Proc. Natl. Acad. Sci. USA
`Vol. 76, No. 11, pp. 5684-5688, November 1979
`Cell Biology
`
`Introduction and expression of a rabbit f3-globin gene in
`mouse fibroblasts
`(DNA-mediated gene transfer/cotransformation/intervening sequences/gene regulation)
`B. WOLD*, M. WIGLERt, E. LACYt, T. MANIATISt, S. SILVERSTEIN*, AND R. AXEL*
`*College of Physicians and Surgeons, Columbia University, New York, New York 10032; tCold Spring Harbor Laboratories, Cold Spring Harbor, New York
`11724; and tDivision of Biology, California Institute of Technology, Pasadena, California 91125
`Communicated by Sol Spiegelman, July 16, 1979
`
`The cloned chromosomal rabbit fl-globin gene
`ABSTRACT
`has been introduced into mouse fibroblasts by DNA-mediated
`gene transfer (transformation). In this report, we examine the
`expression of the rabbit gene in six independent transformants
`that contain from 1 to 20 copies of the cloned globin gene.
`Rabbit globin transcripts were detected in two of these trans-
`formants at steady-state concentrations of 5 and 2 copies per cell.
`The globin transcripts from one cell line are polyadenylylated
`and migrate as 9S RNA on methylmercury gels. These transcripts
`reflect correct processing of the two intervening sequences but
`lack 48 + 5 nucleotides present at the 5' terminus of rabbit
`erythrocyte globin mRNA.
`Cellular genes coding for selectable biochemical functions can
`be stably introduced into cultured mammalian cells by DNA-
`mediated gene transfer (transformation) (1, 2). Biochemical
`transformants are readily identified by the stable expression
`of a gene coding for a selectable marker. These transformants
`represent a subpopulation of competent cells that integrate
`other physically unlinked genes for which no selective criteria
`exist (3). In this manner, we have used a viral thymidine kinase
`(tk) gene as a selectable marker to isolate mouse cell lines that
`are stably transformed with the tk gene along with bacterio-
`phage OX174, plasmid pBR322, or the cloned chromosomal
`rabbit f3-globin gene sequences (3).
`Cotransformed mouse fibroblasts containing the rabbit
`,B-globin gene provide an opportunity to study the expression
`and subsequent processing of these sequences in a heterologous
`host. In this report, we demonstrate the expression of the
`transformed rabbit 3-globin gene generating a discrete poly-
`adenylylated 9S species of globin RNA. This RNA results from
`correct processing of both intervening sequences, but lacks
`approximately 48 nucleotides present at the 5' terminus of
`mature rabbit 3-globin mRNA.
`
`MATERIALS AND METHODS
`Cell Culture. Murine Ltk- aprt- cells are adenine phos-
`phoribosyltransferase-negative derivatives of Ltk- clone 1D
`cells (4) that were originally isolated and characterized by R.
`Hughes and P. Plagemann. Cells were maintained in growth
`medium and prepared for transformation as described (5).
`Transformation and Selection. The transformation protocol,
`selection for tk+ transformants, and maintenance of transfor-
`mant cell lines were as described (5).
`DNA Isolation. DNA was extracted from cultured L cells
`as described (5). Recombinant phage containing the rabbit
`fl-globin gene in the X phage vector Charon 4A were grown and
`
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be hereby marked "ad-
`vertisement" in accordance with 18 U. S. C. §1734 solely to indicate
`this fact.
`
`purified, and DNA was isolated as described (6). The herpes
`virus DNA fragment containing the tk gene was purified from
`total DNA of herpes simplex virus strain F (7). Intact herpes
`virus DNA was digested with the restriction endonuclease Kpn
`I and fractionated by agarose gel electrophoresis, and the
`5.1-kilobase pair (kbp) fragment containing the tk gene was
`extracted from the gel as described (8).
`RNA Isolation. Total RNA was isolated from logarithmic-
`phase cultures of transformed L cells by successive extractions
`with phenol at pH 5.1, phenol/chloroform/isoamyl alcohol
`(25:24:1, vol/vol), and chloroform/isoamyl alcohol (24:1,
`vol/vol). After ethanol precipitation, the RNA was digested
`with DNase (9) and precipitated with ethanol. Nuclear and
`cytoplasmic fractions were isolated as described (5) and
`RNAs were extracted as described above. Cytoplasmic poly-
`adenylylated RNA was isolated by oligo(dT)-cellulose chro-
`matography (10).
