Complementary DNA Libraries and Neurological Disease

BASIC SCIENCE SEMINARS IN NEUROLOGY

SECTION EDITOR: HASSAN FATHALLAH-SHAYKH, MD Complementary DNA Libraries and Neurological Disease

Benjamin Joseph, BA; Henry Furneaux, PhD

he DNA in our cell nuclei contains all the information necessary to direct our biologi- cal functions. The gene is 1 unit of such information that usually specifies the expression of 1 protein product. A detailed knowledge of each gene will enable us to understand disease at the molecular level. To study each gene we must purify and study it in Tisolation. This process is usually described as gene cloning. However, this is a difficult task since most of the human genome is junk DNA that does not encode genes. Messenger RNA (mRNA), on the other hand, is representative of only that DNA that has been transcribed. More to the point, within each tissue or cell type, only mRNA that is important for the function of the selected cells will be present. Indeed, different cell types, developmental stages, and many disease states arise because of differential gene expression. The problem is that mRNA itself cannot be easily manipulated to clone genes. On the other hand, DNA can be digested with restriction enzymes and propa- gated in a bacterial vector. Therefore, to isolate specific genes, DNA (which can be easily manipulated) is made from the mRNA by a process called reverse transcription.1 The resulting DNA is called complementary DNA (cDNA). When the mRNA from any specific cell type is used to gen- erate cDNA, that collection of cDNA corresponds to the genes that are expressed in those cells at that time. This is especially valuable for the cloning of genes that are expressed specifically in a particular cell type or expressed differentially between normal and disease cells.

CLONING A GENE Editor’s Note: In this issue, the ARCHIVES introduces a new se- USING A cDNA LIBRARY ries, “Basic Science Seminars in Neurology,” to be published ap- proximately every other month. The seminars are intended to fa- The first step in cloning a gene is the pro- miliarize readers with technologies in basic sciences that have duction of a cDNA library.2 A library in this applicability to the practice of neurology. Fortunately, the “De- case is simply a collection of different cade of the Brain” has delivered significant methods. These basic cDNA molecules that can be physically technologies are not only crucial for our understanding of the bio- separated and manipulated. Since cDNA logical basis of neurological disease, but have also become essen- is made from mRNA, it is imperative that tial for patient care by providing novel diagnostic assays and treat- mRNA of the gene of interest be highly ex- ment modalities. Authors have been instructed to illustrate laboratory pressed in whichever tissue type is se- methods and portray basic concepts clearly and accurately using lected to serve as the starting material for nontechnical language, and emphasize their applicability to the un- the library. When the mRNA has been iso- derstanding, diagnosis, and treatment of neurological disease. The lated and prepared, the first strand of the seminars, by bridging clinical and basic sciences, are designed to cDNA is produced. In generating a cDNA assist in the translation of basic technologies to patient care, and library, reverse transcriptase, an RNA- to keep readers abreast of novel laboratory advances that have po- dependent DNA polymerase, is used to tential clinical applicability. catalyze the reaction (Figure 1). First- strand cDNA synthesis is followed by sec- Hassan Fathallah-Shaykh, MD, Section Editor ond-strand synthesis. To do this, the mRNA template is removed and a second, complementary strand of DNA is syn- From the Program in Molecular Pharmacology and Therapeutics, Memorial Sloan thesized using a DNA-dependent DNA Kettering Cancer Center, New York, NY. polymerase.

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First-Strand Synthesis by Reverse Transcription

mRNA First-Strand DNA

mRNA Is Removed From DNA

First-Strand DNA

Second-Strand Synthesis

Second-Strand DNA First-Strand DNA

cDNA Is Ligated Into the λ DNA

λ DNA With } cDNA Insert λ DNA Is Packaged Into Reassembled Phage Particle

Assembled Phage Particle Containing λ DNA With cDNA Insert A

Bacteriophages Are Plated Onto Bacterial Lawn DNA-DNA Annealing Cloning an Antigen Interaction Cloning

