MSU LlBRARlES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. au- - ---t.. PART I. STUDIES ON THE HAMSTER RIBONUCLEOTIDE REDUCTASE GENES PART II. CONSTRUCTION OF MUTATIONS IN THE CHICKEN ADULT ALPHA GLOBIN GENES By Paul F. Bates A DISSERTATION Submitted to Michigan State University in partial fulfiilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1985 ABSTRACT I. STUDIES ON THE HAMSTER RIBONUCLEOTIDE REDUCTASE GENES II. CONSTRUCTION OF MUTATIONS IN THE CHICKEN ADULT ALPHA GLOBIN GENES By Paul F. Bates 1. Studies were conducted in an attempt to isolate the hamster ribonucleotide reductase genes. A majority of the work described utilized DNA-mediated gene transfer techniques in an attempt to transfer and then specifically isolate the ribonucleotide reductase gene. The donor DNAs in the gene transfer experiments were from either hamster HUT-2 cells which contain an altered ribonucleotide reductase activity which confers a dominant hydroxyurea-resistant phenotype or hamster L3-107 cells which contain elevated ribonucleotide reductase levels and have an aphidicolin-resistant phenotype. Recipient cell lines in the gene transfer studies included chinese hamster ovary cells, hamster V79 tk‘ cells, rat 3 tk’ cells, mouse L tk‘aprt‘ cells, and mouse LM cells. Neither the hydroxyurea-resistant nor the aphidicolin-resistant phenotype was transferred in these studies using any of the recipient cell lines or donor DNAs. Based on the number of cells transfected it was estimated that the transformation frequency of the hydroxyurea-resistant phenotype is less than 2 x 10'9. It was estimated from parallel studies with thymidine kinase phenotype transfer, that the frequency of hydroxyurea-resistant phenotype transfer is at least 1.5 x 103 fold lower than that for thymidine kinase phenotype transfer. In other studies cloned DNAs containing the herpes simplex virus I and II ribonucleotide reductase genes were used as hybridization probes in Southern blotting experiments with hamster genomic DNA. Although many different stringencies were employed in these studies, specific hybridization of the herpes virus ribonucleotide redcuctase probes to the hamster genomic DNA was not seen. These experiments suggest that the hamster and herpes virus ribonucleotide reductase genes do not share sufficient homology at the nucleic acid level to be detected by hybridization. II. Nucleotide sequence analysis has not allowed identification of the promoter elements of the chicken adult alpha globin genes, aA and up. Therefore, several series of deletion mutants of the putative promotor regions of these two genes were constructed using plasmid clones. These deletions extend into the putative promoter regions from both the 5' and 3' directions, and were constructed in such a manner that they can be recombined for use in so-called linker-scanning mutagenesis studies. -To my parents, for the genes and the environment that sparked my interest in science. -To John Boezi, for his infectious enthusiasm and love for research. -To Karen, for the love and encouragement that pushed me over the top when things were looking bleak. ii ACKNOWLEDGEMENTS Persons who deserve acknowledgement Jerry Dodgson - intellectual and financial support, Friday afternoon beer Ed Frtisch - "the" cloning course, enthusiasm in lab Hsing-Jien Kung - ideas and basketball scores John Burczak - make life in E.L. shall we say "memorable" Dan Robinson - ibid Wynne Lewis - tissue culture help, discussions on life, “my dad said..." Karen Friderici - lots of general discussions, great food and parties MaryBeth Raines - great volleyball and lots of fun in lab and out Paul Boyer - late-night lab partner, constant supply of munchies Michelle Fluck - only member of my committee to "stick it out" for 5 years Sue Conrad - new insights on old problems, list of SF restaurants "Sticky Ends" - allowed me to play shortstop (maybe short"drop") "Fubars" - 2nd place isn't so bad “the lab group" - Dave 6., Dave B., Corrinne, Mark, JD, Sara, Dave B., Claudette, Larry, David H., Kristen, etc. Julie Doll - Somehow deciphered my writing Theresa Fillwock and Betty Brazier - cut through the red tape many times iii TABLE OF CONTENTS Page DEDICATIONOOOOOOOOO 0000000000000000000000000 O OOOOOOOOOOOOOOOOOOO OOOOii ACKNOWLEDGEMENTS... ......... .... ................... ........... ..... iii TABLE OF CONTENTS.. ...... ....................... ..... ...............iv LIST OF FIGURES. ...... .... ........................................... v LIST OF TABLES ............ . ..... ...... ......... . ................ .....i PART I LITERATURE REVIEW...... ....... .. .......................... ..... ...... 1 History of Project..............................................1 Ribonucleotide Reductase........... .............. . .............. 2 Gene Amplification.............................................10 Gene Transfer..................................................13 MATERIALS AND METHODS.................. ..... ........................19 Materials......................................................19 Cell Culture...................................................19 RNA Extraction........................... ......... .............20 DNA Extraction.................................................21 cDNA Synthesis.................................................21 Enrichment of Aphidicolin-Resistant Cell SpECific CDNA sequenCESOOOOOOOOOOOOOOOOOOO0.000.000.00.000.000021 Lamda Library Construction and Screening.......................22 DNA MediatEd Gene TranSferOOOOOOOO0.000.000.0000...0.000.000.0022 RESULTSOOOOOOOOOOOOOO...O0....O...0.00....0.0.0.0.00.00.00.00000000024 IntrOdUCtionooto0000onoooooooooooooooooooooooo00000000000000.0024 iv Page cDNA Enrichment................................................24 Differential Screening of Southern Blots.......................27 Differential Screening of Lamda Libraries......................27 Gene Transfer Experiments............................... ..... ..30 Gene Transfer: Selection for APH Resistance....................35 Gene Transfer: Selection for HU Resistance.....................37 Isolation of Higher Level HUT-2 Mutants........................4O Gene Transfer: Experiments with HUT-2 (300) DNAOOOOO0.0......0.00.00.00.00...0.0.0.0....OOOOOOOOOOOOOOO0.0.43 Serial Selection with G418 then HU.............................45 Herpes Simplex Virus Ribonucleotide RadUCtase PrObESOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.000... ..... .046 DISCUSSIONOOOOOOOOOOOOOOOOOOOOOOOOO. ...... .00...OOOOOOOOOOOOOOOOOOOOSO REFERENCESOOOO...OOOOOOOOOO0.0.000.00.0000...OOOOOOOOOOOOOOOOO0.0.0.55 PART II LITERATURE REVIEwOOOOOOOOOOOOOOOOOOO000......OOOOOOOOOOOOOOOOO0.00.059 BaCkground.OOOOOOOOOOO...O...0.00.0...0..0.00.00.00.0000000000059 ChiCkenaGIObin GeneSoooooooooooooooonooncoco-00.000000000000060 Eukaryotic Polymerase II Promoters.............................62 LE Vitro MutageneSiSooo00000000000000.000000000000000000.00000066 MATERIALS AND METHODSOOOOOOOOO0.....0...00......0.0.0.0000000000000070 Materia15000000000000000......OOOOOOOOOOOOOOOOOOOOO0.0.00.00.0070 MethOdSOOOCOOOOIOOOOOOOOOOOOOOOOOOOOOO0.000.0.0000000000000000070 RESULTSOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOO0..0.0.0.000000000000000071 up: Construction of 5' Deletions...............................71 Sequencing of a0 5' Deletion Mutants...........................74 Location of Linkers in A5 and A10 Clones.......................79 an: Construction of 3' Deletions...............................86 v Page on: Sequencing 3' Deletion Mutants.............................89 Construction of “A 5' Deletions................................92 DISCUSSION............ ....... . ................... ... ............. ..100 REFERENCES.........................................................104 APPENDIX I........... .............. .... ........ .. ............... ...106 APPENDIX II.............. ..... ........... ..... ... ........... . ...... 120 vi Figure Ohm->0) LIST OF FIGURES PART I Proposed model for the structure of the mammalian ribonucleotide reductase.......................4 Model for the allosteric regulation of ribonuc'IEOtide rEductaSEOOOOO0.0.00.0.0...0.0.000000000006 mRNA:CDNA hYbridization SChemeOO.......OOOOOOOOOOO0.0.0.26 Enriched cDNA: Southern blots...........................29 cosmid rescue SChemEOOOOOOOOOOOOOOOOO0.000.000.00000000034 Comparison of mouse L tk'aprt' and CHO cell plating efficiencies in aphidicolin.....................39 Herpes simplex virus 1 and 2 ribonucleotide redUCtase mapSOO0.0000000000000.00.0.0000......0.0.0....49 PART-II Chromosomal arrangement of the chicken a 910bin geneSOOOOOOOOOOOOOOO0.0.0.0.00...0.00.00.00.00061 Typical eukaryotic RNA polymerase II transcription unitOOOOOOOOOOOOOOOO00......0.0.0.0000000064 Example of linker scanning mutagenesis..................68 Scheme for construction of an 5' deletions..............72 Strategy for sequencing up 5' deletion clones using a single sequencing reaction......................76 Sequencing gel of a0 5' deletion clones by sequenCing reaction..................................COO78 Scheme for mapping up 5' deletion clones: One fragment length determination...........................81 Sequencing gel of a0 5' deletion clone Xho I/Pvu II Iabe11ed fragmentSOOOOOOOOOOO000.00.000.00.000000000083 vii Figure Page 9 Summary of o0 5' deletions..............................85 10 Construction of a0 3' deletion mutants..................87 11 Sequencing strategy for up 3' deletion clones...........91 12 ‘Summary of o0 3' deletions..............................94 13 Construction of aA 5' deletion mutants..................96 14 Estimated locations of aA 5' deletion clone endpolints in both direCtionSooooo0000000000ooooooo00000.99 viii Table LIST OF TABLES 3393 PART I Gene amplification in drug resistant cells..............11 Gene amplification in tumors and tumor ce111ines.00.00.000.000....OOOOOOOOOO0.0 00000 O. 00000000 14 Lamda library construction data.........................31 DNA mediated transformation: APH selection results......36 Hydroxyurea sensitivity of various cell lines...........41 DNA mediated gene transfer: Hydroxyurea SEIECtionSoooooooooooo00000000000000.0000 0000000000 0.0.042 DNA mediated gene transfer with HUT-2(300 DNA...............0............OOOOOOOOOOOOOO0.00.00.00.44 ix PART I STUDIES ON THE HAMSTER RIBONUCLEOTIDE REDUCTASE GENES LITERATURE REVIEW History of Project In prokaryotic systems mutations have been used extensively to dissect the processes involved in DNA replication. Similarly, it was believed that generation of mutations in the genes involved in DNA replication in eukaryotic cells would aid in analysis of their mode of DNA replication. To this end Dr. John Boezi's laboratory isolated a series of Chinese hamster ovary (CHO) cell mutants resistant to the drug aphidicolin (APH). Since APH had been shown to be a specific inhibitor of DNA polymerase o (Ikegami gt 11., 1978), it was thought that the APH resistant mutants would contain alterations in the polymerase enzyme. It was hoped these mutants would help clarify the role of polymerase a in replication and/or repair. Upon characterization, these APH resistant cells showed no change in polymerase a activity, but instead appeared to have increased levels of the enzyme ribonucleotide reductase (for a detailed review see Appendix 1). This led to the following suggestion regarding the mechanism of APH resistance in these cells: APH inhibits o-polymerase via competition with dCTP (Oguro gt_gl., 1979); ribonucleotide reductase (RdRase) activity controls the cellular pool sizes of all the four dNTP's (Thelander and Reichard, 1979); thus by increasing the activity of RdRase in the cell and raising the pool sizes of the dNTP's (especially dCTP) the inhibition of o-polymerase by APH is relieved by dCTP competition. In agreement with this theory, increased resistance 2 to APH was correlated with larger dNTP pools and higher levels of RdRase activity (Sabourin gt 21- (1981). Although there are several mechanisms whereby the enzymatic activity of RdRase could be increased, the manner in which these APH resistant mutants were selected suggested a specific mechanism might be responsible for the increased RdRase levels. CHO mutants resistant to high levels of APH were isolated by a series of selections in gradually increasing levels of the drug. Attempts to isolate such mutants by exposure to a single large dose of drug were unsuccessful (Sabourin gt .21., 1981). For numerous drugs such stepwise selection has been shown to cause gene amplification (see section on gene amplification for detailed review). The intriguing possibility existed that these cells had amplified the RdRase genes; therefore it was decided to attempt to characterize these APH resistant mutants at the molecular level, specifically by isolation of the RdRase genes. When this work was initiated only two documented cases of gene amplification existed in mammalian cells and nothing was known of the mechanisms involved in amplification. Also veny few genes coding for low abundance (sometimes called ”housekeeping") messages had been cloned. The RdRase gene was interesting since it offered the possibility of research in both of these areas. Ribonucleotide.Reductase Ribonucleotide reductase (ECI.17.4.1) is responsible for the conversion of all four ribonucleoside diphosphates to the corresponding deoxynucleotides required for DNA synthesis. The level of enzyme 3 activity is tightly coupled to DNA synthesis (Peterson and Moure, 1976) and has been shown to be cell cycle regulated (Kucera gt 31., 1983). Evidence suggests that this activity is the rate limiting step in DNA replication, and modulation of RdRase activity plays a key role in the regulation of cell division (Cory gt 21- 1975, Lewis et 31., 1977). RdRase levels are very low in non-dividing cells or tissues and are elevated in rapidly growing tissues such as regenerating liver (Larsson, 1969) or malignant growths (Elford gt 31., 1970). The results of experiments with protein synthesis inhibitors suggest that the half-life of RdRase activity is only two hours, and the level of RdRase activity is at least partially controlled by turnover of the enzyme (Turner gt 31., 1968; Elford, 1972). In nearly all organisms examined, the structure of the RdRase enzyme consists of two non-identical subunits with a molecular weight of between 250,000 and 280,000. Each subunit is a dimer of identical polypeptides, thus the enzyme is an an, as complex (see Figure 1). The on dimer is generally referred to as the M1 or 81 subunit in the mammalian and bacterial systems respectively. Similarly the as dimer is called the M2 or 82 subunit (for a review see Thelander and Reichard, 1979). Not only is the overall architecture of RdRase highly conserved, but the proposed subunit functions are also very similar between the bacterial and mammalian enzymes. The M1 subunit is thought to bind the allosteric effectors which regulate the activity and specificity of the enzyme. M2 contains the tyrosine free radical essential for enzyme activity (Thelander gt 31., 1980). It is unclear whether the catalytic site is contained on one of the subunits or whether it lies between Ri~v lib]. Figure 1. Substrate Opacificity (ATP, dATP, d‘l’TF: dCTP ) M1 Activity (ATP, dATP) Proposed model for the structure of the mamalian ribonucleotide reductase. The M1 (a,a) subunit contains the two effector binding sites. The substrate specificity site controls the selection of the NDP to be reduced, while the activity site controls the overall catalytic activity of the enzyme. The M2 (8.8) subunit contains the tyrosine free radical required for enzyme activity. (From Thelander and Reichard, 1979). 5 them. Photoaffinity labelling of RdRase with [32P]CDP shows that M1 contains a site involved in substrate binding (Caras gt 31., 1983). The role of M2 in the formation of the substrate binding catalytic site may be direct or indirect (for example M2 may induce a conformational change in M1). The fact that M2 contains the tyrosyl free radical required for RdRase activity argues for a more direct role of M2 in catalysis. RdRase exhibits a complex set of strict allosteric controls based upon the levels of ribo- and deoxyribonucleotides in the cell (Figure 2). (For a review see Thelander and Reichard, 1979). Models for the allosteric effects support the theony that the RdRase contains at least two types of effector binding sites that are distinct from the catalytic site. Studies with the E. 2211 enzyme suggest that one of these effector sites controls overall enzyme activity while the other determines substrate specificity (Brown and Reichard, 1969). Photoaffinity labelling of the effector sites of the mammalian enzyme suggests they are located on M1 (Caras and Martin, 1982). It is noteworthy that the allosteric control mechanism of RdRase from E, ggflj_ and mammals is almost indistinguishable (Thelander and Reichard, 1979). The E. ggll_ribonucleotide reductase has been purified to homogeneity (Erikson gt 31., 1977) and the structural genes (nrd A and nrd B) have been defined by mutational analysis (Fuchs, 1977). Recently the g. 2211 nrd A and nrd B genes have been cloned and their nucleotide sequence completely determined (Carlson.gt‘gl., 1984). Interestingly, it has been shown that perturbations in E, £211_DNA replication lead to increased levels of the polycistronic nrd mRNA Figure 2. COP UDP ADP ,ANP ‘» dCDP ——-§ dCTP 1 _‘ wouop +++cmP “are '''''' :J-‘YH' " 'l ' 'Lp (MD? ——> one A —-> DNA Model for the allosteric regulation of ribonucleotide reductase in mammalian cells. The broken lines represent positive effectors whereas the open boxes are negative effectors. For example, ATP stimulates GDP and UDP reduction while dATP has a negative effect on reduction of all four NDPs. (From Thelander and Reichard, 1979.) suggesting that in E. .2211 the RdRase activity is controlled at the level of mRNA transcription (Hanke and Fuchs, 1983). The mammalian RdRase has proven very difficult to purify. Only recently have pure preparations of the M1 subunit been obtained (Thelander st 31., 1980). The M2 subunit has never been purified and appears to be very labile (Thelander gt_gl., 1980). This instability of M2 may be due to degradation of the protein itself or destruction of the tyrosine free radical in M2 which is required for enzyme activity (Akerblom gt 21., 1981). Purification schemes which avoid separation of the M1 and M2 subunits always yield M2 in low, substoichiometric amounts (Thelander gt 21., 1980) suggesting that the M2 protein and not just the free-radical is unstable. In fact, two separate studies have suggested the M2 is the RdRase rate controlling subunit and regulates the overall levels of activity. In one report, immunoprecipitation of M1 showed that the levels of this subunit were fairly constant over the cell cycle, while M2 activity was decreased in 61 cells and high in S phase cells (Eriksson and Martin, 1981). The other study used a mutant mouse 3T6 cell line iwhich showed a 15-fold increase in RdRase activity. Extracts from these cells showed no increase in M1 protein when compared to wild type 3T6 cells. Only the M2 activity in the mutant cells was increased. Also the amount of M2 tyrosine free radical, as measured by electron paramagnetic resonance (EPR) spectroscopy, was greatly increased in the mutant cells (Akerblam gt 31., 1981). Many viruses which require a supply of dNTP's for replication have been shown to either code for their own RdRase or induce the cellular €"Z.Yme. These viral enzymes also exhibit many of the similar 8 properties observed in both the bacterial and eukaryotic RdRase enzymes. In the g. £211 system, the T4 bacteriophage-encoded RdRase has been described (Berlund, 1972). Like the E. ggli and the mammalian enzymes, it also has an an, 38 configuration, and the subunit sizes of 160,000 (on) and 80,000 (68) are also very similar. Unlike the other enzymes, T4 RdRase responds to only positive allosteric effectors (affecting substrate specificity). It does not respond to dATP as a negative regulator (Berglund, 1972). This pattern of response can be explained if the T4-encoded RdRase lacks the activity sites on the model in Figure 1. Another similarity demonstrated by the T4 RdRase is the presence of a tyrosine free radical in the B2 subunit. Hyperfine EPR spectra show the geometry of the tyrosine free radical is very similar between T4, E. £331, and mamalian RdRase (Graslund 31331., 1982). Infection of mammalian cells with herpes simplex virus (HSV) also induces RdRase activity. This RdRase has recently been shown by genetic (Bachetti, 1983) and biochemical data to be encoded by the viral genome (Lankinen gt 31., 1982). Like the T4-encoded enzyme, the herpesvirus RdRase appears to lack the negative allosteric effector sites. The EPR spectra of HSV infected cells was similar to, but distinct from, that of either the T4 or mammalian enzymes (Lankinen gt ‘31., 1982) suggesting a similar environment around the tyrosine free radical. Genetic mapping studies place the HSV RdRase genes in a region of the genome which codes for two polypeptides; one of MN 140,000 and the other of MN 38,000 (Draper gt 31., 1982). Recently the homologous region of the Epstein-Barr virus (EBV) genome has been $93. 