f! ‘. IIO- .. . , . ‘ ‘ ‘31! ) .Mfinfihfln. IL... A"? film... . . . . A .. . . . . v . . . ‘ .p: ..ua~:!5.0 v .n.snlblo\bv‘0i . n 7'. 1W-W"’; ”ZLVP'm-l’ ' v- v. 5! "run— I KIT-”"1” ’* T317 fiSIS .- .-‘.1.' *. -' yiéfghf ‘31:} :1 y .’a" it“) ’ ' . Pg! 1‘ i ‘. fl . ‘. ' 1““!‘r‘ " ‘ - i 'v \." ‘-‘ ‘unh’gl‘ v' 'hn _;, .3... - ,_, _ ~ ~ ,. .> -| ‘ ‘ I ' ~ . -"'~‘ ~ .jp $1. ’ ' 1... _ . . 1_ I Q U"! 1|: .‘3‘ J «I 4;- a" 'a , I' l 7-; ff’uu ... m. use.) ‘9". 'Wfiwnwzazgpy,“r This is to certify that the thesis entitled ISOLATION AND CHARACTERIZATION OF CHICKEN MYOSIN HEAVY CHAIN GENES presented by Kevin James Barringer has been accepted towards fulfillment of the requirements for M.S. degree in Food Sc1ence , 4371!” / Major professor Date May 17, 1983 0-7539 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES ”- 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. ISOLATION AND CHARACTERIZATION OF CHICKEN MYOSIN HEAVY CHAIN GENES By Kevin James Barringer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1983 ABSTRACT Seventeen individual recombinant chicken genomic DNA clones encoding myosin heavy chain (MHC) protein were isolated by virtue of their cross-hybridization with a cDNA clone that contains 3' sequences of a quail fast skeletal muscle MHC gene. Partial restriction mapping and orientation of transcription within five randomly selected clones indicated that they contained unique (but related), non-overlapping MHC sequences. This suggests that the original seventeen clones contain a variety of different MHC genes. Studies on the expression of these seventeen clones were performed by DNA dot blot hybridizations to radioactive cDNA templated by various cellular RNAs. The extent of homology of these clones to sequences expressed in the chicken tissues was tested at both high and low hybridization stringency. Thirteen clones appeared to be specific for embryonic and/or adult fast skeletal muscle MHC, two hybridize strongly at high criterion to both fast and slow skeletal muscle cDNA, and two show strong specific hybridization to both non-muscle (brain) and muscle cDNA samples. It is concluded that the chicken MHC gene family is extremely complex, containing upwards of 10 to 20 genes accounting for the adult fast skeletal and embryonic muscle MHCs alone. ACKNOWLEDGEMENTS I would like to thank Dr. Jerry B. Dodgson of the Department of Microbiology and Public Health for his invaluable help and support during the performance of this research project and the writing of this thesis. I would also like to thank Dr. Ronald B. Young of the University of Alabama at Huntsville for his getting me involved in this research and his assistance in supplying me with RNA samples and critical reading of this manuscript. I would like to thank Dr. A.M. Pearson of the Department of Food Science and Human Nutrition for serving as my major professor and for his continued support during my research, Dr. Maija H. Zile for serving on my committee, and to Ms. Theresa Fillwock for typing my thesis. ii TABLE OF CONTENTS List of Tables . . . . . . . . . . . . . . . . . . . . . List of Figures. . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . II. Materials and Methods. . . . . . . . . . . . . . . A. Methods. . . . . . . . . . . . . . . . . . . . 1. Screening of a recombinant chicken genomic Iibrary. O O O O O O O O O l O O O O O O O 2. Amplification and isolation of recombinant phage DNA. . . . . . . . . . . . . . . . . . . . 3. Isolation and purification of plasmid DNA. . . . 4. Isolation of cellular RNA. . . . . . . . . . . . 5. Restriction endonuclease mapping of DNA. . . . . 6. Blotting of electrophoretically separated DNA. . . . . . . . . . . . . . . . . . . . . . . 7. Construction of DNA and RNA dot blots. . . . . . 8. Synthesis of complementary DNA (cDNA). . . . . . 9. Labelling of double-stranded DNA . . . . . . . . 10. Southern blot and dot blot hybridizations. . . . 11. Purification of gel-fractionated DNA fragments. . . . . . . . . . . . . . . . . . . . 12. Subcloning of DNA. . . . . . . . . . . ..... 13. Transformation of HBIOI with recombinant plasmid DNA. . . . . . . ............ B. Materials. . . . .................. III. Results. . . . . .................... 1. Outline of protocol. . . . .......... 2. Testing the hybridization probe. . . . . . . . . 3. Screening the recombinant DNA library ...... 4. Restriction and hybridization mapping of clones ..................... 5. Differential expression of MHC phage clones. . . 6. Subcloning of MHC coding regions ........ Page vi 11 11 35 35 37 43 49 57 73 Page IV. Discussion. . . . . . . . . . . . . . . . . . . . . . . . 77 V. suma ry O O O O O O O O O 0 O O O O O O O O O O O O 0 O 0 89 VI. Appendices. . . . . . . . . . . . . . . . ..... . . . 90 1. Appendix I. . . . . . . . . . . . . . . . . . . . . . 90 2. Appendix II . . . . . . . . . . . . . . . . . . . . . 94 3. Appendix III. . . . . . . . . . . . . . . . . . . . . 95 VI I. References. 0 O O O O O O O O O O O O O O O O O O O O O O 97 iv LIST OF TABLES Table Page I Summary of DNA Dot Blot Experiments. . . ...... 63 II Summary of Cloned DNA Melting Experiments. . . . . . 67 71 III Summary of RNA Dot Blot Experiments. . . . . . . . . Figure 1. 2. LIST OF FIGURES Page Genomic DNA blots hybridized with cC128 plasmid. . . . 39 Partial restriction map of the cC128 quail fast skeletal muscle MHC cDNA clone. . . . . . . . . . 41 Autoradiographs of nitrocellulose filters pulled from each stage of the library screening . . . . . . 45 Ethidium bromide stained gel of a Sma I endonuclease mapping experiment. . . . . . . . . . . . 53 Partial restriction maps of five randomly selected chicken MHC genomic clones. . . . . . . . . . 55 Autoradiograph of cloned MHC genomic DNA dot blot experiment. . . . . . . . . . . . . . . . . 61 Autoradiograph of cloned MHC genomic DNA dot blot melting experiment. . . . . . . . . . . . . 65 Autoradiograph of myogenic cell RNA dot blots hybridized against cloned chicken MHC genomic DNA. . . . . . . . . . . . . . . ....... 69 Fine structure restriction map of pl.8H3.A4b1. . . . . 75 INTRODUCTION The process of myogenesis is a convenient system for the study of gene expression since it involves the coordinate expression of many different proteins. These proteins are often encoded by specific members of large gene families and so the process of myogenesis not only involves the activation of different genes but also the repression of other genes within the same gene family. The genes coding for the myosin heavy chains represent one such gene family. The myosin heavy chain (MHC) peptide constitutes up to 25% of the total mass of protein present in fully differentiated muscle fibers. The various MHC types are encoded by a large gene family that comes under diverse controls. Specific genes expressed in certain developmental stages are modulated by specific hormones (e.g., thyroid hormone), physiological stresses, direct neuronal contact, and by tissue specific types of control. Examples of each of these types of control over the MHC type present will be discussed below. In the chicken, different MHC isotypes have been detected in muscle tissue by virtue of differences in their electrophoretic mobility and peptide maps (1). The myosin heavy chain proteins isolated from fast white or slow red skeletal muscle, when partially digested with the protease papain, show distinct peptide maps as well as differing electrophoretic mobilities in sodium dodecyl sulfate (SDS) polyacrylamide gels. In addition, MHC isolated from presumptive embryonic fast skeletal muscles shows a very similar but not identical peptide map to that of adult fast white skeletal muscle. MHC isolated from fast skeletal muscle of dystrophic chickens also showed a similar peptide map with some apparent differences from both the adult fast skeletal and the presumptive embryonic fast skeletal muscle MHC isotypes. All three of these MHC isotypes were very different in their peptide maps from that of slow red skeletal muscle MHC. The same situation was shown to be present in rabbit muscle as well. Hoh and Yeoh (2), using cyanogen bromide peptide mapping, showed that the peptide maps obtained from MHC isolated from fast twitch or slow twitch skeletal muscles had distinct peptide maps, as was a fetal MHC isotype distinct from the adult MHC. Rat muscle tissues also contain distinct fast, slow, and embryonic MHC isoproteins (3), as shown by peptide mapping and gel electrophoresis in two dimensional systems. Nhalen et al. (4) also showed that as many as three MHC isozymes are expressed during the development of rat muscles. Both peptide mapping and immunological data indicate that in rat muscles, there is a transition from an embryonic form of the MHC present in fetal tissues to a neonatal form in muscle tissues (approximately 7-11 days old), and finally to the adult forms. The process that governs the switch of myosin isotypes (involving the regulation of different heavy chain and light chain isoproteins) in skeletal muscle is the pattern of neuronal innervation. Needs et al. (5) isolated whole myosin from cat muscles that had been cross-innervated in vivo. Upon examination of the myosin proteins isolated from those muscles by SDS-polyacrylamide gel electrophoresis they determined that when a slow-twitch type muscle is cross-innervated by a nerve from a fast-twitch type muscle, the myosin light chain pattern is similar in composition to a fast-twitch muscle. The converse is also true when a fast-twitch muscle is cross-innervated with a slow-twitch nerve. Rubinstein et al. (6), using antibodies that were specific for either slow or fast twitch muscle myosins, showed that when a nerve innervating a fast-twitch rabbit muscle was chronically stimulated at a frequency simulating a slow-twitch neuron, there was a decrease in the amounts of myosin reacting with the fast-twitch antisera, and a corresponding increase in myosin reacting with slow-twitch antisera. They determined that normal muscle fibers prior to the chronic stimulation contained either fast myosin or slow myosin, but not both. During the course of their experiment, however, these authors found that there was a gradual change-over in muscle fiber reactivity such that both anti-fast and anti-slow myosin antibodies reacted with the same muscle fiber. This implied that a change in gene expression was taking place and not an atrophy of one type of muscle fiber to be replaced by another of a different type. Rubinstein and Holtzer (7) showed that when chicken pectoralis muscle cells (a fast-twitch muscle) or those from anterior latissimus dorsi (a slow-twitch muscle) are cultured lg vitro in the absence of any neural innervation, all fibers reacted with an affinity purified anti-fast MHC antibody. Taken together, these studies suggest that the pattern of gene expression in fully differentiated skeletal muscle is still plastic in that the genes specific for fast or slow twitch myosin heavy and light chains can be turned off or on after muscle differentiation has been completed. Differing isoforms of MHC also exist in cardiac muscle tissues. Sartore et al. (8), on the basis of immunochemical reactions of myosin preparations from the ventricles and atria of chicken hearts against anti-sera raised against myosin from ventricles or fast skeletal muscle, showed that the myosins from the ventricles of the chicken heart were immunologically distinct from those of either atrial or fast skeletal myosins, while the atrial and fast skeletal muscle myosins were immunochemically very similar. The atrial myosins did not react with anti-slow muscle myosin antibodies. These results were confirmed by conjugating their antibodies to fluorescein isothiocyanate and performing direct immunofluorescence assays on tissue sections. Alteration of myosin isoforms in the ventricles of rabbits can be accomplished by thyroxine administration to induce a state of thyrotoxicosis in the animals (9-10,12,13). Flink et al. (9), after injecting rabbits for two weeks with 100 pg/kg body weight thyroxine, isolated the myosin from the ventricles of thyrotoxic rabbits and compared its two dimensional peptide map generated by cyanogen bromide (CNBr) digestion to myosin isozymes obtained from the ventricles of normal (euthyroid) rabbits and those from fast-twitch and slow-twitch skeletal muscles. The peptide maps of the thyrotoxic and euthyroid myosins showed several distinct differences that apparently were localized in the heavy chains since purified heavy chains contained the same differences in their maps. In addition, the euthyroid ventricular myosin had, with the exception of one peptide, an identical peptide map with slow-twitch skeletal muscle myosin. Neither cardiac isoprotein had similar CNBr peptide maps with myosin isolated from fast twitch skeletal muscle. Again, these differences were localized in the MHC proteins. Direct evidence of the expression of myosin isoforms was first provided by Hoh and coworkers (10,11) who identified three myosin isoforms in rat ventricles by non-dissociative electrophoresis, each form differing in its heavy chain content (10). This group proposed that the three forms were characterized by the various combinations of two MHC isoforms, designated H6“ and H68, two myosin isoforms being composed of the homodimers of each heavy chain, V1, the HCa homodimer, and V3, the HCB homodimer, and the