. . . o . _. .o n . ‘ . , . o.< ,. a K“~ r“ C u- o 0‘ ‘. .‘fii'r‘i. {4 .it. )3 ."‘\ -;,}- 3:2“;3'7. Mum-m- 1.: :4 6 \ Q‘- - . ’0 0 r" .v- \ ---~-". v- u» o c~~QQ.-. .-— . . . v -. . ’ ’ a a D" l - o . I I . _ . I .- I ' . . . . A ‘ l’ .. - .I 5. . _. n ' I . . . . . . - I v. . STUDIES ON MESSENGER RM Thesis for the Degree of M. .8.- MICHiGAN STATE UNIVERSITY ' o . o a .. a ,-,. I: --or.- .--— -.v -..\‘...- ..b-¢oq'u-1“‘ I““ IN Artemia saiina CYSTS DIANA K. iCE FQLNER- 1973 v v a h a o O n a . ' .' I. — n . e a ' l . . - . . . . o. . . . ‘ .. . - o I o . - . ' _ I, u a . . . . ~ - 'r . i- . . ' . - ' -3. r .' ' no u . . . _. .. . ' _ ( . / ' a . o .‘.4 .- . . . (I . a a_ - _ ,. . ‘ . - , I , ' ' ' r. . ’ o I ,II‘ A - - I . ' I .’o l _ r. I -1 U ‘l- ._ . ' ' ’e . . . _ ' .. . .. , . t . ' I 0 .a .- .- D . . . a .. I I -u' a »r " 7" IV‘ .‘_ -— 35.". I ‘ma, =’ non & 3313' soon amnznv .. mm BINDERS Iii-uni? 'IIOIIQA .- n I g“. , ‘Iw-wuvm- I... ABSTRACT STUDIES ON MESSENGER RNA IN Artenia saline CYSTS By Diane K. Filner A poly dT-cellulose chrolatography system was developed to detect and isolate poly A-containing RNA ( presumed to he messenger RNA) fro. total RNA preparations. The poly dT-cellulose wes prepared and its retention prOperties characterized using total RNA extracted tron Novikott ascites cells. Approxilately 1.5x of Novikoff cell RNA interacted as poly A RNA. or this material around 7: wes Tl RNase- resistant ( poly A ) es assayed by sephadex 6-50 or poly dT-cellulose chromatography. Preliminary results for Arteeia saline indicated that 0.6% of the total RNA extracted tron developing cysts contained poly A sequences, and that the percent in dor-ant cysts is not zero. Atteopts were also nade to isolate nRNA in polysone preparations. aade tron incubated Arte-is cysts. Up to 50% of the 260nn absorbing naterial in a nooinal ribosone-polysoee pellet wes RNase-sensitive and sedilented as polysole area material ( BAH ) in a sucrose gradient. however spectral examination and EDTA sensitivity of this material revealed a high degree of contalinatlon. STUDIES ON MESSENGER RNA IN Arte-la saline CYSTS By Diane K: Ice Filner A THESIS Sublitted to Michigan State University in partial fulfillment of the require-ents for the degree of MASTER OF SCIENCE Depart-ent of Biochemistry 1973 TO 1.28 WHO ENCOURAGED, PHILIP WHO ENDURED,AND T0 DANIEL BAER AND MVID “(HARD WHO WILL APPRECIATE ii ACKNOWLEDGMENT The author wishes to thank her sajor advisor, Fritz M.Rottnan, for patience and support during the course of this study; and R.A.Ronsio and L.L.Bieber for serving on the guidance cos-ittee. She gratefully acknowledges assistance of various natures from Joseph Abbatte, Brian Dunlap, Galvin H.5wift. and Karen Priderici. The author is especially grateful to Lee M. Pike both for fruitful discussions and for the interest, tile and effort he spent adapting his G.L.C. technique for the purposes of this study. Last but not least the author wishes to thank her parents, Jane and Hoeerd Ice. for investing faith, hope and war bonds: and her husband, Philip. for babysitting. typing the final draft and for financial support e ill TABLE OF CONTENTS DEDICATIONe O O O O O O O O O O O O O O O O O O O O O O O O O ACKNOWLEDGMENT . . ABBREVIATIONS LIST or “all” C O O O O O O O O O O O O O O O O 0 O O O O O 0 LIST OF FIGURfi O O O O O O O O O O O O O O O O O O O O O O O O I mODUCT IO" 0 O O O O O O O O O O O O O O 0 O O O O O O O O O 0 MATERIALS AND METHODS RESULTS Part I 3 Part II: Part I 2 Part II: Polysone Isolation . . . . . . . . . . . . . PretrCCmHt of cy3t8 e e e e e e e e e e e e Inwmtion Of CYSCB. e e e e e e e e e e e e PolyBOIa Pt.”f‘t3°n e e e e e e e e e e e e Discontinuous Gradient Centrifugation . . . Polysoee Assay by Continuous Sucrose Gradient Centrifugation . . . . . . . . CCICUICtIOI‘ Of YICId e e e e e e e e e e e e IDTA SOn‘ICIVEEY 0: PAN a e e e e e e e e e Poly dT-Cellulose Chromatography . . . . RNA Preparation fro. Novikoff Cells . . . . RNA Preparation fro. Arteeie saline . . Preparation of Poly dT-Callulose . . . . Fraction Collection During Poly dT-Cellulose Chronatography eoeeeeeeeeee T RNese Digestion of Fraction fro- Poly dT-Cellulose Chroletography . . . Preparation of RNA Sasples for G.L.C. Analysis, En'yutic DISOOCIOR e e e e e e e e e e Preparation of Seaples for G.L.C. Analysis, Alhltne “Ydr01y.i‘ e e e e e e e e e e 8". ”tic An.ly.i‘ by GeLeCe e e e e e e e 6.1 BIOCtrophor..i. e e e e e e e e e e Isolation of Polysones Effects of Freezing Yields........o EDTA Sensitivity of RAM Spectral PrOperties . . . Poly dT-Cellulose Chromatography . . . . . . RNAPTCP.r.t10nseeeeeeeeeeeee. Characterisation of Poly dT-Cellulose . . . Characterisation of Poly dT-Cellulose. BF’.Ctloneeeeeeeeeeaeee Page ii iii vi vii viii 12 12 12 13 14 16 15 15 l6 17 18 19 20 21 21 22 22 22 2h 25 25 36 36 38 38 60 AA RESULTS Part 11: DISCUSSION Part I: Part 11: Poly dT-Cellulose Adsorbing RNA in Total RNA from Artemia saline . . P01?!“ ISOl.tion e e e e e e e e e Yieldeeeeeeeeeeeeeee Characteristics of PAH . . . . . . . SISHIficmc. Of PAM e e e e e e e e Poly dT-Cellulose Chromatography . . . Characteristics of Poly dT-Cellulose . cn.r&crer1‘t‘c. or B Fr.CtSon e s e e FraCtionAweeeeeeeeeeeee Novikoff Ascites Cell RNA . . . . . . Arte-I. ICIII“ RNA e e e e e e e e e a MCI-L’s ION C O O O O O C O O O O O O O O O O O O O O O O 0 REFERENCES . 54 55 55 56 57 58 58 59 61 62 63 64 65 ATP DNA EDTA G G.L.C. HnRNA mRNA PAH Poly A poly U poly dT poly A RNA RNA rRNA RNase RNP SDS TCA Iris ABBREVIATIONS adenosine adenosine triphosphete cytidine deoxycholate deoxyribonucleic acids ethylenedianine tetraacetic acid guanosine gas-liquid chromatography heterogeneous nuclear RNA messenger RNA polysome area material, as assayed on a sucrose gradient homopolyler of adenylic acid homopolymer of uridylic acid homopolymer of deoxythymidylic acid RNA covalently bound to poly A ribonucleic acids ribosomal RNA pancreatic ribonuclease, specific for pyrimidines ribonucleoprotein sodium dodecyl sulfate Takadiastase,type 1. a ribonuclease specific for guanylic acid trichloroacetic acid tris(hydroxymethyl) eminomethane firidine vi Table l. 2. 3. 4. 5. L IST OF TABLES Page Range of A26 Background Observed During 39 Blank Runs on Poly dT-Cellulose Columns. Adsorption Properties of Poly dT-Cellulose 41 as e Phnction of RNA Source. Duplicate G.L.C. Response Ratios Obtained A7 for Novikoff Ascites Cell RNA Previously Fractionated by Poly dT-Cellulose Chrome- tography. Sephadex 6-50 Analysis of T1 Ribonuclease 49 Digestion Products of RNA Fractions Obtained by Poly dT-Cellulose Chromatography. RNA Prepared from Artenia saline: Characteris- S3 tics of its Adsorbance on Poly dT-Cellulose as a Function of Stage of Development (length of time incubated at 28°). vii LIST OF FIGURES Figure Page 1. Sucrose Density Gradient Profiles of 26 Polysome Preparation from Incubated Artemia Cysts: With or without Treatment with Pancreatic RNase. 2. Sucrose Gradient Resolution of Polysomes 28 from Artemia Cysts Incubated l2 h at 28°. 3. Sucrose Gradient Profiles of a Polysone 30 Preparation from Incubated Artemia Cysts: EDTA Sensitivity of PAH, Hith or without Treatment with Pancreatic RNase. 4. Typical Absorption Spectre Observed for 32 Various Fractions During Artemia Polysome I‘Ol.t‘0ne 5. Sucrose Gradient Profile of Artenia Polysome 3A Preparation P25: RNaae and EDTA Sensitivity of IX Pellet Material. 6. G.L.C. Analysis of Novikoff Ascites Cell 45 Total RNA Prep R1 and of Fraction B as Obtained by Poly dT-Cellulose Chromatography Of PTOP R1 RNAe 7. Representation of Results of Gel Electro- 50 phoresis Analysis of Selected RNA Samples. viii I NTRODUCT ION The species Artemia saline. (Crustacea, Phyllopoda. subclass BranchiOpoda. order Anostraca) commonly known as the brine shrimp. presents a potentially interesting organism for the study of morpho- genesis and cell differentiation. particularly in terms of control mechanisms. Embryonic development can proceed via two pathways after fertilisation occurs in the ovisac of adult females. Meiosis and blastulation are apparently normal. but gastrulation either occurs normally with the release of the free-swimming nauplius larvae from the ovisac or it may be arrested Indefinitely by the formation of a cyst shell (Finamore and Clegg. 1969). Morris and Afselius (1967) have extensively studied the structure of this shell. It was found to be composed of a In thick hypochlorite-sensitive chorion of maternal origin which is at least 50 1 protein in composition. Below the chorion lies an outer cuticular membrane of unknown origin that is resistant to most reagents including hypochlorite and trypsin: and an embryonic cuticular layer containing chitin and protein. The encapsulated cysts are believed to be at least partially dehydrated in the ovisac and then released to the saline medium in which the species lives. The cysts float on the water surface and are eventually blown to shore where they are further dehydrated. It is thought that complete desiboation dries and consequently destroys an outermost cellular membrane covering the chorion. which may be responsible for maintaining minimal dehydration during the trip to shore (Morris and Afzelius. 1967). This "air drying" activates the gastrulae which are then dormant only because they lack water (Dutrieu. 1960). Emerson (1963) found no evidence for oxygen consumption in this encysted dormant state. which may last up to 23 years without affecting the cysts' viability. One can rehydrate the cysts at 0 - 6° without inducing resumption of metabolism, a situation which makes it possible to biochemically analyse the dormant cyst (lwasaki. 196A; Clegg et al.. 1967), but metabolic activity is resumed within five minutes of addition of water at 28° (Finamore and Clegg. 1969). A rehydrated encysted gastrula stage embryo undergoes morpho- genesis aad after 8 - 15 hours emerges as a prenauplius larva which is still bound by a hatching membrane ( Finamor and Clegg. 1969). The prenauplius continues to develop into a free-swimming larva resembling a tiny shrimp which then undergoes about 16 melts in as little as two weeks to reach adult stage (Anderson. 1967). The hydrated encysted embryo of the brine shrimp is quite re- markable in that metabolism may still be reversibly halted by complete redesicostia (Finamore and Clegg. 