cloning of total mrna populations from adult and embryonic mice

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 223, No. 1, May, pp. 140-148, 1983

Cloning of Total mRNA Populations from Adult and Embryonic Mice

MICHAEL J. TOCCI,’ KENNETH A. FLEMING,2 AND JOHN J. MONAHAN

Roche In-stitute of Molecular Biology, Department of Cell Biology, Nutley, New Jersey 07110

Received November 18, 1982

Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated -400,000 for the 13th day embryo and neonatal mouse and -610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the “G-C tailing” method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by “nick translation” using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back- hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs.

Very little is known about how growth and development is controlled at the molecular level in developing animals. The continuously changing nature (as the embryo develops) of the source material often has made even superficial molecular analysis difficult. Studies in the past were often confined to the measurements of rates of RNA synthesis during embryogenesis, the content of RNA/cell, or the effects of RNA inhibitors on the process of embryogenesis. Recently, some progress has been made in the isolation and analysis of poly(A)- RNA from early mouse embryos using classical techniques (l-5). Quantitative as well as qualitative differences were seen as the embryo developed. However, fur-

’ Present address: Department of Biochemical Ge- netics, P.O. Box 2000, Merck Sharpe & Dohme Re- search Laboratories, Rahway, New Jersey 07065.

’ Present address: University of Oxford, Depart- ment of Pathology, John Radcliffe Hospital, Head- ington, Oxford, 0X3, 9DU, England.

3 To whom all correspondence should be addressed.

ther progress along these lines will prob- ably be slow because of the limited supply of starting material and the monumental amount of work involved in carrying out such studies.

The application of recombinant DNA techniques has brought new hope to the possibility of unraveling the complex process of multigene expression during embryogenesis. With this technology specific RNA species can be obtained and at least some attempt can be directed toward searching for nearby regulatory DNA sequences. Perhaps equally significant, cloning methodology provides an essentially unlimited supply of cloned DNA from a small supply of initial starting material. For a study of mammalian embryogenesis and development often only minute quan- tities of material can be isolated practi- cally. We have chosen the mouse embryo as a model system for studying the molecular biology of embryogenesis. It is well characterized in terms of the systems morphological development, embryogene-

0003-9861/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved

140

EMBRYONIC AND ADULT RNA CLONE BANKS 141

sis can proceed to a reasonably advanced stage in vitro (7-lo), and a number of in- teresting developmental mutants exist (10-11).

MATERIAL AND METHODS

Animals. Embryos and neonatal mice were obtained from SWR/J X SJ/L matings (The Jackson Laboratory, Bar Harbor, Me.). Embryos (13th day) were dissected from adult female mice, trimmed free of extraembryonic membranes, rinsed twice in phos- phate-buffered saline, and frozen immediately in liquid nitrogen. Neonatal mice were obtained on the morning of their birth and treated similarly. All animals were stored at -79°C until used.

Isolation of poly(A)-RNA. Total RNA was isolated from la-day-old and neonatal embryos as follows: approximately 20 g of tissue were homogenized in 200 ml of 20 mM Tris-HCl, pH ‘7.6, 1 mM EDTA, 2.0% SDS,’ and 0.5 mg/mi proteinase K (E. Merck, Darm- stadt) which had been preincubated at 45°C for 20 min. The material was homogenized for 60 s in a Po- lytron homogenizer (Brinkman, Westbury, N. Y.) incubated at 45°C for 1 h and then extracted with an equal volume of buffer-saturated phenol. The aqueous phase was ethanol-precipitated, the nucleic acid pellet dissolved in 10 IIIM Tris-HCI, pH 7.6,5 mM EDTA, 0.01% SDS, and dry CsCl was added until a final density of 1.70 g/cm3 was achieved. Total RNA was pelleted by centrifugation at 30,000 rpm for 24 h at 10°C in a Beckman 70Ti rotor. After centrifugation, the supernatant was decanted, the RNA pellets dissolved in water and the solution made up to 0.5 M