9 sequenced. Coding regions for 35 and 93 kilodalton (Kd) proteins were found (Baer gt 51., 1984). These proteins were highly homologous to the 38 and 140 Kd HSV proteins (Gibson gt 31., 1984). Comparison of the amino acid sequences of the EBV and E, 9211 polypeptides has shown that the 93 Kd protein shares extensive homology with the B1 subunit (nrd A gene product). However no high degree of homology could be demonstrated between the 38Kd EBV protein and the 82 subunit (nrd B gene product) (J. Campbell, personal communication). RdRase activity can also be induced in mammalian cells by infection of non-dividing tissue culture cells with DNA tumor viruses that stimulate DNA replication (Linberg gt 31., 1967) or by treatment of growth arrested cells with certain mitogens (Tyrsted, 1984). In the case of viral induction, the virus must be inducing the cellular enzyme since the viral genome contains no coding regions for its own RdRase. Induction of other enzymes involved in DNA replication, including thymidine kinase, DNA polymerase a, thymidylate synthetase, and dihydrofolate reductase, has also been seen with both viruses and mitogens (Tyrsted, 1984; Linberg gt gl., 1969). The mechanism by which viruses induce enzymes has not been elucidated. Use of mutants of RdRase in mammalian cells has allowed some preliminary information on this enzyme to be deduced. In general two main classes of RdRase mutants have been described in mammalian cells. These include: a) mutants with altered allosteric control such that they are insensitive to dATP inhibition (Meuth gt 31., 1976; Eriksson gt 91., 1981), and b) mutants which are resistant to the drug hydroxy- urea (HU) (Lewis and Wright, 1974; Akerblom gtugl., 1981). Hydroxyurea is a specific inhibitor of RdRase and is thought to act by destroying 10 the tyrosine free radical of the M2 subunit. Lewis and Wright have isolated a CHO cell line which by genetic and biochemical data appears to produce an altered RdRase M2 subunit which is more resistant to H0. In cell fusion studies this RdRase gene acts as a dominant marker. This cell line is called HUT-2 (Lewis and Wright, 1978) and is resistant to low (75 ug/ml) concentrations of HU. Another type of H0” mutant was isolated by Akerblom 35 21. (1981). Unlike the HUT-2 mutants, these mutants are resistant to very high levels of H0 (about 450 ug/ml). These highly resistant mutants could only be selected by continuously culturing the cells in slowly increasing HU concentrations over a period of two years. These mutants appear to overproduce a normal RdRase. Gene amplification In the mid 1970's mammalian cells were used increasingly for the study of gene expression. Many of these studies used resistance to specific inhibitors of enzymes essential for cell growth to select cell lines which overproduce the target enzyme. In several cases the drugs being used as selective agents were cancer chemotherapeutic drugs, and resistance to the drug was found in both cultured cells and cells originating from human neoplasias. In 1978, Alt gt 31. reported that resistance of a cell line to the drug methotrexate was caused by a selective amplification of the genes encoding the target enzyme, dihydrofolate reductase (DHFR) and the resulting overproduction of DHFR. Since then numerous examples have been demonstrated of cells in which there is a substantial elevation in the levels of an enzyme in response to selective pressure (Table I). In I I f 4‘ fl.‘ I! ..iLl II; I', IE ‘I I. Table 1. Gene amplification in drug-resistant cellsa ‘F‘".D ,~,.‘..-~I.. .o‘iv~to'. ~~~\r\r'\’ ‘ 11 0‘. ‘0'. v Drug(s) Enzyme or binding protein Methotrexate *DHFR (two distinct genes) Methotrexate *Bifunctional thymidylate synthetase dihydrofolate reductase (Leishmania) PALA *CAD Cadmium *Metallothionein I Copper *Copper-chelatin (yeast) 6-Azauridine or pyrazofurin Adenosine, alanosine, and thymidine ("HAT") Compactin Methionine sulfoximine 5-Fluorodeoxyuridine a-Methyl-or a—difluoromethyl- ornithine Hydroxyurea or deoxynucleotides Aphidicolin Aphidicolin Adenine and coformycine Albizziin or a-aspartyl 'hydroxamate Mycophenolic acid Tunicamycin Borrelidin Multiple drugs Canavanine *UMP Synthetase *Adenosine deaminase phosphoribosyl transferases *3-Hydroxy-3-methylglutaryl coenzyme A reductase *Glutamine synthetase Thymidylate synthetase Ornithine decarboxylase Ribonucleotide reductase DNA polymerase a (Drosophila) Ribonucleotide redhctase AMP deaminase Asparagine synthetase IMP-5'-dehydrogenase N-Acetyl glucosaminyl transferase Threonyl-tRNA synthetase 170K P-glycoprotein, 19K cytoplasmic protein Argininosuccinate synthetase *Genes have been proven to be amplified by molecular analysis. a) Adapted from Stark and Wahl (1984). 12 about fifty percent of these cases gene amplification has been conclusively demonstrated as the molecular basis for the drug resistant phenotype (for a review on amplification see Stark and Wahl, 1984). One common feature of cell lines which contain amplified genes is that all have been selected by increasing the concentration of the inhibitor in small increments or steps. In unselected populations of cells there are at any given time cells with varying levels of the target enzyme, and, it is believed, low levels of amplification of the gene coding for this protein (Zieg gt 31., 1983; Johnston gt 91., 1983). By initially using low levels of inhibitor, cells with slightly increased levels of enzyme (and presumably slightly amplified genes) can be selected. After allowing for re-amplification of these same genes, mutants with higher levels of enzyme can gradually be selected in a series of steps. Amplification can apparently occur spontaneously at many (or all) locations in the genome; however, not all sites appear to be amplified at the same rate (Wahl 35 $1., 1984). It has been estimated from genetic (Luria-Delbruck fluctuation analysis) and biochemical (fluorescent activated cell sorting) studies that a single DNA segment may be amplified as often as 10"3 to 10'4 times per cell per generation (Zeig gt 31., 1983; Johnston gt 11., 1983). Another feature of gene amplification is that generally large regions surrounding the selected gene are amplified. The amount of DNA co-amplified with the selected gene can be as large as 3000 kilobase pairs (kb) and is generally at least 500 kb (Cowell, 1982). This amplified DNA may be located within chromsosomes in expanded regions termed homogeneously staining regions (HSRs), Or it may be on small IL) 13 acentric chromosome fragments called double minutes (DMs). Generally, amplifications on HSRs are stable whereas DMs tend to segregate randomly during cell division because of a lack of a centromere and are often lost in the absence of selective pressure. It remains unclear whether DM5 and HSRs are interconvertible or are distinct genetic elements (for a review see Cowell, 1982; Biedler gt_al,, 1983). DM5 and HSRs have also been observed in numerous tumors and tumor cell lines implying that amplification may play a role in tumorigenesis (Varmus, 1984). Table 2 lists several known oncogenes which are amplified in tumors or tumor cell lines. Overproduction of normal cellular gene products is thought to be the basis fOr tumorigenesis in several current models (Varmus, 1984). Amplification may allow overproduction of factors which allow cells to proliferate more rapidly, escape immune surveillence, or increase motility for invasion of surrounding tissue. In light of the randomness and size of the regions amplified, the process of “gene amplification“ might more aptly be called “sequence amplification". Clearly if amplification occurs at a rate of 10'4 and the region amplified is 103kb long, then there is a high probability of having a region amplified in a cell with a genome complexity of 3 x 106 kb. Thus it is not surprising that so many examples of amplification have been found. GenenIransfer A recently developed technique allows the efficient transfer of exogenous DNA into eukaryotic cells. This procedure, called DNA mediated gene transfer, relies on the ability of cells in culture to 14 omezm .ocwp Ppmu meo:_ucmu copoo a xo~-e Esooot eee coooe_e . .copou .mcap eo masocwucmu xgmewca e mm: xoo-om Aeu . zo xoe-om Aeu = xofi-m oeoeeexp _emese noosoe_->mu . mm:\zo x0e Ameee.ea>v moe__ ppmuloEo:_ocmu meap p_mu pmem e mm=\ze xoe owmoooo .oee_ _Poo eeoo=e< . mm: xom Loss» xeee_ea nee coo: .mcw_ —pmu owsmx=m_ o_u»uopmxeoca e a xmm-e~ Aozev oo-e= e em: x~m-e~ Amzv own case = so xum Amzv o~m oboe oxe-o mm: xom tease eee moe._ ppwu msoc_ucmu asap _Fmo ppuEm e mm:\zo xoo~-oH noose» steepee oee .me» .oep_ _Peo eeoemepooewoom . mm=\zo xoofl-m A>H new SHH omeomv mueoumopnocsm: xngFLa e Amefivmmz xom um-mzH . a xoofi-om ¢< . so eee Afifiemfivmmz xome-oofi sz as: e Aemefiv mm: xo~-mH sz ~m-azH . 2a nee Ammeovmmz xowa-oofl sz oNH-e=u . Ae~.eevem= me-o~ sz emH-e=u . Amfieavemz xm~-om sz do: . AMHUNH .ofiemvmmx xoefi-o~fi sz eez . Aemfivmmz xoo~-oo~ sz »_.ox ose-z newuwooar.#030mo.=ogzu £0353.» :95. 0.50 9.3.. flame so uosah mammooco mo mucous 69.: ~30 LOEau Ucm 95:59 E. concur: ~95 wcmw .N m—nmh 15 Aemmfiv assee> oee Acmefiv _ee: oee xeeom eoee oooeeo< (\o G. (\o (\o w mmz\zo p t O) xom Hme< .eew_ __oo eeoe_oeeo e_o5eoewem onim mvsmxsm— upopmxs msuo< on mo~\Ho~o4ou .mm:__ ppmu meccvucwu co_ou xofiim Nomx map. ppmu oweoxamp cFopmas upcocgu xofiim smut .mcw— neocpucmo xumsamz xooiom H> .m:_P ppmu aeocpucmu puupucouocmcv< anemic pasta nxsiu Panic mmciz muciwxiu 16 take up DNA which is added as a calcium phosphate-DNA precipitate. The use of this procedure to incorporate DNA into cells was first described by Graham and Van der Eb in 1973 and was used to infect cells with adenovirus DNA. More recently, total cellular DNA was used to transfer the tk+ phenotype to tk' mouse cells. These experiments demonstrated that single copy genes in the eukaryotic genome could be efficiently transferred with this technique (Wigler e£_al., 1978). The efficiency with which single copy genes are transferred using DNA mediated gene transfer varies somewhat, but is usually 0.1 to 10 tranformants per 20 ug donor DNA per 1 X 106 recipient cells (Wigler .gt.al., 1978; Wigler 23.21:, 1979). In other words, with this procedure one in 105 to 107 cells become stably transformed and is isolated by biochemical selection. Since the reversion rate of many mutations is in the range of 10'7-10'8, it should be feasible to use DNA mediated gene transfer for isolation of genes for which there is a biochemical selection. One factor which has been shown to greatly influence the transformation efficiency is the cell line used as the recipient (Graf 35 31., 1979). Mouse L cells and mouse NIH 3T3 cells have been used extensively in transfer experiments because of their high efficiency of transfer. Another factor affecting transformation efficiency is the size of the donor DNA. Genomic DNA fragments greater than 50 kb generally yield the best results. This is apparently due to the high degree of recombination and rearrangement that occurs at the ends of the linear molecules. Perucho gt 31. (1980) demonstrated that upon transformation the host cell ligates the incorporated DNA into :an:. the .‘flfl WW 5p}: u I m '1.) “.8 | J's E39. In: r'f 17 concatameric structures which may be as long as 2000 kb. They coined the term "pekelasome" to describe this concatameric structure. The fact that the transferred DNA is ligated into a single large concatamer bgjggg integration into the host genome, allows transformation of cells with unselectable genes. Inclusion of a selectable marker (often a cloned gene) with the donor DNA makes possible the transfer of genes which by themselves have no phenotypic marker (Wold gt 91., 1979). The application of DNA mediated gene transfer for the isolation of eukaryotic genes relies on two criteria. First, the gene of interest must be selectable or linked to a selectable marker. Secondly, the donor DNA sequences must be discernable against the background of the recipient genome; that is, they must be "tagged" in some fashion with either a physical or biological marker. For example, the donor DNA can be ligated to or transfected with (since ligation will occur in the recipient cell) pBR322 plasmid DNA. After DNA mediated transfer, the replication origin and drug resistance genes of pBR322 may be used to "rescue“ the adjacent donor DNA by their ability to replicate in E. £211 (Lowy gt alL, 1980). Alternatively, naturally occurring repetitive sequences, such as the Alu repeats in human DNA, may be used as a hybridization probe to identify the transferred DNA (Perucho gt 31., 1981). Using procedures such as those described above, numerous eukaryotic genes have been isolated with DNA mediated gene transfer. These include several human oncogenes (for a review see Bishop, 1983), the human, hamster, and chicken thymidine kinase genes (Lewis gt 11., 1983; Perucho gt 31., 1980b; 5. Conrad, personal communication), the 18 hamster and mouse adenine phosphoribosyl transferase genes (Lowy gt .31., 1980; Sikela gt 31., 1983), human DNA repair enzyme genes (Rubin .££.El-: 1985) and many others. Clearly this technique will continue to be useful in the isolation of eukaryotic genes. MATERIALS AND METHODS Materials The aphidicolin used in these studies was supplied by the Developmental Therapeutics Program, Chemotheraphy, National Cancer Institute. G418 was from Scherring Pharmaceuticals. Restriction enzymes were from New England Biolabs, Bethesda Research Labs, or Boehringer Mannheim. Mouse L tk'aprt' cells were from Michael Wigler (Cold Spring Harbor Laboratory). V79tk' cells were from William Lewis (University of Toronto). HUT-2 cells were from James Wright (University of Manitoba). Rat 3 and human 293 cells were from Sue Conrad (Michigan State University). The herpes simplex RdRase plasmid clones were from Sandy Weller (Dana Farber Cancer Institute). Cell Culture Growth of parental-type chinese hamster ovary cells and growth of aphidicolin resistant mutants was as described (Sabourin gt 11., 1981). L-tk',aprt' cells were cultured in Dulbecco Modified Eagle's Medium (Grand Island Biological 00., Grand Island, NY) supplemented with 10% calf serum. Maintenance of the aprt‘ phenotype required addition of 50 ug/ml diaminopurine. Tk+ cells were selected in HAT medium (15 ug/ml hypoxanthine, 0.2 ug/ml aminopterin, 5 ug/ml thymidine). All other cell lines were maintained in DME with 10% calf serum except for HUr-Z and V79 tk' cells which were kept in aMEM + 10% fetal calf 19 20 serum. G418 selections were done in regular media supplemented with 500 ug/ml G418. Hydroxyurea was dissolved in sterile 10 mM Hepes pH 7.0 at a concentration of 35 mg/ml and added to the media at the concentrations indicated. Plating efficiences were determined by plating 2 x 102 to 5 x 105 cell per plate at various concentrations of aphidicolin. Surviving colonies were scored by staining with methylene blue. Growth in HU was determined by plating 5 x 105 cells per 100 mm plate and scoring growth on an arbitrary scale (-,+/-, +, ++). RNA Extraction Total cellular RNA was prepared from pelleted cells by lysis in guanidinium thiocyanate. Five volumes of 4 M guanidinium thiocyanate, 1% sodium lauryl sarcosinate, 0.15 M a-mercaptoethanol, 10 mM EDTA, 50 mM Tris, pH 7.6 were added to the cell pellet at room temperature. The cell pellet was dissolved and DNA released from the cells was sheared by homogenization with a tight fitting dounce homogenizer. One gram of CsCl was added per 2.5 ml of homogenate. The homogenate/CsCl solution was layered over 1.2 ml of 5.7 M CsCl in 0.1 M EDTA in a Beckman SW 50.1 polyallomer tube. RNA was pelleted for 12 hrs at 35,000 rpm at 20°C in a SW 50.1 rotor. The supernatant was poured off and the RNA was resuspended in 10 mM Tris pH 7.5, 5 mM EDTA, 1% SDS (TES). The cloudy mixture was extracted once with an equal volume of CHCl3:Butanol (4:1). The organic phase was re-extracted with an equal volume of TES. The aqueous phases were combined and RNA was ethanol precipitated. Polyadenylated RNA (Poly-A+) was prepared using 21 an oligo dT cellulose column. In all work, baked glassware and diethylpyrocarbonate-treated, autoclaved solutions were used. DNA Extraction High molecular weight DNA for use in A library construction and DNA mediated gene transfer experiments was prepared as described by Blin and Stafford (1976). The DNA obtained was routinely at least 100 kb in length as judged by agarose gel electrOphoresis in a 0.2% gel. Plasmid DNAs were prepared by the methods outlined by Maniatis gtggl. (1982). Fragments to be used as probes were excised with the appropriate restriction enzymes, eluted from agarose gels (Girvitz g3 31., 1980) and labeled by nick-translation (Maniatis gt 21., 1982). cDNA Synthesis cDNA was synthesized from poly-A+ RNA by standard procedures using avian myeloblastosis virus reverse transcriptase. In some synthesis reactions an RNase