May 4, 1979 - 1 and 2). By contrast to the other crystallins, 6-crystallin is present only in ... dGTP and dTTP, 500 ,uCi (1 Ci = 3.7 X 1010 becquerels) of ... endonucleases were purchased from Bethesda Research Lab- ... EcoR-I Bam H-I. Pst-I.
Proc. Nati. Acad. Sci. USA Vol. 76, No. 7, pp. 3299-3303, July 1979 Biochemistry
Molecular cloning and partial characterization of b-crystallin cDNA sequences in a bacterial plasmid (mRNA/pBR322/recombinant DNA/cellular differentiation/chick lens)
SURAJ P. BHAT AND JORAM PIATIGORSKY Section on Cellular Differentiation, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20205
Communicated by Bernhard Witkop, May 4, 1979
ABSTRACT Double-stranded cDNA synthesized from 5crystallin mRNA isolated from lens fiber cells of 15-day-old embryonic chicken was cloned in Escherichia coli X1776 in the Pst I site of the plasmid pBR322 by using the oligo(dC);'oligo(dG) joining procedure. Twelve Amps Tetr transformants contained sequences complementary to purified 6-crystallin [32P]cDNA. One of the recombinant clones (pbCr-2) had an insert of 1241 + 240 base pairs, as judged by R-looping analysis with purified -crystallin mRNA. The inserted cDNA represents at least 69% of the 6-crystallin coding sequences. pbCr-2 was further characterized by restriction analysis, protection of -crystallin [3H]cDNA from digestion by SI nuclease, and hybrid-mediated arrest of -crystallin mRNA translation in vitro. p6Cr-2 provides an invaluable probe for additional analysis of the primary structure, gene organization, and regulated synthesis of &-crystallin, the principal protein synthesized during lens differentiation in the chicken embryo.
The major constituents of the vertebrate lens are four classes of structural proteins termed crystallins (a, 3, y, and 3) (see refs. 1 and 2). By contrast to the other crystallins, 6-crystallin is present only in avian and reptilian lenses (see 2). 6-Crystallin has been most intensively studied in chickens, in which it represents 60-80% of the protein in the embryonic lens and about 50% of the protein in the adult lens (3-5). Chicken b-crystallin is a polymeric protein (6, 7) of about 200,000 daltons (8). The b-crystallin subunits have similar tryptic peptides and can be resolved by electrophoresis into two bands of 48,000 and 50,000 daltons (9, 10). Chicken 5-crystallin is particularly suited to developmental studies because it is the first crystallin to appear in the embryonic lens (3, 11, 12), and its rate and location of synthesis within the lens change during development (4, 5, 11-13). Moreover, chicken lenses can be readily obtained from embryos at known stages of development, and lens cell differentiation involving 6-crystallin gene expression can be studied in tissue (5, 11, 14) and cell (15, 16) culture. In the present communication we have taken advantage of our ability to purify 6-crystallin mRNA (17, 18) in order to synthesize and clone a double-stranded (ds) 3-crystallin cDNA. MATERIALS AND METHODS Synthesis of ds cDNA. [3H]cDNA was synthesized from 45 ,gg of b-crystallin mRNA (17) with 200 units of avian myeloblastoma virus reverse transcriptase (a gift of J. W. Beard, Life Sciences, St. Petersberg, FL) in a reaction mixture of 500 II containing 100 mM Tris-HCI (pH 8.3), 2.5 mM Mg acetate, 40 mM dithiothreitol, 10 ,ug/ml oligo(dT), 750 tiM each of dATP, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