third (V2) containing the heterodimer (11), with the synthesis of the MC“ isoform being stimulated by the administration of thyroid hormone (and the concomittant increase in the V1 myosin). This change of MHC composition has also been shown to occur in rabbits in studies from two groups (12-13). Sartore et al. (13), showed a change upon thyroxine administration of myosin types by indirect immunofluorescence. This group, using antibodies raised in rabbits specific for bovine atrial MHC, showed that thyroxine administration in rabbits elicited a change in MHC isotypes present in the rabbit ventricles, an analogous situation to that of Hoh and coworkers (11) in rats. They observed an increase in a myosin isoform called Va that increased upon thyroxine administration implying that their antibody recognized a MHC form analogous to the HCa of Hoh et al. Chizzonite and coworkers (12) prepared monoclonal antibodies to the H6“ and H68 isoforms identified by Hoh et al. (11). Using these antibodies they were able to determine that in normal euthyroid rabbits the MC“ makes up approximately 50% of the MHC at birth and decreases steadily to 10-12% by 12 weeks, HCB then being the predominant MHC isoform. In thyrotoxicosis of adult rabbits, HCQ once again becomes the predominant species of MHC to such a level that HCB could no longer be detected. Peptide mapping of the MHC variants isolated by the antibodies confirmed their differences in primary structure. It has also been shown that a chronic increase in the loading of the heart can lead to a shift in myosin isozymes. Using micro-comple- ment fixation Lompre et al. (14) determined that hypertrophy of the rat heart induced by chronic overloading induced a shift in the myosin isozymes present. These isozymes of myosin were the V1, V2 and V3 isozymes identified by Hoh et al. (10). Chronic hypertrophy led to the V3 form being the predominant form (this is the HCB homodimer containing myosin isoform, ref. 11). Removal of the pituitary gland mimicked the cardiac hypertrophy, whereas thyroid hormone administration led to the V1 (HCa homodimer) being predominant. Early studies indicated that the myosins from non-muscle tissues were structurally distinct from muscle myosins. Burridge and Bray (15) analyzed peptide maps of MHC proteins from various chicken tissues. Papain digests of MHC from smooth muscle (gizzard) tissues indicated that, based upon this criterion, the smooth muscle form was identical to that of non-muscle MHC proteins. when the proteolytic enzyme tryp- sin was used, myosins from gizzard, leg muscle, platelets, brain and fibroblasts were clearly all different. Differences in secondary and tertiary structure of the proteins could not be ruled out as a cause of this behavior, however. when they used 2-nitro-5-thiocyanobenzoic acid to cleave cysteine residues in fully reduced and denatured myosin proteins so that any differences observed would presumably be due to differences in the primary structure of the proteins, five types of patterns were observed. They were typified by breast muscle, heart muscle, gizzard, platelet, and brain. Other tissues gave patterns that could be mixtures of the above, for example, the peptide map of fibroblast myosins generated by this cyanylation reaction was identical to a mixture of brain and platelet forms. These authors concluded that the non-muscle cytoplasmic MHC isoforms could be characterized by their peptide maps to two groups, a brain type and a platelet type, which were different from each other. Nillingham et al. (16), using antisera to whole myosins from a fibroblast cell line (mouse L929 cells), smooth muscle (mouse uterus), and mouse skeletal muscle, established the immunological non-identity of each of these isoforms with each other. Cytoplasmic (fibroblast) myosin was localized to the cell surface. All of the above studies point to the existence of multiple genes that encode the MHC proteins. The degree of similarity of all of these isoforms at the protein level (with the possible exceptions of smooth muscle and non-muscle forms) is such that, even though such techniques as peptide mapping and immunological studies can indicate some differences, more subtle differences that exist at the DNA level may go undetected. For example, two MHC isoforms which any react with the same antibody and share identical peptide maps may in fact be products of different genes. In fact a problem of this sort, although not exactly as stated above, recently arose. It was postulated on the basis of peptide mapping experiments that fast twitch skeletal muscles in rabbits expressed two different MHC isozymes in different amounts in a muscle-specific manner (27,28). when the mRNA was extracted from these tissues and translated in a cell-free translation system (29), the synthesized MHC proteins had identical peptide maps, indicating that the original researchers were probably misled by post-transla- tional modifications of the proteins that affected the proteolytic reactions they observed. Therefore, to more accurately determine the size of the MHC gene families, as well as to study the expression of their genes and its control, it is necessary to examine them at the nucleic acid level. Some work at the RNA level has been done but the characteristics of the MHC mRNA have made such studies very difficult. MHC proteins are encoded by very large mRNA molecules; the bare minimum length of a mRNA necessary to direct the synthesis of the MHC protein (approxi- mately 1550 amino acid residues) is almost five thousand nucleotides. In fact, the MHC mRNA has been found to be approximately five to seven thousand nucleotides in length (17-21) depending upon the organism and tissue, and anywhere from 26-335 in sedimentation rate (17,22-24,26). The size of this molecule makes it very susceptible to cleavage by nucleases and a poor template for the viral reverse transcriptase (RNA dependent DNA polymerase) enzyme. As a result, most attempts to analyze the MHC gene family through the use of cDNA clones (see Appen- dix 1) have been limited to the 3' end of the genes containing the carboxy terminal portions of the MHC proteins (21,23-26). This region of the mRNA has been most extensively studied because it is the portion of mRNA sequence adjacent to the typical oligo(dT) primer for reverse transcriptase, the enzyme used to prepare MHC cDNA clones (Appendix 1). From the nucleotide sequence analysis of these clones and the resultant MHC amino acid sequence, and from the use of the clones in hybridiza- tion studies, a relatively high level of conservation of the MHC across its isoforms and across species is revealed. For example, recombinant cDNA clones encoding the a and B isoforms of the cardiac ventricular MHC (26) from rabbits were shown to be 79% and 83% homologous, respec- tively, to the amino acid sequence of a rabbit fast skeletal muscle MHC, and they showed 90% homology with each other. Amino acid sequence comparison of the cardiac isoforms with the rabbit fast skeletal muscle MHC indicated that the highly homologous region between the two species was interrupted by an extremely divergent region. This type of organi- zation, highly homologous regions interspersed with widely divergent regions, is also found by comparison of MHC sequences expressed in different tissues as determined by direct sequence analysis (30) or by R-loop analysis (31). R-loop analysis is an electron microscope analy- sis of the duplex structure formed by the annealing of two different clone DNA strands or by cloned DNA annealed with RNA. Homologous areas between the two nucleic acid strands form tight duplexes, whereas divergent areas show up as areas of nucleic acids that are "looped out" and not annealed. Hydro et al. (31) used this technique on isolated rat MHC genomic clones to observe areas of homology between their individual clones. This technique is also used to locate regions of intervening sequences (non-coding blocks of DNA internal to coding regions of a gene) in genomic DNA by annealing a selected genomic clone DNA with its complementary mRNA under conditions that favor DNA:RNA annealing over DNAzDNA. Intervening sequences at the genomic level are 10 "looped-out" since the corresponding mRNA does not carry that sequence and thus cannot form a complementary duplex structure. Hybridization analysis of cloned MHC cDNA further revealed the cross species conservation of MHC coding sequence DNA. Using the rat embryonic MHC cDNA clone pMHCZS (17), Nguyen et al. (32) showed that the MHC coding sequence is conserved across metazoan evolution. The sequence in pMHC25 was seen to hybridize to nitrocellulose blots (see Appendix II) of restriction enzyme digested genomic DNA from such diverse species as nematodes (Caenorhabditis elegans), sea urchins (Strongylocentrotus purpuratus and Lytechinus pictus), goldfish (Carassius auratus), insect (Drosophila melanogaster), chicken, mice, hamsters, and man. The MHC gene family is an interesting family for study from several viewpoints. It is a large gene family with several members under non-coordinate control with respect to each other, but controlled coordinately during myogenesis along with other myogenic proteins (33-34). To further study this gene family we decided to screen a recombinant chicken genome library to obtain the clones containing portions of some of the chicken MHC genes. MATERIALS AND METHODS A. Methods Screeningof a recombinant chicken genomic library - A library of chicken genomic DNA contained in A phage Charon 4A previously con- structed (35) was screened for MHC gene sequences by the method of Benton and Davis (36). A sufficient aliquot of the combined library to yield a total of approximately 1.2 x 106 plaque forming units (PFU) for the initial, high-density screening was added to 4.0 ml of an over- night culture of the E; 5911 strain DP50 supF (dap', thy', supE, supF) grown in LB medium supplemented with diaminopimelic acid (DAP) to 0.01% and thymidine to 40 ug/ml (LB/DT medium). Aliquots of 0.21 ml of this mix were placed in 20 separate, sterile 13 x 100 mm culture tubes, and the phage were allowed to preadsorb to the bacteria for 20 minutes at 37°C. At the end of this incubation period, 6 ml of LB/DT top agar kept liquid at 55°C was added to each tube, mixed, and poured onto the surface of individual 150 mm diameter petri dishes containing LB/DT standard agar. The tap agar mixture was allowed to solidify, and the plates were inverted and incubated overnight at 37°C. After incubation, nitrocellulose filter replicas of the plaques on each plate (approximately 60,000 PFU/plate) were obtained. Each plate was overlaid with a 135 mm diameter nitrocellulose filter (Millipore type HA, 0.45 pm, or Schleicher and Schuell BA85, 0.45 uM), the filter was 11 12 allowed to wet thoroughly and then marked for orientation on the plate by puncturing the filter with a syringe loaded with dye in an assymet- ric fashion through the filter into the agar. This filter was removed, and a second filter was placed on the plate and marked in the identical positions by puncturing the filter in the positions marked in the agar by the dye. Duplicate filters were pulled from all the plates and the adhering phage were lysed by immersing all of the filters in 500 ml of IX Southern denaturing solution (0.4 M NaOH, 0.8 M NaCl) for 2 minutes at room temperature. The filters were neutralized in the same volume of IX Southern neutralization solution (0.5 M Tris, pH 7.5, 1.5 M NaCl) under the same conditions (plates were stored at 4°C until needed). The filters were then air-dried and baked under vacuum at 80°C for two to four hours (because of differential shrinkage of the two brands of nitrocellulose filters upon drying, they were never mixed in the dupli- cate sets). These nitrocellulose blots were probed with the nick-- translated (see Materials and Methods) 0.37 Kb Pst 1 fragment of cC128 (21) according to a slight modification of the method of Nahl et al. (37). Filter blots were incubated at 65°C in 1 liter of 1.0 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA (pre-prehybridization solution) for 30 minutes. The pre-prehybridization was drained off the filters and the filters pre-hybridized overnight at 40°C in 250 ml of 50% formamide, 0.1 M Tris, pH 8.0, 0.05 M sodium phOSphate, pH 6.8, 1 mM EDTA, 0.5% SDS, 5X Denhardt's (1X = 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 10 pg/ml poly(A), 10 ug/ml denatured E; coli DNA and 10 ug/ml E; coli tRNA. The pre-hybridization fluid was removed. Remaining pre-hybridization solution was removed by washing the filters one time in 50% formamide, 10% dextran sulfate, 1X 13 Denhardt's, 0.75 M NaCl, 0.12 M Tris, pH 8.0, 12.5 ug/ml poly(A), and 10 ug/ml each of E; ggli_DNA and E; ggli_tRNA. 240 ml of hybridization solution (50% formamide, 10% dextran sulfate, 0.75 M NaCl, 0.12 M Tris, pH 8.0, 1 mM EDTA, 50 mM NazHP04, 0.1 mg/ml yeast RNA, 3.5 ug/ml (rC)n, 12.5 ug/ml (rA)n, and 8 ug/ml denatured salmon sperm DNA was added. Nick-translated probe (specific activity = 2 x 107 cpm/pg, 1.7 x 107 cpm total) was heated in 10 ml of the above solution at 90°C for 5 minutes, and cooled rapidly to room temperature. The denatured probe was added to the dish containing the filters and hybridization solution, and allowed to hybridize for 48 hours at 40°C in a rotary shaker water bath with gentle