Volume 6 Number 11 1979

Nucleic Acids Research

Identifi'cation of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels

Norman Rave, Radomir Crkvenjakov and Helga Boedtker Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, USA Received 20 March 1979

ABSTRACT Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro al collagen cDNA clones, pCgl or pCg54, or to the nick translated pro a2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro a2 collagen mRNA was shown to be slightly larger than pro al mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.

INTRODUCTION The identification of specific RNAs, even when present at low concentrations, has been significantly facilitated by Alwine et al's procedure for transferring RNA from agarose gels to diazobenzyloxymethyl

(DBM) paper (1) and by the availability of highly specific probes from molecular cloning. We report here the extension of Alwine's procedure to formaldehyde agarose gels which are as fully denaturing as methyl mercury hydroxide agarose gels (2) but not as toxic. By hybridizing nick translated pCgl or pCg54, pro al collagen cDNA clones (3) and nick translated pCg45, a pro a2 collagen cDNA clone (4) to chick calvaria poly A containing RNA, we have been able to identify and to determine the molecular weights of pro al and pro a2 collagen mRNAs as well as two minor RNAs that hybridized weakly to pCg54 and were larger than pro al collagen mRNA.

MATERIALS AND METHODS

Poly A containing RNA was prepared from the calvaria of 16 day old

C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research chick embryos as described previously (5,6). Poly A containing RNA was also prepared from the sterna of 16 day old chick embryos by the same method except that the phenol-chloroform-isoamyl alcohol extraction was carried out at room temperature rather than at 60°C (7). Electrophoresis was carried out in either cylindrical tube gels (0.5 cm diameter, 7 cm long) or on horizontal slab gels (12 by 14 cm) containing 0.75% agarose ("Seakem" LE agarose obtained from Marine Colloids, Inc.), 6% formaldehyde (Mallinckrodt analytical reagent), 0.018 M Na2HPO4, and 0.002 M NaH2PO4 (E buffer). The gels were prepared by dissolving 0.75 g of agarose in 50 mls distilled deionized water by heating in an autoclave for five minutes. 50 mls of 12% formaldehyde in 2X E buffer were added after the agarose had cooled to 600. Before electrophoresis, RNA samples were dissolved in 50% deionized formamide, 6% formaldehyde in E buffer, reacted at 600 for five minutes, fast cooled and one half volume of 50% glycerine 50% buffered deionized formamide containing bromphenol blue dye was added. The tops of the tube gels were cut with a razor blade to provide a flat surface before the samples were applied. Electrophoresis was carried out for 15 to 17 hours at 0.5 mamps per tube, or 30 volts for slab gels, in E buffer containing 3% formaldehyde. After electrophoresis, the gels were stained with ethidium bromide (1 ig/ml in 0.1 M NH4 acetate) for thirty minutes and then destained in distilled water for either four hours at room temperature or 17 to 20 hours at 4°. Before transfer to DBM-paper, the gel was heated in water at 60° for 5 minutes to reverse the formaldehyde reaction. The gels were fast cooled to room temperature and then hydrolyzed for 20 minutes in 50 mM NaOH, using 200 mls for slab gels and 20 mls for each tube gel. After alkaline hydrolysis, the gels were neutralized with 0.2 M potassium phosphate (1:1) for ten minutes and then equilibrated with 0.025 M potassium phosphate for five minutes, as described by Alwine et al (1). DBM-paper (tradename, Enzobound, Enzo Biochemical Company, New York, N.Y.) was diazotized immediately before transfer as described by Alwine et al (1). Aluminum foil containing a window the size of the gel was placed over the gel to isolate the blotting paper under the gel from the DBM-paper and blotting paper over it. Transfer was usually carried out for 17 to 20 hours with at least four changes of paper. At the end of the transfer, the gels were paper thin. The DBM paper was then preincubated for four hours at 420 in 1 to 3 mls of 50% formamide, 0.75 M NaCl, 0.075 M Na citrate, 0.02% (w/v) bovine serum albumin, 0.02% ficoll, 0.02% polyvinyl pyrrolidone, 1.0 mg/ml denatured