1969) and in that considerable cell differentiation occurs in the cyst capsule stage without any evidence for cell division (Nakanishi at al.. 1962). The last feature is especially interesting since it impliesthat translational changes can be studied independently of cellular variations due to the mitotic cycle. The feature of prime concern in this study is the appearance of the protein synthesis apparatus during the first 5 - 10 minutes of hydration in dormant cysts apparently (but not definitely) devoid of pre-formed polysomes. Hultin and Morris (1968) found no evidence for pro-formed polysomal structures in electron micrographs of dormant gastrulae. but they did find that the cytOplasm was densely packed with free ribosomes and a lesser amount of membrane-bound ribosomes. They studied cell-free amino acid incorporation with microsomal fractions. but the levels of incorporation seem too low to be of any real significance (20 -50 cpm) and their techniques did not rule out beeterial contamination. The free ribosomes were capable of incorp- orating phenylalanine in a reaction dependent upon poly U as template, with low endogenous levels of incorporation (around 2500 cpm over background per 50 ug RNA) and they concluded that free or bound polysomes were at best present at very low levels in dormant cells, although potentially active ribosomes were abundant. Clegg and Golub (1969) found low amounts of RNAasecsensitive A260 absorbing materials (presumably polysomes) sedimenting more rapidly than 81 S monosomes in both dehydrated and rehydrated dormant cysts maintained at 4°. Based on qualitatively convincing sucrose gradient profiles both of extract and the cell-free amino acid incorporating system made from the extract. they defined these materials as polysomes. They were. however. reading RNAase-dependent differences in the 0.04 - 0.08 A260 range and in the 2 - 20 cpm range of radioactivity. Cell-free protein synthesis in homologous systems fell within the 50 - 100 cpm/mg protein range. except when additional poly U was added (1200 cpm/mg protein). The rapidly sedimenting A254 material as assayed by sucrose gradient analysis was often not completely sensitive to RNAase digestion. Other studies (Finamore and Clegg. 1969) showing low levels of polysomes in extracts made from dormant cysts also exhibit very low levels of incorporation. Clegg and Golub (1968) found a relatively dramatic increase in so-called polysomal material after incubating prehydrated cysts three minutes at 28°. and after only 15 minutes in non-prehydrated cysts. As the cysts are incubated at 28°. polysomes detected by sucrose gradient analysis increase rapidly; protein synthesis activity ig.!1§§g_ increases concomitantly. The question. therefore. becomes one involving the mechanism of rapidly implementing protein synthesis in an organism almost completely devoid of preformed polysomes. Specifically. what may be the developmental significance of any real polysomes present in dormant cysts. and. given a population of apparently active ribosomes. what is the source of the mRNA found in the polysomes formed so soon after resumption of metabolism 1 Is it stored in some manner in the dormant cysts or is it formed rapidly upon rehydration and incubation ? One would suspect that any preformed polysomes in the dormant cysts may represent a random selection of protein synthesis activity. However. they may represent specific synthetic activity involved in the formation of the cuticular membranes . which Morris and Afselius (1967) believed to be of embryonic origin. If the latter case is true. it may be possible to synthesise specific proteins from cell-free systems using dormant cyst polysomes. provided an adequate quantity of polysomes could be isolated. The question of mRNA stored in dormant Artggia cysts invokes the possibility of "masked messenger” or Spirin's “informosome” (Spirin. 1966). a RNA-protein complex which is suggested as the trans- portable form of messenger RNA or as the RNAase-resistant non-ribosome bound form which can be stored in the cytoplasm ( Lee and Brawerman,l97l). Recent developments in RNA chemistry have produced more evidence in support of the existence of such template active ribonucleoprotein (RNP) particles ( Toennesen and Toenne. 1973; Infante and Nemer.1968; Perry and Kelley. 1968). Heterogeneous nuclear RNA ( HnRNA) proposed as precursor to mRNA ( Jelinek et a1. 1973; Imaizumi et a1. 1973). is I found almost entirely in RNP particles ( Lukenidin. 1972); rapidly labeled RNA is released from polysomes by EDTA treatment as RNP particles ( Adesnik et a1..l972). The association of both HnRNA and mRNA-bound poly A sequences with HnRNA cleaving enzyme and poly A synthetase (Niessing and Sekeris, 1973) and with poly A protector and cleaving enzymes (Rosenfeld et al.. 1972; Blobel.l973) suggests that the protein portion of RNP particles is indeed strongly linked with the fate of the RNA portion. One might expect to find any mRNA or precursor for mRNA stored in dormant cysts as e RNP particle. It was the purpose of this work to investigate the possible existence of a fraction of non-ribosome bound DNA-like RNA in dormant cysts and if found to determine whether or not it is mRNA, and if possible to isolate a quantity of it to use in more detailed characterization. This project seemed especially feasible since it was reported that the dormant cyst is free of RNase (Uarner.l97l). The first prerequisite for such a study is an operational definition of mRNA. By strictest definition mRNA must be characterized by the ability to direct the polymerization of amino acids in specific sequences. i.e. the synthesis of proteins ( Zomzely-Neurath and Moore. 1973). With the exceptions of globin messenger. which is essentially pure in red blood cell extracts. and of myeloma heavy chain messenger. which is apparently specifically precipitated by myeloma protein 5563 HZLZ (Blobel. 1973). potential templates have not been isolated to purity. Total message is isolated. usually using poly A techniques that will be discussed later. and proteins synthesized in_!1££g are identified by gel electrophoresis (Stevens and Williamson. 1973: Zomzely-leureth and Moore. 1973) or by immuno- chemical precipitation (Schutz et al.. 1970). Generally. incorporation of labeled amino acids into acid precipitable counts in a cell-free system has been accepted as in- dicative of the presence of non-specific mRNA. but one must consider that other RNAs may have activity also or may activate endogenous mRNA of the ribosomal system (Kruh et al.. 1966; Scheffer et al.. 1964). Historically. however. mRNA has been defined as rapidly-labeled RNA with a high turnover rate that is associated with polysomes (Lee and Brawerman. 1971a; Clever and Storbeck. 1970). It has a DNA-like nucleotide composition (Evans and Lingrel. 1969). Such RNA has been shown to be released from polysomes by EDTA treatment (Adesnik and Darnell. 1972; Penman et al.. 1968) or by puromycin-induced term- ination of polypeptides (Blobel. 1973). There is great danger in using this criterion as sole defining condition for mRNA however. as non-Rlbase-sensitive RNA contaminants have frequently been observed co-sedimenting with polysomes in sucrose gradients (Ilagemann. 1969; Rabat and Rich. 1969: Perry and Kelley. 1968). The contaminating particles can be distinguished from polysomes by equilibrium gradient centrifugation (Olsnes. 19703 Penman at al.. 1970): and by lowering of their density gradient S-value by ionic detergent treatment. but not by EDTA (Olsnes. 1970) Penman et a1. 1968). Within the last few years it has been found possible to identify and isolate mRNA as defined by one or more of the above criteria. on the basis of covalently linked polyadenylio acid sequences. The poly A sequences have been shown to be covalently linked to the 3'-0H end of the mRNA (Nakazato et al. 1973) in essentially all eukaryotic and viral RNA's tested thus far (Green and Cartes.1972). except for histone message (Jelinek et a1. 1973). No poly A has been found in E. 221; (Green and Cartas. 1972). Poly A RNA has been found associated with both membrane-bound (Faust at al. 1973) and free polysomes (Jelinek et al.. 1973; Adesnik et al.. 1972). with both strands of double- stranded (;n_!;££g) SV-hO specific RNA (Aloni. 1973); with 36 - 39 1 of mitochondrial RNA of Lettr‘ Ehrlich ascites tumor cells (Avadhani et al.. 1973). Furthermore. 4.8 X of rabbit embryo total RNA was found to be poly A RNA (Schultze et al.. 1973). Poly A RNA isolated from polysomes is usually found to represent 2 - 3.5 X of polysomal 32F counts (Schutz et al.. 1970; Swan et al.. 1972; Green and Cartas, 1972). Between 12 and 20 Z of a poly A RNA fraction is found to be RNAase resistant. depending on the species and length of labeling period (Beast. 1973; Nakazato et al.. 1973). Yeast rRNA has less than 0.06 1 which fits the poly A criteria (McLaughlin et all. 1973). At least 20 Z of HnRNA has also been shown to contain poly A sequences 150-250 nucleotides long (Greenberg and Perry. 1972; Jelinek et al.. 1973). Nakazato et al. (1973) prOpose that no more than 40 z of HeLa cell HnRNA has poly A. NnRNA has for some time been thought to be a precursor of mRNA (Darnell. 1968); it has now been rather conclusively shown to be so. based on poly A data (Greenberg and Perry. 1972; Jelinek et al.. 1973). Nakazato et al. (1973) have recently found an additional poly A sequence of approximately 20 nucleotides in HeLa cell HnRNA that is not at the 3'-OH terminus. Except for yeast with about 50 nucleotides (McLaughlin et al.. 1973). most of the poly A sequences are 6-5 8, 150 -250 nucleotides long (Adesnik et al.. 1972: Edmonds et al.. 1971; Lee et al.. 1971b) and seem to be relatively homogeneous in size in any given species. as judged by gel Analysis. dowever. Stevens and Williamson (1973) found two distinct gel bands for cytoplasmic immunoglobulin heavy chain mRNA that had been specifically precipitated with 5563 N2L2 protein. The two fractions differed at least in the length of their poly A sequences. Swan et al.. (1972) also found two poly A RNA fractions differing in the leng§N'of poly A sequences. using poly dT cellulose chromatography on myeloma (MOPC-hl) membrane-bound polysome RNA. These two poly A RNA fractions may represent two stages of adenylation. The poly A sequences seem to be added after transcription of the NnRNA is completed (Jelinek et al.. 1973; Philipson et al.. 1971). Poly A synthetases have been shown. as early as 1960 by M. Edmonds. Niessing et a1. (1973) have demonstrated the presence of synthetase in ascites cell nuclei and Raff and Walker (1973) have found two distinct poly A polymerases in yeast nuclei. Polymerase I is active in low salt and is dependent on primer without poly A; polymerase II is active in high salt and requires poly A primer. These enzymes may account for the suggestion of Slater et al. (1972) that mRNA's transcribed before fertilization in sea urchin eggs may have short poly A sequences attached ( type 1 enzyme has been active) while longer stretches of poly A are added after fertilization in the absence of concomitant transcription ( type 11 enzyme has been active). Such a two-stage adenylation process would provide a convenient control point in organisms which