Glycoprotein methods protocols - biotechnology
Mucin cDNA Cloning 29729724Mucin cDNA CloningJean-Pierre Aubert and Nicole Porchet1. IntroductionMuch information on the structure, organization, and expression of many genes hasbeen acquired by using the powerful technique of cDNA cloning (1–5). cDNA clonesdiffer from genomic DNA clones in that they represent a permanent copy of an mRNAand are representative of the parts of a gene that are expressed as mature RNA. Thiscomparison of cDNA clones with their genomic homologs has resulted in the discov-ery of introns in most eukaryotic genes. Amino acid sequence of gene products canonly be obtained from full-length cDNA sequences. cDNA clones can also be used toexpress the protein products of genes in prokaryotes.A cDNA library must be created by a series of enzymatic reactions. The first oneconsists in generating a first DNA strand complementary to RNA followed by a re-moval of the RNA strand and replacement by a “second strand” of DNA. The newdouble-stranded DNA is ligated into an appropriate vector. Two kinds of vectors canbe used: plasmid or bacteriophage cloning vectors. Most of the cDNA cloning experi-ments of human mucins have been done in bacteriophage λgt11 and/or Lambda ZAP®Vector. Both are protein expression cDNA vectors. They are designated to generatecDNA libraries from small amounts of DNA (between 5000 and 10,000 clones pernanogram of cDNA). cDNA inserts are cloned into the 3' end (λgt11) or the 5' end(λZAP®) of the β-galactosidase gene-coding region, allowing blue/white color screen-ing of clones with inserts in the presence of X-Gal and isopropyl-1-thiol-β-D-galactopyranoside (IPTG).Due to the large size of the mRNA of mucins (superior to 20 kb) described, it isnearly impossible to obtain a full-length cDNA by screening cDNA libraries. There-fore, methods have been developed to amplify DNA sequences from an mRNA tem-plate between a known internal site and the unknown sequences of either the 3' or the5' end of the mRNA. These methods were first described by Frohmann et al. (6) andare referred to as RACE (rapid amplification of cDNA ends).From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 298 Aubert and PorchetThis chapter describes the protocols that have been successfully used in our labora-tory to clone three cDNAs corresponding to MUC4, MUC5AC, and MUC5B (7).2. Materials2.1. Biological Materials1. Mucus-secreting cultured cells (e.g., LS174T, HT29 MTX) or mucosae from human oranimals (e.g., tracheobronchial, gastric, intestinal) are snap-frozen in liquid nitrogen andstored at –80°C until used.2.2. Monoclonal or Polyclonal Antibodies1. Antibodies must be directed against the peptide moiety of mucins. A chemical degly-cosylation of whole mucins or of mucin glycopeptides is needed. The quality of the anti-bodies must be checked by immunohistochemistry before screening (7–10). See Chapters29 and 30.2.3. Buffers1. Phenol/chloroform: Add a volume of chloroform equal to that of TE (see item 3) andsaturated phenol and mix. Allow the phases to separate. Store in dark bottles at 4°C.2. Chloroform: Prepare isoamylalcohol by mixing 24 vol of chloroform with 1 vol of isoamylalcohol. Store at room temperature in a dark bottle.3. TE buffer: Dissolve 1.21 g of Tris base and 0.37 g of EDTA-disodium salt per litre.Adjust to pH 8.0 with HCl. Sterilize by autoclaving.4. STE buffer: Dissolve 5.84 g of NaCl, 1.21 g of Tris base and 0.37 g of EDTA-disodiumsalt per liter. Adjust pH to 8.0 with HCl. Sterilize by autoclaving.5. SM buffer: Dissolve 5.8 g of NaCl, 2 g of MgSO4-7H2O, 6.05 g of Tris base, 5 mL of 20 ggelatine per liter. pH to 7.5 with HCl. Sterilize by autoclaving.6. TBS buffer: Dissolve 6.05 g of Tris base and 8.76 g of NaCl per liter. Adjust pH to 8.0with HCl. Sterilize by autoclaving.7. TBST buffer: Add 0.5 mL of Tween-20 to 1 L of TBS. Sterilize by autoclaving.8. 