dGTP and dTTP, 500 ,uCi (1 Ci = 3.7 X 1010 becquerels) of [3H]dCTP (New England Nuclear, 16 Ci/mmol). The [3H]cDNA was fractionated on an alkaline sucrose density gradient (18) and the fractions containing half-size to full-length transcripts were pooled and used for the synthesis of ds DNA using DNA polymerase (19). The ds cDNA was tailed with dCTP by terminal transferase (Miles) and subjected to electrophoresis on a 1% agarose gel. After autoradiography, ds cDNA longer than approximately 1000 base pairs (bp) was eluted by electrophoresis, concentrated by DEAE-cellulose chromatography, and precipitated with ethanol at -200 C. Construction of a Recombinant cDNAkpBR322 DNA. The restriction endonuclease Pst I was used to linearize the pBR322 DNA (Bethesda Research Laboratories, Rockville, MD). The linear plasmid DNA was tailed with oligo(dG) in the presence of 1 mM CoCl2 by terminal transferase (19), extracted with phenol, and precipitated with ethanol. The oligo(dG)-tailed plasmid DNA and the oligo(dC)-tailed ds cDNA were hybridized at 65°C in 10 mM Tris-HCl, pH 7.5/100 mM NaCl/1 mM EDTA for 2 hr. The reaction tubes were left at room temperature for 6 hr and then kept at 4°C until used to transform Escherichia coli X1776. Transformation of E. coli X1776 and Identification of Clones. All procedures were conducted in a P3 physical containment facility in compliance with National Institutes of Health Guidelines for Recombinant DNA Research. E. coli X1776 was transformed by a described procedure (20). Cultures (100 ml) were grown to an A5s0 of 0.5 in L-broth, supplemented with diaminopimelic acid (100 ,ug/ml) and thymidine (50 tg/ml). The cells were chilled, centrifuged, and washed by centrifugation in 0.01 M NaCl. The washed pellet was resuspended in 10 ml of 40 mM sodium acetate, pH 5.6/70 mM MnCl2/30 mM CaCl2, kept on ice for 20 min, centrifuged, and resuspended in 3 ml of the same buffer; 0.2 ml of this cell suspension was added to sterile tubes containing 50-100 ng of the hybrid plasmid DNA. The mixture was incubated on ice for 20 min and then at 37°C for 3 min. Supplemented L-broth (0.5 ml) was added and the tubes were incubated at 37°C for 30 min. The cells were plated and Tetr transformants that appeared after 48 hr of incubation at 37°C were transferred to Millipore filters. The hybrid clones were identified by a modified colony hybridization procedure of Grunstein and Hogness (21). Hybridization was performed in sealed polyethylenepolyester bags at 70°C (22). Strongly positive colonies were replated and single colonies were grown in liquid culture. For crude analysis of different clones, DNA was isolated by combined procedures of Meagher et al. (23) and Guerry et al. (24). For further analysis DNA was purified from the clones by banding in CsCl. Plasmid DNA was amplified by culturing the cells in the presence of chloramphenicol (200 ,tg/ml). Abbreviations: ds, double stranded; bp, base pairs; SV40, simian virus 40.
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Electron Microscopy. In general, R-loops were formed by hybridizing 25 jug of p6Cr-2 DNA per ml (predigested with BamHI) with b-crystallin mRNA at 50-75 ,jg/ml for 12-14 hr at 52°C in 70% formamide/0. 1 M N[tris(hydroxymethyl)methyliglycine (Tricine)-NaOH, pH 8.0/0.5 M NaCl/10 mM EDTA (26). R-loops which were used for hybridization to simian virus 40 (SV40) DNA tailed with poly(T) or polybromodeoxyuridine were formed by using only 2.5-10 ,ug of 6crystallin mRNA per ml; SV40 poly(T) or SV40 polybromodeoxyuridine was added to a final concentration of 5 jig/ml and hybridization was performed for 4 hr at 52°C. After hybridization, samples were diluted 1:20 to 1:30 with the hybridization buffer, spread on a hypophase of water, transferred onto Parlodion-coated grids, shadowed with platinum/palladium and carbon, and viewed with a Philips 300 electron microscope.
