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Aging and protein synthesis : serine and leucine transfer RNA genes in Drosophila melanogaster Dartnell, Vicki June 1988

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AGING AND PROTEIN SYNTHESIS; SERINE AND LEUCINE TRANSFER RNA GENES IN DROSOPHILA MELANOGASTER by VICKI JUNE DARTNELL B.Sc, University of British Columbia, 1983  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1988 (c) Vicki June Dartnell, 1988  In  presenting this  degree  at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  D-ge.  2-2-,  l^gg  1 1 .  ABSTRACT  This thesis consists of two distinct parts.  Part I describes  preliminary studies undertaken to investigate whether defective tRNA molecules may be at least p a r t i a l l y responsible for the general decrease in protein synthesis a b i l i t y seen with advancing age. Part I I describes the cloning of three recombinant plasmids,  each containing at least Ser  Ser  one putative gene for one of Drosophila melanogaster tRNA^b ,  tRNAuCA'  or tRNA^gg. To approach unfractionated approximately  the f i r s t  Drosophila  problem from an i n v i t r o  perspective,  melanogaster tRNA was either degraded by  four nucleotides or elongated by one nucleoside 3',5'—  diphosphate at the 3'-terminus, and these defective tRNAs were added to a rabbit reticulocyte lysate protein synthesis system i n varying amounts.  I t was found that these molecules did not produce appreciable  inhibition of protein synthesis i n this cell-free system u n t i l they were present i n quantities similar to the estimated amount of endogenous tRNA present.  This finding suggests that such defective molecules  would not play an appreciable role i n the age-related protein synthesis decrease seen i n vivo, as i t i s highly improbable that defective tRNA molecules would accumulate at levels approximating  the levels  of active tRNA i n the c e l l . A search was conducted for p a r t i a l l y 3'-degraded tRNA molecules among the entire tRNA population and among both ribosome-associated from such organisms.  isolated  from aging  Drosophila,  and non ribosome-associated  tRNAs  These tRNA samples were treated with alkaline  1 1 1 .  32  phosphatase and 5'-labelled with [tf- P]ATP and polynucleotide kinase. Differential labelling of tRNA bands from the aged population could indicate the presence of partially 3'-degraded tRNA in this population; this was not observed, however, other than for one band which appeared to label somewhat more darkly in the older population in two independent experiments.  The  significance of this band is not clear, but i t  does not appear abundant enough to affect protein synthesis, on the basis of the in vitro results described. Analysis of whole tRNA isolates from both young and aged Drosophila by two-dimensional polyacrylamide gel electrophoresis did not reveal consistent differences between these two tRNA populations. In Part Ser tRNA^  II, two  oligonucleotides complementary to the known • •  sequence were used to screen pUC 13 recombinant DNA libraries  containing inserts of Drosophila genomic DNA purified by size.  One  of these oligonucleotides, GT8, hybridized to a 5.1 kb Hindlll restric• • Ser •• tion fragment containing tRNA^ , & 3.6 kb EcoRI fragment containing Ser •• an apparent tRNA^^ gene, and a 3.6 kb EcoRI fragment containing Leu a tRNAQjG gene. The entire structural genes for both tRNA^b and tRNAQjQ were Ser sequenced, as well as the 56 3'-nucleotides of the putative tRNAjjcA structural gene. The sequence data suggests that examples of genetic Ser Ser microheterogeneity sequence of the  are seen here for both tRNA2^ and tRNAy^.  tRNA^yg  The  gene corresponded exactly to that of a previousGlew  ly cloned Drosophila tRNAcuG" S ( et al. (1986) Gene 44, 307-314), but the flanking sequences of these two clones were different. Thus, . Leu • • * a second copy of this tRNA gene was obtained m this work. e n e  IV.  In derive  situ  hybridization studies  from chromosomal sites  66B (tKNA^gg).  88A  showed  the three  fragments to  (tRNA^ ), 58AB (tRNA^^), and  V.  TABLE OF CONTENTS  Page Abstract  ii  Table of Contents  v  List of Figures  viii  Abreviations  ix  Acknowlegements  xi  Dedication  xii PART I  I. Introduction  .  II. Materials and Methods i. ii. iii. iv.  2 . 12  Isolation of tRNA from Drosophila - Method I  12  Isolation of tRNA from Drosophila - Method II  13  Sephadex® G-100 Gel Filtration of RNA  13  Isolation of tRNA from Post-Ribosomal Supernatant and from Salt-Washed Ribosomes  14  v. Partial Enzymatic 3'-Degradation of tRNA  15  vi. vii. viii.  3'-Elongation of. tRNA  16  Reticulocyte Lysate Protein Synthesis Assays  16  Determination of Reticulocyte Lysate Endogenous tRNA . . 18  ix. One-Dimensional Polyacrylamide Gel Electrophoresis . . . 18 x. Two-Dimensional Polyacrylamide Gel Electrophoresis . . . 20 xi.  32  5'-Labelling of-tRNA with P  III. Results and Discussion i. ii.  Partial Enzymatic 3'-Degradation of tRNA 3*-Elongation of tRNA  20 22 . 22 26  VI.  Page iii.  Inhibition of Protein Synthesis by Defective tRNA . . . 29  iv. Two-Dimensional Electrophoresis of tRNA from Young and Aged Drosophila v.  35  5'-Kinasing Studies of Young and Aged tRNA Populations  39  a) Whole tRNA Extracts  41  b) Free and Ribosome-Associated tRNA  46  PART II I. Introduction II. Materials and Methods i. ii. iii.  vi. vii.  55  Agarose Gel Electrophoresis  56  Restriction Endonuclease Digestion  56  III.  57  Purification of DNA Fragments of Selected Size  58  Oligonucleotide Preparation and Radioactive Labelling  58  DNA Ligation, Bacterial Transformation, and Colony Screening  viii.  55  Isolation of Genomic DNA  iv. Genomic Southerns and Filter Hybridizations v.  49  Plasmid Isolation and Purification for Sequencing  . 59 . . . 61  ix. DNA Sequencing  62  Results and Discussion  65  i.  tRNA^ Gene Localization  65  ii.  Cloning of tRNA^, tRNAJj^, and tRNA^g  68  iii.  Sequencingr and in situ Hybridization of tRNA^ , tRNAj^, and tRNA^ Clones  73  VI1.  Page r  a) tRNA^  74  References  90  Appendix  95  V l l l .  LIST OF FIGURES  Page PART I Figure  1.  Comparison of unaltered and partially 3'-degraded tRNA  Figure  2.  Figure 3. Figure 4. Figure Figure  Figure  Figure  5. 6.  7.  8.  24  Comparison of unaltered and 3'-elongated tRNA . . . . 27 Effect of high levels of whole and partially degraded tRNA on protein synthesis Effect of low levels of partially degraded tRNA on protein synthesis Effect of high levels of elongated tRNA-pCp on protein synthesis  . . . 31 32 34  Two-dimensional electrophoretic comparison of tRNA populations from newly-hatched and 32 day old Drosophila  37  Comparison of 5'-kinasing susceptibility • of tRNA from newly-hatched and 30 day old Drosophila  42  Comparison of 5'-kinasing susceptibility of ribosome-associated vs. free tRNA from newly-hatched and 25 day old Drosophila  44  PART II Figure  9.  Ser Cloverleaf structure of tRNA^ Ser  53  Figure 10.  Genomic Southern analysis of tRNA^ genes  66  Figure 11.  Comparison of tRNA gene sequences  70  Figure 12. Figure 13. Figure 14.  In situ hybridization of the tRNA^ gene Cloverleaf structures of cloned tRNA genes as predicted from the DNA sequence data • • • Ser In situ hybridization of the t R N A y c A gene  Figure 15.  In situ hybridization of the tRNAQjQ gene  77 79 82 86  IX.  ABBREVIATIONS  ^60 Bis-MSB bp BSA ddNTP DMSO  —  - p-bis-(O-methylstyryl) benzene - basepairs - bovine serum albumin (Fraction V) - dideoxynucleoside triphosphate - dime thylsulfoxide deoxynucieoside  dN DNA dNTP DTT EDTA EGTA EtBr HEPES HSP HSS kb mRNA OAc PAGE pCp PPO Y  RNA rRNA  absorbance at 260 nm  - deoxyribonucleic acid - deoxynucleoside triphosphate - dithiothreitol - ethylenediamine tetraacetate (disodium salt) - ethylene glycol bis-^S-aminoethyl ether)-N,N'-tetraacetic - ethidium bromide - N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid - high speed pellet - high speed supernatant - kilobasepairs - messenger ribonucleic acid - acetate - polyacrylamide gel electrophoresis - cytidine 3 ,5 -diphosphate - 2,5-diphenyloxazole - pseudouridine - ribonucleic acid 1  —  1  ribosomal ribonucleic acid  acid  X.  SDS  - sodium dodecyl sulphate  TEMED  - N,N,N',N'-tetramethylethylenediamine  tris  - tris(hydroxvmethyl)aminomethane  tRNA  - transfer ribonucleic acid  1 x Denhardt's solution = 0.02% each of Ficoll®, polyvinylpyrollidone, and BSA (Fraction V) 1 x SSC = 0.15 M NaCl / 0.015 M Na citrate 3  1 x TBE = 80 mM t r i s / 80 mM boric acid / 1 mM EDTA 1 x YT =1% Difco bactotryptone / 0.5% Difco yeast extract / 0.5% NaCl  xi. ACKNOWLEGEMENTS I would sincerely l i k e to thank my supervisor, Gordon Tener; without h i s enduring patience and support t h i s work could not have "been carried out. The members of h i s laboratory have also been extremely h e l p f u l throughout:  Shizu Hayashi, who most generously contributed the  i n s i t u h y b r i d i z a t i o n studies and greatly helped me with the i n t r i c a c i e s of the UBC computer, Don S i n c l a i r , who always had a smile and an u p l i f t i n g word when the gremlin would s t r i k e , Ian Gillam, J e f f Leung, Craig Newton, and Nina Seto, a l l of whom contributed protocols, discussion, and much encouragement. of you.  My sincere gratitude goes to each  I am also thankful to my parents f o r a l l t h e i r  encouragement, and to my mother f o r most kindly typing this manuscript.  F i n a l l y , I g r a t e f u l l y acknowlege receipt of  a Medical Research Council  Studentship.  xii.  To Heather  1.  Part I  2  I.  .  INTRODUCTION  The quest for immortality, the "fountain of youth", has probably dwelt  i n the human race since the dawn of our a b i l i t y to reason.  In this century, the quest has been continued i n the s c i e n t i f i c i n v e s t i gations of many researchers. deal at  These researchers have amassed a great  of information, sometimes conflicting, on age-related changes the biochemical, c e l l u l a r ,  and higher  in a large number of tissues and organisms.  levels  of  organization,  I t i s unclear, but probable  that most changes are a result of, rather than a cause of aging. To find the primary "trigger" of the aging process, assuming there is  one, or a few, i s an important research goal.  also important to use any accumulated  However, i t i s  information to ameliorate the  effects of aging as far as possible by delaying the occurrence or lessening  the severity of the degenerative diseases common i n o l d  age. Many diverse theories on the basis of aging have been constructed from this array of information. These theories can be roughly divided into two broad categories: programmed developmental  Theories regarding aging as a genetically  stage, and those concerning the deleterious  effects of accumulation of random sub-lethal damage over time.  Propo-  sals of the f i r s t type have tended to suffer from a scarcity of evidence for  specific mechanisms by which  they may operate, and also from  a lack of convincing theoretical arguments supporting the evolution of such a genetic program. One model, based on a study of c e l l senescence i n v i t r o , has  3. involved the eventual production of an inhibitor of DNA replication (Smith, 1984).  Intriguing research supporting the programmed release  of a "death hormone" from the pituitary has also been carried out (Bolla, 1982).  It was  shown that in rats hypophysectomized early  _  in life (at 3 6 months), and maintained on minimal hormone replacement therapy, a number of parameters which normally decrease with age remained at "young" levels, even in 18~24 month old rats.  These  parameters include total RNA synthesis, initiation of RNA synthesis, liver aldolase synthesis, and a number of thyroid dependent physiological parameters. On  the theoretical  side, i t is difficult  to rationalize the  existence of "genetically programmed" death as a way of preventing species overcrowding in light of the fact that most organisms, including humans until recent times, die of environmental causes before they live  long enough to senesce.  This would eliminate any selection  pressure toward programmed aging (Kirkwood, 1984). Perhaps the strongest theory in this direction is the "disposable soma" theory (Kirkwood and Holliday, 1979) which suggests that energy is conserved and allocated toward growth, reproduction, and repair in such a way  that  somatic cell processes are not maintained at the level of accuracy they would require for extended survival.  Thus, reproduction and  continuation of the species is promoted at the expense of somatic immortality. The second category of aging theories, those concerning accumulations of unrepaired sub-lethal damage, have a much wider base of experimental data to support them.  These theories address damage  which occurs over time to the integral components of the cell such  4. as DNA (Burnet, 1974) and protein (Orgel, 1963). One example is the error catastrophe theory put forward by Orgel (1963). of  He postulated that errors could accumulate in the proteins  the protein synthetic machinery over time, thus making i t more  prone to synthesizing defective proteins.  Some of these, in turn,  form part of the new synthetic machinery and thus give rise to even more unfaithful translations.  Eventually protein synthesis becomes  so error-prone that a "catastrophe" ensues causing, presumably, cell death.  Although i t is now  generally believed that such a dramatic  escalation of errors does not take place during aging, age-related changes to such cellular  components as chromatin and  protein are  observed by some investigators (Makrides, 1983; Thakur, 1984).  Orgel  later amended his proposal to allow for a steady-state error frequency during aging, as opposed to a catastrophic escalation (Orgel, 1970). Experiments designed to support or refute the error theories of aging have been carefully  reviewed  (Laughrea, 1982)  and  the conclusion  drawn that a modest increase in error frequency does accompany advancing age.  It is not clear, however, whether such damage is a major or  significant cause of aging  in organisms, or of cell senescence in  model fibroblast systems. A major type of damage to cellular components is thought to be caused by free radicals. ionizing 02 The  radiation, and  Free radicals arise from exposure to  from both enzymatic  (during reduction of  to water) and nonenzymatic (02 with organic compounds) reactions. theory proposes that these could negatively affect cells in a  variety of ways. These effects include oxidative damage (i.e., crosslinking) to long-lived molecules such as collagen, elastin, and chroma-  5. tin;  oxidative  degradation  of  mucopolysaccharides;  production  lipofuscin  (age pigment) by oxidative polymerization of lipids  proteins;  lipid  peroxidation  of  of and  organelle membranes; post-injury  blood vessel damage by peroxidation of serum and vessel wall components (Harman, 1981). Living organisms have developed a number of defense mechanisms against oxygen free radicals, including antioxidants (i.e., tocopherols and  carotenes), heme-containing peroxidases  (i.e., catalase), gluta-  thione peroxidase, superoxide dismutase, and DNA repair enzymes (Harman, 1981).  