pass through a dormant stage for postponing any function dependent on the presence of poly A. Various functions have been proposed for the poly A sequences: 1) control of transport of mRNA to the cytoplasm (Darnell.1971b; Lee et al.. 1971b): 2) recognition signals for cleaving of HnRNA to create mRNA (Edmonds et a1..1971); 3) control the efficiency of mRNA utilisation - Sussman's "ticketing" hypothesis (Sussman.l970); A) directing activity of binding of 3N-met-tRNA to ribosomes (Rosenfeld et a1..1972). An influence of poly A on translational activity is suggested by the report of Swan et a1. (1972) that poly A RNA fractions with longer poly A residues are more active in directing protein synthesis in_vitro than the RNA fraction with shorter poly A segments. It is most likely that the total mechanism in a normal cell will prove to be a combination of all of these functions. Since neither the function of poly A in normally developing cells nor the mechanism of reversible dormancy in Artemia saline cysts are known it would be difficult to make a prediction concerning poly A RNA in dormant cysts. Dormant cysts are apparently devoid of preformed polysomes but do exhibit protein synthesis activity within five minutes of onset of metabolism, a fact which indicates either that the dormant organism contains a substantial pool of free mRNA or that extensive transcription is initiated immediately in the incubated cyst. The latter case seems least likely since the cysts contain a nucleotide pool lacking in ATP and heavily weighted toward guanine-containing nucleotides; are incapable of de novo purine synthesis ( Finamore and Clegg. 1969); are impermeable to nucleotides or their immediate precursors; have. at best. only inactive RNase ( Warner. 1969). It is much more likely that Artemia cysts do contain a population of preformed I a mRNA or of mRNA-precursor in the form of NnRNA. In either of these cases the RNA may be protected and/or inactivated in the dormant cyst by q specifically bound proteins or by adenylation or lack of adenylation. Whatever the state of mRNA stored in dormant cysts. one may assume on the basis of data obtained from the eukaryotic species examined so far that mRNA in incubated metabolizing cysts do contain poly A segments. In summary. the purpose of this work was to look for an RNA species in dormant brine shrimp that could be defined as mRNA by one or more of the criteria discussed. All experiments based on radioactive incorporation were immediately ruled out as there are reports, not supported by published data. that brine shrimp are impermeable to all commonly used radiolebeled compounds except 1"003'2 (Warner and Finemore. 1965). The major effort of this work was directed toward isolating polysomes from rehydrated incubated cysts. It was hoped that the polysomes could be dissociated to yield ribosomal subunits and the RNA-template fraction. The latter would be extremely useful in defining the sensitivity of a cell-free protein synthesis system assay for any presumed mRNA isolated from dormant cysts. An mRNA from incubated cysts would also have intrinsic interest with respect to its composition. i.e. protein content. and to its other biological characteristics. Determination of these would 10 facilitate the search for mRNA in dormant cysts. Difficulties were encountered in the isolation of polysomes from incubated cysts; consequently when reports of poly A RNA appeared, effort was directed toward isolating the mRNA via poly A techniques. Specifically poly dT-cellulose was made and characterized and an experiment was run to determine whether poly A-linked RNA exists in dormant or incubated cysts. ll _r-:V MATERIALS AND METHODS Unless noted. all chemicals used were reagent grade. All water used was double distilled or deionised distilled. Part I: Polysome Isolation Pretreltment of 91sts All procedures were carried out at 4°. DU! costs. 26 grams. r (Longlife Aquarium Products) were pre-hydrated for 3 h in a 2 Z NaCl solution to which had been added 0.6 ml concentrated Vescodyne (Vest Chemicals Co.) per 100 ml solution. The cysts I were drained by decantation and subsequently soaked in 100 ml freshly prepared antiformin (100 ml fresh commercial bleach. 7.8 g NaOH, 3.2 g Na2003,diluted 7:93 with water) for 30 minutes. The dechorinated cysts were then drained and rinsed thoroughly with sterile 2 1 1.01. 'Buring the rinsing process, the cysts were decanted away from the heavy residues and stones always found in commercial batches of dry cysts. The sterile cysts were then hydrated for 6 h in sterile 2 1 NaCl to which I x urea had been added to insure complete removal of residual antiformin. Prior to incubation, the cysts were drained and rinsed with a total of 2 liters sterile 2 1 NaCl after which excess liquid was aspirated off. Incubation of Cysts Five g drained cysts were added to 800 ml sterile growth medium (0.528 M NaCl, 0.0096 M KCl, 0.0254 M M559“, 0.0227 M MgClz, 0.0014 M CaClz. 0.0005 M NaH003) resembling the composition of water in the Great Salt Lake region (Warner and McLean, 1968), in sterile 2800 ml Fernbach flasks. 1000 units Penicillin G per ml medium, 17 X Zephiran (Winthrop Labs) to make the medium 10"“9 X in Zephiran and 0.1 g streptomycin were added and the flasks were inc- 12 13 ubated at 28° on an Eberbach reciprocal shaker at slow speed. Cysts were collected on a luchner funnel and rinsed with 500 ml water at room temperature. Cysts were either immediately placed in ice cold grinding buffer for polysome extraction or wrapped in the filter paper and aluminum foil, immediately frozen in a dry ice-acetone bath and stored at -70°. One flask was allowed to continue incubation as a control for normal development of the cysts and as a control for contamination. gglyscme Preparation ' All procedures were carried out as quickly as possible at 6°. Solutions containing sucrose were pre-treated with diethyl pyro- carbonate (ethoxyformic anhydride. or laycovin from Bayer, Inc.) at 5 drops per 200 ml solution, heated 1 h to drive off the reagent, and stored at 4°. All glass equipment was treated with 1 M KOH to insure freedom from RNAase contamination. Incubated cysts from two flasks were added to 5 volumes (50 ml) IO % sucrose-buffer solution. The buffer used was 0.01 M Tris HCl. pH 7.5: 0.01 M MgClz. 0.25 M KCl. 0.007 M beta mercaptoethanol (TKMh buffer) unless designated differently. This high ionic strength buffer stabilizes polysomes and increases their concentration relative to monosomes (Heywood et al, 1968). 0.1 2 Nonidet P-40 (NP-60, Shell Oil 00.). a nonionic detergent, was added to the homogenizing buffer when noted. The cysts were then broken in Dounce homogenizers using first 2-3 passes in a 50 ml capacity homogenizer with an "A" pestle.which had been hand-ground to give a minimal passage to the cysts, and then with one pass, down only, in a 12 ml homogenizer with a normal "A" pestle. The use of the Dounce 16 homogenizer results in gentle breakage of the cells with minimal rupture of the nuclei ( Penman et al. .1963). Such treatment rendered the cysts 80 -901 broken as judged by visual examination. The homogenate was centrifuged in a Sorvall centrifuge, 55-36 rotor, for 30 minutes with the rotation increased from 2,000 to 10,000 g after 10 minutes, and then to 16,000 g after 15 additional minutes with a 5 minute spin at 16,000 g. This procedure was designed to minimize mechanical damage to the polysomes (Evans, 1970). The top layer of orange lipid material was aspirated and 70-80% of the remaining supernatant was drawn off as the 16,000 g supernatant. Qigcontinuous:gradient centrifugation Five ml of 0.6 M sucrose-buffer was layered over 5 ml 1.6 M sucrose-buffer and pre-cooled 2-6 hours. TMKh was used unless otherwise noted. Approximately 15 ml of 16,000 g supernatant was layered over the gradients and centrifugation carried out for 7 h at 28,000 rpm (or equivalent) in a type 30 rotor in a Beckman L2 ultracentrifuge. The supernatant was aspirated, the clear pellet at the bottom was rinsed 3 times with 0.5 ml portions of ice cold 10% sucrose solution and dissolved in 500 ul of the same solution ( 1x pellet). ggiysome Assay_by Continuous Sucrose Gradient Centrifugation Linear gradients ( 6.6 mls ) from 15 to 60 1 sucrose were poured at room temperature over a 0.2 ml cushion of 60% sucrose. Gradients were equilibrated for at least 2 h at 6° before use. Unless noted, 0.5 ml of sample was layered over the gradients and centrifugation was carried out for 50 minutes at 38,000 rpm in the Beckman SH 39 rotor at 6°. Gradients were either collected by hand or run through a Gilford 0.2 cm flow cell mounted in a Gilford spectrophotometer, using a Buchler polystaltic pump to pull 15 the solutions through the system, and a Sargent recorder to trace the changes in absorbency. Calculation of Yield The yield of 260 nm absorbing material sedimenting in the polysome area of the 15 - 60 1 gradient will hereafter be referred to as polysome area material or PAM. The polysome area is defined as the section of the gradient below the level of the ribosome peak. The ribosome peak represents around 30 - 90 Z of the 260 nm absorbing material in Artemia extracts and sediments almost at the same rate as g. £21;_ribosome standards. The RAM was quantitated as the percent of total absorbency falling below the lower margin of the ribosome peak, as extrapolated to the baseline ef the gradients. Total ab- sorbency at 260 nm is taken as the sum of the absorbencies above baseline in the monosome and polysome regions. In calculation of RAM yield, the baseline was established by the absorbency in the polysome area of an identical sample which had been treated with 5 ul pancreatic RNAase for 10 minutes at 0 - 6° prior to centrifugation. when the automatic flow cell system was used, background absorbency was observed for blank gradients, i.e. with .5 m1 portions of 10 1 sucrose solution applied in lieu of sample. This level of true background absorbency was used as baseline for calculating percent PAM remaining after RNAase treatment. Areas were computed by weighing cut out graph areas. EQTA Sensitivity of RAM 1 x pellets were dissolved in 0.01 M Tris HCl, pH 7.6, 0.01 M 16 Mg 012. 