KCI, 20 mM Tris-HC1, pH 7.6, and 5 mM EDTA. Poly(A)-RNA was isolated by two cycles of oligo(dT)- cellulose column chromatography as described by Aviv and Leder (13). The poly(A)-RNA was then ethanol- precipitated and stored in water at -7O’C. Samples of RNA were monitored throughout the purification for degradation and DNA contamination by electrophoresis in 1.5% agarose gels containing 10 mM

CHsHgOH as described by Bailey and Davidson (14). Total RNA from adult mice was isolated by phenol

extraction (15) and purified as described above. Synthesis qf‘ single-stranded complementary DNA

Iss-cDNA). The method for synthesizing ss-cDNA has been described previously (16). In general -7-15% of the poly(A)-RNA was converted to ss-cDNA in

4 Abbreviations used: AMV-RT, avian myelobas- tosis virus reverse transcriptase; TDT, terminal deoxynucleotidyl transferase; CFU, colony forming units; ss-cDNA, single-stranded cDNA; SDS, sodium dodecyl sulfate; ds-cDNA, double-stranded cDNA; NTBP, nucleotide base pairs, Tc’ tetracycline-resistant; Amp”, ampicillin-sensitive.

these experiments. For cloning purposes it was usually necessary to generate -10 fig of ss-cDNA.

Synthesis of double-stranded cDNA (ds-cDNA). Af- ter hydrolysis of poly(A)-RNA with 0.3 M NaOH, the ss-cDNA was converted to double-stranded material using reverse transcriptase (17). Approximately 40- 60% of the ss-cDNA was coverted to ds-cDNA as monitored by S,-nuclease sensitivity.

Removal of hairpins from d.wDNA by S,-nuclease. Hairpins were cleaved from ds-cDNA preparations by digestion with S,-nuclease. The conditions used were critical. For optimum yields, the reaction was carried out in 20 mM NaOAc, 300 mM NaCI, 4.5 mM

ZnClz, pH 4.5. Digestion was for 20-30 min at room temperature using 100 units of S1 nuclease (Sigma Chemical Co., St. Louis, MO.), per microgram of ds- cDNA at a concentration of 1200 units/ml. The reaction mixture was diluted twofold with 0.1 M EDTA, phenol-extracted, and the aqueous phase precipitated with ethanol. Under these conditions 35-40% of the ds-cDNA was resistant to Si nuclease digestion. Sam- ples of ds-cDNA were assayed on 1.5% agarose-10 mM CH3HgOH gels before and after S1 digestion to determine the size of the cDNA and to ascertain whether or not the hairpins had been cleaved (16). ds-cDNA treated in this manner generally ranged from 200 to 1800 NTBP, with the average size being approximately 600 NTBP in length.

PuriJication of ds-cDNA by sucrose gradient centrifugation. Prior to “dCTP-tailing,” it was necessary to remove various small oligonucleotides from cDNA preparations by sucrose gradient centrifugation. Ap- proximately 1-5 Kg of S,-cleaved ds-cDNA in water was layered onto a 5-25% neutral sucrose gradient and centrifuged in a Beckman SW-40 rotor at 35,000 rpm for 20 h at 5°C. After centrifugation the gra- dients were collected and fractions containing ds- cDNA greater than 300 NTBP were pooled. These fractions were precipitated with ethanol at least twice. After precipitation the cDNA pellet was dissolved in water.

Terminal addition of dCTP to ds-cDNA. ds-cDNA was tailed with dCTP using calf thymus terminal deoxynucleotidyl transferase (TdT). The reaction mixture contained 100 PM dCTP, 1 mM CoCl,, 0.2 M K+ cacodylate, pH 6.9, and lo-20 Kg/ml ds-cDNA. Reaction mixtures were preheated to 37”C, 10 units of TdT per Fg of ds-cDNA was then added and the mixture incubated at 37°C for 3-5 min. The reaction mixture was then heated at 60°C for 2 min and frozen at ~70°C. Under these conditions 20-30 dC residues are added per end as determined by the incorporation of a-%PdCTP into acid-precipitable material.