inhibitor RNasin (Promega Biotec, Madison, WI) or a vanadyl-ribonucleoside complex was included in the cDNA reaction mix. Enrichment of Aphicolin-Resistant Cell Specific cDNA Sequences A cascade enrichment scheme similar to one described by Timberlake (1980) was used to selectively enrich for mRNA sequences found at higher copy number in aphidicolin-resistant cells than in parental-type CHO cells. Figure 3 outlines the procedure used. In short, poly-A+ RNA from high level aphidicolin resistant cells was used to make 32P-labeled cDNA. This cDNA was then hybridized to a 5-fold mass excess of CHO cell mRNA in 0.42 M phosphate buffer with 0.2% SDS. At ii I .w 22 various times corresponding to Cot (nucleotide moles x seconds/liter) values of 0, 150, and 500 the non-hybridized cDNA sequences were isolated by chromatography on hydroxylapatite at 68°. These cDNAs were then used to probe either Southern blots of restriction endonuclease digestions of CH0 and L3-5 genomic DNA or an L3-5 lamda library. A Library Construction and Screening Genomic libraries were constructed in the A vector Charon 28 by the method of Maniatis gt 31. (1982). These libraries were screened by the method of Benton and Davis (1977). DNAdMediated»Gene.Transfer The transformation protocol was as described by Wigler 35 31. (1979). Briefly, 24 hours prior to transformation cells (CHO or L-tk’aprt') were plated at 5 x 106 cells per 100 mm dish. The medium was changed approximately 4 hours prior to transformation. Sterile, high molecular weight eukaryotic DNA was dissolved in 1 mM Tris (pH 8), 0.1 mM EDTA. The DNA solution was adjusted to 250 mM CaClz with sterile 2 M CaClz. The DNA concentration in this DNA/CaClz mix was 40 ug/ml. The DNA/CaClz mix was added dropwise to an equal volume of 2X Hepes-buffered saline (280 mM NaCl, 50 mM Hepes, 1.5 mM NazHPO4, pH 7.10 1_0.05). While adding the DNA/CaClz, air was bubbled through the 2X HBS to gently mix the solutions. After addition of the DNA/CaClz mixture the DNA-calcium phosphate precipitate was allowed to form for 40 minutes without agitation. The precipitate was then mixed by gentle pipetting and 1 ml of precipitate (20 ug DNA) was added to the 10 ml of median on each plate of ‘ m 0v Tater, t'n liiase SE ,g'il tn; se'ect j resistai ef‘u'm v in? to 23 plate of recipient cells. After 12 hours of exposure to DNA, the medium over the cells was replaced with fresh medium. Thirty six hours later, the medium was replaced with selective medium. For thymidine kinase selection, HAT (15 ug/ml hypoxanthine, 0.2 ug/ml aminopterin, 5 ng/ml thymidine) medium was used. Aphidicolin resistant cells were selected with 0.4 ug/ml aphidicolin added to the normal medium. G418 resistant cells were selected in 500 ug/ml of that drug. The selective medium was changed every 2 to 3 days. Resistant colonies were visible in 2 to 3 weeks. RESULTS Introduction Several approaches have been utilized in our efforts to isolate the hamster RdRase genes. Initially, the putative amplification of the RdRase genes was used as a selection for the gene. More specifically, cDNA enrichment schemes and gene transfer experiments utilizing the APH resistant cell line L3-5 (and later L3-107) were used. More generalized techniques which did not rely on RdRase gene amplification were attempted subsequently. These included gene transfer of a dominant RdRase marker and screening of genomic blots with heterologous probes. cDNA Enrichment Schemes which utilize hybridization of cDNA and mRNA to various extents have been previously used to enrich a cDNA population for sequences expressed abundantly in one cell type and expressed to a lesser extent in a closely related one (Timberlake, 1980; Mather et 31., 1981). As shown in Figure 3, the process used here involved hybridization of APH resistant cell (L3-5) 32P-labelled cDNA to a 5-fold excess of parental type (CHO) mRNA. Hybridizations were carried out under conditions such that any sequences common to both L3-5 and CHO cells should have hybridized. Sequences unique to, or enriched in, L3-5 cells should have remained unhybridized. These unhybridized 24 25 Figure 3. mRNA:cDNA hybridization scheme. This procedure was used to selectively enrich for APH-resistant cell (L3-5) cDNA sequences. Details of the procedure are given in the text. 26 L3-5 POLY A+ RNA AMV reverse transcriptase a-32P dNTPs 32 P cDNA Hybridize to lO-fold mass excess of CHO poly A+ RNA. Final Cot = 500 mole-sec./liter Non—hybridized 32F cDNA isolated by chromatography on hydroxylapatite Use this enriched 32? cDNA as a hybridization probe Figure 3 27 cDNA's were isolated by hydroxylapetite chromatography and were used in the hybridization experiments detailed below. Differential"Screening,of.Southern.Blots Genomic DNAs from L3-5 and CHO cells were digested with various restriction enzymes, electrophoresed on agarose gels, and blotted to nitrocellulose filters. These filters were then hybridized to the L3-5-enriched cDNA probes described in section 1a. Figure 4 shows the autoradiogram obtained using L3-5 and CHO cDNA's isolated at the indicated Cot values. Any mRNA sequences which were enriched in the L3-5 cDNA should appear as bands not seen with the control CHO cDNA. Also, if gene amplification has taken place, these bands should be more intense in the L3-5 DNA lane compared to the CHO lane. As can be seen, there is no discernable difference in the banding pattern at the Cot values tested. The bands which are seen in the cDNA's hybridized to moderate (175) and high (500) Cot values are probably highly abundant messages which haven't been completely hybridized with the CHO driver RNA. In no case is any difference seen between the L3-5 and CHO DNA lanes. Differential Screening of Lamda Libraries Since the negative results obtained when screening Southern blots with the enriched cDNA probes might be due to the low sensitivity of this method, we attempted to use these cDNA probes for screening lamda libraries. Bacteriophage libraries were constructed for use in differential screening experiments with the enriched cDNA probes. It was hoped the added sensitivity of screening phage libraries would Figure 4. Enriched cDNA:Southern Blots. Results of hybridization for enriched cDNA's to Southern blots of genomic digests. 32F labeled cDNA probes (CHO or L3-5) were enriched by hybridization with CHO mRNA to a Cot of 500 (top) or 175 (bottom) and used to probe Eco RI restriction endonuclease digests of CHO or L3-5 genomic DNA's. Each lane is labeled with the type of genomic DNA used. 29 Cot 500 L3-5 cDNA CHO cDNA 0 H o L3-5 ! ’ Cot 175 L3-5 cDNA CHO cDNA 0 L's-5 CHO 1.3-5 Figure 4 3O allow identification of the putative amplified cDNAs in the enriched L3-5 probe. The factors contributing to added sensitivity of phage screening, compared to Southern blotting, are a) increased hybridization due to higher amounts of filter bound DNA acting as driver in the hybridization reaction, and b) more discernable isolated signals on a phage screen versus the general smear obtained when screening Southern blots with cDNA. Using a differential screening procedure similar to this, except that un-enriched probes were used, St. John and Davis were able to clone genes for messages which represented at most 0.01% of the mRNA population (1979). Complete libraries from both CHO and L3-5 genomic DNA were constructed in the bacteriophage vector A Charon 28. Pertinent data about these libraries is given in Table 3. Phage from the amplified L3-5 library were plated at the density of 5 x 103 plaques per 100 mm plate. Filters replicated from these plates were hybridized with the enriched cDNA probes (Cot 500) described above. A total of 6.5 x 104 phage were screened. Many plaques which appeared to hybridize preferentially to the L3-5 cDNA 'probe were noted, but upon re-screening none of these were found to contain sequences enriched in L3-5 cDNA (data not shown). GenenTransfer.Experiments Because we had been unable to obtain any positive results with the enriched cDNA probes, we decided to explore other methods for isolating the RdRase gene(s). Several genes have recently been isolated by DNA mediated transformation of eukaryotic cells. The major requirements for isolation of a gene using this procedure are a) a strong, positive 31 .ozu to; an oo— x n ea o~.m deacon a mosamm< o~mm meccam o~.m atom: i . ao.o-~ c~ w cosmos woman .02 cowueaam as» so woua_:u_ou .xcetn.— ugu c. oucmacom L~_=u.ucen c mc_uc*» co au.p.ncnocq woo mcv>og an uo:.umv xyocn*p ouupneoua mo_ x ~.e -o_ x ~.m ¢o~ x on._ eo~ x o.~ ax m.m~ mo— x s.— con x s.~ w.~ min; mc~ x #.m N—c~ x ~.e as" x wn.— me” x o.o as o.c— wow u o.~ mod x o.n o.~ ozu cc_uou.e_ae< cmcosouoz cm.»._ne< c»cocn.4 mum—aeou utomc_ A 100 kb) was prepared from HUT-2 cells. Before gene transfer experiments were conducted, it was necessary to characterize the growth of the recipient cells in HU. As shown in Table 5, various cell lines tested had widely different sensitivities to HU. It was surprising that Ltk'aprt‘ and their parent cell line LM had such different sensitivities to H0. In general it appeared that 38 Figure 6. Comparison of mouse Ltk'aprt' and CHO cell plating efficiencies in aphidicolin. The cells were plated as described in the text. Relative plating efficiency was determiend as described by Sabourin gt El: (1981). Relative Plating Efficiency 39 d C) I ' L-ceN 15".. CHO X o 0.1 0.2 0.3 024 Concentration Aphidicolin (uglml) Figure 6 4D the two hamster lines, CHO and V79 tk‘, were the most sensitive to HU. The HUr-Z cells appeared to grow well in HU concentrations up to 75 ug/ml. DNA mediated transformations were carried out using both hamster and mouse cell lines as well as a rat 3 tk' cell line as recipients. The donor DNA in these experiments was high molecular weight (>100kb) HUr-2 genomic DNA. All selections were done at 50 ug/ml HU, except the Ltk‘aprt‘ transformants which were selected with 100 ug/ml HU. The data in Table 6 shows that no HUF colonies were seen on any of the transfected plates. Each of the tk' cell lines was tested for the ability to transfer the HUT-2 tk+ marker. As noted previously, the tk+ phenotype was transferred with high efficiency (Table 6). No tk+ colonies were seen when mouse L tk' cell DNA was used as the donor DNA (data not shown). Isolation of Higher Level HUr-Z Mutants In order to increase the possibility of being able to transfer the mutant RdRase gene in subsequent experiments, a step-wise selection scheme was employed in an attempt to amplify this RdRase gene in the HUr-Z cells. As stated previously such step-wise selections have almost always yielded cells with amplified genes. The HUF-Z cells were selected in steps using 100, 150, 200, 250, and 300 ug/ml of HU. The cells were not cloned at each selection level, and were kept in the presence of HU continuously throughout the selection scheme. At each step, approximately 0.1-0.3% of the cells remained viable (approximate frequency of 10'4). After each selection step, cells were allowed to adapt to the higher level of drug for 2-6 weeks of logarithmic let'n “is + SC +I. Table 5. Hydroxyurea Sensitivity of Various Cell Lines 41 ‘ Celluline CHO pro V79 tk LM Ltk aprt Rat 3 tk cos 7 293 Hu 2 Hu 2 (300) V“. Growth of cell lines was determined as described in Materials and Methods. +++ +++ +++ +++ 30- +/- +++ ++ ++ ++ +++ +++ +++ Vigorous cell growth ++ slowed growth + some limited replication +/- most cells dead, but much cell debris and enlarged cells attached to plate +++ (HU) uglml 15 +/- ND ND ND +++ +++ - plates clean with little or no debris left N.D. Not determined 29.9 ND ND ND +++ 1.52 ND ND ND +++ 3a ND ND ND ND ND ND ND ND +++ 42 Table 6. DNA mediated gene transfer: Hydroxyurea selections i ‘ w Total No. plates No. HUT Average No. Recipient Cells Donor DNA transfected clonies tk+ colonies per plate CHO (hamster) HUr2 25 0 N.D. V79 tk' (hamster) HUEZ 50 0 N.D. V79 tk' HUr2 5 N.D. 3 rat 3 tk' HU'"2 25 o N.D. rat 3 tk‘ Hurz 5 N.D. 8 LM (mouse) HUr2 50 0 N.D. Ltk'aprt' (mouse) HU" 2 50 o N.D. Ltk'aprt' WI 2 5 N.D. 52 43 growth before proceeding to the next step. The highest level mutants, HUT-2 (300) appeared normal by microscopic examination and had a slightly longer doubling time than the HUT-2 cells (data not shown). Gene Transfer Experiments with HUT-2.(300) DNA High molecular weight DNA isolated from the HUT-2 (300) cells was used to transform the three cell lines which were most sensitive to HU. One hundred plates of LM cells, ninety-five plates of V79tk' and fifty plates of CHO cells were transfected with 20 pg per plate of HUT-2 (300) DNA. Unlike the previous experiments, in this set of transformations after absorption of the DNA for 16 hours, a 30 minute shock with dimethylsulfoxide (DMSO) was used to facilitate uptake of the donor DNA. Experiments with the hamster V79tk' and mouse Ltk‘aprt' cells selected for the tk+ phenotype showed this DMSO shock caused approximately a two fold increase in the V79tk' transformation efficiency, while the efficiency in mouse cells seemed unaffected (data not shown). As shown in Table 7 no colonies were obtained after selection with 35 ug/ml HU for 3 weeks. It should be noted that this level of H0 is lower than that used in previous experiments but still yielded no HU resistant colonies. All the plates were checked for colonies first by microscopic examination then by staining with methylene blue. In all cases the H0 selection appeared to be very stringent and no cell debris remained attached to the plate. The V79tk‘ cells were also selected for transformation to the tk+ phenotype. An average efficiency of 3 colonies per 20 ug DNA per 44 Table 7. DNA mediated transformations with HUr-2 (300) DNA. Total No. plates No. HUr Average No. Recipient Cells Donor DNA transfected clonies tk+ colonies per plate V79 tk' HUVZ (300) 5 N.D. 3 V79 tk‘ HUr2 (300) 95 0 N.D. CHO HUr2 (300) 50 0 N.D. 0 LM HUr2 (300) IOO N.D. 45 1 x 106 cells was observed. This corresponds to a frequency of approximately 3 x 10‘5. Serial Selection with G418 then HU Since the direct selection of the HU resistant phenotype had proven unsuccessful, it was decided to try a more indirect approach to transfer the RdRase gene. Mouse L tk‘aprt' cells were used in this procedure since our results with tk phenotype transfer indicated this cell line was 20 fold more efficient at DNA mediated gene transfer than the hamster cell lines (6 x 10'5 vs 3 x 10'5). L cells were transfected with 20 ug of HUT-2 (200) DNA plus 5 ug of linearized pSV2-neo plasmid DNA. This plasmid contains the early promoter region and splicing and polyadenylation signals of SV-40 fused to the bacterial neomycin resistance gene. Incorporation of pSV2-neo into eukaryotic cells confers resistance to the drug G418 (Southern and Berg, 1982). Transfected L cells were first selected for resistance to 6418. Since the pSV2-neo DNA was added in a vast molar excess compared to the genomic DNA fragments, most cells which had taken up and expressed exogeneous DNA survived the selection. Approximately 1.6 x 103 colonies were obtained per plate after G418 selection. After 3 weeks these colonies which were expressing the donor DNA were then trypsinized and replated in 80 ug/ml HU at a density of 5 x 105 cells per 100 mm plate (since each colony had on the average about 250 cells, the trypsinized cells were split 1:2 when plating in HU). After an additional 3 weeks of selection in HU, no colonies were observed. 46 Herpes Simplex Virus RdRase Probes While the gene transfer experiments were in progress, information about the possible location of the HSV-1 and HSV-2 RdRase genes became available (S. Weller, personal communication). Since the architecture of the RdRase subunits had been so well conserved throughout evolution, it was decided to attempt to use these heterologous RdRase genes as probes for the hamster gene(s). The strategy employed in these experiments was similar to that used in the cDNA enrichment Southern blots in that DNA from CHO cells and the putative amplified cells lines (L3-5, HUT-2 (200)) was digested with restriction enzymes, run in parallel on agarose gels, and Southern blotted to nitrocellulose filters. RdRase gene fragments which are within the amplified region of DNA should hybridize to the HSV probe more intensely in the L3-5 and HUT-2 (200) lanes (assuming of course that amplification has taken place). These same fragments should be present in the CHO lanes but at a decreased signal strength. Since the degree of homology between the HSV and hamster RdRase genes was unknown, the hybridizations were carried out at several different stringencies to maximize the signal while keeping the background low. The stringencies were adjusted by varying the percentage of formamide in the hybridization solution (20 to 50 percent), temperature of hybridization (37 to 42 0°), the salt concentration of the washing solution (30 to 300 MM NaCl), and the temperature of the washes (room temperature to 50C°). Generally the filters were hybridized at various stringencies then all washed at low stringency (300 mM NaCl, room temperature). These blots were then 47 exposed and subsequently rewashed at higher stringency as necessary and re-exposed. The probes used for these experiments are shown in Figure 7. Also shown is the region of HSV which was found to hybridize strongly to repetitive sequences in the hamster genome (data not shown). The Bam T probe of HSV-2 contained nearly all the sequences for the 38kd (M2) polypeptide and only the carboxyterminal end of the 140kd coding region. The Bam 0 probe for HSV-1 contains nearly half the 140kd coding region and all of the 40kd coding region. Bam 0 and Bam T nick-translated probes were used in hybridizations witfli Southern blots of CHO, L3-5, and HUr-2 (200) DNAs. No bands were seen above background with either of the probes at any criteria (data not shown). Control experiments with the hamster aprt gene probe yielded the expected banding pattern with bands of approximate equal intensity in all lanes (data not shown). Figure 7. 48 Herpes Simplex Virus 1 and 2 ribonucleotide reductase Maps. The regions thought to contain the HSV 1 and 2 RdRase genes are shown. Both viruses code for two polypeptides in this region. The coding regions are shown as dashed boxes, and the mRNA transcripts as wavy lines. The numbers refer to the map coordinates of the respective viruses. The probes used are drawn below each map. The restriction enzyme sites are as follows: B=BamHI, Bg=BglII, P=PvuII. 49 HSV-1 .sgs‘ f f j I f I '3' , —l :M—rf'o-k “3;. 5M W 7 °" ‘ ‘ ' ' - - -“ o‘er". L...- -.a L Bam 0 probe ‘ \ fl HSV-2 B Bg B B L l l J .54 .60 . ‘ ‘“ A. “A AM—‘AML --- ‘ rwr w v“ f.— V .----.-q r. L__.139.k.