0.1 M KCl, 0.02 M EDTA, 0.005 M dithiothreitol and analyzed over l5 - 60 X sucrose gradients made in TEKh buffer (0.01 M Tris ac: pH 7.4, 0.01 M son, 0.25 M Kai). Part II: Poly dT-Cellulose Chromatography Techniques for isolating poly Aalinked RNA's are based on the following characteristics of poly A (Raskas and Bhaduri, 1973): l) poly A is resistant to pancreatic and T1 RNAases but not to T2 RNAase; 2) it binds to millipore filters in high but not in low salt; 3) the poly A segment clipped from the RNA by T1 RNAase migrates as 6 -7 S material in polyacrylamide gel electrophoresis; 6) it has few or no uridine residues. Various methods of isolation have been used, including collection on millipore filters (Raskas and Bhaduri, 1973); retention on poly dT-celluloae (Edmonds et al, 1971a), poly dT-sephsrose (Adesnik et al.,(l972a) or poly U-cellulose (Katee, 1970): and poly U hybridization followed by hydroxyapatite chromatography to separate out the double and triple stranded UsA complexes (Avadhani et al.. 1973). These techniques are dependent on proper initial extraction of the RNA (Perry et al., l972). The poly A-protein complex property of the RNA results in apparent selective loss of poly A RNA when high ionic strength buffers are used at pH 7 (Lee et al., 1971b). It is believed that this RNA aligns perpendicular to the phenol-aqueous interface with the poly A - protein section in the phenol phase and the remaining nucleotides in aqueous phase. The result is either shearing of the poly A sequences with their loss in the phenol, or loss of the entire poly A RNA at the interface which is normally discarded(Perry et al., 19721. Endonucleolytic cleavage also leads to loss of poly A sequences from poly A RNA during isolation (Adesnik et al., l972a). These problems can be overcome by phenol extracting 17 at neutral pH at 60 ° (Edmonds, 1971a): or by extracting at pH 9, room temperature or below; or by using a chloroform: phenol mixture at pH 7 (Perry at al. ,1972; Schuts, 1970). Although mRNA molecules may not all include covalently bound poly A sequences, and although those that do may have sequences of different lengths (McLaughlin et al., 1973), extraction techniques based on the unique properties of poly A have thus far proved to be, by far, the most successful methods for ‘«‘I isolating RNA fractions from eucaryotic cells that can direct the f w synthesis of specific proteins in vitro. _w‘ 1 RNA Preparation from Novikoff Cells Hale Sprague Dawley rats were injected intraperitoneally with 0.5 ml Novikoff ascites cells. Seven or eight days later they were killed by ether inhalation and ascites fluid was collected by syringe, after opening the abdominal cavity surgically. Fluid volume was measured and cells were collected by 8 minutes of centrifugation at 150 -l66 g, 6°. The supernatant was aspirated and the cells were washed twice with l fluid volume of ice cold 1 1 NaCT solution. The layer of red blood cells on top of the strawbcolored ascites cells was aspirated and one fluid volume of 0.1 M acetate buffer pH 5.0 (0.1 N NaAcetate pH 5.0, 0.001 M EDTA, 0.5 1 sodium dodecyl sulfate) and an equal volume of phenol reagent (275 ml distilled, water-saturated phenol, 0.25 g 8 hydroxyquinoline, 35 ml m-cresol) was added. The mixture was vortexed intermittently at room temperature for 20 minutes and then hand-shaken in a 650 water bath for 10 minutes. The 650 phenol method results in an increase in RNA yield with less degradation and less DNA contamination (McClean and warmer, 1971). The solution was chilled one h and then centrifuged for 35 minutes at 25,600 g. 18 The phases were separated and reextracted at 65° for 10 minutes: the aqueous phase by an equal volume of phenol reagent with 0.1 1 sodium dodecyl sulfate, and the phenol phase by acesama buffer. These extraction mixes were chilled and centrifuged as above. Aqueous layers were combined and RNA precipitated by two volumes of acetone at -20°. The precipitate was collected at 12,000 g in 20 minutes, rinsed with absolute ethanol, dissolved in 10 ml 0.1 M acetate buffer, pH 5.0 -=; without sodium dodecyl sulfate and reextracted mtth 1 volume of phenol ‘ reagent at 6°. The phases were separated by spinning at 12,000 g for 30 minutes and the aqueous layer cleared by spinning it at 20,000 g for 30 minutes. The RNA was precipitated dram the aqueous phase by the addition of two volumes of absolute ethanol at -20°. Residual phe- nol was removed by three washes with 1.5 volumes ethyl ether, -20°._ The RNA was precipitated overnight in absolute ethanol and redissolved in the high salt buffer to be used of the poly dT cellulose column. RNA Preparation from Artemia saline 5.5 g portions of prepared cysts were incubated for 8 hours in 2 L growth medium with forced aerationat room temperature before being collected, frozen and ground as below. 11.5 g portions of cysts were frozen immediately in dry ice-acetone and then ground in an ice-chilled mortar and pestle. 10 ml 0.1 M acetate buffer pH 5.0 that was 1.2 X in sodium dodecyl sulfate and 10 m1 phenol reagent were added to the slurry and the mix was ground for an additional 2 minutes. An additionl 20 ml of buffer and of phenol reagent were added and the entire mix was shaken by hand at room temperature for 5 minutes. The mix was then incubated for 30 minutes in a 65° water bath with occasional swirling l9 and subsequently chilled on ice for l h. The aqueous phase was removed after 50 minutes of centrifugation at 12,000 g and the RNA was precipitated by two volumes acetone, -20°. The RNA was further treated as with the Novikoff ascites cell RNA. Preparation of Poly_dT-Cellulose Two batches of poly dTbcellulose were prepared according to m t“. "thOd 9‘ Gilhal (1966). Thymidine 5' monophosphate (2 mmoles) fl as the sodium salt (Rayle Labs) was converted to the free acid by passing it over a Dowex-50 column in the H+ form. The eluate was ’ evaporated to dryness at least 3 times in the presence of 10 m1 dry pyridine. Five g What-an column chromedia CFl fibrous cellulose powder #11011, predried over P205 in an evacuated desiccator at 1000 was used. The poly dT-cellulcse was stored at 6° under aqueous solution of 1 mM NaN3. Batch 11 was made with the following alter- ations: 1) no glass beads were added, 2) 1.5 ml dry dimethylformamide (Aldrich Labs) was added to the reaction, 3) an additional 0.8 g dicyclohexylcarbodiimide (Aldrich) was added to the second step, 6) cellulose had been dried over KOH at 80° and appeared white in contrast to the brown color of batch I. . Columns were made by packing a quantity of poly dT-cellulose in 5 ml luer-lok syringes lined on the bottom with a small glass wool plug. New columns were washed with 1 M NaCl until background absorbency fell below 0.03 at 260 nm. Prior to each sample application, the columns were washed with 50 ml water and 50 ml 1 M NaCl at room temperature, and then with 25 ml buffer A (0.1 M NaCl, 0.01 M Tris HCl pH 7.5) at 6°. The column was temperature equilibrated at 6° for 2 h 20 and the column head was brought to a minimum volume before adding the RNA sample in 500 - 900 ul buffer A. Unless noted, the poly dT— cellulose and sample were well mixed at least twice during the 30 minutes following application of sample, taking care to leave a 0.5 cm layer of packed cellulose undisturbed at the bottom of the column. Fraction Collection During Poly dT-Cellulose Chromatography Fractions were collected manually as follows, using acid-washed, tared tubes for collections A1 - the head from the settled column plus two 1 m1 rinses of buffer A (0.1 M Tris NCl pH 7.5, 0.1 M NaCl); Axx‘ remainder of 50 I1 Buffer A wash at 6°. The column was then taken to room temperature and allowed to equilibrate for 30 minutes. Approximately 10 ml of buffer A were allowed to wash through at room temperature, the first 1 - 2 m1 forming fraction Aw. The volume at the top of the celumn was brought to a minimum and buffer B (0.01 M Tris HCl, pH 7.5) at room temperature was usiddfor the next elution step. The first 3 ml of Buffer B eluant formed fraction 8. Fractions were kept on ice as much as possible and frozen at -20° for future use as soon as the A260 units had been determined. Columns were regenerated immediately by washing with an additional 25 m1 Buffer 8 at room temperature and with 10 ml of 0.001 M NaNa. Columns were used repeatedly with no loss of poly A retaining capacity. In later meriments 0.1 M illi;.iiC03 was substituted for buffer A and 0.01 M NH4HCO3 for Buffer B in order to reduce residual salt in the lyophilized samples to be used for base analysis. There was no change in the retention characteristics when NH4HCO3 buffers were used. 21 £1;_NAase Digestion of Fractions from Poly dT-Cellulose Chromatography Selected samples (approximately 10 A260 units/ml) were treated with 0.1 volume of Tl-RNAase (100 units/ml) in 0.05 M Tris HCl pH 7.5, 0.05 N NHQHC03 for 20 minutes at 37°. Fractions were brought to 0.1 M NH4H003 before applying to Sephadex G-50 1n columns or reapplying to poly dT-cellulose columns in the normal j fashion. Fractions from these poly dT-cellulose runs were lyophilized S in preparation for analysis on G-50 or for base analysis by G. L. C. Preparation of RNA Samples for G.L.C. Analysis; Enzymatic Digestion ' Samples were precipitated by addition of 2 m1 ice cold 5 X trichloroacetic acid and collected on Nhatman 3 mm filter paper (2.6 cm) presoaked in 5 Z trichloracetic acid. Filters were washed with 6 - 8 ml cold 5 Z trichloroacetic acid, 6 - 5 ml cold ethyl alcohol, and at least 2 ml of cold ethyl ether before drying under a lamp. Dry filters were incubated in 25 ml Erlenmeyer flasks with 1.5 - 2 ml of enzyme solution at 37° for approximately 3 days, including at least 26 h of shaking on a Dubnoff shaker. The enzyme solution used was either Enzyme I (0.05 mg bacterial alkaline phosphatase (BAPSF, salt fraction, Horthington)/m1: 2.0 ug pancreatic RNAase (Sigma)lml; .05 mg Crotelus venom phosphodiesterase (Northington)/ml, all in 0.01 N (NN4)2C03, 0.001 M MgC12,)or Enzyme II (0.1 mg BAPSF/ml, 2 ug pancreatic RNAase/ml, 0.5 mg venom phosphodiesterase/ml in 0.01 M (NN4)2003, 0.001 M NgAcetate2 , 0.001 M NaN3). Reaction solutions were removed by pipet and filter and flask were rinsed with water. Combined rinses were lyophilized from water repeatedly until residual salts were minimal. The samples were stored 22 lyophilized until needed for G.L.C. analysis. Preparation of Samples for 955:9. Analysis, Alkaline Hydrgylsis Samples of standard synthetic polymers or nucleotides were treated with l N KOH for 26 hours at 37° at concentrations of 5 - 8 A260/m1. The solutions were made slightly acidic with l N perchloric acid and centrifuged at high speed after 30 minutes, in a clinical centrifuge, to collect the precipitate. The supernatant was made slightly alkaline with 1 N(NH4)2CO3 pH 8.75 and 60 ul alkaline phosphatase was added to each supernatant. The reactions were incubated 7 h at 65 ° and then 1y0philized several times from water to remove residue. Base Ratio Analysis by Gas-Liquid Chromatography (G.L.C.) Lyophilized samples were analyzed on a Hewlett-Packard F a N 602 High Efficiency Gas Chromatograph, according to the method of Pike (1971). The column was composed of 2 Z OV-l7 on silanized Gas Chrom Z. Samples were treated with 100 ul of N,0~Bis(Trimethyl silyl)acetamide (Regis Chemical Co.) for 2 h at 120° before injecting 3.9 ui of trimethylsilyl derivative into the column at 200°. Results were analyzed by weighing cut out peaks from on. chromatograph. W222 Selected samples were analyzed by gel electrophoresis using a vertical electrophoresis cell, model EC 670, E. C. Apparatus corp.). 