The question might be asked whether individuals or species  with relatively higher amounts of these protective agents in their tissues would age more slowly. One very interesting result has been found regarding superoxide dismutase. A variety of mammalian species (both rodents and primates, including humans) were tested for superoxide dismutase levels in both liver and brain.  An excellent linear relationship was found  between the quotient of superoxide dismutase/specific metabolic rate vs. lifespan potential for each species (Cutler, 1983). relationships between superoxide dismutase and  Similar  lifespan have also  been observed for the housefly, Musca domestica (Sohal et a l . , 1986 and  1987).  This proportionality  between potential lifespan and  superoxide dismutase content gives considerable experimental support to the free radical theory of aging, as the only known function of superoxide dismutase is to inactivate the highly toxic superoxide free radical. The theory also suggests that dietary ingestion of antioxidants could potentially retard the aging process.  This has, in fact, been  6. found to be the case in a number of species including fruit flies, nematodes, mice, and rats (Harman, 1981).  Antioxidants were able  to increase the average, but not the maximum lifespan of various species  studied.  The lifespan benefits of excessive  antioxidant  consumption by humans have yet to be shown. A dietary manipulation  that has yielded dramatic results is  food restriction (reviewed by Masoro, 1986).  It has been possible  to extend the maximum lifespan of rats 50% by reducing their caloric intake to 60% of the normal ad libitum level from the time of weaning (Yu et al., 1982). peroxidation  Food restricted rats show markedly reduced lipid  of liver  subcellular organelles  compared to control  rats, at ages 6, 12, and 24 months (Laganiere and Yu, 1987). Restricted mice show increased  liver  catalase activity  of approximately 50%  at 12 and 24 months, and less lipid peroxidation at both ages, although the 13% difference at 24 months was not statistically significant (Koizumi et al., 1987). Furthermore, mice show less lipofuscin deposition in the hippocampus and frontal cortex of the brain (sites commonly affected in Alzheimer's disease), and better learning and performance in a maze test than control mice after 12 months of a restricted diet (Idrobo et al., 1987).  Results like these seem to suggest that  restricting caloric intake while ensuring full consumption of vitamins and minerals  reduces the cumulative amount of oxidative damage to  tissues, dramatically slowing  the aging process and extending not  only the average but also the maximum lifespan.  This gives support  to the theories on oxidative damage and aging, and also suggests possible strategies for slowing the aging process in humans. It is well established that the rate and amount of protein synthe-  7. sis decreases during aging, in a wide variety of organisms (Gabius et al., 1982; Makrides, 1983). The reason or reasons for this decrease may hold an important key to the mystery of aging, as proteins are so integral to the structure and function of cells and their organelles. In vitro protein synthesis systems derived from aged organisms also show decreases in synthesis ability (Webster and Webster, 1979; Gabius et al., 1983; Khasigov and Nikolaev, 1987).  Such a system  from Drosophila melanogaster exhibited a 70% drop in rate of synthesis by just 14 days after eclosure (Webster and Webster, 1979).  This  system has been studied intensively to determine the step or steps which show the greatest decrease (Webster and Webster, 1979, 1981, and 1982; Webster et al., 1981).  It was seen (Webster and Webster,  1982) that the elongation stage of protein synthesis, and more specifically, binding of the aminoacyl tRNA to ribosomes, showed the greatest rate decrease with age:  60-70% after 20 days.  This same step of-  the elongation process also showed the greatest rate decline with age (30~40%) in an analysis of cell-free protein synthesis from rat liver and kidney (Gabius et a l . , 1983).  Further, elongation also  showed the greatest rate decrease in cell-free systems from mouse brain, liver, kidney, and skeletal muscle (Blazejowski and Webster, 1984), and rat liver and brain cortex (Khasigov and Nikolaev, 1987). In Drosophila, at least part of this decline may be the result of a precipitous drop in the level of EF-1 mRNA available for translation (Webster and Webster, 1983 and 1984), which happens about five days previously to the decline.  Experiments on a cell-free system  from rat liver also show EF-1 from aged rats 30-40% less active than EF-1 from young rats (Moldave et a l . , 1979), however this has not  8. been consistently observed (Sojar and Rothstein, 1986). A soluble factor from high-salt washes of young rat liver ribosomes has been found to significantly stimulate old ribosome preparations; the active factor does not appear to be present in high-salt washes from old ribosomes (Sojar and Rothstein, 1986).  A similar situation  has been found in the study of ribosomal material from young and old rabbit reticulocytes (Rowley et al., 1971).  However, a proportion  of the ribosomes of senescent nematodes are inactive and do not show stimulation by material obtained from salt washes of young ribosomes (Egilmez and Rothstein, 1985).  The binding of the ternary complex  EF-1 • GTP • aminoacyl tRNA is found to be reduced for these inactive ribosomes, but not for the aged rat liver system described above. It is seen, therefore, that the nature of the decline in protein synthesis is s t i l l somewhat obscure.  It appears that ribosomes can  become inactivated in aged organisms, but that soluble factors such as EF-1 can also play a role in the decreased ability to synthesize protein. There has been some evidence suggesting the buildup during aging of defective material which co-purifies with tRNA, possibly being an inactive form of this vital adaptor molecule.  Indirect evidence  for this is the significantly decreased ability of nine different tRNAs from 35 day old Drosophila to be aminoacylated by aminoacyl-tRNA synthetases from either young or old flies (Hosbach and Kubli, 1979). The ability of eight tRNAs was reduced 10-25%, even using young synthetases, and for tRNA  a 50% reduction was seen.  Further, tRNAs  from aged rat liver were found to be somewhat less efficient than young tRNAs from the same source in a Krebs ascites cell tRNA-dependent  9. cell-free  protein  synthesis  system  (Mays-Hoopes et al.,  1983).  Possibly even more informative is the following data on aminoacylation of young and old rat liver tRNA:  On comparison of amino acid  acceptance of young and old whole tRNA extracts, testing for 17 amino acids, old tRNA isolated from whole tissue showed an average 64% decreased acceptance, whereas old tRNA isolated from the post-ribosomal supernatant (HSS) only showed an average decrease of 7% a l . , 1979; Lawrence et al., 1979).  (Mays et  This suggests the presence of  defective or inhibitory material, which co-purifies with tRNA, bound to the aged ribosomes.  Possible candidates could be partially degraded  or otherwise damaged rRNA and/or  tRNA molecules.  On analysis of  the material bound to young and old ribosomes (HSP), i t was found that material derived from old ribosomes had only 64% of the acceptance capacity of young ribosome-derived material. During purification of Drosophila melanogaster tRNA isoaeceptors for sequencing in our laboratory, a contaminant was found co-purifying S  with the tRNA5^ fraction.  On sequencing, this contaminant was found  r  to be a tRNA^ ^ molecule which had lost five nucleotides from the 3'-end (Cribbs, 1982).  Even though the molecule was only present 32  in very low amounts, i t labelled at the 5'-end with [tf - P]ATP and polynucleotide kinase to give a significantly darker band than the s  much more abundant intact tRNA^ the absence  also present, presumably  due to  of steric hinderance from the missing 3'-nucleotides,  creating a one-nucleotide 5'-overhang.  It is not clear whether this  partially degraded species is an artifact of the tRNA purification process or whether i t is actually present in live Drosophila, but this points to the possibility of such defective but largely intact  10. tRNA molecules being present in vivo, possibly due to lowered activity of degradative enzymes. These molecules could form part of the material which causes lowered amino acid acceptance of tRNA from aged organisms. If such material tended to bind tightly to ribosomes, i t would be largely removed from the post-ribosomal supernatant, accounting for the increased acceptance of tRNA from this fraction mentioned earlier. •  *  Met  Finally, i t has been shown that unprocessed tRNA precursors are stably present nucleotides and  nine  Similar molecules may bound RNA  and tRNA  XJGUI  in HeLa cells, with four extra 5'-  extra 3'-nucleotides  (Harada et a l . , 1984).  also become candidates for defective ribosome-  during aging, i f the activity of processing enzymes was  decreased, causing a significant increase in their abundance. The research contained in this thesis is a preliminary investigation  of  the hypothesis  that tRNA partially degraded or elongated  at the 3'-end could bind to ribosomes and synthesis. stranded  interfere with protein  tRNA has been partially 3'-degraded by removing the single  nucleotides using  snake venom phosphodiesterase,  elongated by one nucleotide using pCp and RNA ligase.  and  3'-  These modified  molecules were added to a rabbit reticulocyte lysate protein synthesis system, but  inhibition was  not seen until  the ratio of defective  : whole tRNA was almost 1:1. 32 Further, 5'-labellmg studies using [# -  P]ATP, alkaline phospha-  tase, and polynucleotide kinase, designed to detect 3'-degraded tRNA in whole extracts from young and old Drosophila melanogaster, showed no marked differences between the two tRNA populations, using this assay. Further, young and old tRNA from both post-ribosomal supernatant (HSS) and ribosomal bound fractions (HSP) showed a similar labelling  11. p r o f i l e i n the tRNA range. ential  labelling  i n two  Only one minor band possibly showed d i f f e r independent  experiments.  The  conclusion  i s drawn, from these preliminary studies, that p a r t i a l l y 3'-degraded or elongated tRNA does not to be a significant machinery  appears  have the  potency or i n vivo abundance  inhibitor of protein synthesis. able  to  discriminate  The  synthesis  e f f i c i e n t l y between whole  and defective tRNA i n these i n v i t r o assays, only becoming inhibited with very high levels of defective tRNA.  12.  II.  MATERIALS AND METHODS  All enzymes and lysate were obtained commercially, and the suppliers given where used exclusively. grade.  A l l chemicals used were reagent  Drosophila melanogaster, strain Oregon R, were grown by Dr.  G.M. Tener, and were a gift from him. i.  I s o l a t i o n o f tRNA from Drosophila - Method I  For  isolation  of tRNA, freshly killed Drosophila melanogaster  were preferable, but the tRNA of such flies frozen at -70°C for a few days or weeks also appeared intact by PAGE.  The method was scaled  down or up to cover weights of flies between a fraction of a gram and 100 gm. In a typical experiment by this method, derived from the method of Roe (1975), 1 gm of flies was ground in a 15 ml Corex® tube for 3 minutes with a Polytron® apparatus at setting "4" in a 1:1 mixture of 0.14 M NaOAc pH 4.5 (HOAc) : 88% phenol (total volume = 2.8 ml). This was centrifuged at 1,000 x g^^. for 15 minutes.  The aqueous  layer was further extracted twice with phenol and applied to a 3 ml DEAE-cellulose column equilibrated with 0.14 M NaOAc pH 4.5 (HOAc) (= buffer A). ^260  =  The column was washed with buffer A until the eluant m  f)>  t n e  column buffer was then changed to buffer  A / 0.3 M NaCl and the column washed until the eluant  = 0.03  (20 ml); finally, the tRNA was eluted with 8 ml buffer A / 1.1 M NaCl, and ethanol precipitated.  tRNA isolated in this manner contained  considerable 5S rRNA, as well as larger rRNAs.  Where necessary,  a Sephadex® G-100 column was used to remove these, as described below.  13. For isolation of tRNA from 100-200 flies, the method was scaled down as follows:  The  flies were ground in 600 pi of 1:1 buffer A  : phenol in a v-shaped tube 1.3 cm wide, with four passes of a PotterElvehjem homogenizer pestle at setting "4". at 77 x g^^  for 5 minutes.  aqueous phase was  This was  centrifuged  After two more phenol extractions, the  applied to a 1 ml DEAE-cellulose column as above  and eluted step-wise with 30 ml buffer A, 24 ml buffer A / 0.3 M NaCl, and 2 ml buffer A / 1.1 M NaCl. The latter eluate, which contains the tRNA, was desalted using an Amicon® centricon 10 microconcentrator unit and dessicated. ii.  I s o l a t i o n of tRNA from Drosophila - Method I I  To  isolate tRNA from 1 gm  of Drosophila by this method, the  flies were ground for 1-2 minutes with a Polytron® apparatus at setting "4", in 2.5 ml of 2% SDS / 0,14 M NaOAc pH 4.5 (HOAc) / 1.5 M NaCl, plus 2.5 ml of 88% phenol. for 5 minutes.  This was  The aqueous phase was  centrifuged at 1,000  x  ^  further extracted three times  with phenol, back extracting the phenol phase with H20 and pooling the resulting aqueous layers.  The  each time,  final aqueous phase  was  ethanol precipitated, taken up in 3 ml buffer B (see below) /  10%  sucrose, and  chromatographed directly  on  the  Sephadex®  G-100  column described below. iii.  19  Sephadex  The  G-100  Gel F i l t r a t i o n of  RNA  tRNA isolated from Drosophila was. contaminated with 5S  as well as  significant amounts of larger rRNAs.  contaminants where relatively pure tRNA was  RNA  To remove these  needed, gel filtration  14. using Sephadex® G-100 proved to be effective. A Pharmacia 1.6 with Sephadex® G-100 97 cm.  The  cm x 100 cm column was used.  (fine grade) at 65 cm pressure to a height of  column buffer was  1% methanol / 0.02%  NaN^  0.75  M NaCl / 0.01 M HOAc pH 5.0 /  (= buffer B).  The  sample was  3 ml of buffer B with 10% sucrose added; this was ml "cushion" of 15% sucrose /buffer B. through a 0.45  This was packed  loaded in  followed by a 4  Both solutions were filtered  u Millipore® filter before loading.  