‘l- -J I'LJ=::‘P r ‘3 t. 3.8- 125.3 4 Ben E probe ‘ I—E T_:;Bam T probe 44 Figure 7. DISCUSSION The major focus of this project was the isolation of the hamster RdRase gene, or more specifically the gene for the M2 subunit of RdRase. The experiments detailed here failed to attain that goal. However several conclusions about the hamster gene can be surmized from this work. First, the frequency of DNA mediated gene transfer with the hamster RdRase gene is very low. In our experiments nearly 500 plates of cells were transformed with DNA containing the dominant, selectable marker for the M2 subunit from HUT-2. No HU resistant colonies were found on any of these plates. Thus, the frequency of transformation with this gene must be less than 2 x 10'9. Using the same recipient cells and donor DNA's, the tk+ phenotype was transferred with a frequency of 5 x 10"5 or 3 x 10'6 in mouse and hamster cells respectively. Recently Lewis and Srinivasan (1983) were able to transfer the HU resistance marker using chromosome mediated gene transfer at a frequency of 10'6 using the V79tk‘ cells as recipients and HUr-Z cell chromosomes. This means that the frequency hfith DNA mediated versus chromosome mediated transfer is at least 500 fold lower. Lewis and Srinivasan (1983) also attempted DNA mediated transfer of the HU-resistant phenotype with no success (W. Lewis, personal communication). One positive aspect of the DNA mediated transfer experiments which should be noted is the high efficiencies 50 51 which we attained in the tk transformations. Our best transformation efficiencies of 5 x 10"5 (mouse cells) and 3 x 10'6 (hamster) are 25-fold and 30-fold higher respectively than previously reported values for these cells (Abraham gg‘gl., 1982; Wigler gt‘gl., 1979). It is intriguing to consider why we have been unable to transfer the RdRase gene using the DNA/CaP04 method. One possibility is that the hamster RdRase gene is too large and does not remain intact during the transfer process. Very large DNA (2 100kb) was used to form the CaP04/DNA precipitates. If one assumes that mechanical (shearing) and biological (recombination and nuclease digestion) factors act on the DNA during the precipitation and cellular uptake, then the average size of the intact transferred sequences is probably on the order of 50kb. It is not inconceivable that the RdRase gene is larger than 50kb and cannot be efficiently transferred with the DNA/CaPO4 method. The high efficiency seen with chromosome mediated transfer may be explained by the length of contiguous sequence transferred using chromosomes, which is generally on the order of 7000 kb (McBride and Peterson, 1980). It is interesting to note how our perceptions of eukaryotic gene structure have changed recently. A few years ago it was generally assumed, based mainly on the globins and other abundantly expressed genes, that the length of most eukaryotic genes would be on the order of 20kb or less. Several lines of evidence have recently shown this is not the case. In somatic tissues, or before recombinational joining has taken place, the immunoglobulin genes are spread over very long distances (probably hundreds of kb, Marco and Cooper, 1982). In Drosophila, the bithorax complex has been mapped to a region over 100 52 kb in length. Analysis of cDNA clones for this region suggests certain messages are derived from sequences nearly 100 kb apart (W. Bender, personal communication). Finally the human thyroglobulin gene has been cloned and was shown to extend over 200 kb. One intron in this gene is nearly 60 kb in length (G-J VanOmmen personal communication). These examples clearly demonstrate that eukaryotic genes can be much larger than originally anticipated, thus it is possible that the RdRase gene is over 50 kb in length. A second possible reason for our inability to transfer the RdRase gene by DNA mediated transfer is that the RdRase gene remains intact during the transfer but the gene cannot be expressed. This could occur if some stable chromatin structure or transcription complex were needed to activate expression of the RdRase gene. This structure or complex would be present in the chromosome mediated transfer procedure, but would be stripped off when making DNA for CaP04 transfer. A similar explanation has been suggested for the unregulated expression of human a globin genes introduced into mouse erythroleukemic cells. In this case, however, it was theorized that a structure or complex was required to repress, rather than activate, a globin expression in undifferentiated MEL cells; thus the a globin genes introduced as naked DNA were always expressed (Charnay gt 21., 1984). In a more clear-cut example of the effect of structural features on expression, Xenopus 5S oocyte genes are inactive unless specifically assembled in an active, stable transcription complex. When assembled in a regular nucleosome structure in somatic cells, these genes are inaccessible to polymerase (for a review of stable transcription complexes see Brown, 1984). 53 Although the majority of the work described here involved attempts to transfer the RdRase gene by DNA mediated gene transfer, two other methods were used in our efforts to clone this gene. The HSV RdRase gene was unable to specifically identify any fragments in the hamster genome when used as a heterologous probe. After these experiments were completed, the nucleotide and deduced amino acid, sequences of the E. 2211, HSV-2, and Epstein-Barr virus (EBV) RdRase M2 (or B2) genes were published (Gibson gt 31., 1984; McLauchlan and Clements, 1983; Carlson .££.21°: 1984). Comparison of these sequences shows that although there is a high degree of conservation of the amino acid sequence among all these genes, they diverge strongly at the nucleotide level. In fact there is only a slightly discernible nucleotide homology between two of the herpes virus M2 genes (EBV versus HSV-2) (Gibson 35 21., 1984). In light of these results, it is not surprising that the HSV RdRase probes failed to detect the hamster gene. A cDNA enrichment procedure was also tried as a method to isolate the RdRase gene. Using both Southern blotting and lambda library screening we were unable to identify the RdRase gene with enriched cDNA probes. There are several probable reasons for this result. First, the protocol used to synthesize the cDNA did not employ actinomycin D to inhibit hairpin loop formation in the cDNA; thus unhybridized cDNAs, which should not have bound to the hydroxylapatite, in fact may have contained small double strand loops and might have been removed in this chromatography step (M. Davis, personal communication). Second, the cells used to prepare the mRNA's were not synchronized; consequently most to them were not in S phase when harvested. 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Robert de Saint Vincent, B., DeRose, M.L. (1984) Nature 333, 516-200 Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. (1978) Cell 33, 725-731. Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979) Cell 33, 777-785. Wold, B., Wigler, M., Lacy, E., Maniatis, T., Silverstein, S., Axel, R. (1979) Proc. Natl. Acid. Sci. USA 33, 5684-5688. Zieg, J., Clayton, C.E. Ardeshir, F., Giulotto, E., Stark, G.R. (1983) M0]. C61]. BIO]. 3, 2089-980 PART II CONSTRUCTION OF MUTATIONS IN THE CHICKEN ADULT ALPHA GLOBIN GENES L” m r ‘3 n- l") l anaTy to be gene genes orgar and f thall Kazaz is is gene 0f la the c are l ChrOr and t (Lars Iota: genes LITERATURE REVIEW Background The recent advent of molecular cloning techniques has allowed the analysis of eukaryotic genes at the molecular level. One of the first to be isolated, and consequently one of the most thoroughly studied gene systems is that of the globins. The mammalian a and e globin genes have been extensively analyzed both in structure (gene organization, linkage, nucleotide sequence, and nuclease sensitivity) and function (evolutionary conservation of flanking sequences, thallasemias, and in vitro mutations). (For a review see Orkin and Kazazian, 1984; Efstratiadis 33.33., 1980). Another well-studied system is that of the chicken globin genes. As is the case for the mammalian genes, much is known about the avian gene structure; however, not as much work has been done on the function of various elements within the chicken genes. For example, although the complete nucleotide sequences of all three chicken a globin genes are known (Dodgson and Engel, 1983; Engel 23 33., 1983), their chromosomal linkages have been determined (Engel and Dodgson, 1980), and their pattern of nuclease sensitivity during development is known (Larson and Weintraub, 1982), very little has been learned about the location or function of elements controlling expression of these genes. 59 60 Specific DNA sequences and/or cellular factors that control globin expression within the erythroid cell have not yet been identified; however, progress has been made at defining the elements necessary for constitutive expression (i.e. expression in heterologous cells in a nonregulated fashion) of the mammalian globin genes (reviewed in Orkin and Kazazian, 1984). Since there is little homology between the mammalian and chicken a globin promoter regions, sequence comparisons have not been useful in defining analogous sequences necessary for constitutive expression of the chicken o globin genes. One of the long term goals of this laboratory is to better understand the molecular basis for the differential expression of the chicken globin genes during erythropoesis. As a prelude to this, we have begun studies aimed at defining the promoter elements of the chicken adult o globin genes which are necessary for constitutive expression of these genes. The work presented here describes the 39 13359 mutagenesis procedures which we are employing to answer this question. Chicken-e.globin.gggg§ The chicken a globin locus is shown in Figure 1. It consists of three closely linked genes which are all located within a 6.8 kb region. All three genes are transcribed in the same direction and arranged 5'-:-ap-aA-3' (Dodgson and Engel, 1983). The up and aA genes are expressed in both primitive (embryonic) and definitive (adult) red blood cells (RBC), whereas the w gene is active only in embryonic blood cells. In adult RBCs the aA and up genes are expressed at a 3:1 ratio. In other tissues these genes are inactive. Unlike some mammals which contain two adult a globin genes which are 61 _La _.PD_) ‘1' A v v :3: J F? I. ma: J. J. ’1‘ 11‘ T $00!: if P Figure 1. Chromosomal arrangement of the chicken a globin genes. The two adult (oA, a0) and the embryonic (a) genes are shown. The arrows indicate direction of transcription. Exons are shown as black boxes and introns are closed boxes. Restriction enzymes used in analysis of the locus are it, Eco RI; 1‘, Barn Hl;v, Hind 111;.1, Taq1;6, Sac I. 62 almost identical in sequence, the chicken adult a globin genes are widely divergent, both in coding and flanking sequences. The GA and on genes contain little homology in the 5' flanking region, except for the TATA box located 28 and 29 bp respectively upstream from the mRNA start sites. EukagyotiC~Promoters A prototypic eukaryotic RNA polymerase II transcription unit is shown in figure 2 (for a review see Nevins, 1984). Classically, the signals which control transcription have been called promoters or promoter elements. Analysis of genes transcribed by polymerase II has revealed a complex set of elements which can interact to control the frequency and location of mRNA initiation. In general the definition of eukaryotic promoters has relied upon two methods: a) Sequences from various genes have been compared, and conserved, presumably important, sequences identified; and b) IBM and 191.1212 generated mutations have been used to assay the function of these presumably important sequences. The most clearly defined element of the polymerase II promoter is the TATA or Goldberg-Hogness box located 25-30 base pairs upstream from the transcription start site (Goldberg, 1979). This element is present in nearly all polymerase II structural genes examined. It is thought to function by identifying the location where mRNA transcription will start (also called the CAP site). Deletion of TATA results in the use of multiple start sites, but does not always decrease the rate of transcription from the region. In other words the total number of 5' 63 Figure 2. Typical eukaryotic RNA polymerase II transcription unit. The various elements which have been observed in the polymerase II transcription unit are indicated. In cases where the exact role of a particular element has yet to be elucidated, the element is marked with a question mark (?). 64 mufim coaumamcmvmuaoa Tllllli coaumcwahmu a Ammocmncmv w '5 ln‘... um mmumm m3 'i‘. l" A sound“ Aumocmxcov s N ouswwm Mon ov no Hmcoaho: Til.» Ammocmncmv ~ 65 RNA ends may be unchanged, but the location of the ends is heterogenous in the absence of TATA (Orkin and Kazazian, 1984). Other elements of polymerase II promoters are not so clearly defined. Sequence comparisons have identified a pentanucleotide sequence, CCAAT, located 80 to 100 base pairs upstream from the cap site of many eukaryotic structural genes, most notably all of the normal mammalian globin genes (Benoist 33_33., 1980; Efstratiadis 33 33,, 1980). This so called “CAT" box is not found at a similar location in any of the avian adult a globin genes examined (Dodgson and Engel, 1983). It is also absent in several other genes and therefore its function as a promoter element is unclear. Another distal transcription signal which has the sequence PuCPuCCC (Pu = purine) has been identified in the -80 to -100 region of the mammalian a globin gene. This element has a striking homology to transcriptional control elements defined for constitutive expression of the herpes simplex virus thymidine kinase gene and the SV 40 early region (Dierks 33 33., 1983). Scott and Tilghman (1983) also suggest that 60 rich regions, similar to PuCPuCCC, flanking the CCAAT pentamer are important in transcriptional control. A much more detailed study of the herpes simplex thymidine kinase gene draws the same conclusion (McKnight and Kingsburg, 1982). A final element whose importance in transcription control has recently been defined, is called an enhancer or activator. These elements seem to act in a position-independent, orientation-independent manner to increase transcription in 333 from promoters located at distances up to at least 10 kb (for a review see Khoury and Gruss, 1983). Many eukaryotic viruses, including DNA viruses and 66 retroviruses, have been shown to contain enhancers. It has also been shown that enhancers often act in a cell-type or tissue - specific manner suggesting they may be intimately involved in differential control of gene expression. For example, the immunoglobin heavy chain gene contains an enhancer in the major intron. Upon rearrangement of the inmunoglobin gene, this enhancer, which is active only in lymphocytes, is brought into a region near enough to the immunoglobin promoter to activate transcription (Gillies 33.33., 1983). In.vitro.Mutagenesis The ability to construct and analyze specific changes in a defined DNA sequence is central to the study of transcriptional regulatory elements. This procedure of 33H33353 construction and subsequent analysis of specific mutations has often been called “reverse genetics" (for a review on directed mutagenesis, see Shortle 33H33., 1981). In general, "reverse genetic" analysis of regulatory elements is accomplished in a two step procedure. Initially, defined sections of DNA are deleted from or inserted in the region of interest to roughly map the sections important in regulation. Secondly, specific base substitutions are used to precisely define the elements localized by the first set of mutants. Recently McKnight and Kingsbury (1982) developed a system for studying eukaryotic promoters called "linker scanning" mutagenesis. In this system two series of deletions, terminating with a synthetic restriction endonuclease site (linker), are constructed from either end of the promoter region (see Figure 3). By using the linker for recombination of the appropriate 3' and 5' deletions, clusters of point Figure 3. 67 Example of linker scanning mutagenesis. The region to be mutagenized is depicted as the hatched box in A). Various deletions are constructed using restriction enzyme sites X and Y as starting points. Each deletion has a linker (boxed region) at its terminus. For example, the deletion of clone Y4 starts at Y and extends just into the target region. The X1 deletion extends from X exactly the length of the linker past the Y4 deletion endpoint. Recombination of X1 and Y4 at the linker gives the clone shown in B). Recombination of two mutants whose deletion endpoints overlap (Y2 + X2) results in an internal deletion in the target area as shown in C). 68 m ouowfim r _l \ a L «x + N» l r H NHL 1 a r _l \ M N. l mxT :xT * mxT i NxT i fix") .ln' 0 _ N N K N Ix > _N \ N \L. 1 me I 2 m I a H l m> H 4.1 i 2 3% it (I. 69 mutations can be generated. Mutants with endpoints that are spaced exactly the length of the linker will recombine to give only a cluster of point mutations in the linker region. (Of course some of the endogenous nucleotides in the region where a linker is substituted may be identical to the corresponding bases in the linker, or the complete linker sequence may differ from the normal sequence.) The remainder of the reconstructed gene is identical to the natural gene. A major advantage of linker scanning mutagenesis is, that unlike simple deletion mutations, the orientation of sequences in the reconstructed gene, outside of the linker itself, is identical to the natural gene. If one recombines deletions with endpoints which are further apart or closer together than the length of the linker, then the resultant recombinant will yield point mutatations plus deletions or insertions respectively. As with other mutagenesis procedures, initially the 3' and 5' deletions are used to define the specific area(s) of interest by assaying their promoter activity, and then the appropriate deletion mutants are recombined. base p173: vhic‘ Nani; using Irodl'f letho. lithe ! 