2 X acrylamide-0.5 % agarose gels were prepared according to the procedure of Dingman and Peacock (1968), using cyanogum 61 (Fisher Scientific Co.). a prepared mixture of acylamide and N,Ni-meth!lene- bis acrylamide in 1981 proportion, and Seekem agarose (Bausch and Lamb). 23 Samples were applied with bromphenol blue dye markers and run 1 h at 250 V, 6°. Gels were stained with Stains-Ali (Stock No. 2718, Eastman Organic Chemicals) and the results were recorded by polaroid photography. 26 RESULTS Part 1: Isolation of Polysomes There are formidable biological difficulties encountered when working with.Artemia gaging.cysts obtained from commercial sources. Such cysts are apparently randomly collected from shores of various salt lakes and pressure sealed in cans without regard for sand and other environmental contaminants. The biological state of such cysts could be expected to vary with time on shore. weather conditions, etc. Indeed, both hatching efficiency and incubation period for the cysts varied considerably (5 - 90 X hatching, first hatching 11 to 20 hours after commencement of incubation) depending on the particular can and the length of time it had been opened. Fungal contamination, class- ified as Scopglariopgis brevigglus by Dr. Ellingboe of the Michigan State University Botany Department, was difficult to avoid3.1t often developed after hatching commenced even when the most stringent sterile precautions were observed, unless Zephiran at 1 ppm was included in the growth media. One might almost suspect that there was perhaps an occasional fungal spore present within the cyst shells which was released when hatching occurred. The hatching pentncls for batches used in these experiments exhibited some hatching after 15 hours, 20 1 after 20 hours and 80 - 90 s after 36 hours. There was no visual evidence for either fungal or bacterial contamination after 5 days' incubation at 28°. Breaking the tough cyst walls open with a Dounce homogenizer was physically quite difficult and the amount of cysts that could be homogenised during any reasonable period was thereby limited to 15 grams. The use of detergents (NP-60 or DOC) did not facilitate this 25 process noticeably. It was hoped that a certain amount of experimental consistency could be obtained, without sacrificing yield, by storing large batches of incubated cysts at<185° and using portions Cor several homogenizations. Effects of frggzing Cyst homogenates were made at various times from the same F? batch of incubated cysts, designated P2°, and subjected to various " periods of -135“ storage. Freshly prepared homogenate (l X pelleted) exhibited 68 X of ribosomal material as RAMs there appeared to be i only slight decrease in PAN as a function of storage either of incubated cysts (63 Z in frozen cyst homogenate) or 1 X pellet at -185° (65 z in frozen pellet made from fresh cysts, 36 I in frozen pellet from frozen cysts) for up to 6 weeks. Time at 6° does seem to lead to loss of this material (13 I after 6 hours at 6°). There was generally more variability in RAM between preparations from different incubated cyst batches (30 - 53 2) than between preparations from the same batch exposed to A” storage at various stages, and large batches of incubated cysts stored at ~1'85°were routinely used in these experiments. use The yield of 260 nm absorbing material in the 16,000 g supernatant was generally 70 - 100 A260 units per gram of drained incubated cysts. Of this,7 - 10 1 was found to pellet through discontinuous gradients. The yield of PAN, as determined from continuous gradient centrifug- ation of l X pellet material, was either in the 0 - 10 1 range or the 30 - 50 2 range, depending on fungal contamination of the incubated cysts and on cyst viability. Typical profiles for preparations in the 26 Figure 1. Sucrose Density Gradient Profiles of Polysome Preparation from Incubated Artemia cysts: with or Hithcut Treat- ment with Pancreatic RNAase. 1X pellet material prepared as described in text from cysts that had been incubated for 12 h at 28° and then stored at -1850 for 3 weeks prior to homogenization: 6.2 A260 without RNAase treatment and 6.2 A360 treated with 5 ul pancreatic RNAase for 10 minutes at 0 - 6 . Semples layered on 15 - 60 l.sucrose gradients in 0.01M Tris pH 7.6, 0.01M NgClz, 0.25M KCl and centrifuged at 38,000 for 65 minutes. Gradients were collected by the flow cell system: top of gradient is to the right of the .me 3.0 OON< 28 Figure 2. Sucrose Gradient Resolution of Polysomes from Artemia Cysts Incubated 12 h at 28°. 1X pellet was prepared as described from non-frozen cysts. THKh homogenization buffer ( 0.25 H K01) included 0.1% NP-60. A 3.7 A26ozample was applied to a 15-6OX sucrose gradient and centrifugation carried out at 32,000 rpm for 1.5 h. Gradient was collected by flow cell system: top of gradient is to right of scan. Figure 3. Sucrose Gradient Profiles of a Polysome Preparation from Incubated Artemia Cysts: EDTA Sensitivity of PAN, With or without Treatment with Pancreatic RNAase. Cysts were incubated for 12 h at 28° and homogenized in 0.01 N tris pH 7.6, 0.1 M KCl, 0.01M MgClz. 0.02 M EDTA, 0.001 M Dithiothreotol, 10% sucrose. 16 A260 of 1X pellet material and 16 A260 of pellet material treated with RNAase for 10 minutes at 6° were layered on 15 ~60% sucrose gradients in 0.01 M tris pH 7.2, 0.25 M KCl, 0.01M EDTA. Centrifugation was at 38,000 rpm for 2 h. Gradients were collected by flow cell system: top of gradient is to right of scan. Profiles were similiar for 1X pellet material after storage at -185° for 6 days. ceu< 32 Figure 6. Typical Absorption Spectra Observed for Various Fractions During Artemia Polysome Isolation. Type A spectrum: observed in most 16,000 g supernatants and many 1X pellet samples. Type 8 spectrum: observed in many 1! pellets, a few 16,000 g supernatants and in all RAN samples. Type C spectrum: observed for fractions 1,2, and 3 of figure 5 sucrose gradient scan. 260 280 240 >oconeoen< ciao—om Figure 5. Sucrose Gradient Profile of Artemia Polysome Preparation P25: RNAase and EDTA - Sensitivity of 1X Pellet Material. Polysome preparation P25 exhibited type B spectrum for both 16,000 g supernatant and 1X pellet material. Fractions marked 1,2, and 3 exhibited protein spectra ( type C spectrum, figure 6 ). Fraction 6 exhibited a type B spectrum( figure 6 ). 1X pellet was prepared in 0.01 M Tris pH 7.3, 0.05 M KCl, 0.01M NgClz, 0.005 N dithiothreotol. Samples, each 2.9 A260, were centrifuged at 38,000 rpm for 1.5 h on 15-60 X sucrose gradients made in 0.01 M tris pH 7.6, 0.01 M KCl, 0.01M HgClz ( THK ) : made in TMK brought up to 0.02 M EDTA : or made in TMK with pre-treatment with RNAase as described in methods . 36 upper range are shown in Figure 1 ( 63 X PAN). Very similar profiles have been obtained from continuous gradient centrifugation of 16,000 g supernatant (36 1). Some evidence for gradient resolution of polysome peaks was seen in flow cell profiles of the 26 1 X freshly prepared P2° l X pellet (Figure 2) and even in P pellet stored at-iBflq’for 16 hours prior to gradient analysis. The letter resolution was blurred but recognizable. EDTA s t vi of RAM EDTA treatment usually resulted in complete disappearance of the ribosomal peak in sucrose gradient profiles of Artemia extracts and in the appearance of two slower sedimenting peaks assume! to represent subunits. As can be seen in Figure 3, there is PAN under certain conditions which is neither EDTA-sensitive nor RNAase- sensitive. The percent RAM in EDTA goes from 23 x to 17 I after RNAase treatment as calculated using a blank sucrose gradient profile to establilh baseline. Spgctral Examination Figure 6 illustrates typical spectra of homogenate fractions. The abnormal spectra which display a high degree of and absorption (type B spectrum) were observed in at least 3 different batches of 25. P2°) for l X pellet material and in batch (P2°) in cysts (P50, P the crude l6,I00 g supernatant. In all these cases, 260 nm absorbency profiles of 15 - 60 1 linear sucrose gradients differed from the typical profiles (Figure l) in that they indicated ample PAN, a minimal ribosome peak, a significant peak of material sedimenting faster than that of the presumed ribosomes. RNAase treatment moved essentially all of these 260 nm absorbing materials to the top of the 37 the gradient: EDTA treatment resulted in two peaks sedimenting slower than ribosomes (Figure~5). The spectrum of the heaviest of these EDTA peaks was checked once and found to have extremely high and absorption. There did not appear to be any correlation between these unusual spectra and any particular experimental condition, including the use of NP-60, which exhibits high absortion at 275 nm and below 265 nm. The apparent biological condition of the cysts varied from preparation to preparation, in that the lipid fraction was either salmon colored (common) or bright red-orange. In all experiments in which 1 X pellet material gave abnormal spectra, a layer of pigmented material (salmon colored) was found over the clear 1 X pellet on the bottom of the centrifuge tube. This top layer was removed as well as possible before dissolving the l X pellet. It was found on one examination to have an abnormal (type 8) spectrum with slight absorption at 257 nm superimposed. In this work it was only possible to take spectra of continuous sucrose gradient fractions when manual dripping was employed. In no case (6 homogenizations) was an RNA spectrum found for PAM fractions, ewen fps one cast batch where the 16,000 g supernatant and l X pellet. spectra, and the sucrose gradient profile itself ( 58 1 PAN) had all been normal. In one cyst batch (P25, which had abnormal 16,000 g supernatant and l X pellet spectra) spectra from three representative fractions of the unusual sucrose gradient all gave protein spectra (type C spectrum) with a maximum at 285 nm, minimum at 265 nm, and 260/280 ratio of .56, 285/265 rationof 2.7. Polysomes should have an A26OIA280 ratio of about 1.85 (Vessey and Keck, 1970). 