The use of recombinant DNA technology to study gene alteration

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Mutation Research, 153 (1985) 13-55 Elsevier

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ICPEMC Publication No. 11 T h e u s e of r e c o m b i n a n t D N A t e c h n o l o g y to s t u d y gene alteration Ph. Mekler a, J.T. Delehanty b P.H.M. Lohman c, J.Brouwer d P. v.d. Putte d p. Pearson e, P.H. Pouwels c and C. Ramel e a Preclinical Research, Sandoz S.A., Basel (Switzerland), b Burroughs Wellcome Co., Research Triangle Park, N.C. (U.S.A.), c Medical Biological Laboratory -- TNO, Rijswijk (The Netherlands), d Department of Anthropogenetics, University of Leiden, Leiden (The Netherlands), e Department of Biochemistry, University of Leiden, Leiden (The Netherlands), and f Wallenberg Laboratory, University of Stockholm, Stockholm (Sweden) (Received 1 October 1984) (Accepted 4 October 1984)

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 1. Recombinant D N A techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Cutting and joining D N A molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Cloning vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Introduction of foreign D N A into cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Cloning in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 2. Progress in mutagenesis and carcinogenesis research related to recombinant methodologies . . . . . . . . . . . . . 2.1. Structure of the eukaryotic genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Repeated D N A sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Globin genes: an example of a multigene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Excision and incision of D N A sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Gene mapping and the analysis of genetic disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Restriction fragment length polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Molecular analysis of hemoglobin disorders in man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Transposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Bacterial transposition: elucidation of illegitimate recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Transposons in Drosophila: their occurrence and use as recombinant vectors . . . . . . . . . . . . . . . . . . . . 2.4. Cellular transforming oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Mutagenic events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Chromosomal rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

All contact and reprint requests should be addressed to the Secretary of ICPEMC: Dr. J.D. Jansen, Medical Biological Laboratory - - TNO, P.O. Box 45, 2280 AA Rijswijk, The Netherlands. Tel. (0)15-138777, telex 38034 pmtno nl.

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ICPEMC is affiliated with the International Association of Environmental Mutagen Societies and is sponsored by the Institut de la Vie.

0165-1110/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

14 2.5. A n a l y s i s of gene a l t e r a t i o n s at the m o l e c u l a r level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. The use of c l o n i n g a n d D N A s e q u e n c i n g in m u t a t i o n research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2. Site-directed m u t a g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C h a p t e r 3. Topics of biological interest a n d i m p o r t a n t a p p l i c a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Short-term m u t a g e n i c i t y testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. U n t a r g e t t e d m u t a g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Polygenic m u t a t i o n a n d gene a m p l i f i c a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Differentiation a n d evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. D i f f e r e n t i a t i o n in a n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. D i f f e r e n t i a t i o n in p l a n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. Interspecies gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. M o v e m e n t of o r g a n e l l a r D N A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Reversed genetics as an a n a l y t i c and synthetic tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. A n t i g e n i c d e t e r m i n a n t s a n d the d e v e l o p m e n t of s y n t h e t i c vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements ....................................................................... Glossary .............................................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Preface DNA technology has drastically changed the way biologists investigate genetic problems. Within the last decade, radically new insights have been made in the organization and processing of genetic material. The detection of genetic alteration has markedly improved, and the ability to monitor alterations in the human genome may soon be realized. In this overview, Task Group 8 of ICPEMC sought to describe briefly recombinant methodology, highlight its application in certain areas, and select rather subjectively facets of biology on the verge of important progress. In the course of its analysis, Task Group 8 reached a general consensus about the impact of recombinant D N A technology on mutagenesis research. There are several points in this consensus that are worth stressing. We encourage continued direct analysis of the human genome. Restriction site polymorphism may represent an accurate and noninvasive monitor of human genetic variability and mutational load. The diagnosis of specific genetic diseases, for which gene probes are available, has already been performed in utero. To apply this approach to nonspecific genetic alterations may reduce our dependence on the extrapolation of animal and in vitro genetic data to human health risk. However, technical improvements must be made before screening of large populations becomes practical. We encourage the continued application of re-

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combinant technology to short-term mutagenicity tests. These tests are indispensable but imperfect tools in genetic toxicology. By improving their detection powers with DNA sequencing methods and expanding their extrapolation powers by introducing mammalian components, investigators may create even more powerful diagnostic aids for genotoxicity. We encourage attempts to understand the apparent mobility of genetic material and its relation to mutagenesis and carcinogenesis. This effort has already shown to be highly relevant. Transposons exist in plants and animals capable of disrupting genetic stability. Some animal tumor viruses or proviruses also possess structural features of transposons and may migrate within the genome by transposition-like mechanisms. We encourage the development of data bases and software to assimilate and analyze the rapidly accumulating DNA sequence data. Computer centers in the U.S.A. and Europe already serve as repositories of sequence information and compendiums are routinely published. Introduction

Until recently, the analysis of genetic organization and cellular differentiation largely depended upon ingenuity in devising mutant selection schemes. Without efficient means for probing mutation at the molecular level, geneticists were restricted to exploiting the phenotypic and meta-

15 bolic consequences of mutation. To analyze mutation in detail, fine structure mapping utilized complementation and recombination experiments. In the most successful cases, elucidation of the primary structure of mutated gene products (protein or RNA) allowed an indirect correlation between phenotypic alteration and the underlying change in the DNA. Paradoxically, many facets of gene regulation and genome organization that were discovered initially in prokaryotes were found not to exist in eukaryotes. Major recent advances in eukaryotic genetics stem not from previous means of experimentation, but from new methodologies to which Brown (1981) refers as 'genetics by DNA analysis'. New approaches have already been designed to study human genetic diseases and to provide new tools for antenatal diagnosis and identification of monogenic diseases (Davies, 1981). For example, remarkable insights into oncogenesis have recently been made as a consequence of these new molecular techniques. One culmination has been the discovery and characterization of cellular cancer genes - - oncogenes (Bishop, 1981). The techniques have also been applied to problems of cellular differentiation and gene evolution. The combination of techniques in recombinant DNA and site-directed mutagenesis offers powerful possibilities for specifically altering DNA segments and introducing them into cellular genomes. Motulsky (1983) has discussed the educational and ethical dilemmas raised by these technical advances. In particular, he makes the historical point that efforts to manipulate human and agricultural genetic characteristics are ancient. The new molecular techniques follow this tradition but do so with remarkable speed and power. Recombinant DNA technology is a tool and not an end in itself; it must be evaluated in many settings, and its potential value awaits further exploration in the multidisciplinary interfaces of medicine, public health, genetics and molecular biology, and related fields. To stimulate activity in mutagenesis and carcinogenesis research, ICPEMC formed a task group (Task Group 8) to review the major advances in DNA recombinant techniques and to encourage the application of molecular techniques to mutagenesis and carcinogenesis research. The ultimate intent of this paper is to

promote direct, molecular analysis of genetic material that leads to studies on the propagation of mutation and to new epidemiological approaches to carcinogenesis research.

Chapter 1. Recombinant DNA techniques The term 'recombinant DNA technique' often means different things to different people. We agree with the definition given by the British Genetic Manipulation Advisory Group (GMAG): 'the formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their amplification and then incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation'. Although conceptually simple, the methodology is technically complicated, but divisible into a set of basic components: (a) a method for joining two DNA molecules from different sources, giving rise to a 'hybrid' or 'recombinant' DNA molecule (e.g., vector + insert DNA); (b) a DNA vector that replicates in host cells after foreign DNA has been inserted into this vector; (c) a host cell able to take up, replicate, and maintain 'foreign' recombinant DNA molecules; (d) a means to introduce the hybrid DNA molecule into a suitable host cell (by DNA transformation or transfection); (e) a method to screen, select, and propagate those cells which have replicated the desired DNA molecule. Many excellent references are available that detail the methods of recombinant DNA technology. Among the best are Vol. 65, 68, 100, and 101 of Methods in Enzymology, Vol. 1-5 of Genetic Engineering: Principles and Methods and Molecular Cloning: A Laboratory manual (see References).

1.1. Cutting and joining DNA molecules Recombinant technology depends heavily upon the use of restriction endonucleases to cleave DNA

16 at specific sites. An endonuclease recognizes a particular oligonucleotide sequence in duplex D N A and cleaves within or near that sequence. At present more than 350 different restriction enzymes (representing at least 85 different recognition specificities) have been described. M a n y produce D N A fragments with protruding single-stranded 5'- or 3'-termini with specific nucleotide sequence. Because some single-stranded regions can form hydrogen bonds with complementary overlapping oligonucleotide sequences, they are referred to as 'cohesive' or 'sticky' ends. Other restriction endonucleases create D N A with no cohesive ends; such D N A fragments are called 'flush-' or 'blunt-ended'.

It is important to realize that the biologic origin of D N A is irrelevant when D N A is digested with restriction enzymes. If the D N A contains the proper consensus site, a terminus may be produced that is chemically complementary to D N A from other sources (e.g., see Fig. 1). There are several methods for covalently joining D N A fragments in vitro. One depends on the ability of D N A ligases to catalyze phosphodiester bond formation between D N A strands after the cohesive ends have base-paired with complementary single-stranded oligonucleotide sequences. Another method depends on the ability of D N A ligase from bacteriophage T4-infected E. coil to

DNA fragments

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m e c h a n i ca i shearing

d u p l e x cDNA synthes I s

direct chemical synthes I s

join

homopolymer tailing

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blunt-end llgatlon (no linker)

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transfectlon with recombinant phage/eukaryotlc virus DNA

\/ select ion

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transformation with recombinant p l a s m i d DNA

in vitro packaging Into phage coat: transduction with recombinant phage or cosmld

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Fig. 1. General cloning strategies. Boxesat left refer to desired end points, At right are various techniques to achieve the endpoint. The selection of a particular technique depends on many considerations, as discussed in the text. Arrows denote the possible interactions that may occur when techniques are used sequentially as end points evolve.

17 catalyze the formation of phosphodiester bonds between blunt-ended fragments, i.e., without prior oligonucleotide base-pairing. Cohesive ends can be generated in blunt-ended DNA by using enzymatically made, terminal homopolymer nucleotide sequences or by adding specific oligonucleotide 'linkers'. These artificial cohesive ends may then be annealed with complementary single-stranded oligonucleotide sequences of other fragments and sealed with a DNA ligase. 1.2. Cloning vectors

The ability to join specifically DNA from diverse sources in vitro is one prerequisite for recombinant DNA technology. Another important condition is the ability to introduce and maintain this exogenous DNA within the cells. Although many attempts had been made to propagate foreign DNA in prokaryotic and eukaryotic cells, experiments had been relatively unsuccessful. Even though exogenous DNA may be taken up, it will not be retained unless it replicates in the host cell. To replicate, foreign DNA may integrate into the host cell genome, and this occurs only very rarely. Alternatively, exogenous DNA may contain sequences that permit autonomous replication (i.e., origins of replication). Replication is mediated in two ways: one way involves specific, self-replicating vectors, i.e., plasmids (such pBR322), and the second entails integration vectors that insert into the host genome and then replicate under the control of the host cell genome (see Fig. 1). Because replication is essential for maintaining foreign DNA within a host cell, plasmids are key tools. To assemble the recombinant molecule, the exogenous DNA must be joined to a plasmid containing an origin for replication, and the hybrid plasmid must retain the ability to replicate autonomously in host cells. DNAs with these properties (autonomous replication and the ability to carry exogenous DNA) are called cloning vehicles or vectors. Vectors used in recombinant DNA technology are derived from bacterial plasmids, bacteriophages, and animal viruses. Vectors usually either have a single restriction site at which foreign DNA can be inserted (i.e., 'insertional vectors') or a pair of restriction sites defining a fragment that can be removed and replaced by

foreign DNA (i.e., 'replacement vectors'). An ideal cloning vector should have the following properties: (a) it is stably inherited either extrachromosomally without sequence homology to the host cell genome, or intrachromosomally in the host genome; (b) it is genetically stable and capable of conferring selectable phenotypic traits on host cells; (c) it is of low molecular weight for easy handling, can be amplified readily, and, thus, may be synthesized in many exact copies; (d) it contains DNA restriction sites for one or more endonucleases, permitting insertion of foreign DNA at these sites, preferably within genes of readily scorable phenotype (e.g., antibiotic resistance). Many prokaryotes and lower eukaryotes possess plasmids that code for antibiotic resistance or enterotoxin production and are stably inherited extrachromosomally. Because many natural plasmids are not efficient cloning vectors, many cloning vehicles are artificial derivatives of natural plasmids. The most widely used are the ampicillin/tetracycline resistance E. coli plasmid pBR322 and its derivatives (Bolivar et al., 1977a, b; Bolivar, 1978). Other plasmids have also been developed in Bacillus (Bingham et al., 1980), non-enteric Gram-negative bacteria (Bagdasarian et al., 1979), and yeast (Beggs, 1978; Struhl et al., 1979). Bacteriophages and viruses are natural parasites of prokaryotic or eukaryotic ceils, respectively. For cloning purposes, they must not inactivate and/or lyse the host cell unless under controlled conditions and should be taken up easily by the cell. The best bacteriophage vectors are phage X and its derivative DNAs (Blattner et al., 1977; Leder et al., 1977; Davison et al., 1979) and the singlestranded phage M13 (Messing et al., 1977, 1981). In contrast to plasmids, bacteriophage-derived hybrid DNAs may be packaged in a phage coat and participate in subsequent phage infection, which results in about a 100-fold higher efficiency of transfection as compared to DNA transformation. Cosmids are phage-derived vectors that contain the 'cos' site (i.e. natural cohesive ends) of A DNA (Collins and Brianing, 1978; Collins and Hohn, 1979). Because of this cos site and of the in vitro packaging system, cosmids are very efficient clon-

18 ing vectors. Packaged, recombinant, cosmid DNA can be injected and circularized like phage DNA. It replicates as a normal plasmid without expressing any phage functions. Animal viruses have the potential for serving as cloning vectors for eukaryotic systems; SV40 and polyoma DNAs are among the most popular viral vectors (Mulligan et al:, 1979; Elder et al., 1981). Combining phage, plasmid, or viral DNAs allows the construction of so-called 'shuttle vectors', which can be replicated (and possibly expressed) in either prokaryotic or eukaryotic cells. In terms of insert size, plasmid vectors can accommodate up to 10 kb, phage or virus vectors up to 20 kb, and cosmids up to 45 kb of DNA. At the moment, cosmids provide the only efficient means of cloning very large pieces of foreign DNA. They are therefore particularly attractive for constructing libraries of eukaryotic genomes. 1.3. Introduction of foreign D N A into cells