`cDNA Synthesis. Rabbit and mouse cDNAs were prepared
`by using avian myeloblastosis virus reverse transcriptase
`(RNA-dependent DNA polymerase) (obtained from J. W.
`Beard), as described (11).
`DNA Filter Hybridizations. Cellular DNA was digested
`with restriction endonucleases, electrophoresed on agarose slab
`gels, transferred to nitrocellulose filter sheets, and hybridized
`with 32P-labeled DNA probes as described by Wigler et al.
`(5).
`Solution Hybridizations. 32P-Labeled globin cDNAs (spe-
`cific activities of 2-9 X 108 cpm/,gg) were hybridized with
`excess RNA in 0.4 M NaCl/25 mM 1,4-piperazinediethane-
`sulfonic acid (Pipes), pH 6.5/5 mM EDTA at 750C. Incubation
`times did not exceed 70 hr. Rots were calculated as moles of
`RNA nucleotides per liter times time in seconds. The fraction
`of cDNA rendered resistant to the single-strand nuclease S1 in
`hybridization was determined as described (10).
`RNA Filter Hybridizations. RNA was electrophoresed
`through 1% agarose slab gels (17 X 20 X 0.4 cm) containing 5
`mM methylmercury hydroxide as described by Bailey and
`Davidson (12). The concentration of RNA in each slot was 0.5
`ug/,ld. Electrophoresis was at 110 V for 12 hr at room tem-
`perature.
`RNA was transferred from the gel to diazotized cellulose
`paper as described by Alwine et al. (13) by using pH 4.0 citrate
`transfer buffer. After transfer, the RNA filter was incubated
`for 1 hr with transfer buffer containing carrier RNA at 500
`,ug/ml. The RNA on the filters was hybridized with cloned
`DNA probe at 50 ng/ml labeled by 32P nick translation (14) to
`specific activities of 2-8 X 108 cpm/,g. Reaction volumes were
`
`Abbreviations: tk, thymidine kinase; kbp, kilobase pairs; Pipes, 1,4-
`piperazinediethanesulfonic acid; Rot, product of RNA concentration
`(moles of nucleotide per liter) and incubation time (seconds).
`
`5684
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`Merck Ex. 1034, pg 1109
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`

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`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`5685
`
`Charon 4A (Fig. IA) (unpublished data). The purified tk gene
`was mixed with a 100-fold molar excess of intact recombinant
`DNA from clone R3G1. This DNA was then exposed to mouse
`Ltk- cells under transformation conditions previously described
`(5). After 2 weeks in selective medium, tk+ transformants were
`observed at a frequency of one colony per 106 cells per 20 pg
`of tk gene. Clones were picked and grown into mass culture.
`We then asked if the tk+ transformants also contain rabbit
`/3-globin sequences. High molecular weight DNA from eight
`transformants was cleaved with the restriction endonuclease
`Kpn I. The DNA was fractionated by agarose gel electropho-
`resis and transferred to nitrocellulose filters, and these filters
`were then annealed with nick-translated globin [32P]DNA [blot
`hybridization (17)]. Cleavage of this recombinant phage with
`the enzyme Kpn I generates a 4.7-kbp fragment that contains
`the entire adult 3-globin gene, along with 1.4 kbp of 5' flanking
`information and 2.0 kbp of 3' flanking information (Fig. 1A).