Preparation of pBR322 DNA. The methods for pu- rifying and preparing dG-tailed, PstI cut pBR322 DNA have been described elsewhere (16). Briefly 200 Fg of pBR322 DNA were cut to completion with PstI restriction endonuclease. The DNA was phenol-ex-

142 TOCCI, FLEMING, AND MONAHAN

tracted, ethanol-precipitated, and dissolved in water. Terminal addition of dGTP to P&I. pBR322 was

tailed with dGTP using TdT under the same reaction conditions described above, except that the dGTP concentration was increased to 1.0 mM and the reaction allowed to proceed for 2 h at 37°C. The DNA was then phenol-extracted, ethanol precipitated, and dissolved in water. The dG-tailed DNA was centrifuged through a 5 to 25% neutral sucrose gradient using an SW40 rotor at 4°C and 38,000 rpm for 13 h. The gradient fractions were collected and assayed as previously described (16). Material with low trans- forming activity (i.e., linear-tailed DNA) was pooled, ethanol-precipitated, and used for transformation of E. coli.

Annealing dC-ds-cDNA and dG-pBR.322 to form chi- me& plasmids. ds-cDNA and pBR322 DNA were mixed such that the molar ratio of ends of the DNAs were approximately 5:l in 0.15 M NaCl, 10 mM Tris- HCl, pH 7.6, 1 mM EDTA, at a total DNA concentration of 4 fig/ml or less. The DNA was allowed to anneal at 58°C for at least 1 h in water and then held on ice (18).

Transfwmatimz of cells. Cells for transformation were prepared as described previously (18-19). Ap- proximately 1.5 ml of E. coli RR1 cells were grown to ODSN = 0.3, washed, and resuspended in approximately 6.5 ml of Ca++ medium. The competent cells were then mixed with 3.3 ml (-10 pg of DNA) of chimeric plasmid DNA, held on ice for 1 h with oc- casional shaking, heat-shocked for 3-4 min at 37”C, and l.O-ml aliquots plated onto large pans (-900 cm’) containing L media with 10 rg/ml tetracycline-HCl. The pans were then incubated at 37°C for 24-48 h. Recombinant clones were scraped off the agar surface into L broth containing 10 pg/ml each of tetracycline and streptomycin. The cells were washed once by centrifugation resuspended in 100 ml of L broth containing 5% (w/v) glycerol, and frozen in 2- to 3-ml, aliquots at ~70°C.

Transfomnation eficiency. The efficiency of transformation for these experiments ranged from 50,000 to 200,000 colony forming units (CFU)/Fg ds-cDNA. The average transformation efficiency was approximately 100,000 CFU/gg ds-cDNA.

Ampli$cation and isolation of plasmid DNA. The method for isolation of pBR322 and recombinant pDNAs has been described before in detail elsewhere (16).

Restriction endonuclease analysis qf cloned mouse cDNA. All restriction digests were performed in 6 rnM Tris-HCI, pH 7.6, 6 mM p-mercaptoethanol, and 6 mM MgClz. The salt concentration of each digestion was adjusted with the appropriate salt based upon the supplier’s recommendations and the enzyme used. All reactions were incubated at 37°C and analyzed by agarose-gel electrophoresis as described elsewhere (16).

Purijication of cloned cDNA inserts. Plasmid DNAs from each clone bank (-500 pg) were digested to completion with PstI restriction endonuclease (0.1 pg of DNA). The DNA was then phenol-extracted, ethanol-precipitated, and stored in water. PstI digested pDNA was layered onto a 5-25s neutral sucrose gradient and centrifuged in an SW40 rotor at 38,000 rpm for 20 h at 4°C. The gradient was collected and those fractions containing cDNA inserts but not pBR322 DNA were pooled and ethanol-precipitated.

Nick translation of cloned-cDNA inserts. Purified PstI-digested insert DNA used for complexity analysis was nick-translated as described by Rigby et ~1. using @P]dATP as label (20).