A recombinant DNA molecule can be introduced into a host cell by transformation, when the vector is plasmid DNA, or transfection, when the vector is phage or virus DNA. Early attempts to achieve DNA uptake into E. coli were unsuccessful. However, eventually it was found that E. coli cells took up naked DNA from bacteriophages (Mandel and Higa, 1970) or from plasmids (Cohen et al., 1972) when it was first treated with CaC12. CaC12 causes the formation of a calcium-DNA precipitate that is required for efficient uptake by the host cell. The efficiency of transformation is not very high: up to 10 6 transformants//~g of vector D N A , representing only one DNA molecule/103 molecules added. When high transformation efficiency (108 transformants//~g of DNA) is required, in vitro packaging of either phage or cosmid DNA may be favored. Because transformation is not very efficient, only a minute proportion of the total DNA introduced into the host cells may be target D N A . Powerful selection techniques exist to identify a desired recombinant from a population of transformed or transfected bacteria. Three main selection strategies are available: genetic selection, immunochemical selection, and selection by nucleic acid hybridization. When combined with microbiological techniques, genetic

selection is a very powerful tool because it can be

applied to large populations of cells. All useful vectors carry at least one selectable genetic marker. Selection may be for drug resistance, nutritional markers, or plaque formation (in the case of biologically active phage vectors). If a foreign gene is actually expressed, then selection for the expressed insertion sequence may be the simplest method for isolating the clone (e.g., expression of the yeast 'his' gene in E. coli hisB mutants, Ratzkin and Carbon, 1977). Conversely, the insertion of a DNA sequence may also be detected by inactivation of a genetic marker in the vector. Clones have been successfully identified by the immunochemical detection of new protein coded for by the hybrid DNA. An advantage of this method is that genes can be detected and selected that are actually expressed after introduction into the host cell, but which do not confer a selectable phenotype. Thus an antibody must be prepared against the desired gene product. An in situ detection method using lysed bacterial colonies and a solidphase sandwich-type immunoassay has been applied widely (Broome and Gilbert, 1978; Reiser and Wardale, 1980; Kaplan et al., 1981). Immunochemical methods may also detect polypeptides with slightly differing amino acid sequences if the altered antigen does not prevent interaction with the antibody. This property may be particularly interesting in cases of fused gene products or of novel genetic constructions (VillaKomaroff et al., 1978). Another powerful screening procedure detects DNA sequences by hybridization in situ with specific radioactive 'probe' RNA or DNA (Grunstein and Hogness, 1975). This procedure can determine rapidly which colonies in large bacterial populations contain the desired DNA sequence. The great advantage of the hybridization method is its generality. It does not require expression of inserted DNA sequences and can be applied to any oligonucleotide sequence if a suitable radiolabeled nucleic acid probe is available. Recombinant DNA technology has solved two long-standing problems involving use of hybridization methods. It provides pure probes, making possible extremely sensitive and specific reactions, and it permits the production of large amounts of homogenous DNA fragments.

19 Once a recombinant molecule carrying a desired DNA sequence has been isolated, further characterization is often required. In the simplest case, this may extend only to confirming that the required DNA has been cloned. More often, however, nucleotide sequencing may be required. The size and partial restriction map of a newly isolated recombinant plasmid can be determined electrophoreticaUy (Barnes, 1977; Telford et al., 1977). To determine if a cloned DNA encodes a particular protein(s), the hybrid-arrest translation method (Paterson et al., 1977) is very useful. It is based upon the fact that a m R N A cannot be translated in a cell-free system if the m R N A has been hybridized to complementary DNA. Another important method, R-loop mapping (Thomas et al., 1976; White and Hogness, 1977), makes use of the fact that RNA can hybridize to double-stranded DNA by displacing the corresponding DNA strand. For example, the topographies of R loops observed in the electron microscope show that many eukaryotic genes consist of noncontiguous coding regions split by one or more noncoding intervening sequences. It should also be noted that R loops, representing regions of single-stranded DNA, are important targets in site-directed mutagenesis.

ses constraints on the length and the expression of inserted DNA sequences. Hybrid DNA is often non-replicating, but can be introduced into a variety of eukaryotic cells together with a helper virus. The helper virus supplies vital functions to the SV40 hybrid and ensures the replication and the maintenance of the SV40-hybrid DNA. To overcome constraints encountered when using replication-defective viruses, Wigler and coworkers (1979a, b) developed an alternative method, co-transformation. In this procedure, two physically unlinked genes (e.g., a thymidine kinase gene and the gene for rabbit fl-globin) are introduced into the host cell with pBR322 or ~X174 bacteriophage DNA. One gene serves as the basis of selection; the other for successful transformation. Successful transformants (obtained by selection for the genetically scorable component) are assayed for the other (genetically non-scorable) component. Theoretically, the co-transfornaation system permits the introduction and stable integration of virtually any defined gene into a variety of mammalian cultured cells without using viral vectors.

1.4. Cloning in mammalian cells

2.1. Structure of the eukaryotic genome

From the preceding sections it should be apparent that there are many plasmid and phage vectors for cloning in prokaryotic and lower eukaryotic cells. This is somewhat in contrast to the situation with mammalian cells, where the principal vectors are derived mostly from papova and papilloma viruses. SV40 vectors in particular have been used to introduce bacterial and eukaryotic genes into monkey cells in culture (Mulligan et al., 1979). SV40 is an attractive cloning vector for several reasons: (a) the genome consists of a single, small DNA of known nucleotide sequence; (b) the viral genome can multiply either autonomously or as part of chromosomal DNA; (c) the replication and expression of the SV40 genome are well-studied. The use of SV40 DNA and its derivatives impo-

The eukaryotic genome is a mixture of singlecopy and repeated DNA sequences (Britten and Davidson, 1969). Within the limitations of defining sequence repetition by DNA hybridization techniques, it appears that most DNA-coding sequences are single copy. Estimates of the total number of genes present in man vary between 3 x 10 4 and 1 x 105 and are based on the following considerations: - - the 23 chromosomes of the human haploid set contain 3 x 109 bp. If one assumes that the average coding sequence is about 1 x 103 nucleotides (giving a polypeptide length of 333 amino acids), then 5 x 107 nucleotides are involved in gene transcription. This represents only 2% of the total genomic DNA; - - the total number of RNA species detected in the human tissue culture cell line HeLa is approximately 4 x 104 (Bishop, 1974);

Chapter 2. Progress in mutagenesis and carcinogenesis research related to recombinant methodologies

20 the human genome contains 10 times as much DNA as Drosophila, for which firm estimates of 5 x 103 genes have been made (Judd et al., 1972); the predicted tolerable dominant-lethal mutation load in man corresponds to an upper limit of about 5 x 104 structural genes (Ohta and Kimura, 1971); - - the number of genes in the mouse, whose genome is the same size as man's, has been estimated to be not less than 3 x 104, based on total meiotic recombination distance and calculation of the number of genes within recombinationally well-defined genetic regions (Ruddle, 1981; McKusick and Ruddle, 1977). About 60% of the human genome consists of unique-sequence and 40% of repetitive-sequence DNA, and it is accordingly clear that the large portion of single-copy DNA is not involved in gene transcription. However, certain genes are repeated sequences of structurally and functionally related gene clusters known as multigene families. Several have now been analyzed in detail, including mammalian globin, immunoglobulin, histone, actin, and ribosomal RNA. Some aspects of these will be mentioned later. -

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-

2.1.1. Repeated DNA sequences In general, repeated sequences can be divided into those with low-repetition (2-2 x 102 copies), mid-repetition (2 x 102-1 x 104 copies) and highrepetition frequencies (> 1 x 10 4 copies). Lower eukaryotic species such as yeast usually have few repeats and differ in their sequence organization from most higher eukaryotes. The most detailed analyses of genome structure in higher eukaryotes have been carried out in Drosophila, sea urchin, and Xenopus (for review see Britten, 1981). Detailed measurements of Xenopus and sea urchin DNA have shown that most single-copy DNA occurs in segments averaging 1 kb, interspersed with repeats of a few hundred nucleotides in length (Britten, 1976). Although this has come to be known as the Xenopus pattern of interspersion, analysis of many other species indicates that the pattern is wide-spread (Goldberg et al., 1973). Jacob and colleagues (Brulet et al., 1983) have discovered an interesting family of moderately repetitive sequences in the mouse genome. This

6-kb-long repetitive sequence possesses transposon-like characteristics, and its RNA transcripts are found in undifferentiated ceils but not in differentiated cells. Quantitative variations in mid-repetitive sequences, presumably unrelated to heterochromatic regions, have been observed in colon tumors (Humphries, 1981) and may eventually be the basis for a sensitive method for assaying tumorigenic changes. The reasons for these variations are speculative, but unequal mitotic crossing-over and aneuploidy are the most likely mechanisms. The latter possibility has gained credibility with the evidence that some repeat sequences are chromosome-specific and vary quantitatively when individual chromosomes are lost or gained following transformation (Willard et al., 1983). An example of an ubiquitous, highly repeated sequence in man is the Alu family. The name derives from the fact that the restriction endonuclease Alu 1 cleaves human DNA into many genetically homogeneous, 300-bp-long fragments. Estimates of the number of Alu copies in man vary between 3 X 105 and 5 x 105, representing 3-6% of the human genome (Jelnick and Schmid, 1982). DNA changes involving both Alu repeats and sequences flanked by Alu have been observed in leukemia cells, in lymphocytes of aged patients, and in cell lines maintained in vitro for long periods of time (Shmookter Reis et al., 1983; Robberson et al., 1983). The main change is an increase in the number of copies and appears to involve excision of Alu sequences and associated regions to form small circles of DNA that persist extrachromosomally within the nucleoplasm. The function of repetitive DNA is unclear. Although RNA transcripts have been detected for several species of repetitive sequence, such as the Alu family, there is no known protein product (Schmid and Jelinek, 1982). Because many repetitive DNA sequences demonstrate a remarkable genetic divergence between organisms, they give little or no cross-hybridization when used for probing the genome of other organisms. Gusella et al. (1982) have made use of this fact to analyze small fragments of a single human chromosome in somatic-cell hybrids of Chinese hamster and human cells. They noted that the human repetitive sequence used in their experiments was distributed

21 along the entire length of chromosome 11 in a characteristic fashion that enabled the detection of small deletions. This work indicates the possibility of rapidly characterizing individual human chromosomes by identifying and analyzing small chromosome fragments retained in hybrid cells. Furthermore, the use of hybrid ceils to characterize repetitive DNA permits the correlation of molecular data, measured in tens of kb, with chromosomal data corresponding to thousands of kb. This is important because the smallest chromosomal rearrangement that can be identified visually on human metaphase chromosomes is approximately 5000 kb (Ruddle, 1981). Satellite DNA is a particular type of short repetitive DNA that separates from main-band DNA after density gradient centrifugation usually because of an increased GC (heavier) or AT (lighter) content. First analyzed by Corneo et al. (1973), human satellite DNAs occur on the heterochromatic regions of chromosomes, particularly the paracentric regions of chromosomes 1, 9, 16, and the long arm of the Y. These chromosome regions contain large polymorphic variations in human populations and in human cells in culture. The presumptive mechanism for these changes is unequal crossing-over in somatic tissues, which must be an extremely common occurrence to account for the large variations (Hoehn and Martin, 1973). Mitotic labeling of half-chromatids with 5-bromodeoxyuridine showed that about 10 sister-chromatid exchanges occur in each normal human cell per cell generation (Latt, 1974), thus providing ample opportunity for unequal crossing-over and accounting adequately for induction of quantitative changes in repetitive sequences. The evidence that satellite and other highly repetitive sequences can evolve extremely quickly, both quantitatively and qualitatively, is abundant. For example, the major satellite fraction present in the African green monkey is absent or qualitatively very different in closely related species (Manuelides, 1978). Similarly, of the 4 satellite D N A fractions present in man, satellite II is absent in the chimpanzee, satellite IV is absent in the orangutan, and satellites I and III are present in all great apes (Jones et al., 1972; Gosden et al., 1977). Variants of particular repetitive DNA families

have now been recognized, and the major human satellites can be split up into different subfamilies, many of which are chromosome-specific. Presumably each type has evolved independently within each chromosome after its establishment and subsequent dispersion throughout the genome (Beauchamp et al., 1979; Bostock, 1980). Willard et al. (1983) identified a 2.0 kb Bam H1 sequence localized at the centromere region of the X chromosome and repeated about 5000 times. This particular repeat sequence accounts for 5-10% of X chromosome DNA and constitutes the major sequence component of the pericentric region of the human X. 2.1.2. Globin genes: an example of a multigene family

The mammalian globin genes are a small multigene family in which the genes are structurally related and linked. Unlike immunoglobulin genes they do not rearrange to be expressed, but evidence of their evolution by recombinational mechanisms has profound implications for those interested in mutational events. The fl-like globin genes occur fairly close together in a single stretch of DNA, 60 kb in length. This greatly exceeds the total length of the primary transcripts, which are estimated to be considerably less than 10 kb (Orgel and Crick, 1980). Complete nucleotide sequences of the 5 human globin genes have been determined (Lawn et al., 1980; Spritz et al., 1980; Baralle et al., 1980; Slightom et al., 1980). Recent reviews (Efstratiadis et al., 1980; Proudfoot et al., 1980; Leder et al., 1980) present this information and discuss the evolutionary complications. The genes are arranged linearly in the order of their expression, and individual genes are expressed at different developmental stages. A common feature of all globin gene dusters is the occurrence of duplicate, but not identical, genes that are adjacent to each other and are coordinately expressed. The human G#-Gs, Gv-A v, and ed-a2 genes are examples. The coding regions within these gene pairs are highly conserved, whereas noncoding regions tend to diverge. This has been taken as evidence for selective mutational pressure, and models for inter- and intrachromosomal gene conversion have been proposed. Maintenance of homology within a family of