agitation. At the end of the hybridization reaction, the hybridization solution was removed (solution was reserved at -20°C for re-use) and the filters washed two times at room temperature in 250 ml of 0.3 M NaCl, 10 11M Tris, pH 7.5, 1 mM EDTA, 0.1% SDS (w/v), 0.1% Na4P207 (w/v) with agitation for 15 minutes each wash. The filters were then washed a total of four times at 60°C in 250 ml of the above prewarmed buffer, 15 minutes each wash, in a rotary water bath set at 110 rpm. The filters were air dried for at least one-half hour and then exposed to X-ray film (Kodak XAR-5) with an intensifying screen (DuPont Cronex Lightening Plus) at -70°C for one week. Plaques were determined to be positive by the occurrence of a signal on both duplicate filter autoradiographs. Tracings were made of the autoradiographs and used to orient the original plates. Two to three millimeter circles were picked out of the agar in the areas corresponding to positive plaques with sterile capillary pipets. Picked agar was placed into 1 ml of sterile SM buffer (0.1 M NaCl, 50 mM Tris, pH 7.5, 5 mM M9504, 0.01% gelatin 14 (w/v)) contained in sterile 1.5 ml microfuge tubes. The tubes containing the individual picked plaques were vortexed briefly and phage allowed to diffuse out of the agar by storage overnight at 4°C. Each plaque was then titered by "spot-titration" (making dilutions into SM buffers and and plating aliquots of the dilutions onto plates containing bacterial lawns (DP50 supF). Secondary, low-density screening of the positive plaques from the first, high-density screening was performed essentially as above. Aliquots from each resuSpended positive plaque sufficient to yield about 150 PFU per 100 mm diameter petri dish were preadsorbed separately to 200 pl of an overnight culture of DP50 supF (in LB/DT) for 20 minutes at 37°C. 3.5 ml of LB/DT top agar (45°C) was added to each tube, mixed and overlaid onto LB/DT agar plates (100 mm diameter), allowed to cool, and incubated overnight at 37°C. Single nitrocellulose filters (100 mm diameter) were pulled from each plate and treated as before. These filters were pre-treated as before (except prehybridization volume was 200 ml) and washed with hybridization solution (without probe). The reserved hybridization solution from above was heated to 70°C (to denature any reannealed probe), cooled in an ice water bath, and added to the prehybridized, washed filters. An additional 300 ng of denatured, nick-translated probe was added, and the filters were hybridized at 40°C with agitation for three days. Filters were washed as before, dried, and exposed to X-ray film for 48 hours at -70°C with single intensifying screens. Tracings were made from the autoradiographic exposures, and used as before to pick positive plaques. 15 The final screen was performed by transferring, with sterile toothpicks, viral plaques that were positive from the secondary screen directly to a gridded, 150 mm diameter LB/DT plate containing a lawn of DP50 supF. This plate was incubated overnight at 37°C to allow large area lysis to occur within each grid. A single nitrocellulose filter was pulled from this plate the next morning and processed as described above. The filter was probed and washed as before (except prehybridi- zation and hybridization volumes were 20 ml). The dried filter was exposed to X-ray film for 21 hours at -70°C with one intensifying screen. Plaques that were positive from this third screening were picked from the appropriate secondary screen plates into 1 ml of SM buffer. Five microliters of each of the resuspended phage were added to separate sterile 13 x 100 mm culture tubes containing 50 ul of a DP50 supF overnight culture, and preadsorbed for 20 minutes at 37°C. 4 Inl of LB/DT top agar was added to each tube, mixed, and poured onto separate, 100 mm diameter LB/DT plates. The plates were incubated overnight at 37°C to obtain complete lysis of the bacterial lawn. The plate lysates of each amplified, individual recombinant genomic clone were scraped off with sterile microscope slides into separate, sterile polypropylene tubes (Falcon 2005) containing 4 ml of SM buffer. Several drops of chloroform were added to each tube to kill any remain- ing viable bacterial cells, and the tubes were mixed on a vortex mixer. The tap agar was removed by centrifugation at 1500 rpm for five minutes in a table-top centrifuge, and the supernatant removed to new sterile tubes. Phage concentration of each supernatant was determined by Spot-titration, and the plate lysates were stored at 4°C. 16 ,Amplification and isolation of recombinant phage DNA - The DNA from the recombinant phage was isolated by a modification of the large-scale PDS procedure of Yamamoto et al. (38) or by a modification of a mini-- prep procedure (T. Kost and S. Hughes, personal communication). For the large-scale PDS procedure, 500 ml overnight cultures of the §;_coli strain DP50 supF were grown in NZCYM/DT liquid media. The cells were spun down at 5K rpm in a Sorvall GSA rotor for 10 minutes, the super- natant drained off, and the cells resuspended in 20 ml of NZCYM/DT. Absorbance of a suitable dilution (usually 1:50) at 600 nm was deter- mined and the concentration of cells present calculated (assuming 1A600 unit = 8 x 108 cells). Phage were preadsorbed to 2.5 x 1010 cells at a 50-100 1 cell to phage ratio for 20 minutes at 37°C and used to innoculate 600 ml of prewarmed NZCYM/DT medium contained in sterile, two liter flasks. Incubation was at 37°C for 12 hours with vigorous aeration. One to two ml of chloroform were added to lysed cultures, and the cultures were then shaken for 15 minutes at 37°C to kill any remaining viable cells. Cell debris was removed by centrifu- gation in 500 ml sterile polypropylene centrifuge bottles at 5000 rpm for 5 to 10 minutes on a Sorvall GSB rotor. The supernatants contain- ing the amplified recombinant phage were transferred to new containers and 0.5 ml of a DNAEES I/RNAEEE A solution (3.6 mg/ml each in 10 mM Tris, pH 7.5, 1 mM EDTA) was added to degrade bacterial nucleic acids. This reaction was allowed to proceed for one hour at room 'temperature. Thirty-five grams of NaCl was then added to each flask, followed by 0.1 gram per ml of culture polyethylene glycol (PEG) flakes (Carbowax 6000). These mixtures were shaken at low speed at room temperature for one hour (or until the PEG had dissolved). The 17 cultures were then incubated at 4°C for at least two hours to precipi- tate the phage. Precipitated phage were spun down at 7000 rpm for 10 minutes in a Sorvall GS3 rotor, the supernatants removed, and the pellets drained completely. The pellets were resuspended in 15 ml of SM buffer by repeated pipetting, and spun down in a Sorvall SS34 rotor at 10,000 rpm for ten minutes. The supernatants were decanted to fresh tubes and made up to 20 ml with SM buffer. 16.4 grams of CsCl were added to each tube and dissolved. These solutions (0 = 1.5 gm/ml) were transferred to Ti50.2 tubes and spun in a Ti50.2 rotor at 30-35 K rpm for at least 24 hours to isolate the phage. Translucent phage bands were collected, and dialyzed against one liter of SN buffer at 4°C for at least 4 hours. The dialyzed phage were then collected, the solution adjusted to 0.2% SDS, Proteinase K was added to 50 ug/ml, and the phage coats digested at 60°C for one hour. Recombinant phage DNA was puri- fied by two to three phenolzchloroform extractions (0.5 volumes each) and one CHCl3 extraction (one volume). The aqueous phase was dialyzed against one liter of 0.1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA in distilled H20 at 4°C for at least four hours, followed by two changes of 10 mM Tris, pH 7.5, 1 mM EDTA (TE buffer) at 4°C for the same length of time, and then once against two liters of 10 mM Tris, pH 7.5, 1 mM EDTA in double distilled H20 at 4°C for at least four hours. When the DNA had to be concentrated for use NaCl was added to 0.1 M, two volumes of isopropyl alcohol were added, and the DNA preci- pitated at -70°C for one hour or -20°C overnight. DNA was spun down at 10,000 rpm at 4°C for 20 minutes in a Sorvall $334 rotor, the superna- tant removed, the pellet drained, dried and resuspended in 1 ml of TE buffer. The DNA concentration was determined by absorbance at 260 nm (1 AA260 UHIt = 50 pg Of DNA). 18 Alternatively, a mini-prep procedure for isolation of phage DNA (T. Kost and S. Hughes, personal communication) was used. Recombinant phage were preadsorbed to E; 2911 strain 803supF at a 50 to 100:1 cell to phage ratio as in the PDS procedure, except that 1 ml of 10 mM MgCl2, 10 mM CaClz (Mg/Ca buffer) was included in the preadsorption mix. The preadsorbed phage were used to innoculate 30 ml of NZCYM medium in sterile, 125 ml flasks. The flasks were incubated with vigorous shaking for 12-16 hours at 37°C. 0.5 ml of CHCl3 was added to each flask, mixed, and left to stand at room temperature for 5-10 rninutes. Cell debris was removed by centrifugation at 10,000 rpm for 15 minutes in a 5534 Sorvall rotor. The supernatants were decanted to fresh tubes and then layered on glycerol step gradients. The step gradients were prepared by pipetting 6 ml of 40% glyerol in 50 mM Tris HCl, pH 7.5, 10 mM M9504 (TM buffer) in the bottom of a 5N27 poly- allomer tube and layering 8 ml of 5% glycerol in TM on tap. The step gradients were pre-cooled to 4°C, and the phage supernatants were care- fully layered on top of the step gradients. Phage were pelleted through the step gradients by centrifugation in a 5N27 swinging bucket rotor at 25 K rpm for two hours at 4°C. The supernatant was decanted and the translucent phage pellet resuspended in 0.5 ml of TM buffer. The resuspended phage solutions were made to 0.2% with SDS and the phage protein coats digested with proteinase K at 50 ug/ml at 37°C for one hour. The phage DNA was purified by two phenolzchloroform extrac- tions (0.5 volumes of each) and further purified by dialyzing the aqueous phage against two changes of 0.1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA at 4°C for 3_4 hours, and then two changes of TE buffer at 4°C 19 for Z 4 hours. DNA was precipitated as before, and the concentration measured by absorbance. Isolation and,purification of plasmid DNA - Bacteria containing recombinant plasmids were amplified with chloramphenicol, the plasmid DNA isolated by a slight modification of the alkaline lysis (ref. 39, p.88, p.90-91) procedure and purified by centrifugation through ethidium bromide (EtBr)-containing CsCl gradients (ref. 39, p.93). §;_ggli containing plasmid DNA (recombinant or otherwise) were grown in a 10 ml overnight culture of LB medium containing the appropriate antibiotic (as defined by which antibiotic resistance gene is expressed by the plasmid) at 37°C overnight. Overnight cultures (100 HT) were used to innoculate 25 ml of antibiotic containing LB media in sterile, 125 ml flasks. The innoculated cultures were allowed to grow until the optical density of the medium at 600 nm corresponded to the late log phase (which is approximately an 0.0. of 0.6). The 25 Inl cultures were then used to innoculate 500 ml cultures of pre-warmed antibiotic containing LB medium, and incubated at 37°C with vigorous aeration for exactly 2.5 hours. At the end of this incubation, chloramphenicol at 34 mg/ml in 100% ethanol was added to 170 ug/ml. Amplification of the plasmid DNA was allowed to proceed overnight at 37°C with constant shaking. The bacteria were pelleted after amplification by centrifugation at 5000 rpm for 10 minutes. The bacterial pellet was resuspended in 3 ml of 0.05 M glucose, 0.025 M TriS°HCl, pH 8.0, 0.01M EDTA and then 1.2 ml of a fresh 20 mg/ml solution of lysozyme in the above buffer was added, the solutions mixed together and incubated at 0°C for 10-15 minutes. The solutions were transferred to 30 ml polypropylene 20 centrifuge tubes (DuPont), 8 ml of 0.2 N NaOH, 1% 505 (cold) were added to each tube, and the contents mixed by inversion and incubated on ice for 15 minutes. At the end of this incubation, 3.4 ml of cold 5M potassium acetate (pH 4.8) were added to each tube, the contents mixed by'inversion, and incubated on ice for 10-15 minutes. The cellular DNA and debris were removed by centrifugation at 17,000 rpm for 30 minutes at 4°C in 5534 Sorvall rotor. The supernatants were transferred to fresh 30 ml Corex tubes, 0.6 volumes of isopropyl alcohol added to each, mixed and the plasmid DNA allowed to precipitate at room temperature for 15 minutes. Plasmid DNA was pelleted at 10K rpm for 30 minutes and the supernatant removed. The pellet was dried and resuspended in 15 ml of TE buffer. One gram of CsCl was added for every milliliter of solution, dissolved (1.55 g/ml final density) and transferred to 25 ml Oak Ridge tubes. 1.2 ml of a 10 mg/ml EtBr solution was added to each tube, mixed, and the plasmid DNA isolated by equilibrium centrifugation at 35K rpm at 20°C for 24-48 hours. Banded plasmid DNA was visualized by UV illumination and collected with a Pasteur pipet. EtBr was removed by repeated extraction with equal volumes of isobutanol equilibrated with a saturated solution of CsCl until all color has disappeared from the aqueous phases. The aqueous phase was dialyzed against two changes of TE at 4°C for 2.4 hours (2 liters each change), concentrated to 1.0 ml by butanol extraction, and precipitated by addition of NaCl to 0.1 M and two volumes of isopropyl alcohol. The precipitated DNA in 1.5 ml microfuge tubes was Spun down for 10 minutes at 4°C, the plasmid DNA pellet dried and resuspended in 1 ml TE. The DNA concentration was determined by the absorbance at 260 “HID Eth an W- 40.. 