3911f) IPQate MATERIALS AND METHODS Materials. Plasmids pHRa5-4.3, and pBRo7-1.7 have been described by Dodgson and Engel (1983). Plasmid pBo5-0.9 is a subclone of the 900 base pair Bam HI fragment of a9 constructed by J. Dodgson. pATBRa7-1.7ANIZO is a clone of the eA gene derived from pBRa7-1.7b in which the internal Nae I fragment has been deleted and replaced with two synthetic Xho I linkers. This oA gene is cloned in a pBR322 derivative in which the Nae I fragments between base pairs number 401 to 1283 (relative to the Eco RI site) have been deleted and thus contains no Nae I or Nar I sites (constructed by J. Dodgson). All restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, and calf intestinal alkaline phosphatase were from the following sources: PL Biochemicals, BRL, New England Biolabs, Promega Biotec, International Biotechnologies, or Boeringer Mannheim.Nuclease Bal 31 fast and slow isomers were from International Biotechnologies, Bal 31 (mixed) was from BRL. Methods. In general all cloning techniques followed those outlined by Maniatis, Fritsch and Sambrook (1982). Sequence analysis was performed using the chemical degradation method of Maxam and Gilbert (1980) as modified by Smith and Calvo (1980). Plasmid DNA minipreps used the method of Ish-Horowicz and Burke (1981). Nuclease Bal 31 digestions were generally performed according to Maniatis 33H33., (1982) but modified slightly as follows: Five micrograms of linearized DNA was treated with the amount of Bal 31 indicated at 30° for the length of 70 71 time indicated. Digests were stopped by chelation of calcium with EGTA, then the samples were deproteinized with phenol. The DNA was ethanol precipitated and resuspended in 10 mM Tris, 1 mM EDTA. One half of the DNA from each timepoint was digested with the appropriate restriction enzyme and run on a 5% acrylamide gel to assay the extent of nuclease digestion. The DNA of appropriate time points was made blunt-ended with DNA polymerase Klenow fragment and ligated with T4 DNA ligase to synthetic XhoI linkers. Excess linkers were removed with XhoI and the plasmids were recircularized by ligation in dilute solution. The plasmids were then transformed into 3, coli HBIOI. RESULTS 00: Construction of 5' deletions. The strategy outlined in Figure 4 was used to generate the deletions extending into the 5' end of the putative up promoter region. This assumes that like other genes studied to date, the up promoter lies within 120 bp of the mRNA initiation site. Plasmid pHRaS-4.3 contains 3 ApaI sites at the positions indicated. The 3' most site is 120 bp upstream (-120) of the up mRNA start site (the numbering system employed designates the mRNA start site as zero, sequences preceeding it are negative, and those following it are positive). Five micrograms of ApaI digested pHRa5-4.3 was treated with a very low level of nuclease Bal 31 (0.2 unit/5 ug) for times varying between 0.5 and 10 minutes. The extent of nuclease digestion was assayed by cleaving the DNAs with BstXI and analyzing the products on a 5% acrylamide gel. The BstXI site is located at +352 and the ApaI site is at -120; therefore the BstXI digest will generate fragments between Figure 4. 72 MNDIII Eco RI 1) Linearize pHR 5-4.3 with Apa I 2) Digest with nuclease Bal 31 for various lengths of time 3) Assay extent of deletions with Bst XI digests 4) Klenow polymerase fill the DNA ends & ligate with synthetic Xho I linkers 5) Remove excess linkers & recircularize plasmids by ligation in dilute solution 6) Transform E. coli & screen mini DNA preps with Xho I + Bst XI digests Scheme for construction of o0 5' deletions. Plasmid pHR05-4.3 contains a 4.3 kb chicken DNA insert (thin line) cloned in the vector pBR322. The up transcription start site and direction of transcription are shown. The deletions were constructed as described above and in the text. The area deleted is indicated by the arrows outside the plasmid. 73 350 and 470 bp in size if the endpoints of the deletion fall in the putative promoter region. Gel analysis showed the DNAs from the 5 and 10 minute time points contained deletions of approximately 0 to 30 and 50 to 120 base pairs respectively. Deletions of this length would be expected to lie in the -120 to -40 and -70 to CAP regions. All other time points contained no detectable deletions. One feature of the Bal 31 digestions which was noticed was the appearance of distinct bands imposed on the smear expected from the nuclease digestions. These bands suggest that there are preferred stop sites for the Bal 31 nucleolytic digestion. After attachment of linkers and plasmid recircularization the 5 and 10 minute time point DNA samples were used to transform 3, 3333 HB101. Approximately 500 and 62 colonies were obtained with the 5 and 10 minute DNAs respectively. Some of the 10 minute time point DNA was presumably lost during the manipulations. One hundred and eighty of the five minute time point colonies and all 62 of the ten minute digest colonies were picked and plasmid minipreps were prepared. The five and ten minute time point clones were designated A5 and A10 clones respectively. The approximate location of the linkers in these clones was determined by digestion of one fifth of each of the DNAs with Bst XI and XhoI followed by analysis on 5% polyacylamide gels versus pHRa5-4.3 ApaI plus BstXI digested DNA. Based on the size fragments obtained from XhoI + BstXI digests, 88 A5 and 32 A10 clones were chosen for further study. The A5 clones which were discarded all appear to have no detectable deletions, whereas the thirty A10 clones which were discarded were deleted well beyond the CAP site. 74 SequencingoD 5'.deletion mutants. The strategy used for determining the exact location of the XhoI linker in the up 5' deletions is shown in Figure 5. Plasmid DNA was isolated from 50 milliliter cultures of various clones using a scaled up miniprep procedure. These DNAs were then digested with Pvu II and the fragments slightly smaller than 1.2 kb were gel purified. These fragments were prepared for sequencing by treatment with calf intestinal alkaline phosphatase followed by labeling with polynucleotide kinase. The labeled DNA was digested with Sin I to cleave the 5' labelled end, and the large fragment was purified by electrophoresis on a 1.2% agarose gel before being subjected to chemical degradation sequencing using all four reactions (A>G, G, C + T, C). The sequences of four A5 clones were determined using this method (data not shown). Determination of the linker location by performing all four sequencing reactions was not favorable for two reasons; first, the sequencing reactions themselves were laborious and also required four lanes per clone on a sequencing gel, thus decreasing the number of clones that could be sequenced at one time. Secondly, the labelled sample was divided into four reactions; thus a significant (>100 cps) number of counts were needed to obtain the sequence of each clone. Since such a large number of counts were needed, large (50 milliliter) cultures had to be prepared for each clone to be sequenced. For these reasons we attempted to simplify the sequencing procedure by only performing one reaction for each cloned DNA, namely the AG or formic acid reaction. These AG reactions were compared to a sequencing ladder of the wild type (or parental) DNA labelled at the same Pvu II site to Figure 5. 75 Strategy for sequencing up 5' deletion clones using a single sequencing reaction. Miniprep DNAs of clones containing deletions starting from the Apa I site in pHR a5-4.3 were subjected to the sequencing scheme outlined above and discussed fully in the text. The restriction enzyme sites are as follows: A = Apa I, P = Pvu II, S = Sin 1, X = Xho I linker. The numbers in parenthesis are the locations relative to the up CAP site. 76 S A A CAP P l 1 __,L l X... l J fier I I (-120) "" (0) (+40) Digest deletion clones with Pvu II Gel purify 1.0 kb fragment End label DNA 8 A A i CAP *CTG‘Ifi/Il l L I 7fL H GTC* J’ Second cut with Sin I Gel purify 0.9 kb fragment A A J’ c P I X i EL crc* Figure 5 Sequence using AG reaction Electrophoresis on sequencing gel & compare to pHRaS-4.3 Pvu II 1.2 kb ("parental") sequence 77 Figure 6. Sequencing gel of 00 5' deletion clones:0ne sequencing reaction. Autoradiograph of a 6% acrylamide, 7M urea sequencing gel of 11 different up 5' deletion clone DNAs prepared for sequencing as shown in Figure 5. The control lanes marked G, A + G, C + T, C are the "parental“ pHRo5-4.3 Pvu II 1.2 kb DNA sequencing reactions. Deletion endpoints are determined by comparing clone and parental DNA AG reactions. The A + G and C + T reactions on the right are both reactions run with clone A5-46 DNA to confirm the validity of this method for determining deletion endpoints. 78 ..h s '5. nil-sis. .... t he. 5? 8 Figure 6 79 determine the location of the linker (Figure 6). Since the sequence of the XhoI linker was known (5'-CCTCGAGG-3'), it was relatively easy to place the linkers by scanning the sequencing autoradiograph for the 4 base gap in the AG lane left by the CCTC sequence of the linker. Sequencing reactions using this procedure could routinely be performed using the DNA from a five milliliter culture. Although the single reaction method for placing the linkers was fairly simple, it still required a significant amount of time and labor to perform each of the AG sequencing reactions. An alternative scheme was devised to make placement of the linkers even simpler. As outlined in Figure 7 this method takes advantage of the fact that the restriction enzymes Pvu II and Xho I leave blunt and 5'-protruding ends respectively, and that after labelling at the PvuII/XhoI sites, the resulting fragments which differ in length by 4 bp can easily be resolved on a sequencing gel. As shown in Figure 8, comparison of the labelled fragment from each strand of the clone DNA with a sequencing ladder of the parental DNA labelled at the Pvu II site allows simple placement of the linkers in each clone. Subtraction of 2 nucleotides from the shorter fragment (labelled at the Pvu II end) or 6 nucleotides from the longer fragment (labelled at the Xho I end). Using this method, up to 12 deletion mutants can be analyzed on a single sequencing gel. Location of linkers in-A5 and A10 clones. A summary of the locations of the linkers in all of the up 5' deletion clones sequenced to date is shown in Figure 9. The exact location of the Xho I linker has been determined for fourty six of the 5' deletion mutants; thirty five of the A5 clones and eleven of the A10 Figure 7. 80 Scheme for mapping up 5' deletion endpoints by fragment length determination. The position of the deletion endpoint was mapped by accurately determining the length of both strands of the fragment obtained by digesting up 5' deletion clone DNAs with Xho I and Pvu II. Symbols are as in Figure 5. Details are given in the text. 81 P A A CAP P J l l 1" l l (-120) / (0) (4340) ’ l Digest deletion clone I DNA with Pvu II / l / / Gel purify 1 kb I fra ent / gm ' / / Digest w/ Xho I : End label DNA / l / , I *TCGAGG CAG CC 3130* Determine fragment of labelled clone DNA using sequencing ladder of pHRa5-4.3 1.2 kb Pvu II fragment Figure 7 82 Figure 8. Sequencing gel of an 5' deletion Clone DNAs Xho I/Pvu II labelled fragments. Clone DNAs prepared as in Figure 7 were analyzed on a sequencing gel versus of sequencing ladder of the "parental“ pHRo5-4.3 Pvu II 1.2 kb fragment labelled at the 3'-most (+40) Pvu II site. Additional A + G parental sequencing reactions were run to accurately size the two strands of each clone. \\ 63+) 83 m+> —l+n rd 5'. e a. ~‘ili‘1 ‘dafi‘l' Figure 8 m+> In Figure 9. 84 Summary of o0 5' deletions. The locations of the linkers, and consequently the deletion endpoints for the up 5' deletions are summarized. In each case the sequence of the linker is shown in the position relative to the wild type sequence. Wild type sequences are designated by a line. The top line is the wild type sequence of the -120 to CAP region of an. m musmfim 8595”. @3859 MW 8388 $38.59 All 858.8 9938.8 allow/$8.8 03.859 .||.|l @385 <9uUu5o$5upuuuo8+ooRI PBOS-O.9 1) Linearize pBoS—O.9 with Bst XI 2) Digest DNA with nuclease Bal 31 for various lenths of time ' 3) Assay extent of deletion with Bam HI digests 4) Fill ragged DNA ends with polymerase Klenow fragment 6 blunt and ligate to synthetic Xho I linkers 5) Digest with Xho I to remove excess linkers 6 recircularize plasmid by dilute ligation 6) Transform E. coli & screen minipreps by Xho I + Eco RI digests Figure 10. Construction of a0 3' deletion mutants. The thin lines represent cloned chicken sequences. Thick lines are plasmid pBR 322 DNA. on transcription start site (CAP) and direction and region of transcription are indicated by arrow inside plasmid. The arrow outside the plasmid denotes the direction of deletion from the Bst XI site. The restriction sites are as indicated and cloned sequences are to scale. 88 XhoI linkers were added to the 12, 15, and 18 minute Bal31 digests and the recircularized plasmids were transformed into‘g..3333. The 12, 15 and 18 minute digests yielded 500, 1000, and 100 colonies respectively. It appears that fewer colonies were obtained with the 18 minute digest DNA because the 3' endpoints of some of the Bal 31 deletions extended into the adjacent pBR322 sequences and disrupted the origin region needed for plasmid replication. Miniplasmid DNA preparations were made from 336 and 24 of the 15 and 18 minute colonies respectively. These 1.2 ml culture plasmid DNA preparations were very crude and contained large amounts of RNA and chromosomal DNA; therefore restriction enzyme fragments below 200 bp in size were very difficult to visualize with ethidium bromide staining of 5% acrylamide gels. Because of this, the Bam H1 site at -254, which would be expected to yield fragments of 130 to 250 base pairs when used in a double digest with XhoI, could not be used to screen these deletion mutants. Instead the Eco RI site located in the pBR322 DNA 630 base pairs 5' to the CAP site was used. Fragments extending from the Eco RI site to the linkers should range from 500 to 630 base pairs in length if the linker falls in the putative promoter region. These size fragments are fairly difficult to size accurately on acrylamide gels since this is near the nonlinear portion of the gel's partitioning range. Also, the large amount of RNA in the miniprep can make the fragments run anomalously. Therefore, the linker locations obtained in these experiments could only be considered approximations. Double digests of miniprep DNA with Xho I + Eco RI showed that all of the 18 minute colonies contained deletions well past the -120 region. These clones were not further characterized. Eighty three of 89 the 15 minute digest colonies were shown to have deletions which appeared to fall in or near the region between -120 and the CAP site. These clones were designated 815-1 through 815-83. Although the EcoRI + XhoI digest had roughly positioned the linkers in the 815 clones, it was decided to more precisely map these linkers before proceeding with sequence analysis. DNA was prepared from 5 milliliter cultures (called "midipreps") of all 83 815 clones. Care was taken while preparing these DNAs to rid the samples of chromosomal DNA and RNA and thus avoid the problems described previously. Gel analysis of Xho I + 8am HI digests of a small sample of the midiprep DNAs confirmed that 42 of the 83 815 clones contained deletions in the -120 to CAP region. The other 41 clones contained deletions slightly outside the putative promoter region, usually within 20 base pairs of the -120 to CAP region. a0: Sequencing 3' deletion mutants. The strategy outlined in Figure 11 was used to exactly position the linkers in the 815 clones. This scheme took advantage of the fact that the Bal 31 deletions had extended beyond the 8am H1 site at the 3' end of the up gene. Therefore the 815 mutants contain only one 8am HI site at position -254. Linearized midiprep DNA (three quarters of each sample) was sequenced from this 8am H1 site. Similar to the procedure used for the A5 clones only one sequencing reaction, the AG reaction, was performed on the labelled 815 DNAs. The 815 reactions were run in parallel on a long 6% sequencing gel with a sequencing ladder of the parental 8am HI labelled p8a5-0.9 DNA. The linker positions were located by examining the autoradiograph 90 Figure 11. Sequencing strategy for up 3' deletion clones. The steps shown above were used to map the location of the linker in the up 3' deletion clones (designated 815 clones). The deletions have removed the 8am HI site 3' to the 8st XI site in Figure 10 leaving only one 8am HI site in each clone. The sequenced DNAs were run on long 6% sequencing gels. 91 Eco RV Bst XI deletion clones Bam HI (deleted in mutants) Bam HI digest Gel purify linearized DNA End label DNA Eco RV Eco RV digest l l l l Gel purify large fragment Sequence using AG reaction Figure 11 92 for the location where the mutant and parental DNAs diverged (data not shown). Using this procedure the deletion endpoints suilmarized in Figure 12 were mapped. A procedure similar to the fragment sizing protocol used for the A5 clones (Figure 7) was attempted with the 815 Clones. In this case the 815 clone DNAs were digested with Bam HI and Xho I and labelled at both 5' ends. The labelled DNAs were run on a sequencing gel versus the parental DNA sequencing reactions. This procedure could not be used to exactly locate the linkers in the 815 clones for two reasons: First since Bam HI and Xho I both leave a 4 bp 5' protruding end, the labelled fragments from both strands are the same length and migrate as a single, somewhat diffuse, band on the sequencing gel. Unlike the A5 clones where there are two bands separated by 4 bp, the DNA fragment lengths of the 815 clones cannot be double checked to ensure proper positioning of the linker. The second, and probably most critical, reason this method could not be used with the 815 clones was the distance between the labelled BamHI site and region where the linkers were located. The 8am HI site lies 130 to 250 base pairs upstream of the putative promoter region; thus the labelled fragments being sized are located in a region of the sequencing gel where the bands are very close together (less than one millimeter between bands). Therefore it was not possible to definitively align the labelled 815 clone fragment bands with a single band in the parental sequencing reaction lanes. Construction-of~aA.5'udeletions. Plasmid pBRa7-1.7 DNA was used to construct deletions extending into the 0A promoter region from the 5' direction using the protocol 93 Figure 12. Summary of o0 3' deletions. The location of the aP 3' deletions are shown. Details are as in figure 9. 94 ooqouhuwli NH muswfim woquHUU Moueuueuooeuoeuuueoueooqoqaeeee mu Omi ooqouhuu ooquHuu oum a u _ zmx mic 8am; 1. 88¢ . 