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Nucleic Acids Research calf thymus DNA (Sigma) and 1% glycine in a Sears and Roebuck "Seal-N-Save" bag. The preincubation mixture was discarded and replaced by an identical solution minus glycine containing 0.5 to 10 x 106 cpm (32P)-labelled procollagen plasmid pCgl, pCg54 (3) or pCg45 (4), labelled by nick translation (8) using E. coli DNA polymerase I, a gift of Yvonne Chow. Hybridizations were carried out for 40 to 60 hours at 420, after which the paper was washed at 420 for four hours in 50% formamide, 0.75 M NaCl, 0.075 M Na citrate with six changes of washing buffer. The paper was blotted dry, covered with Saran wrap and exposed to Kodak XR-5 film at room temperature for one to twenty hours as indicated. The nick translation and hybridization of recombinant plasmids were carried out under P1 containment conditions in compliance with the National Institutes of Health Guidelines for Recombinant DNA Research [Federal Register (1976) 41, 27902-27943].

RESULTS Calvaria poly A containing RNA was electrophoresed on two lanes of a

formaldehyde agarose slab gel, stained and photographed and then blotted to DBM-paper.

The paper was cut in half and one half was hybridized to nick translated pCgl while the other half was hybridized to pCg45. The two halves were then autoradiographed. The resultant autoradiographs as well as the photograph of the lanes of the gels that were transferred are shown in Figure 1. While 27S and 18S rRNA are the major stained species visible in the photograph of both lanes, two faint bands with mobilities less than 27S rRNA, presumed to be procollagen mRNAs, can also be seen. Together they probably represent only 1% of the 5 ig of poly A containing RNA applied to each lane (see below). Nevertheless, both nick translated pCgl and pCg45 hybridized most strongly to a band of identical mobility approximately comigrating with the two faint procollagen mRNA bands. Within the resolution obtained in this experiment, the molecular weights of the two procollagen mRNAs appear to be the same. Similar results were obtained when nick translated pCg54 was used to identify pro atl collagen mRNA (data not shown). The large extent of hybridization, expecially of pCgl, to RNAs having mobilities greater than that of intact procollagen mRNAs, is probably the result of a large amount of degraded procollagen mRNA in this poly A containing RNA preparation. To show that the hybridization was not due to the nonspecific hybridization of nick translated pBR322 sequences to chicken poly A

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.01~: . Rm

procollogen

:~~~~~~

~

RNA

1Bs 8S ----gE

Transfer of calvaria poly A containing RNA fron formaldehyde agarose slab gel to DBM paper. From left to right: photograph of ethidium bromide stained gel; autoradiograph of DBM paper hybridized to 0.5 pg pCgl (specific activity 1 x 106 cpm/ig; 7 x 103 cpm/cm2 of paper) for 40 hours at 420; photograph of ethidium bromide stained gel; autoradioyraph of DBM paper hybridized to 0.5 pig pCg45 (1 x 106 cpm/lig, 5 x 103 Cpm/cm1-LL of paper. DBM papers were exposed to Kodak XR-5 film for 20 hours at room temperature.

Figure 1.

containing RNA, nick translated pBR322 (6 x 106 cpm) was hybridized to poly A containing RNA transferred to DBM paper from a formaldehyde agarose gel. As shown in Figure 2, no radioactivity was localized on the paper to which the RNA was transferred even though there were 1.3 x 105 cpm/cm2in this hybridization compared to 7.0 and 5.0 x 03cpm/cm2used in the hybridization shown in Figure 1. To obtain a clearer picture of the procollagen mRNA bands and to reduce the amount of rRNA in the poly A containing RNA, the latter was rechromatographed over oligo(dT)-cellulose and 8.8 pig of twice bound RNA was then applied to a formaldehyde agarose tube gel. A tube gel was used rather than a slab gel because the former permits loading of more RNA than the latter. Following electrophoresis, the gel was stained with ethidium bromide, destained and transferred to DBM paper as described in Materials 3562

Nucleic Acids Research

Figure 2. Autoradiograph of DBM parer hybridized to 0.5 vig pBR322 (specific activity 1. 2 x 107 cpm/lag; 1. 3 x 109 cpm/cm2 of paper) for 47 hours at 420. DBM paper was exposed to Kodak XR-5 film for 14 hours. The part of the paper that was exposed to the gel is outlined. and Methods.