22

evolving genes in a species has been termed 'concerted evolution' (Zimmer et al., 1980). The loci for the globin chains of mammalian hemoglobin usually occur in duplicated, nonallelic pairs, differing in their relative expression at different stages of development. As Slightom et al. (1980) point out, the similarity between the products of these duplicate d globin genes varies, but in many cases the products of a pair of duplicated genes within a species are more similar to each other than either to the comparable pair of globins in other species. Great similarity between members of a duplicated pair within a species usually is taken to indicate a short evolutionary time since the last duplication. A lack of similarity between duplicated pairs in two species is interpreted to mean that the last duplication occurred after the species had diverged. However, knowledge of the sequences of these gene pairs and their flanking regions indicates that such simple interpretations cannot suffice. Apparently, selective pressures are exerted unequally on different regions. Leder et al. (1980) have termed this phenomenon 'evolution at two speeds'. The same situation has been observed in the mouse globin system. The problem is to understand how duplicated genes and regulatory flanking regions continue to share many features between species, while the more peripheral flanking regions show little evidence of a common origin. Leder and colleagues (1980) have cloned and sequenced the two mouse fl-globin genes. The minimal amino acid differences between the genes (9/146) suggest that they arose by duplication. Both genes are embedded in nonhomologous segments of DNA, but have conserved not only their coding regions but a few hundred bases bordering the structural genes and the junction regions of the intervening sequences. The region of nonhomology, like that of the human fetal T-globin genes, corresponds to a large segment of the second intervening sequence. It appears that the conserved regions differ from each other mainly by point mutations and have changed very slowly - - one base at a time. Deletions or insertions that would affect protein structure or cause out-of-phase missense do not appear or have not been propagated. However, the second intervening sequences have diverged

greatly, propelled by many small- and large-scale mutations. Because the noncoding sequences in the fl-globin regions are highly divergent, they may act as barriers to intergenic recombination. As the lengths and perhaps numbers of such sequences increase, their effectiveness as barriers rises. Leder et al. (1980) proposed that one role of intervening and flanking sequences would be as modulators of intergenic recombination. For the number of gene copies to vary but remain homologous, it is thought that there may be mechanisms promoting 'concerted evolution' - - a tendency of a family of repeated genes to evolve in unison. Similar arguments have been proposed by Hood et al. (1975) and by Baltimore (1981) for the immunoglobulin genes. Differences between the a-globin amino acid sequences of several primates are consistent with sequence drift. However, intraspecies comparisons show much less divergence, indicating that aglobins within a species have been corrected against one another, i.e., a-polypeptides have been evolving in concert. Zimmer et al. (1980) conclude that lengths of noncoding regions in the two a-globin genes are determinants of the rates at which genes are gained or lost by intragenic recombination. This observation, in conjunction with the evidence for a-globin gene duplication in most vertebrates, indicates that the duplication is ancient and favors the existence of a process that leads to sequence matching between a-globin genes within a species. Among the many surprises to come from cloning and sequence analysis of eukaryotic gene clusters is the discovery of pseudogenes. These were first identified in the 5S gene cluster of Xenopus (Jacq et al., 1977). A pseudogene displays significant homology (75-80%) to a functional gene but contains mutations which prevent expression. In every mammalian globin cluster thus far characterized, a pseudogene is found between the embryonic (or fetal) genes and the adult genes. For those interested in mutagenesis, pseudogenes offer an advantageous setting in which to observe DNA alterations. Each mammalian a- and fl-globin pseudogene contains a variety of point mutations and small deletions or insertions that alter the translational reading frame. Of equal importance are alterations in exon-intron junctions, which disrupt common splicing signals for

23

RNA maturation. In the B A L B / c mouse, pseudogene a4 contains point mutations that substitute Tyr for His at position 58. Were it not for a second mutation that introduces a premature termination codon, a4 would direct the synthesis of a well-characterized pathologic hemoglobin (Leder et al., 1980). Leder points out that pseudogenes have revealed an unexpected range of mutational mechanisms which operate on apparently unselected sequences of DNA. The most striking example is the structure of the mouse pseudogene ct3. In contrast to every known globin gene, this pseudogene lacks intervening sequences. It is of particular importance that intervening sequences are missing at the 3 ' - G T . . . AG-5' termini, the characteristic boundaries of intervening sequences thought to represent the RNA splicing signals. Multiple deletions of such precise regions of DNA cannot be coincidental. A possible mechanism for the absence of introns in pseudogenes is given in the next section. 2.1.3. Excision and incision of DNA sequences Numerous hypotheses have been proposed for the evolution of the intron-exon architecture of the eukaryotic gene (Lewin, 1983c). Two models account for the role of introns in the evolution of eukaryotic proteins. One (Craik et al., 1982, 1983) requires the fusion of exon-encoded protein domains by intron-mediated recombination, as in the formation of immunoglobulins. Thus, the origin of the gene would depend on exon shuffling. In another model (Go, 1983) introns would disrupt a gene or exon. By being located on the protein surface, intron-exon junctions may tend to produce positive or neutral changes in protein function. More recently, several groups have been examining the excision of DNA sequences that are moderately to highly repeated in the genome (Lewin, 1983a, b, c). Evidence suggests that Alu RNA, transcribed by RNA polymerase III, is reverse-transcribed to Alu DNA and inserted back into the genome by a transposition event. Current thinking dictates the participation of reverse transcriptase activity and accounts for the elimination of intervening sequences in some pseudogenes by reverse transcription of messenger RNA that had

undergone splicing (Sharp, 1983). Most Alu DNA show tandem direct repeats of DNA, thus signifying a transposable element. Also, because Alu contains an internal pol III promoter, inserted Alu copies retain the ability to be transcribed (Jogadeeswaran et al., 1981; Sharp, 1983). With reference to mutagenesis mechanisms, it is noteworthy that Alu-related sequences have been discovered as major intron sequences. This implies that insertion of an Alu sequence can create an intron. Weiner and colleagues (Bernstein et al., 1983) have investigated the ability of human small nuclear RNA (snRNA)U3 to create pseudogenes by reverse transcription and transposition. The ratio of snRNA pseudogenes to genes is approximately 10:1 (a multigenic family). The pseudogenes are truncated, possibly as a result of an internal reverse transcriptase initiation site starting at nucleotide 74 in the U3 gene or possibly because of their RNA secondary structure. Because snRNA genes are transcribed by RNA polymerase and thus form up-stream promoter sequences, no promoter sequences are present in the U3 RNA. The resultant reverse-transcribed gene would also lack a promoter and would be, by definition, a pseudogene. Analysis of snRNA pseudogene and Alu flanking regions indicates that a transposition insertion may not always occur. Bernstein et al. (1983) have speculated that creation and sequence maintenance of multigene families could occur by gene conversion. If a nuclear RNA can be transcribed to a complementary DNA (eDNA), it might undergo homologous pairing and subsequent conversion of the chromosomal copy. Because the eDNA resembles the processed RNA progenitor, it contains no introns. If a homologous chromosomal segment were converted, it could lose its introns. One conversion in such a multigene system could explain how a large number (200-1000) of immunoglobulin variable genes (v genes) is maintained. Because maintenance of sequence identity is critical to v gene function, gene conversion could permit the selection of a few indispensable v regions to be distributed throughout the genome. Most importantly, gene conversion could combine parts of two homologous genes to create a new one.

24 TABLE 1

TABLE 1 (continued)

ASSIGNED DNA HYBRIDIZATION PROBES FOR HUMAN STRUCTURAL LOCI Chrom. 1

Amylase-1 Anti-thrombin 3 C-reactive protein Muscle a-actin Nerve growth factor Renin U1 small nuclear RNA Chicken viral oncogene homolog

AMY-1 AT3 CRP ACTA NGF REN RNUI

Propiomelanocortin Immunoglobulin r Glucagon Propiocortin

POC IGK GCG POC

Chrom. 3

Somatostatin A a-Fibrinogen

SST FGA

Chrom. 4

Albumin Alcohol dehydrogenase class I a-Feto-protein B fl-Fibrinogen

ALB ADH AFP FGB

Chrom. 5

Dihydrofolate reductase

DHFR

Chrom. 6

Prolactin HLA-A HLA-B HLA-DR Cellular homolog to meyeloblastosis Chorionic gonadotropin, a subunit Methionine transfer RNA Kirsten oncogene-1

PRL HLA-A HLA-B HLA-DR

Collagen type 1, a 2 Collagen type 1, a 1 Histones Carboxypeptidase Trypsin-1

COL1A2 COL3A1 H1, H2A, H2B CPA TRP1

Moloney sarcoma virus Myelocytomatosis virus Carbonic anhydrase II Thyroglobulin

C-MOS C-MYC CA2 TG

Interferon - - fibroblast Interferon - - leucocyte Arginosuccinate synthetase Abelson MulV homology

IFN-beta IFN-alpha ASS ABL

Parathyroid hormone Murine sarcoma Harvey virus Insulin

PTH

Chrom. 2

Chrom. 7

Chrom. 8

Chrom. 9

Chrom. 11

fl-Globin complex Hormone receptor Apolipoproteins A-I Apolipoprotein C-III Lactate dehydrogenase

NAG M4F2 ApoA1

Chrom. 12

Kirsten rat sarcoma Phenylalanine hydroxylase a I (I)-like collagen Elastase-1 Immune interferon Lactate dehydrogenase

KRAS2 PAH COLL ELA1 INF-gamma LDHB

Chrom. 13

Ribosomal genes

RNR

Chrom. 14

a 1-Antitrypsin Immunoglobulin heavy chains Ribosomal cistrons Purine-nucleoside phosphorylase

AAT

Feline sarcoma virus Ribosomal genes ~2-micro-globulin Cardiac actin

FES RNR

SK

MYB

Chrom. 15

Chrom. 16

Chrom. 17

CGA TRM2 KRAS1

HRAS1 INS

Chrom. 19

a-Globin complex Adenine phosphoribosyl transferase Chymotrypsinogen B Haptoglobin Growth hormone Placental lactogen Collagen type I a I Collagen type 4 a 4 Chorionic somatomammotropin Myosin heavy chain skeletal Thymidine kinase

LDAa

IGH RNR NP

ACTC HBA APRT CTRB HP GH PL COL1A1 COL4A4 CSH MYHS TK

Complement component 3 Luteinizing hormone-fl Apolipoprotein Chorionic gonadotropin a Chorionic gonadotropin, 13 × subunit

C3 LHB APOE CGA

Chrom. 20

Avian sarcoma virus Adenosine deaminase

SRC ADA

Chrom. 21

Ribosomal cistrons Superoxide dismutase-1

RNR SOD

Chrom. 22

Simian sarcoma virus Immunoglobulin 21

SIS IGL

LHB

25 TABLE 1 (continued)

X Chrom.

Glucose-6-phosphate dehydrogenase Hypoxanthine-guanine phosphoribosyl transferase Phosphoglycerate kinase Factor 9 Actin Harvey sarcoma virus Mitochondrial cytochrome b

G6PD

HPRT PGK CF9 ACT HRAS2

Conversion as a mutagenesis mechanism has also been proposed in the formation of products of the major histocompatibility complex (MHC) (Bodmer, 1981). In this regard, Pease et al. (1983) have analyzed spontaneous mutants from the H-2k locus of the major histocompatibility complex in mice. Structural predictions for these partially characterized H-2k mutants (bml, bm3, and bm8) were made, leading to the following conclusions: (i) In all cases in which an amino acid substitution in the k b mutants were found, the identical amino acid variant was present in an homologous position in another member of the class; (ii) a majority of the mutants contained multiple substitutions; (iii) the multiple substitutions in the k b mutants were found in the same array as in other class I sequences; and (iv) these mutants were not random. The relationship between polymorphic characters in the laboratory and in the wild population was unclear. However, the H-2 complex loci K and D differed by 20% in amino acid sequence and resembled each other as much as themselves. The interaction of MHC genes through a copy mechanism permits the introduction of multiple nucleotide base substitutions by a single genetic event. Thus, gene conversion is suggested by Pease et al. (1983) as a possible explanation of the large structural divergence among MHC gene loci and of their extensive polymorphism in mice. Weiss and colleagues (1983) have also found evidence of polymorphism in H-2 genes caused by a gene conversion. They conclude that gene conversion may be a principal mechanism in the generation of new alleles of H-2 genes. It is of interest that they propose conversion between nonallelic genes, different haplotypes, and different chromosomes in a heterozygous mouse.

Baltimore (1981) has addressed the problem of maintaining sequence homology in multigene families in relation to the immunoglobulin (Ig) genes. Both constant and variable regions of all Ig domains appear to be derived from a single ancestral gene by several duplications. Sufficient structural homology between nonallelic constant region genes exists to question whether gene conversion might not have been a very frequent event during the evolution of the immune genes. This argument extends to Ig variable regions, wherein internal 'sub-regions of homology' have been described. The homology is so strong that these sequence homologies have been designated as 'minigenes'. As Baltimore (1981) points out, because variable-region genes contain no intervening sequences for minigenic joining, recombinational mechanisms for variable region production are unlikely. However, prior gene conversion events not requiring somatic assembly could account for the variable region arrangements. 2.2. Gene mapping and the analysis of genetic disease Of the estimated 5 x104 human structural genes, abou L 500 have been mapped. 116 of these (26%) are on the X chromosome, an assignment that is entirely disproportionate to the relative size (6%) and presumptive structural gene content of the X chromosome. This assignment may be due to the greater ease of establishing sex-linked than autosomal inheritance for human genetic disorders. The latest edition of McKusick's catalogue, Mendelian Inheritance in Man (McKusick, 1983), lists some 3500 genetic disorders. Although the genetics of many of these entities is ill-defined, we may assume that inherited abnormalities for approximately 3% of all human loci have been observed. Recombinant DNA technology has started to play a major role both in increasing the speed and precision of gene mapping and in defining the nature of disease mutations (Kurnit and Hoehn, 1979; Davies, 1981). Table 1 gives a list of those human gene probes which have been mapped as of July 1983. This modest list of approximately 100 individual cloned gene sequences or gene clusters will increase enormously over the next few years.

26 Even in the one year since this listing was compiled, the number has grown. The Committee on Human Gene Mapping by Recombinant DNA Techniques for the 7th Symposium on Human Gene Mapping (Skolnick et al., 1984) reports 134 human genes have been cloned, and 116 of these have been mapped. Restriction fragment length polymorphisms (RFLPs - - see next section) have been described for 47 of the 134 cloned genes. A large number of chromosome-specific and -nonspecific repeated DNA probes have also been identified and mapped. The committee estimated that in the next 2 years over 2000 probes will have been mapped and polymorphic linkage markers will be available for most chromosomes. This progress will certainly lead to improved diagnosis of genetic disorders, particularly of those in which an abnormal, primary gene product can be detected. Newmark (1984) recently reviewed progress in diagnosing medical abnormalities by molecular techniques. Experience suggests that genetic changes of a structural nature, such as deletion, duplication and translocation, can easily be detected by restriction enzyme analysis. Base substitutions are less tractable and can be recognized only if the nucleotide sequences involved are disclosed by a known restriction enzyme. When sequence information is available for a particular nucleotide substitution, synthetic polynucleotide primers can be constructed to permit recognition of one or two nucleotide differences in a sequence of 17-19 bp.