The column was  run against gravity at a flow rate of 16 ml/hr, and 1.4 ml fractions were collected. Totally excluded material, such as large rRNAs, eluted in the fractions from 63 ml to 91 ml; 5S RNA eluted from 91 ml to 122 ml; degraded, elongated, and whole tRNA eluted from 122 ml to 160 totally included molecules eluted from 189 ml to 245 ml.  ml;  The desired  fractions were pooled and ethanol precipitated. iv.  Isolation of tRNA from Post-Ribosomal Supernatant and from SaltWashed Ribosomes  This method is modified from two Stafford, 1970;  Webster et a l . , 1981).  previous methods (Pelly and D. melanogaster (1 gm)  was  ground for 25 seconds with a Polytron® apparatus in a 30 ml Corex® tube, in 20 ml ribosome homogenization buffer: mM  tris pH 7.2  (HCl), 100 mM KC1,  0.25 M sucrose, 50  6 mM Mg(0Ac)2, 1 mM EDTA, 7 mM  y2 -mercaptoethanol, and 10 pg/ml polyvinyl sulfate.  The mixture was  centrifuged at 4°C for 10 minutes at 3,500 x ^ 5 this pellets nuclei and mitochondria (material > 40,000 S). The supernatant was centrifuged at 4°C for 90 minutes at 106,000  15. x gjj^-  The resulting "high speed supernatant" (HSS) and "high speed  pellet" (HSP) were treated as follows:  The HSS was phenol extracted  three times, ethanol precipitated, and chromatographed on a Sephadex® G-100 column as described.  The HSP was resuspended in 10 ml buffer  A / 1.1 M NaCl, phenol extracted, ethanol precipitated, and chromatographed as for the HSS.  v. Partial Enzymatic 3'-Degradation of tRNA Drosophila  tRNA was treated with snake venom phosphodiesterase  (phosphodiesterase  I) to selectively remove the four single-stranded  nucleotides at the 3'-terminus of the molecule (Zubay and Takanami, 1964; von der Haar et a l . , 1971; Sprinzl et a l . , 1972; Addison, 1982). The enzyme was purchased from P-L Biochemicals as a 20 U/mg lyophilized powder.  It was stored at -20°C in a 10 mM Mg(0Ac)2 / 10% glycerol  solution at 1 mg/ml.  The enzyme is stable for four months under  these conditions (Miller et a l . , 1970). The reaction contained 1.0 jug/ul D. melanogaster tRNA, 0.1  pg/}x\  snake venom phosphodiesterase, 10 mM tris pH 7.6 (HC1), and 10 mM MgCl2.  This was incubated at 37°C for 30 minutes.  Extensive phenol  extraction was carried out to remove a l l traces of enzyme (this enzyme would itself  inhibit protein synthesis), and this was followed by  repeated ether extractions to remove phenol. The degraded tRNA was dessicated using a Rotovap apparatus and applied to the Sephadex® G-100 gel filtration purification.  column used for tRNA  This species elutes in the same fractions as intact  tRNA. The eluted fractions containing pure degraded tRNA were pooled, ethanol precipitated and taken up in H20 at a concentration of 2  16. mg/ml; the degraded material was then analyzed by PAGE. vi.  3'-Elongation o f tRNA  tRNA was elongated by one nucleoside 35'-diphosphate 3'-terminus  by the following method (Cribbs, 1982).  at the  The reaction  contained 1.6 pg/ul D. melanogaster tRNA, 2 mM pCp, 2 mM ATP, 15 mM MgCl2, 10 jug/ml BSA, 5 mM DTT, 50 mM HEPES pH 8.0 (KOH), 20% v/v DMSO, and 0.1 ug/ul T4 RNA ligase, in a volume of 160 ul. was incubated overnight at 17°C.  The mixture  It was then ethanol precipitated,  taken up in 100 ul H20, and a portion was analyzed by PAGE. The remainder was extracted three times with 1:1 phenol  : chloroform,  three times with ether, and ethanol precipitated. The tRNA-pCp was applied to the Sephadex® G-100 gel filtration column used for purifying tRNA.  Two peaks eluted from this column:  The tRNA-pCp peak expected at a similar elution volume as tRNA, and a peak of totally included material, likely a mixture of residual phenol and pCp, widely separate from the tRNA-pCp peak. The tRNA-pCp fractions were pooled, ethanol precipitated, taken up in H20 at a concentration of 50 jug/ml, and stored at -20°C. vii.  Reticulocyte Lysate Protein Synthesis Assays  To detect inhibition of protein synthesis by defective (degraded or elongated) tRNA, a rabbit reticulocyte lysate protein synthesis assay was used. Promega Biotec nuclease untreated lysate was aliquotted on arrival and stored at -70°C.  Activity decreased somewhat after  _  4 5 months' storage. A cocktail containing 105 mM creatine phosphate, 525 jug/ml creatine  17. kinase, 53 mM HEPES pH 7.5 (KOH), 13 mM DTT, 0.42 mM ATP, 0.11 mM GTP, 19 unlabelled amino acids (excluding leucine) at 0.21 mM each, 0.13 mM hemin, 484 mM KOAc, and 5.72 mM Mg(OAc)2 was prepared and stored at -20°C (method from Promega Biotec, with minor modifications). Before each experiment, a premix was made containing cocktail, 3 H-leucine (2 mCi / 0.0017 mg / 1.0 ml, New England Nuclear), Promega Biotec RNasin® For  (32 U/pl), and H20  in 5:2:1:4 proportions  (v/v).  a typical time course experiment with 10 time points, each tube  contained 15 ul lysate and 18 jul premix.  Any extra whole, degraded,  or elongated tRNA and Mg(0Ac)2 to be added had already been dessicated in the appropriate tube before the lysate and premix were added. All tubes were incubated at 25°C for the indicated time. 3 Determination of H-leucine incorporation was performed as follows: At the approximate time, 3 p.1 of the reaction mix was withdrawn and added to 1 ml 1 N NaOH / 2.0% H202 preincubated at 37°C.  After a  10 minute incubation at 37°C, 4 ml ice cold 25% cone. HCl / 3.5% glacial HOAc / 2% casamino acids was added and the mixture was put on ice for 30 minutes.  It was then precipitated onto Whatman® GF/C  2.4 cm filters under vacuum; the filters were washed liberally with ice cold 10% cone. HCl / 1.4% glacial HOAc and dried by washing under vacuum with acetone.  Filtration results were much more consistent  when the initial vacuum drawing the mixture through the filter was applied gently and intermittently (i.e., 5% error with intermittent vacuum vs. 17% error with continuous vacuum). The dry filters were covered in scintillation vials with 700 ^il  Protosol® and incubated either overnight at room temperature or  for 3 hours at 37°C. 58 pi glacial HOAc and 10 ml of a 0.4% PPO / 0.01%  18. Bis-MSB toluene-based fluor solution were added, and the vial contents were mixed well.  The samples were then counted 2 minutes in an Isocap®  300 scintillation counter, discriminating for the 0.5 to 18 KeV energy range. viii.  Determination o f Reticulocyte Lysate Endogenous tRNA  Determination of the amount of tRNA endogenous to the rabbit reticulocyte lysate used was performed as follows:  544 pi lysate  was diluted with 600 pi H20 in a siliconized glass tube, and 169 pi  (= 22 ug) large rRNA purified on a Sephadex® G-100 column was  added as a carrier.  This was extracted twice with 700 ^il phenol,  conserving the entire aqueous layer each time.  The phenol was back  extracted with 700 ul H20, and the pooled aqueous phases were extracted with 1.4 ml ether. The aqueous phase was chromatographed on a Sephadex® G-100 column as described, and the amount of rabbit tRNA was quantified by integrating the area under the A ^ Q peak to give an estimate of 0.11 /ig endogenous tRNA/ul lysate. A previous tRNA determination on less lysate (239 pi), which included one more phenol extraction step (with greater losses at the interface), no carrier RNA, and ethanol precipitation prior to chromatography, gave a result 45% less than the more rigorous determination described above. ix.  One-Dimensional Polyacrylamide Gel Electrophoresis  A 42.9% acrylamide / 2.1% methylene bis-acrylamide stock solution was made and stored at 4°C.  This was used to make 10% or 20% polyacryl-  amide gels (Fradin et a l . , 1975). Acrylamide at the desired concentra-  19. tion, 4 M urea, 1 x TBE, 0.05% ammonium persulfate, (and for some gels 1 pg/ml EtBr) were mixed and degassed under vacuum.  TEMED was  added according to the acrylamide concentration and the presence or absence of EtBr:  For a 10% gel, 0.1% TEMED was used, and for  a 20% gel, 0.03% TEMED was used; these amounts were doubled i f EtBr was present.  The mix was poured between 35 cm x 20 cm glass plates  (one plate siliconized), and allowed to set approximately one hour at room temperature.  The gels were run in 1 x TBE (and 1 /-tg/ml EtBr  if present in the gel). For 1.5 mm thick gels with 9 mm wide slots, up to about 75 jag of tRNA could be loaded in each slot; the tRNA sample was dessicated and taken up in 6 pi 50% (v/v) formamide / 13% sucrose / 0.3% xylene cyanol ff / 0.04% bromophenol blue loading solution.  0.4 mm thick  gels were used when autoradiography was needed, to increase resolution. In this case, 3 pi samples in 9 mm slots gave good bands. tRNA was spread over a larger range i f the 10% gels were run at 4°C, as opposed to room temperature.  In these gels, xylene cyanol  runs at the leading edge of the tRNA.  Maximum separation of tRNA  was obtained by running the gel at approximately 450 V for > 20 hours, until the xylene cyanol was 1-2 cm from the edge of the gel. For 20% gels, tRNA was spread well through the middle range of the gels i f they were run at room temperature, 600-1,000 V, for 24 hours.  If dye markers were desired throughout these 20% gel runs,  15 pi of loading solution could be applied to the top of the gel as the initial xylene cyanol in the sample reached the bottom. Electrophoresis was terminated when the second xylene cyanol band was about 3 or 4 cm from the end of the gel.  20.  x.  Two-Dimensional Polyacrylamide Gel Electrophoresis  tRNA could be typically  separated into  > 70 spots by two-  dimensional PAGE, using 10% acrylamide / 4 M urea in the first dimension and 20% acrylamide / 4 M urea in the second dimension (Fradin et al., 1975; Mazabraud and Garel, 1979). For these gels, the first dimension was run as for one-dimensional electrophoresis using a 1.5 mm thick 10% gel and 75 ug tRNA, until the leading edge of the tRNA was 1 cm from the end of the gel.  A  12 cm x 1 cm strip was cut from the lowest 12 cm of the lane where the tRNA was run (using a cutter 1 cm wide made of sharpened plexiglass for this purpose), carefully lifted with a spatula, and laid horizontally at the top of one of the second-dimension plates.  The gel  apparatus was assembled, and care was taken to keep the gel strip moist so that no bubbles formed between i t and the glass plates. The 20% acrylamide / 4 M urea gel was poured around the strip, allowed to set for approximately 1 hour, and run at about 550 V for 48 hours. New dye was added during the run as described, and the run was terminated when the second xylene cyanol band was 1-2 cm from the end of the gel. xi.  5'-Labelling o f tRNA with  32  P  The 5'-terminal phosphate group was removed from the tRNA to be  studied using calf  intestinal alkaline phosphatase (Boehringer,  28 U/jul) (Cribbs, 1982).  The enzyme was diluted by 1/1,000 in water  immediately before use.  A reaction tube with 0.25 /Jg/jul pure D.  melanogaster tRNA (larger RNA removed with Sephadex® G-100 gel filtra-  21. tion), 0.003 U/jul alkaline phosphatase, 20 mM  tris pH 8.0  (HCl),  and 0.2 mM EDTA (total volume 5 to 10 pi) was incubated 15 minutes at  55°C; 1 pi 50 mM nitrilotriacetic  acid pH 7.0 was added to stop  the reaction, and the mix was dessicated at room temperature. To  label  the 5'-terminal nucleotide of susceptible tRNAs, T4 . . 32  polynucleotide kinase  (Boehrmger, 4.5  U/pl) and  [fl - P]ATP  (New  England Nuclear, 250 juCi / 25 pi / 87 pmol) were used (Cribbs, 1982). A premix containing 40 mM tris pH 8.0 (HCl), 9 mM MgCl2, 10 mM DTT, 0.01 mM ATP, 0.1 mg/ml BSA, 125 mM KCl, 0.2 U/pl T4 polynucleotide 32 • kinase, and 0.5 pCi/ul [X - P]ATP was  added to the dessicated mix,  the volume being equal to that of the alkaline phosphatase reaction. This was mixed well and incubated for 30 minutes at 37 °C. The reaction was stopped by the addition of %-volume of 25% sucrose / 0.2% xylene cyanol ff / 0.1% bromophenol blue in 98% formamide. resulting sample was 0.4 mm  thick).  at 4°C for 3 days.  3 jul of the  analyzed by PAGE (20% acrylamide, 4 M urea,  The gels were autoradiographed  (Curex X-ray film)  22.  III.  RESULTS AND DISCUSSION Studies were undertaken, both in vitro and on tRNA isolates from  young and old Drosophila to attempt to determine the likelihood of partially  3'-degraded or elongated  tRNAs accumulating during aging  and acting as significant inhibitors of protein synthesis, by such mechanisms as blocking the binding of aminoacyl tRNAs to ribosomes. The  in vitro  degradation defective  studies involved enzymatic modification (partial 3'-  or elongation) of Drosophila. tRNA and addition of this tRNA to a rabbit reticulocyte  lysate protein synthesis  system to study its effect on protein synthesis.  Studies on tRNA  extracts took the form of two-dimensional PAGE of young and old tRNA, and  5'-labelling of young and old tRNA to detect exposed 5'-ends  of molecules partially degraded from the 3'-end. i.  P a r t i a l Enzymatic 3'-Degradation of tRNA tRNA  folded  in the characteristic  typically has four  single-stranded nucleotides  (see Figure 9, Part II). diesterase  "cloverleaf"  preferentially  conformation  at the 3'-terminus  It has been shown that snake venom phosphoremoves  the single-stranded  nucleotides  at the 3'-end of tRNA, hydrolyzing only very slowly past this point unless  the reaction is forced (Zubay and Takanami, 1964; von der  Haar et a l . , 1971; Sprinzl et_al., 1972; Addison, 1982). A whole tRNA extract from Drosophila was used for degradation by this enzyme. Reaction conditions were chosen to maximize degradation of  the four single-stranded nucleotides  (somewhat elevated enzyme:  tRNA ratio and 37°C temperature), and to minimize degradation beyond  23. this point (30 minute incubation), thus creating tRNA molecules essentially  intact,  but defective  at the aminoacyl acceptor  These partially degraded tRNAs were intended fragment discussed  site.  to mimic the tRNA^^  (Introduction), where only five nucleotides have  apparently been removed from the 3'-end (Cribbs, 1982). 10% PAGE was used to assay for the extent of tRNA 3'-degradation after  treatment with  snake venom phosphodiesterase.  The results  showed a distinct shift of the entire tRNA profile downward in the gel, consistent with removal of a small number of nucleotides from the majority of tRNAs in the sample (Figure 1). Further digestion did not increase this shift, unless the reaction was forced, implying that the digestion stopped after removal of the four single-stranded nucleotides. For comparison, tRNA digested with polynucleotide phosphorylase was also visualized by 10% PAGE (data not shown).  This enzyme is  known to function by a processive mechanism (Thang et a l . , 1967), whereby digestion of an entire tRNA molecule takes place before the enzyme proceeds to the next molecule.  In this case, a slight downward  shift of the entire tRNA profile, as seen for snake venom phosphodiesterase digestion, was not apparent.  