38 Part II: Poly dT-Cellulose Chromatography RNA Prgpgrations Four different Novikoff ascites cell RNA preparations were used in the experiments designed to characterize the poly dT - cellulose column behavior. The total RNA preparation defined as R1 was made from cells taken from rats which had been injected with ascites cells 8 days previously. Its spectrum exhibited a 2601280 absorbency ratio of 2.0. RNA preparations R2 and R5, made from rats injected 7 days previously, had 260/280 ratios of 2.2 and yields of roughly 20 “260 units per gram ascites fluid. Preparation Ra from 7 day cells had a 260/280 ratio of 2.5 and a yield of around 10 A260 units per gram ascites fluid. The procedure used during preparation R4 differed in that the initial phenol extraction mix was chilled 5 hours before centrifugation and in that the combined aqueous phases of the reextraction step were shaken with l/3 volume phenol for 3 minutes at room temperature before precipitatingsthe RNA in acetone. All RNA preparations exhibited absorbency maxima at 258 nm with minima between 228 (R4) and 233 (R5) nm. None of the preparations showed significant degradation of ribosomal RNA on 2 X polyacrylamid e gels. Artemia ggling_RNA prepared from dormant cysts had a 260/280 absorbency ratio of 1.75 with a maximum at 256 nm and a minimum at 230 nm. Approximate yields were 960 5260 units from preparation I dormant cysts (83 A260 units/ gram damp cysts) and 810 units from preparation II, incubated cysts (76 A260 units/ gram hydrated cysts). 39 coapavcoo comm page: mpcmEopsmwms cop pwmma pm pcommuama mmmcmm .vo>uomno hocmonownm mo Ho>oH new coapasnnopcw mo camcoa cmmzuon cowpmaopuoo poouap mm: phone .mopscas o: op ounces H scum umwnm> sodas 30am cadaoo ca mcoapashumpca popmw dogma ohms mcoauomum pmuam .mamemm ham mo coapmowammm poama psonpwx mace ponduommm mm mmuznoooum canamumopmsonno vumucmpm wm>ao>ca mash xcmam maesaoo omo s Hmonau haom no mean xcmam mcausn nm>mompo choumxomm < mo mmcmn .H mHnma mmo.uoao. mmo. u oao. omo.ndao. coapomnm pecan maasmm sovmmu nao.uaoo. mac. n Hoo. No.|doo. .casaoo madccsn H00. H00. H00. ummmzp vmmscs .nsop goon no o: 52.0 3.5 28.0 343 5.0 mo 2H0.o .msmp soon make mao.o «823:3 a 3:3 < .833 coapownm Characteriggtion of Poly dT-Cellulose Since the poly dT-cellulose technique was intended to be an assay for extremely small quantities of mRNA, it was first necessary to define the cellulose°s limits of adsorption. Background absorbency characteristics of the columns were monitored during blank runs (no samples). Ranges for at least 10 measurements under each condition are reported in Table l. The capacity of each poly dT-cellulose preparation to adsorb and release poly A was determined by passing various amounts of poly A over several columns made from each batch. Column A with a 1.4 cc bed volume of cellulose cmntainmd 0.65 g cellulose after drying 24 h at 100°. 5260 units/g dry cellulose were calculated using this factor. The data for A260 units of poly A reversibly adsorbed demonstrated that poly dT-cellulose columns could be expected to retain up to 6.5 A260 units of poly A per g dry cellulose (batch I) or up to ll units (batch II). regardless of buffer system used. This is roughly 0.3 mg poly A/g dry cellulose and 0.4 mg/g respectively. using a specific absorbency coefficient of 25 A260 units per mg poly A. Since poly A covalently bound to native RNA would have, theoretically, up to 7 times as much A26o absorbency per poly A sequence as synthetic poly A. the columns may be expected to retain up to 28 A260 units of poly A RNA per g dry cellulose. Aviv and Leder (1972) give a value of 0.5 g dry weight per 2 ml of their preparations of poly dT.cellulose, which is glee! dense than the preparation used in this work. One g of their preparation retained 160 . 200 ug poly A (using a 0.5 M KCl buffer for application) which corresponds to the retention capacity of these columns. Edmonds fiflh.1‘.;al 41 .oopooaaoo mcoapowum_s< on new mamemm one owoasaaeo mo madman oz u s 60382000 52 .8 8565 £33 0&3...“ 3503 How savages... mm .A80Cfiaé a henna no 0053 8&8 .0395 58050 Qfigfiafiooéonfiugxofioaum .5 053: 03338.8 new oonmmsnpaeu mm: «zm 039 .m ma pm accuse new: ouspaaoaaop soon pm oopoonpne 3: «gm H33. .052 .00. eBaopoom no cflepomé an 008 we... see, 030308? .onspamoaaov soon as Aways x.ro.ovm moundm ca oopooaaoo mmx m coapomum .ouspwuomaop goon no 4 momnsm ca :4 833$ «00 3 32.0 a 5.0 42 z 70: .335 5 038.38 mm: 4 0338.0 oopsom 43m no compocsm m we omOHdHHoonABovhHom «o eoapmooomm coavmmomo< .N eanma 0.3 m; 00.. 8; 0.09 .09 J 08m .maaoo 00000.4 mm m.p mm.w 00.0 m..mw .omp «moxdpoz um I o. 9.0 o.wmw .wa mm 3.0 :0. e mm.® N.0w mm 3.0 No. a d.: 0.m Somaaoohz flnmmyoomm. o p «km m < oomammfl «an oo~< mo m pcmoaom acoavoaum ca owm< cm~< mo cannon 42 and Caramela (1969) however report the binding of l - 2 mg poly A (applied in 0.1 M NaCl) by l g dry weight poly dT-cellulose. The total recovery of applied RNA ranged from 76 to 114 X with 94 - 100 1 being most common. Edmonds et al.(i97l) report 80 - 90 2 recovery of poly A. Faust et a1. (1973) report that 1.5 Z of poly A applied was never eluted from poly dT-cellulose except upon washing with 0.1 M KOH, but that 92 X eluted in 0.01 M Tris, 25°. The extremes of recovery range observed in the present work may be due to the use of uncalibrated micropipets or to drop losses occurring during manual collection of fractions. values for percent RNA reversibly adsorbed (8 fraction) as a function of RNA source are reported in Table 2. Values for percent RNA eluted in buffer 8 are not considered significant relative to experimental variation when the sum of absorbency units in the total 8 fractions fell below 0.1 A260 unit. Exception was made when the fraction‘s volume was less than 1 m1 and eXperimental A260 values in individual fractions was above 0.04 Azoolml, the upper limit of background absorbency (Table I). By this criterion, no significant mycoplasmic RNA was found to bind to the poly dT-cellulose. Myco- plasm, as a procaryotic organism, was not expected to have poly A sequences in its RNA population (Green and Cartas, 1972). Novikoff ascites cell RNA prepared as stated, was found to have around 1.2 - 1.8 X of its total 5260 units adsorbed to polyde'cellulose. These experiments demonstrate that RNA is probably not being non-specifically and reversibly adsorbed to the poly dT-cellulose columns. The efficiency of polymer adsorption by poly dT-cellulose was 43 evaluated by passing various fractions over the alumna a second time. If adsorption is indeed specific for poly A sequences (or any other characteristic), one would eXpect to find no RNA from an A fraction adsorbed by the column in a second pass, and no RNA from a 8 fraction not adsorbed. Results show a definite trend in this direction. RNA preparation R1 shows 1.3 X in the 8 5 fraction after one pass, while the fraction 8 obtained when the ‘ fraction A of the first pass was run through the column a second ‘ time, showed only 0.2 Z retention. Total RNA preparation R4 had 1.3 1 in the 8 fraction, but preparation Ra's A fraction had only 0.1 X in the 8 fraction obtained by a second column pass. Fraction 8 from the original pass of preparation R4 shows 111 X in the 8 fraction after a second pass. However, in the case of passing fraction 8 RNA over the column for a second time, 46 % of the total applied A260 units appeared in the nequ fraction, giving a total recovery of 157 X, a phenomenon which is hard to explain. Faust et a1. (1973) found that 98.6 X of rRNA (mouse myeloma tumor) was not adsorbed by poly dT-celluloae, while 1.6 % of 18 5 RNA and 0.8 X of 28 S rRNA are reversibly adsorbed and there is 1 X contamination of the 8 fraction by rRNA. It is not known if this indicates the presence of poly A sequences in rRNA or rather non-specific adsorption by the cellulose. Novikoff ascites 8 fractions (poly dT adsorbing RNA released at room temperature with low salt) was isolated for use in standardizing methods needed to characterise anticipated Artemia saline 8 fraction RNA. There was a high degree of reproducibility of 1 8 fraction obtained from different columns using a given RNA preparation when the calculation was based on the A260 units recovered in fraction A. The assumption was made that "A260 units applied" is the less accurate figure due to the effect of systematic errors involving highly concentrated samples. The ranges of percent 8 fraction observed were, for different RNA preparations: R1,l.3 - 1.4 X: R2,0 - 0.25 1: R4, 1.4 - 1.6 13 R5,1.8 - 1.9 74. Characterisation of poly dT-Cellulose 8 Fraction Preparation for base analysis'iy collection of RNA on filters j followed by enzyme digestion: It was necessary first to establish the effectiveness of the method; that is, the completeness of RNA ll‘c-labeled digestion by the enzyme solutions. 1.16 A260 units of 5, £2;;_RNA (49,000 cpm in Bray's counting solution) were precipitated with trichloroacetic acid and counted directly or treated by ensyme solution or non-enzyme buffer as described. Ensyme treatment removed essentially all counts (20,500 cpm before digestion(contrel) ;to 111 cpm after digestion). Buffer washing was nearly as effective, averaging 347 cpm for duplicates. The first 2 ml of ethanol rinse contained: 201 cpm and it can be concluded that essentially all counts have gone into the buffer solution. Since more buffer wash also removed the precipitated RNA from the filters, it was necessary to assure the effectiveness of the enzyme digestion. A test digestion of 112 A260 units of Pseudomonas putida rRNA was carried out according to the method described, using enzyme solution 11. At various times samples of the mix were spotted on Hhatman 3 mm paper and electrophoresed in 0.05 M Tris acetate pH 8.75, at 4 me for 1.5 h. After 60 h, all visible A26o absorbing material was 7. I .. . p: 1. :. J .. . .. .. .p. “o. 1. . .. J. 0. .. .. .2 .. . .5 . ... .. ._ .. . o.‘ .. .._ .. E I. .. .o. _..o .. .e ._ ; . ._. 1. : .y .* . . . . - a. .a .n r. . ... .. q. as . . .. _.. _ o '. ... ... _... ... .. ._ .. .. .n 2. .‘k .. . ._ .. .. ... __.: 3 A: a: y. _. .. I. . . ... 8.. . e .. _.o u.. =- .. . ...... .. 3. u... .. .4 .. . ... .. .. . . . O .. .0. .. . Ta. =. .M . . .2 .: .-. .... .J . .. . ... L . .. .W .1”: u .. .. .g n. _._.._ ... ._ .. .H .2 .7” .. ... .. .. r” ... 9. .. 4:. ---,-- -O-cs'--. . . t. .t .. t. z. .3 . . z. .. .. 0.. e... H. . 4.... u ._" u ... 2 n." a .u .. n. .. .... . g . m.“ .u .3.“ ._ z. .r. .._. H.” ___. i r. ,. .2... oh ....o.§\ .0‘ I, .3 ".aw ... v. 2%. 2 .._ .; _. .._. .... . .. r. . H." . .: i . ... ..._ ... . . l. :H .. a. e .. . .2 .. . . I... .2 .2 .. . .. .. .. ... .H .H .2 J... .. _ 2. ..... .. .. h. u... h. 0,. . m... a 0...... 2 .H . “3 “J ‘. :. :. .gu. Relative Response —+ Retenilo n 'l'lme —->- 47 .noomosvom <_haom Gamecoo op woesmonm on one .onsvmnoasop Soon .nommsn camconpm canon 30H ma pcwsao Heaven. on» mm: m coapomnm .0: .amnnsn newsman. once. gene an mamas» Hansen. one mm: < coneomnm ..m moapenmaonn was com: «at Hmpoe hemmnmopmsonso omoasaaoo.so haom hp oopmcoapomnm hamsowsonm 42d Haoo mopaom< nnoxn>oz non condense moapwe omcoammm .o.q.m memonaeso .m magma 0.m :.H A.H N awn m.a 3.0 0.0 a awn m coneomnm e consume. «we Hmpon moaaemm 0.0 \ 0.