3 M sodium acetate: Dissolve 408.1 g of sodium acetate 3 H2O in 800 mL of water.Adjust pH to 5.2 with glacial acetic acid. Adjust volume to 1 L. Dispense in aliquots andsterilize by autoclaving.9. 0.25 M EDTA: add 9.3 g of disodium EDTA-2H2O to 80 mL water. Adjust to pH 9.0 with10 N NaOH and adjust the volume to 100 mL. Sterilize by autoclaving.10. 10 mM magnesium sulfate (MgSO4-7H2O): Dissolve 7.4 g of magnesium sulfate/100 mLof distilled water. Sterilize by autoclaving.11. 0.5 M calcium chloride (CaCl2-2H2O): Dissolve 7.4 g of calcium chloride/100 mL ofdistilled water. Sterilize by autoclaving.12. 1 M magnesium chloride (MgCl2-6H2O): Dissolve 20.3 g of magnesium chloride/100 mLof distilled water. Sterilize by autoclaving.13. PEG/NaCl solution: Dissolve 20 g of polyethylene glycol 6000 and 11.7 g of NaCl/100 mLof SM buffer pH 7.5. Sterilize by autoclaving.14. 20% maltose: Dissolve 10 g of maltose/50 mL of distilled water. Filter sterilize. Store at 2–8°C.15. Ampicillin stock solution: Dissolve 0.25 g of ampicillin/10 mL of distilled water. Filtersterilize. Store at –20°C.16. Luria-broth (LB): Dissolve in 800 mL of H2O, 10 g of tryptone, 5 g of yeast extract, and10 g of NaCl. Adjust to pH 7.0 with 5 M NaOH and increase volume to 1 L. Sterilize byautoclaving immediately. Mucin cDNA Cloning 29917. M-broth + ampicillin : just before use, add 20% maltose, to a final concentration of 0.4%and ampicillin, to a final concentration of 50 µg/mL in L-broth.18. L-amp plates: Add 15 g of agar per litre to L-broth, prior to autoclaving. Cool to approx50°C, add 2 mL of ampicillin stock solution, and pour 25 mL into each 90 mm sterilePetri dish (see Note 1).19. L-top agar: Add 0.8 g of agar per 100 mL of LB before autoclaving (see Note 2).20. M-top agar + ampicillin: Melt L-top agar, cool to 45°C and add 20% maltose to a final con-centration of 0.4 M and ampicillin (25 mg/mL stock) to a final concentration of 50 µg/mL.21. Color-selection reagents: 100 mM IPTG; dissolve 23.8 mg isopropyl β-D-thiogalacto-pyranoside/mL in water. Filter sterilise. 2% X-gal: Dissolve 2% (w/v) 5-bromo-4-chloro-3-indoly-β-D-galactopyranoside in dimethyl formamide. Add 10 µL of each solution/mLof top agar just before plating at 45°C.22. Denaturing solution: Dissolve 87.66 g of NaCl and 20 g of NaOH in 800 mL H2O slowlywith care. Adjust volume to 1 L.23. Neutralizing solution: Dissolve 175.32 g of NaCl in 500 mL of 1 M Tris-HCl, pH 7.0.Adjust to 1 L with water.2.4. Kits and Modules1. Poly A tract® mRNA isolation system (Promega, Charbonnieres, France).2. mRNA purification kit (Pharmacia Biotech, Orsey, France).3. cDNA synthesis module (Amersham,Les Ulis, France).4. cDNA rapid adaptor ligation module (Amersham).5. cDNA rapid cloning module (Amersham).6. 5'/3' RACE kit (Boehringer Mannheim, Roche Diagnostics, Meylan, France).2.5. Equipment1. Several water baths at a range of temperatures from 12 to 90°C and incubators at 32, 37, and 43°C.2. Microcentrifuge.3. Horizontal gel electrophoresis apparatus.4. Thermocycler.3. Methods3.1. Preparation of mRNA1. Total RNA is isolated from cultured cells or human mucosae as described in Chapter 25.In spite of the fact that the purification yield of poly A+mucin fraction is very poor, thepurification step of polyA+RNA from total RNA cannot be omitted prior to cloning (care-ful elimination of ribosomal RNA is needed).2. Different technologies are available for the purification of mRNA. We have successfullyused the poly A tract mRNA isolation system from Promega