Figure 2. Three methods of screening a complementary DNA (cDNA) library. After the bacteria that have been infected with phages form plaques, a Plaque nitrocellulose filter is placed on top of the bacterial lawn. The cell lysate in the plaque binds the nitrocellulose with high affinity, creating a precise spatial replica of the plaques in the Petri dish. The cDNA library can now be screened by a number of methods. (1) Screening by DNA-DNA annealing. Synthetic-labeled DNA oligonucleotides are incubated with the nitrocellulose filter. Only those oligonucleotides that are specifically bound to the cDNA will remain and give a positive signal on an autoradiograph after the Figure 1. Creating a complementary DNA (cDNA) library from messenger nitrocellulose is washed. (2) Screening for an antigen with serum. The filter RNA (mRNA). When the mRNA has been isolated and prepared, the first is incubated with serum and the irrelevant antibodies are washed off. The strand of DNA is produced by reverse transcription. This is followed by the bound antibody is detected using iodine I 125 protein A. (3) Screening by removal of the mRNA template so the second strand of DNA can be made. interaction cloning. The filter is incubated with a radiolabeled protein. The The double-stranded DNA is then inserted into the ␭ DNA, which is then unbound protein is washed off and the positive plaques identified using reassembled to form a bacteriophage particle that can infect bacteria. The autoradiography. phages are then diluted and spread over a bacterial lawn of Escherichia coli in a Petri dish. Each plaque is the result of the infection of bacteria with a single phage. Since each phage particle contains only 1 cDNA molecule, each plaque represents a single cDNA clone.

Once double-stranded cDNA lated. The vector most commonly se- ferred material in the exact same has been synthesized, the collec- lected for use in cDNA libraries is place on the nitrocellulose. The Petri tion of molecules must be inserted bacteriophage ␭, usually phage ␭ dish containing the phage is kept and into vectors so that they can be fur- ZAP or phage ␭ gt11. The phage ␭ used as the source of the phage. As ther manipulated. Vectors, most of- DNA is cut so that it can accept a for screening the library deposited ten bacteriophages, are merely self- cDNA insert. Once the phage ␭ DNA on the nitrocellulose filter, there are replicating entities. They contain the has incorporated a cDNA insert and many variations, and it is useful to information necessary to repro- has been religated, it can be as- look at a few actual examples of the duce themselves, as well as any for- sembled easily into a phage particle different strategies that have been eign stretch of DNA (eg, cDNA) that that is ready to infect a bacterium. adopted (Figure 2). has been inserted into their own ge- When the collection of phage nome. A single bacteriophage par- has been made, it can be screened CLONING BY DNA ticle can be detected by the produc- for the desired clone. First, the li- ANNEALING: ISOLATION tion of a plaque (the result of cell brary is plated onto a bacterial lawn OF THE SCRAPIE GENE lysis) on a bacterial lawn (Figure 1). of Escherichia coli in a Petri dish. The Therefore, if a phage suspension is bacteria that are infected with phage One common method of screening greatly diluted and spread over a bac- form plaques. Next, a piece of ni- involves specific DNA-DNA anneal- terial lawn, individual phage clones trocellulose is placed on top of the ing. In this approach one must know can be detected and isolated. Each plate. The entire cell contents in the the amino acid sequence of the pro- phage particle contains only 1 cDNA plaque bind nitrocellulose with high tein gene product. With this infor- molecule; thus, by isolating a single affinity, so wherever there is a plaque mation, synthetic DNA can be made phage, a pure cDNA clone is iso- on the plate, there will be trans- that will be complementary to the