`This fragment was purified by. gel electrophoresis and nick
`translated to generate a hybridization probe. Blot hybridization
`experiments showed that the 4.7-kbp Kpn I fragment con-
`taining the globin gene was present in the DNA of six of the
`eight tk+ transformants. In three of the clones (Fig. 2, lanes E,
`F, and H), additional rabbit globin bands were observed, which
`probably resulted from the loss of at least one of the Kpn I sites
`during transformation. The number of rabbit globin genes in-
`tegrated in these transformants was variable: some clones
`contained a single copy of the gene (Fig. 2, lanes J and K),
`whereas others contained up to 20 copies of the heterologous
`gene. It should be noted that the fl-globin genes of mouse and
`rabbit are partially homologous. However, we do not observe
`hybridization of the rabbit f3-globin probe to Kpn-cleaved
`mouse DNA, presumably because Kpn cleavage of mouse DNA
`leaves the fl-gene cluster in exceedingly high molecular weight
`fragments not readily detected in these experiments (Fig. 2).
`These results demonstrate the introduction of the cloned
`chromosomal rabbit f-globin gene into mouse cells by DNA-
`mediated gene transfer.
`
`Cell Biology: Wold et al.
`25 Ml/cm2 of filter. Hybridization was in 4X standard saline
`citrate (0.15 M NaCI/0.015 M sodium citrate)/50% formamide
`at 570C for 36-48 hr.
`After hybridization, filters were soaked in two changes of 2X
`standard saline citrate/25 mM sodium phosphate/1.5 mM so-
`dium pyrophosphate/0.1% sodium dodecyl sulfate/5 mM
`EDTA at 370C for 30 min with shaking to remove formamide.
`Successive washes were at 680C with 1X and O. X standard
`saline citrate containing 5 mM EDTA and 0.1% sodium dodecyl
`sulfate for 30 min each.
`Berk-Sharp Analysis of Rabbit #-Globin RNA in Trans-
`formed Mouse L Cells. The hybridizations were carried out
`in 80% (vol/vol) formamide (Eastman)/0.4 M Pipes, pH 6.5/0.1
`mM EDTA/0.4 M NaCI (15, 16) for 18 hr at 51°C for the 1.8
`kbp Hha I fragment and 49°C for the Pst I fragment. The
`hybrids were treated with S1 nuclease and analyzed by a
`modification of the procedure described by Berk and Sharp
`(16).
`
`RESULTS
`Transformation of mouse cells with the rabbit
`f,-globin gene
`We have performed cotransformation experiments with the
`chromosomal adult rabbit fl-globin gene, using the purified
`herpes virus tk gene as a biochemical marker. The addition of
`the tk gene to mutant Ltk- mouse fibroblasts results in the ap-
`pearance of stable transformants that can be selected by their
`ability
`to grow in hypoxanthine/aminopterin/thymidine
`(HAT) medium. Cells were cotransformed with a #-globin gene
`clone designated RJG1, which consists of a 15.5-kbp insert of
`rabbit DNA carried in the bacteriophage X cloning vector
`
`TI
`
`5 1
`
`30
`
`A
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`K ilnhiici
`rmiiooases.-
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`§
`I.
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`I
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`I
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`A
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`B
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`C
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`E
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`F
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`G
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`H
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`J
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`K
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`L
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`1 46 222
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`221
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`t10
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`438
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`2
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`Rabbit
`globin mRNA
`
`Transformant
`globin RNA
`(A) Structure of the rabbit 3-globin genomic clone Rf3G1.
`FIG. 1.
`The solid box represents the mRNA coding sequence in the adult
`13-globin gene. The clear regions bounded by coding sequence indicate
`the intervening sequences within the f3-globin gene. The larger 3'
`intervening sequence is about 600 base pairs long and the smaller 5'
`sequence (shown only in the lower map) is about 125 base pairs long.
`Restriction sites are indicated by arrows: v, Kpn I; 0, Pst I. (B)
`Structure of the cDNA clone pBG1 and rabbit f3-globin mRNA. The
`Hha I restriction fragment of p3G1 is shown. The heavy black lines
`indicate pMB9 plasmid vector sequence and the thin straight line
`indicates rabbit mRNA sequence: * Hha I sites. The map of rabbit
`globin mRNA shows the 438-nucleotide translated region bounded
`by the 5' 56-nucleotide untranslated region and the 3' 95-nucleotide
`untranslated region. The bottom map is of cytoplasmic polyadenyl-
`ylated rabbit globin RNA from transformant cell line 6, which lacks
`approximately 48 nucleotides of 5' mRNA sequence (see Results).