The paper was first hybridized to nick tranislated pGg54 and autoradiographed for 1 and 18 hours.- The hybridized DNA was removed by washing the paper four times in deionized formamide at 600 for five minutes. The paper was then rehybridized to nick translated pCg45 and autoradiographed for 1, 8, and 22 hours. The photograph of the ethidium bromide stained gel of the twice oligo(dT)-cellulose bound RNA, and of a parallel gel containing TMV RNA and 27S and 23S rRNA as size markers are shown in Figure 3 (lanes 2 and 4 respectively) together with the autoradiographs (lanes 1 and 3). The two procollagen mRNA bands migrating more slowly than 27S rRNA are now the major stained species clearly visible in the photograph shown in Figure 3, lane 2. Both pCg54 and pCg45 hybridized most strongly to bands that had mobilities similar to that of the two procollagen mRNA bands. In each case there was relatively little hybridization to more rapidly migrating RNA species. Evidently this twice oligo (dT)-cellulose bound RNA species had less degraded procollagen mRNA than the one used in the experiment shown in 3563

Nucleic Acids Research

_~~~4eTMV RNA pro aI 2

,

+ 27S rRN A

iit

12423S rRNA

1

2

3

4

Figure 3. Transfer of calvaria RNA twice bound to oligo(dT)-cellulose from a formaldehyde agarose tube gel to DBM paper. Lane 1, autoradiograph of DBM paper hybridized to 0.1 pg pCg54 (specific activity, 108 cpm/lg, 2 x 105 cpm/ cm of paper) for 60 hours at 420, paper exposed to Kodak XR-5 film for 1 hour; lane 2, photograph of ethidium bromide stained gel that was transferred; lane 3, autoradiograph of DBM paper hybridized to 0.1 pg pCg45 (specific activity, 107 cpm/kg, 2 x 104 cpm/cm2 of paper) for 60 hours at 420, paper exposed to Kodak XR-5 film for 8 hours; lane 4, photograph of ethidium bromide stained gel of TMV RNA (1 pg), 27S chick rRNA (2 pg) and 23S E. coli rRNA (1 pg).

Figure 1. pCg54 hybridized to a band having a slightly greater mobility than the band to which pCg45 hybridized (Figure 3, lane 3). This suggests that the molecular weight of pro a2 collagen mRNA is somewhat larger than that of pro al and that the two stained bands in the photograph of the gel are pro a2 and pro al collagen mRNA as indicated. Using TMV RNA and 27S rRNA as size markers, we estimate the procollagen mRNAs to have molecular weights of

1,700,000 and 1,770,000 corresponding to 4.9 and 5.1 kb respectively. The breadth of the bands in the autoradiographs shown in lanes 1 and 3 in Figure 3 results from the relatively large amount of RNA in each band. Alwine et al-obtained a band of comparable breadth in the hybridization to

Drosophila melanogaster rRNA when 100 ng of RNA was applied to the gel but obtained three very narrow bands when only one ng of RNA was applied to the gel (1). Since the specific activity of their probe (2 x 107 cmp/pg, 2 x 104 3564

Nucleic Acids Research cpm per

cm2),

and their 5 hour exposure time were similar to those used in

our experiments, our results compare favorably with theirs assuming the amount of RNA added was about 100 ng per intact procollagen mRNA band. We estimate that this is the case as follows: of the 8.8 pg RNA applied to the gel, only 10% or 880 ng, sediments at 27-30S on an 85% Me2SO 5-20% sucrose

gradient; assuming a purity of 30% (20% rRNA, 50% non-collagen poly A containing RNAs), this fraction contains about 260 ng intact procollagen mRNA or 130 ng/band. While this rough calculation could be off by a factor of two, it could not be off by a factor of ten. Therefore, the breadth of

the hybridization bands in the autoradiographs in Figure 4 are largely the result of overloading the gels. To obtain narrow bands, the amount of RNA added must be reduced by a factor of 100. A large amount of poly A containing RNA was used in this experiment not only to better visualize procollagen mRNAs but to identify possible precursors to procollagen mRNAs present at low concentration. Two weakly hybridizing bands, marked by unlabelled arrows, can be seen in lane 1, Figure 4. They correspond to RNAs 6.4 and 5.6 kb long. To more clearly establish the existance of these minor RNA species, the paper was autoradiographed for 18 hours. The photograph of the resultant overexposed autoradiograph, shown on the left of Figure 4, clearly verifies the existance of a 6.4 kb RNA and at least suggests the existance of a 5.6 kb RNA containing pro al collagen mRNA sequences. Since pCg45 hybridized with almost equal intensity to a band including a range of RNA lengths extending from 5 to about 5.6 kb, but to nothing larger, even after a 22 hour exposure (right side, Figure 4), we can draw no conclusions about minor RNA species containing pro a2 collagen mRNA sequences. Sternum poly A containing RNA was transferred to the right half of the DBM paper whose autoradiographs are shown in Figure 4. Neither pCg54 nor pCg45 appear to hybridize to sternum poly A containing RNA. This surprising lack of cross hybridization between type I procollagen cDNA clones and type II procollagen mRNA was unexpected since al(I) and al(II) collagen chains have about 70% sequence homology (9). Either there has been considerable change in the third base of the codons in the two RNAs, or type II procollagen mRNA is very poorly transferred to DBM paper.