83; Fun on... t1. Tc _ _ 2 c zax s-a ~_ =>a H: sea «to DISCUSSION The experiments described here are designed to produce mutants of the putative chicken adult alpha globin promoters 33.33333. The regions on which this study has concentrated lie within 120 bp upstream of the mRNA transcription start sites of the a0 and o5 genes. The reasons for confining our work to these regions of the an and up genes stems from the detailed study of three other eukaryotic promoters, namely the rabbit a globin, herpes simplex virus thymidine kinase, and chicken ovalbumin promoters (Dierks 33 33., 1983; McKnight and Kingsbury, 1982; Knoll 33H33., 1983). In all three cases the DNA sequences necessary for initiation of transcription were defined by creation of specific mutations jgnliggg followed by characterization of the effects of these mutations 33 1333. All three of these studies used heterologous host cells in which the genes were expressed in a nonregulated fashion, and in all three cases the DNA sequences necessary for constitutive expression were located within 110 bp of the mRNA start site. In fact, besides the TATA box at -25 to -30, the sequences which had the most dramatic effects on the level of expression were located between -60 and -110. We have chosen to use the linker scanning method of McKnight and Kingsbury (1982) to generate the aA and on promoter mutations. Initially, we have concentrated on construction of 5' deletions mutants which should allow us to roughly define the distal sequences necessary 100 101 for transcription. Other studies have found the 5' deletions are generally the most useful for the preliminary mapping of the promoter elements (for example see Dierks 33.33., 1983). The results presented for the locations of the 5' and 3' deletion. endpoints in the up promoter region in Figure 9 and 12 clearly demonstrate that we are now in a position to use these mutations to identify the promoter elements of this gene. The collection of 5' deletions nearly saturates the area between -120 and CAP with only a region between ~60 and -40 lacking any mutants. We are presently constructing deletions using the slow isomer of Bal 31 and extending the deletion from the Xho I linker at :78 in one of the mutants. These new deletions Should cover this -60 to -40 region. Also, twenty additional A10 Clone ONAs which have deletions which map in the -60 to CAP area have been prepared and are being labeled at the Xho I and Pvu 11 sites for analysis on sequencing gels. It is expected that among the new deletions being constructed and these A10 clones, 5' deletions covering every base between the -120 and CAP sites of on will be available. Although the collection of 3' «D deletions presented in Figure 12 is not as extensive at the 5' deletion collection, it should be adequate for definition of the up promoter. In addition to the rilones shown in Figure 12, twenty-four more 815 clone DNAs have been larepared for sequencing and are presently being analyzed. The linkers in all of these new clones have been mapped to the -120 to CAP region, and hopefully they will add significantly to the regions in c0 covered by the 3' deletions. 102 The reconstruction experiments, in which the appropriate 5' and 3' deletions will be recombined to generate an intact on gene unth a cluster of point mutations, have not yet been initiated. Before proceeding with the reconstructions, lflulilfl expression experiments using various 5' deletions spaced throughout the -120 to CAP region will be conducted. These experiments should define which areas of the -120 to CAP region are most important in transcriptional control, and, thus, where the linker reconstruction experiments should be done. Studies on the expression of deletion mutant and normal control a globin genes are presently underway using both transient and long term expression systems in mammalian cells. Preliminary results with the intact o0 and aA genes suggest that it may be necessary to introduce an enchancer element (for example, a retrovirus long terminal repeat) into the cloned DNAs to get measurable expression of the chicken a globin genes in these mammalian cell systems. Construction and sequencing of the aA gene deletions has not progressed as far as the up mutants. Several deletions extending into the putative 0A promoter region from the 5' end have been mapped. These are all located at least 70 bp upstream of CAP. One of these (K7-3) has been used to extend the deletions further into the aA promoter region; however, these new mutants have not yet been mapped. Technical problems have precluded construction of the 3'aA deletions, although recent experiments may have solved these problems. Another aspect of the reSearch described here which deserves some mention is the development of the strategies for using small culture (5 ml) for precise localization of the deletion endpoints. These methods use either a single sequencing reaction (Figure 5) or alternatively, precise fragment length determination on sequencing gels (Figure 7) to 103 locate the linker in the deletion mutants. A major requirement for either of these procedures is that a restriction enzyme site which can be used for labeling the DNA is located within approximately 250 bp of the deletion endpoints. This restriction site must lie on the opposite side of the region to be mutated from the restriction site used for the Bal 31 deletions. For example, in Figure 3 site X can be used to sequence (or size) the deletions which are initiated at site Y (Y1, Y2, Y3, etc.). Another requirement of the sequencing scheme, but not of the fragment length determination scheme, is that there is another restriction enzyme site available on the labelled DNA fragment which can be used for the second cut before DNA sequencing (this yields the uniquely labelled DNA end for sequencing). In fact the linker can probably be used as the second cut site. Since both fragments are utilized in the fragment length determination method. There is no need for a second restriction enzyme site. With the deletion clones which have already been sequenced, the clones which have been constructed but not sequenced, and the methods developed for rapid localization of deletion endpoints, we may soon be ready to use these mutations to define the constitutive promoters of the chicken adult a globin genes. REFERENCES Benoist, C., O'Hare, K., Breathnach, R., and Chambon, P. (1980) Nuc. Acids. Res. 3, 127-142. Breathnach, R. and Chambon, P. (1981) Ann. Rev. Biochem. 33, 349-383. Dierks, P., Ooyen, A.V., Chochran, M.D., Dobkin, C., Reiser, J., Weissmann, C. (1983) Cell 33, 695-706. Dodgson, J.8. and Engel, 0.0. (1983) J. Biol. Chem. 333, 4623-4629. Efstratiadis, A., Posakony, J.W., Maniatis, T., Lawn, R.M., O'Connell, C., Spritz, R.A., DeReil, J.K., Forget, 8.G., Weissman, S.M., Slightom, J.L., Bechl, A.E., Smithies, 0., Baralle, F.E., Shoulders, C.C. and Proudfoot, N.J. (1980) Cell 33, 653-668. Engel, J.D. and Dodgson, J.B. (1980) Proc. Natl. Acad. Sci. USA 13, 2596-2600. Engel, J.D., Rusling, D.J., MCCune, K.C., and Dodgson, J.8. (1983) Gillies, S.D., Morrison, S.L., 0i, V.T., Tonegawa, S. (1983) Cell 33, 717-728. Girvitz, S.C., Bacchetti, S., Rainblow, A.J., Graham, F.L. (1980) Anal. Biochem. 333, 492-496. Goldberg, M. (1979) Dissertation (Stanford Univ, Stanford, CA). Ish-Horowicz, D. and Burke (1981) Nucl. Acdis. Res. 3, 2989-2998. Khoury, G. and Gruss, P. (1983) Cell 33, 313-314. Knoll, B.J., Zarucki-Schulz, T., Dean, D.C., O'Malley, B.W. (1983) Larsen, A. and Weintraub, H. (1982) Cell 33, 609-622. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory, NY). Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. 33, 499-560. McKnight, S.L. and Kingsbury R. (192) Science 331, 316-324. Nevins, J.R. (1983) Ann. Rev. Biochem. 3, 441-466. Orkin, S.H. and Kazazian, H.H. (1984) Ann. Rev. Genetics 33, 131-171. 104 105 Orkin, S.H. and Kazazian, H.H. (1984) Ann. REv. Geneticsi33, 131-171. Scott, R.W. and Tilghman, S.M. (1983) Mol. Cell. Biol. 3, 1295-1309. Smith, D.R. and Calvo, J.M. (1980) Nucleic Acids Res. 3, 2255-2274. Shortle 0., DiMaio, 0., and Nathans, D. (1981) Ann. Rev. Genetics 33, 265-294. APPENDIX I Somatic (cl! Generics. Vol, 7, No. 2, 798]. pp. 255—268 Selection of Aphidicolin-Resistant CHO Cells with Altered Levels of Ribonucleotide Reductase Carol L. K. Sabourin,‘ Paul F. Bates,’ Louis Glatzer.” Chia-Cheng Chang.2 J. E. Trosko,2 and John A. Boezi' 'Dcparrmcnl of Biochemistry and 2Department of Pediatrics and Human Development, Michigan State University. East Lansing. Michigan 48824 Received l0 September I980—Final 25 November I980 Abstract—Chinese hamster ovary cells were initially selected for resistance to aphidicolin at 0.3 rig/ml. Serial cultivation with aphidicolin at concentra- tions up to 5.0 ug/ml yielded a series of mutants with increasing resistance. The most resistant mutant isolated was 44 times more resistant to aphidi- colin than the parental C H 0. The (ii-polymerases, assayed in the cytoplasmic extracts of the mutants, did no! increase in specific activity or di'fler from the parental CHO in their sensitivity to aphidicolin. When cultured in the presence of dcoxylhymidine. deoxyadenosinc. and l-B-D-arabinofuranosyl cytosine (araC) the mutants showed considerably more resistance to these inhibitors than did the parental CHO. The intracellular pools of all four dcoxynucleosr'dc triphosphates (dNTPs) in the mutants increased with increasing resistance to aphidicolin. The elevated dNTP pools in the mutant most resistant to aphidicolin appear to be the result of a 4— to 8-fold increase in the level of ribonucleotide reductase (Z-deoxyribonuclcosidc diphos- phatc.'oxldlzed thioredoxr'n I-oxidoreductasc. EC 1.17.4.1). INTRODUCTION Aphidicolin, a tetracyclic ditcrpenoid produced by the fungus Ccphalo- sporium aphidicola, is cytotoxic to eukaryotic cells. The cytotoxicity results from a block in cell division (I, 2). Aphidicolin is a potent inhibitor of DNA synthesis, but it does not inhibit RNA or protein synthesis (1-3). One of the target enzymes for aphidicolin appears to be DNA polymer- ’Pcrmancnt address: Department of Biology. University of Toledo. Toledo. Ohio 43606. 255 mamas/31 /omozsssos.oo/o o 1981 Plenum Publishing Corporation 106 107 256 Sabouriu et al. use a. Purified a-polymerasc. but not d-polymerasc or 7-polymerasc. is effectively inhibited by aphidicolin (I. 2. 4-6). With the purified a- polymerase aphidicolin functions as a competitive inhibitor of dCTP (7). Reactions catalyzed by the purified enzyme that do not use dCTP as a substrate, for example the poly(dA) - oligo(dT) directed polymerization of dTTP. are not effectively inhibited. The mode of inhibition by aphidicolin on DNA synthesis in isolated nuclei. however. appears to be somewhat difTerent from that with the purified a-polymerase. Noncompetitive inhibition with dCTP. rather than competitive inhibition. is observed (8. 9). It is assumed that the a-polymerasc is still the target enzyme but that in the nucleus the polymerase is part of a replication complex with somewhat different catalytic properties. Mammalian cell mutants of DNA metabolism are needed for studies of the mechanism of replication. repair. and recombination. In this report aphidicolin was used to isolate resistant mutants of Chinese hamster ovary cells. Characterization of the mutants isolated indicates that the basis of resistance to aphidicolin is through oversynthesis of the dNTPs brought about by overproduction of ribonucleotide reductase. MATERIALS AND METHODS Reagents. Aphidicolin was supplied by the Developmental Therapeutics Program. Chemotherapy. National Cancer Institute. The aphidicolin was dissolved in dimethyl sulfoxide (DMSO) and sterilized by filtration using MilIipore-type FG filters. The concentration of DMSO added to the culture was always less than 0.1%. Other reagents were from sources previously described (IO) or from the usual commercial sources. Cell Culture. The parental Chinese hamster ovary (CHO) cell line which is auxotrophic for proline was obtained from Dr. Louis Siminovitch, University of Toronto. The experiments described here were carried out with one subclone. The methods used in culturing were as previously described (I I). The cells were routinely maintained in suspension culture at 37°C in MEM alpha medium, containing ribonucleosides and deoxyribonucleosides (01+) and 10% fetal calf serum (FCS). The cells were mutagenized with ethyl methane sulfonate (EMS) at 150 ug/ml. This concentration of EMS was sufficient to reduce the plating efficiency by about 50%. After I6 h the cells were washed twice with phosphate-bufi‘ered saline and resuspended in fresh medium. After allowing two days for recovery, the cells were plated in medium containing 0.3 ug/ml aphidicolin. The colonies which appeared were picked with a Pasteur pipet and put into 24-well dishes. The relative plating efficiency for Fig. I was determined by plating 2 x n—s 108 Altered Levels of Ribonucleotide Reductnsc 257 103-2 x IO‘ cells per 60-mm dish or 2 x l0‘~5 x IO“ cells per I00-mm dish at various concentrations of aphidicolin. The concentration of drug reducing the plating efficiency to l0'x'i ofthc control (LD,,.) was determined by plating I x l03-2 x l0‘ cells in the medium indicated in Table l. in 35-mm dishes. Dishes were incubated at 37°C for 7—14 days and then stained with I”? methylene blue in 70% isopropanol. The sensitivity to ultraviolet irradiation was determined by plating 2 x lot-8 x 10“ cells per IOO-mm dish. The cells were allowed 3.5 h for attachment. With the medium removed. the attached cells were exposed to ultraviolet light from a germicidal lamp which was positioned to deliver 2.0 J/mz/sec. After one week the surviving colonies were stained as previously described. Deoxyribonuclcosidc Triphosphate Pool Measurements. The intraccl- lular dNTPs were extracted from cell monolayers in the log phase of growth with 609? ice-cold methanol. The pools were measured using the defined copolymers poly d(l-C) - poly d(l-C) and poly d(AoT) - poly d(A-T) (P-L Biochemicals) and E. coli DNA polymerase I (Boehringer Mannheim) as previously described (I2. l3). The amount of DNA pcr culture was measured in the methanol precipitate (l4). Assay of DNA Polymerization Reaction. The cytoplasmic extract was prepared and assayed for the a-polymerase as previously described (IO). The concentration of DMSO added to each assay was 2.5%. Protein determina- tions were done using Coomassie brilliant blue G-250 as described by Bradford (25). Assay of Ribonucleotide Reductase Activity. The ribonucleotide reduc- tase assays were performed with crude extracts of parental CHO or mutant cells. These extracts were prepared by rupturing 0.035 g of cells per I ml of extraction buffer. IO mM HEPES (N—Z-hyroxyethylpiperazine-N-Z-ethane- sulfonic acid). pH 7.2. and 2 mM dithiothreitol. with 20 strokes of a dounce homogenizer at 4°C. The homogenate was centrifuged at 40,000 rpm in a type-50 Beckman rotor for l h. The clear supernatant was decanted off. This fraction. called the crude extract. generally contained l0 mg protein per ml. Before assaying. the crude extract was extensively dialyzed against the extraction buffer to remove endogenous nucleotides. Assays of the crude extract contained. in a final volume of 300 pl. 50 mM HEPES. pH 7.2. 6 mM dithiothreitol. 6 mM ATP. IO mM MgClz. IOO uM ["C]CDP (6.7 mCi/mmol)'plus the designated amount of protein. The samples were incubated for I h at 37°C. after which the reactions were stopped by boiling for 3 min. The reaction was determined to be linear with time. The precipitated protein was pelleted by centrifugation. and the samples were digested from the nucleotide to nucleoside forms with 25 lag/assay Crotalus atrox venom phosphodiesterase (Sigma) and 25 ug/assay 109 258 Sabourin et al. bacterial alkaline phosphatase (Sigma). The ["C]deoxycytidine and ["C]cy- tidine were assayed by the method of Sleeper and Steuart (l5). After digestion the samples were applied to 0.7 x 4.5-cm Dowex 50—boratc columns. The deoxycytidinc was first eluted with 4.5 ml of H30 and then cytidine was eluted with 5 ml of saturated KZB‘O7. The radioactivity in each peak was determined by scintillation counting in Formula-963 (New England Nuclear). Electrophoresis. Electrophoretic analysis of extracts was carried out by the method of Laemmli (16). using an 8.75% acrylamide separating gel and 3% acrylamide stacking gel. Gels were stained with Coomassie blue. Densi- tometric scans were performed on a Ortec dcnsitometer model 4310 at 540 nm. 0—0.3 OD full scale. RESULTS Selection of Aphidicolin-Resistant C H0 Cells. In the presence of 0.3 ug/ml of aphidicolin the plating efficiency of wild-type CHO was reduced to 8 x lO' 5. Resistant colonies were selected when mutagenized cells were plated in 0.3 ug/ml aphidicolin. One of these mutants. L3. was used in a stepwise manner to isolate mutants that have increasingly greater resistance to the drug. Without additional mutagenesis L3-2. which was about IO times more resistant to aphidicolin than the parental CHO. was isolated from L3; L3-3a. which was about I9 times more resistant than the parental CHO. was isolated from L3-2; and L3-5. which is about 44 times more resistant to the drug than the parental CHO. was isolated from L3-3a (Table I). Figure I shows the effect of increasing concentrations of aphidicolin on the plating efficiency of parental CHO. L3. L3-2. L3-3a. and 1.3-5. Mutants with high resistance to aphidicolin (>0.5 ug/ ml) could not be isolated in single-step isolations. The phenotype of the resistant clones remained stable over a period of many months when the mutants were maintained in the absence of aphidicolin. Characterization of Aphidicolin-Resistant CHO Cells. The doubling time of L3. L3-2. L3—3a. and L3-5 increased compared to the parental CHO when grown in suspension culture in the absence of aphidicolin (Table 2). When L3-3a was grown in 2.5 ug/ml aphidicolin and L3-5 in 5.0 ug/ml aphidicolin. the concentrations at which they were selected. the doubling time increased twofold. In addition L3-3a and L3-5 in suspension culture doubled once and then stopped dividing. although the cultures retained their viability for at least two days. as determined by trypan blue staining. The doubling times in monolayer culture of L3-3a grown in 2.5 ug/ ml aphidicolin and L3-5 grown in 5.0 ug/ ml aphidicolin were the same as those for the suspension cultures but the monolayer cultures grew from a low density until the cultures were confluent. 110 259 Altered Levels of Ribonucleotide Reductasc tugg- ’ 63: was mu... veg-Ev 48. ...:3 “8.:qu.33. ..e. 63.: was mug 8°. 5:: 83989.33 +e. .2: .552; of .3 so.— 2: c. on: .5252 2: 5.. 2...; so.— 2: ..o 2...... 2: 2.. c8885 .2 85339. 3:53. aggro? can V2522 E Engage ma B5883“. v.5: .938 2: ..o cec— S 35659 nets... 2: 9:258 mac .3 533.528“. of .29... m . .. co. V». N. m_r. ca... 3 a.» ed «A; E. C»... ad v._ v... cm... a. v6 ON «Tn.— c_. ca. no, V.T., a... car ed N... o._ «A.— 2. co... x.~ c. o.— mnd «N 9.6 M... n.— c._ cm: 0.. mg. C.— Zd o._ Ed OIU 85:16.. A25. 8557.2 :53 35.772 :ERE 35:32 Cains SEES on: =oU 3:33. 22-. 3:3:— ._:._ 3:53. so; 2:23. an: 5.823. .8 . . 5.3.2.150 .oEvccov—wieneo 352821.252 .UE< .:=8_E:Q< :o:«..:=3:oU 353:2 .c...77e~.-c__:u€£c< 9:" OIU 22:85. .6 352.0...— .— «Sch. 111 260 \ ‘\. \ >~ U S, o 9 E O E E» a. O 2 E: o 0 o: \ D L 1 i 1 Aphidicolin (pg/ml) 4 5 6 7 8 Sebourin et al. Fig. I. Relative plating efficiency of parental CH0 (0) and the aphidicolin-resistant mutants. L3 (0). L3-3 (A). l.3-3a (’1') and L3-5 (A) with varying concentrations of aphidicolin. L3. L3-2. L3-3a. and L3-5 were examined to see if the basis of drug resistance was due to a change in the ctr-polymerase. Using cytoplasmic extracts of these cells. no differences in specific enzymatic activities of the cit-polymerase or sensitivities of the a-polymerase to aphidicolin were observed compared to parental CHO enzyme (Table 3). Thus. the resistance to aphidicolin in the mutants is probably not due to a structurally altered a-polymerase or because of an overproduction of the a-polymerase. The sensitivity to ultraviolet irradiation of L3 and L3-2 was examined. Table 2. Effect of Aphidicolin on Doubling Time of Parental CH0 and Aphidicolin—Resistant Mutants‘ Doubling time (h) in aphidicolin Cell line Oug/ml |.0 tag/ml 2.5 ug/ml 5.0ug/ml CHO 13 J — —— L3 I5 — — — L3-2 I7 20 -— -— L3-3a 27 36 62 — L3-5 20 27 29 44 ‘Cells were grown in suspension culture in 01+ supplemented with IO‘Z FCS. The number of cells/ml was determined using a Coulter counter model 28]. ‘ — indicates that the cells will not grow at this concentration of aphidicolin. 