21 nm. A scaled down version of the above procedure was also utilized as described (39, p.368-369) in the preparation of plasmid mini-preps. Isolation of cellular RNA - Total cellular RNA from the tissues of adult chickens was isolated by the guanidinium-hot phenol method of Feramisco et al. (49). Tissue fragments from a freshly killed chicken were homogenized in a Polytron (Brinkmann Instruments) homogenizer in the presence of 5 volumes of 4.0 M guanidinium thiocyanate, 0.14 M 2-mercaptoethanol, 50 mM Tris, pH 7.5, 10 mM EDTA, and 2% Sarcosyl. The tissue homogenates were heated to 60°C and the liberated chromo- somal DNA was sheared to reduce the viscosity by drawing the homoge- nates repeatedly through a sterile syringe fitted with an 18 gauge needle. An equal volume of preheated phenol (60°C, containing 0.1% 8-hydroxyquinoline and equilibrated with 10 mM Tris, pH 8.0) was added to each tissue homogenate and passed through the syringe again. One-half volume of 0.1 M sodium acetate, pH 5.2, 10 mM Tris, pH 7.5, 1 mM EDTA and one volume of a 24:1 mixture of chloroform and isoamyl alcohol were added to the homogenates and the mixtures were incubated at 60°C for 10-15 minutes with vigorous shaking. After cooling the mixtures on ice, the organic and aqueous phases were separated by centrifugation at top speed in a table-top centrifuge (IEC) for 10 minutes at 4°C. Aqueous phases were re-extracted once with an equal volume of phenol:chloroform, and twice with equal volumes of chloro- formzisoamyl alcohol. Two volumes of 100% ethanol were added to the extracted aqueous phases and placed at -20°C for one to two hours. The ethanol precipitates were placed into sterile baked 30 ml Corex tubes and the RNA recovered by centrifugation at 10,000 rpm for 20 minutes at 4°C in a Sorvall 5534 rotor. RNA pellets were dried and resuspended in the Iris 1‘4: huh rco— e;.e 173,1 22 the original starting volume used to homogenize the tissues of 0.1 M Tris, pH 7.5, 50 mM NaCl, 10 mM EDTA, and 0.2% SDS. Proteinase K (pre-digested) was added to 200 ug/ml and allowed to digest for one to two hours at 37°C. The Proteinase K-digested RNA solutions were heated to 60°C, and one-half volume of preheated phenol was added and the mixture shaken. One-half volume of 24:1 chloroform/isoamyl alcohol was added to the phenol-containing solutions and shaken again for 10 minutes at 60°C. The mixes were cooled on ice and centrifuged as before (4°C, 10 minutes) to separate the phases. The aqueous phase was extracted once more at 60°C with phenol/chloroform/isoamyl alcohol as before, and twice with equal volumes of chloroform/isoamwl alcohol at room temperature. Two volumes of 100% ethanol were added to the aqueous phases, the RNA precipitated out by cooling at -70°C for one hour (or -20°C overnight) and spun out as before. RNA pellets were washed once with 70% ethanol, then dried and resuspended in sterile, RNA3§§ free water. RNA concentrations were determined by absor- bance at 260 nm (1 A260 unit = 40 ug RNA) and stored at -70°C. Polysomal RNA was isolated from cultured cells by the method of Young et al. (47). Muscle or fibroblast cells cultured in 10 cm dishes were collected at appropriate times by scraping the cells in a minimal volume (54.5 ml) of sterile, ice-cold polysome isolation buffer (PIB = 0.25 M NaCl, 10 mM MgClz, 10 mM Tris, pH 7.4). Triton X-100 was added to the isolated cells to final concentration of 0.5% and the cells lysed by aspiration twenty times with sterile pasteur pipets. Cell lysates were centrifuged at 12,000xg for 10 minutes, and the supernatant (5 ml/gradient) layered on top of a 15-40% sucrose gradient in PIB. The gradients were centrifuged in a SN27 rotor at 24.5 K rpm .‘h— for‘ nan. gra: cent care was ",I -E Vom- 23 for two hours at 2°C to fractionate polysomes (>4 ribosomes) from non-polysomal material. Isolated polysomal fractions from the sucrose gradients were placed into sterile Ti60 tubes, filled with P18, and centrifuged at 30 K rpm overnight. Pelleted material was washed once carefully with P18, and the pellets dissolved in 0.5 ml of P18. 505 was added to 0.5%, proteinase K to 200 ug/ml, and digestion proceeded at 37°C for two hours. The samples were transferred to sterile 1.5 ml microfuge tubes, one volume of phenol/chloroform added to each, and mixed vigorously at room temperature. The phases were separated by centrifuging for one minute in a microfuge and the aqueous phases transferred to fresh, sterile 1.5 ml microfuge tubes. One volume of isopropyl alcohol was added to each tube, mixed, and stored overnight at -20°C. RNA was recovered by centrifugation at 10,000 rpm in a JA-21 rotor for 10 minutes at -20°C, the supernatants removed, and the pellets drained thoroughly. The pellets were taken up in a 200 pl of RNAEEE free double-distilled water. Sodium acetate was added to 0.1 M final concentration, one volume of isopropyl alcohol was added, and the RNA precipitated at -20°C overnight. The RNA was collected by centrifugation as before, the dried pellet resuspended in sterile, RNAEEQ-free water, and the absorbance at 260 nm taken to determine the RNA concentration. RNA samples were stored frozen at -70°C. Restriction endonuclease mapping of DNA - All restriction enzyme diges- tions were performed under the conditions specified by the manufac- turers. In reactions where more than one enzyme was used, reactions were allowed to proceed for several hours at the optimum temperature and conditions of one enzyme, the buffer conditions altered to suit the secom the I" the C requi 9e tr 24 second enzyme, and the reactions allowed to proceed. Alternatively, if the reaction conditions of two enzymes were sufficiently similar, then the digestions were performed under the conditions of the enzyme requiring the lower ionic strength condition, in the presence of both enzymes. Routinely, 0.25 ug of recombinant genomic or plasmid DNA was digested with restriction endonucleases, and the reactions terminated by the addition of 0.1 volumes of 1.0 M NaCl, 0.25 M EDTA. Two volumes of isopropyl alcohol were added to each terminated reaction, and the DNA fragments precipitated out of solution at -70°C for 0.5 to 1 hour. The precipitated DNA was recovered by centrifugation in a microfuge for 10-15 minutes at 4°C, the pellets drained, dried, and resuspended in 40 mM Tris-acetate, 2 mM EDTA, 5% ficoll, 0.05% bromophenol blue, and 0.05% xylene cyanol for electrophoresis in agarose gels, or in 100 mM Tris-borate, 2 mM EDTA, 5% ficoll, 0.05% each of bromphenol blue and xylene cyanol for polyacrylamide gel electrophoresis. Restricted DNA fragments from recombinant genomic clones, along with DNA size standards were generally electrophoresed in vertical, 0.8% agarose gels for 250-300 volt-hours in recirculated 40 mM Tris-acetate, 2 mM EDTA, pH 7.5. Restricted plasmid DNA was separated by electrophoresis in vertical 5% polyacrylamide gels for 320-400 volt-hours in 100 mM Tris-borate, 2 mM EDTA, pH 8.3. Selection of electrophoretic systems was based upon the expected sizes of the DNA fragments generated by restriction endonuclease cleavage. The electrophoresed DNA fragments were visualized by soaking the gel in 0.2 ug/ml ethidium bromide in distilled water for thirty minutes at room temperature. Destaining of the gel was generally not needed at this level of ethidium bromide. The stained DNA was caused to neat mi 11. (the IUXE filtl 25 fluoresce by ultra-violet light illumination, and the gel photographed using Polaroid 667 film and a red filter. From the photographic record, a standard curve was constructed from the migration of the DNA standards of known sizes, and used to calculate the sizes of the DNA fragments generated by restriction enzyme digestion of the cloned genomic or plasmid DNA. The pattern of sizes produced by such digestions by enzymes, alone or in combination with others, allowed a physical map of the cloned DNA samples to be generated. Blotting of electrophoretically separated DNA - Gels containing fractionated DNA were soaked in 0.4 M NaOH, 0.8 M NaCl for 30 minutes at room temperature to denature the DNA in sjtg. The gels were neutralized by immersing them in 0.5 M Tris, pH 7.5, 1.5 M NaCl for 30 minutes at room temperature. The gel was placed on a wick of 3MM paper (Hhatman) which had both ends placed in a buffer reservoir containing IOXSSC (1X55C=0.15 M NaCl, 0.015 M sodium citrate). A sheet of nitrocellulose cut to the dimensions of the gel was pre-soaked, first in double-distilled water, and then in IOXSSC. The pre-wetted paper was laid on top of the gel carefully to omit any air bubbles. Four to five sheets of 3MM paper, cut to the same approximate dimensions of the nitrocellulose, were placed on top of the nitrocellulose, followed by paper towelling (also cut to the same approximate size) to a depth of 3-4 inches. Tension was applied either by placing a weight on top of the towelling, or by wrapping polyethylene film tightly over the transfer set-up. Transfer was accomplished by the wicking of the buffer through the gel, with subsequent movement of DNA into the nitrocellulose, where under these salt conditions, the DNA binds. The transfer was left to proceed from 12-16 hours at room temperature. 0f IOr COO Merl We- the of p VBCU 26 After the transfer process was finished, the wells of the gel were marked, any adhering pieces of the gel were rinsed off with 3XSSC, the blots were air-dried and then baked in a vacuum oven at 75-80°C for 2 to 4 hours to irreversibly bind the DNA to the nitrocellulose. Construction of DNA and RNA dot blots - DNA dot blots were constructed by denaturing and neutralizing in a batch reaction, followed by aliquoting out of the batch for each dot. DNA (0.5 ug DNA/dot) in TE buffer was denatured in 0.4 N NaOH for ten minutes at room temperature. Equimolar amounts of HCl were added to neutralize the DNA, along with additional Tris/HCl (pH 7.5) to help stabilize the pH. NaCl was added to 1.5 M, and aliquots containing 0.5 ug of denatured DNA were placed in separate wells of a Hybri-dot apparatus (BRL, Inc.) containing pre-wetted nitrocellulose paper. The DNA was drawn onto the nitrocellulose by suction, and each well was washed three times, with two volumes per wash of 1.5 M NaCl, 0.5 M Tris, pH 7.5. The dot blots were air dried, and the DNA irreversibly bound by baking under vacuum at 75-80°C for 2-4 hours. RNA dot blots were constructed by a modification of the procedure of White and Bancroft (50). Known amounts of RNA were denatured in 6% formaldehyde and 6XSSC for 10 minutes at 60°C using RNA§§§ free conditions. Aliquots containing identical amounts of denatured RNA were applied to the wells of a Hybri-dot apparatus containing pre-wetted nitrocellulose paper and vacuum applied to draw the RNA onto the nitrocellulose. Each well was washed three times with two volumes of RNAQiQ free 6XSSC. The RNA dot blot was then air dried and vacuum-baked at 75-80°C for 2-4 hours. (f) tr as fr e111 pH ei‘. 27 Synthesis of complementary DNA (cDNA) - Two to six pg of RNA were used as a template in the presence of 50 mM Tris, pH 8.0, 60 mM NaCl, 6 mM MgCl2, 5 mg/ml calf thymus DNA primer (or 20 ug/ml oligo(dT)12_18primer), 16 mM dithiothreotal (DTT), [o-32PJdNTP at 8-10 uM, unlabelled dNTPs at 33 pM, and 32-80 units of AMV reverse transcriptase in a volume of 20-25 ul per reaction. Reactions were assembled on ice in sterile 1.5 ml microfuge tubes using RNAEEE free technique. Each reaction was incubated at 37°C for 1-2 hours, and terminated by the addition of an equal volume of 50 mM EDTA. RNA templates were degraded by the addition of NaOH to 0.5 N and heating at 90°C for 5 minutes. The reactions were neutralized by the addition of equimolar amounts of HCl (approximately 5 ul was withdrawn to check the pH on pH paper) and unincorporated dNTPs were separated from the cDNA either by chromatography over Sephadex G-50 columns equilibrated in 0.1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, or by centrifugation through 1 ml Sephadex G-50 spun columns (equilibrated in 0.1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA) contained in 1 ml tuberculin syringes. Typical specific activities of 2-3 x 109 cpm/pg cDNA were obtained in this manner. Labelling of double-stranded DNA - Double-stranded DNA was labelled by nick-translation. DNA (0.5 to 1.0 pg) was incubated in the presence of 50 mM Tris, pH 7.8, 6 mM MgClz, 50 ug/ml bovine serum albumin, deoxyribonuclease I at 0.5 ng/ml, [o-32PJdNTP at 2 uM, unlabelled dNTPs at 16.5 pM, and 12-24 units of E. coli DNA polymerase I in a reaction volume of 100 pl for 1-2 hours at 16°C. The reactions were terminated by the addition of 0.1 volume of 0.5 M EDTA and extracted vi l‘E Se sea PEr left 28 with one volume of phenol/chloroform. Unincorporated dNTPs were removed from the labelled DNA by Sephadex G-50 chromatography or by Sephadex G-50 spun columns. Typically, specific activities of 107 to 108 cpm/pg DNA were obtained. Southern blot and dot blot hybridizations - DNA nitrocellulose blots were pre-wetted in double-distilled water, then in 1.0 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA at room