2.2.1. Restriction fragment length polymorphism Since specific gene probes became available, restriction fragment length polymorphism (RFLP) has been used in the antenatal diagnosis of human monogenic disease. Kan and Dozy (1978), Little and colleagues (Little et al., 1980; Little, 1981), and Orkin et al. (1978, 1982), have investigated hemopathologies associated with the fl-globin gene. In particular, sickle-cell anemia and/3-thalassemia have been correlated with certain DNA polymorphisms. Populations have been screened and antenatal diagnoses have been made utilizing this linkage. The most successful application of the RFLP approach has been with pure gene probes. Besides probes for abnormalities in the fl-globin gene family, however, quite a few other gene probes

have been used to identify polymorphisms with possible metabolic consequences. In contrast to using structural gene probes, Botstein et al. (1980) have developed an indirect method of determining human genetic linkage that identifies altered sites in human DNA. The method identifies restriction fragment length polymorphisms (occurring through the mutational loss of a nucleotide recognition sequence) that occur frequently in natural populations. These sites can correspond to or be proximal to loci responsible for genetic disease. Such variety has been inferred from previous analyses of protein primary structure, and Wyman and White (1980) have recently shown that loci for restriction fragment polymorphism do exist in human DNA and that these features can be traced in a pedigree analysis. In characterizing an RFLP 5' to the human antithrombin III gene, Bock and Levitan (1983) discuss in general the origins of DNA length polymorphism. Polymorphisms that evolved by DNA insertion and flanked by direct repeats and their inserted DNA segments are nonhomologous with surrounding sequences. As shown by Kreitman (1983) in Drosophila, local amplification events may also yield DNA length polymorphisms. DNA secondary structure may be an important factor in the evolution of polymorphism, and it has been postulated that inserted repeat structures may themselves assume secondary structures that serve as recognition sequences. Such structural considerations may influence the regulation of gene expression as in the case of MHC-related oncogenes. Botstein and colleagues estimate that about 150 polymorphic loci linkage fragments must be characterized to define all possible linkage overlaps for mapping purposes. Differences in the length of any fragment can result from many kinds of genetic alterations, and these changes can be detected by mobility shifts in agarose gels. Rather than depend on pure gene probes to identify the fragment polymorphism, this technique can use single-copy human DNA. Many human recombinant DNA libraries have already been constructed. However, it is important that clones from these libraries are free of repetitive sequences that would obscure the interpretation of hybridization analysis. Singlecopy DNA libraries have been constructed, including one from total messenger RNA (Botstein et al., 1980).

27 By hybridizing a single-copy D N A clone to a population of human restriction fragments, the relative position (i.e., the map) of that single-copy D N A can be determined. If the restriction fragment(s) that hybridizes to this probe exhibits significant and heritable polymorphism (which may exist within the probe or proximal to it), one has a means for tracing its heritance within a human pedigree. All polymorphic restriction fragments are expected to map as Mendelian co-dominant alleles. Thus, without knowing the function of the sequence present in the single-copy D N A probe and in the restriction fragment, heritable human m u t a t i o n can be observed and m a p p e d . D'Eustachio and Puddle (1983) have described the success of using gene- and sequence-specific probes to map mammalian chromosomes in somatic cell hybrids. Human D N A markers have been used to map not only multigene families and other repeated DNA sequences, but also single-copy genes for insulin, collagen, fl-interferon, and families of cellular oncogenes. R F L P has been a critical tool for these mapping analyses. Another perhaps more universal approach makes use of R F L P within noncoding DNA sequences to search for linkage to inherited diseases (Botstein et al., 1980). This method is applicable to diseases in which no abnormal gene product has been identified, precluding the possibility of isolating the mutant sequence itself. Many clinical disorders fall into this category, including inherited forms of muscular dystrophy and neurological disorders such as Huntington's chorea. The chance of detecting linkage between a randomly selected probe and a particular disease is extremely small. It has been calculated that up to 1500 random RFLPs would be required to detect tight linkage for disease genes in all parts of the human genome (Langer and Boehnke, 1982). Fortunately, it is possible to use probes that have been mapped to particular chromosomes to ensure that as wide a net as possible is spread. The chromosomal location of genes is known for some diseases such as X-linked muscular dystrophy of Duchenne or autosomal dystrophia myotonica. The latter is loosely linked to the loci for secreter and to C3 complement, which themselves had been allocated to chromosome 19, implying that the locus for dystrophia myotonica is itself on chromosome 19.

When a chromosomal location for a disease is known, linkage to RFLPs need only to be tested for DNA probes that derive from that chromosome. Establishment of chromosome-specific D N A probe banks, either by cloning individual chromosomes isolated with a cell sorter (Krumlauf et al., 1982) or D N A from a hybrid cell containing a single human chromosome (Gusella et al., 1980), is proving to be a powerful tool for determining linkage within particular chromosomes. For example, linkage has been recently detected on the short arm of the X chromosome and between two RFLPs and the putative gene for Duchenne muscular dystrophy, indicating that the locus for this disease is located in the center of the short arm. This allows the possibility of isolating other RFLPs from that region. Prenatal screening of this disease in male children of proven carriers may be possible (Wieacker et al., 1983). An almost identical linkage relationship with RFLPs has been found with another form of X-linked muscular dystrophy (Beckers), which had been believed to be a separate clinical entity. This new information prompts reconsideration of the status of these two forms of muscular dystrophy and of whether they may represent allelic mutations (Kingston et al., 1983; Davies et al., 1983). However, endonuclease mapping may not be applicable yet to monitoring of all human genetic defects in all populations. Panny et al. (1981) analyzed more extensive population samples than did Kan and Dozy (1978) and concluded that the degree of association of sickle cell with RFLPs in all populations may not be sufficiently great. Asmussen and Clegg (1982) studied population genetic models at two loci and the linkage disequilibrium between them. In this mathematical exercise, tightly linked neutral marker loci, such as RFLPs, gave a higher predictive value for overdominant selection than for recessive genes. Thus, tightly linked neutral markers are less useful to predict the transmission of genes when selection acts against recessive genes rather than for overdominant selection. Suarez (1983) claimed high linkage between restriction fragments and type II diabetes for affected sib pairs. Helentjaris and Gesteland (1983) have used a battery of restriction endonucleases on an intentionally small number of individuals to determine the usefulness of ran-

28 domly isolated human single-copy cDNA clones. Their conclusion was that RFLPs may not be very predictive if the clones are single-copy genes, but may be an accurate gauge of polymorphism if the clones are from gene families. Barker et al. (1984) have examined 31 arbitrary unique loci in human DNA to determine the relative efficiency of different restriction enzymes and to compare polymorphisms caused by genetic rearrangement versus nucleotide changes. They found 9 of 10 polymorphic sites had in common the dinucleotide CpG, indicating that methylated cytosines may have a role in such polymorphisms. The authors surmise that their data support the argument that methylated cytosines are hotspots for mutation in mammalian DNA, because many cytosine residues in CpG dimers are methylated in human DNA. All the polymorphisms detected in this study resulted from point mutations or small deletions. The analysis of hemoglobin production and of sickle-cell anemia is historically important as the first description of a human genetic disorder at the transcriptional-translational level. As mentioned earlier interest in the hemoglobin system has continued, and other genetic disorders of hemoglobin synthesis have been examined. 2.2.2. Molecular analysis of hemoglobin disorders in man Thalassemia is a genetic disorder of human hemoglobin synthesis characterized by defective production of either the a- or fl-globin component of the adult hemoglobin. Two general classes of thalassemia are known: those that produce no detectable polypeptide and those that produce only small amounts. Recent evidence indicates that the former class either lacks the appropriate globin m R N A or contains mRNA that is not translated into protein. Individuals with small amounts of a particular polypeptide often contain insufficient globin m R N A for that polypeptide, either because the expression is reduced or because a globin gene has been lost. Kantor et al. (1980) and Maquat et al. (1980) have detected mRNA-processing defects in certain heterogenous fl+-thalassemias (conditions wherein fl-globin is reduced). Kantor has shown that in several patients fl-globin mRNA sequences accumulate to high levels. From these patients large

m R N A precursors have been isolated. One of these precursors hybridized to a fl-globin probe containing the large intervening sequence. Maquat et al. found similar mRNA precursors from patients with fl+-thalassemia and surmised that alteration of DNA at the intron splicing site was responsible. Neinhuis and colleagues (Humphries et al., 1981; Goldsmith et al., 1983) more recently explored this hypothesis in an exciting set of experiments. They found a human fl÷-thalassemia gene that contained a silent nucleotide substitution (i.e., a substitution that retained the same amino acid codon recognition). However, the consequences of the mutation created a new RNA splicing site within the coding region of the gene and reduced fl-globin m R N A accumulation by 75%. Treisman et al. (1983) discovered other mutations in fl-thalassemia genes that cause aberrant splicing patterns and reveal cryptic splicing sequences. Some mutations abolish splicing sites, whereas others create new sites and reveal secondary ones that elicit an array of normal and abnormal globular proteins. In addition, Proudfoot et al. (1980) have performed in vitro transcription of mutant fl-globin genes of patients with thalassemia. They found a normal rate of transcription of fl+-mutant globin genes and, in some cases, of fl°-globin genes (genes that produce no fl-globin) as well. They conclude that mutations in structural genes are far more prevalent than in regulatory sequences. Williamson (1981) summarized the results of a symposium on thalassemia. One report of a fl0_ thalassemia, which has now been published (Busslinger et al., 1981), showed that a point mutation occurred at the 5' intron-exon junction of the large intron so that processing could not take place. A study of a fl+-thalassemia condition indicated that a mutation in the small intron created a preferred, new splicing site. This resulted in a reduced level of normal fl-globin. Deficient production of a-globin (a-thalassemia) has also been studied by cloning and sequence analysis. Several instances are known in which one or both of the a-globin genes on human chromosome 16 have been deleted. This suggests the participation of non-homologous recombination and gene conversion. Higgs et al. (1980) have described a Welsh family in which three et-globin genes exist on one

29 chromosome and two on its complement, a-Globin mRNA in these people was elevated, probably producing the mild fl-thalassemia they exhibited. Goossens et al. (1980) have identified 12 individuals who are heterozygous for a chromosome with three et-globin genes. Apparently an unequal crossing-over occurred, because the homologous chromosome contained only one locus for the a-globin gene. Lauer et al. (1980) have reported remarkable results. They sought to determine the arrangement of human a-globin genes from a bacteriophage library of human DNA. During the propagation of these phages in E. coli, deletions in several a-globin genes occurred, corresponding to regions of unequal intra- or interchromosomal crossing-over. The locations of the breakpoints that occurred in the cloned DNA are indistinguishable from those shown by Orkin et al. (1979) to be associated with a-thalassemia 2. a-Thalassemia 2 results from a deletion of one of the two a-globin genes. Orkin et al. (1978) and Spritz and Forget (1983) have reviewed the use of endonuclease mapping to diagnose thalassemias caused by globin-gene deletion. More recent improvements (Orkin et al., 1982) are based on selecting the optimal size of a DNA restriction fragment for linkage studies. Boehm et al. (1983) have used DNA polymorphic markers for prenatal diagnosis in pregnancies in which the fetus was at risk for hemoglobinpathy. The prenatal diagnosis was correct in all 78 cases that could be confirmed by subsequent clinical examination. In an analogous situation, Rotwein and colleagues (1983) have identified a polymorphic 5' flanking region of the human insulin gene that was associated with non-insulin-dependent diabetes. Based on length, the polymorphism at the 5' flanking region which followed simple Mendelian inheritance, was thought to be a consequence of variation in the number of short repeat units of DNA and appeared to produce a gene dosage effect. Others (Bell et al., 1981) have found similar properties in the 5' flanking region of the insulin gene.

2.3. Transposition McClintock (1934, 1944) provided the first evidence for the transposition of DNA segments in her genetic studies on maize. Transposition studies

at the molecular level in prokaryotes became possible in the early sixties through the application of new techniques such as heteroduplex analysis of DNA molecules, which could be visualized in the electron microscope and studied by genetic experiments in vivo. These methods were precursors of recombinant DNA techniques in vitro that evolved approximately 10 years later. One class of transposable element was discovered in studies of polar mutations in the gal operon of E. coli (for reviews: Calos and Miller, 1980; Kleckner, 1981). Mutations were 'cloned' in vivo using bacteriophage )~ and heteroduplexes constructed from mutant and wild-type phage DNA. Polar mutations were caused by insertion of discrete DNA elements, called IS (insertion) elements, into the gal operon. The size of these elements ranged between 800 and 1500 bp. Initially, 5 of the elements were identified in E. coli, and with the exception of IS4, the elements seemed to integrate randomly. At about the same time, two other classes of transposable elements were discovered. One type elicited the classic pattern of transposition, because it existed on antibiotic resistance factors termed R factors. Several of these transposons (Tn5, Tn9, Tnl0, e.g.) are composed of 2 IS elements, one at each end of an antibiotic resistance marker. A second class of transposon was characterized by inverted repeats of 38-40 bp at both termini. Members of this class exhibit great similarity in organization and comprise the 'Tn3 family'. In addition to these prokaryotic transposons, another exists that has only 2 known members: bacteriophage/~ and the related phage, D108. Here, transposition is a part of the replication machinery of the bacteriophage and, therefore, is extremely efficient. Transposition is clearly not restricted to prokaryotes; indeed, transposition may run the entire phylogenetic spectrum from bacteria to man. Because of their mobility and ability to transfer unrelated genetic information, transposons are considered important sources of genetic instability. For example, a major source of spontaneous mutation in Drosophila is thought due to transposition. Most eukaryotic transposons share structural similarities with prokaryotic transposable elements.

30 An important discovery that greatly stimulated interest in transposition was that DNA copies of R N A tumor viruses became integrated in chromosomes of animal cells. These DNA proviruses display s t r u c t u r a l similarities to bacteriophage/~ and can potentially be considered viral transposons. Also, they are similar to prokaryotic transposons that contain 2 IS elements, because every D N A copy of an RNA tumor virus has 2 long, direct-repeat sequences at their termini. These repeats are called LTRs (long terminal repeats). LTR elements share characteristics of IS elements in that both have short inverted repeats at their ends (for review see: Varmus, 1982). LTRs, like IS elements, carry promoter sequences capable of initiating transcription of genes located outside the IS or L T R elements (Ciampi et al., 1982). Cellular genes located on either side of the promoter can be activated in this way. If the cellular gene is associated with growth regulation, activation may lead to carcinogenesis. By possessing promoter sequences, transposons can do more than act as insertional mutagens and may influence gene expression in their vicinity. The properties of retroviruses have two important characteristics: one concerns the mechanism of carcinogenesis and the other involves a common ancestral mechanism from which transposition vectors may have arisen. Temin and colleagues (Shimotohno et al., 1980; Shimotohno and Temin, 1981) were among the first to recognize that retroviruses resemble bacterial transposons and may have evolved from bacterial elements. In particular they identified a 569-bp direct repeat at each end of retroviral DNA. This repeat is flanked by the terminal dinucleotides 5'-TG • • • CA-3' and by a 5-bp direct repeat of cellular D N A next to a 3-bp inverted repeat of viral DNA. However, ancestral elements have not been detected in eukaryotes and may no longer exist. Recently, the hypothesis of the evolution of retroviruses from transposable elements found support from analyses of other biological systems (Finnegan, 1983). Shiba and Saigo (1983) reported the striking resemblance between retroviral-like particles (VLP) and the transposable element copia in Drosophila. They established a structural link between copia and VLP. The latter also contained reverse transcriptase activity but was not infec-

tious. This work extends the earlier findings of Flavell and Ish-Horowicz (1981), who showed that covalently closed circles of copia contain one or two copies of a terminal direct repeat. As Finnegan (1983) states, the similarities between retroviruses and copia-like elements are not just structural, but include their transcription into long polyadenylated RNAs that are initiated at one terminal direct repeat and end at the other. However, as Ising and Ramel (1976) have shown in Drosophila and others have found in bacteria, transposition is surprisingly resistant to induction by mutagens and carcinogens. Thus, the significance of the structural similarities between transposons and retroviruses should not be exaggerated until mechanistic studies are complete. Among others, Elder et al. (1983) and Eibel et al. (1981) have found similar structural resemblances between retroviral RNA and the RNA from the yeast transposable element Tyl. TyI-RNA is an abundant poly(A)RNA with ends that closely resemble those of retroviruses. Long terminal repeat sequences and short inverted repeats are distinctive features of both structures. It is also interesting to note that a tRNA~ et contains a region of homology with Tyl that is analogous to the retroviral tRNA-binding site.