`
`Rabbit ,B-globin genes in transformed mouse L cells. High
`FIG. 2.
`molecular weight DNA from eight independent cotransformant clones
`was digested with Kpn I and electrophoresed on a 0.7% agarose gel.
`The DNA was denatured in situ and transferred to nitrocellulose
`filters, which were then annealed with a 32P-labeled 4.7-kbp fragment
`containing the rabbit f3-globin gene. Lanes A and L, 50 pg of the
`4.7-kbp Kpn fragment of RfG1; lane B, 15 ,ug of rabbit liver DNA
`digested with Kpn; lane C, 15 ,ug of Ltk- aprt- DNA; lanes D-K, 15
`,ug of DNA from each of eight independently isolated tk+ transfor-
`mants.
`
`Merck Ex. 1034, pg 1110
`
`

`
`5686
`
`Cell Biology: Wold et al.
`Rabbit ,3-globin sequences are transcribed in mouse
`transformants
`The cotransformation system we have developed may provide
`a functional assay for cloned eukaryotic genes if these genes are
`expressed in the heterologous recipient cell. Six transformed
`cell clones were therefore analyzed for the presence of rabbit
`3-globin RNA sequences. In initial experiments we performed
`solution hybridization reactions to determine the cellular
`concentration of rabbit globin transcripts in our transformants.
`A radioactive cDNA copy of purified rabbit a- and 3-globin
`mRNA was annealed with a vast excess of cellular RNA. Be-
`cause homology exists between the mouse and rabbit globin
`sequences, it was necessary to determine experimental condi-
`tions such that the rabbit globin cDNAs did not form stable
`hybrids with mouse globin mRNA but did react completely
`with homologous rabbit sequences. At 750C in the presence of
`0.4 M NaCl, over 80% hybridization was observed with the
`rabbit globin mRNA, whereas the heterologous reaction with
`purified mouse globin mRNA did not exceed 10% hybridiza-
`tion. The RotI/2 of the homologous hybridization reaction was
`6 X 10-4, a value consistent with a complexity of 1250 nucle-
`otides contributed by the a- plus f-globin sequences in our
`cDNA probe (10).
`This rabbit globin cDNA was used as a probe in hybridization
`reactions with total RNA isolated from six transformed cell lines
`(Fig. 3 and data not shown). Total RNA from transformed clone
`6 (Fig. 2, lane H) protected 44% of the rabbit cDNA at com-
`pletion, the value expected if only f-gene transcripts were
`present. This reaction displayed pseudo-first-order kinetics with
`an Rot12 of 2 X 103. A second transformant (Fig. 2, lane E)
`reacted with an Rot112 of 8 X 103 (data not shown). No signifi-
`cant hybridization was observed at Rots > 104 with total RNA
`preparations from the four additional transformants.
`We have characterized the RNA from clone 6 in greatest
`detail. RNA from this transformant was fractionated into nu-
`clear and cytoplasmic populations to determine the intracellular
`localization of the rabbit globin RNA. The cytoplasmic RNA
`
`V0
`.N 20
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`40
`<60
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`2
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`3
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`4
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`5
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`-1
`o
`log Rot
`Hybridization of rabbit globin cDNA with RNA from a
`FIG. 3.
`mouse L cell transformant containing R3G1 sequences. The curves
`represent single pseudo-first-order kinetic components fit to the data
`l)y least-squares methods. 0, Hybridization of rabbit globin
`I:'2P]cDNA (7-9 X 108 cpm/mg) with excess globin template RNA. At
`termination, 80% of the cDNA is in hybrid. The rate constant is 1.1
`X 10() M- I sec-1. v, Hybridization of rabbit globin cDNA with mouse
`globin mRNA. &, Hybridization of excess polyadenylylated cyto-
`plasmic RNA from transformant 6 (see text) with rabbit globin cDNA.
`The rate constant is 2.8 X 10-2 M-1 sec-1. The extent of reaction was
`43% after normalization for the 70% reactivity of the cDNA at the time
`of this measurement. 0, Hybridization of excess total cellular RNA
`from transformant 6 with rabbit globin cDNA. At termination, 43%
`of the [:32P]cDNA was in hybrid. The rate constant is 13.5 X 10-4 M-1
`sec-1. o, Hybridization of excess nuclear RNA from transformant
`6 with rabbit globin cDNA. The SI resistance of cDNA at zero time
`has been subtracted from all hybridization values. These background
`values were 5% and 14% for the cDNA preparations used in this ex-
`periment.