Rather, the tRNA bands remained  fixed and became more faint as digestion conditions became more vigorous . Thus, on the basis of published studies on snake venom phosphodiesterase and the distinct downward shift seen on 10% PAGE, i t is concluded  that this tRNA has had only its single-stranded nucleotides  removed by the enzyme, and that the majority of tRNAs present have been altered in this way.  24.  Figure  1.  Comparison of unaltered and partially 3'-degraded tRNA.  Drosophila tRNA partially degraded as described in Materials and Methods (lane a) and whole Drosophila tRNA (lane b) were electrophoresed using a 1.5 mm thick 10% polyacrylamide / 4 M urea gel. Fifteen jig of each RNA sample was loaded, and 430 V were applied for 23 hours at 4°C. Electrophoresis was terminated when the xylene cyanol ff dye (XC) was 2 cm from the end of the plate. The gel was immersed in 40% methanol / 1 jug/ml EtBr for 20 minutes and photographed. The area shown in the figure is from the lower third of this gel.  25.  a b  -«XC  26.  ii.  3'-Elongation of tRNA tRNA molecules elongated by one nucleoside 3',5'-diphosphate  at  the 3'-terminus were constructed by ligating pCp to whole tRNA  extracted from Drosophila.  The reaction stopped after addition of  one monomer to each tRNA, as the enzyme will not use the newly added 3'-phosphate as a substrate.  These molecules were designed to mimic  tRNA precursors to the extent that the acceptor stem is not functional due to the presence of an elongated 3'-terminus, beyond the CCA end. They are also nonfunctional due to the absence of a 3'-hydroxyl moiety. The ligation reaction appears to have modified the major portion of the  the tRNA, according to the moderate, distinct 20% PAGE tRNA profile seen in Figure 2.  upward shift of  The major tRNA bands  have certainly undergone a shift upon elongation, and most of the more faint bands also appear adjusted. The discrete upward tRNA profile shift seen here, and the discrete downward shift of slightly larger magnitude seen for partially degraded tRNA (Figure 1), support each other as evidence that the desired minor adjustments have been made to the average tRNA size in both cases.  Because these samples contain a mix of at least 99 different  tRNA species (White et a l . , 1973), i t is not possible to deduce the shift distance of each individual tRNA.  However, the relative shift  distances, when compared with the length of the tRNA profile itself, appear compatible with the removal of four nucleotides or  the addition  of one nucleotide  3',5'-diphosphate  (Figure 1) (Figure 2).  These gels were run using 4 M urea as a denaturing agent; to fully denature the tRNA, the gels would need to be run at high tempera-  27.  Figure 2.  Comparison of unaltered and 3'-elongated tRNA.  Drosophila tRNA elongated by one nucleoside diphosphate at the 3'-terminus (lane a) and whole Drosophila tRNA (lane b) were electrophoresed using a 1.5 mm thick 20% polyacrylamide / 4 M urea gel containing 1 ug/ml EtBr. Twenty ug of elongated and unaltered tRNA were loaded, and 600 V were applied for 24 hours at room temperature. Electrophoresis was terminated when the xylene cyanol ff dye (XC) was 1 cm from the end, and the gel was photographed. The area shown in the figure is from the middle third of this gel.  29. ture with 7 M urea present.  However, the milder conditions were  chosen because the influence of shape caused the separation of tRNA into a longer profile with more bands than would have been obtained under harsh  denaturing  conditions.  Since individual bands cannot  be matched between whole and altered tRNA profiles on the basis of these gels alone, total denaturation appeared unnecessary, and the wider spread of the tRNA on these partially denaturing gels seemed preferable for this qualitative analysis. These analyses on partially degraded and elongated tRNA suggest that sufficient modification has taken place to render the majority of  tRNA molecules  in each sample ineffective, by altering the  3  I_  terminus. iii.  Inhibition of Protein Synthesis by Defective tRNA A rabbit reticulocyte lysate nuclease untreated cell-free protein  synthesis system was  used to assay inhibition of protein synthesis  by the partially degraded and elongated tRNAs described. This system was  suitable for these initial studies, as i t is readily available  commercially, and i t contains a l l the components necessary for translation, including mRNA. with micrococcal nuclease  Unlike the more widely-used lysate treated to destroy endogenous mRNA (Pelham and  Jackson, 1976), the untreated lysate was quite stable with respect +2 to variations in Mg concentration. Nuclease-treated lysate had +2 an activity peak for a narrow range of added Mg  concentrations  (0.6 mM to 0.8 mM), with the activity dropping off sharply on either side of this range, whereas untreated lysate was equally active from +2 0.6 mM to at least 3.6 mM Mg  30. Micrococcal nuclease is +2 chelation of Ca with excess of Mg+2 present, enough Ca+2  +2 activated by Ca , and deactivated by . . . EGTA. With an increase in the amount •is presumably displaced from chelation  to re-activate the nuclease, causing a decrease in lysate activity J m +9 s +9 [k = 5 x 10 for Ca and = 2.5 x 10 for Mg (Schmid and Reilley, +2 Since tRNA does chelate Mg to a certain extent, extra  1957)]. +2 Mg was added where extra tRNA was present at high levels. +2 a lysate system much more stable with respect to Mg was needed for this series of assays.  Thus,  concentration  The nuclease untreated lysate  was seen to be suitable in this respect, showing no activity change even upon addition of 0.3 ug whole tRNA/ul lysate, with or without +2 extra Mg  (Figure 3).  If the data had suggested that more detailed studies of inhibition should be pursued, a Krebs ascites cell tRNA-dependent protein synthesis system may have been suitable (Boime and Leder, 1972), as the amounts of both functional and non-functional tRNA can be controlled in that system.  As seen in Figure 4 however, levels of exogenous partially  degraded tRNA from 0.006 - 0.06 ,ug/ul lysate produce absolutely no detectable change in lysate activity. result.  This is an important negative  It shows that even exogenous defective tRNA levels well  within an order of magnitude of endogenous tRNA levels (0.11 jug/jul lysate) do not inhibit protein synthesis in this system. Two determinations of the level of endogenous lysate tRNA were made, as described.  The most reliable experiment, in which care  was taken to reduce tRNA losses to an absolute minimum at each stage, gave an estimate of 0.11 jdg tRNA/ul lysate. gave a value of 0.06 pg tRNA/pl lysate.  An earlier determination  This earlier value is well  Figure 5 E f f e c t of High Levels of Whole and P a r t i a l l y Degraded tRNA on P r o t e i n Synthesis . 40000 -i  ;  •  1  Time (minutes)  Figure 4 E f f e c t of L o w L e v e l s of P a r t i a l l y Degraded t R N A on P r o t e i n 30000  Time (minutes)  Synthesis  33. within an order of magnitude of the more rigorously determined value, and  shows quite good agreement with  opportunities  i t considering the increased  for tRNA loss in this earlier experiment (discussed  in Materials and Methods). If partially degraded tRNA were present in vivo at a l l , i t would certainly not be expected to be found in a quantity approximating the level of functional tRNA present, but rather in much smaller amounts.  Thus, the relevance of partially degraded tRNA to protein  synthesis inhibition in vivo appears very doubtful from these data. Higher levels (such as 0.3 pg/pl lysate) of both defective tRNA types do produce appreciable inhibition of protein synthesis (Figures 3 and 5).  This inhibition becomes most apparent after the first  20 to 40 minutes of synthesis, with the inhibited samples attaining . . 3 only about 50% to 65% of the uninhibited  H-Leu incorporation after  2 hours.  The initial synthesis rates appear comparable with or without  defective  tRNA present.  In these preliminary results,  elongated  tRNA-pCp appeared to be a slightly less potent inhibitor than partially degraded tRNA.  This could, however, be affected by the percentage  of tRNA molecules modified in the respective tRNA samples, a percentage which was not measured quantitatively by the PAGE assays which were done. The mechanism of this inhibition, although an interesting study in itself, was not investigated further here, due to the high levels of modified  tRNA needed to produce detectable changes in protein  synthesis, and the resulting apparent irrelevance to an in vivo situation. To approach this study, one could prepare sufficient quantities  Figure 5 E f f e c t o f H i g h L e v e l s o f E l o n g a t e d t R N A - p C p on P r o t e i n 30000 i  _  _  ,  Synthesis _  «o  Time  (minutes)  35. • • Met •• • of both purified tRNA^ and a purified elongator tRNA, partially degrade them, and conduct inhibition studies. It would be interesting Met to see whether pure defective tRNA^  could produce the same type  of inhibition curve seen here, suggesting that these. results signify inhibition at the initiation step. Further, the use of a pure degraded elongator  tRNA species may illuminate any inhibition of elongation Met  possibly masked here by the presence of defective tRNA^ . Elongation, and more specifically the binding of aminoacyl-tRNA to the ribosomes, is the step which shows the greatest activity decrease in  aging Drosophila  (Webster and Webster, 1982) and rat liver or  kidney (Gabius et a l . , 1983).  A defective elongator tRNA would be  a logical candidate as a competitive inhibitor of this step.  Here,  however, the ribosomes and/or elongation factors appear to possess efficient means of discriminating against excess defective tRNA approaching even a 1:1 concentration ratio with respect to functional tRNA; any small amounts of partially degraded tRNAs or unprocessed tRNA precursors  that may be found in vivo would therefore not be  expected, on the basis of these preliminary data, to be at all effective in inhibiting protein synthesis. iv.  Two-Dimensional Electrophoresis of tRNA from Young and Aged Drosophila Whole tRNA extracts  day  (by Method I) from newly-hatched and 32  old Drosophila were compared using  separates  two-dimensional PAGE, which  the samples into more than 70 spots.  This approaches the  99 different major and minor tRNA species found by RPC-5 chromatography of Drosophila tRNA (White et al., 1973).  36. The 32 day old flies used in this study already show clear signs of  aging, such as appreciably decreased vigor, as measured by the  longer time taken to climb upward in a bottle after being forced to the bottom.  Thus, this study was undertaken to see whether any  major or minor tRNA spots can be seen to appear, disappear, or change as flies  age.  The tRNA populations from first- and third-instar  larvae and newly-hatched adult Drosophila have previously been compared by RPC-5 chromatography to assay for developmental et al., 1973).  changes (White  Approximately one-third of the 99 detectable peaks  showed some developmental alteration in that study. In the present study, i t appears that the tRNA population remains essentially unchanged as the flies age, as assayed by two-dimensional PAGE (Figure 6).  The lower limit of tRNA detection with these gels  is estimated at less than 0.5 pg, and the presence of more than 70 spots suggests that the majority of tRNAs are visualized here (bearing in mind that some spots overlap). In  the gel pair shown here, the isolate from aging flies has  about four extra faint spots which run with the tRNA at the front of  the sample (lower part of gel).  These are not seen consistently  however, and in other comparisons of tRNA from young and old flies this area of the gel is identical, and a few extra spots may appear among the larger tRNA which are not present here. The variable presence and position of these extra spots suggests they are probably artifacts resulting  from degradation in some samples during tRNA isolation  or handling, even though care was taken to prevent this. Taken  collectively  therefore,  the data  from  two-dimensional  PAGE suggest that no appreciable change in tRNA population occurs  37.  Figure  6.  Two-dimensional electrophoretic comparison of tRNA populations  from  newly-hatched  and 32 day old Drosophila.  Entire tRNA populations from newly-hatched (part a) and 32 day old (part b) Drosophila melanogaster, isolated by Method I (Materials and Methods), were compared by two-dimensional PAGE. First dimension: 75 jug of each of the two tRNA samples were electrophoresed in the same 1.5 mm thick 10% polyacrylamide / 4 M urea gel containing 1 pg/ml EtBr. 450 V were applied for 19% hours, and 700 V were then applied for 2 hours, a l l at 4°C. Second dimension: The lowest 12 cm of each lane were cut out, rotated through 90° and electrophoresed at room temperature using two matched 20% polyacrylamide / 4 M urea / 1 pg/ml EtBr gels, at 550 V for 45 hours followed by 200 V for 7 hours. New xylene cyanol ff dye was added to each gel when the first aliquot of this dye reached the end of the gel, and each run was terminated when the second aliquot of the dye was 2 cm from the end of each gel (XC, shown in figure). Further staining for 10 minutes in an aqueous 1 pg/ml EtBr solution was performed, and the lowest 23 cm of each gel was photographed and is presented here.  38.  39. as Drosophila age, as assayed by this method. Microheterogeneity  between young and old tRNA populations would  not be detected here.  This is addressed to a certain extent, however,  in the 5'-labelling studies which follow. of RNA  1/100  In those studies, levels  - 1/1,000 times lower than in these spots should be  detected, provided that the species is amenable to labelling at its 5'-end. An  interesting  finding  that emerged during  these studies is  that a much lower amount of tRNA seems to be available for isolation from aged flies than from young ones (typically about 60%).  This  figure is based on  two  old  in one experiment and about 200 in the other,  flies were 102  isolations where the numbers of young and  both performed by the scaled-down version of Method I. using 1-10  gm  of flies  In experiments  i t also appeared that older flies yielded  less tRNA, but these experiments were not as closely matched as to weight of young and old flies used. It  therefore appears either that older flies contain 40% less  tRNA, or that tRNA is much less accessible for isolation in these flies.  The former appears more probable, in view of the appreciable  body tissue wastage and of older flies  greatly reduced protein synthesis ability  (Webster and Webster, 1979).  