< mosam> oumoaaasn 48 found in the nucleotide and nucleoside regions. .jll._ln‘1!lllj Various known standard RNA's and polymers were prepared for base analysis as described, and injected into the 6.1.0. in order to obtain values for response ratios in the G.L.C. system. There was no clear separation of U or G derivatives from those of C (Figure 6) and, in general, the system proved unreliable .~ possibly due to incomplete derivatization of the enzyme digested T] samples which still had a relatively high concentration of salts after lyophiliution. ' . The base ratios 0:0 and GiA varied independently from day to day for any given RNA type as can be seen comparing the difference between Ala and BIG ratios on any given day. For example, Novikoff total RNA on day 1 had an A/G value of 0.4 that of its U/G value, while on day 2 the AIS value was 1.74 times that of the U/G ratio(Table« 3). The only consistent trend in comparison of ratios occurs with Novikodf 8 fraction where the AIS ratio is higher than the U/G value in all samples run. GLC chromatographs (Figure 6) did seem to indicate higher concentrations of adenosine in the RNA in fraction 8. T -RNAese digestion of poly dT-cellulose fractions: A or B1 1 fractions were obtained by poly dT-cellulose chromatography of Novikoff total RNA, subjected to Tl-RNAase digestion as described and re- chromatographed on poly dT-cellulose. A4 RNA (retained by the column in high salt at low temperature only) yields low or insignificant (less than 0.1 A260) reversibly adsorbingA260 units after Tl-RNAase digestion as would be expected if material is primarily ribosomal RNA which is T1-digestible. Gel analysis (Figure7 ) does indicate considerable contamination by rRNA. Fraction 81 (retained in high salt, 69 .oan soomnamm co sonamnmopmeonnoon an: em nopmcmwmew coapomnm one can: vocwmnoo A HH moon V some nmeao.uocos esp mm: N coapomnm .cssaoo omum Xenmcoom op ooaaaam one: .0: .nonnso npmcmnpm canon ewe: ca emoasaaooueo mace no oocwmpon soc Hmwnopms v oceanomnm 4 mcdpasmon one .mmoasaamo-uo haoa co nosnmnmopmsonnoon new woman He ho nopmomao mapcosdomosw coop ow: moan: anamnmopmeonno omoasaaooueo haoq Bonn mcoapomnm m one: H ncoapomnm .meocosaow <.naom caeucoo oa passwona me new unspenonseu soon .spmconem canon 30H pm naco cesaoo owoasaaoogao neon song oopsao mm: m compownm .moomnsmz. xa.0 a. e00 eonaamo mm: neewnmopmeoneo nn< hnqmnmopmsonno encamaaoo-en haom hp occamnpo meowpomnm «an no epoouonm coapmomam omwoaoscoofiu as no mammamc< omum xmnmnqmm .: mason 0n.0 No.0 mu N HH.0 m.m mn H 00.0 s.n me n NH.O OH.: Mn am mn.0 0n.0 0: 0 0n.0 mm.n an n oaamonm om o movmnaom no oopmoman coapmnmaonm uspmemna Amnoeao.nocofiv HH xmom op . on mo owmpm mo coapocsm m we omoasaaoo-en haom co mocmnuomo< mod mo woapmauopownmno "moaamm masopu< seam umumooum 43m .m canoe no.0 00.0 0.0 m.m0m mam m. ®:.0 0.0 H.0 m.mna mam m 53.0 0.0 H.0 n.0NH .03H m 42m Aommv swan empmpaoaH an m .HHH 0.0 0.0 0.0 0.:Hm m.oo~ m. o~.o s.o 3.0 m.HmH m.oom m om.o a.o ~.o H.amH m.maa m Amom v Aommv «we ammo nopmoeocH an m + Awom 0 «am ammo pcmsuom .HH pwoa madame ... mm A o: .nmpmpvanop v um>0 pcmEpom .H 1‘ m coapoaua m .34 < oaaaam ca accouom coapomam ca 00~< oomq cesaoo 54 the oligoeer peak from Sephadex G-50 chro-atography of T‘-digest of ‘1 A“ and the R1 81 fraction. respectively, show only light ( 5 - 9 S) heterodiaperse staining material as one would eXpect from T‘-stable poly A. Poly dTBBellulose Adsorbing;RNA in Total RNA from Artemia aalina RNA preparations fro- doraant cysts and fro- incubated cysts FT (8 h) vere passed over poly dTBcellulose coluans to deter-ine if a there was a poly dTbadsorbing fraction in these RNA populations and ‘ 1 whether or not such fractions would differ quantitatively as a i J function of developeental stage. The doruant Arteuia £3112; cyst RNA was lost due to error but both the 8 hour incubated cyst RNA and the mixed dormant plus 8 hour incubated RNA sanples exhibited real, albeit snail, alounts of poly dT-cellulose adsorbing RNA. The 1 B fraction value for 8 hour incubated cysts is 0.b7 X and for the mixing eaperilent, 0.26 z. a value one would eXpect if the cor-ant cyst RNA (80 x of the total RNA of the seaple) has a B fraction of about 0.1 %. Pre-incubation of the total RNA preparations with poly U results in essentially complete disappearance of the 3 fraction as one would eXpect if this fraction's adsorbanee to the poly dT-cellulose is dependent on free poly A sequences. 55 DISCUSSION Part I: Polysome Isolationn use A maximum yield of RNAase-sensitive RAM of 40 - 50 X of total absorbency could be isolated from incubated Artemia cysts, givenle moles] preparation from normally developing uncontaminated .1 cysts. This means that a l X pellet yield of 7 A260 units per g of wet cysts would produce 40 5260 units of RAM from 15 g cysts. Assum- \ ing that 3AM is entieely polysome material, free of contaminants and r ‘y that mRNA does represent 1 - 2 X of petpeemes' 260 nm absorbing material (Zomselyvet al.. 1970; Evans and Lingrel. 1969), and that 100 1 recovery of the RNA can 'be effected, A - 6 A260 units of mRNA could theoretically be obtained from standard preparations. Yeast sphereplasts have usually 70 - 80 X of total cellular RNA in polysoues (Vessey and Keck, 1970: McLaughlin, et al.. 1973). 5. ggli_have 25 - 80 1 of total ribosomes as polysomes, 60 z of the total lysate “260 as polysomes (Tai, et al.. 1973). Collagen polysomes from 3T6 fibroblasts account for 66 - 77 X of the ribosomal A260 units. (Lasarides, et al.. 1971). The proportionally high percent of ribosomes in the Artemia ribosomal pellets may be due to mechanieal disruption of polysomes during extraction or to cold-induced runoff (Fuhr, 1971i Friedman, et al.. 1969), i.e. completion of already initiated protein molecules with resulting release of ribosomes while further initiation is pre- vented by temperature sensitive factors. In the Arte-is polysome extraction procedure used, the intact cells were by necessity kept at 6° for at least 5 minutes prior to disruption during which period runoff could occur to a substantial degree. Mechanieal disruption would 56 result in formation of monosomestNA complexes still active in protein synthesis. Such complexes are stable at high ionic strength (Zybler, et al., 1970), whereas cold-induced runoff ribosomes or naturally occurring ribosome pools are dissociated. It may be possible to determine whether, in fact, a polysome pepulation has been mechanically disrupted..creating the observed monoribosome population L‘v“ ‘ l by examining the latter in high ionic strength buffer sucrose gradients. Characteristics of PAH Considering merely the optimal yield and the difficulty and 7 nondependabiiity of obtaining such a yield due to variations in hatching and to contamination, Artemia cysts do not seem to be a favorable system, pg;,gg, for extraction of mRNA from polysomes. This becomes even more obvious when one considers the nature of RAM isolated from Artemia cysts as defined by RNAase sensitivity, EDTA dissociation and spectral properties. Remarkably, although RAH appearedtbo be almost completely RNAase sensitive, it was never found to have a typical RNA absorption spectrum. Hith the exception of one experiment(P25H) where it displayed a typical protein spectrum, it mioays gave the unusual end-absorption spectrum (type B, Figurel. ). which was also often found for l x and 16,000 g supernatant fractions. This type of spectrum is assumed to be due to the effects of con- taminants. There is excessive trehalose and glycogen in dormant cysts which may contribute to this absorption. The glycogen probably also accounts for non-RNAase sensitive material observed in sucrose gradient profiles. In some tmdbvidual homogenates RNP particles and glycogen contamination nay account for most of the 260 nm absorption‘ Finamore and Clegg, 1969 ). 57 In these cases a type B spectrum is found for l X pellet or even for 14,000 g supernatant, and RNAase digestion of the RNP particle contaminant yields only very light products that remain at the top of the sucrose gradients ( Figure 5 ). The RNP particles may be largely protein resulting in the observed protein spectrum for RAM. The RNAase-insensitive RAH present in EDTA gradients may also represent excessive amounts of glycogen, RNP particles protected from RNAase by effects of EDTA-induced aggregation ( contaminants largely seem to be RNAase sensitive in Mg‘z buffers), or possibly aggregates of double stranded RNA. Olsnes ( 1970 ) also observed a two phase ribosomal pellet similiar to that sometimes observed here. RNP contamination in this clear bottom pellet varied with centrifugation time but was always present in the upper layer. These contaminants were not affected by EDTA, were rapidly labeled and contained DNA-like RNA. He did not test for RNAase-sensitivity in BDTA. Significance of RAM Since this work was begun McClean and Warner ( 1971 ) have reported a large population of heavy polydisperse RNP particles in Artemia aauplii based on results from methylated albumin on Kieselguhr ( MAX ) column analysis of RNA, extracted at pH 7.5 with phenol at 60°. This fraction varied from 15 to 21 percent in prenauplii and in various stages of aauplius development, had DNA-like base ratio and was rapidly labeled. The hypothetical RNP contamination in the present study may be related to the RNP particles reported in McClean and Harner's work and may be similar to the DNA-like RNA contaminants of Olsnes' system. H— 58 As such, these RNP particlescontaminants may be relevant to the question concerning presence of pre-formed mRNA in dormant Artemia cysts: there is some evidence that RNP particle "contaminants" in otherr systems may be directly related to mRNA metabolism. ‘Aviv and Leder (1972) report template active 18 8 RNA which directs amino acid products identical to those made by 9 S RNA of their system. This 18 S template RNA may well be an intermediate between HnRNA and mRNA as found in polysomes and may yield a smaller piece of protein-bound DNA-like RNA upon conversion. Greenberg and Perry (1972) propose that the RNA more of their RNP-contaminant has a more or less holo- geneous size and that it is only non-specific binding of proteins that results in polydisperse sediementation behavior in sucrose gradients. They found no evidence for poly A sequences on the RNA core of the RNP particles. Part II: Poly dT-Cellulose Chromatography Characteristics of 2211 dT'Cellulose The retention capacity of the poly dT-cellulose preparations was judged to be sufficiently efficient to accommodate the levels of poly A RNA anticipated in natural RNA samples. In fact, essentially all poly dT-adsorbable RNA was adsorbed during the first pass over the column, and essentially none during the second pass (0.2 X). The general background adsorbance of the 8 fraction from poly dT-cellulose columns when non-poly A RNA samples are applied seems to be around 0.2 x of the material applieds RNA preparation R2 3 0.25 X; mycoplasm RNA 0.4 2; fraction A RNA on a second pass: 0.2 X. The nature of this material is not presently known. Faust, et a1. (1973) have found that their 8 fractions are contaminated by 1 % rRNA; 1. 