Complexity analysis of mouse embryo cDNA libraries. Liquid hybridization was carried out at 68°C in sealed Reacti-vials (Pierce Chemical Co.). Linear- ized plasmid DNA (using EcoRI) at various concentrations was mixed on ice with nick-translated probe (-5,000 cpm/vial) in 0.5 M NaCl, 0.1 M Hepes, pH 7.6, and 1 mM EDTA. The DNA was denatured on ice for 10 min by the addition of l/10 vol 1.0 M NaOH and then neutralized with l/10 ~011.0 M HCl. All reaction vials were then incubated for various times until the appropriate Cot values were obtained.

Cross-hybridization analysis. Cross-hybridization experiments were carried out by hybridizing either 32P-labeled ss-cDNA (prepared form embryo RNAs) or nick-translated cloned cDNA inserts (isolated from cDNA libraries) to poly(A)-RNA from different developmental stages, or back to the pDNA plasmid DNA from the cloned cDNA libraries. Hybridizations were incubated to a Cot or Rot of approximately 2500 mol s liter-’ under the same reaction conditions as described above, except that for RNA hybridizations the denaturation step was omitted.

S, n&ease assay. All hybridizations were assayed for S, resistant material in the following manner. To each hybridization reaction (-20 ~1) was added 225 ~1 of Si nuclease buffer containing 300 mM NaCl, 30 mM sodium acetate, 4.5 mM ZnCla, pH 4.6, and 5 ~1 (200 units) of S1 nuclease (Miles Biochemicals). Sam- ples were incubated at 37°C for 90 min and then assayed by binding to DE-81 filter discs (21).

RESULTS

The procedure we have used to synthe- size and clone cDNAs is simply an exten- sion of the methods used to clone unique mRNAs such as hemoglobin and ovalbu- min (22-24). For the isolation of RNA from 13-day-old embryos and neonatal mice the proteinase K digestion procedure gave good yields of poly(A)-RNA. However, with adult mice, yields were poor by this method. Instead, for adult tissue, a


A B

910

600 520 403

281

FIG. 1. Electrophoresis of total adult clone bank plasmid DNA after treatment with EcoRI and BglI. The large smear at position X in lane A, is due to the numerous inserts of cDNA that have been cloned into this region of the PBR322 plasmid. Lane B is the molecular weight marker of pBR322 DNA cut with AlUI.

multiple phenol extraction method (15) was used. In every case the yield and pu- rity of the poly(A)-RNA were carefully monitored by electrophoresis on methyl mercury hydroxide gels. Single-stranded cDNAs was made from poly(A)-RNA preparations using AMV reverse transcriptase as described under Materials and Methods. Usually, about 10% of the RNA could be converted into cDNA. Gel electrophoresis of the 32P-labeled cDNA was used to determine the size of the cDNA. In these studies, the cDNA ranged in size from 200 to 1200 nucleotides in length. Short cDNAs were removed at a later stage in the procedure (see below). Reverse transcriptase was then used to convert the ss- cDNA to its double-stranded equivalent. Approximately 50-60% of ss-cDNA was converted in these experiments.

The critical step in the procedure was cutting of the hairpin loops in the cDNA. The concentrations of both DNA and S1 enzyme were critical. Using the conditions described under Materials and Methods, up to 50% of the DNA was S1 resistant and suitable for cloning. As an assay for complete cutting, the DNA was analyzed on a denaturing gel. A twofold decrease in the overall molecular weight of a band was taken as complete S1 cutting (16). The DNA was then phenol-extracted, ethanol-precipitated, dissolved in dHpO, and sedi- mented through a neutral sucrose gradient. Material 8s and larger was pooled, ethanol-precipitated twice, and tailed with dCTP using TdT. After the addition of approximately 15-20 dC residues to the 3’- OH ends, the ds-cDNA was annealed with dG-tailed Pat1 cut pBR322 (16).

Suitable clone banks were made as described under Materials and Methods. The number of individual clones was counted and the fraction containing no inserts (background) was determined by plating aliquots of the cells on Tc and Amp plates. Only clones that were Tc’ Amp” were scored as containing inserts.