However, even i f the  total, amount of tRNA drops, i t seems from these data that i t is a fairly  uniform drop, such that the tRNA pattern on two-dimensional  PAGE remains relatively unchanged during aging. v.  5'-Kinasing Studies of Young and Aged tRNA Populations One  possible inhibitor of protein synthesis in vivo was thought  40. to be tRNA with a few nucleotides removed from the 3'-end as discussed earlier, although the preceding in vitro data do not support this hypothesis.  To assay for the presence of this partially degraded  tRNA in vivo, radioactive  5'-labelling with polynucleotide kinase  was employed. The preferred substrate for both alkaline phosphatase and polynucleotide kinase is RNA or DNA with an exposed 5'~hydroxyl group; intact tRNA is generally a poor substrate, due to the omnipresent NCCA 3'-extension and the resulting steric hinderance.  This is seen  in the excellent removal of the 5'-phosphate and subsequent kinasing r  of a tRNA^ £  fragment missing five 3'-nucleotides, contrasted with 3  the poor labelling of an intact tRNA^ species also present (Cribbs, Ar£t 1982), as discussed.  The 5 -terminus of the tRNA ° fragment had  a one-nucleotide overhang, and gave a much darker band upon labelling s  than the intact tRNA^ , even though the latter was present in great excess. Because of this  differential labelling, a suitable assay for  the buildup of partially 3'-degraded tRNA in aging Drosophila could be the appearance of tRNA bands which 5'-kinase tRNA population of these aging flies.  intensely in the  This is a sensitive assay,  which should be able to detect picogram quantities of partially 3'degraded tRNA.  As well as being much more sensitive  than two-  dimensional PAGE of non-labelled tRNA, this is a more specific assay, in that i t detects tRNA molecules with at least five nucleotides missing from the 3'-end in particular.  Defects in the 3'-end may  have an increased bearing on protein synthesis, due to the aminoacyl acceptance activity of this end.  41.  a) Whole tRNA Extracts The  entire tRNA population from both newly-hatched and 30 day  old Drosophila were treated with alkaline phosphatase and kinased as described, and then separated by 20% PAGE. The results are shown in Figure 7.  Young tRNA isolated by both methods and old tRNA isolated  by Method II are compared; i t is seen that no prominent dark bands differentially appear in the tRNA fraction as flies age.  The three  bands which do appear slightly darker in the older population are of uncertain significance.  They may  be artifacts, but the middle  one appears to correspond in position to a differentially labelling band from 25 day old HSS  tRNA in Figure 8 (see below); the other  two bands here do not appear to have similar correspondences.  These  bands may represent trace amounts of partially degraded tRNA molecules, but these very low amounts of defective tRNA would not be expected to affect protein synthesis, based on the preceding in vitro data. Elution and  sequencing  of  the band which differentially  labelled  in the two  independent experiments might provide information as to  its origin and possible significance. It should be noted at this point that the major differences between Methods I and II for tRNA isolation are the omission of the DEAE-cellulose chromatography step and  the addition of 2% SDS  1.5 M NaCl to the initial grinding buffer in Method II.  and  The high  salt concentration was added because large rRNA is relatively insoluble in high salt; with the absence of the DEAE-cellulose step, i t was thought that this might decrease the relative amount of large rRNA applied to the Sephadex® G-100  column. However, for most purification  42.  Figure 7.  Comparison  of  5'-kinasing  susceptibility  of  tRNA from  newly-hatched and 30 day old Drosophila. Whole tRNA isolated by Method I from newly-hatched flies (lane a), by Method II from 30 day old flies (lane b), and by Method II 32 from newly-hatched flies (lane c) was 5'-labelled with [tf - P]ATP using alkaline phosphatase and polynucleotide kinase as described. A 0.4 pg aliquot of each sample was electrophoresed using a 0.4 mm thick 20% polyacrylamide / 4 M urea / 1 jig/ml EtBr gel, at room temperature, 1,000 V, for 22 hours. The gel was autoradiographed at 4°C for 3 days. The lower 25 cm of this gel is shown in the figure. Closed triangles (•) mark the boundaries of the size range occupied by whole Drosophila tRNA; open triangles (>) delimit the size range of tRNA degraded by snake venom phosphodiesterase as described; arrowheads (•) show the positions of the three bands differentially seen in tRNA from 30 day old flies (lane b), with a double arrowhead marking the extra band which also appears in Figure 8, lane b.  43.  a  b  c  44.  Figure 8 . Comparison associated  of  5'-kinasing  susceptibility  of ribosome-  (HSP) vs. free (HSS) tRNA from newly-hatched  and 25 day old Drosophila. Susceptibility to 5'-kinasing was studied for tRNA populations derived from the high speed supernatant (HSS) centrifugal fraction of young (lane a) and 25 day old (lane b) Drosophila extracts, and from the high speed pellet (HSP) fraction of young (lane c) and 25 day old (lane d) extracts. The tRNA samples were isolated and 5'labelled as described. A 0.4 ug aliquot of each sample was electrophoresed and autoradiographed as described in Figure 7. The lower 25 cm of this gel is shown in the figure. Closed triangles (•) mark the boundaries of the size range occupied by whole Drosophila tRNA; arrowheads (•) mark the positions of the three bands seen in 25 day old (lane b) but not young (lane a) HSS tRNA, with a double arrowhead marking the band which also appears in Figure 7, lane b.  45.  a b e d  •  46. experiments, i t was found that relatively less high molecular weight material was obtained during gel filtration where a DEAE-cellulose column had been used first.  For these labelling experiments, tRNA  which had not been passed through DEAE-cellulose was included (i.e., Method II tRNA), in order to obtain a tRNA sample with the least chance of removal of any potentially interesting components in the tRNA size range by ion exchange chromatography. b) Free and Ribosome-Associated tRNA It  has been found  that the aminoacyl acceptance capacity of  ribosome-bound tRNA from old rat liver  is about 64% of that from  young specimens (Mays et a l . , 1979; Lawrence et a l . , 1979).  Thus,  a kinasing study was done on tRNA from a salt-wash of young and 25 day old ribosomes  (HSP tRNA), as well as relatively ribosome-free  HSS tRNA from these two groups. lower acceptance  If old ribosome-bound tRNA has 36%  due even partially to appreciable 3'-degradation,  it should, in theory, kinase very efficiently. The results of the study, given in Figure 8, show that aged ribosome-bound tRNA does not appear than young ribosome-bound tRNA.  to kinase significantly better  It seems likely from this result  that extensive 3'-degradation does not exist  among the ribosome-  associated HSP tRNA of aged flies, and i f reduced aminoacyl acceptance were to be seen in this tRNA fraction, as in aged rat liver, i t would have some other cause.  Furthermore, there are no major differences  between the young and old HSS tRNA labelling profiles.  Three bands  do appear slightly darker in the old HSS tRNA fraction, but only one of these apparently coincides with a differentially dark band  47. from aged whole tRNA extracts (as described above).  However, this  band is of similar intensity between the young and old HSP tRNA fractions.  Thus, although the band has appeared darker in the old tRNA  fraction in two independent experiments (Figures 7 and 8), i t does not seem sufficiently intense to be a probable inhibitor of protein synthesis i f , in fact, i t is a partially degraded tRNA, based on the results of the in vitro studies done. Furthermore, dramatic age-related changes are also absent from the labelling patterns of RNA fragments smaller than the tRNA size range.  The largest difference is seen in RNA smaller than tRNA from  HSS (Figure 8a and b), with about four bands in the 50-65 nucleotide range significantly darker in the 25 day old sample.  This difference  is not seen in the young vs. old whole tRNA labelling experiment however (Figure 7), and was not seen consistently in HSS electrophoresis; i t is therefore probably an artifact of this particular electrophoresis experiment. Overall, these data hint at possible alterations in the 5'labelling  pattern between young and old tRNA populations, but no  prominent differences are seen. Defective tRNA molecules with exposed 5'-ends label many times more efficiently than intact tRNA, as a rule, making i t unlikely that any minor changes in band intensity seen here are indicative of defective tRNA molecules abundant enough to significantly alter protein synthesis in vivo.  48.  Part II  49.  I.  INTRODUCTION  The tRNA molecule forms the vital link which matches amino acids with their correct triplet codons in the mRNA during protein synthesis. The range of this function is attested to by the presence of at least 63 major and  39 minor chromatographically  Drosophila melanogaster  distinct tRNA species in  (White et a l . , 1973).  These are thought  to be encoded by approximately 600-750 genes (Ritossa et a l . , 1966; Tartof and  Perry, 1970; Weber and Berger, 1976).  Bearing in mind  that the majority of these minor tRNA species are thought to differ only  in post-transcriptional modification, not  this gives an estimate of 10-13  in genetic  origin,  copies for each tRNA gene contained  in the Drosophila genome. When total 4S RNA  (containing the entire complement of tRNA)  is hybridized in situ to Drosophila polytene chromosomes, i t is seen to derive from at least 54 sites: ones (Elder et a l . , 1980).  26 strong sites, and 28 weaker  Only one major site is found on the X  chromosome (at 12E), and the rest are scattered seemingly at random throughout both arms of the two large autosomes; no tRNA sites are observed on the small autosome.  However, i t is now known that this  procedure failed to detect some gene sites, such as the 19F site of tRNA^S (Newton, unpublished). Cloning and sequencing of regions rich in tRNA genes has elucidated some of The  the  general  features  of  their  chromosomal organization.  largest region examined by these techniques to date is a 94 kb  sequence at 42A on the right arm of chromosome 2, containing a total  50. Asn  8  ys  Ile  of 18 genes for tRNA , tRNA^ , tRNA2 , and tRNA , within 46 kb (Yen and Davidson, 1980).  A second site at 90BC has also been  studied, and is seen to contain 10 or 11 tRNA genes within 31 kb (DeLotto and Schedl, 1984). Val  Pro  These include 6 sequenced genes for  Ala  tRNA , tRNA , tRNA , and tRNA^, with  other  genes present in this area not yet sequenced.  apparent tRNA  Sequencing studies  of these major tRNA hybridization sites, as well as smaller-scale endeavours (summarized by Leung, 1988) have shown that these sites can contain a number of genes for various unrelated tRNA species, that  the genes are present in either transcriptional orientation,  and that identical genes for a given tRNA isoacceptor can be found at more than one chromosomal hybridization site. It has been found that approximately 20% of yeast tRNA genes contain introns, with the vast majority of these eukaryotic introns found thus far occurring one base pair 3' to the anticodon (Johnson and Abelson, 1983).  Exceptions  (Del Rey et al., 1982).  to this position do exist, however  For Drosophila, only eight such genes have  been found to date, suggesting  that  intron-containing tRNA genes  may be somewhat less abundant in this species. Leu • • • Drosophila  tRNA^g genes which derive  Two closely-spaced  from site  50AB (chromosome  2R) contain introns which are nearly homologous, and are 38 and 45 bp  in length (Robinson and Davidson, 1981).  otherwise indentical.  These two genes are  Further, six out of eight otherwise identical  Drosophila tRNA^^ genes  contain  introns  of varying  size (20-113  bp) and sequence (Choffat et al., 1988). A functional role in anticodon base modification seems apparent for those eukaryotic introns studied.  For example, i t has been found  51. that deletion of the intron from a yeast tRNA^^ ochre suppressor gene resulted in a lack of post-transcriptional modification of the middle nucleotide of the anticodon to Y, causing the in vivo suppressor activity  of  this  tRNA to fall  dramatically (Johnson and  Abelson,  1983).  Similarly, deletion of the Drosophila and Xenopus tRNA""^  1  introns prevents modification of the middle position of the anticodon to Y (Choffat et al., 1988). Leu yeast amber suppressor tRNA  Further, loss of the intron from a . . . gene prevented modification of cytosme  in the wobble position of the anticodon to 5~methylcytosine, leading to a reduction of suppressor activity (Strobel and Abelson, 1986). In our laboratory, a study has been undertaken of the Drosophila major isoacceptor tRNA4  (codon UCG)  genes at their most prominent site  and 12DE  tRNAy  (UCA, UCC,  UCU)  on the X chromosome, and  at the three minor sites 23E on chromosome 2L, 56D on chromosome 2R, and 64D on chromosome 3L (Hayashi et al., 1980; Cribbs et a l . , 1987b; Leung, 1988; Dr. D. Sinclair, unpublished).  These two iso-  acceptors differ from each other at only three positions:  #16, 34,  and 77 (#34 being the wobble position of the anticodon), giving 96% homology between these two distinct isoacceptors which utilize different codons.  This is unusual, as functionally distinct tRNA isoacceptors  generally show 10 to 30% sequence divergence (Sprinzl et al., 1987), as is the case with tRNA^b  (AGC, AGU) which only shows 71 to 73% er  er  homology to tRNA^y (Cribbs et a l . , 1987a). Thus, tRNA^ and tRNA^  are thought to exhibit a high degree of concerted evolution. Ser In total, twelve tRNA^ ^ genes have been cloned and sequenced Ser , three for tRNA4 Ser , three genes that thus far: Five for tRNAy Ser Ser consist of hybrids between the tRNA^ and tRNAy sequences, and  52. Ser  •  •  • •  one copy of tRN/A4  showing microheterogeneity with a C to T transition  at position  This study has illuminated interesting features  of  50.  tRNA gene organization and evolution, including  the concerted  evolution discussed above, the suggestion that recombination occurs between non-allelic tRNA genes giving rise to the observed hybrid gene structures, and the observation that mutations can accumulate at sites which are thought to be selectively neutral in tRNA genes which are otherwise highly conserved.  