Restriction Enzyme Analysis of Cloned DNA. Restriction endonucleases were purchased from Bethesda Research Laboratories or New England BioLabs. All enzyme digestions were done at 37'C in 6 mM Tris-HCl, pH 7.5/50 mM NaCI/5 mM MgCl2/5 mM 2-mercaptoethanol/100 .ug of bovine serum albumin per ml. Restriction fragments were analyzed by electrophoresis on horizontal slab agarose (SeaKem, Marine Colloids, Rockland, ME) gels (1% or 1.6%); the electrophoresis buffer was 0.04 M Tris.HCI/0.02 M Na acetate/0.002 M EDTA at pH 7.8. The DNA was transferred to nitrocellulose filters (25) and hybridized with b-crystallin [32P]cDNA in Denhardt's solution (22) at 70'C for 4 hr. The filter was washed three times with 1/10th concentrated standard saline citrate solution containing 0.1% sodium dodecyl sulfate and twice with 1/10th concentrated standard saline citrate alone before autoradiography. 5'
3'
mRNA
AAAA Reverse Transcriptase, dNTP OligodT ~ AAAA - TTTT
cDNA (Q
I ALKALI _____
- TTTT
I DNA Polymerase-l, dNTP _____
_
S -Nuclease
TACGTC G
I
I
TTTT
'fi 3
cccC3c
3GGGGiACGTC
J,
GTC
Heat Denature, Anneal
cDNA CTGCAGGGG G CCCC
CCCC G GGGGACGTC
cDNA
X
G CTGCA 3'
I Terminal Transferase, dGTP
Terminal Transferase, dCTP
3'cccc
Linearized by Pst-I
Transformation Pst-l Sites of E. Coli X1776 * Reconstructed by the Host CTGCA GGGG CCCC TGCAG GGGG|ACGTC ~LGACGT CCCC Pst-l Site Pst-l Site
Transformants (Tet') Identified by Hybridization to / a\
32P-cDNA
cDNA
FIG. 1. Strategy for cloning of b-crystallin cDNA.
G CTGCAGGGG 3'
Biochemistry: EcoR-I 1 2
Bam H-I 3 4
Proc. Nati. Acad. Sci. USA 76 (1979)
Bhat and Piatl"gorsky Pst-I 5 6
Bgl-l 7 8
- 1342 bp - 1078 bp
--
972 bp 601 bp
-
301 bp
-
2
3
4
5
6
7
8
FIG. 2. Restriction enzyme analysis of plasmid p b(r-2. (Upper) Restriction fragments of pbCr-2 DNA as seen by ethiidium bromide staining in the agarose-gel. (Lower) Hybridization of [32P]cDNA to the DNA blot made from the same agarose gel. A Hfae III digest of OX174 DNA was used for molecular weight markers. L anies 2,4,6, and 8 show pBR322 (lacking the insert) cleaved by the specified enzyme. As expected, the DNA in these lanes did not hybrid .ize to the [32p cDNA.
RESULTS
Construction and Identification of Recomibinant cDNA Clones. Fig. 1 gives the steps followed for consitruction of the recombinant cDNA. As starting material, we isolated cytoplasmic poly(A)-containing RNA by oligo(dT)-4cellulose chromatography from purified lens fibers of 15-da v-old chicken embryos. b-Crystallin mRNA represents a minir num of 70% of the RNA in this preparation, and the remainderr appears to be mostly rRNA (17).