2.3.1. Bacterial transposition: elucidation of illegitimate recombination For some time, bacterial geneticists puzzled over the phenomenon that had come to be termed 'illegitimate or illegal recombination'. The process appeared to involve exchange of nonhomologous regions of DNA between the bacterial genome and extrachromosomal DNA. Until the advent of recombinant techniques, the molecular explanation for this type of exchange remained obscure. The following is a description of the basis of bacterial transposition, the event bacterial geneticists had observed earlier. All transposable elements have one property in common: during transposition, a small sequence at the site of integration is duplicated. This has been demonstrated by the integration of transposons into a known DNA sequence (viz. the lacI gene) and by comparing the D N A sequences around the transposon with the sequences before the integration event (Calos and Miller, 1980). From investi-

31 gation of viral transposons (retroviruses in eukaryotes and bacteriophage/~ in E. coli), only a few small segments of DNA appeared important (Galas and Chandler, 1981). These segments are the termini of the transposons and in some cases a few genes involved in transposition or its regulation. The sites have been sequenced, and the gene products involved in transposition studied by cloning them separately on small, multicopy plasmids (Reed, 1981). Recombination during transposition occurs within 2 identical sites termed res. The res sites of Tn3 have been cloned and sequenced. As suggested by Cohen and Shapiro (1980), the transposition and duplication of sites result in the creation of two circles from one. The whole process can be studied in vitro. Transposition can be mediated by the enzyme transposase. The enzyme (transposase R; R = resolvase) has been purified after amplification of its gene by recombinant DNA techniques. The sequence of the binding sites of the resolvase and of the specific sites where recombination takes place has been determined. The investigation of the transposase gene of Tn3 (TnpA) is an example of the use of recombinant DNA techniques for processes that would be otherwise difficult to approach (Reed, 1981; Reed and Grindley, 1981; Grindley et al., 1982). There are strong indications that the regulation of the level of transposase is of utmost importance. Without strict regulation, transposition can cause many genetic rearrangements. This genetic instability could create chaos in a living cell. In the cases of bacteriophage/~ and Tn3, very little transposase is synthesized, and this may be true for most transposons. Moreover, during bacteriophage # transposition, the transposase must be synthesized de novo. Because of their low production, transposases have been difficult to isolate and study in vitro. Very recently, Mizuuchi (1983) developed an in vitro transposition system using bacteriophage/z and purified/~ transposase. A strong promoter has been placed in front of the transposase gene of Tn3 to increase production and facilitate isolation. Although improved, the production was still low, probably because of inefficient translation. In order to improve production further, the regulatory region of TnpA was fused to the lacZ gene. Selection for high expression of lacZ led to the isolation of mutants with more

efficient translation of TnpA message. By recombination, translation signal sequences were placed before the TnpA gene. This led to the overproduction of TnpA, permitting its purification (Casadaban et al., 1982). Although a start has been made towards the characterization of transposases and towards the study of the transposition, other mediating enzymes and transpositionspecific sequences must be identified, isolated, and studied in vitro. 2.3.2. Transposons in Drosophila: their occurrence and use as recombinant vectors

Transposition in Drosophila might conveniently be viewed as an opportunity to investigate problems that are difficult to approach in other higher eukaryotes. Investigators are using transposons in eukaryotes in much the same way as their earlier colleagues .exploited the bacteriophage system. Drosophila has provided a rapidly unfolding picture of the nature of transposition in eukaryotic genomes and has demonstrated its utility in eukaryotic genetic research. Transposition in Drosophila was recognized as the movement of Mendelian genes from their normal chromosomal site to other sites in the genome. First Green (1969) and later Ising and Ramel (1976) detected transposition in Drosophila in cytogenetic analyses, and subsequent study has probably identified the transposon responsible for the movement (Ising and Block, 1981). Green (1980) presented three types of data to demonstrate transposition in Drosophila. Inferred from genetic experiments are two classes of mobile elements: Mendelian genes that spontaneously move from one chromosomal location to another, and discrete DNA sequences that integrate into specific genes to cause genetic unstability. Molecular data identified dispersed repetitive sequences as a third type. The dispersed repetitive sequences comprise 16-17% (2.7000 kb) of the total DNA content of Drosophila. A number of dispersed repeated gene families have been identified (412, 297 and copia). The chromosomal locations of copia-like elements differ greatly among Drosophila strains and are particularly unstable in Drosophila cells in culture. As Spradling and Rubin (1981) discuss, members of a dispersed repetitive gene family do

32 not appear to insert at specific genomic locations. However, their occurrence and location vary tremendously among Drosophila species. The average size of these elements is about 4 kb, and their sequences are conserved and highly nonpermuted at each genornic location. The 412, 297, and copia elements code for abundant poly(A)-containing mRNA (Rubin et al., 1981). The structure of copia-like elements shows common structural features with other very mobile eukaryotic elements (e.g., Tyl of yeast and provirus of retrovirus) and with bacterial transposons (Temin, 1980; Shiba and Saigo, 1983). These similarities involve the terminal nucleotides and internal sequence homologies. Green (1977) postulated that unstable mutations in Drosophila were due to the insertion of discrete D N A fragments. Unstable mutations in natural Drosophila populations arose spontaneously or were induced in hybrid crosses of particular strains carrying isolated chromosomes, the M R chromosomes. The active factor MR could be allocated to a particular site on these chromosomes. MR is only effective if transmitted paternally in a hybrid cross, and recombination in males and high frequencies of recessive lethal mutations and chromosome aberrations occur. Male flies from natural populations have been crossed to females from laboratory strains to examine sterility problems. The pattern of sterility induction (in the females) shows the identical reciprocal cross effect as the effects due to MR. Strains in which the males are capable of inducing sterility are designated P strains. Strains in which the females are reactive to the sterility induction by P-strain males are designated M strains. Presumably, sterility is due to the presence of undefined MR chromosomes contained in P strains. In addition to female sterility, high rates of mutation and chromosome aberration occur in these crosses and the phenomenon in general is designated hybrid dysgenesis. The mechanism of hybrid dysgenesis is somewhat analogous to that of bacterial lysogeny. Engels (1981) used genetic arguments to propose that the characteristics of P - M hybrid dysgenesis were a consequence of transposition. The molecular basis of hybrid dysgenesis became clearer in 1982 (Rubin et al., 1982; Bingham

et al., 1982). These investigators analyzed 4 white eye mutants that were recovered in hybrid crosses using the p - M system. They showed that all 4 P-M-induced white mutations contained a homologous DNA insert that was designated as the P element. During the reversion of these unstable white mutations, the P elements were precisely excised. Further, P strains were found to contain 30-50 copies of the P element, whereas the M strains contained none. P - M hybrids are genetically very unstable, and P elements transpose frequently in them. The DNA sequence (2907 bp) of one complete P element is now known (O'Hare and Rubin, 1983). All P elements derive from this 2.9-kb core, which contains identical 31-bp inverse terminal repeats, and upon insertion (of the P element), an 8-bp sequence is duplicated. Although P elements insert at many chromosomal locations, they apparently do so at the same nucleotide, indicating local site specificity. MR-induced mutations have also been shown to depend on the presence of a P element (Eeken, personal communication). Most spontaneous mutations have been shown at the molecular level to be caused by the insertion of DNA fragments. Transposition may therefore play a major role in the appearance of spontaneous mutations, and the effect of mutagenic/carcinogenic agents on this process is relevant. The interaction of MR with X-rays and DNA-repair deficiencies was studied in some detail (Sobels and Eeken, 1981; Eeken, 1982a; Eeken and Sobels, 1981). Frequencies of sex-linked recessive lethal mutations from irradiated MR-carrying males were significantly higher than the sum of the frequencies recovered from irradiated non-MR males and non-irradiated mR males. This could not be ascribed to an interaction between MR and the maternal DNA-repair system. DNA-repair deficiencies in MR-carrying males increase the frequency of locus-specific mutations at the singed bristle (sn) and raspberry eye color (ras) loci. DNA-repair deficiencies seem to interfere with the MR-dependent transposition as measured by a P element integration event (i.e., induction of unstable mutations). The unstable nature of P element-associated mutations seems to depend on the presence of an active MR for several of the unstable mutations (Eeken, 1982b). The frequency of reversion of such an unstable

33 MR-dependent mutation at s n (sn MR39B1)is about 1.7% in the presence of MR. This reversion has been studied after larval treatment with the chemicals ENU and MMS simultaneously with the production of X-linked recessive lethal mutations. A minor effect of both chemicals on the frequency of singed reversions only occurred at extremely high concentrations, whereas the considerable increase of recessive lethal frequencies indicated that mutagenic concentrations of the chemicals had reached the gonads (Eeken and Sobels, 1983). The partial elucidation of this particular transposition process in Drosophila may be the progenitor of more detailed studies using recombinant DNA technology. Assitionally, it provides a means to clone any gene using P element transposon tagging. Spradling and Rubin (1982) and Rubin and Spradling (1982) utilized P element transposition to study gene regulation and expression. They cloned the 2.9-kb P element and injected it into presumptive germ cells in Drosophila embryos. The DNA integrated into germ-cell DNA and was expressed in the progeny of the injected embryos. Only P element DNA integrated into germ-cell chromosomes. Plasmid DNA without the P element did not integrate. It appears that the 2.9-kb P element contains sequences that code for a trans-acting product, required for transposition. It also seems likely that the P strains synthesize a P element transposition suppressor. Plasmids containing the structural gene for xanthine dehydrogenase (XDH), which has been inserted into a P element, have been used as vectors for P element-mediated gene transfer in Drosophila (Rubin and Spradling, 1982). These authors also have studied how the chromosomal position of a reintegrated gene in Drosophila may influence gene expression. Position effects have been shown to influence gene expression by classical cytogenetic methods and more recently by gene transfer methods. Spradling and Rubin introduced an intact XDH gene by the P element transposon system into a homozygous X D H - (rosy) background. An analysis of 36 strains, each that differed in the chromosomal location of the XDH gene-containing P element, showed that expression of the XDH gene was not influenced qualitatively, but that quantitative expression varied within a 5-fold range. By comparison, the insertion of a

retro~iral provirus in different chromosomal locations does influence proviral gene expression. Maniatis and colleagues (D.A. Goldberg et al., 1983) have also used the P element system to study gene expression (this time with the gene for alcohol dehydrogenase, ADH). Using a set of mutated ADH genes that were inserted in a P element-containing plasmid, the authors showed that a contiguous cis-acting DNA sequence was required for normal temporal- and tissue-specific expression of ADH. Normal activity was gauged by the qualitative levels of ADH activity in larvae and adults, by tissue-specific activity, by the size of ADH mRNA, and by the developmental switch in larval and adult promoter utilization. The Drosophila strains carried the integrated gene at several different sites on all three major chromosomes. Although no evidence of structural instability or unregulated expression was noted, levels of enzyme activity varied more than 3-fold in some strains. Scholnick and colleagues (1983) reintroduced the DOPA decarboxylase gene into the Drosophila genome by means of a P element. The gene was expressed correctly during development despite being inserted at different chromosomal locations. In only one other reported case (Palmiter et al., 1982) in a multicellular organism other than Drosophila has a reintegrated gene been expressed in a regulated manner during development. Although genes located on short DNA segments are sometimes expressed when reintroduced into cells, proper temporal- and tissue-specific expression has been absent. Developmental regulation, as distinguished from transcription, may require the presence of a gene in its normal chromosomal domain.

2.4. Cellular transforming oncogenes That some tumor cells inherit a stable transformed phenotype suggests that oncogenic transformation may result from genetic alteration, and the somatic mutation hypothesis of carcinogenesis has been proposed to account for the relationship. A direct etiology is easier to show for cells infected by certain oncogenic viruses, but what is the role of genetic alteration in naturally occurring tumors? A promising answer to this question lies with the

34

investigation of cellular genes with potential oncogenic properties. Several lines of evidence argue in favor of the somatic mutation hypothesis of carcinogenesis: (a) Cancer usually behaves as an irreversible and transmissible cellular phenomenon. (b) Tumors are monoclonal. (c) Many carcinogens 'and mutagens are largely overlapping sets and are frequently electrophilic. (d) With appropriate metabolic biotransformation, most carcinogens are mutagens, although striking exceptions exist. (e) DNA-repair deficiencies in man, such as xeroderma pigmentosum, lead to an increased cancer incidence. (f) Oncogenic transformations have occasionally been traced to interactions between viral and cellular DNA or to mutational events in cellular oncogenes. This evidence indicates an association between mutagenicity and carcinogenicity but no compelling causal relationship. However, the recent identification and analysis of cellular and viral oncogenes provide direct evidence of mutational changes in carcinogenesis. Understanding the relationship between mutagenicity and carcinogenicity is complicated by the fact that mutagenicity can be regarded as a single event, whereas carcinogenicity seems to be a sequence of several steps. An operational classification of carcinogenicity involves initiation, promotion, and progression. The precise sequence of events may be far more complicated. In particular cell transformation assays indicate that initiation and promotion can be subdivided into several steps. 2.4.1. Mutagenic eoents Acute transforming RNA viruses (retroviruses) contain specific transforming genes called oncogenes, which can cause neoplastic transformation. Viral oncogenes are highly homologous to normal cellular genes, but lack the introns of their cellular counterparts. Approximately 20 retroviral oncogenes have been identified, some of them in several viral strains (for a review see Cooper, 1982). These viral oncogenes can be inserted almost randomly into cellular DNA.