or the mRNA purification kitfrom Pharmacia Biotech.3.2. cDNA Synthesis (see Note 3)Many kits are commercially available. We have used the cDNA synthesis modulefrom Amersham and strictly followed the manufacturer’s instructions.1. Synthesize the first strand cDNA at 42°C for 1 h by the AMV reverse transcriptase.2. Synthesize the second-strand cDNA by the T4 DNA polymerase after hydrolysis of theremaining RNA by the Escherichia coli Ribonuclease H. 300 Aubert and Porchet3. Purify the double-stranded cDNA by phenol/chloroform extraction and ethanol precipitation.4. If a radiolabeled nucleotide has been added during the synthesis of either the first- or thesecond-strand cDNA, the yield of cDNA synthesized can be calculated and the quality ofthe cDNA evaluated by alkaline gel electrophoresis in agarose.3.3. cDNA Cloning (see Notes 4 and 5)We have used the cDNA rapid adaptor ligation module and the cDNA rapid cloningmodule from Amersham and followed the manufacturer’s instructions.1. Ligation of adaptors to cDNA: Each adaptor contains a blunt- end for ligation with cDNAand an EcoRI cohesive end to permit ligation with any EcoRI-digested vector.2. Purification of “adapted” cDNA: Use the spun columns of the kit.3. Phosphorylation of the EcoRI overhangs carrying 5'-hydroxyl groups with T4 polynucle-otide kinase.4. Ligations into λgt11 vector arms: The λgt11 vector arms and the cDNA are ligated togenerate recombinant DNA molecules, which also form linear concatemers by ligation oftheir cos ends.5. In vitro packaging of ligation mixtures: The recombinant DNA molecules are packagedinto infectious phage particles. The necessary cell extracts are supplied with Amershamλ-DNA in vitro packaging module.6. Phage plating cells: Use Y1090 E. coli bacteria.7. Titration of λgt11 recombinants: As predicted by the manufacturer’s instructions, wehave regularly obtained approx 106–107 recombinants per microgram of cDNA.8. Quality of λgt11 cDNA library immunoscreening: The quality of immunoscreeningdepends on the specificity of the antibodies (monoclonal or polyclonal).a. Plate out the λgt11 library at the required plaque density. The best result is obtainedwhen each individual plaque lysis is still well separated from the surrounding plaques.b. Induce the expression by overlaying plates with IPTG-impregnated filters. Incubateat 37°C overnight.c. Mark the filters and then block the nonspecific binding by incubation of the filters in20% fetal calf serum (FCS) for 1h at room temperature.d. Bind protein-specific primary antibodies diluted in TBS + 20% FCS for at least 12 hat 4°C.e. Wash the filters three times with TBS and then TBST.f. Bind peroxidase-labeled second antibody in TBS + 20% FCS for 1 h at room temperature.g. Wash the filters as described in step e.h. Incubate filters with DAB substrate + H2O2 diluted in TBS.i. Align filter and original plate. Identify and pick positive plaque(s). Rescreen to checkand isolate clone(s).9. Characterize the selected clones by purification of phage DNA either by polymerase chainreaction (PCR) using forward and reverse λgt11 primers or by small-scale liquid cultureand phage DNA purification.10. The cDNA inserts can now be subcloned into plasmids or M13 phagemids for sequencing.3.4. 5'/3' RACE PCRSince most human mucin mRNAs are usually very large (up to 24 kb), it is notjudicious to try to obtain the full-length cDNA by screening libraries (see Note 6 and7). We have successfully used the 5'/3' RACE kit from Boehringer Mannheim by scru-pulously following the manufacturer’s instruction. Mucin cDNA Cloning 3013.4.1. 