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We ued the Pst I site of pBR322 for cloning cDNA for two reasons. First, this site is reestablished by the host after transformation if the hybrid molecule is constructed by the G-C tailing procedure (27), as shown in Fig. 1. Reconstruction of the Pst I site on both sides of the insert allows the cDNA sequences to be removed from the plasmid by digestion with Pst I. Second, E. coli transformed with pBR322 is Ampr and Tetr (28). Ampicillin resistance is destroyed by inserting foreign DNA into the Pst I site of the plasmid. This allows selection for Amps Tetr transformants containing the cDNA insert. Twelve colonies of transformed bacteria containing the b-crystallin DNA sequences were identified by hybridization with [32P]cDNA synthesized from b-crystallin mRNA which was purified by sucrose density gradient centrifugation. We estimate that this purified b-crystallin mRNA is at least 90% pure (18). As expected, these colonies were Tetr Amps. Recombinant DNA from different clones was partially purified and screened for the presence of an insert by subjecting it to digestion with Pst I and to electrophoresis on agarose gels (data not shown). On the basis of this preliminary test we selected one recombinant clone (called pbCr-2 for plasmid b-crystallin clone number 2), which seemed to have the largest inserted DNA piece. Partial Restriction Enzyme Mapping of p5Cr-2. pbCr-2 DNA was treated with different restriction enzymes and subjected to electrophoresis on an agarose gel (Fig. 2 upper). b-Crystallin sequences in different DNA bands were identified by hybridization to b-crystallin [32P]cDNA by the Southern blotting procedure (25) (Fig. 2 lower). EcoRI cleaved the inserted DNA once, about 500 bp away from the Pst I site, thus releasing a fragment about 1300 bp long containing part of the inserted sequence (Fig. 2, lane 1). The larger fragment containing the rest of the insert was about the same length as the original pBR322 DNA (without the insert) cut with EcoRI (Fig. 2, lanes 1 and 2). BamHI cut the pBR322 DNA once, as expected, and did not cut the inserted DNA. Thus, the p3Cr-2 DNA restricted with BamHI was larger than pBR322 cut with this enzyme (compare Fig. 2, lanes 3 and 4). Bgl I cut the pBR322 DNA twice, generating two larger fragments and one smaller fragment (Fig. 2, lane 8). Bgl I, like BamHI, did not cut the inserted DNA, which remained attached to the smaller of the two larger original pBR322 fragments (Fig. 2, lane 7). Pst I digestion of p6Cr-2 DNA removed two poorly resolved fragments approximately 550 bp long which contained the inserted DNA and the original linear pBR322 DNA (Fig. 2, lanes 5 and 6). The data are summarized in Fig. 3 which shows these and other restriction endonuclease sites present in the insert. Taken together, these restriction analyses indicate that the inserted DNA is approximately 1300 bp long. Electron Microscopy of pOCr-2 DNA Hybridized with 5-Crystallin mRNA. We have used electron microscopy to characterize further the pbCr-2 DNA. R-loops represent single-stranded DNA displaced by RNA complementary to the coding DNA strand (29, 30) (see Fig. 4 lower). The electron 4 show the presented in Fig. micrographs h R-loops -op made aebby i.4so mcorpspeetdi hybridizing sucrose gradient-purified b-crystallin mRNA to pbCr-2 DNA. At least 90% of the cloned DNAs formed R-loops when hybridized to a 4-fold excess of 6-crystallin mRNA purified from 15-day-old chicken embryo lens fibers either by sucrose density gradient centrifugation or by oligo(dT)-cellulose chromatography. The size and intramolecular distribution of the inserted DNA were very uniforpi in several hundred molecules, which were measured by a Numonics digitizer (data not shown). The mean length of the inserted DNA was calculated from the DNA-RNA hybrids to be 1241 + 240 bp. This agrees with the 1300 bp estimated from restriction analysis. We measured the length of b-crystallin mRNA molecules by
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Proc. Natl. Acad. Sci. USA 76 (1979)