Cellular oncogenes show a high evolutionary stability and must have a normal function in the cells, presumably in connection with cellular differentiation. The oncogenic property of these genes is acquired by some kind of activation. Activation can occur genetically, resulting either in an increase in the normal cellular product or in an alteration of the gene leading to an altered product. The transforming property of DNA from tumor cells has been studied intensively by transfection assays in combination with restriction enzyme analysis. The transfection assay is equivalent to the classic genetic transformation of bacteria with DNA, as first reported by Avery et al. (1944). During a transfection experiment, recipient cells are exposed to donor D N A from tumor cells. The cell line used commonly for this purpose is the NIH3T3 mouse fibroblast cell line, in which a stable integration of DNA occurs with high efficiency (Shih et al., 1979). NIH3T3 is a permanent cell line, which, as do many such cell lines, possesses properties of both normal and transformed states. Whereas transfection with high molecular weight DNA from normal human or animal cells does not transform NIH3T3 cells, transfection with high molecular weight DNA from tumor cells often produces a high frequency of transformation. This event implies a permanent genetic change that is transmitted from the tumor D N A to the recipient NIH3T3 cells and their daughter cells. Recently three research groups (Tabin et al., 1982; Reddy et al., 1982; Taparowsky et al., 1982) have independently shown that the transforming property of cells from two related human bladder tumor cell lines, EJ and T24, depends on a single nucleotide substitution in an oncogene. This oncogene is closely related to the viral oncogenes of the Harvey sarcoma virus (o-ras n) and the human cellular homologue c-ras H. The gene product of the ras oncogene is a protein (p 21) of molecular weight 21 000 located at the inner cell membrane. The transversion of the base pair GC to TA in the oncogene of the human bladder cells designated a corresponding alteration of the p 21 gene product by one amino from glycine to valine. This transversion appears responsible for the transforming property of the bladder tumor DNA

35 used to transfect NIH3T3 cells. An exchange of this amino acid can change the configuration of the protein (Tabin et al., 1982). The importance of this change is illustrated by the fact that in the rat the v-ras H oncogene deviates from the normal rat cellular oncogene by coding for arginine instead of glycine in exactly the same position as p 21 (Tabin et al., 1982) and an equivalent change of glycine to serine is indicated with the closely related v-ras Ki oncogene. Sukumar et al. (1983) have lent supporting evidence to the concept of mutational specificity of oncogene activation. They examined 1 of 9 mammary carcinomas induced in rats by nitrosomethylurea at the H - r a s - 1 gene. The gene possessed a single point mutation in the 12th amino acid codon leading to the substitution of glutamic acid for glycine. The DNA of the other 8 carcinomas contained restriction polymorphisms that suggested a single nucleotide substitution, but was not sequenced. Feinberg et al. (1983) investigated the frequency with which this oncogenic hot spot occurs and discussed the implications of mutagenesis for carcinogenesis. 28 human cancers (10 bladder, 10 lung, 9 colon) were analyzed using as a marker a restriction endonuclease cleavage site in the 12th amino acid codon of the c - H a - r a s gene. None of the 29 genes possessed the specific mutation in codon 12. While not disavowing the possibility of mutation at other sites in the c - H a - r a s gene or other mutational mechanisms, these results challenge concepts of specificity and generality that other studies have suggested. It should also be emphasized that DNA from normal bladder cells can transform cells in transfection assays only when present in small DNA fragments. Moreover, the base substitution found in the bladder carcinoma cells is not a genetic polymorphism but seems to be directly linked to a transforming property. Regardless of their generality, these results on human bladder carcinoma constitute the first direct evidence that a mutational change of one base pair in DNA is responsible for a carcinogenic transformation. The picture is, however, more complicated. Both experimental data and epidemiological considerations strongly point to carcinogenesis as a multistep process. The NIH3T3 cell line used in transfection experiments is already partly transformed, and presumably the transfected DNA from the

bladder carcinoma cells furnishes only one of the properties required for transformation. The NIH3T3 cells are themselves responsible for the remaining step or steps. The involvement of two events in one transformation has been indicated in the induction of B cell lymphoma by avian lymphoid leukosis virus (LLV). In 80% of LLV lymphomas, activation occurs by means of a single insertion of the viral transcriptional promoter in the vicinity of the cellular oncogene, c-myc. However, no integration of a viral DNA sequence has been detected in NIH3T3 cells transformed by B cell lymphoma DNA. A cellular DNA sequence not of viral origin must have been responsible for the transforming property of the transfected DNA (Hayward et al., 1981). LLV does not contain a viral oncogene, and it differs from acute retroviruses, which carry oncogenes, by being only weakly oncogenic and having a longer latency period. Such viruses are termed chronic oncogenic viruses. Recent investigations by Land et al. (1983) and Ruley (1983) have shown that transformation of primary and secondary cultures of rodent fibroblasts require 2 or 3 events. Land and colleagues found that transformation required the combined transfection by the ras oncogene, activated by a base-pair substitution, and the m y c oncogene, activated by DNA rearrangement. Also, transformation was induced by a combination of treatments, involving transfection with activated oncogenes and tumor virus DNA and application of chemical carcinogens. One essential step seems to be the immortalization of the cells, a property already present in the 3T3 cells used in previously described transfection assays. These results on transformation of more normal fibroblast cultures constitutes an important step towards the understanding of the sequence of events leading to in vivo tumor formation. 2.4. 2. C h r o m o s o m a l r e a r r a n g e m e n t s

Cancers of blood cells are generally correlated with chromosomal translocation. It has been suggested that these rearrangements lead to an altered expression of cellular oncogenes (Klein, 1981). There are several lines of evidence to support this view (Marx, 1982; Merz, 1983). Oncogenes have been located close to the break points of transloca-

36 tions, such as the ones associated with myeloblastic leukemia, Burkitt's lymphoma, and chronic myelogenous leukemia (the Philadelphia chromosome). Immunoglobulin genes are also located near the break points of such translocations. Translocations bring the oncogenes close to the highly active transcription promoter and enhancer sequences of the antibody genes. The data indicate that the increased expression of such oncogenes may be responsible for the carcinogenic property of cells carrying these translocations. In vitro activation of an oncogene resulting in increased gene expression has been accomplished by combining a segment of the human oncogene c-ras H with the long terminal repeat of the viral oncogene v-ras H, which functions as a transcriptional promoter. This recombinant produces more p 21 protein and causes oncogenic transformation. As mentioned before, similar activation by insertion of a viral promoter in the vicinity of an oncogene is associated with the induction of B cell lymphoma by avian lymphoid leukosis virus (LLV). Saito et al. (1983) have performed a detailed molecular examination of the consequences of cmyc translocation. They surmise that portions (segments between intron and exon 2 and 3) of the c-myc gene are broken during translocation. The mRNA that results from this truncated gene is thought to lack secondary structures that hinder translation. In the case of the translocated c-myc oncogene that is involved in Burkitt's lymphoma as well as in mouse plasmacytomas, the break point of the translocation is located in the repeated D N A sequence of the heavy-chain coding segment of the antibody gene, which is involved in the normal antibody rearrangements of DNA (Dalla-Favera et al., 1983). It is therefore possible that these translocations are the consequence of the same rearrangements that are normally performed by antibody genes (Robertson, 1983a). However, data also suggest that c-myc activation alone may be insufficient to induce full malignant transformation. The contribution of the c-myc product may be to immortalize the cells, while a second unknown oncogene contributes other properties (Robertson, 1983b). This hypothesis is supported by recent experiments demonstrating the cooperation of transforming genes from RNA and DNA tumor viruses.

Although several investigators have reported an increased expression of oncogenes in connection with translocations (see e.g., Erickson et al., 1983), there are also data correlating translocation with a shorter than normal transcript of c-myc. This indicates an intragenic rearrangement of the c-myc oncogene. That a qualitative change of the gene product can cause transformation is in accordance with the association of a single base substitution in the oncogene with carcinogenesis in bladder cells. 2.4. 3. Perspectives

It is now clear that the acutely transforming retroviruses obtained their oncogenic properties by acquiring cellular 'proto-onc' ('c-onc') genes (Bishop, 1981). Oncogenes are limited in number (Coffin, 1982) and conserved in evolution (Stehelin et al., 1976; Shilo and Weinberg, 1981). These genes probably have an essential role in cellular differentiation or proliferation. An important clue concerning the normal function of an oncogene has recently been reported by Doolittle et al. (1983) and Waterfield et al. (1983), showing that the onc gene of simian sarcoma virus is closely related to the cellular gene for platelet-derived growth factor. Furthermore, Downward et al. (1984) have examined the peptide sequences from human epidermal growth factor (EGF) and part of the deduced sequence of the v-erb-B transforming protein of avian erythroblastosis virus. The transforming protein may be a truncated version of an EGF that lacks external EGF binding, but still contains the peptide domain responsible for cell proliferation. Rigby and his colleagues have identified the class I major histocompatibility (MHC) antigen gene as the active oncogene in mouse oncogenesis (Murphy et al., 1983; Brickell et al., 1983). These investigators found that a set of mRNAs present at elevated levels in tumor cells (induced by a variety of ways) and embryonic cells share a dispersed repetitive element. The repetitive eleme~at is characteristic of the long terminal repeat (LTR) of a transposable element and contains a 12-bp direct repeat at its flanking sequence. The MHC-I gene was identified by comparing the nucleotide sequence of the repetitive element-containing clone from tumor cell DNA with sequences from data bases. As indicated recently (Anonymous, 1983), these results not only implicate transposition in

37 cellular oncogene activation, but suggest how the normal mechanism of differentiation is disrupted in tumorigenesis. Schrier et al. (1983) and Bernards et al. (1983) have also recently found evidence for class I major histocompatibility antigens in the induction of transformation by adenovirus. While one strategy for studying oncogenic function in carcinogenesis has been to identify the gene product, another has been the determination of the number of oncogenes involved in a single carcinogenic episode. Campisi et al. (1984) have performed a very interesting simultaneous comparison of the influence of the c-myc and c-ras oncogenes on cell cycle function, c-myc seems more active during cell proliferation and less active as terminal differentiation ensues. In contrast, the c-ras gene is dependent on a specific cellular cycle between G and S phases. Mutations are most often found outside the myc coding regions and are often related to translocation. Taub et al. (1984) have shown that translation of the c-myc gene that is translocated t(8; 14) in Burkitt lymphoma cells is correlated with somatic mutation. They have discovered a set of lymphomas that contain a translocated gene that is slightly deregulated because of somatic mutation at regulatory sequences 5' to the c-myc gene. However, point mutations are more often found in ras coding regions. These results clearly describe independent functions for the oncogenes and, by implication, a multistep mechanism for carcinogenesis. Once incorporated into the viral genome, the c-onc gene (now termed 'v-onc') is controlled by viral signals and may be expressed at abnormally elevated levels within the infected cell. Alternatively, the transforming v-onc gene can differ in its amino acid coding sequences from its non-transforming cellular c-onc progenitor (Tabin et al., 1982); this change is probably unrelated to increased gene activity. The slowly transforming retroviruses, which do not transduce cellular genes, were shown in some cases (Hayward et al., 1981; Payne et al., 1981) to activate transcription of a particular c-onc gene by integration in an adjacent position on the cellular DNA. The picture is more obscure for the non-virally induced tumors (Blair et al., 1981). By using the DNA-mediated gene transfer approach, it has been possible to assay tumor DNAs for their ability to transform cul-

tured mouse 3T3 fibroblasts. This led to the identification of transforming genes in chemical- and radiation-induced tumors (Shih et al., 1979, 1981; Lane et al., 1981, 1982; Hopkins et al., 1981) and in spontaneous human and mouse tumors (Krontiris and Cooper, 1981; Perucho et al., 1981; Pulciani et al., 1982; Murray et al., 1981). Recently three transforming genes from human bladder carcinomas (Der et al., 1982; Parada et al., 1982; Santos et al., 1982) and one human lung carcinoma (Der et al., 1982) have been identified as altered forms of the cellular oncogenes 'ras H' and 'ras K', respectively. Within the span of this decade, studies on oncogenes have produced some conceptually startling realizations. For the first time, specific genetic alterations were shown definitively to be responsible for cellular transformation. Of even greater importance, viral and cellular genes have been identified as participating in carcinogenesis. Their participation in normal cellular processes will soon be elucidated. Clearly, more than one mutational event occurs during transformation, and the mechanism of this mutation can conceivably include any known genetic alteration. These developments owe their discovery to recombinant D N A technology. 2.5. Analysis of gene alterations at the molecular level 2.5.1. The use of cloning and D N A sequencing in mutation research

Until recently, mutation research in bacteria was based on genetic analysis of base substitution or specific frameshift mutations (Ames et al., 1975; Coulondre and Miller, 1977; Bridges et al., 1967). All of these systems are restricted by the limited number of sites available for mutagenesis and by the limited types of mutations that can be detected. In mutagenicity testing, a compound can be judged nonmutagenic because mutation does not correspond to the specific genetic change being selected for. Especially when related to the specificity of mutagenesis, the limited number of nucleotide sites or types of mutation that can be analyzed are serious experimental drawbacks. The Ames mutagenicity test (Ames et al., 1975) is a widely used short-term testing system in which

38 the induction of reversion to histidine prototrophy is measured in several special mutants of Salmonella typhimurium. The sensitivity of this system was greatly enhanced by the incorporation of the plasmid pKM101 into the tester strains, because many compounds induce mutagenesis only in the presence of this plasmid. All histidine-requiring mutants in this set of Salmonella strains have GC base pairs at the reversion sites. Recently, Levin et al. (1982) constructed strains that have AT base pairs at the critical site for reversion. These strains detect chemical oxidants and other compounds that were not mutagenic or were less mutagenic in the standard tester strains. Many histidine-requiring mutants were screened with several oxidants, and the H i s G 4 2 8 mutant, carrying an ochre mutation generated by a cytosine-tothymidine transition, most readily reverted. To enhance the sensitivity for reversion by oxidants, the H i s G 4 2 8 mutation was cloned into the multicopy plasmid pBR322 and used to construct a strain with an amplified, mutated histidine gene. In this way, many sites became available for reversion to His ÷ via a substitution of an AT bp, and a tester strain was developed for a whole series of oxidative mutagens. The lacI system developed by Coulondre and Miller (1977) has been successfully used to study mutational specificity. This system detects 72 independent transition and transversion events, leading to amber or ochre nonsense mutations in the lacI gene of E. coli. Despite the large number of sites that can be monitored, only one type of mutagenesis can be analyzed, i.e., base-pair substitutions leading to amber or ochre codons. Complete knowledge of the specificity of induced or spontaneous mutagenesis can be achieved only by sequencing the mutated DNA (Farabaugh, 1978). Examples of this strategy will be described for the mutational specificities of the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AAAF) and UV light. Fuchs et al. (1981) examined the mutation induced by N-AAAF, using a plasmid (pBR322) carrying genes for ampicillin (Ap) and tetracycline (Tc) resistance. Plasmid DNA treated with NAAAF was cut into a small and a large fragment with two restriction enzymes. The small fragment (275 bp) was purified and reinserted by ligation

into a non-treated large fragment. In this way, plasmid DNA was obtained that carried N-AAAF lesions only in a distinctive small fragment and could be used to transform E. coli. The 275-bp fragment is part of the tetracycline resistance gene, which detects N-AAAF-induced forward mutations by selecting for an ApRTc s phenotype. Plasmid DNA from ApRTc s colonies was isolated, and the small fragments carrying the mutations were sequenced using the method of Maxam and Gilbert (1977). All N-AAAF-induced mutants were generated by deletion of either a single GC bp or a doublet of adjacent GC base pairs. Furthermore, Fuchs et al. (1981) concluded that alternating GC sequences are likely candidates for mutational hot spots. This approach has also been used by Livneh (1983) to investigate mutations induced by ultraviolet light. Hot spots for induced mutagenesis can be caused by sites in DNA with high affinity for a given mutagen or, alternatively, by sequence-dependent processing of premutational lesions. In the latter case, a hot spot can arise from the inability of error-free repair enzymes to excise a lesion at a given sequence (Fuchs et al., 1981). Therefore, knowledge of the distribution of the lesions in DNA after treatment with a mutagenic agent is important for understanding mutational specificity. Techniques derived from the Maxam-Gilbert sequencing technique have been used for determining lesions in a DNA fragment. For example, compounds such as bleomycin, adriamycin, and neocarzinostatin cause single- and double-strand breaks in DNA. To analyze the breakage, DNA is labeled in vitro at its 5' end and subjected to chemicals that induce strand breaks in the DNA. When degraded DNA is run on a denaturing polyacrylamide gel, it is separated in a series of bands that appear on the autoradiography of the gel. Comparison of the positions of the bands with the sequence of the original DNA fragment run upon the same gel reveals the sequences at which the compound induces strand breakage. In this way, the sequence-specific DNA cleavages produced by neocarzinostatin and bleomycin (D'Andrea and Haseltine, 1978a) and adriamycin (Berlin and Haseltine, 1981) were determined. Sites of modification by compounds that induce