Figure 1 shows the pattern obtained when the total plasmid clone bank (in this case the adult bank) was digested with EcoRI and &II. The discrete fragment that would exist in unmodified pBR322 containing the PstI site was replaced by material that migrated as a broad smear (labeled X in Fig. 1). Figure 2 shows the optical density scan of such digests from each of the three clone banks (13-day-old, neonatal, and adult). The arrow points to the area containing the cDNA inserts. Curve D is the pattern seen for pBR322 digested with BglI and EcoRI. From the area under each relevant peak the fraction of insert cDNA sequences in each clone bank was determined.

To analyze the sequence complexity of each clone bank a small amount of each cDNA insert was labeled with 32P. The total insert population was separated from the plasmid DNA after digestion with PstI by sucrose gradient centrifugation (16).

When the 32P-labeled inserts were hybridized with an excess of plasmid clone


FIG. 2. Optical density profiles of photographs similar to the one in Fig. 6. The inserts position is indicated by the arrow. A, the profile for the adult clone bank. B, the profile for the neonatal clone bank. C, the profile for the 13-day-old embryo clone bank. D, the profile of pBR322 control cut with EcoRI and B&I.

bank DNA (linearized with EcoRI) from each under conditions suitable for a second-order reaction (25), renaturation curves were obtained as shown in Figs. 3- 5. For kinetic standards we used a nick- translated bacteriophage X DNA preparation and nick-translated DNA from three cDNA clones from a mouse liver library (16). From the base sequence complexity of these standards and the data in Figs. 3-5. The sequence complexity of the banks was calculated. This data is summarized in Table I.

Computer analysis of the data as described previously (27), yielded three component curves in each case (Figs. 3-5). The first two columns of Table I show the calculated Coti,z and fractional contribution of each component. The next column across shows the theoretical calculated Coti,a of each component if it could be isolated from the other components (26,27). From these values and the Coti,a of the standards described above, the total nucleotide sequence complexity of each component was caculated. The average size of the cDNA

h

%I -25 -20 -15 -1 0 -05 00 05 IO 15 20 25 30 35 ‘ LOGlO COT

FIG. 3. Hybridization of 32P-nick-trans1ated total clone cDNA inserts from 13-day-old poly(A)- RNA back to an excess of the same DNA. Varying amounts of driver plasmid DNA were incubated with -20,000 cpm of probe to the indicated Cot values. Hybridization was assayed with Si nuclease. The Cot values have been normalized to standard 0.12 M Pi conditions.


I p3.0

I 1 I 2.5 20 15 IO 0.5 0.0 05 IO I5 20 25 30 35 40

LOG,, COT

FIG. 4. Hybridization of 32P-nick-translated total clone cDNA inserts from 13-day-old neonatal poly(A)-RNA back to an excess of the same DNA. Varying amounts of driver plasmid DNA were incubated with -20,000 cpm of probe to the indicated Cot values. Hybridization was assayed with S, nuclease. The Cot values have been normalized to standard 0.12 M Pi conditions

n

t 01 I 1 1 I I -30 -25 -20 -15 -10 -05 00 05 IO 15 20 25 30 35 1

LOGlO COT

FIG. 5. Hybridization of “P-nick-translated total clone cDNA inserts from adult poly(A)-RNA back to an excess of the same DNA. Varying amounts of driver plasmid DNA were incubated with -20,000 cpm of probe of the indicated Cot values. The C values have been normalized to standard 0.12 M Pi conditions.


TABLE I

BASE SEQUENCE COMPLEXITY OF EMBRYONIC cDNA CLONE BANKS

Stage cotm

Fraction of cot Total sequence No. of Diff.

total isolated complexity (NT) cDNA clones

13th Day 0.01790

2.5590

76.3636

0.308

0.381

0.311

0.0055

0.9724

23.7590

Neonatal 0.0175 0.268 0.0047

1.2829 0.391 0.5029

100.0000 0.341 34.4100

Adult 0.0258 0.211 0.0054

0.5324 0.358 0.1901

81.8182 0.431 35.2636

5.998 X lo3

1.060 X lo5

2.591 x lo7

5.125 X 10”

5.484 X lo5

3.752 X lo7

5.888 X lo3

2.073 X lo5 3.845 x lo1

10

1,696

41,453

9

997 68,223

13

460

85,000

inserts in the 13-day-old, neonatal, and adult banks was 625, 550, and 450 nucleotides, respectively. The number of different cDNA species represented in each component could then be calculated from the total sequence complexity of the component (26, 27). This data is shown in Table 1.