`
`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`was further fractionated by oligo(dT)-cellulose chromatography
`into poly(A)+ and poly(A)- RNA. Poly(A)+ cytoplasmic RNA
`from clone 6 hybridizes with the rabbit cDNA with an Rotl/2
`of 25. This value is 1/80th the Rotl/2 observed with total cellular
`RNA, consistent with the observation that poly(A)+ cytoplasmic
`RNA is 1-2% of the total RNA in a mouse cell. Hybridization
`is not detectable with either nuclear RNA or cytoplasmic
`poly(A)- RNA at Rot values of 1 X 104 and 2 X 104, respec-
`tively. The steady-state concentration of rabbit 3-globin RNA
`present in our transformant can be calculated from the Rotl/2
`to be about five copies per cell, with greater than 90% localized
`in the cytoplasm.
`Several independent experiments argue that the globin RNA
`detected derives from transcription of the rabbit DNA se-
`quences present in this transformant: (i) cDNA was prepared
`from purified 9S mouse globin RNA. This cDNA does not hy-
`bridize with poly(A)+ RNA from clone 6 at Rot values at which
`the reaction with rabbit globin cDNA is complete (Fig. 3). (ii)
`Rabbit globin cDNA does not hybridize with total cellular RNA
`obtained with tk+ globin- transformants at Rot values exceeding
`104. (iii) The hybridization we observe does not result from
`duplex formation with rabbit globin DNA possibly contami-
`nating the RNA preparations. Rabbit cDNA was annealed with
`total cellular RNA from clone 6, the reaction product was
`treated with S1 nuclease, and the duplex was subjected to
`equilibrium denfsity centrifugation in cesium sulfate under
`conditions that separate DNA-RNA hybrids from duplex DNA.
`The S1-resistant cDNA banded at a density of 1.54 g/ml, as
`expected for DNA-RNA hybrid structures (data not shown).
`These data, along with the observation that globin RNA is
`polyadenylylated, demonstrate that the hybridization we ob-
`serve with RNA preparations does not result from contami-
`nating DNA sequences.
`Characterization of rabbit globin transcripts in
`transformed cells
`In rabbit erythroblast nuclei, the fl-globin gene sequences are
`detected as a 14S precursor RNA that reflects transcription of
`two intervening sequences that are subsequently removed from
`this molecule to generate a 9S messenger RNA (unpublished
`results). It was therefore of interest to determine whether the
`globin transcripts we detected exist as a discrete 9S species,
`which is likely to reflect appropriate splicing of the rabbit gene
`transcript by the mouse fibroblast. Cytoplasmic poly(A)-con-
`taining RNA from clone 6 was electrophoresed on a methyl-
`mercury/agarose gel (12) and transferred to diazotized cellulose
`paper (13, 18). After transfer, the RNA on the filters was hy-
`bridized with DNA from the plasmid p#G1, which contains
`rabbit 3-globin cDNA sequences (19). Using this 32P-labeled
`probe, we observed a discrete 9S species of RNA in the cyto-
`plasm of the transformant, which comigrated with rabbit globin
`mRNA isolated from rabbit erythroblasts (Fig. 4). Hybridization
`to 9S RNA species was not observed in parallel lanes containing
`either purified mouse 9S globin RNA or poly(A)-containing
`cytoplasmic RNA from a tk+ transformant containing no rabbit
`globin genes.
`We were unable in these experiments to detect the presence
`of a 14S precursor in nuclear RNA populations from the
`transformants. This is not surprising, because the levels expected
`in nuclear RNA, given the observed cytoplasmic concentration,
`are likely to be below the limits of detection for this technique.
`The 5' and 3' boundaries of the rabbit globin sequences ex-
`pressed in transformed fibroblasts along with the internal
`processing sites carq be defined more accurately by hybridizing
`this RNA with cloned DNAs, followed by SI nuclease digestion
`and subsequent gel analysis of the DNA products (16). When
`
`Merck Ex. 1034, pg 1111
`
`

`
`Cell Biology: Wold et al.
