- Email: [email protected]
Vaccines 88: New Chemical and Genetic Approaches to Vaccination (Prevention of AIDS and other viral, bacterial and parasitic diseases)
47 (fractions 1-4) and minor peak (fractions 4-6) consist primarily of rRNA and lesser amounts of non-adenylated mRNA.12 The second major and minor peaks (fractions 13-23 and 23-26) represent the reapplication of the first peaks. Since a larger volume is applied the second time, the peak spans more fractions. However, if peak heights are considered, then the areas of the two major peaks are similar. The next elution buffer (SEB) washes the SDS off the column, which results in a slight shift in the baseline. Following addition of a low ionic strength buffer (TEB) a small peak of polyadenylated m R N A (fractions 41-44) is released from the column. The pooled m R N A is precipitated with ethanol. Since ethanol inhibits translation, 13 the last traces of ethanol must be removed by evaporating under reduced pressure. The precipitate is then redissolved in a small volume of water. An aliquot of the mRNA is added to a commercial in vitro translation kit containing 3Hleucine.13 The translation mixture is incubated for 1 h at 30°C. At higher temperatures a kinase, which inhibits the initiation of translation, is slowly activated. 6 After termination of the reaction, the mixture is bleached with H202 tO reduce color quenching in the scintillation counter. 14 The mixture is then treated with T C A which not only precipitates the newlysynthesized protein, but also hydrolyzes the bond between an amino acid and its tRNA. 6 This helps decrease the contribution of charged tRNA to background precipitable counts. Table 1 shows the results of the translation obtained by five students each using m R N A they had previously purified. The increase in precipitable cpm over the control ranges from about 5-fold to over 10-fold. Table 1 Acid Precipitable cpm Student
SI AA TW SH MB
- mRNA
2779 3047 2503 4948 5341
+mRNA
20 33 28 28 25
267 721 127 798 441
Ratio
7.3 11.1 11.2 5.8 4.8
This project is performed without going to the extremes of dedicated chemicals and baked glassware that are commonplace in nucleic acid research labs to prevent contamination by nucleases. 19 Although such precautions undoubtedly increase the yield of mRNA, they are not practical for most undergraduate labs. Only two simple precautions are taken. Students wear latex gloves at all times as a precaution against 'finger nucleases' and prepare their solutions in DEPC treated water (see Methods). With only these precautions, enough mRNA is purified for a significant increase in protein synthesis over that of the control. The project as described requires approximately 30 hours. As time permits, subsequent studies might include the characterization of either the purified mRNAs or the in vitro translation products by gel electrophoresis.12'20-22
Conclusion Students who enroll in graduate programs in biochemistry will undoubtedly enter a lab that makes use of recombinant DNA techniques. While it is not feasible to have undergraduates cloning genes, it is important that they have practical experience in the handling of nucleic acids. The project described above is an attempt to provide that experience in a stimulating manner.
Acknowledgement I would like to express my appreciation to the students who have participated in this project.
BIOCHEMICAL E D U C A T I O N 17(1) 1989
References 1Hileman, B (1987) Chem Eng News 65, (No: 43) 33-60 2Wolf, E C (1988) Biochem Educ 16, 231-234 3Clark, J M and Switzer, R L (1977) 'Experimental Biochemistry', W H Freeman Co 4Stenesh, J (1984) 'Experimental Biochemistry', Allyn and Bacon 5Plummer, D T (1987) 'An Introduction to Practical Biochemistry' (Third Edition), McGraw Hill 6jagus, R (1987) Methods Enzymol 152, 267-276 7Nienhuis, A, Falvey, A and Anderson, W (1974) Methods Enzymol 30, 621-630 8Wallace, D (1987) Methods Enzymol 152, 33-41 9Mechler, B (1987) Methods Enzymol 152,241-248 1°Wallace, D (1987) Methods Enzymol 152, 41-48 aXJacobson, A (1987) Methods Enzymol 152, 254-261 a2Aviv, H and Leder, P (1972) Proc Nat Acad Sci USA 69, 1408-1412 13New England Nuclear Research Products 14pelham, R B and Jackson, R J (1976) Europ J Biochem 67, 247-256 15Tomlinson, J, Pendergast, A M, Hronis, T and Jurnak, F (1984) Biochem Educ 12, 19-21 16Maniatis, T et al (1982) 'Molecular Cloning - - A Laboratory Manual', Cold Spring Harbor Laboratory 17Krystosek, Aet al (1975) J Biol Chem 250, 6077-6084 laDarnbrough, C et al (1973) J Mol Biol 76, 379-403 WBlumberg, D (1987) Methods Enzymol 152, 20-24 2°Ogden, R and Adams, D (1987) Methods Enzyrnol 152, 61-87 /1jagus, R (1987) Methods Enzymol 152, 296-304 22Merrick, W (1983) Methods Enzymol 101,606-615 23Spindler, S, Siebert, P, Coffman, F and Jurnak, F (1984) Biochem Educ 12, 22-25
Book Review