From the total number of different cDNAs in each clone bank it can be seen that there is a clear increase in the sequence complexity of the banks for the 13- day-old embryo to the adult. This implies that there are sequences in the adult bank that are not present in the banks from the earlier stages. We have confirmed this by the data shown in Table II where 32P nick- translated probe from the cloned inserts of banks were cross-hybridized to them- selves and the other banks. We see, for example, that probe from the adult bank hybridized only to an extent of 70% with driver from the 13-day-old bank compared to a level of 93% when back-hybridized to itself.

inversely proportional to the sequence complexity of the DNA clone bank (16,25- 27). However, since all the original poly(A)- RNA sequences were not present in equal proportions, the interpretation of the hybridization data becomes complicated by the fact that while any one cDNA species will react with a Cot value proportional to its base sequence complexity, the extent of the hybridization reaction is proportional to the fraction of the total clone bank DNA representing it. For this reason it is im- portant to extend these hybridization reactions until the class of least abundant components has hybridized to completion. This class usually constitutes a major fraction of total cellular poly(A)-RNA and any underestimate of this class would se- riously compromise any determination of the number of RNA species in the cloned

TABLE II

CROSS HYBRIDIZATION OF I3-DAY-OLD, NEONATAL, AND ADULT MOUSE CLONE BANKS USING NICK-

TRANSLATED cDNA INSERTS

DISCUSSION

Before discussing our interpretation of the hybridization experiments, it may be appropriate to consider the general valid- ity of the methods used in this analysis. Using conditions of plasmid clone bank DNA excess, the rate of hybridization of 32P nick-translated DNA probe should be bNot done.

pDNA Driver

=P Probe 13th Day Neonate Adult

13th Day 92.0” NDb ND

Neonate 78.0 96.0 ND

Adult 70 83.0 93.0

u. % Sl, resistant material.

DNA population. Fortunately, with plas- quences. For example, we would like to mid DNA sequences there is no problem identify those clones corresponding to the in obtaining enough DNA to drive these new mRNAs that are produced as the reactions essentially to completion. Any mouse embryo progresses from the 13-day remaining unhybridized sequences would stage to the adult. From the data in Table by definition be extremely rare, and would II, we see that hybridization of a nick- be present at a frequency of less than 1 translated adult cDNA probe to 13-day-old molecule per -100 cells (25, 27). clone bank material yields a value of only

The data in Table I, summarize our ob- 70% (compared to 93% when the probe is servations of the sequence complexity of hybridized to its own adult clone bank the DNA in the 13th day, neonatal, and DNA). The remaining -20% of unhybrid- adult mouse banks. As can be clearly seen ized DNA has provided us with suitable from column five of Table I, there is a pro- radioactive probes to identify what we call gressive increase in the number of differ- “difference clones.” There are clones cor- ent sequences in the banks from 13-day- responding to new mRNAs that appear as old embryo to the adult, the adult total the embryo develops. Such “difference complexity being almost twice that of t,he clones” sequences are presently being uti- 13-day-old embryo. The number of se- lized in assays involving in vitro hybrid- quences in the class of most abundant se- ization and autoradiography on tissue of quences is similar in all three cases. It is the developing embryo. tempting to speculate that these corre- spond to structural proteins that are produced by many cells such as actin, tubu-

ACKNOWLEDGMENTS

lin, myosin, collagen, laminin, and fibro- We thank Carolyn Fimiani for her excellent tech-

nectin. nical help during the course of this work and Dr.

In the case of the intermediate abun- Michael I. Sherman and Jay Haron for many valuable

dance class there is an almost fourfold re- discussions.

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cloning of total mrna populations from adult and embryonic mice

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