6 z of applied 18 SyRNA and 0.8 X of 28 S rRNA was found to be reversibly 59 adsorbed and eluted at low K01 concentration. McLaughlin et al. (1973) have found less that 0.06 x of yeast rRNA binds and that 0.5 x of poly A RNA doesn't bind. Whether this rRNA has long poly A sequences (McLaughlin et a1.c1aim that poly dT retains poly A sequences longer than 20 nucleotides) or whether it is retained on the basis of secondary structure interactions with the eellulose, is not known. f] Other classes of RNA, such as those in RNP particles, may also be involved in this apparent adsorption to poly dT. The variance in q total recovery (90 - 115 X) and literature reports of consistent r loss of 3 - 19 X of the applied “260 units (Float et al., 1973) indicates that there may be some non-specific binding phenomenon occurring on the cellulose, which may account fbr the binding of some rRNA. Characteristics of 8 Fraction RNA The poly A content of the reversibly adsorbing 8 fraction RNA was not definitely established due to difficulties with the GLC base analysis system. Only a weak indication dor higher.A:G ratios in 8 fractions was observed. One of the most serious difficulties was the small amount of material with which to work. Given a 3‘A260 8 fraction sample, at most 20 X (Faust et al., 1973), more likely 12 - 14 X (Raskas and Bhadari, 1973; McLaughlin et al., 1973) or 0.36 “260 could be expected to be RNAase-stable, i.e. poly A. Since radioactive labels could not be employed, carrier RNA methods could not be used to facilitate isolation of this fraction from contaminating nucleoside digestion products. In this study, several attempts were made to determine the presencelof oligomera after T1 RNAase digestion of 8 fraction RNA 60 under conditions where poly A is known to be stable. Around 15 - 17 X of the T1 digested fraction was seen to run as oligomer through the 6-50 Sephadex column, but considering the background of 10 1 remaining after these digested samples were cleared of poly A by poly dT.. cellulose chromatography, one concludes that only 5 - 7 X of this fraction is resistant to T1 RNAase. Assayed directly by poly dT.. cellulose, the Ti digested 8 fractions show 7 X resistance. Literature reports commonly cite 12 - 15 1 Tl-RNAase resistance for poly dT.- cellulose adsorbed fractions (Raskas and Bhaduri, 1973) McLaughlin, 1973), but these selues generally are based on 1“C-adenine labels and may not correspond to ratios determined on the basis of A260 . 32 Phust et a1.(l973) cites 5 - 6 x of the total P counts or 2.5 1 of the A. units of the total applied RNA as the percent of mouse 260 myeloma RNA retained after Tl-RNAase digestion. Values of percent RNAase-resistance observed here might be affected by exonucleolytic digestion of poly A sequences at some stage of isolation,naking them too shoot to bind, or is naturally occurring shorter poly A sequences in Novikoff ascites cells. The gel patterns (Figure 7, slots 1 and 2) strongly indicate presence of low'molecular weight resistant species in T1 RNAase- digested fractions. The highly heterogeneous nature of this material may also be an indication of artificial degradation of the poly A RNA during isolation, as other investigators consistently find distinct banding in the a - 6 S regions of their gels. The gel patterns of 8 fraction RNA not treated with RNAase (slots 3 and A) display heavy heterogeneous species which may indicate aggregation of poly A RNA 61 (usually 6 - 7 S) or of degradation products. The poly dT-cellulose reversibly adsorbing material (8 fraction) from Novikoff ascites cells had been shown, at this point, to include approximately 7 Z of 5260 units~es TI RNAase resistant sequences. 8y commonly accepted convention, it can be said to contain 7 Z in poly A sequences and is presumed to be mostly in mRNA (Faust, et al.. 1973; Jelinek, et al., 1973). Fraction 5" The nature of the Novikoff ascites cell A'.fraction RNA (released from poly thcellulose in Buffer A, at room temperature) has not been investigated. Synthetic poly A also produceszthis fraction and it is possible that.A' represents specific poly A adsorption to the columns when natural RNA samples are involved. Tablet. results indicate that there is considerably less RNAase-resistant poly dT adsorbable RNA, percentage-wise, in A. RNA than in 8 RNA. Since gel analysis (Figure 7 ) revetls considerable contamination by rRNA, the AV RNA may depresent a mixture of rRNA's retarded by the columns' structure and poly A RNA weakly adsorbed by the column. Faust et a1. (1973) find up to 2 % contamination by rRNA even in 8 fractions; they also observed an A'-like fraction which was too small to analyse. These authors theorized that the weak binding of po1y A RNA in this fraction was due largely to shorter poly de sequences on the cellulose. However, Swan et a1. (1972) also observed two fractions eluting at low salt and they propose that the difference in binding strength depends on the length of the poly A segment of the RNA. They find that the two fractions direct ig_yi§gg protein synthesis of the same product but that the 8 fraction with, theoretically, longer poly A sequences is more active (Swan et al., 1972). ‘ufa‘... ' up. .0. \. ._~ ' hm: 62 One should be able to distinguish between these two theories by comparing S values of T1 RNAase-digested samples by gel analysis or by rechromatographing fractions from each on the poly dT-cellulose column. According to the theory of Swan et a1. (1972), the 8 fraction should elute as 100 X 8 fraction during the second pass. .A 8 fraction should fractionate between.A',and B if it is dependent on poly dT length. The results reported here, although complicated by recovery of excessA26o units, indicate that fraction 8 RNA rechromatographed does not yield significant A260 units in the A' fraction (0.2 X), *9? is plateau}! We’d in the new I fruition. Due to ambiguities about its nature, A. fractions were in general ignored for the purpose of this study. Novikogf‘Aacites Cell RNA This RNA was shown to have between 1.3 and 1.8 I of the isolated total RNA in the reversibly adsorbed 8 fraction, depending on the particular RNA preparation. This is low compared to most other systems reported in the literature: 2.5 x for mouse myeloma (Faust, .u .1., 1973); 4.8 x of 14c poly A in rabbit embryo mm 1‘‘c poly A in yeast RNA (McLaughlin (Schuts et al.. 1973): 2.4 - 4 I et al.. 1973). It is possible that the Novikoff ascites cells are naturally low in poly A $2.21!!) but the variation of 1.3 - 1.7 X among various preparations suggests that experimental artifacts such as endonucleolytic digestion or loss into the phenol layer, are the more likely cause of the lower percentages. 63 Artemia saline RNA The former experiments sought to establish the reliability of the poly dT-cellulose system. Unfortunately there was little time at this point to devote to the application of the method to the central questions concerning Artemia ggligg_mRNA. One poly dT- cellulose experiment was run with.Artemia RNA samples to compare 'dormant cyst RNA with incubated cyst RNA. The results (Table 5) show that there are extremely low but real levels of poly A RNA in such samples. The levels of percent 8 fraction are not, at face value, significantly above the system's background of 0.2 x but prior complexation with poly U eliminates fraction 8 completely, indicating specificity for poly dT sites. The percent fraction 8 seems to increase with incubation time. Experimental conditions may have resulted in loss of poly A in the same manner that Novikoff poly A RNA seems to be lost, and the.A;tggia results may be spuriously low. Of course, there is no guarantee that all Artemia RNA's carry poly.A (McLaughlin et al., 1973), or that the poly A RNA would be fully adenylated in dormant cysts. That is, the poly A semencu may not be long elimgh to effect rut-don by the poly dT'cellulose. If preformed message is present in the dormant cysts, it could need processing or activating by adenylation, or its dormancy may be solely a question of non-availability of the ribosome binding site due to specific protein complexing as Lee et a1. (1971) suggest for mouse sarcoma 180 ascites cells, that are translationslly inactive due to amino acid starvation. If there is no stored message, one would expect to find rapid metabolism of nucleotides in the dormant cyst entering incubation since the cysts at this stage are impermeable to 64 nucleotides since they are virtually devoid of ATP and free guanine ( 90% of all nucleotides in the cyst contain guanine: Finamore and Clegg, 1969 ). Conclusion On the basis of the data reported here one may infer that there probably is a small amount of poly A RNA in dormant Artemia cysts and that the amount increases as metabolism is resumed. This could indicate either a small adenylated-template pool ( mRNA plus HnRNA ) in dormant cysts or simply a lack of adenylation. Contamination of the acetone-precipitated RNA preparations by guanosine dinucleotides and the naturally occuring high proportion of rRNA in all Artemia RNA preparations ( Finamore and Clegg, 1969 ) may account for the low level of poly A RNA ( 0.1.1 of total RNA A26oapplied ) observed in ca... mu ‘ preps from incubated Artemia cysts. Total RNA preparations were used in poly dT-cellulose chromatography rather than the more customary polyscmal RNA preparations since intact polysomes could not be obtained with any consistent regularity or purity from Artemia cysts. Further efforts should be directed at maximising RNA extraction efficiency and minimising nucleolytic artifacts. It would then be of interest to determine the variations in percent poly A RNA in Artemia as a function of metabolic state ( dormant versus incubated )3 as a function of RNA localisation ( nuclear RNA versus cytoplasmic RNA )3 and as a function of RNA species, e.s. HnRNA. It would also be interesting to examine changes in poly A length with respect to these variables. Ultimately the cell free protein synthesis activity of poly A RNA, and possibly RAH-derived RNA, should be investigated to determine both the sise of the mRNA pool in dormant cysts and the percentage of potentially template-active RNA which is adenylated. 65 REFERENCES Adesnik,M. and J.E. Darnell, 1972, J. Mol. Biol. 21. 397 Adesnik, M., M, Salditt, H. Thomas and J.E.Darnell, 1972.. J. Hal. Biol. 11. 21 Aloni, Yosef, 1973, Nature 24; , 2 Anderson, D.T., 1967, Australian J. Zool. 12 , 47 Ascione, R. and R.8. Arlinghaus, 1970, Biochem, Biophys.Acta 294, 478 Avadhani, N.G., M Kwan, P. 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