electron microscopy in order to determine the proportion of the b-crystallin mRNA that is represented in the pbCr-2 DNA. The b-crystallin mRNA was spread -on the grids in 70% formamide/4 M urea in order to minimize secondary structures. The mean length of about 400 molecules was 1909 + (SD) 530 nucleotides. This is in reasonable agreement with earlier studies using formamide/polyacrylamide gel electrophoresis and cDNA-mRNA hybridization kinetic analysis (18). On the average, then, the cDNA insert is 65% of the length of b-crystallin mRNA. Fig. 4 upper shows three p3Cr-2 DNA molecules with Rloops having RNA tails at the short (arrow a) and long arm (arrow b) of the hybrid. Close examination of the third R-loop reveals two RNA tails in the same hybrid. In order to determine which RNA tail represents the 3'-poly(A) end of the b-crystallin mRNA, we hybridized the R-loops with SV40 DNA that was tailed with poly(T) or polybromodeoxyuridine. Fig. 5 shows that the SV40 DNA tailed with poly(T) hybridized to the RNA projecting from the junction of the short arm and the R-loop in p3Cr-2 DNA. A comparable result was obtained with SV40 tailed with polybromodeoxyuridine. We conclude from this experiment that the 3'-poly(A) sequence of 3-crystallin mRNA is not represented in the cDNA clone. Moreover, this experiment allows us to orient the insert within the plasmid, as indicated in the legend to Fig. 3. The cDNA is inserted in a reversed orientation with respect to the direction of transcription of the pBR322 DNA. Further Evidence That the Inserted DNA Contains Crystallin Sequences. Two additional experiments were conducted to test whether the pbCr-2 DNA contains sequences complementary to b-crystallin mRNA. First, pbCr-2 DNA was tested for its ability to arrest the cell-free translation of purified
f
I_
t!./~~~~~~~. 3.(pdr-2b { .0Kb \ a2h-
Hae-ll
Hif-
i\-\
Bgl-l
//
FIG. 3. Partial restriction map of recombinant plasmid p5Cr-2. The restriction endonuclease cleavage sites of the cDNA insert were determined by comparing the electrophoresis patterns of pOCr-2 DNA and pBR322 DNA (28, 38) after digestion with the indicated restriction enzymes. The bold line indicates the inserted DNA. Not all the sites of Hae II, Hha I, Tac I, and HinfI enzymes have been shown on the parent (pBR322) DNA. The exact order of the sites for these enzymes in the insert is not certain. We believe that there is another Pst I site on the insert which is not indicated, because the combined size of the 6-crystallin sequences in the Pst I fragments of the plasmid is only about 1100 bp and the total size of the insert is approximately 1300 bp (see text). The ends of the insert complementary to the 5' and 3' regions of 5-crystallin mRNA are indicated on the figure. This orientation was established as given in Fig. 5.
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*
.
i
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a
r
7
.a*
-}SS DNA DNA.RNA
between p6Cr2 DNA FIG 4. R.loops Electron micrographs of and 5-crystallin mRNA (R-loops). (Upper) RNA tails, at the short arm (arrow a) and the long arm (arrow b) of the plasmid DNA. (Lower) R-loop at a higher magnification to illustrate clearly the displaced single-stranded DNA (SS DNA) and the DNA-RNA hybrid. An RNA tail is indicated by an arrow. The bar in Upper represents 650 bp and that in Lower, 340 bp.
b-crystallin mRNA, as given by Paterson et al. (31). Translation of the mRNA in a reticulocyte lysate after hybridization with p*Cr-2 DNA was inhibited by 70-80%, as judged by an immunoprecipitation assay. By contrast, 3-crystallin mRNA hybridized with pBR322 DNA under the same conditions was translated as effectively as mRNA that was not hybridized to any DNA. Second, [3H]cDNA synthesized from cytoplasmic poly(A)-containing RNA of embryonic lens fibers, which contains at least 70% 3-crystallin mRNA, was protected from 51 nuclease digestion by hybridization to pbCr-2 DNA by 50% and 70% in duplicate tests. DISCUSSION The present data, involving restriction analysis, hybridization 5-crystallin cDNA, electron microscopy, and hybrid-mediated arrest of 3-crystallin mRNA translation, provide strong evidence that the ds cDNA inserted-into pBR322 has DNA sequences complementary to b-crystallin mRNA. Determination of the nucleotide sequence of the inserted DNA would not yet establish that it codes for b-crystallin, because there are no amino acid sequence data available for b-crystallin. We anticipate, rather, that sequence studies of this cloned DNA will contribute importantly to our knowledge of the primary structure of 3-crystallin.
to
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