CONCLUSION The relatively efficient transfer of RNA from formaldehyde agarose 3565

Nucleic Acids Research

--

i:

.o

--q

pr_tI-0

Figure 4. Overexposed autoradiograph of DBM paper to which two gels, one containing calvaria and the other containing sternum poly A containing RNA had been transferred. Left, DBM paper hybridized to pCg54 as described in legend to Figure 3, but autoradiographed for 18 hours; right, DBM paper hybridized to pCg45, as described in legend to Figure 3 but autoradiographed for 22 hours. Unlabelled arrows on side of transferred gel locate major pro al and pro a2 collagen mRNA bands and the minor 6.4 kb and 5.6 kb minor RNA containing pro al collagen sequences. Arrow pointing downward to lane to which sternum RNA was transferred.

gels to DBM paper reported above has been confirmed by Pederson and Davis. They find that 36-42% of (32P)-labelled 28S and 18S HeLa rRNA was transferred from formaldehyde agarose gels to DBM paper using our method for pretreatment of the gels (T. Pederson and N. Davis, unpublished data). This compares favorably with the 30-60% transfer from methyl mercury hydroxide agarose gels obtained by Alwine et al (1). By adapting the gel transfer to the DBM-paper method to formaldehyde agarose gels, we have been able to identify pro al and pro a2 collagen mRNAs and to assign them molecular weights of 1,700,000 and 1,770,000 respectively. Because these two molecular weights are so similar, the two mRNAs can only be seen as separate species on denaturing gels providing very high resolution 3566

Nucleic Acids Research in this molecular weight range. The hybridization of nick translated procollagen plasmids to calvaria RNA transferred to DBM-paper also has resulted in the definite identification of a 6.4 kb RNA containing pro al collagen mRNA sequences and the tentative identification of another RNA about 5.6 kb long containing pro al collagen mRNA sequences. Experiments to determine if these RNAs are of nuclear origin and are mRNA precursors, are underway. Finally the possibility of transferring RNA from formaldehyde agarose gels to DBM paper provides a safe alternative to methyl mercury hydroxide gels for those interested in identifying RNA present at low concentrations, such as mRNA precursors, but concerned about routine handling of toxic chemicals.

ACKNOWLEDGEMENTS

We want to thank John Wozney and Tricia Bredbury for doing the nick translations of the plasmids and Yvonne Chow for her gift of E. coli DNA polymerase I. This research was supported by the National Institutes of Health Grant HD-01229 and a grant from the Muscular Dystrophy Association, Inc.

REFERENCES

1.

2.

Alwine, J., Kemp, D., Stark, B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354. Lehrach, H., Diamond, D., Wozney, J., Boedtker, H. (1977) Biochem.

16, 4743-4751. 3.

4. 5. 6. 7. 8.

9.

Lehrach, H., Frischauf, A. M., Hanahan, D., Wozney, J., Fuller, F. and Boedtker, H. (1979) Biochemistry, in press. Lehrach, H., Frischauf, A. M., Hanahan, D., Wozney, J., Fuller, F., Crkvenjakov, R., Boedtker, H. and Doty, P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5417-5421. Boedtker, H., Frischauf, A. M., Lehrach, H. (1976) Biochem. 15, 765-770. Frischauf, A. M., Lehrach, H., Rosner, C. and Boedtker, H. (1978) Biochem. 17, 3243-3249. Boedtker, H. and Rosner, C., manuscript in preparation. Rigby, P. W. J., Dieckman, M., Rhodes, C. and Berg, P. (1977) J. Mol. Biol. 113, 237-251. Fietzek, P. P. and Kuhn, K. (1976) Int. Rev. Connect. Tissue Res. 7, 1-60.

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