39 alkali-labile lesions in DNA can be determined in the same way. A DNA fragment can be treated with such an agent and incubated with 1 M piperidine at 90°C to convert the alkali-labile lesions into strand breaks. Sites of strand scission can be determined by sequence analysis. In this way, the sites of DNA modification by aflatoxin B (D'Andrea and Haseltine, 1978b; Muench et al., 1983), by the anti-diol epoxide of benzo[a]pyrene (Haseltine et al., 1980), and by nitrogen mustard (Grunberg and Haseltine, 1980) were identified. The methods described above are limited to agents that induce strand breaks in DNA either directly or at lesions that can be subsequently converted into strand breaks. The use of exonuclease III (exolII) of E. coli has extended the methodology. This enzyme degrades doublestranded DNA, starting from the 3' end of the molecule. When a DNA fragment is treated with a compound that induces stable adducts in the DNA, exoIII digestion of this fragment will stop at the site of the modification, probably as a consequence of local distortions at the lesion. When an experiment is carried out with a DNA fragment labeled at only one 5' end, the exoIII digestion produces a mixture of labeled DNA molecules. The lengths of the fragments are determined by the sites where exoIII digestion stops and, thus, by the sites of the lesions. The sites of modification can easily be detected b y running the partial exoIII digest on a denaturing gel, together with the reaction products of the Maxam-Gilbert reactions. Using this approach, the sites of lesions induced by UV light (Royer-Pokora et al., 1981) and by cis- and trans-Pt(NH3)2C12 (Royer-Pokora et al., 1981; Brouwer et al., 1981) were determined. 2.5.2. Site-directed mutagenesis Site-directed mutagenesis offers an entirely new approach for studying the effects of DNA alterations. Because it is now possible to alter specifically small fragments or particular base sequences in closed, circular DNA, one can theoretically determine the effects of mutation in any DNA sequence that can be isolated and inserted in a vector. One can make a predetermined assessment of the effects of any genetic damage before a mutagenic agent is tested. Nathans and colleagues (Peden et al., 1980; Shortle et al., 1981) have

provided important reviews describing experimental approaches of this kind. One approach is to delete or insert small DNA fragments in a cloned molecule. These methods all involve an initial incision in the circular plasmid at a particular restriction endonuclease-sensitive site in the DNA. The single-stranded ends produced by this incision can be trimmed back or small fragments containing terminals that are complementary to those in the nicked plasmid can be inserted at the incision site. The ends can be ligated and a newly mutated plasmid thus can be created and cloned. Deletion of small fragments (usually less than 50 nucleotides) from each end can be accomplished by limited exonuclease digestion. Green and Tibbetts (1980) have developed a technique for producing deletions in closed, circular DNA without having to incise near the region to be excised. Rather, they were able to displace this region by producing a D-loop (a structure formed when a complementary single-stranded DNA fragment hybridizes to that region in the plasmid). A D-loop can serve as a target for digestion with the single-strand specific $1 nuclease. The level of mutagenesis obtained by this procedure is high enough to render selective growth and to make screening procedures unnecessary. Within 1-2 weeks, several hundred new mutants can be prepared at selected regions. Abelson and co-workers (Wallace et al., 1980) have used this approach to remove an intervening sequence from a yeast tRNA gene to study the processing of tRNA primary transcripts. In addition, intervening sequences of several DNA tumor viruses have been removed in the same manner (see Peden et al., 1980; Shortle et al., 1981). In theory, any small fragment of DNA may be preferentially excised from a plasmid. The fragment could include regulatory sequences, genes involved in DNA repair, and mutator or antimutator regions. Thus, geneticists could study regions which might influence the rates of mutation directly as well as regions affecting mutation rates by post-transcriptional mechanisms. It is possible both to delete and to insert fragments into a plasmid molecule. The purposes of insertion may be to provide new restriction-site sequences (linkers) or to provide a new functional element or a precisely altered copy of a pre-existing one. Several

40

techniques are available for producing insertion mutants. If the plasmid incision fragment terminals and those of the insertion fragments are complementary (i.e., if they possess cohesive ends), direct end-to-end ligation is possible. Methods exist not only for removing or inserting DNA fragments, but also for producing base substitutions in small fragments or at specific sites. Fragment mutagenesis is usually performed by isolating a particular fragment as part of a plasmid or phage molecule, subjecting it to a mutagenic agent, and reintroducing it back into its original genome. The reintroduction, which normally relies on recombination, is vital to the procedure because it permits the fragment to be tested functionally within its proper genome. The chemical method used most frequently for accomplishing site-specific mutagenesis is the bisulfite reaction. Taking advantage of the fact that bisulfite will deaminate cytosine to uracil in a single-stranded polynucleotide, this transition can be generated once a circular duplex molecule has been nicked by an endonuclease. Thus, if a region of interest possesses an endonuclease-sensitive site, one nick can be created to produce a small singlestranded gap. This gap becomes the target for the bisulfite-catalyzed deamination. If a region does not contain an endonuclease-specific sequence, one can be introduced. The specificity of several site-directed mutagenesis techniques depends on the introduction of a single nick at the desired site. The number of sites can be potentially increased by a two-step procedure that permits a nick to be placed in any DNA segment for which a corresponding single-stranded fragment can be isolated (Shortle et al., 1980).

Chapter 3. Topics of biological interest and important application This Task Group recognizes the pervasiveness with which recombinant DNA methodology has entered experimental biology. The overview of techniques and their application presented here is only a glimpse of the knowledge made available by the new approaches. Yet, it is representative of how problems have been viewed and experiments designed. It is representative of how biologists are beginning to think.

The purpose of this final chapter is 2-fold: one is to mention how recombinant techniques could possibly be applied to specific areas of interest to ICPEMC and the audience it serves. Second, in this chapter the Task Group broadens its scope to discuss basic research concerns in molecular biology and genetics. Without trying to include everything, the group wishes simply to point out a few truely exciting discoveries that are sure to have profound consequences. 3.1. Short-term mutagenicity testing It is hard to imagine the impact of recombinant technology being any greater in genetic toxicology than that of the general development of short-term mutagenicity tests. The application of short-term tests has occupied a large part of ICPEMC's evaluations, as well as the efforts of other organizations. One major weakness that has been mentioned repeatedly in these evaluations is the lack of definitive interpretation of short-term data, especially microbial data and its correlation with mammalian genetics and carcinogenicity. In his introduction to the first issue of Science devoted entirely to recombinant DNA, Abelson (1980) concludes that gene cloning and DNA sequencing are the two methods that mainly comprise recombinant DNA technology. Short-term testing has already been the beneficiary of these two general approaches. Thus, on the purely technical side, DNA lesions can be located with absolute precision and their chemical nature identified by sequence analysis. With the ability to manipulate large blocks of genetic material by transposition or with vectors, the analysis of specific mutational mechanisms and the vulnerability of particular DNA segments can be studied with ease and accuracy. One particularly fertile area may be in the analysis of mutagenic hot spots. For example, as described by Coulondre and Miller (1977), mutation induction is not random in bacteria, but favors certain areas of a gene. This phenomenon has been studied with different mutagens in prokaryotic and eukaryotic organisms. Even primary sequence analysis is insufficient to establish a general pattern for hot spot mutagenesis. Some of the more insightful research is directed towards examining

41 secondary and tertiary DNA structure for clues to vulnerability. At the mammalian level, Ehling and Neuh~user-Klaus (1984) demonstrated that the distribution of mutations among 7 loci in mice depends on the noxa. Because specific-locus mutations in mice are germ-cell mutations, the application of recombinant techniques to mouse germ-line DNA should be very instructive. If somatic and germ-cell mutation can be shown to differ qualitatively or quantitatively, this will have an important influence on short-term mouse testing. Another important issue is that of determining threshold effects of mutagenic and carcinogenic agents in mammals. A direct analysis of D N A sequence can provide definitive answers. Indeed, even definitive data on only a few chemicals will be a major contribution in establishing the phenomenon of genotoxic thresholds. The determination of thresholds is imperative for the proper use of genetic risk assessment (Ehling et al., 1983). Cloning eukaryotic genes in bacteria may simplify studying them and, thus, the biological range of short-term tests can be greatly expanded. For example, cellular and viral oncogenes can be analyzed in vitro or on isolated plasmids. The effect of chemicals on gene products can also be assessed without the need to purify them or select for their activity among total cellular products. The extrapolation power of short-term tests also should be greatly improved by including eukaryotic genes in microbial systems. Thus, eukaryotic repair systems, genes governing chemical metabolism, and other specific features can be assessed independently. For example, the intricate and powerful mammalian DNA-repair processes are being dissected and perhaps may be introduced in mutagenicity testing schemes. Also, translocation and recombination mechanisms require analysis. Both are intimately linked to mutation, gene amplification, and carcinogenesis.

3.2. Untargetted mutagenesis Evidence exists in both prokaryotes and eukaryotes that mutations do not always occur in DNA at the site where a lesion is formed. Paradoxically, it is possible that when a lesion is formed in one gene sequence,mutations are formed in

remote sequences (untargetted mutagenesis). Untargetted mutagenesis is often attributed to the action of error-prone DNA repair mechanisms. Genes coding for error-prone DNA-repair mechanisms increase spontaneous and mutagen-induced reversions in Salmonella typhimurium and E. coli (McCann et al., 1975; Mortelmans and Stocker, 1976). Ames and co-workers (1975) exploited this effect to develop a sensitive short-term test for screening mutagenic and carcinogenic agents. However, if error-prone DNA repair induces untargetted mutagenesis but is dependent on the kind of chemical tested, it will be important to understand the mechanism behind this effect. For instance, Mattern et al. (1984) indicate that errorprone DNA repair may influence untargetted mutagenesis. By restriction endonuclease analysis of point-mutated revertant DNA from E. coli, they showed that genes coding for error-prone DNA repair (muc genes) stimulated untargetted mutagenesis.

3.3. Polygenic mutation and gene amplification Unorthodox properties, including a remarkably high mutation rate, have been reported for polygenes involved in quantitative traits (for review, see Ramel, 1983). In Drosophila the spontaneous mutation rate of polygenes has been estimated to be about 20 times the mutation rate of single genes (Crow and Simmons, 1982; Mukai, 1979). Nomura (1982) reported a conspicuously high rate of X-rayand urethane-induced germ-line mutations in mice leading to cancer in their offspring. Perhaps these observations can be tied to similarly obscure polygene mutations. Considering the potential importance that polygenic systems may have to man it seems to be of crucial importance to elucidate the properties and mechanism of action of polygenes at a molecular level. Analysis of oncogenes indicates that their activation can occur either by a qualitative change resulting in an abnormal gene product or by a quantitative increase of the gene product. The latter can be acquired by translocations through which oncogenes are displaced to the neighborhood of active promoters or enhancers of immunoglobulin genes. However, many solid tumors are associated with oncogene amplification (for a

42

recent overview see Marx, 1984). These amplifications can appear as homologously staining regions (HSR) in chromosomes or as double-minute chromosomes. The cytogenetic evidence for amplification often corresponds with an increase in an oncogene product. For example, Kohl et al. (1983) discovered an active oncogene in human neuroblastoma. By means of transposition, this oncogene is greatly amplified and can be found in homologously staining regions or double minutes. Such amplification may be relatively common in cancers of neural ectoderm as a consequence of normal neuroblast differentiation. Furthermore, Wahl et al. (1984) have examined the general phenomenon of the amplification in relation to chromosomal position. Using a cellular model not related to oncogenesis, they found that position effects had a predictable influence on the amplification of the target gene and also on the frequency of chromosomal rearrangements.

3.4. Differentiation and evolution 3.4.1. Differentiation in animals Recombinant DNA and sequencing techniques have been invaluable when directed towards specific end points such as the genetic analysis of carcinogenicity. Thus, before cellular and viral oncogenes could be identified, discrete DNA fragments had to be isolated, purified, and subsequently screened in animal cells in culture. On an even more complicated scale, recombinant techniques have begun to unravel the biological puzzle of differentiation. An example of this progress is a recent review entitled 'Cloning the genes that specify fruit flies' (North, 1983). These gene sets have been combined in novel ways to show how complex multigene interactions may influence development. Yet, even these elegant experiments suffer from the same disadvantages of other in vitro systems: they do not pertain directly to differentiation and embryonic development in higher animals. A new tactic for analyzing these phenomena resides in the creation of transgenic organisms. In this experimental sense, transgenesis depends on the injection of an unrelated DNA fragment into animal embryos and the integration of the exogenous DNA into germ-line chromosomes. Alternatively, the natural host-parasite

relationship for hybrid DNAs derived from viral vectors can be used. As Hogan and Williams (1981) report, efforts to integrate DNA into germ-line genomes have been far more successful than originally expected. Two recent studies have been especially important. Palmiter and colleagues (1982) isolated a DNA fragment containing a mouse metallothionein-I promoter fused to the structural gene of the rat growth hormone and injected it into the pronuclei of fertilized mouse eggs. 7 of the 21 mice which developed from these eggs carried the exogenous DNA fragment. Moreover, and of equal importance, the rat hormone was expressed in 6 mice that grew almost twice as large as normal mice. This experiment even produced dose-response data; the mice with the most rat gene copies (19-35 per cell) grew fastest and weighed the most. Growth was also correlated with amounts of rat hormone messenger RNA. However, the experiment only worked if the injected DNA was placed under the influence of an endogenous control system, which was the liver cell in Palmiter's experiments. Rubin and Spradling (1982, 1983) overcame the genetic instability that often accompanies transgenic experiments by using a transposable element as a vector. Although not strictly a transgenic system because all components came from the same organism, the structural gene for xanthine dehydrogenase (XDH) was inserted into a P element. The fused P element was injected into Drosophila embryos, of which 8% survived treatment. Approximately 39% of the survivors contained the transposon. These reverted from rosy eye color to wild-type as a consequence of the active XDH gene. The transposition seemed to insert randomly in germ-line chromosomes. The reversions and integrations were stable in subsequent generations.