Other examples of this micro-  heterogeneity are found in Drosophila, with putative gene sequences which differ from corresponding tRNA sequences at 1 to 6 nucleotides Mot-  being seen for tRNA-^ al  1984), tRNA4  Vol 1  (Sharp et a l . , 1981), tRNA^  (Addison et al., 1982), tRNA^ Glu  1982), and tRNA  (Leung et al.,  (DeFranco et a l . ,  (Hosbach et al., 1980). The relative transcriptional  activity for each of these allogenes in vivo is not known; however, all  of the genes whose activities have been assayed using various  in vitro systems have shown activity in such systems (Leung et a l . , 1984). The purpose of the present study was to extend this knowlege Ser • Ser of the tRNA genes in D. melanogaster. The tRNA2b isoacceptor has previously been highly purified by BD-cellulose / Sepharose® 6B / RPC-5 chromatography  (Hayashi et al.,  1982), and sequenced in our  laboratory (Cribbs, 1982; Cribbs et al., 1987a).  The sequence and  cloverleaf structure of this molecule is given in Figure 9.  This  tRNA responds to the codons AGC and AGU, and is thus functionally er  er  distinct from both tRNA| and tRNAy . By making use of this RNA sequence information, two DNA oligoSer nucleotide probes were constructed and used to detect the tRNA2^  53.  c G - U C-G G-C 5-A-U G~C P  - s o  A  15  G  D  A  U  l 0  D  n A A D  A  m  G  lG 2  20  70  UACCC  GGC  G  .  c  U  A  m A 1  - *  ' , ,CG 11 U G,' *-A G^ 3r tjl C I J m°U A  1 1  r  m  U  -50  G-C  G-C  30-  -40  mC  A  U  mt A  3  6  GcU • 35  t RNA  S e r  2 b  Figure  9.  Cloverleaf structure of  tRNAou  [from Cribbs, 1982].  54. gene among fragments of the Drosophila One such gene was isolated and sequenced. future  genome selected by size. It is to be used in the  to study the distribution, organization, and architectural  features  Ser Ser of the tRNA2b branch of the tRNA gene family in D.  melanogaster. In addition, one of these probes detected two other genes: A putaSer tive  tRNAucA  g  ene  lacking correspondence to any tRNA characterized  to date, and a tRNA^g gene which appears to correspond to tRNA2 , according to in situ hybridization results.  55.  II.  MATERIALS AND METHODS E. coli strain DH5<*[F~, recAl, endAl, gyrA96, thi-1, hsdR17(r^,  m*), supE44, X~, relAl, d> 80dlacZAM15 (Leung, 1988)] was a gift from J. Leung.  Cloning vector pUC 13 (Viera and Messing, 1982) was a Ser  gift  from N. Seto.  The tRNA2b  specific oligonucleotides GT8 and  GT9 were synthesized by T. Atkinson in the laboratory of M. Smith. Sources of enzymes, chemicals, and Drosophila are as specified in Part I of this thesis. i.  Isolation of Genomic DNA High molecular weight  genomic  DNA  was  obtained from adult  Drosophila melanogaster (Ore R) by the method of McGinnis and Beckendorf (1983), as modified by J . Leung in our laboratory (Leung, 1988). Yields were typically in the range of 0.5 mg/gram of flies.  This  DNA was taken up in 0.5 ml TE [10 mM tris pH 8.0 (HCl) / 1 mM EDTA] and heated at 65°C for 30 to 60 minutes to aid dissolution.  The  genomic DNA was assessed by electrophoresis using 0.3% agarose gels (described below), which fractionate the 60 to 5 kb DNA size range (Maniatis et al., 1982).  Genomic DNA  isolated by this method was  found to occupy a size range > 50 kb. Before digestion with restriction endonucleases, this DNA  was  treated to remove contaminating RNA as follows: Pancreatic ribonuclease (Sigma, 95% type A / 5% type B), dissolved in TE at a concentration of 10 mg/ml, was added to a final concentration of 120 jug/ml. This was  incubated for 2-3 hours at 37 °C.  The DNA was then precipitated  56. by addition of 0.6 volumes of a 20% polyethylene glycol / 2.5 M NaCl solution and incubation on ice for no more than 15 minutes.  After  centrifugation and dessication, the genomic DNA was taken up in half the volume of TE i t had previously occupied, to allow for dilution during restriction enzyme digestions. ii.  Agarose Gel Electrophoresis Agarose submarine gels of the desired concentration (0.3% - 2.0%)  were poured, allowed to set, and run in 0.5 x TBE, with EtBr variously present at 0.5 pg/ml in either the gel, the buffer, or both. A table of DNA fractionation ranges is given  in Maniatis  et a l . , (1982).  Mini gels (10 cm long) were run at 100 V, generally for 1-2 hours. Large gels (25 cm long) were usually run at 50 V, 19 to 24 hours for a typical 0.9% gel.  DNA was visualized and photographed using  short wavelength (254 nm) ultraviolet light, except as noted. iii.  Restriction Endonuclease Digestion Genomic DNA treated for RNA removal as described was digested  with the restriction endonucleases Hindlll, EcoRI, or PstI (Pharmacia) as  follows:  The reactions typically contained  0.3 mg/ml genomic  DNA, 5 U enzyme/pg of genomic DNA, 100 pg/ml BSA, and 1 x Pharmacia H (EcoRI) or M (Hindlll and PstI) buffers.  This was incubated at  37°C for 3 to 5 hours, or, in the case of more delicate enzymes such as PstI, incubated  1 to 2 hours and given a repeat aliquot of the  enzyme before further digestion for 1 to 2 hours. was  extracted  with  1:1  phenol/chloroform  The digested DNA  and ethanol/NH^OAe pre-  cipitated. Plasmid DNA was digested less vigorously, with 3 U enzyme/ug  57. of DNA, 0.1 mg/ml DNA, and 2 hours incubation. Such vigorous digestion  conditions, with 2  to 3-fold excess  of enzyme over that required for plasmid digestion, and significantly longer digestion times, appeared necessary for genomic DNA digestion. This achieved fairly complete digestion, as assayed by 0.6 agarose gel  to  0.9%  electrophoresis, whereas less vigorous conditions only  gave partial digestion.  Thus, a compromise was made between relatively  complete digestion, and  the risk of damage to single-stranded ends  by any contaminating exonucleases. iv.  Genomic Southerns and F i l t e r Hybridizations Genomic DNA digested with restriction endonucleases was electro-  phoresed using large 0.7% per by  lane.  The  agarose gels as described, loading 20 ug  electrophoresed DNA  was  denatured and  standard procedures (Maniatis et a l . , 1982), and  Hybond®-N The  (Amersham) according filter  was  to  prehybridized  Denhardt's solution, and  0.2%  SDS,  neutralized  transferred to  the manufacturer's instructions. in a  solution of 2  at 60 ul/cm  6 x SSC,  5 x  of filter, for 3  to 18 hours, at 37 to 50°C, in a sealed bag. The prehybridization solution was then poured off and the hybridi2  zation solution added at 40 pl/cm of filter. of 6 x SSC, 50 mM  This solution consisted  NaH2P04 pH 7 (HC1), 20 ug/ml E. coli tRNA, 5 x  Denhardt's solution, 0.5% SDS, and 4 pmol oligonucleotide/ml hybridiza32 • •• tion solution, 5'-labelled with [Xzation was The  carried out  P]ATP as described below. Hybridi-  in a sealed bag  following day, the filter was  6 x SSC / 0.1% SDS.  overnight at 50 to 55 °C.  washed with five changes of  The first three washes were done at room tempera-  58. ture, and the last two were done in a water bath heated to the specified temperature, for at least 15 minutes each.  The washing solution  was preheated to the appropriate temperature for these latter washes. The washed filter was autoradiographed at -70°C, using an intensifying screen, for 3 to 10 days. v.  P u r i f i c a t i o n of DNA Fragments of Selected Size  Genomic DNA  digested with  the desired restriction endonuclease  was electrophoresed on large 0.9% - 1.0% agarose gels with EtBr present, covering to exclude fluorescent light. jag DNA  each were run, and the DNA was  (366 nm) 6~7  mm  DNA , was  Four or five lanes of 20 viewed with long wavelength  ultraviolet light, as briefly as possible. wide containing DNA  of the desired size was  electroeluted (Maniatis et a l . , 1982)  A gel slice excised.  The  in 0.5 x TBE using  Spectropor.l dialysis tubing, at 100 V for 30 minutes to 1 hour depending on the size of the DNA  fragments.  Following electroelution,  the current was reversed and a 30 second pulse of 100 V was applied. The  eluant was  removed under subdued light, extracted with butanol  until the volume was approximately 200 p i , extracted with 1:1 phenol/ chloroform, and twice precipitated with ethanol/NH^OAc. was  taken up in 20 pi TE, giving the DNA  The pellet  fragments an approximate  concentration of 0.1 pg/pl. vi.  Oligonucleotide Preparation and Radioactive Labelling  Two  synthetic oligonucleotides were used both to screen for,  and to sequence the cloned tRNA genes.  GT8 consists of nucleotides  Cpv  #1  to 23 of tRNA^k , and GT9  complements nucleotides #62  to 82 of  59. the same tRNA (see Figure 9). For purification, the crude oligonucleotide was electrophoresed for 3 hours at 1,600 V in a 20% acrylamide / 7 M  urea, 0.5  The  mm  thick polyacrylamide  desired band was  gel as described  in Part I.  excised and eluted by soaking overnight in a  minimal quantity of 0.5 M NH^OAc / 10 mM MgOAc. The eluant was filtered through siliconized glass wool and loaded onto a C^g Sep-Pak® column. The  column was  washed with  5 ml H20,  and the oligonucleotide was  then eluted with 20% HPLC grade acetonitrile.  The first 2 ml of  the acetonitrile wash was retained, dried 2% hours in a Savant Speed Vac® apparatus, taken up in H20  at a concentration of 0.02 jug/pl,  and stored at -70°C. For hybridization experiments, the oligonucleotides were labelled 32 at the 5'-end with [8"- P]ATP and polynucleotide kinase.  The reaction  contained  (HCl), 9 mM  40 pmol oligonucleotide, 40 mM  MgCl2, 10 mM DTT, New  tris pH 8.0  32  100 pCi [» - P]ATP (250 pCi / 25 pi / 87 pmol,  England Nuclear), and  1 p i polynucleotide kinase at 5-10  (Pharmacia), in a volume of 20 p i .  This was  U/pl  incubated 45 minutes  at 37°C, and heated to 65°C for 10 minutes to inactivate the enzyme. This labelling mix was  added directly to 10 ml hybridization fluid  and used without further purification. vii.  DNA Ligation,  pUC  13  Bacterial  Transformation,  (Viera and Messing, 1982)  was  and Colony Screening  used as the vector for  cloning genomic fragments containing tRNA genes. The vector, propagated in E. coli strain DH5°< (Leung, 1988), was centrifugation with  in CsCl/EtBr  the appropriate  as  purified by equilibrium  described below.  It was  digested  restriction endonuclease, and the 5'-phosphate  60. groups were removed by adding approximately 1 U/pg  calf intestinal  alkaline phosphatase (Boehringer Mannheim, 19 to 25 U/ul), and incubating at 37°C for an additional 30 minutes.  This limits recirculariza-  tion of the vector during ligation. Ligation efficiency was found to show extreme variation according to  the  relative concentrations of  enzyme.  vector, insert, and  especially  Levels found to be effective were as follows:  50-100 ng  vector DNA,  20-100 ng insert (genomic DNA  scribed), and  1.5  volume of 20 p i . experiments.  - 4 U T4 DNA  ligase (Pharmacia, 7.5 U/ul), in a  Optimum levels within these ranges varied between  Ligation was  6.6  mM  MgCl2, 10 mM  150  mM  NaCl.  The  fragments purified as de-  DTT,  carried out 0.4  mM  reaction was  ATP,  in 66 mM  tris pH 7.6  (HC1),  10% polyethylene glycol, and  incubated at 4°C overnight (12-20  hours). E. coli [strain DH5°< (Leung, 1988)] was used for transformation by the ligated DNA.  Bacterial cells were made competent and transformed  by the high efficiency method of Hanahan [Table 7 of Hanahan (1985)]. The cells could be frozen in 210 pi aliquots at ~70°C for a few months without appreciable loss of efficiency.  The entire 20 pi of ligation  mix was used to transform a 210 pi aliquot of competent cells. ligation and and  transformation efficiency was  such that at least half,  sometimes a l l of the transformed cell mix was  dish.  The  plated per Petri  This was done by briefly centrifuging the cell mix, resuspending  the  pellet in 100-200 pi SOB  one  or two  the  plating medium to select against non-transformed cells.  85 mm  plates.  (Hanahan, 1985), and plating this on  Ampicillin at 50 pg/ml was  included in IPTG  and X-Gal were added to the plates to provide an indication of the  61. number of transformants Typical  yields  were  containing  cloned  100-600 white  colonies  inserts  (Leung, 1988).  (generally containing  plasmids with inserts), and 100-300 blue colonies (containing uncut or recircularized plasmids) per plate. This corresponds to a transform4  ation efficiency of approximately 1 x 10 /ug DNA; cells transformed with uncut pUC 13 by this method gave an efficiency two to three orders of magnitude higher. Duplicate colony lifts  of each plate were made, using 82 mm  Hybond®-N discs (Maniatis et al., 1982).  The DNA was denatured with  1.5 M NaCl / 0.5 M NaOH for 5 minutes, neutralized with 1.5 M NaCl / 1.0 M tris pH 7.5 (HCl) for 2 x 5 minute intervals, and bonded to the filters by irradiation with 254 nm wavelength ultraviolet light for 4 minutes.  Prehybridization, hybridization, and washing were carried  out as described.  Ten ml of hybridization fluid was used for up  to 16 filters. Autoradiographs were exposed at ~70°C with an intensifying screen for 2 to 4 days. viii.  Plasmid Isolation and Purification for Sequencing Small-scale plasmid isolations were done by the alkaline lysis  procedure of Maniatis et al., (1982).  Plasmid DNA prepared by this  method, and treated with ribonuclease and polyethylene glycol as described  for genomic DNA, was sufficiently clean for restriction  digests and agarose gel electrophoresis. However, for sequencing,  further  purification  centrifugation through CsCl gave the best results.  by equilibrium Plasmid DNA was  obtained, from 250 ml of 2 x YT medium containing 50 ug/ml ampicillin, by a scaled-up version of the above isolation procedure.  After KOAc  62. precipitation, 0.7 volume of isopropanol was added to the supernatant to precipitate the nucleic acids.  The pellet was taken up in 10  mM tris pH 7.5 (HC1), 10 mM EDTA, 0.84 g/ml CsCl, and 0.6 mg/ml EtBr, and centrifuged in a Beckman vTi65 rotor at either 65,000 rpm for 4 hours or 55,000 rpm for 17 hours. This was repeated, and the purified plasmid DNA was then extracted four times with butanol, and ethanol/ NH^OAc precipitated three times.  The final pellet was taken up in  0.5 ml TE to give 3~4 mg/ml of highly purified DNA. ix.  DNA Sequencing  Double-stranded  plasmid DNA, which had been purified by CsCl  centrifugation as described, was used as the template for sequencing. For each sequencing reaction, approximately 20 pg DNA was denatured in 20 pi of 0.15 M NaOH at room temperature for 10 minutes, and ethanol/ NH^OAc precipitated. Sequencing was carried out by the dideoxynucleotide termination method (Sanger et al., 1977), using two different procedures.  The  majority of sequencing was performed using the Sequenase kit (U.S. Biochemicals), by the protocol supplied with the kit.  