112 Altered Levels of Ribonucleotide Reductase 26f Table 3. Characterization of a-Polymcrase Activity in Cytoplasmic Extract of Pa rental CH0 and Aphidicolin-Resistant Mutants Specific enzymatic activity" 509? inhibition’ Cell line (units/mg protein) (uMl CHO 9.4 6.5 L3 8.9 7.5 L3-2 9.3 6.0 L3-3a lb 7.8 L3-5 I2 8.8 ‘Nanomolcs of [’HldTMP incorporated into activated calf thymus DNA/3O min/mg protein. The u-polymcrase was assayed as described in Materials and Methods. ’Concentration of aphidicolin which results in a 5097 decrease in the picomoles of [’H]dTMP incorporated into activated calf thymus DNA/30 min in the absence ofaphidicolin. L3 showed no significant differences from parental CHO in the ultraviolet sensitivity in 0+ supplemented with 10% FCS. The results were similar for L3 and L3-2 when compared to parental CHO in MEM alpha medium lacking ribonucleosides and deoxyribonucleosides (n—). supplemented with 10% dialyzed FCS. Therefore the defect in the mutants which brings about resistance to aphidicolin is probably not related to the enzymes involved in DNA repair. The effect of added araC on colony formation of the aphidicolin- resistant and the parental CHO was examined. The mutants showed greater resistance to araC than the parental CHO (Table I). There are two known mechanisms by which a cell can become resistant to araC: a deficiency in the deoxycytidine kinase activity would not phosphorylate araC to its toxic form. araCTP. or an expanded pool odeTP would confer resistance by diluting the araCTP thereby reducing the inhibition of the DNA polymerase (17). To determine the mechanism whereby the mutants became resistant to araC. the effect of deoxythymidine and deoxyadenosine was investigated. The mutant cell lines were more resistant to both deoxynucleosides than the parental cell line (Table I). Exogenous deoxythymidine and deoxyadenosine are converted to their respective triphosphates which in turn act allosterically to diminish the activity of CDP reductase. The supply of deoxycytidine nucleotides then becomes inadequate for DNA synthesis. The resistance to exogenous nucleosides demonstrated by the mutants could be related to modifications of the ribonucleotide reductase activity that would increase the supply of deoxycytidine nucleotides. The above results. coupled with the knowledge that aphidicolin is a competitive inhibitor of dCTP with the purified tit-polymerase (7). suggest a possible mechanism of resistance to aphidicolin involving enhanced synthesis of dCTP. The dNTP pools of L3-2. L3-3a. and L3-5 were measured and 113 262 Sabouri- et II. Table 4. Deoxyribonuclcosidc Triphosphate Pools in Parental CH0 and Aphidicolin-Resistant Mutants‘ Aphidicolin ”mm/“g DNA Cell line (pg/ml) d(‘TP dTTP dATP dCTP CHO 0.3 9.3 1.9 is L3-2 2.1 7.5 6.6 L: L3-3a I66 94 26.1 4.8 1.34.. 2.5 53.: 361 I52 IS I L3-5 85.7 15.8 57.7 10.2 L3-5 5.0 I86 69.9 I63 36.8 ‘Cultures grown exponentially in in supplemented with 10"; FCS at the indicated concentra- tion of aphidicolin and assayed as described in Materials and Methods. The values of each determination are the average of four separate determinations. compared to those of the parental CHO in a+ supplemented with IO’Z FCS. The results are given in Table 4. The pools of the four dNTPs increased with increasing resistance to aphidicolin. The dNTP pools of L3-3a and L3-5 grown in the presence of aphidicolin were larger than their pools when grown in the absence of aphidicolin. Pool expansion was also obtained for L3-3a when compared to parental CHO for cultures grown in a— supplemented with I0% dialyzed FCS. Ribonucleotide Reductase Activity of Aphidicolin-Resistant C H0 Cells. Since the increased pools of dNTPs observed in the aphidicolin- resistant mutants could result from qualitative or quantitative changes in the ribonucleotide reductase. crude extracts of L3-5 and parental CHO cells were assayed for CDP reductase activity. The L3-5 mutant showed a marked increase in CDP reduction compared to the parental CHO (Fig. 2). There was a 4- to 8-fold increase in the specific activity of the CDP reductase. Both the L3-5 and parental C HO extracts showed a nonlinear dependence of CDP reduction on protein concentration. This nonlinear protein dependence has previously been shown for the ribonucleotide reductase of mammalian systems (l8). Also. there was a further increase in CDP reductase activity when the L3-5 cells were grown in the presence of aphidicolin. This increased activity parallels the increase in dNTP pool sizes when the cells were grown in the presence of aphidicolin. The increased CDP reductase activity in the crude extracts was stable when the cells were cultured for several months in the absence of aphidicolin. Loss of allosteric regulation by dATP would result in an overproduction of all four dNTPs as is seen in the mutants. In order to determine whether the increased activity of the reductase in the mutant cells was due to loss of allosteric control. the inhibition of the C DP reductase by dATP was assayed. Both the L3-5 and parental CHO extracts were equally sensitive to dATP 114 Altered Levels of Ribonucleotide Reductase 263 n.“ O : |0 — A/ g ./ 2 8 ~ 0 E on / .E U 4 y... z A > ‘/ o g 2 '- ,A/ o O l 1 l 0.5 l0 LEE 2 0 Protein (ug x IO'3) Fig. 2. CDP reductase activity in extracts of parental CH0 (0) and L3-5 (A). Varying amounts of each extract were assayed for CDP reductase activity as described in Materials and Methods. Each point represent the average of five separate experiments inhibition of CDP reduction with a 50% inhibition of IO “M for both L3-5 and parental CHO extracts. Since alterations in ribonucleotide reductase can apparently produce an aphidicolin-resistant phenotype. assays were run to determine if aphidicolin inhibits this enzyme. Even at IOO uM. fifteen times the value for 509? inhibition of the a-polymerase. the CDP reductase of the parental CHO showed no significant inhibition by aphidicolin (Table 5). Polyacrylamide Gel Electrophoresis of Crude Extracts. From the results of the CDP reductase assays. it appeared that it may be possible to visualize the increased levels of ribonucleotide reductase by comparing L3-5 and parental CHO cell extracts on SDS polyacrylamide gel electrophoresis. The only significant differences in the gels were an increase in staining of the Table 5. Effect of Aphidicolin on the CDP Reductase in Parental ChO Specific activity‘ Addition to the assay (pmol/h/mg protein) IOO uM aphidicolin in DMSO‘ 500 DMSO‘ 620 None 767 ‘CDP reductase activity was determined as described in Materials and Methods. ‘The final concentration of DMSO in the assay was I679}. 115 264 Sabourin et al. bands corresponding to polypeptides of molecular weight 55.000. 72.000. and 80.000 (Fig. 3). The values of 55.000 and 80.000 corresponded well with the subunit molecular weights of 55.000 and 84.000 for calf thymus ribonucleo- tide reductase (l9). These two bands were found using several different preparations of the L3-5 cells. The 72.000-dalton polypeptide was not observed in all preparations. and may be a degradation product of a 76.000-dalton polypeptide. A densitometric scan of the gel indicated that the 76.000-dalton peak was smaller in the L3-5 extract than the corresponding peak in the parental CHO extract (Fig. 4). Integration of thc 72.000 and 76.000 peaks in the L3-5 extract yielded a value equal to that of the 76.000 peak in parental CHO extract. The densitometric scan also showed that the 55.000 and 80.000 bands of the L3-5 extract were increased in approximately equal amounts. as would be expected for an increase in ribonucleotide reductase. These results suggest that thc ribonucleotide reductase may be overproduced in the aphidicolin-resistant mutant. Parental l3- 5 C HO Fig. 3 SDS-polyacrylamide gel electrophoresis of parental C HO and L3-5 crude extracts prepared as described in Materials and Methods. The parental CH0 and L3-5 lanes contain 30 ug each of protein. Lane 3 contains 8 pg each of the molecular weight standards phosphorylase A (94.000). bovine serum albumin (68.000). and catalase (56.000). 116 Altered lxvels of Ribonucleotide Reductase 265 PARENTAL cm Fig. 4. Densitomctric scan of a region of the SDS-polyacrylamide gel of parental CH0 and L3-5 extracts. DISCUSSION This report describes the biological and biochemical characteristics of a series of CHO mutants which are resistant to aphidicolin. The resistance of these mutants does not appear to result from a structurally altered or overproduced (ii-polymerase. The resistance of the mutants seems instead to be due to an alteration in the levels of ribonucleotide reductase which results in the overproduction of the four dNTPs. The aphidicolin-resistant mutants. when cultured in the presence of deoxythymidine. deoxyadenosine. and araC. showed considerably more resis- tance to these inhibitors than did the parental CHO. Deoxythymidine. deoxyadenosine. and araC in high concentrations all act to depress DNA synthesis. Deoxythymidine and deoxyadenosine are phosphorylated to the triphosphates which allosterically interact to inhibit the ribonucleotide reduc- tase activity. The supply of deoxycytidine nucleotides then becomes inade- quate for DNA synthesis. The large dCTP pool expansion which occurs in the mutants confers resistance to deoxythymidine and deoxyadenosine to the cell since the dCTP supply does not become a limiting factor for DNA synthesis. The additional dCTP produced also dilutes the araC nucleotide pool. thereby reducing the inhibition of the DNA polymerase by araCTP. the toxic form of araC. The increased pools of the four dNTPs can be related to an increase in the ribonucleotide reductase activity. Two possible alterations in the ribonu- cleotide reductase which would account for the increased dNTP pools were investigated. Increasing the amount of ribonucleotide reductase in the cell is one mechanism by which the activity of the enzyme can increase. We found a 4- to 8-fold increase in the crude extract specific activity of the CDP reductase activity for L3-5 over the parental CHO. In addition. the electro- 117 266 Sabourili et al. phoretic analysis of the parental CH0 and L3-5 extracts indicates that the two polypeptides of molecular weights similar to those reported for calf thymus ribonucleotide reductase are significantly overproduced in the L3-5 extracts. Modification of the ribonucleotide reductase activity could also result if the allosteric control mechanism was disrupted. Dcscnsitization of the enzyme to dATP. a general allosteric inhibitor of the ribonucleotide reduc- tase. would cause increased activity and overproduction of all four dNTPs. If the mechanism for increased ribonucleotide reductase activity in the step- wise-selected mutants was a loss of allosteric inhibition by dATP. one would expect that L3-5. the mutant with the largest dNTP pools. would be the least sensitive to dATP compared to the parental CHO cells. We found no significant differences in the sensitivity to dATP of the L3-5 and parental CHO CDP rcductascs. Alternatively a structural alteration in the enzyme. not involving the allosteric sites. could also produce a more active enzyme. Such a structural alteration. affecting the intrinsic specific activity of the enzyme. would not be detected by the experiments conducted. Meuth and Green (20) have demonstrated that mutants selected for resistance to araC also showed resistance to deoxythymidine and deoxyadeno- sine. These araC-resistant mutants were shown to have expanded dNTP pools (2|). The alteration in ribonucleotide reductase activity showed a 4- to lO-fold higher level of enzyme specific activity but. unlike our aphidicolin- resistant mutants. also showed a partial desensitization of the enzyme to the allosteric negative effector dATP (20). Enzyme levels of ribonucleotide reductase have been shown to be under strict cell cycle control (22). In mammalian cells the level of ribonucleotide reductase increases during the S phase of growth and falls during the 62 phase. If this cell cycle regulation were lost. the apparent effect in a nonsynchronized culture of cells would be an increase in the amount of ribonucleotide reductase present. Alternatively. the cells may retain cell cycle regulation of enzyme levels. but overproduce the enzyme during the S phase. Our studies do not distinguish between these two possible mechanisms for overproduction of ribonucleotide reductase. The results for L3—5 in the CDP reductase assays coupled with the gel electrophoresis indicate that the aphidicolin-resistant mutants described overproduce ribonucleotide reductase. Each mutant isolated in a stepwise manner has increasingly higher resistance to aphidicolin. larger dNTP pools. and presumably has more reductase. The possibility of isolating mutants with an even greater overproduction of ribonucleotide reductase remains. Massive overproducers of this enzyme will be a great aid in studies characterizing the catalytic and structural properties of this enzyme. Our polyacrylamide gel 118 Altered Levels of Ribonucleotide Reductase 267 results suggest that two polypeptide chains are overproduced. lending credence to the hypothesis that the enzyme is composed of two polypeptide subunits. Further studies of these mutants may also answer questions concerning the control of biosynthesis ofan enzyme composed of two different polypeptide chains. The fact that mutants resistant to high concentrations of aphidicolin are only obtained by stepwise selection indicates that resistance to high aphidi- colin concentrations could be due to overproduction of ribonucleotide reduc- tase through gene amplification. Overproduction of ribonucleotide reductase could be linked to amplification of the genes programing the synthesis of two polypeptide chains. Amplification of genes coding for proteins with one polypeptide chain has been observed in methotrexate-resistant variants (23) and N-(phosphoacctyl)-L-aspartate-resistant variants (24). Highly resistant mutants could only be obtained in this stepwise manner. Mutants of mammalian cells that overproduce a protein composed of two different polypeptides could provide a system for answering many questions concerning the control of their synthesis. ACKNOWLEDGMENTS During the final phases of this work Dr. John A. Boczi died. John was both a dedicated scientist and educator. His enthusiasm and creativity were and still are an inspiration to those who knew him. This research was supported by grants from NIAID (Al-M357) to J.A.B.. from NCI (CA- 2l l04) to J.E.T.. and from NIEHS (ES-01809) to C.C.C. Michigan Agricul- tural Experiment Station Journal Article Number 9625. LITERATURE CITED l. Ikegami, S., Taguchi. T., Ohashi. M.. Oguro. M.. Nagano. H.. and Mano. Y. (I978). Nature 275:458-460. 2. Longiaru. M.. lkeda. J-E.. Jarltovsky. Z.. Horwitz. 8.8.. and Horwitz. M.S. (I979). Nucleic Acids Res. 6:3369—3386. 3. Bucknall. R.A., Moores. H.. Simms. R., and Hesp. B. (I973). Antimicrob. Agents C hemother. 4:294- 298. 4. Ohashi. M.. Taguchi. T., and Ikegami, S. (I978). Biochem. Biophys. Res. Commun. 82:l084-IO90. 5. PedralioNoy. G., and Spadari. S. (I979). Biochem. Biophys. Res. Commun. 88:l I94- I202. 6. Wist. E.. and Prydz. H. (I979). Nucleic Acids Res. 6:1583—l590. 7. Oguro. M.. Suzuki-Hori. C., Nagano, H.. Mano. Y. and Ikegami. S. (I979). Eur. J. Biochem. 97:603—607. 8. Habara. A., Kano. K.. Nagano, H., Mano. Y.. Ikegami, S.. and Yamashita. J. (I980). Biochem. Biophys. Res. C ommun. 92:8-l 2. 268 l2. l3. l4. l5. l6. l7. l8. I9. 20. ll. 22. 23. 24. 25. 119 Sabourin et al. Oguro. M.. Shioda. M.. Nagano. IL. and Mano. Y. (I980). Biochem. Biophys. Rm Commun. 92zl3- l9. Sabourin. C.L.K.. Reno. J.M.. and Boezi. J.A. (I978). Arch. Biochem. Biophys. ”7:96 l0l. Thompson. L.H., and Baker. R.M. (I975). In Methods in Cell Biologi'. lb! 6. (ed.) Prescott. D.M.. (Academic Press. New York). pp. 209 28l. Skoog. L. (I970). Eur. J. Biochem. I7z202- 208. Lindberg. U., and Skoog. L. (I970). Anal. Biochem. 34zl52- I60. Giles. K.W.. and Myers. A. (I965). Nature 206:93. Sleeper. J.R.. and Steuart. C.D. (I970). Anal. Biochem. 34:]23-130 Laemmli. UK. (I970). Nature 227:680—685. Dc Saint Vincent. D.R.. and Buttin. G. (I979). Somat. Cell Genet. 5:67—82. Hopper. S. (I972). J. Biol. Chem. 247:3336- 3340. Engstrom. Y.. Eriksson. 8.. Thelander. L.. and Akerman. M. (I979). Biochemistry l8:294l-2948. Meuth. M.. and Green. H. (I974). Cell 31367-374. Meuth. M.. Aufreitcr. E.. and Reichard. P. (I976). Eur. J. Biochem. 71:39—43. Thelander. L.. and Reichard. P. ( I979). Annu. Rev. Biochen1.48:l33-l58. Alt. F.W., Kellems, R.E.. Berttno. J.R.. and Schimkc. R.T. (I978). J. Biol Chem 253zl357‘ I370. Wahl. G.M.. Padgett. R.A.. and Stark. G.R. (I979). J. Biol. Chem. 254:8679-8689. Bradford. M. (I976). Anal, Biochem. 72:2487 254. APPENDIX I I (lane. 26 (I983) I.W-I-Ifi I.“ Llscvier GITNI- 905 Double cos site vectors: simplified cosmid cloning ° (Genome libraries: chromosome walking; eukaryotic selectable marker; Herpes virus; thymidine kinase; HAT selection; retrovirus LTR) Paul F. Bates and Robert A. Swift Dcymrtments oth‘iit'ltt’ittt'.strt‘ and (if/llit'robiologv and Public Health. Michigan State University. East Lansing. MI 48824-1101 (U.S.A.) Tel. (517) 353-5024 (Received February 5th. I983) (RCHSIOI‘I received August llth. I983) (Accepted August 3lst. I983) SUMMARY A new vector for construction of cosmid libraries is described. Cosmid c2XB contains restriction sites for use in the insertion of foreign DNA and two 7. cos sites separated by a blunt-end restriction site. The presence of two cos sites on a single plasmid eliminates the need to prepare two separate cosmid arms. and the internal blunt-end restriction site prevents cosmid concatemerization. Thus. a double restriction-enzyme digestion is sufficient to prepare the vector for subsequent ligation with DNA fragments which are dephosphorylated to prevent their self-ligation. The use of this vector system allows efficient cosmid cloning (I x 105 colonies per pg insert DNA) and eliminates background due to vector self-ligation. Furthermore. the procedure is so rapid as to eliminate the need to amplify cosmid libraries for storage and reuse. Also described is a cosmid vector for use in construction of cosmid libraries which are to be introduced into cultured eukaryotic cells. This vector contains the Herpex simplex virus thymidine kinase (H SV tk) gene as a selectable marker and a retroviral long terminal repeat (LTR) region as an enhancer sequence. INTRODUCTION Several recent studies of eukaryotic gene organiza- tion and /or expression demonstrate the need to clone large segments of DNA. In some cases the size ‘ This is Journal Article No. 10755 from the Michigan Agri- cultural Experiment Station. Abbreviations: bp. base pairs; c. cosmid; DTT. dithiothreitol; HAT. hypoxanthine-aminopterin-thymidine; HSV. Herpes sim- plex virus; kb, kilobase pairs; LB. Luria broth; LTR. long terminal repeat; tk, thymidine kinase; U. units. 