3.4.2. Differentiation in plants Appropriately, a gene transfer system is being developed for plants, particularly in maize, in which McClintock discovered transposition nearly 40 years ago (Marx, 1983). McClintock's maize transposon system, termed Ac-Ds, apparently behaves like other eukaryotic transposons such as the P element of Drosophila. The Ac and Ds elements

43 have recently been isolated (Federoff et al., 1983). This system could complement the dicotyledenous plant systems, which uses the Ti plasmid from the bacterium, Agrobacterium tumefaciens (Ream and Gordon, 1982). The Ti plasmid system has already been used for introducing genetically stable, bacterial antibiotic-resistant genes into plant hosts. Dell-Chilton and colleagues (Barton et al., 1983) analyzed the transforming properties of the Ti plasmid related to the conversion of infected, tumorous masses into healthy, intact plant tissue. They have shown that the introduction of DNA markers into the 'rooty' locus of the plasmid causes attenuated crown gall tumors, whose cells can regenerate as normal plant cells. Willmitzer et al. (1983) studied Ti transcripts and found that several independent promoter regions in the plasmid are probably recognized by the plant RNA polymerase II. Joos et al. (1983) found that no single T-DNA region controls stable tumor formation. Rather, 3 plasmid genes have been identified that contribute to tumorous growth. Two of these genes suppress shoot formation and stimulate root formation and one gene does just the opposite. The authors suggest that the crown gall system is unique in that plant development seems to be controlled by a few plasmid genes. Such tight regulation may offer important clues for understanding the proper development of the host plant. As reported by Drummond (1983), Herrera-Estrella et al. (1983) have achieved a major developmental breakthrough by constructing chimeric genes in Ti plasmids that are expressed in normal plant cells. The genes are a combination of a bacterial or plant structural gene and the promoter sequence for nopaline synthetase. This was the first successful demonstration of the transfer and expression of foreign genes in plant cells.

3.4.3. Interspecies gene transfer Evolutionary biology has never been a tranquil discipline, and a recent molecular finding will keep debate well fueled. Lewin (1982) has reported that horizontal gene transfer among species may occur in natural populations. Interspecies transfer has been documented in several biological systems. One is the system just discussed, the Ti plasmid of A. tumefaciens and crown gall tumor. Another system stems from the work of Birnstiel (Buss-

linger et al., 1982) and Britten and Davidson (Jacobs et al., 1983) with the sea urchin, a wellstudied experimental organism in molecular biology. Both groups have suggestive evidence in various species of strong homology for certain repeat sequences. Although nothing definitive is yet known, viral translocation is being investigated as a possible mechanism. Interspecies genetic transfer is not confined to the nuclear genome. Using endonuclease restriction fragment analysis, Ferris et al. (1983) have shown that the wild Scandinavian mouse, Mus musculus, possesses the mitochondrial DNA of a neighboring species, M. domesticus.

3.4.4. Movement of organellar DNA Organellar DNA may be quite mobile within the cell (Lewin, 1982, 1983b) and has earned the designation 'promiscuous DNA' to account for this mobility. Stern and Lonsdale (1982) found a 12-kb sequence of DNA to be common in both the chloroplast and mitochondria of maize. The extensive homology between the two organellar sequences suggests that recombination or transposition had occurred. Evidence also suggests that virus-like DNA may act as vectors. Both linear transposon-like elements and small, circular DNAs have been found in maize chloroplasts. Farrelly and Butow (1983) have discovered a contiguous DNA sequence in the nuclear genome of yeast that is highly homologous to non-contiguous yeast mitochondrial DNA (mtDNA). No transcripts of this DNA have been discovered, and the sequence contains numerous termination signals in many reading frames. The authors speculate that petite mtDNA contributed the nuclear component. Moreover, because of the proximity of the nuclear mtDNA to two tandem copies of the yeast transposon Ty, which accounts for the sequence polymorphism observed, vector-mediated mobility is a distinct possibility. 3.5. Reversed genetics as an analytic and synthetic tool The ability to clone genes in microorganisms such as E. coli and to reintroduce them in the original organism has permitted the production of precisely defined mutations in structural or regula-

44

tory sequences. This process is called reversed genetics and allows directed alterations of the gene products or their regulation. Because transformation or transfection has potential industrial importance (Chater, 1982), strain improvement using these methods has become practical and desirable. After a gene is cloned in a microorganism, it may be amplified in that host and manipulated in vitro. Such manipulations may cause deletions, insertions, or base substitutions. Base substitutions can be produced by treating DNA with specific mutagens in vitro or in vivo, or by replacing existing nucleotides (Weiher and Schaller, 1982). In this context, it is important to mention that recent achievements in solid-phase technology make it possible to synthesize in one step defined oligonucleotide sequences up to 20-30 nucleotides long. By synthesizing a number of complementary oligonucleotides and enzymatically joining the fragments, entire genes can be assembled (Smith et al., 1982; Edge et al., 1981; Brousseau et al., 1982). Indeed, using a variety of techniques, Murray and Szostak (1983) have constructed a 55-kb artificial chromosome in yeast. The chromosome is a linear plasmid that contains cloned genes, replicators, centromeres, and telomeres. Deletions or insertions can be created by introducing cuts at specific sites in the DNA by sitespecific restriction enzymes and removing or inserting specific sequences where the DNA was cleaved. Correlation of changes in the properties of a protein with alterations in cell structure and function may deepen our insights into the relationships between proteins and the properties of cells. Alterations of small regions in proteins by the technique of reversed genetics or by replacement of larger regions by in vitro DNA recombination are expected to yield proteins which are better suited for specific purposes than are the parental proteins. Site-directed mutagenesis of regulatory sequences may be of great value in understanding control mechanisms in higher organisms and improving production rates of desired proteins. For some proteins, relatively large regions may be deleted or substituted without loss of function. Such alterations may, however, affect the specificity of action. By genetic manipulation of genes coding for the human a-interferon (IF-a), hybrid IF-a molecules have been obtained with properties

different from those of the parent molecules. These results may have very practical applications. The IF-a family comprises 8 or more species with only slightly different amino acid composition, each having a characteristic specificity of anti-viral activity on particular target cells (Nagata et al., 1980; Goeddel et al., 1981). By constructing hybrid IF-a molecules Streuli et al. (1980) and Weck et al. (1981) have shown that the specificity for human cells resides in the NHz-proximal half of the IF molecule, whereas for mouse cells the specificity appears to reside in the COOH-proximal half of the IF molecule.

3.6. Antigenic determinants and the development of synthetic vaccines Recombinant DNA techniques combined with the chemical synthesis of oligopeptides have already been applied to immunology. The production of antibodies against pathogenic bacteria and viruses has often been difficult. Pathogens may be difficult to isolate and culture and their proteins may be even more difficult to purify. A pathogen's defenses against a host's immunological attack lead to further difficulties, especially when antigenic determinants fluctuate. Lerner and colleagues (Lerner et al., 1981; Lerner, 1982, 1983) have developed an interesting strategy for producing viral vaccines from synthetic peptides whose amino acid sequences have been deduced from DNA sequence data. Nucleotide sequences of viral capsid genes (and regions with unknown coding capabilities) predict the amino acid sequences of viral capsid proteins. Small peptides as short as 7 amino acids have been synthesized by solid-state methods, attached to an immunogenic protein carrier, and injected into animals. The results from this strategy have had both practical and theoretical impact (Lerner, 1982). An example of the latter is the finding that most globular proteins contain fewer than 5 antigenic sites, and these antigenic determinants depend on tertiary structure that brings remote regions of protein chains together. Lerner and colleagues have shown that small synthetic peptides that collectively may represent most of the globular surface of a protein can elicit antibodies to that parent

45 protein. The conclusion of this group is that the immunogenicity of a globular protein is less than the sum of its fragments. Thus, even during a vigorous immunological response against a virus, the regions of a capsid protein represented by these synthetic peptides may not be immunogenic. By immunizing with synthetic peptides, one can create an antibody with a specificity that is not generated in vivo. Invariant regions of viral capsid proteins that would not be subject to mutational (and, thus, antigenic) change because of functional properties would be particularly important targets. Bittle et al. (1982) have made practical contributions using this strategy. They sought to generate serotype-specific antibodies to the foot-andmouth disease virus by using chemically synthesized peptides whose amino acid sequences were deduced from the nucleotide sequence of the viral genome. The group took advantage of knowledge of the complete sequence of the viral genome and of one of the viral capsid proteins (VP1), which was known to carry an important antigenic determinant. A set of synthetic peptides was constructed by reading the nucleotide sequence of the VP1 gene. One peptide, which corresponded to the middle of the protein and was known to display marked variation among serotypes, produced neutralizing antibody and protection of animals against infection.

Acknowledgements The authors would like to acknowledge the help of Dr. F.H. Sobels and Dr. J.C.J. Eeken in preparing the section on transposition phenomena in Drosophila. Glossary Amplification: (i) treatment (e.g., chloramphenicol) designed to increase the proportion of plasmid DNA relative to genomic DNA; (ii) replication of a gene library in bulk; tandem increase of DNA sequences thought due to recombination processes Annealing: see: hybridization Blunt (flush) ends: DNA molecules containing double-stranded termini which can be joined end-to-end with DNA ligase bp: base pairs

Capped 5' ends: the 5' ends of eukaryotic mRNAs are modified post-transcriptionally to the general structure: mTG(5')ppp(5')Nmp .... where m7G represents a 7-methyl-guanosine residue and Nm a 2'-O-methylated nucleoside cDNA (complementary DNA): a single-stranded DNA that has been synthesized from an RNA template as an exact copy by the enzyme reverse transcriptase; used for cloning or as a specific hybridization probe Cistron: a DNA fragment that codes for a particular polypeptide Codon: a group of 3 adjacent nucleotides that codes for an amino acid Cohesive ends: DNA molecules with singlestranded ('overhanging') complementary ends Cut: a double-strand scission in the polynucleotide duplex, in contrast to the single-strand 'nick' Ends: due to the inherent polarity of nucleic acid single strands, two different types of ends (defined relative to the carbon atoms in the sugar moiety) exist: the 5' end (usually carrying a phosphate group) and the 3' end (normally carrying a hydroxyl group) Excision: the enzymatic removal of a nucleotide or polynucleotide fragment from a nucleic acid polymer Exon: portion of DNA that codes for the translated mRNA Gap: a region in a double-stranded DNA molecule where at least one nucleotide in one of the strands is missing Gene library: random collection of cloned DNA fragments in a set of vectors that ideally includes all the genetic information of a given species (sometimes called shot-gun collection) Hairpin loop: a region of double-stranded nucleic acid formed by base-pairing within a single strand of DNA or RNA that has folded back on itself Heteroduplex: a DNA molecule formed by basepairing between two strands that are not completely complementary Hybridization: process of forming double-stranded nucleic acid structures between two single strands with complementary nucleotide sequences Integration: insertion of a DNA fragment by recombination; usually used to describe insertion of viruses or transposons

46 Intron: intervening sequence; a portion of a gene that is transcribed but does not appear in the translated m R N A transcript kb: kilobases (10 3 bp) Ligase: catalyzes the formation of a phosphodiester bond at the site of a single-strand break in a duplex D N A (RNA can also act as a substrate to some extent); enzyme is used for splicing D N A molecules from various sources via cohesive or blunt ends Linker: a small fragment of synthetic D N A that has a restriction site useful for insertion of an exogenous piece of DNA Nick: a region in double-stranded DNA where the phosphate-sugar backbone is hydrolyzed, but with all the complementary nucleotides still in place Nick translation: procedure for labeling D N A in vitro using D N A polymerase I, which initiates synthesis at single or multiple nicks Ori (origin): point or region where D N A replication begins Plasmid: extrachromosomal, autonomously replicating, circular DNA segment Polyadenylation: non-transcriptive addition of a polyadenylate sequence to the 3' end of eukaryotic m R N A Polymerase: enzyme that catalyzes the assembly of nucleotides into RNA and of deoxynucleotides into D N A Primary structure: linear nucleotide sequence of a nucleic acid polymer Probe (hybridization): a small ( < 1000 bp) labeled D N A or RNA molecule used to detect complementary nucleic acid sequence by hybridization Pseudogene: a nucleotide sequence highly homologous to a functional gene; it appears to have no phenotype and could have originated by duplication of that functional gene Restriction enzyme: a nucleotide-sequence-specific endonuclease Restriction site: a specific nucleotide sequence (in general 4 - 6 base pairs with dyad symmetry) recognized and cleaved by a restriction enzyme Retrovirus: oncogenic RNA-containing virus that replicates through a DNA intermediate necessitating the use of an RNA-dependent D N A polymerase (i.e. reverse transcriptase) R-loop: a three-stranded structure formed by the

displacement and annealment of one strand of duplex D N A by complementary RNA, leaving a DNA loop with a characteristic appearance in the electron microscope (if the displacing nucleic acid is a piece of DNA, the corresponding structures are called 'D-loops') Splicing: (i) gene splicing: manipulations which attach one DNA molecule to another; (ii) RNA splicing: removal of introns from mRNA precursors Tailing: non-transcriptive addition of homopolymeric nucleotide sequences to the 3' end of DNA with the aid of the enzyme terminal transferase Transcription: formation of RNA from a DNA template Transduction: transfer of genetic material from one cell to another by means of a viral vector Transfection: transfer of genetic information into a cell by a phage (virus) Transformation: transfer of D N A into a cell by physico-chemical means Translation: the process in which the genetic code contained in m R N A directs the order of amino acids in the formation of polypeptide Transposon: a D N A element that can insert into plasmids or the cellular chromosome independently of the host cell recombination system; transposons carry genes involved in insertion and (in bacteria) antibiotic resistance Vector: an agent that replicates autonomously and may be used to transfer and amplify particular genes or DNA segments

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