This was found  to give somewhat clearer data than sequencing done with the Klenow fragment of DNA polymerase; the latter was used, however, where i t was  desirable to read sequences very close to the primer, and to  support data generated by the Sequenase method. The  GT8 and GT9 oligonucleotides  purified  as described were  used as primers to sequence a l l cloned tRNA genes in both directions wherever possible, with both sequencing methods. For  sequencing using the Klenow fragment (Newton, 1984), 16 ng  63. oligonucleotide was annealed to the denatured DNA  in 50 mM tris pH 7.5  (HCl) / 10 mM NaCl / 10 mM MgCl2., in a volume of 8 p i , by heating to 75°C for 5 minutes and cooling gradually to 37°C. To this annealed 35  DNA was added 2 pi 15 pM dATP, 3 pi [o<- S] dATP (New England Nuclear, 250 pCi / 20 pi / 190 pmol), and 5 p i of a premix consisting of 1 mg/ml BSA,  100 mM  I^HPO^ pH 7.5,  DTT,  and  the Klenow fragment of  DNA polymerase (Pharmacia, 5.0 - 7.5 U/pl)  at  0.5 - 1.0 U/pl.  this mix was  10 mM  3 pi of  dispensed per G, A, T, and C reaction, and extension  was started by adding 1 pi of the appropriate ddNTP/dNTP mix (Appendix). This was incubated at 37°C for 10 minutes; 1 pi of a solution containing 0.5 mM in each of dGTP, dATP, dTTP, and dCTP was added to each reaction, and a further incubation of 37°C for 10 minutes followed.  Finally,  6 pi of a solution of 10 mM EDTA, 0.1% xylene cyanol f f , and bromophenol blue in 98% reactions.  deionized formamide was  0.1%  added to stop the  The samples were either electrophoresed immediately after  heating to 75°C for 3 minutes, or frozen at -70°C for up to two weeks, heated as above, and electrophoresed. Electrophoresis  was  performed using  6%  polyacrylamide  / 7  M  urea wedge gels, mixed as described in Part I, with minor modifications: 0.08%  TEMED was used, and the gels were run at 1,600  1 hour, until the bromophenol blue dye was plates.  The  wedge was  mm  thick spacers, 11 cm and  2-3 pi of sample was  1 cm from the end of the  constructed by using 0.4 mm  along the entire length of the gel, and  V for just over  thick spacers  then adding two  extra  0.3  5 cm long, at the bottom of the gel.  loaded in each 5 mm wide slot.  Sequencing gels  were fixed in 10% acetic acid / 12% methanol for 45 minutes to remove urea, and  dried under vacuum for 1 hour at 80°C.  Autoradiography  64. took place with the film in direct contact with the dried gel, at room temperature, for 1 to 3 days.  65.  III.  i.  RESULTS AND DISCUSSION  tRNA^ Gene Localization r  Ser tRNA^  was  previously highly purified  and sequenced (Cribbs, 1982; Cribbs et al.,  (Hayashi et al.,  1982)  1987a) in our laboratory.  The sequence of this molecule is shown in Figure 9.  From the known  sequence of this tRNA, two oligonucleotide probes were synthesized: GT8, a 23-mer identical to the 5'-end, with the sequence p-GACGAGGTGGCCG AGTGGTTAAG, and GT9, a 21^ner complementary to the 3'-end, with the sequence p-CGACGAGGATGGGATTCGAAC. These probes were used to identify the genomic restriction fragSer ments likely to contain copies of the tRNA2^  gene.  Genomic DNA  from Drosophila melanogaster (Ore R) was digested to completion with the  restriction endonucleases Hindlll, EcoRI, and PstI, and probed  with the above oligonucleotides (Figure 10). the  When GT9 was used as  probe (Figure 10a), three major bands were seen in each lane;  these were of dark, medium, and light intensity. For the hybridization using GT8 as a probe (Figure 10b), these same three bands were seen, and an extra dark band also appeared in each lane. Ser contain either a fragment of tRNA2|-)  This band must  from the 5'-end which lacks  any homology to GT9, or a sequence very closely related by chance to GT8. In situ hybridization studies (Hayashi et al., 1982) show purified Ser • • tRNA-^ binding to three sites m the polytene chromosomes: It binds  66.  Figure 10.  Genomic Southern analysis of tRNA^ genes.  Drosophila genomic DNA was digested with Hindlll (lane H), EcoRI (lane E), and PstI (lane P) and electrophoresed using a large 0.7% agarose submarine gel at 50 V for 16 hours.  The DNA fragments were Ser transferred to Hybond-N and probed with the tRNA^ -derived oligonucleotides GT9 (part a) and GT8 (part b), which were 5'-labelled as described. Washing was done in 6 x SSC / 0.1% SDS, at 63°C for GT8 and at 60°C for GT9. Autoradiograms were exposed at ~70°C with an intensifying screen for 10-11 days. Size markers are derived from a Hindlll restriction digest of lambda DNA, and are indicated at the left of the figure in kilobasepairs. Positions corresponding to regions which were targeted for cloning are indicated by size (kilobasepairs) in the middle of the figure. Arrowheads mark the three bands which hybridize to GT8 but not to GT9.  H  kb  E  P  H  68. with similar intensity at sites 86A and 88A, and with slightly increased intensity at site 94A, a l l on the right arm of chromosome 3.  These  results suggest the presence of at least three or four (or multiples Ser thereof) copies of the tRlN^^ gene in the genome.  ii.  Cloning of trafefb** tRNAucA> and tRNA^JQ  The  data  obtained  from genomic Southerns was  used to locate  the size ranges of genomic digests to target and purify when attempting Ser • . . . to clone the tRNA^  gene.  When DMA  is purified in this way before  attempted cloning, the chances of cloning the desired gene are greatly increased, as most extraneous genomic fragments are eliminated by this procedure. to  Based on the ratio of the size of gel slice taken  the length of the entire track of electrophoresed DNA, by a very  crude estimate the gene in question would be purified approximately 25-fold using this procedure.  Thus, theoretically, to clone a specific 8  4 kb fragment from the 1.4 x 10 an estimated  6,500 colonies would have to be screened  a 99% probability of success by  this  kb Drosophila genome by this method, to achieve  (Kaiser and Murray, 1985).  However,  calculation, even screening 3,000 colonies would give an  estimated 88% probability of success. Initially, the gel slice containing EcoRI fragments of 1.9 to 2.1 kb, and that containing 3.6 kb fragments were cut out, and the associated DNA was electroeluted and cloned (see Figure 10). of  A total  2,700 white colonies containing the 1.9 - 2.1 kb fragments were  screened, but no tRNA genes were found.  This is not surprising in  retrospect, as the choice to clone this region was based on early  69.  genomic Southerns probed only with GT8.  later found that Ser the strong band at 1.9 kb does not, in fact, contain an intact tRNA^b Ser  gene, as discussed earlier. in the faint 2.1 but  since only  It was  One might have expected to find tRIN^^  kb region which hybridizes to both GT8 and  GT9,  2,700 colonies were available for screening, such  a tRNA sequence could have been missed by chance. Very interesting results were obtained from the cloning of the 3.6 kb EcoRI region, however. A total of 4,900 white colonies derived from this region were screened using GT8, and two putative tRNA genes were cloned:  a tRNAQjQ, and a  tRNAycA-  Neither of these two tRNA  genes has close homology to tRNAgb • tRNAQjQ was Ser  sequenced fully,  and has only sporadic homology to tRNA2b , other than an 18 nucleotide exact match through the TVC  arm  of tRNA^), Figure 11a and c]. Ser  [#56  to 73 of tRNA^ (#57 to 74  At the 5'-end of tRNA^ only 16/23  bp match GT8 (the tRNA^b probe that picked i t out during screening), but a block of 12 homologous nucleotides in this region must have been sufficient to bind GT8 under the relatively less stringent conditions used (discussed below). Ser Only the 3'-portion of subcloning, as GT9  tRNAycA  could be sequenced without further  did not bind to this gene well enough to permit  sequencing in the 5'-direction (only 14 to 16/21  bp homology with  GT9, with no blocks of homology to this probe longer than 8 to 10 Ser bp, Figure lib). Of the region of tRNA^Q^ that was sequenced, a Ser 66 to 71% homology to tRNA2b was found. These results suggest that Ser the 5'-end of tRNAy^ is probably only partially homologous to GT8, as was the case for tRNA^g. However, sufficient homology must exist in the region for GT8 to bind the gene during screening and sequencing.  70.  F i g u r e 11.  Comparison of tRNA gene sequences.  Ser The sequences of the nontemplate strands for a) tRNA2b , b) SGTT XJGU. tRNAy^, and c) tRNA^UG are given, 5'-3' left to right. Structural genes or portions thereof are shown in bold print. underlined.  Anticodons are  R = purine; Y = pyrimidine; N = undetermined nucleoside.  a) b) c)  Ser-2b Ser-UCA Leu-CUG  a) b) c)  Ser-2b Ser-UCA Leu-CUG  a)  Ser-2b  -50 -40 -30 -20 -10 A A G A A A G T G G T A G T T A T G GAGTGTANGA A A T N G T N A T C G A T T T N G T G G A A A A G A A A G T  10 20 30 40 GACGAGGTGQ CCGAGAGGTT AAGGCGTTGG A C T G C T A A T C ATGG A C T T G A A A T C T A A C A GTCAGGATGG CCGAGTGGTC TAAGGCGCTG C G T T C A G G T C  50  60  70  80  90  100  110  120  130  CAATGTGCTC TGCACGCGTG GGTTCGAATC CCATCCTCGT CGAGTGGAAT TTTTTTNTTT TTTCCATTNG AATATTAACN CAAAAAAGCC CATTGGGTTC TACCCGCRCA GGTTCRARTC CTGTCCGCAG CG GCAGTCTACT CTGTAGGCGT GGGTTCGAAT CCCACTTCTG ACAATRNYTT TTTNTCCNAT TT  150 160 170 180 190 TACTTTATGG AATATTTTAA CTTGAATTTA ACTTTCATTT TATACAATAT ATCTA  140  ATGTGTGAAT  72. The  fact Ser  that these two • •  genes were unexpectedly found using . . .  the tRNA^ -specific probe GT8  is interesting, considering the lack  of homology between the three genes. enough stringency (58°C, 6 x SSC) these divergent though the  sequences.  theoretically  Screening was  to allow hybridization of GT8 to  This washing temperature was calculated temperature was  oligonucleotide (Meinkoth and Wahl, 1984) iliarity with  done at a low  used even  69°C for this  because of initial unfam-  the probe and its behaviour at various stringencies,  and because i t was  desirable to obtain and test about 20 positives  per screening. Ser The failure to find tRNA2b among these 4,900 colonies derived from 3.6 kb EcoRI fragments is probably explained by the randomness of the search.  The gene does appear to be contained within this  region, according to the strong hybridization there of both GT8 GT9  (Figure 10).  and  Using the earlier calculation, the screening of  4,900 colonies would give an estimated 97% chance of obtaining the gene (Kaiser and Murray, 1985). However, the best presumption appears Ser to be that the tRNA^ gene was missed by chance in these screenings. Possible, but  less likely, is that the gene contained in this  3.6  kb EcoRI region is not clonable. The 5.1 kb region of a Hindlll genomic DNA digest was targeted Ser for  tRNA^  cloning, after repeated failure to obtain this gene from  the 3.6 kb EcoRI region.  This region was chosen because, in addition  to the strong band at 5.1 kb, there is also a band of lesser intensity at 5.6 kb which hybridizes to both GT8 gel  slice containing these two bands was  eluted and  cloned.  and GT9  (Figure 10).  cut out, and the DNA  The was  A total of 3,700 colonies were screened, and  73. Ser one was found to contain a putative tRNA2b gene. In summary, three fragments  containing at least one copy of  a tRNA gene each were cloned into pUC 13: containing a  5.1  iii.  tRNArjuQ,  kb Hindlll  Sequencing  and  A 3.6 kb EcoRI fragment  a 3.6 kb EcoRI fragment containing fragment containing  i n situ  tRNA;?b  Hybridization of  tRNAycA?  (codons AGC,  tRNAf^s  a n  AGU). a n  tRNAycAj  &  d  tRNAcuG Clones  Preliminary  sequence data  (Figure  11) was  obtained from the  three pUC 13-based recombinant plasmids isolated as described. Double stranded DNA  sequencing was used, and the oligonucleotides GT8 and  GT9 were used as primers to sequence in opposite directions where possible. As mentioned earlier, this was not possible with the putative .Ser Ser UCA gene, due to insufficient homology with GT9; thus, only the tRNA™. r 3'-portion of this gene was sequenced, using GT8 as a primer. tRNA2§ and  tRNAQjQ were able to be fully  sequenced using both primers.  Thus, data was only obtained from one strand at the two ends of the genes where these primers hybridize, and in the flanking regions. Ser The anticodon region was sequenced on both strands for both tRNA^b and  tRNAcuG-  Except for positions -58 to -2 and 98 to 195 of the  Ser tRNA2b  flanking regions, the data for a l l areas which were not se-  quenced on both strands was confirmed by at least two to four repeated experiments.  These data appear quite reliable, according to their  excellent correspondence to other known sequences, where such sequences exist (i.e., tRNA2b  an  d tRNAQJGj  a s  discussed below), and the exact  agreement of a l l data generated from complementary strands.  74. None of the three cloned genes has encoded in the DNA  sequence.  the tRNA CCA 3'-terminus  This is universally found to be the  case for eukaryotic genes, the three terminal nucleotides being added post-transcriptionally by nucleotidyl transferase.  A l l three genes  do show good agreement with the 5'- and 3'-internal promoters described in the literature (Galli et al., 1981), there being differences at only two  positions at most.  The  putative internal promoters run  from nucleotides 8 to 18, and 61 to 72 (62 to 73 for tRNAQJQ), Figure 11a to c. GT8  and  It is not surprising that these include the areas where  GT9  showed maximum cross-hybridization between the  three  genes. r  a) tRNAfb  Ser The sequence of the cloned tRNA2b  gene is shown in Figure 11a.  This gene had the actual tRNA sequence data available for comparison (Cribbs, 1982;  Cribbs et a l . , 1987a; Figure 9).  The gene cloned  here agreed with the tRNA data at every position except #16. nucleotide, which appeared as a most unambiguous "dA"  in the  This DNA  sequence data of this clone, appeared quite clearly as dihydrouridine Ser in the tRNA2b sequence data (Cribbs, 1982), which would give a "dT" in the DNA at this position. If both sets of data are, in fact, Ser correct, i t appears that the tRNA2b gene cloned here represents an allogene with a T to A transversion at position 16. Ser of the tRNA^  Subcloning  gene contained in the 5.1 kb Hmdlll fragment, and  sequencing of both strands would be necessary to confirm absolutely the existence of a sequence discrepancy at this nucleotide.  75. The occurrence of tRNA allogenes is well documented (see Introduction).  Ser For example, in previous studies of the tRNA^^ gene cluster  at 12DE on the X chromosome, as discussed earlier, a gene was found Ser which was identical in sequence to tRNA4  except for a C to T transi-  tion at position 50 (Cribbs et al., 1987b). Ser • • tRNA2k  gene m  Both this gene and the  question have tracts of oligo(dT) residues a short  distance downstream . from the 3'-end of the gene.  These are thought  to function as signals for termination of transcription (Adeniyi-Jones et a l . , 1984), and their retention may  suggest that such genes are  actively transcribed, the single mutations being neutral to natural selection. The fact that allogenes do show in vitro transcriptional ability where these assays have been done, raises questions as to why  the  corresponding tRNA sequences show no variation at the microheterogeneous nucleotides.  