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 mRNA. However, because the trip- hamster brains. Hamsters were CLONING AN ANTIGEN: let genetic code is degenerate, great used as experimental hosts because THE PARANEOPLASTIC care must be taken in designing the they have the shortest incubation ENCEPHALOMYELITIS DNA. Although any 3 bases corre- period. First, prions were purified ANTIGEN spond to only 1 amino acid, 18 of from scrapie-infected hamster the 20 amino acids can be encoded brains. These prions were ob- Another use of cDNA libraries is us- by more than 1 set of 3 bases. Thus, served to contain 1 major protein ing human serum to screen for spe- the DNA can be designed either to constituent, called PrP 27-30, in cific antigens. A slightly different kind be short but with a high degree of which an amino acid sequence of library than the one discussed ear- homology to the cDNA in ques- was determined via gas-phase lier has to be constructed. In what is tion, or it can be somewhat longer microsequencing. To ensure that termed an expression library, vec- with a lesser degree of homology.3 an abundant supply of the mRNA tors are used that can express the Both strategies have their advan- coding for this disease-related cDNA as fusion proteins. That is, the tages and drawbacks, and in the end protein would be present in the cDNA sequence is inserted within the it is generally smart to make a mix- starting material, scrapie-infected coding region of another known pro- ture of different oligonucleotides to hamster brain tissue from the tein so that the final product is the try under varying conditions. exponential phase of prion forma- known protein attached, or fused, di- At this point, the nitrocellu- tion (35 days after inoculation) rectly adjacent to the peptide en- lose membranes with the cDNA was used as a basis for the library. coded by the cDNA. This screening bound to them are incubated with A set of 32 icosameric oligo- technique is similar to that de- radiolabeled DNA, which should nucleotides was synthesized to scribed earlier. In this case, after re- only anneal to cDNA. The mem- screen the library. These oligo- moval from the Petri dish the nitro- branes are then washed to remove nucleotides were derived from the cellulose filter is blocked to prevent unbound DNA and autoradio- reverse translation of a 7 amino nonspecific adsorption of proteins. graphed. The autoradiograph is acid sequence of PrP 27-30. A The blocked filter is then incubated aligned with the original plate and library containing 150 000 cDNA with the human serum and washed any positive phage selected. These clones was screened, yielding 1 to remove nonspecific interactions. plaques are replated until all the positive clone. Any bound antibodies are visual- phages are positive. Finally, the The next question to be an- ized by incubation with labeled pro- DNA is purified from the positive swered was whether the isolated tein A and autoradiography. Posi- phage and digested to release the cDNA was derived from cellular tive clones are selected and replated cDNA insert. DNA or from an infectious agent. until all phages are positive. This type of approach was DNA from healthy and scrapie- This technique was used in the taken by the group that cloned the infected hamsters was digested with study of the neurological disorder gene for the human prion protein restriction enzymes and annealed paraneoplastic encephalomyelitis (PrP).4 Prions are transmissible with a radiolabeled copy of the sensory neuronopathy.6 A clinical pathogens now known to be cDNA insert. No difference was ob- observation was made that certain involved in neurodegenerative dis- served between these 2 prepara- patients with small cell lung cancer eases of animals and humans. In tions, suggesting that a single PrP developed this disorder, which is animals, the predominant disease gene exists in the hamster genome. characterized by dementia, sensory is scrapie, while in man the group In addition, no difference was ob- loss, and other neurological disor- includes Creutzfeldt-Jakob dis- served in hamster PrP mRNA lev- ders. The sera of most of these pa- ease, Gerstmann-Stra¨ussler syn- els harvested at different times af- tients reacted with a group of anti- drome, and kuru. These diseases ter inoculation. The information gens that are expressed specifically are all transmissible and have gathered in these experiments was in small cell lung tumors and neu- common histopathological and used to detect and isolate related se- rons. The antibody responsible for clinical features. Primarily, spon- quences in human and mouse DNA, these reactions is called anti-Hu. giform degeneration of central and eventually led to the cloning of Cloning of the gene that encodes nervous system neurons is ob- the human PrP gene.5 these proteins has enabled a more served, along with intense restric- The most important conclu- rigorous analysis of the antigens’ role tive astrocytic gliosis and amyloid sion was that PrP is encoded by a cel- in this disorder. plaque formation. Prions contain a lular gene. Thus, PrP is present in In this case, the method for protein, called PrPSc, that is healthy cells. Knowing the se- screening a library included using required for infectivity. Initially, quence of PrP has allowed research- the serum of a patient with the dis- the origin of PrPSc was unknown; ers to study its biochemical proper- order, which contains the anti-Hu whether it was the result of a viral ties in depth and determine what antibody. For the purposes of this or bacterial infection, or something other factors are involved in these experiment, an expression library altogether different, had not been neurodegenerative diseases. The cur- was made from cerebellar tissue, in determined. rent hypothesis is that these neuro- which mRNA for the antigen in To investigate the origin of degenerative diseases are caused by question would be in relative abun- PrPSc, a cDNA library was con- a conformational change in the dance. When the library of 1 mil- structed from scrapie-infected structure of PrP. lion clones was screened with the