5' RACE PCR (Fig. 1)1. Synthesize first-strand cDNA from poly (A+) mRNA using a cDNA-specific primer (des-ignated from the first cDNA sequences obtained) and avian myeloblastosis virus (AMV)reverse transcriptase.2. Used terminal transferase to add a homopolymeric A-tail to the 3'-end of the first strand.3. Then amplify tailed cDNA by PCR using an oligo dT-anchor primer and a second spe-cific primer of the cDNA of interest (nested primer).4. Clone the PCR product in a vector such as pCR2.1 (Invitrogen) and sequence.3.4.2. 3' RACE PCR (Fig. 2)1. Initiate first-strand cDNA synthesis at the natural poly(A+) tail of mRNA using an oligodT-anchor primer.2. Then directly perform amplification using the PCR anchor primer and a specific primerdesignated from the first cDNA sequence obtained.4. Notes1. L-amp plates may be stored inverted in plastic bags at 4°C.2. Sterile L-top agar may be stored solid and melted in a microwave oven before use.3. To optimize the yield of cDNA synthesis, the enzymatic steps must be initiated using astemplates freshly purified poly(A+)RNAs.4. Perform at least three separate cDNA-containing ligation reactions to find the optimalcloning efficiency. We use generally 20-, 50-, 100-, and 150-ng linkered cDNA for 1 µgof λgt11 arms.Fig. 1. 302 Aubert and Porchet5. To obtain the best yield of recombinants, a crucial step is the speed with which the extractsare mixed.6. Such cDNA libraries can be screened with oligonucleotide or DNA radiolabeled probes.7. In the case of large human mucin cDNAs, it will probably not be possible to obtain thefull-length 5'-end cDNA in one attempt. From the new sequence determined, the 5'-endRACE procedure can be repeated.AcknowledgmentsThe authors wish to thank Dr. M. Crépin for his collaboration and D. Petitprez forher excellent technical assistance.To the memory of G. Vergnes who contributed to selection of the first recombinantclones of human tracheobronchial apomucins.References1. Davis, R. V., Botstein, D., and Roth, J. R. (1980) Advanced Bacterial Genetics, ColdSpring Harbor Laboratory, Cold Spring Harbor, NY.2. Williams, J. G. (1981) Genetic Engineering, vol. 1, (Williamson, R., ed.), Academic Press,London, pp. 1.3. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY.4. Huynh, T. V., Young, R. A., and Davis, R. W. (1985) DNA Cloning : A PracticalApproach, vol. 1 (Glover, D., ed.), IRL, Oxford, UK, pp. 49.5. Watson, C. J. and Jackson, J. F. (1985) DNA Cloning : A Practical Approach vol. 1(Glover, D., ed.), IRL, Oxford, UK, pp. 79.Fig. 2. Mucin cDNA Cloning 3036. Frohmann, M. (1994) Beyond classic RACE. PCR Methods Appl. 4, 540–558.7. Aubert, J. P., Porchet, N., Crépin, M., Duterque-Coquillaud, M., Vergnes, G., Maz-zuca, M., Debuire, B., Petitprez, D., and Degand, P. (1991) Evidence for different humantracheobronchial mucin peptides deduced from nucleotide cDNA sequences. Am. J. Respir.Cell Mol. Biol. 5, 178–185.8. Crépin, M., Porchet, N., Aubert, J. P., and Degand, P. (1990) Diversity of the peptidemoiety of human airway mucins. Biorheology 27, 471–484.9. Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S.(1989) Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evi-dence for genetic polymorphism. J. Biol. Chem. 264, 6480–6487.10. Toribara, N. W., Robertson, A. M., Ho, S. B., Kuo, W. L., Gum, E., Hicks, J. W., Gum, J.R., Byrd, J. C., Siddiki, B., and Kim, Y. S. (1993) Human gastric mucin. Identification ofa unique sequence by expression cloning. J. Biol. Chem. 268, 5879–5885. . 23.8 mg isopropyl β-D-thiogalacto-pyranoside/mL in water. Filter sterilise. 2% X-gal: Dissolve 2% (w/v) 5-bromo-4-chloro-3-indoly-β-D-galactopyranoside. the β-galactosidase gene-coding region, allowing blue/white color screen-ing of clones with inserts in the presence of X-Gal and isopropyl-1-thiol-β-D-galactopyranoside