0378—Ill9/83/SO3OO © I983 Elsevier Science Publishers 120 of a gene requires a large insert if the gene is to be isolated intact. Studies of gene families or of closely linked genes which are coordinately expressed may also require cloning of large DNA fragments. Fur- thermore. the study of extensive regions of a eukaryotic genome by serial isolation of a number of overlapping clones (i.e. genome walking) is facilitated by cloning fragments as large as possible. Both plasmid and .1 bacteriophage vectors are often inadequate for cloning large DNA fragments. The ). vectors available can only accommodate inserts up to about 20 kb. Theoretically. plasmids have the capacity to accommodate fragments of unlimited ms 121 size; however, the transformation efficiency of large plasmids is so low that their use in the construction of genomic libraries is not practical. Presently the primary system used to clone large DNA fragments efficiently is that of cosmid vectors (Collins and Hohn, 1978). These vectors are modified plasmids which contain a plasmid replicon. a selectable drug resistance marker, and the 1. cos site. Cosmids can accept inserts of 30—45 kb, and the 11 in vitro packag- ing system selects for large-size inserts and allows efficient introduction of the DNA into bacterial cells. However, because of several problems associated with cosmid cloning, this system has not yet been widely employed. Three main problems which have been encountered are vector concatemerization re- sulting in cosmids lacking inserted eukaryotic DNA, recombinational rearrangements caused by multiple inserts ligated into a single cosmid, and differential grth of cosmid clones causing misrepresentation of sequences in amplified cosmid libraries. A cloning system which overcomes the first two of these problems has been described (lsh-Horowicz and Burke, 1981). Concatemerization of the vector was avoided by using a multiple enzyme digestion procedure involving four separate restriction enzyme digestion steps and two 81 nuclease treatments. The problem of insert rearrangement was minimized by treating the DNA to be cloned with phosphatase prior to its ligation to the vector; this prevents the insert DN As from ligating to one another, and elimi- nates the need to purify insert DNA of the proper size (i.e. 30—45 kb). Because the Ish-Horowicz and Burke (1981) cloning scheme requires a considerable number of steps it becomes tedious when large numbers of cosmid libraries must be constructed. Furthermore, since differential growth of cosmids alters the representation of specific fragments in amplified cosmid libraries, it would be preferable to be able to rapidly construct a new cosmid library for each individual screening, thereby eleminating any amplification steps. » We describe here cosmid vectors which are easy to prepare and use, and avoid the problems of insert rearrangement and cosmid concatemers. This has been accomplished by constructing vectors contain- ing two cos elements of }. separated by a blunt-end restriction enzyme site (see Figs. 1 and 3). The use of two cos sites on a single plasmid eliminates the need to separately prepare two cosmid arms, each of which contains only a single cohesive end. After cleavage at the blunt-end restriction site. cosmid concatemerization does not occur during the subse- quent ligation step when high ATP concentrations are employed (Ferretti and Sgaramella. 1981). We have also constructed a cosmid vector for use in the transfection of cosmid libraries into eukaryotic cells. This vector uses the HSV 1k gene as a selectable marker for cells which have incorporated cosmid DNA. A retroviral LTR is also contained on this vector as an enhancer sequence to aid in expression of the ik gene and adjacent cloned sequences. MATERIALS AND METHODS (3) Bacterial strains and enzymes Escherichia coli HBIOI, rB’ mB ’ recA ' (Boyer and Roulland—Dussoix, 1969) was used for transfor- mation and growth of most of the plasmids con- structed. Those plasmids containing tandem repeats were grown in the strongly recA ‘ strain DHI (Maniatis et al., 1982). Another recA ' strain, E. coli 1046 (Cami and Kourilsky, 1978) was used for the transduction and grth of the cosmid libraries. Restriction enzymes, T4 DNA ligase, T4 DNA poly- merase, and Klenow fragment of E. coli DNA po- lymerase I were from New England Biolabs or Bethesda Research Labs. Calf intestinal phospha- tase was from Boehringer. [at-”PldCT P was from : New England Nuclear. (b) Nucleic acids - Preparative plasmid DNA extractionsemployed the alkaline lysis method (Bimboim and Doly, 1979). Analytical plasmid and cosmid DNA extractions (1.5 ml) followed the method of lsh-Horowicz and Burke (1981). Genomic DNA for use in construction of cosmid libraries was prepared from chicken erythrocytes by the method of Blin and Stafford (1976). DNA prepared by this method was generally greater than 150 kb in size as judged by electro- phoresis in 0.2% agarose gels. 122 (c) Plasmid constructions Details of the plasmid constructions are given in RESULTS AND DISCUSSION. sections a. d and e. In general. all restriction enzyme digestions were per- formed as recommended by the supplier. Cohesive ends were filled in using T4 DNA polymerase. DNA was incubated with T4 DNA polymerase (3 U/pg DNA) in the absence of added deoxynucleoside triphosphates for 5 min at 37°C. All four deoxynucleoside triphosphates were added to 100 pM and the reaction was continued for 30 min at 37°C. A probe specific for the 2 cos site was prepared by incubating Charon28 DNA (Rimm et al., 1980) with T4 polymerase for 70 min before adding [1-32P]dCTP to 10 pM and the other dNTPs to 100 pM and incubating for an additional hour. The Charon28 DNA was then digested with Bglll and Aval to give a labeled 2. end-specific probe. Partial end-filling reactions were performed with Klenow fragment of E. coli DNA polymerase I (0.] U /pg DNA) and 200 pM of the required deoxy- nucleotide used (e.g., 200 pM dATP to partially fill EcoRI ends). (I!) Preparation of insert DNA for library construc- tion Insert DNA for construction of cosmid libraries was prepared by partial digestion with either Mbol or EcoRI. 200 pg of chicken erythrocyte DNA was digested at 37 °C for 60 min in 0.5 ml with an amount of enzyme optimized to yield 30—50 kb fragments. The reaction was stopped by incubation at 70°C for 15 min. The pH was adjusted with 50 pl of 2 M Tris - HCl pH 8.5 and calf intestinal phosphatase (2.5 U) was added. The DNA was dephosphorylated for 30 min at 37°C, and the phosphatase was inacti- vated at 70°C for 1.5 h. The DNA was then either precipitated with spermine (H00pes and McClure, 1981) or used in Iigations without further treatment. (e) Preparation of vector DNA for cosmid library construction c2XB DNA (20 pg) was digested with Smal (40 U) and BamHI (20 U) or EcoRI (20 U) at 37°C for 4 h to ensure complete digestion. The enzymes were inactivated by incubation with 0.1"/o diethyl- I39 pyrocarbonate at 70’C for 15 min. The DNA was precipitated with spermine. washed with ethanol. and resuspended in 20 pl of 10 mM Tris. pH 7.5. 0.1 mM EDTA. (f) Ligations Two difTerent ligation conditions were used de- pending on whether blunt-end ligation was to be inhibited or not. To join cohesive and/or blunt ends. a buffer consisting of 66 mM Tris pH 7.5, 10 mM DTT. 5 mM MgCl2 and 0.25 mM ATP was employed. Inhibition of blunt end ligation employed the same buffer except ATP was used at 5 mM. (g) Packaging extracts, DNA packaging Packaging extracts were prepared by protocol ll of Maniatis et al. (1982) using strains BHBZbSS and 8H82690 (Hohn, I979). The efficiency of these extracts was I x 10" plaques/pg A DNA. 5 pl (1.5 pg) of ligated DNA was packaged per reaction using a procedure detailed by Maniatis et al. (1982). Packaged cosmids were stored in phage buffer over CHCI3. Cosmids were transduced into E. coli 1046. A saturated culture of 1046 grown in LB + 0.4‘3/o maltose was pelleted by centrifugation and resus- pended in an equal volume of 10 mM MgSO,,. Up to 100 pl of packaged cosmids was added to 100 pl of the resuspended bacteria. Adsorption was allowed to take place for 20 min at 37°C, then 500 pl of LB was added, and the incubation was continued for 30 min at 37°C. The mixture was spread on a 9-cm plate of L agar containing 75 pg/ml ampicillin. (b) Other methods Standard published procedures were used for gel electrophoresis, transfer to nitrocellulose paper, nick translation, and hybridization (Maniatis et al., 1982). Elution of DNA fragments from agarose gels was by the method of Girvitz et al. (1980). Bacterial transformations were by a modification of the CaCl2 procedure for E. coli 11776 (Maniatis et al., I982; Hanahan, D., personal communication). Eukaryotic cell transformations were done by the calcium phos- phate precipitation technique using Ltk ’ aprt ’ cells and the procedure described by Wigler et al. (1979), using HAT selection (Szybalska and Szybalski, I962). 123 I-tti fieEEBfit :39 .0: an: 3% ...... of ..c 2.52.5.5 2:75.? 2; .3595 use 2 8273 E3, «.37. EC: cezficutc 3:28 2: 286:. mBCt< .539 02% c. .70 ..c 3:“ 35% can _xeom of 2:. v2.20 3 £033 _Omuho 22:2. :2: «2.56:. oz: .93; 2; .a 5:63. .mh-=.~mmx 2: E coin 8a 5.6.5.538 05.6 $800 .mxmu .38.» 2.5.8 Cc 5:935:09 E com: 82» 3.3.535 EEwSD ._ .wE A3939 rot AEOaOUV :00... ..eo a». at! o ... ouch...- :.. oeo loco... .....o .9; .9; .7230 ii I i 2 ..Ex: 2.6 ..ot (86 Ch ...... .. ... \ 1‘ 4 :00: Deon—ea... a 009/ . EK::>A .2051 3.0.0... . .KOuw 0. 8. 2.30... .3: «to v» ...-.9 3.5.3.3 Iii l I” ...t I \ .00 E .1 m .3... r0u\\ cuhuv Quezwv a. aEchEw 300: 0:01:33 o ...?o .3: >20 5.... 3:... Reduce; ....oeo loco... 5.2:: :30 ...;n. .COuw :- 06.1 .9; ..ea «:0 v» .329 2.- l1 124 RESULTS AND DISCUSSION (a) Construction of vector c2XB The construction of cosmid vector c2XB was per- formed as shown in Fig. l. A 560-bp DNA fragment containing the J. cos site was isolated from the cosmid vector MuaS (Meyerowitz et al., 1980) after digestion with Pvul + Pstl. Ends of the fragment were filled in with T4 DNA polymerase. and it was inserted by blunt-end ligation into either the Xhol or Bgl ll site of the plasmid pKC7 (Rao and Rogers, 1979) after these sites had also been blunt-ended with T4 poly- merase. After transformation. colonies with plas- mids containing the 2. cos site were identified using ”P-labeled Charon28 DNA (Rimm et al., I980). DNA was prepared from several colonies with posi- tive signals and digested with EcoRI + Smal or BamHI + Smal. In all the plasmids examined only a single cos-containing 560—bp fragment had been inserted (results not shown). These plasmids were named ch and CEO] for those with the cos site at the Xho] or Bglll site. respectively. The ex} and cBGI plasmids were then combined to make the double cos site vector c2XB. A triple enzyme digestion scheme was designed to enrich for recombinants with two cos sites. cBGI DNA was digested with XhoI and the sticky end was then partially filled with DNA polymerase I Klenow frag- ment using d'l'l'P only. Partial end filling ensures that the XhoI cohesive ends will be unable to correctly base pair and will be unable to religate (see Fig. 1). This DNA was then digested with Xmal and EcoRI. This triple enzyme digest of cBGl yielded three fragments but only the XmaI-EcoRI fragment containing the cos site had both cohesive ends intact and available for ligation. Similarly, ch DNA was digested with Hindlll, partially filled with dATP, then digested with EcoRI and Xmal. The ex} and CEO] digestion products were mixed and ligated. After transformation of E. coli DH], only plasmids with i. cos sites at both the XhoI and Bglll sites were isolated. For c2XB to be packaged in vitro the l. cos sites must be in the same orientation relative to one another. as shown by the arrows in Figs. 1 and 3. To test the relative orientation of the cos sites, plasmid DNA was isolated from ten colonies and tested for its utility for cosmid library construction. After NI 5 5 a: E m \ \ _E~—— ‘g\\\ -‘\§EE lzzEmmms Fig.2. Restriction enzyme mapping of c2XB cosmid vector. Electrophoregrams of single. double. and triple restriction digests of c2XB are shown in lanes 2—7 (RI = EcoRI). Lanes 1 and 8 contain 1 Hindlll fragments as size markers. Electrophoresis was carried out on 6.5 x 9 cm gel of0.7°,, agarose in Tris-acetate buffer for l h at 75 V. digestion with EcoRI and Smal the plasmid DNA was ligated to chicken DNA that had been partially digested with EcoRI, packaged in vitro, and trans- duced into E. coli. Four of the ten plasmids tested gave rise to ampicillin-resistant colonies, and so were judged to have the 1. cos sites in the same relative orientation. DNA from one of these plasmids was prepared and the structure of c2XB shown was con- firmed by restriction endonuclease mapping (Fig. 2). Single sites in c2XB, which can be used for cloning. are BamHl. Clal, EcoRI, and Hindi". The BamHI and Clal sites are the most useful sites for cosmid cloning. since these enzymes leave ends complimen- tary to the 4-bp recognition sequence of enzymes Mbo] and Taql, respectively. (b) Construction of cosmid libraries with c2XB A general method for construction of cosmid libraries with c2XB is shown in Fig. 3. A double 125 I42 IIDNI SM“ SUM in! Bar“ A _-—. WW Dot 5"‘3' at.” “onion ””1th g Ila-1W (2.5-unto) msmt DNA S-ro 0 GI! Cronaoto auto. I coal ..MHI mnom Cut Inaon tonota 32~ as-c JI-SJ a t. oats pacing-B. not-mum 0 who ooclao' @t osm-o Debi." y Fig. 3. Scheme for use of c2XB in construction of cosmid libraries. After digestion with BamHI and Smal. c2XB DNA is ligated to phosphatase-treated insert DNA partially digested with Mbo]. Note that BumHl and Mhol have the same cohesive GATC ends. A high ATP concentration is used during the ligation reaction to prevent the joining ofthc Smal blunt ends (sec RESULTS. section b) digestion prepares the vector for cloning. Digestion of c2XB with Smal creates a linearized molecule with blunt ends. Digestion with a second restriction enzyme, for example BamHl, cleaves the vector into two fragments. each containing a 2. cos site and each having one blunt and one sticky end. The vector DNA is then mixed in a 2:1 vector to insert mass ratio with partially digested (Mbol), dephos- phorylated insert DNA. The DNA is ligated at a DNA concentration of 300 pg/ml with a high ATP concentration to inhibit concatemerization of the vector by blunt end ligation at the Smal end. It has recently been demonstrated that an ATP concen- tration of 2.5 mM effectively inhibits blunt-end liga- tion but does not affect cohesive end ligation (Ferretti and Sgaramella, 1981). After ligation, the DNA is packaged in vitro, transduced into E. coli 1046, and ampicillin-resistant colonies are selected. The cloning efficiency obtained with this vector is routinely l x 105 colonies per pg insert DNA. This efficiency is slightly lower than that reported for some other cosmid cloning systems (Ish-Horowicz and Burke, 1981; Hohn and Collins, 1980), but this is probably due to the lower efficiency of our packaging extracts (l x 108 plaques/pg A DNA vs. 5 x 108 plaques/pg A DNA obtained by others). With an average insert size of 37 kb (see below) only 1.2 x 105 cosmid clones would be required to have a 99"/o probability of cloning any sequence in the chicken genome. In this system only 1.2 pg of partially digested insert DNA would be needed to generate a complete chicken library. Since the insert can be phosphatase treated, and need not be size fractionated to prevent insertion of multiple frag- ments. both insert and vector DNA can be prepared for library construction in one day. Because libraries can be constructed so easily there is no need for amplification prior to screening. Thus, problems due to unequal growth of cosmid clones during amplifica- tion, causing unequal sequence representation in the library, are avoided. (c) Characterization of cosmids Independent colonies from a cosmid library con- structed using c2XB and chicken DNA partially digested with EcoRI were picked randomly for further analysis. DNA prepared from these colonies was digested with EcoRI and analyzed by gel electro- phoresis (Fig.4). Insert sizes were determined by summing the bands. The average size was 37.6 i 7.5 kb (n = 12). As expected, all five colonies examined showed different restriction patterns of their inserted chicken DNA (Fig. 4A), but only a single 5.1-kb vector band. The latter implies that a single c0py of the vector is contained in each of the cosmids. We noticed that the average insert size is smaller than expected. The packaged vector occupies only 5.1 kb of DNA (6.8-kb c2XB minus 1.7-kb fragment between cos sites lost during packaging; Fig. 3), and we expect that fragments up to 45 kb should be inserted. However, the average insert size obtained was only 37.6 kb. Similar results have been noted previously by others with the vectors p188 and Homer 1, with average insert sizes of 38.8 kb and 126 .0.-. a Fig. 4. EcoRI digests of cosmid DNA clones, A cosmid library of EL'URI partially digested chicken DNA was prepared DNA was prepared from several randomly picked clones and digested with EcoRI and run on a 08”,, agarose gel. Lanes 1 and 7 contain 2. Hmdlll fragments used as size markers. Panel A: Ethidium bromide-stained gel. Panel B: Autoradiograph of Southern transfer ofgel. hybridized Wllh "zP-labclcd pKC7 DNA (see Fig. I). Arrowheads indicate 5.1-Rh vector bands. 36 kb. respectively (Ish-Horowicz and Burke, 1981; Chia et al., 1982). Possibly the smaller insert sizes obtained reflect the dearth of larger fragments result- ing from our partial digestion procedure, since Meyerowitz et al. (1980) have obtained inserts of 45.5 kb average size using sheared DNA. Altema- tively, the in vitro packaging system used may be the cause of the smaller inserts. The same packaging system was employed in all three cases where small insens were obtained, whereas Meyerowitz etal. (1980) used the system described by Blattner et al. (1978). With other cosmid cloning systems, background colonies containing plasmids with insert DNA resulting from vector concatemerization are often a problem. The c2XB vector provides a simple system for checking vector background. As can be seen in Fig. 3, the Kan’ gene should not be packaged. How- ever, if the vector and recombinant have been ligated at the Smal site, cosmids containing more than one vector molecule and, therefore, a Kan' gene, will be packaged. No kanamycin-resistant colonies have been detected in any of the libraries examined thus far. Other experiments in which no insert DNA is added to the ligation reaction also suggests that under the ligation conditions used vector concateme- rization is not a problem. Ligation of the vector alone produced no ampicillin- or kanamycin—resistant colonies after packaging and transduction (results not shown). (d) Construction of c2RB We have constructed a derivative of c2XB for use EcoRI Hinqll XOIII \c" Sun I ’0: Banotl Fig. 5. Construction of the c2RB “walking“ vector. The stippled segment indicates that portion of p.138 cloned into c2XBJB to give c2RB. 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