temperature. The filters were then placed in seal-a-meal bags and prehybridization solution consisting of 50 mM sodium phosphate, 10 ug/ml poly(A), 13.6 ug/ml D; melanogaster total poly(A)- RNA, and 10 ug/ml denatured salmon sperm DNA (sometimes §;_ggli tRNA and denatured g, ggli DNA at 10 ug/ml each were substituted for the eucaryotic nucleic acids mentioned above) was added to the bag. Usually 10-15 ml of prehybridization solution was used for 1-4 filters. The blots were pre-hybridized at the identical temperature of the hybridization reaction (32-37°C for heterologous probes, 40-44°C for homologous probes) for at least 4 hours with rocking. After the prehybridization incubation, denatured probe (by heating at 90°C for 5-10 minutes, and quick-cooling) was added to the seal-a-meal bag containing the prehybridization solution to 2105 cpm per ml of solution. Hybridization of the probe to the blotted DNA was left to proceed overnight with rocking at the apprOpriate temperature. At the end of the hybridization reaction, the solution was removed (solution could be stored at -20°C for re-use) and the blots washed according to the following protocol. Two room temperature washes in 50 mM NaCl, 10 mM Tris HCl, pH 7.5, 1 mM EDTA, 0.05% SDS, and 0.05% Na4P207 were performed with agitation to remove excess 29 formamide-containing hybridization solution. Three to four washes at 65°C in the same wash-solution were performed to remove any probe not specifically bound. Exceptions to the wash buffer composition are noted in the text. Excess wash fluid was drained off and the blots exposed while still damp to X-ray film. After exposure to X-ray film, bound probe was stripped off the blot by washing at 65-70°C for one-half hour in two changes of glass-distilled water and dried. The blot was then ready to be reprobed with a different probe following the same prewetting and pre-hybridization protocol described. RNA containing blots were treated according to the procedure of Thomas (51). RNA blots were prewetted, first in RNAEEE free glass distilled water, then in RNAEEE free 0.75 M NaCl, 10 mM Tris HCl, pH 7.5, 1 mM EDTA. RNA blots (in seal-a-meal bags) were prehybridized at 42°C with rocking in 50% formamide, 1X Denhardt's SXSSC, 50 mM sodium phosphate, and 250 ug/ml denatured salmon sperm DNA (RNAEEE free) for 8-20 hours. Probe (denatured as described) was added to the bags to the same level as the DNA blots, and allowed to hybridize overnight at 42°C. Hashing was performed in the same manner as for the DNA containing blots. RNA containing blots were stripped of bound probe by washing in 0.1x to 0.05X wash buffer (1X wash buffer contains 50 mM Tris, pH 8.0, 2 mM EDTA, 0.05% Na4P207, and 1X Denhardt's) for 1-2 hours at 65°C. Purification of gel-fractionated DNA fragments - DNA fragments separated by agarose gel electrophoresis were purified by electroelu- tion into 3 MM paper backed by dialysis membrane (Girvitz et al. (54)). 3O Digested DNA was electrophoresed in a horizontal agarose gel containing 0.2 to 0.5 ug/ml ethidium bromide, and the DNA visualized by illumination with ultraviolet light. A slice is made in front of the DNA fragment of interest and a piece of 3 MM filter paper (cut to the approximate dimensions of the slice and just wider than the thickness of the gel) backed by a piece of dialysis membrane was inserted into the slice, with the 3 MM paper between the band and the dialysis membrane. Electrophoresis was continued until the DNA had migrated into the paper. The 3 MM paper and dialysis membranes were then placed into 1.5 ml microfuge tubes that had had holes pierced in the bottom of them with hot 25 ga. syringe needles, and these were placed into 13 x 100 mm test tubes. The DNA was recovered by washing the paper and dialysis membrane with 200 pl of 0.2 M NaCl, 50 mM Tris HCl, pH 7.5, 1 mM EDTA, spinning the wash into the 13 x 100 mm collecting tube (2-3 minutes, top speed of table top centrifuge), repeating these steps 3-4 times. The contents of the collecting tubes were transferred to a fresh centrifuge tube, two volumes of isopropyl alcohol were added, and the DNA precipitated as described before. If a fragment had been fractionated by polyacrylamide gel electrophoresis, then the DNA band of interest was excised out of the polyacrylamide gel with a scalpel, the gel piece was placed on a horizontal gel plate, molten agarose is poured around it, and a slice was made in the agarose after it was solidified. Electroelution was then performed as described. Subcloning of DNA - DNA fragments generated by restriction enzymes to be sub-cloned were isolated by electroelution (see above). Approxi- mately 100 ng of vector DNA cut with the appropriate restriction enzyme 31 was treated with 0.3 pl calf alkaline phosphatase (0.3 U/pl) in 50 mM Tris HCl, pH 8.0, in a 1.5 ml microfuge tube at 37°C for one-half hour to remove the 5' phosphate groups. Reactions were terminated with the addition of a fifth volume of 1.0 M NaCl, 0.25 M EDTA and extraction two times with one volume of phenol. The aqueous phase was extracted twice with two volumes of ether to remove phenol. The isolated restriction fragment was combined with the phosphatase treated pBR322 plasmid DNA in a 2:1 insert to vector molar ratio (insert refers to the restriction fragment that is to be inserted into the vector pBR322 plasmid DNA). Ethanol (2.5 volumes) was added to the mixed DNA and precipitated at -70°C for one hour. The DNA was spun down in a microfuge for 15 minutes at 4°C, and the DNA pellet was drained and dried. The pellet was then resuspended in 15 pl of 1x ligase salts (50 mM Tris HCl, pH 8.0, 10 mM MgCl2, 1 mM ATP, 200 pg/ml gelatin), 15 mM DTT and 120 units of T4 DNA ligase (New England Biolabs) were added. The reaction was incubated at 15°C for five minutes. An additional 285 pl of 1X ligase salts, 15 mM DTT was added after this incubation, and incubation proceeded an additional two hours at 15°C. The ligation reaction was stopped by adding 30 pl of 1.0 M NaCl, 0.25 M EDTA. Two volumes of i50propyl alcohol were added, and the DNA precipitated at -70°C for one hour. The DNA was pelleted in a microfuge as before, the pellet was drained and dried completely, and taken up in 15 pl of TE buffer. Transformation of H8101 with recombinant,plasmid DNA - Plasmids containing insert DNA (see above) were used to transfect the E; coli St!“ per RaC the rat by 1111* ac ti re tu mi OV 32 strain HB101 by the RbClz transformation technique (D. Hanahan, personal communication). Tubes containing 200 pl aliquots of H8101 at 3'0A600 units/ml in 45 mM MnClz, 60 mM CaClz, 40 mM potassium acetate, 100 mM RbClz, and 15% sucrose, pH 5.7 (stored at -70°C until needed) were thawed and placed in an ice bath for 5 minutes. The tubes were refrozen by immersion in a COzlethanol bath and then thawed quickly by swirling in hot tap water. The tubes were placed on ice for 30 minutes. At the end of this period, 7 pl of dimethylsulfoxide was added, swirled gently into solution, and placed on ice for an addi- tional 5 minutes. Five pl of solution containing the resuspended recombinant plasmid DNA (see above) was added to the bacteria, the tubes swirled to mix them, and placed back on ice for 15 minutes. The tubes were quick-frozen in COZ/ethanol, and quickly thawed to 0°C by shaking in hot tap water. When the thaw was completed, the tubes were immediately placed on ice for 30 minutes. The tubes were then incubated for exactly two minutes at 37°C after which 200 pl of LB broth medium was added. Incubation at 37°C then continued for 30 minutes. The transformed bacteria were then spread on LB agar plates containing the appropriate antibiotic and the plates incubated at 37°C overnight. Colonies containing recombinant plasmids were identified by the colony hybridization technique of Grunstein and Hogness (52). Transformant colonies were transferred with sterile toothpicks, first to a numbered grid plate (LB + antibiotic), and then to the identical position on a numbered and gridded nitrocellulose filter placed on the surface of a antibiotic containing LB plate. Both types of plates were 33 incubated at 37°C overnight to allow the bacteria to grow. The gridded nitrocellulose filter containing bacterial growth was then removed, the colonies lysed and the DNA denatured by placing it on top of several sheets of 3 MM paper that had been soaked in 1X Southern denaturing solution. The filter was allowed to soak for 5 minutes at room temper- ature, and was then transferred to sheets of 3 MM paper soaked in 0.5 M Tris, pH 7.5, for 5 minutes. They were then placed onto 3 MM sheets soaked in 1X Southern neutralization solution for 5 minutes more, air dried for 1/2 hour, and vacuum baked at 70-80°C for 2-4 hours. The colony filters were hybridized and washed as previously described for Southern blots, using as probe labelled insert DNA. The colony blots were dried, and the transformant colonies containing the correct insert DNA were identified by autoradiography. Presence of the correct insert was confirmed in those transformants that were positive by the above by growing plasmid mini-preps (see above), cutting the isolated plasmid DNA from the mini-preps with the correct restriction enzyme, and analyzing the digestion products on agarose gels. Large scale plasmid isolations were performed on the transformants that were positive by both of the above criteria. B. Materials Restriction endonucleases were obtained from Bethesda Research Labs, Biotec, and New England Biolabs and used according to their Specifications. AMV reverse transcriptase was provided by J. Beard of Life Sciences, Inc. through the Office of Program Resources and 34 Logistics, Viral Cancer Program, National Institutes of Health. 'E; .ggli_DNA polymerase I was from Biotec or New England Biolabs. T4 polynucleotide kinase was purchased from Collaborative Research. T4 DNA ligase was from New England Biolabs. Calf alkaline phosphatase was obtained from Boehringer and further purified by column chromatography. Deoxyribonuclease I, ribonuclease A, and ribonuclease T1 were all purchased from Millipore Corporation. Lysozyme, ampicilin, tetracycline, and chloramphenicol were all from Sigma. Proteinase K was obtained from Bethesda Research Labs. [o-32PJDeoxynucleotide triphOSphates were purchased from New England Nuclear or Amersham, and [y-32PJATP was from ICN. All X-ray film and supplies were from Eastman Kodak. RESULTS Outline of Protocol - The isolation of chicken MHC chromosomal DNA sequences can be outlined as described below. A cDNA clone (see Appendix I) denoted c6128 was obtained from Dr. C.P. Emerson, Jr. (University of Virginia). This cDNA clone had previously been shown to code for the 3' end of a quail fast skeletal muscle MHC mRNA (21). It contains a 600 base pair (bp) fragment inserted into the Pst I site of the plasmid vector pBR322. This clone shows considerable derived amino acid homology with chicken MHC (C.P. Emerson, Jr., personal communication) as well as with other published amino acid sequences (24). Thus the cDNA clone plasmid DNA was expected to hybridize specifically to any recombinant phage DNA that contains coding sequences for the 3' portion of a member of the chicken MHC gene family (at least those of the sarcomeric MHC type). These chicken MHC gene-containing clones were isolated from a pool of recombinant DNA phage, each of which contains a 15-20 kilobase pair (kb) long fragment of chicken DNA. Together, these clones are referred to as a library. Approximately 5 x 104 to 1 x 105 such clones should contain a genomic equivalent of chicken DNA. Therefore, each individual clone should contain, on average, enough DNA to correspond to a typical single-copy gene. Each clone is identified and purified by its specific hybridization to the complementary labeled nucleic acid 35 36 sequences in the probe (see below). Of course, a multigene family will result in more positive clones in proportion to its multiplicity per haploid genome. If the probe contains a highly repeated sequence then a high percentage of the recombinant phage will be positive in the hybridization assay. 