and delivery of short interfering RNA ( siRNA) directly, rather than using the longer dsRNA that is cleaved by dicer to produce the siRNAs. The precise delivery of siRNA may also prove troublesome, depending on the cell or tissue used, and RNA instability is still a major issue to be fully addressed.

The production of short hairpin RNA ( shRNA) from sequences cloned and expressed from plasmid vectors introduced into the cell line or organism has also proved beneficial. The choice of which dsRNA to use largely depends on the organism, for example mammalian cells have a process whereby the introduction of long dsRNA evokes an unwanted interferon-based immune response, whereas this is decreased when using synthesised siRNA. RNAi technology holds enormous promise in the biotechnology industry and also medical fi elds where, for example, viral infections may be addressed by gene knockdown of specific mRNA targets in HIV-1, hepatitis B and C. Indeed, RNAi-based therapy may have the potential to treat certain types of cancers where aberrant levels of oncogene mRNA such as MYC are found, leading to a highly specifi c form of treatment.

CRISPR/cas9-Based Genome Editing

One major gene editing system is CRISPR (clustered regularly interspaced short palindromic repeats) involving RNA-guided engineered nucleases (Figure 8). The system was first identified as an adaptive immunity pathway in prokaryotes providing resistance to bacterial phage and analogous to the RNA interference process found in eukaryotes. It has since been modified and adapted for the engineering of genomes. Essentially, CRISPR may be thought of as a programmable restriction enzyme system. It utilises double-stranded sequence-specific breaks and repair using HDR (homology directed repair) via homologous recombination. The availability of a vast amount of sequence information has allowed the development of the system into an essential molecular biology technique.

Fig8. The CRISPR/Cas9 gene editing system. The nuclease Cas9 is targeted to DNA by a guide RNA consisting of a specific 20 nucleotide spacer/targeting sequence (crRNA) that identifies the DNA to modified, linked to a trans-activating (tracrRNA) sequence necessary for the recruitment and stability of Cas9 nuclease. DNA is then digested by the Cas9 nuclease at specific points on both DNA strands. Homologous recombination to repair the break and incorporate the new DNA sequence then follows.

The system consists of two parts, a short synthetic guide RNA (gRNA) and a non- specific double-stranded endonuclease termed Cas9 (Cas: C RISPR- as sociated). The guide RNA has a specific 20 nucleotide spacer/targeting sequence (crRNA) that identifies the genomic target to be identified and modified, linked to a trans-activating (tracrRNA) sequence necessary for the recruitment and stability of Cas9 nuclease. The gRNA sequence and Cas9 nuclease elements are constructed into a plasmid expression vector that can be used to transfect cells under study. Transcription of the gRNA and Cas9 results in crRNA that binds to the DNA to be altered, which is held in the Cas9 nuclease with the aid of the tracrRNA. Indeed, it is the fact that the target sequence can be reprogrammed simply by changing the 20 nucleotides in the crRNA that makes the system so elegant. DNA is then digested by the Cas9 nuclease at specific points on both DNA strands. A donor DNA sequence may be included with a desired feature, such as a base change, insertion or deletion. The cell then employs homologous recombination to repair the break and incorporate the new DNA sequence. It is also possible to program Cas9 with multiple guide RNAs to allow multiplex site-specific editing for designing large deletions, inversions and translocations. Further refinements of the editing system have allowed the targeting of protein domains for transcriptional regulation and epigenetic modification.

اخر الاخبار

اخبار العتبة العباسية المقدسة

أساتذة المجمَع العلمي يشرفون على تحكيم المسابقة القرآنية في جامعة النهرين

قسم الشؤون الدينية ينشر صناديق مخصصة لزكاة الفطرة داخل مرقد أبي الفضل العباس (عليه السلام) وخارجه

شعبة التوجيه الديني النسوي تختتم مجالسها العبادية لإحياء ليالي القدر المباركة

قسم الشؤون الفكرية ينظم ندوة توعوية ثقافية في محافظة ذي قار

المزيد

الآخبار الصحية

تأثير اللياقة البدنية على الدماغ

تجربة سريرية تظهر فوائد اللوز للمصابين بالسمنة

اكتشاف طريقة غير متوقعة لتقليل خطر الإصابة بالسكري

خمسة مؤشرات صحية مهملة قد تنقذ حياتك

المزيد

الآخبار التكنلوجية

علماء الجيولوجيا يفكون لغز الصدع العملاق تحت الماء في المحيط الأطلسي

تحذير جديد: الاحترار العالمي يتسارع وقد نكسر حاجز 1.5 درجة قبل 2030

الصين تكشف عن ألياف كربون تفوق قوة الفولاذ بـ10 مرات

الجهاز المناعي يظهر دورا غير متوقع في صحة العظام

المزيد

مواضيع ذات صلة

Screening Gene Libraries

Hybridisation and Gene Probes

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity

Interphase Polytene Chromosomes Arise by DNA Amplification

Cloning Vectors : Cosmid-Based Vectors

Cloning Vectors : Phagemid-Based Vectors

Cloning Vectors : Virus-Based Vectors

Cloning Vectors : Plasmids

The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA

Chromosome Painting and DNA Sequencing Reveal the Evolution of Chromosomes