Perhaps the variant genes form minor chromatographic  species which are separable from the major tRNA peaks studied; otherwise, i t is possible that sequence microheterogeneity the post-transcriptional modification of in such a way  could affect  the variant tRNA species  as to cause their rapid degradation, as suggested by  Sharp et al . (1981). The flanking sequences of this clone do not appear to show homology to any  consensus sequence other  than the 3'~oligo(dT) discussed,  as is the general case for eukaryotic tRNA genes. In previous studies, however, regions around tRNA genes have been found to be quite AT-rich (Addison et a l . , 1982; DeLotto and Schedl, 1984; Glew et al.,  1986;  Suter and Kubli, 1988), and this is clearly seen here in the 64% AT  content of the 5'-flanking sequence and the even higher 77% AT  76. content of the 3'-flanking sequence. Ser In situ hybridization results (Figure 12) show that the tRNA2b clone described appears to derive from site 88A on the right arm of chromosome 3, one of the three chromosomal sites to which the Ser . . . tRNA2b species also binds (Hayashi et a l . , 1982; Dr. S. Hayashi, Ser Ser pers. comm.). As both functional tRNA^b and this apparent tRNA2b allogene appear to derive from 88A, i t is possible that this chromosomal Ser site may  code for two  or more tRNA2b -related genes, or this  represent  cross-hybridization.  may  This site does not appear enriched  for tRNA sequences, having less than four according  to (Elder et  a l . , 1980), but further molecular cloning and sequencing of the region Ser would contribute to a comprehensive study of the tRNA2b  group of  isoacceptors. b).  t R N A g  Ser The  partial sequence of a putative tRNArj^ gene is given in  Figure l i b . standard  The sequenced portion is seen to fold readily into a  tRNA cloverleaf structure (Figure 13b).  This tRNA would  be expected to recognize the codon UCG, in addition to UCA. Interestingly 16/21  enough, there  are at least 14/21  matches between this gene and GT9  (and possibly  (positions 62 to 71, 74 to  76, 78, 81, and 82; Figure lib) similar to the situation for the cloned tRNAQJQ gene. It could not, however, be clearly sequenced . . . . . Leu using this oligonucleotide as a primer, as could tRNAQjQ. Perhaps this is because the block of homology at the 3' -end of GT9 is only 8 to 10 nucleotides in length here, with the other 6 matches more dispersed.  77.  Figure 12.  In situ hybridization of the tRlN^^ gene.  . . Ser The pUC 13 recombinant plasmid containing the tRNA2b gene withm a 5.1 kb Hindlll restriction fragment was labelled by nick translation with biotinylated dUTP (Bio-ll-dUTP, Bethesda Research Laboratories), and hybridized to Drosophila third instar larvae salivary gland polytene chromosomes in 50% formamide / 6 x SSC at 37°C for 24 hours. Site of hybridization was determined using the BluGene nonradioactive detection system from BRL. Hybridization is seen at site- 88A on the right arm of chromosome 3. Figure courtesy of Dr. S. Hayashi.  78.  A i-G-C T -A C - G -so A-T G-C _ GA  J  G  /  10  "  r  A  CCG^  /  G-80  70  c 1  A C G C  1  T  1  A  A  U _ 60  T A  T ? 3  T  ' / ,  C T  A  G  T  C '  C  G Q  1  GTGGG  I I I  J  W  G C  t  RR  T  c  so-G-C  C-G G-C T T T G CAG  50  30-  A-T G T-A G-C G ~ C - 40  60  G  G  A AorG  T  T  /  C  1  T  T  T  C  C A T A TQA  (a) c-tRNA J . e  c  T G T C C ACAGG C:°°<G " G,  1  A or  70  G  (b) Sequenced portion of c - t R N A ^ r  (numbered as tR N A ^ , F ig. 1 1) Figure 13. Cloverleaf structures of cloned tRNA genes as predicted from the DNA sequence data.  )°  80. The 5 -end of the gene proved to have enough similarity to tRNA2D to allow GT8 to be used for its cloning and sequencing, but the amount of homology was not discovered, as this portion of the gene could not be  sequenced from this recombinant plasmid.  Again, subcloning  and sequencing in both directions would be desirable here. There is 66 to 71% homology between the known part of the sequence Ser • • Ser of tRNAy^ and the corresponding portion of tRNA^ (Figure 11a and . . Ser b), which is similar to the 71 to 73% level of homology between tRNA2b Ser and  tRNA^ j  (Cribbs et a l . , 1987a), and  in agreement with the 10  to 30% sequence divergence generally found between functionally distinct tRNA isoacceptors (Sprinzl et a l . , 1987).  Further, there is also Ser Ser  59 to 66% homology between the known part of  an  tRNAycA  d tRNA^y  (Cribbs et al., 1987b). By contrast, the only notable homology between •  the  sequenced portion of  SGI"  XJGIJ. an  tRNAucA.  d  the tRNAQjQ clone described  is a 9 to 11 nucleotide match in the region of the T¥C loop from positions 62 to 72 of tRNA^UG (61 to 71 of tRNAycA) > Figure lib and Ser c. The heightened homology between this putative t R N A u c A gene and Ser • . Ser tRNA^ ^ -j suggests that i t may be an active member of the tRNA gene family. The sequence data extending beyond the 3'-terminus of the tRNAuc^ structural gene appears quite degenerate (data not shown). finding suggests  that two  or more copies of the  Ser tRNAucA  This  gene may  be present in this 3.6 kb EcoRI fragment, with these genes possessing divergent flanking sequences. There is further evidence that two or more slightly different versions of the gene may be present: At three positions within the Ser tRNAy^ gene sequence, i t is unclear whether the nucleotide is a.  81. G or an A (Figure lib).  It appears more likely that these anomalies  represent G to A transitions than sequencing artifacts, given the absence of such artifacts in other sequencing done here, their clear Ser presence in each experiment carried out on tRNAyrj^, and the suggestion of the presence of more than one gene by the overlapping flanking sequence data above. Thus, this clone may contain yet another example of tRNA gene microheterogeneity. It should be noted that a dT-rich area extends from a position approximately 8 to 30 nucleotides past the 3'-terminus of the putative Ser tRNAyjcA  two  ene  s  g ( ) (data not shown). Ser  The sequence data from at least . . .  en  tRNAucA  g es appears to overlap in this region as described  above, however, and i t is therefore unclear what oligo(dT) termination signal each individual gene possesses. In a study of the four major serine accepting tRNA peaks (#2, 4, 5, and 7), separated by BD-cellulose chromatography, i t was found that none of these major peaks bound significantly to the triplet Ser UCA, except for a very low level of binding observed with tRNAy (White et a l . , 1975). It appears, therefore, that this serine accepting Ser tRNA may  form one  of the more minor tRNA  peaks, being present  in the cell at lower levels than the major serine isoaccepting species. The  pUC  13  recombinant plasmid  containing this 3.6  kb EcoRI  fragment was also analyzed by in situ hybridization to D. melanogaster polytene chromosomes (Figure 14).  It was found that this clone hybri-  dized at 58AB on the right arm of the second chromosome.  This is  not a major site of tRNA genes, according to in situ hybridization experiments, being thought to have less than four putative tRNA genes altogether (Elder et a l . , 1980).  However, the fact that tRNA does  82.  Figure 14.  In situ hybridization of the tRNA^^ gene.  The pUC 13 recombinant plasmid containing at least one putative Ser • • • tRNAy^ gene withm a 3.6 kb EcoRI restriction fragment was labelled, hybridized to polytene chromosomes, and detected as in Figure 12. Hybridization is seen at site 58AB at the extremity of the right arm of chromosome 2. Figure courtesy of Dr. S. Hayashi.  84. bind at this site suggests that the clone obtained could correspond to a tRNA product which is uncharacterized at present. This chromosomal site also shows significant hybridization  to tRNA^^ (Hayashi et a l . ,  1982). Overall, the significant levels of homology between the sequenced Ser ene portion of the tRNAjjcA g cloned here and the known members of Ser • Ser the tRNA gene family, the apparent presence of more than one tRNAycA sequence in this plasmid, and the possibility of microheterogeneity between these genes, a l l suggest that further study of this clone, and of the chromosomal region 58AB to which both i t and tRNAgQ^ hybridize, could provide an interesting addition to the body of knowlege Ser already accumulated on the evolution of the tRNA  gene family in  Drosophila, and on tRNA gene organization within the genome of this organism. c) tRNAJfg The sequence data for the tRNA^g gene contained in a cloned 3.6 kb EcoRI fragment is given in Figure 11c. The cloverleaf configuration of this sequence is shown in Figure 13a, suggesting that i t is able to fold into a viable tRNA structure. As noted earlier, i t is interesting that this gene was found • • Ser when .screening with  GT8, an oligonucleotide specific for tRNA2^ •  There is a 12 bp block of homology from positions 8 to 19 (Figure 11a and c, bearing in mind that GT8 has a "dT" at position 16), and four matches to the sides of this block at positions 1, 3, 6, and 22, giving a total of 16/23 hybridizing nucleotides.  The relatively  85. low washing stringency used (58°C, 6 x SSC), combined with the block of 12 homologous nucleotides, apparently created conditions sufficient to clone and sequence this gene using GT8. It also proved to be possible to sequence this gene in the reverse direction using GT9, due to a fortuitous block of homology 12 nucleotides long between tRNA^uQ and the 3'-end of GT9 74 of tRNAQjQ, Figure 11c).  Two  (positions 63 to  other nucleotides at positions 80  and 82 also show homology in this area, giving a total of 14/21 matches between GT9 16/21  and tRNAQjQ.  As mentioned earlier, tRNAjj^ had 14 to  nucleotide homology to GT9, but could not be sequenced with  that oligonucleotide.  The  was 8 to 10 nucleotides.  longest block of homology in that case  It thus appears that the 2 to 4 extra nucleo-  tides in the block of homology between tRNArjijQ and GT9 contribute greatly to its ability to form a viable hybrid with that oligonucleotide under the conditions used. It was impossible to obtain sequence data well into the flanking regions with this clone.  This may indicate the presence of two or  more identical copies of the tRNAQjQ gene in this recombinant plasmid, Ser with somewhat divergent flanking sequences as' discussed for the clone.  tRNAycA  When the plasmid was digested with Alul, at least three bands  of approximate sizes 340, 390, and 500 bp hybridized to GT8  (data  not shown). Attempts to subclone these fragments proved unsuccessful, but their existence does suggest the presence of more than one tRNAQjQ gene in this plasmid. In situ hybridization showed the clone to derive from site 66B on the left arm of chromosome 3 (Figure 15); this is also the major 11  site of hybridization for tRNA^ , suggesting the possible correspond-  86.  Figure 15. In situ hybridization of the tRNA^g gene. The  pUC  13 recombinant plasmid containing at least one copy  of the tRNA^g gene withm a 3.6 kb EcoRI restriction fragment was labelled, hybridized to polytene Figure 12.  chromosomes, and detected as in  Hybridization is seen at site 66B on the left arm of  chromosome 3. Figure courtesy of Dr. S. Hayashi.  (  88. ence of this clone to that tRNA (Hayashi et al., 1982; Dr. S. Hayashi, pers. comm.).  As  the tRNA2  isoacceptor has not been sequenced  to date, this cannot yet be verified.  However,: further support for  this isoacceptor assignment comes from the fact that minor hybridization was  occasionally observed at sites 44E (2R) and 79F (3L), the other  two significant sites of tRNA2  hybridization (Hayashi et al., 1982;  Dr. S. Hayashi, pers. comm.).  This hybridization is not visible  in Figure 15. A  Leu.  tRNAcuQ  • • • gene from D. melanogaster identical to this one has  previously been cloned (Glew et al., 1986).  However, comparing the  available flanking sequence data of the present clone (Figure 11c) with that of the previous one shows that the two genes lack homology in both the 5'- and 3'-flanking regions, with the exception of the oligo(dT) terminator.  Lack of flanking sequence homology between  otherwise identical Drosophila tRNA genes has been a common finding (Robinson and a l . , 1984).  Davidson, 1981;  DeLotto and  Schedl, 1984;  Leung et  It thus appears that at least two identical but distinct  copies of the Drosophila tRNAQjG gene have now  been cloned.  These  two tRNA^y^ genes both derive from the 66B region (Figure 15; Glew et a l . , 1986).  This region was  seen to be one of the major sites  of total tRNA hybridization to Drosophila polytene chromosomes, being thought to contain four or more tRNA genes (Elder et al., 1980). The  tRNAQJg genes described show an extremely high degree of  homology (93 to 95%) to tRNA^g genes or their tRNA transcripts cloned from a variety of organisms, including those from Xenopus laevis, mouse, rat, and a l . , 1987).  cow mammary gland  (Glew et a l . , 1986;  Sprinzl et LyS  This is similar to the situation found for tRNA  iso-  89. acceptors, where the tRNA^g genes from two widely divergent species, Drosophila and rabbit, have been found to be  identical (Silverman  et a l . , 1979; Raba et a l . , 1979), and the tRNA^ genes from these same species differ by only 5% (Cribbs, 1982).  A fairly high degree  of sequence conservation, 82%, also exists between these intron-less Drosophila tRNA^g genes and the mtron-contammg Drosophila tRNA^g genes described previously (Robinson and Davidson, 1981), with a l l but  one  of  the nucleotide changes occurring within the  anticodon  stem and loop, and in the extra arm [sequences compared by Glew et a l . , (1986)]. •  •  •  •  I_i6U.  The finding here of a second distinct tRNA^g gene copy deriving from the chromosomal region 66B, along with the high degree of sequence Leu • conservation between this gene and various other tRNA isoacceptors, and the major tRNA hybridization to this site, suggests that the . . . . Leu chromosomal region 66B may contain an interesting cluster of tRNA genes, and possibly other tRNA genes as well.  90.  REFERENCES  Addison, W.R. (1982) Ph.D. Thesis, University of British Columbia. 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(1982) J. Gerontol. 37, 130-141. Zubay, G. and Takanami, M. (1964) Biochem. Biophys. Res. Comm. 15, 207-213.  95.  APPENDIX  Dideoxy/deoxynucleotide Mixes  G mix  A mix  T mix  C mix  ddGTP  158  ddATP  -  114  ddTTP  -  -  549  ddCTP  -  -  -  546  dGTP  15.4  109  158  158  dATP  -  dTTP  158  109  15.4  158  dCTP  158  109  158  14.3  (a) concentrations given  in pM..  Originally obtained courtesy of  Dr. Joan McPherson (see Newton, 1984) with some modifications.  

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