37 Testing_the Hybridization Probe - The c0128 plasmid DNA hybridization probe was first tested for its hybridization to total chicken genomic DNA digested with a particular restriction enzyme, electrophoresed on an agarose gel and blotted to nitrocellulose (Appendix II). A suitable probe should give approximately as many specific bands as there are corresponding genes in haploid chromosomal DNA. Chicken sperm genomic DNA was digested to completion with the restriction endonuclease Eco RI (A phage DNA was included in the digestions as an internal control for the completeness of digestion and as an internal molecular weight marker), electrOphoresed on horizontal 0.5% agarose gels, and then transferred to nitrocellulose paper (see Appendix II and Materials and Methods). These genomic blots were hybridized to the intact cC128 plasmid that had been labelled by nick-translation (see Materials and Methods). Approximately five fragments were labelled by the probe. These fragments had approximate lengths of 3.8, 4.1, 6.2, 12.5, and >21 kb as estimated by the use of the internal Eco RI digested x DNA as size markers (Figure 1A). As shown in Figure 2, the restriction map of c0128 contains no Eco RI sites (C.P. Emerson, Jr., personal communication). Therefore, assuming that this coding region is not interrupted by non-coding intervening DNA sequences at the genomic level which contain Eco RI recognition sites, each band represents one or more different genes homologous to the c0128 insert DNA. These bands resulted from fairly homologous hybridizations as autoradiographs of duplicate genomic blots washed at low stringency (300 mM NaCl, 65°C) versus those washed at higher stringency (50 mM NaCl, 65°C, see Materials and Methods for complete 38 make-up of wash buffers) showed retention of these bands at similar relative intensities (Figure 1B). When the cC128 insert DNA was liberated by digestion with the restriction endonuclease Pst I, purified away from the pBR322 vector sequences, nick-translated, and used to probe genomic blots for an extended period (2.5 days), the resultant autoradiograph showed a dark, uniform exposure over the length of the blot (data not shown). This result could be explained by the presence of a sequence in the quail MHC cDNA sequence that is repeated in the chicken genome but represents only a small percent of the overall complexity of the intact cC128 plasmid. When the intact recombinant plasmid was used as a probe on genomic blots for short hybridization times (12-16 hours), the small ((1%) percentage of the probe complexity represented by the putative repeated sequence apparently did not hybridize to completion. However, when the isolated insert DNA was used as a probe, the percentage of the total sequence represented by the proposed repeated sequence is much greater. This, combined with the longer hybridization period, allowed it to hybridize to completion, or near-completion. To localize this putative repeated sequence in the c0128 insert DNA, the cC128 plasmid was digested with the restriction enzymes Pst I and Hind III (alone or in combination), the fragments isolated, labelled via nick-translation, and used as probes on genomic DNA blots. This procedure localized the probable repeated element upstream of the internal Pst I site (see Figure 2) in the 230 bp Pst I fragment. Therefore, the 370 bp Pst I fragment that includes the extreme 3' end of the MHC mRNA and the 3' untranslated region of the mRNA was used as a probe for the genomic library. 39 Figure 1 Genomic DNA blots hybridized with the cC128 plasmid. Autoradiographs of chicken genomic DNA cut with the restriction enzyme, EcoRl, electrophoresed, transferred to nitrocellulose paper and hybridized to the nick translated cC128 plasmid. Blots were exposed for 17.5 hours at -70°C with a single intensifying screen. Figure 1A is a genomic blot washed at low stringency (300 mM added NaCl), and Figure 18 is a blot washed at high stringency (50 mM added NaCl). The numbers refer to the approximate lengths (in kilobase pairs) of the indicated bands as determined by the use of the included A DNA as size markers (see Materials and Methods). 40 nut—cw-vv 7..- 7 7H- 12.5 — in II i I i i 1 l l is 1 r‘/ 41 Figure 2 Partial restriction map of the cC128 quail fast skeletal muscle MHC cDNA clone (C.P. Emerson, Jr., personal communication). ommwmm A .s>t.bib.s»-pi».>->p.mp5. 54 and: Figure 4 55 Figure 5 Partial restriction maps of five randomly selected chicken MHC genomic clones. Symbols and scales are as indicated in the figure. faibv cozgcoto 3:23.535» I one... .553 5.3 acifitnz .0: 3.3609: (20 U manic. :mu 5:.» cache 9300 5:; 9:53.92 3:05am: (20 \\\\\\\\\\\ «020.3 (290 “.255 (20 9:525»; 2:25;: <20 _:uc::r .95 I .153. .ochV .coom. and... mzun onunozx o.m “ 630m IileI Bo <01: 56 . * +1 III Nov 51.2 Ucom '. ...‘ ', ‘ ‘ .‘~ «.- ' ' _ . .5 - ' ' I ' ' p ’7 I - . I r'. I b n ‘ . I I I l A x H I . Figure 6 63 Table 1 Summary of cloned MHC genomic DNA dot blotting experiments. The dot numbers refer to dots on Figure 6. Symbols used are +, relative degree of hybridization; +-, positive hybridization upon over exposure of autoradiograph; o, no hybridization detectable. 64 0.0.50 02> .5 002 0363320 03.0.. .00 350.0 3 0.: 0302030 03.0.. .0. 3:00.. 32> mOCwOm o. 002) >9... 03.0.3... .36: 3.30.0 )9... 03.0.3: 030.: 3:00.. >05. 03.020: )9... 03.010: >9... 03.0.8: UO~ .* 0030030. 003).: 3.0. .0... 00-04%”..020 o.-m-.M.N..cmn_m ”Show... .. 2.10:... + ++ + +++ + 1 + 1 o m. 2.10 >3» +++ +++ + ++ + I + I + I o. 2.19.3. +++ +++ + +++ + I + I + I a. 2.10:?» + +++ + I + + I + I + I m. 2.10;». + + + I ++ + I + I + I m. 2.13.2: +++ ++ +++ ++++ + I + 1 + I u. 2.10 >03 ++ + ++ +++ +++ ++ ++ m. 2.10:6. ++ + +1 + o o o m. ZIO>.wo_ + + +1 o o o o .0. 2.1053. +1 +1 +1 + o o o 2. 2.10 imam ++ ++ ++ +++ +1 +1 o 3. 2.1058» +1 +++ + + ++ + ++++ .0. 2.10 .30..» +++ +++ ++++ +++... + I +1 0 212.107.?» +1 ++ +1 o +1 +1 +1 5. 2.1053: .7... ++ +l + o o o .0. 2.1053. ++ ++ +++ ++++ ++ ++ +1 o 00.3 +1 + + +++ o o o a > o o o o o o o Table I 65 Figure 7 Autoradiographs of replicate cloned MHC genomic DNA dot blots hybridized to cDNA preparations templated by RNA from various chicken tissues and washed at different stringencies. Each dot represents 0.5 pg of cloned DNA. Panel A have been washed with buffer containing 300 mM NaCl. Dot blots in panel B have been washed with buffer containing 12.5 mM NaCl (see text for complete details). Dots 1-19 are explained in Table 11. RNA sources of cDNA are: a, 14 day embryonic chick leg muscle polysomal RNA; b, adult chicken thigh muscle total cellular RNA; c, adult chicken breast muscle total cellular RNA; d, adult chicken cardiac muscle total cellular RNA; e, adult chicken gizzard muscle total cellular RNA, and f, adult chicken brain total cellular RNA. 66 O.» a...» O.» O.» 41'- 1'1“" Figure 7 67 @1911 Summary table of the melting experiment shown in Figure 7. The dot numbers refer to the dots shown in Figure 7, and the capital letters above each column refer to the replicate dot blots in Figure 7. Symbols used are +, relative degree of hybridization; +-, hybridization observed after overexposure of autoradiograph; o, no detectable hybridization. 68 > 22 amk macsk1 o=.n 02.02 2mm acmndm 202ac.a 02.020: A2122 acmn.m n=2> moc2nm >22.” 02.nrm: cxmmmn acmn.m >gc.fl n:.nrm: nmxa.mn acmn.m >acdfi n22nxm: c.-qu acmndm >22.” n22nxm: oxm23 aofim2 30. 22> aono. 22> ”can. 22> fiofim. 22> flown. 22> 22> con 2 22> woos: H~.m53 woos: H~.maz wooaz Hm.msz woos: Hm.maz woos: H~.maz woos: Hm.maz H randyHmH + + + +1 + +1 0 o. o o o o m zznymam ++++ ++++ ++ + +++ +++ +1 +1 3 0 +1 +1 w :mdyavw +++ + +++ + +++ ++ +++ + + +1 +++ + 2 320220» +++ ++ +++ + +++ ++ ++ +1 + o ++ +1 m zrnymmp ++++ ++++ ++ + +++ +++ + a. +1 0 + +1 m zznyacw +++ ++\~ + o + +1 +1 o o o +1 o N 322200» +++ ++ +++ + +++ ++ +++ + + +1 +++ + m xxnymcw + + + +1 ++ + + o +1 o + o c 22952 ++ + + o + +1 +1 d o o +1 0 Ho zxnywanw +++ +++ ++ + +++ ++ +1 .10 o 0 +1 0 HH zrnywmvm +++ +++ ++ + +++ ++ +1 0 o 0 +1 0 H2 zznyHmnm +1 o ++++ ++++ ++++ ++++ ++++ +++ ++ + ++++ ++++ Hw ardywmmw ++++ ++++ ++ ++ ++++ ++++ +1 0 o 0 +1 0 H2 zznywumm +1 0 ++ + ++ +1 +++ + + o +++ + 3. 21195.: +++ ++ + + +++ ++ +1 o o o +1 0 gm zxnyumnp ++ + ++++ +++ ++++ +++ ++ +1 + o +++ +++ H2 zuquomw +++ ++ +++ + ++++ +++ ++ +1 + o +++ + Hm nnwmm + +1 + +1 + + o o o o o 0 Ho 2 o o o o o o o 110 o o o o ht CIEI‘S ‘1' I in I a U Table II 69 MNCXZb Figure 8 NHCM Analyses of expression of selected MHC genomic clones in cultured “HCM myogenic cells. Autoradiograph of RNA dot blots hybridized against nick translated cloned DNA. Each dot represents 2.5 pg of RNA. RNA preparations are Fb/p, polysomal RNA from cultured enbryonic chick I ““C) epidermal fibroblasts, pr/p, polysomal RNA from presumptive myoblasts, 3d/p, polysomal RNA from myotubes 3 days post-fusion, and 6d/p, polysomal RNA from myotubes 6 days post-fusion. .uHC ‘1 l' l L11... ; I l 2 y E l a; 7O Ira/p. pr/p 3% 0% ggfl 1am uncxzbz ' _ I 111,11, menu 5 Ill-16).“: , mucus-1 ‘1 f . . 13:: 1 , ’ A_, a ' 1.1-L - D O . . . ‘..c. 1' ' ~ ' . +2 I‘- 0 ‘ UHCXOM ‘ ‘“ " . . "o. - I O ' O . o ' , ‘ ‘ . ‘ ‘ y 0 ‘9 3 _‘ A . I. , _ a . '- 1 ‘ . _ _o'- 1 '- ‘ o ‘ . ‘ ' . ' ' . I 1 . - . ‘1 i K. . -1 L I ' . . M.UJ‘ ”61.-.... “Is c I. -“‘l "..'n‘~.-‘.- M \i- 11‘. “001’ M‘qu$“-‘M-L&:W “ L‘;‘ t a" _ ‘ 5 ' o ' '0 . 1 I .1 ,1 . 1 ° J ., w '. i . ' . . - . ‘ 1 . . ‘ - .. ; ‘ ' . ( 'r . ’n m.‘ “.1 1" \.. ‘ . Adi —L—‘: ‘ ‘~--. '3 *‘H—f' D 6‘2“)?“ {—L-‘JJ '1 9“ 3.41”“! m. 'Mn-N , ‘— nth 1‘. L‘ uni.-. .b- ‘ D '.‘ U A‘ ‘i - Figure 8 71 M Summary table of RNA dot blot experiment shown in Figure 8. RNA preparation designations are as described in the legend of Figure 8. Symbols used are 0, no detectable hybridization; -, +-, hybridization barely detectable (+->-), and +, relative degree of hybridization. 72 100cm 2v\2 Wz> wwmw>w>HHoz 2332 wa\2 oa\1 m. OOHH sz> Smoymvm zmoybvw zmnxbnm zmnVomH zmnwmdp nopmm zmnvpmnw zmowwmoH o +++ +7 ++ +7 ++ +OOO+ Table III 73 Subcloning of MHC Coding‘Regions - To further study the chicken MHC genes, the DNA restriction fragments from the five mapped clones that bound the cC128 plasmid probe (therefore containing the 3' ends of the genes) were eluted from gels (see Materials and Methods) and ligated to the plasmid vector pBR322 cut with the appropriate restriction enzyme (see Materials and Methods). These recombinant plasmids were used to transfect the g; 2211 strain H8101. The fragments subcloned contained all of the cC128 specific regions of DNA and were the 6.0 kb Eco RI fragment of MHCA2b2, the 1.8 kb Hind III fragment (fragment adjacent to the 9.0 kb Hind III piece, see Figure 5) of MHCA4b1, the 4.4 kb Eco RI fragment of MHCA4c2, the 5.9 kb Eco RI fragment of MHCAGal, and the 3.6 kb 8am HI fragment of MHCAGbl. These recombinant plasmids were amplified, the DNA isolated (see Materials and Methods), and characterized by fine structure restriction endonuclease mapping. Fine structure mapping of subcloned DNA is used to generate a map in which the average DNA fragment size is small, on the order of 400 bp in length. This is necessary in order to determine the nucleotide sequence of the DNA since 400 bp is the approximate maximum length of nucleotide sequence that can be obtained in one step using most current techniques. Fine structure mapping of plasmid DNA is distinguished from the previously described restriction mapping in that the enzymes used in fine structure mapping recognize specific nucleotide sequences in the DNA that are only 4 to 5 bp in length and thus cut much more frequently than the enzymes used above (which recognize specific nucleotide sequences 6 bp or longer). The cleavage sites recognized by these 4-5 "hitters" are therefore more closely spaced and provde a more detailed map of the DNA. Since more sites must be identified, fine 74 structure mapping requires more data analysis and is generally only performed on subcloned DNA fragments. An example of such a study done on one of these subclones (pl.8H3.A4b1) is shown in Figure 9. Studies to determine the nucleotide sequence of this particular subcloned fragment of DNA using the chemical degradation technique of Maxam and Gilbert (53) are currently being performed. 75 Figure 9 Fine structure restriction map of the subclone p1.8H3.A4b1 containing two 1.8 kb Hind III fragments of MHCA4b1 aligned in a head to tail arrangement in the Hind III site of the plasmid vector pBR322. 76 UmeNMDZ>NL a a a a at a 1 > rummwmmozey mooc . mom2w w» n 2 6039 > .um. _ 1 Emu _ 1 15a 5 e moon _ & m=4_ 41T::‘_ Figure 9 DISCUSSION Seventeen individual recombinant DNA genomic clones have been isolated by virtue of their hybridization to a cDNA clone specific for a quail fast skeletal muscle MHC mRNA (21). This particular cDNA clone encodes a MHC peptide shown to have extensive homology with MHC peptides from several species, including chicken (21, C.P. Emerson, Jr., personal communication). Five of these recombinant clones were chosen at random to characterize by restriction mapping. As can be seen in Figure 2, comparison of these maps allows the five clones to be placed into three groups based upon map homologies within the DNA fragments that are specific for the 3' ends of the genes. The clones MHCAZbZ and MHCA6al both show similar Hind III restriction site patterns within an Eco RI fragment of similar size. The clones MHCA4c2 and MHCAGbl show the same type of homology with respect to the Hind III and Eco RI sites, plus an equivalent Bam HI site. The clone MHCA4b1, however, has a restriction map very different from the other four clones, indicating that MHCA4b1 represents a quite different MHC gene. Furthermore, the restriction maps of all five clones are sufficiently divergent outside of the homologous 3' ends to demonstrate that they encode five separate, though related, genes. Allelic differences would not be expected to lead to the extent of restriction map differences that are observed outside of the 3' coding 77 OVE' bei any the int fir lev con mac arm wag re‘ Th. f i SEI DN Shl hor the CDT. 78 regions in these clones. Similarly, none of these clones appear to be overlapping clones. The twelve remaining unmapped clones are currently being mapped with restriction enzymes. Since five randomly selected clones showed no identical recombinants, there are probably few, if any, identical clones among the seventeen. The DNA dot blot experiments further demonstrate the diversity of the coding sequences contained in these genomic clones. In interpreting these results several things must be kept in mind. The first is that the only valid comparison that can be made