Vaccines 88: New Chemical and Genetic Approaches to Vaccination (Prevention of AIDS and other viral, bacterial and parasitic diseases) Edited by H Ginsberg, F B r o w n , R A L e r n e r and R M C h a n o c k . pp 396. Cold Spring H a r b o r L a b o r a t o r y . 1988. $95. ISBN 0-87969-310-X This text comprises a collection of original research articles, written by scientists from various disciplines, summarising papers presented at the Cold Spring Harbor Meeting, September 1987. The articles are grouped into five sections on Immunology (9 papers), Parasitology (8), Bacteriology (6), Virology (21) and AIDS (20). In general, the articles reflect how technological advances in molecular biology are being utilised for vaccine development. Strategies include (1) the use of existing attenuated vaccines (eg vaccinia) as vectors for introducing replicating antigens into the host, (2) the use of recombinant DNA technology to produce subunit vaccines, and (3) the development of molecular construct vaccines containing both T-cell and B-cell epitopes. Teachers of advanced courses in a variety of biological sciences may find this a useful reference text to update their knowledge of research in vaccine development, especially AIDS. However, the papers will only be appreciated by those with a sound understanding of modern molecular biology and immunology. The emphasis of each contribution is on experimental results and conclusions. Knowledge of the various pathogens and technologies utilised is taken for granted. E Ingham
SI AA TW SH MB
- mRNA
2779 3047 2503 4948 5341
+mRNA
20 33 28 28 25
267 721 127 798 441
Ratio
7.3 11.1 11.2 5.8 4.8
This project is performed without going to the extremes of dedicated chemicals and baked glassware that are commonplace in nucleic acid research labs to prevent contamination by nucleases. 19 Although such precautions undoubtedly increase the yield of mRNA, they are not practical for most undergraduate labs. Only two simple precautions are taken. Students wear latex gloves at all times as a precaution against 'finger nucleases' and prepare their solutions in DEPC treated water (see Methods). With only these precautions, enough mRNA is purified for a significant increase in protein synthesis over that of the control. The project as described requires approximately 30 hours. As time permits, subsequent studies might include the characterization of either the purified mRNAs or the in vitro translation products by gel electrophoresis.12'20-22
Conclusion Students who enroll in graduate programs in biochemistry will undoubtedly enter a lab that makes use of recombinant DNA techniques. While it is not feasible to have undergraduates cloning genes, it is important that they have practical experience in the handling of nucleic acids. The project described above is an attempt to provide that experience in a stimulating manner.
Acknowledgement I would like to express my appreciation to the students who have participated in this project.
BIOCHEMICAL E D U C A T I O N 17(1) 1989
References 1Hileman, B (1987) Chem Eng News 65, (No: 43) 33-60 2Wolf, E C (1988) Biochem Educ 16, 231-234 3Clark, J M and Switzer, R L (1977) 'Experimental Biochemistry', W H Freeman Co 4Stenesh, J (1984) 'Experimental Biochemistry', Allyn and Bacon 5Plummer, D T (1987) 'An Introduction to Practical Biochemistry' (Third Edition), McGraw Hill 6jagus, R (1987) Methods Enzymol 152, 267-276 7Nienhuis, A, Falvey, A and Anderson, W (1974) Methods Enzymol 30, 621-630 8Wallace, D (1987) Methods Enzymol 152, 33-41 9Mechler, B (1987) Methods Enzymol 152,241-248 1°Wallace, D (1987) Methods Enzymol 152, 41-48 aXJacobson, A (1987) Methods Enzymol 152, 254-261 a2Aviv, H and Leder, P (1972) Proc Nat Acad Sci USA 69, 1408-1412 13New England Nuclear Research Products 14pelham, R B and Jackson, R J (1976) Europ J Biochem 67, 247-256 15Tomlinson, J, Pendergast, A M, Hronis, T and Jurnak, F (1984) Biochem Educ 12, 19-21 16Maniatis, T et al (1982) 'Molecular Cloning - - A Laboratory Manual', Cold Spring Harbor Laboratory 17Krystosek, Aet al (1975) J Biol Chem 250, 6077-6084 laDarnbrough, C et al (1973) J Mol Biol 76, 379-403 WBlumberg, D (1987) Methods Enzymol 152, 20-24 2°Ogden, R and Adams, D (1987) Methods Enzyrnol 152, 61-87 /1jagus, R (1987) Methods Enzymol 152, 296-304 22Merrick, W (1983) Methods Enzymol 101,606-615 23Spindler, S, Siebert, P, Coffman, F and Jurnak, F (1984) Biochem Educ 12, 22-25
Book Review
Vaccines 88: New Chemical and Genetic Approaches to Vaccination (Prevention of AIDS and other viral, bacterial and parasitic diseases) Edited by H Ginsberg, F B r o w n , R A L e r n e r and R M C h a n o c k . pp 396. Cold Spring H a r b o r L a b o r a t o r y . 1988. $95. ISBN 0-87969-310-X This text comprises a collection of original research articles, written by scientists from various disciplines, summarising papers presented at the Cold Spring Harbor Meeting, September 1987. The articles are grouped into five sections on Immunology (9 papers), Parasitology (8), Bacteriology (6), Virology (21) and AIDS (20). In general, the articles reflect how technological advances in molecular biology are being utilised for vaccine development. Strategies include (1) the use of existing attenuated vaccines (eg vaccinia) as vectors for introducing replicating antigens into the host, (2) the use of recombinant DNA technology to produce subunit vaccines, and (3) the development of molecular construct vaccines containing both T-cell and B-cell epitopes. Teachers of advanced courses in a variety of biological sciences may find this a useful reference text to update their knowledge of research in vaccine development, especially AIDS. However, the papers will only be appreciated by those with a sound understanding of modern molecular biology and immunology. The emphasis of each contribution is on experimental results and conclusions. Knowledge of the various pathogens and technologies utilised is taken for granted. E Ingham