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 anti-Hu serum, 8 positive clones Huntington disease, a rat brain work, such as picking clones and were revealed, none of which were cDNA expression library was sequencing them. Computers are positive when screened with nor- screened for interacting proteins us- responsible for lining up and orga- mal human serum.6 Three of the ing the polyglutamine repeat do- nizing overlapping sequences and clones encoded 1 gene, which was main. A new gene was discovered, arranging the genes into likely func- called HuD. The other 5 encoded an- called huntingtin-associated pro- tional classes. These databases are of other highly similar gene, HuC. tein 1 (HAP1),8 which has been great benefit to researchers who can The primary advance that has found to bind to mutated hunting- quickly ascertain the complete arisen directly from this work is a di- tin with greater affinity than to the sequence and likely function of the agnostic test for paraneoplastic en- wild-type protein. After rat HAP1 gene of their interest. In addition, it cephalomyelitis sensory neuronopa- cDNA had been isolated, the human permits scientists to compare the thy. In this test, the HuD protein is gene was cloned from human cau- sequence of the genes they have mass produced in bacteria and then date tissue using polymerase chain cloned with similar genes from other purified, and a patient’s serum can reaction. Human HAP1 is most con- species. For more than 20 years, be checked using Western blot analy- sistently expressed in those parts of cDNA libraries have played an inte- sis to see if it contains antibodies the brain that are primarily affected gral role in biomedical research. In against HuD, signifying a positive di- in Huntington disease, namely cau- light of the exciting developments agnosis. Of particular importance is date and cortex. Since huntingtin is in the field, they promise to be of the finding that most patients with expressed ubiquitously throughout even greater importance in the paraneoplastic encephalomyelitis the body, the selective expression of future. sensory neuronopathy also have a tu- HAP1 may explain the neural speci- mor, so a neurologist whose patient ficity of Huntington disease. Fur- Accepted for publication February 13, has a positive anti-Hu result should ther screening of cDNA libraries 1998. search the patient for an occult tu- using this protein-protein inter- The databases of the Human Ge- mor. It is important to note that the action approach may yield other nome Sciences and The Institute for recombinant protein used in this as- proteins involved in Huntington dis- Genomic Research are available at say gives a definitive result that could ease, leading to a greater understand- www.hgsi.com and www.tigr.org. not be obtained by using cellular ex- ing of the molecular mechanisms Reprints: Henry M. Furneaux, tracts. Such extracts could contain a that underlie this disorder. PhD, Box 20, Laboratory of Molecu- different 36-kd protein that may be lar Neuro-Oncology, Memorial Sloan recognized by other antibodies, pro- SUMMARY AND FUTURE Kettering Cancer Center, 1275 York ducing a false-positive result. DEVELOPMENTS Ave, New York, NY 10021 (e-mail: [email protected]). INTERACTION CLONING: In summary, screening cDNA librar- THE HUNTINGTIN- ies is a highly valuable approach for ASSOCIATED PROTEIN discovering important information REFERENCES about neurological functions and In many hereditary diseases the diseases. As illustrated herein, the pathogenic gene has been identi- advantages of knowing a gene’s se- 1. Efstratiadis A, Kafatos FC, Maxam AM, Maniatis fied but the function of its protein quence are profound and numer- T. Enzymatic in vitro synthesis of globin genes. product is unknown. One way to ous. Cloning a gene also provides Cell. 1976;7:279-288. 2. Sambrook J, Fritsch EF, Maniatis T. Molecular clarify the function of a disease gene therapeutic opportunities. A gene Cloning: A Laboratory Manual. 2nd ed. Cold Spring product is to identify other pro- that helps prevent or cure a disease Harbor, NY: Cold Spring Harbor Laboratories; teins that interact with it. This is can be overexpressed in cells. Alter- 1989. called interaction cloning.7 The natively, antisense DNA vectors can 3. Wallace RB, Johnson NJ, Hirose T, Miyake M, Kawashima EH, Itakura K. The use of synthetic oli- genes encoding interacting pro- be constructed that may block ex- gonucleotides as hybridization probes. Nucleic Ac- teins can be cloned by screening a pression of a gene that is respon- ids Res. 1981;9:879-894. cDNA library with a radioactively la- sible for causing a disease. 4. Oesch B, Westaway D, Walchli M, et al. A cellular beled protein. One recent development in the gene encodes scrapie PrP 27-30 protein. Cell. This method has been used to field is the undertaking of 2 groups 1985;40:735-746. 5. Kretschmar HA, Stowring LE, Westaway D, identify genes that are involved in to sequence as many cDNA clones Stubblebine WH, Prusiner SB, Dearmond SJ. Mo- Huntington disease. This autoso- as possible in the hope that they will lecular cloning of a human prion protein cDNA. mal dominant neurodegenerative become medically important at some DNA. 1986;5:315-324. disorder usually has its onset in the point. Human Genome Sciences and 6. Szabo A, Dalmau J, Manley G, et al. HuD, a para- fourth or fifth decade of life and is The Institute for Genomic Research, neoplastic encephalomyelitis antigen, contains RNA binding domains and is homologous to Elav marked by motor disturbance, cog- both located in Rockville, Md, have and Sex-Lethal. Cell. 1991;67:325-333. nitive loss, and psychiatric manifes- set up facilities where the sole func- 7. Blanar MA, Rutter WJ. Interaction cloning: identi- tations. Huntington disease is caused tion is to produce cDNA libraries fication of a helix-loop-helix zipper protein that in- by an expanded polyglutamine re- from different starting tissues, teracts with c-fos. Science. 1992;256:1014-1018. 8. Li X-J, Li S-H, Sharp AH, et al. A huntingtin- peat in the gene that codes for the human or otherwise, and then associated protein enriched in brain with impli- protein huntingtin. To study the role sequence these cDNA strands. cations for pathology. Nature. 1997;378: of huntingtin in the progression of Robotic machines do much of the 398-402.

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