regarding the levels of expression of each of these cloned sequences is the relative amount of hybridization of an individual clone to the cDNA of each tissue tested. Since we do not currently have an estimate of the actual amount of DNA that codes for mRNA in any of these clones, comparisons between different clones versus the same tissue cannot be made, since any differences observed nay be due solely to the actual amount of coding sequence contained within a given clone. In fact, the way this particular experiment was conducted, we can estimate the relative amount of coding sequence available in each of these clones. The dot blotting experiment was performed under conditions where the filter-bound DNA was in gross excess over the amount of homologous sequences found in the cDNA preparations used as a probe. Therefore, all MHC sequences in the cDNA probes complementary to the filter-bound DNA will have reacted, and the strength of the autoradiographic signal should be proportional to the amount of filter-bound DNA sequences homologous to the cDNA probe sequences. The degree of homology between the cloned DNA sequences and their counterparts in the various tissue cDNAs may also influence the level of hybridization observed. However, 79 from the melting experiment it appears that at least at the lowest criterion shown most cross-hybridizing sequences indeed formed stable hybrid structures. Using the data shown in Table II in the 300 mM NaCl wash column of adult chicken breast muscle (column C) as an estimate of the amount of coding sequence in each clone available to complementary probes, the seventeen genomic clones can be placed into four groups. The largest extent of MHC sequences seem to be contained in the clones MHCA15c2, MHCA16a2, MHCA18cl, and MHCAl9al. The second largest amount are contained within clones MHCA2b2, MHCA4b1, MHCA4c2, MHCA6al, MHCAGCZ, MHCA14cl, MHCA15b2, and MHCAl8a1. The third group in order of MHC coding capacity include the clones MHCA8b1 and MHCA17e2. The smallest apparent RNA coding capacity in the seventeen recombinant genomic DNA clones is found in clones MHCAlal, MHCAGbl, and MHCA13c1. Since the cC128 plasmid encodes a MHC judged to be specific for fast skeletal muscle tissue, it should be expected that the majority of the isolated clones would also correspond to fast skeletal muscle MHC genes. Furthermore, since Nguyen et al. (32) determined that all MHC nucleic acid sequences present in rat sarcomeric muscle tissue (skeletal and cardiac) were able to cross-hybridize with each other under very stringent conditions we should also expect that our chicken MHC recombinants might also contain genes expressed primarily in either (or both) type of striated muscle tissue. The data obtained in the dot blot experiment suggests that most of our clones do indeed hybridize to sequences expressed preferentially in striated muscle tissue. The melting experiments indicate some other rather surprising data. Under low stringency conditions (300 mM added NaCl columns of Table 11), all skeletal muscle cDNA preparations 8O hybridized to the cloned DNA, which is as expected. However, even under low stringencies, only nine of the cloned sequences showed homology to cardiac tissue RNA (these were MHCA4b1, MHCA4c2, MHCAGal, MHCAGcZ, MHCASbl, MHCAlScZ, MHCAl7e2, MHC118cl, and MHC119a1). Hhen high stringency washes were performed (12.5 mM added NaCl columns of Table II), a clear pattern begins to emerge. Practically all of these clones contain MHC sequences specific for embryonic skeletal muscle (14 day) tissue or adult fast skeletal muscle (breast) tissue, as seen by the selective retention of signals at high stringency from these tissues as opposed to loss of signal at high stringency in all other tissues (the only exceptions being the clones MHCAIScZ and MHCAIBcl). Binding to the cDNA preparation from a predominantly slow phenotype muscle (adult thigh muscle, column B, Table 2) was reduced greatly, although not entirely at high stringency. The clones MHCAGal, MHCAl4cl, MHCAleZ, and MHCAl7e2 showed an approximate 50% loss of relative binding at high versus low stringency, whereas the clones MHCAlScZ, MHCAlGaZ, MHCAl8al, and MHCAchl showed little (25% or less, MHCAchl) to no decrease in binding. All other clones were reduced in relative binding 70% or more. Cardiac tissue cDNA was almost completely washed off at this high stringency (the only exception were the clones MHCA4b1, MHCA6c2, MHCA15c2, and MHCA17e2). This is in contrast to the pattern oberved in the rat (24,31-32) where an embryonic rat skeletal muscle cDNA clone pMHC25 (17) was able to cross-hybridize with all rat striated muscle tissues (31-32). Two isolated cardiac MHC sequences were able to duplicate those hybridization patterns (24). Jakowlew et al. (23), using an embryonic cardiac-Specific MHC cDNA clone from chickens, found that their clone 81 would hybridize only poorly to skeletal muscle samples under stringent conditions but hybridization became more pronounced under less stringent conditions (their probe did not hybridize to adult rat heart RNA). Our data support these data in a converse sense, in that our clones are relatively specific for the fast skeletal muscle, and do not hybridize well at high criteria with cardiac muscle RNA. As expected, non-striated (smooth) muscle tissue cDNA showed virtually no binding to any of the 17 clones. At high stringencies only the two unusual clones (MHCAIScZ and MHCAchl) showed any hybridization to non-muscle (brain) cDNA. Some tentative conclusions nay be drawn from the DNA dot blot data. In general, most, if not all, of the seventeen clones isolated can be categorized as containing members of the sarcomeric gene family as termed by Hydro et al. (31) and Nguyen et al. (32). This chicken gene family differs from the rat gene family studied by the above authors in that the sequences specific for embryonic and skeletal muscle of the fast phenotype are more divergent from cardiac and slow skeletal muscle types than are the corresponding rat MHC genes. This is indicated by the fact that most of these clones showed appreciable cross-hybridization to cDNA probes from the slow and cardiac muscles only at low stringency. The rat genes, however, maintained their binding to slow and cardiac MHC sequences even at high stringency (15 mM NaCl, 32). Even though the embryonic skeletal muscle tissue tested was of a presumptive slow adult phenotype, it has been reported that embryonic muscles share a common phenotype (2-3,40) that changes only after innervation of the muscles is completed. Our results tend to confirm this proposal and suggest that the common embryonic MHC genes 82 expressed are more closely related to the fast adult MHC genes (at least near their 3' termini). The strong hybridization of the clones MHCAISCZ and MHCAchl (and to a much lesser extent several others, see Table II) to non-muscle cDNA is more difficult to explain. No other groups of investigators have reported hybridization of muscle MHC coding sequences to non-muscle MHC coding sequences, either using MHC cDNA clones as probes (17,24,32) or genomic clones (31). Several possibilities exist to explain these hybridization patterns. One possibility is that there are regions in these clones that are highly conserved, and therefore common to most or all chicken MHC genes. This might explain the observed data since the clones that hybridized to the non-muscle probe seemed also to hybridize to some extent to every probe used (see Tables I and 11). However, the strength of the binding of cDNA from cardiac, fast, and slow muscle tissues to MHCAlScZ was approximately equal to brain cDNA binding, as was the case with MHCAchl (excepting the cardiac cDNA). This makes the above possibility seem unlikely, since if a conserved sequence is responsible for the cross-hybridization observed, then all the signals observed from tissue containing non-homologous MHC sequences should be approximately equal to each other due to the cross-hybridization of only the conserved portion, but they should be much less than the signal from the tissue expressing the homologous sequence. The presence of just such a conserved sequence somewhere else in the MHC transcription unit not represented in these clones cannot, of course, be ruled out entirely. None of the studies referred to above used probes (cDNA or genomic) that were full-length, 83 nor do we feel that any of our cloned genomic MHC genes are full-length for reasons to be discussed later. A second possibility is that transcribed repeat elements nay be present on these clones which are expressed in relatively high frequency in other non-muscle tissues. Keeping in mind that we used total cellular RNA (transcribed repeat sequences are often less prevalent in cytoplasmic RNA populations) as a template, and random primers for the cDNA synthesis, any transcribed repeat elements contained within one of our clones would result in hybridization. We have done a preliminary experiment to try to determine the presence of repeated DNA on the clone MHCAlScZ (data not shown) by digesting the cloned DNA with several restriction enzymes, blotting the agarose gel of the digest, and hybridizing to nick-translated total chicken genomic DNA. This experiment did indicate the presence of a repeated sequence element on this particular clone. We do not know if this repeat element is transcribed, and, if so, if that transcription is regulated in a manner compatible with our dot blot data. Some repetitive DNA sequences found in cellular RNA are known to vary in a developmental and/or tissue specific manner in a variety of eucaryotic organisms (see reference 41 for a review), however, chicken repetitive sequences differ from those of many other organisms. Eden and Hendrick (42) determined by reassociation kinetics that repetitive DNA in the chicken represents only 13% of the chicken genomic complexity, the remaining 87% being exclusively single-copy DNA. Two-thirds of this repetitive DNA is represented by a family of repeats with a reiteration frequency of 1500, whereas the remaining fraction is repeated on the order of 10 to 20 times. They determined that 84 approximately one-half of the total chicken genomic DNA is made up of single-copy DNA not interrupted by any repeated sequences and averaging 17.5 kb in length. The rest of the genome consists of single-copy DNA interpersed with repetitive DNA, the single copy portion being about 4.5 kb in length, and the repetitive DNA being at least 2 kb long. Transcription of these repeats was analyzed near the B-globin gene cluster in chickens by Villeponteau et al. (43). This group detected several families of transcribed repeats that were regulated in a developmental and tissue specific manner. They detected one class of repeats transcribed in the e-globin gene region that was also transcribed in brain tissue. Furthermore, these transcribed repeat units were not part of any B-globin transcription unit. They formed an independent transcription unit transcribed by RNA polymerase II. It is conceivable that some of the hybridization observed might be due to hybridization of transcribed repeat sequences, although it would be difficult to assign all of it to those sequences. To do so, one would have to assume that the transcribed repeat sequence is present at roughly equal levels in the RNA of brain tissue to those of MHC coding sequence present in muscle tissue. As discussed above, MHC proteins represent a very high fraction of total muscle protein and the MHC RNA should also represent a relatively large fraction of muscle cellular RNA. The third possibility, and the one that seems most plausible, to explain this apparent non-muscle cross-hybridization is that there is another linked gene on these two clones that binds non-muscle cDNA. The MHC genes in the rat are known to be linked, since several genomic clones isolated have been shown to contain more than one gene (31), 85 however, no data regarding non—muscle MHC genomic or RNA sequences are currently available. Therefore, a much more detailed study of these two clones is needed to determine the cause of their unusual hybridization pattern. The dot blot data as a whole seem to suggest that of the seventeen clones tested, thirteen are homologous to MHC sequences expressed primarily in embryonic and/or adult fast skeletal muscle. All five of the mapped clones (which code for distinct MHC genes or pseudogenes) fall into this first group). Two clones (MHCAlGaZ and MHCAIBal) are strongly homologous to sequences present in both slow and fast muscle RNA (but not brain RNA). These clones nay therefore code for either fast or slow muscle-specific (or both) MHC genes. Although avian leg muscle is primarily of a slow phenotype, it is also known to contain fast fibers in addition to the slow, so either possibility cannot be excluded yet. Two clones MHCA15c2 and MHCAIBcl showed surprisingly strong hybridization to non-muscle RNA. As discussed above, further study will be needed to explain their behavior. None of the clones are clearly specific for cardiac or smooth muscle MHC genes. Since the final seventeen clones were selected for strong hybridization to c0128, clones containing MHC genes which differ considerably in their 3' region from the cC128 sequence nay have been lost in the multiple screening process. Therefore, these clones probably do not represent an entire MHC gene family. The RNA dot blot experiments, shown in Figure 8 and summarized in Table III, also yield some information on the expression of several of the isolated chicken MHC genomic clones. MHC mRNA appearance through myoblast differentiation into myotubes in vitro has been quantitated by 86 several groups (44-45). In the chicken, Dym et al. (44), using a MHC cDNA probe, determined that MHC mRNA constitutes approximately 21.5% of the poly(A) containing RNA in heavy polysomes after 40 hours in culture (when 20% of the nuclei were in multi-nucleated myotubes). After 72 hours in culture, when the fusion index reached 80%, MHC mRNA reached almost 60% of the total poly(A) containing RNA in the heavy polysome fraction. Benoff and Nadal-Ginard (45) determined in rat myoblast differentation that MHC mRNA constituted up to 0.4% of the total cytoplasmic RNA by the time the myoblasts had essentially differen- tiated completely into myotubes at day 7 in culture. This mRNA data corresponded nicely with MHC protein appearance. Two of our cloned DNA samples show peak expression in myotubes 3 days post-fusion (MHCAISCZ) and 6 days post-fusion (MHCAIBcl). MHCA15c2 seems to be represented in the RNA samples for only a short period, whereas, MHCAchl first appears 3 days post-fusion, and reaches an apparent maximum at 6 days although expression after 6 days was not monitored. Neither of these two clones, which had bound nonmuscle cDNA in the DNA dot blot experiments, showed any homology with the nonmuscle (fibroblast) RNA sample used here. This does not rule out the possibility that these clones may contain a nonmuscle MHC coding sequence as earlier studies (15, see Introduction) suggested that nonmuscle MHC peptides nay be an extremely diverse family of proteins and that the brain MHC nmy be quite different from the fibroblast MHC. It is interesting to note that MHCA4b1 (and to a lesser extent MHCA6al) bound strongly to presumptive myoblast polysomal RNA. Several groups of researchers have reported that MHC mRNA exists in pre-fusion myoblasts in the form of stored, untranslatable messenger ribonucleoprotein (mRNP) particles 87 (46-48) and that these mRNP particles serve as precursors to the MHC mRNAs being actively transcribed in the polysome fraction of myotubes (48). These mRNPs would not be found in the polysomal RNA preparations since they would be excluded by the sucrose gradient fractionation step. These results probably indicate formation of a few precocious myotubes leading to contamination of the presumptive myoblast polysomal RNA with MHC sequences. Neither MHCAGbl nor MHCA4c2 bound to any of the tissue culture RNA samples, even though the DNA dot blotting data suggested strong homology to sequences present in yiyg in embryonic muscles. The RNA sequences present in the 1!.XIEEQ polysomal RNA used in the RNA dot blot experiment could be of a different gene product than those found in the embryonic leg muscles. Alternatively, the hybridization of MHCAGbl and MHCA4c2 to embryonic leg muscle cDNA may be artifactual due to transcribed repeats, but this is extremely unlikely from the fact that they both bind c0128 and share restriction maps somewhat similar to other MHC clones. Also interesting is the lack of binding of the c0128 plasmid to any of the in yitgg myogenic polysomal RNA samples. This is probably due to the lack of homology of the adult MHC encoded in the cC128 with the MHC sequences expressed in the tissue culture cells. It has been shown that cultured cells often express an embryonic form of MHC (3) and this may be the reason for the lack of c0128 hybridization to any RNA samples (note that c0128 showed little homology to any embryonic muscle cDNAs in the DNA dot blot experiment). In general, the clones tested with RNA dot blots showed expected hybridization patterns (with the exception of MHCA6b1 and MHCA4c2) in that the strongest binding occurred to myotube polysomal RNA samples when both MHC mRNA and protein appearance is peaking. 88 By analogy with other organisms, it is doubtful whether any of the seventeen individual recombinant clones studied here contains a full length MHC gene. None of the rat genomic clones studied by Hydro et al. (31) were full length although some contained the 5' ends of linked MHC genes. Analysis of the coding regions (exons) of these genes showed that they had an average length of 200 bp (range of 100 to greater than 1000 bp) with the non-coding intervening sequences (introns) ranging in size from 100 to 1000 bp and a reported intron:exon ratio of 1.6:1. This would make an average rat MHC gene 18.5 kb in length containing as many as 30 introns. A full length Drosophila MHC gene has been shown to be included in a transcriptional unit of 19 kb in length and to contain nine introns (19). Further study of our clones will indicate whether any of them contain full length genes. Although the average size of our inserts makes it feasible that one might contain an entire gene, no data is yet known on the intronzexon organization of the chicken MHC genes. It will also require further study to determine how many of the clones we have isolated contain overlapping fragments of the same gene, or if any contain pseudogenes (related DNA sequences that cannot express functional protein). SUMMARY In summary, we have isolated 17 recombinant chicken genomic clones encoding MHC isoforms. We have shown these clones to be relatively specific for embryonic and adult fast skeletal muscle tissue. Each of these 17 clones probably represent different MHC genes on the basis of the nonidentity of the restriction maps of five randomly selected genomic clones. 89 APPENDICES APPENDIX I Reverse transcriptase (RNA-dependent DNA polymerase) is an enzyme encoded by RNA tumor viruses. The most often used enzyme is the one encoded by the avian myeloblastosis virus which consists of two polypeptide chains with three enzymatic activities, the most important of which for recombinant DNA studies is its DNA polymerase activity. This activity is important for two reasons, 1) it allows the generation of a specific single-stranded complementary DNA (cDNA) copy of a mRNA, thereby (using radioactive nucleotide(s)) making a labeled hybridization probe specific for sequences identical to that mRNA, either DNA or RNA, and 2) by copying that cDNA molecule into a double-stranded complementary DNA molecule, either with reverse transcriptase or DNA polymerase, one can generate a cDNA copy of the original mRNA suitable for cloning into a vector DNA. Amplification of that cDNA-containing vector DNA in the appropriate host organism will then generate large amounts of a specific cloned sequence that initially may only have been present in a very impure state. One can then either characterize the cDNA physically by determining the nucleotide sequence of the mRNA which allows the derivation of the amino acid sequence of the encoded protein, or use it as a specific probe to monitor the expression of a particular gene. 90 91 The polymerase activity is dependent upon three things: 1) divalent magnesium cations, 2) a RNA (or DNA) template, and 3) a RNA or DNA primer with a free 3' hydroxyl that the enzyme can utilize to extend a complementary copy of the template molecule. Experimentally, the primers are usually supplied exogenously in two ways. The first takes advantage of a post-transcriptional modification of most eucaryotic mRNA molecules, which is the addition of polyadenylic acid residues on the 3' termini of the molecules. Synthetic oligonucleotides containing a free 3' hydroxyl made up of thymidylic or uridylic acid residues will specifically bind to this poly(A) tract and allow the polymerase activity to copy the mRNA. This method, however, makes the resultant cDNA p0pulation skewed towards copies of the 3' ends. The second is the use of random primers, usually eucaryotic genomic DNA that has been digested with deoxyribonucleases down to oligonucleotide length. The diversity of these molecules theoretically guarantees that the resultant cDNA molecules will be a representative population of sequences equivalent to that of the template mRNA. The reverse transcriptase polymerase reaction is represented diagrammatically below. 92 Diagram, Appendix I 0ligo(dT)primer molecules with free 3' hydroxyl nPPi Priming of the cDNA for second strand synthesis in prepara- tion for cloning is simplified by the fact that, in vitro, the first strand cDNA forms a "hair-pin" structure at the 3' end of the cDNA. This hairpin can act as a primer for the second strand synthesis. DNA Polymerase I (Klenow Fragment, see Appendix III) HOIIII mRNA AAAA""An 5' 3' Reverse transcriptase Mg++ dNTP 3' 5' cDNA < IIIT mRNA AAAA°°'An + 5| Jr 3| 3' 0H 5' cDNA C III! mRNA *‘1 AAAA...An 5' 3' alkali digestion of mRNA cDNA 3&1 TTTTS dNTPs Mg++ 5| 3| second , strand 7 3' TTTT SI SI nuclease 3| 3| 51 The "hairpin" loop is opened by use of a single strand specific nuclease, usually SI nuclease. 93 Diagram, Appendix I Continued 1) 5' ‘0H 3' 3' H0 5' ATP, Mg++ DNA Ligase linkers GAATTCC GGAATTC CTTAAGG CCTTAAG Eco RI AATTCC 00 PP uu CCTTAA This fragment is annealed to the plasmid vector cut with the same restriction enzyme, the annealed DNAs are sealed with DNA ligase and used to transfect a host microorganism, usually E; coli. 2) 5' *** 0H 3' H0‘** 5' Terminal transferase Mg++ ndNTP 3| 5' 2—NNNNn nNNNN 5‘“ 3' 5' +nPP1 The double stranded cDNA (dsc DNA) is now prepared for insertion into a vector molecule, usually a plasmid (double stranded, circular, extrachromosomal DNA) by two generally used methods. 1) Ligation of synthetic restriction endonucleases recognition sites onto the ends of the cDNA. These are small, double stranded DNA molecules that contain a single recognition site for a particular restriction enzyme. For example, Eco RI linkers are of the structure 5' GG‘I'IATTCC 3' 3' cameras 5' (Eco RI makes an asymmetric cut at the arrows). Usually the synthetic linkers are specific for restriction endonucleases which cut only once in the vector molecule. 2) Homopolymer tailing of the dscDNA, along with homo- polymer tailing of the vector with the complementary base of the "tailed" dscDNA by the use of the enzyme terminal transferase which adds nucleotides sequentially to the 3' hydroxyl ends of DNA molecules. The two tailed DNA fragments are allowed to anneal together by complementary duplex formation with their "tails", followed by transfection of the recombinant plasmid into the host organism. APPENDIX 11 Detection of specific DNA and RNA sequences that have been separated by gel electrophoresis has been simplified by the procedure of "blotting“ and binding these fragments onto a cellulose support, usually nitrocellulose paper. This procedure was first developed by Southern (55) for DNA and adopted for RNA by other researchers. Basically, this procedure involves the fractionation of DNA that had been digested with some type of restriction nuclease by gel electrophoresis. The DNA in the gel is denatured in situ with alkali, neutralized, equilibrated in high salt-containing soltuions and transferred out of the gel to a nitrocellulose support. The DNA (denatured in the gel) is moved from the gel directly onto the nitrocellulose to which single-stranded nucleic acid binds. Binding of the DNA to the nitrocellulose is rendered essentially irreversible by heat. This “blot" then forms a re-usable replica of the original gel that can be hybridized to any Specific radioactive probe to locate that specific sequence in a mixed sample of DNA. An analogous procedure is used for separation and detection of RNA. RNA blots are sometimes referred to as "Northern" blots as opposed to DNA blots which are referred to as "Southern" blots. 94 APPENDIX III When one has obtained a specific DNA sequence, either through molecular cloning or electroelution from a gel, this DNA can be labelled in vitro to a high specific activity and used as a probe for nucleic acid sequences identical, or very similar to it, by the tech- nique of nick-translation. This techniques involves taking advantage of the multiple enzymatic properties of the enzyme DNA polymerase I (g; .9911) and the judicious use of deoxyribonuclease I (DNAEEE I). E; coli DNA polymerase I contains in its single polypeptide chain three separate enzymatic activities. One is the 5' to 3' directional polymerase activity, and the other two are 5' to 3' and 3' to 5' exonuclease activities. The polymerase activity requires a single-- stranded template and a primer molecule with a free 3' hydroxyl group. By using DNA239 I at sufficiently low levels to permit the enzyme to make random breaks or "nicks" in the phosphodiester backbone of a double-stranded DNA molecule, free 3' hydroxyls are generated for use as a primer by DNA polymerase I. The polymerase enzyme then can make its own single strand template by virtue of its endogenous 5' to 3' exonuclease activity, and subsequently produce a radioactive copy of the template strand by inclusion of a radioactive nucleotide mix for use by the polymerase activity. Specific activities in excess of 108 cpm per pg DNA can routinely be obtained in the probe generated by this 95 96 reaction. Nick-translation is a misnomer in that the process involved is a transcriptional one rather than a translational one. 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