`
`A B C
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`D
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`E
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`28S-
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`18S-
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`9s-
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`4S-
`
`Sizing of cytoplasmic polyadenylylated rabbit globin
`FIG. 4.
`transcripts from transformant 6. RNA was electrophoresed in a 1%
`methylmercury/agarose gel and the RNA was transferred to diazo-
`tized cellulose paper. The positions of 28S, 18S, and 4S RNAs on the
`gel were determined optically after staining with ethidium bromide.
`The RNA on the filter was hybridized with 32P-labeled plasmid DNA
`(pfGl) containing the rabbit f-globin cDNA sequence. Lane A, 1 ng
`of purified 9S polyadenylylated RNA from rabbit reticulocytes, plus
`25 mg of carrier chicken oviduct RNA. Lane B, 50 pg of purified 9S
`polyadenylylated RNA from rabbit reticulocytes, plus 25kg of carrier
`chicken oviduct RNA. Lane C, 1 ng of purified 9S polyadenylylated
`RNA from mouse reticulocytes plus 25 Mg of carrier RNA. Lane D,
`:30 Mg of polyadenylylated cytoplasmic RNA from transformant 6.
`Lane E, 30 Mg of cytoplasmic polyadenylylated RNA from a trans-
`tormant containing no rabbit globin genes.
`
`/-globin mRNA from rabbit erythroid cells was hybridized
`with cDNA clone pfGl (Fig. 1B) under appropriate conditions,
`the entire 576-base pair insert of cDNA was protected from Si
`nuclease attack. When this cDNA clone was hybridized with
`RNA from our transformant, surprisingly, a discrete DNA band
`was observed at 525 base pairs, but not at 576 base pairs (Fig.
`5). These results suggest that, in this transformant, rabbit globin
`RNA molecules are present that have a deletion in a portion of
`the globin mRNA sequence at the 5' or 3' termini. To distin-
`guish between these possibilities, DNA of the X clone, R3G1,
`containing the chromosomal rabbit /3-globin sequence hy-
`bridized with transformed fibroblast RNA. The hybrid formed
`was treated with S1 nuclease, and the protected DNA fragments
`were analyzed by alkaline agarose gel electrophoresis and
`identified by Southern blotting procedures (17). Because the
`rabbit /-globin gene is interrupted by two intervening se-
`quences, the hybridization of mature rabbit mRNA to RfG1
`DNA generates three DNA fragments in this sort of analysis:
`a 146-base pair fragment spanning the 5' terminus to the
`junction of the small intervening sequence, a 222-base pair
`internal fragment bridging the small and large intervening
`sequences, and a 221-base pair fragment spanning the 3'
`junction of the large intervening sequence to the 3' terminus
`of the mRNA molecule (Fig. 1A). When transformant RNA was
`analyzed in this fashion, we observed a 222-base pair fragment
`and an aberrant fragment of 100 base pairs but no 146-base pair
`fragment (Fig. 5); Hybridization with a specific 5' probe
`showed that the internal 222 base pair fragment was present
`(data not shown). The sum of the protected lengths equaled the
`length of the DNA fragment protected by using the cDNA
`clone. Taken together, these results indicate that although the
`intervening sequences expressed in transformed mouse fibro-
`blast are removed from the RNA transcripts precisely, they5'
`termini of the cytoplasmic transcripts we' observe do not contain
`
`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`5687
`
`A
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`1
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`2
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`3
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`B
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`1800-
`
`576- ow
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`EA
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`^ -525---
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`221 222-
`146-
`100-
`
`Characterization of rabbit f3-globin RNA in transformed
`FIG. 5.
`mouse L cells. Numbers of base pairs are given beside the autora-
`diograms. (A) Both total rabbit reticulocyte RNA and poly(A)+ RNA
`purified from cell line 6 were hybridized to the 1.8-kbp Hha I fragment
`from plasmid pf3G1 (Fig. 1B) and analyzed as described by Berk and
`Sharp (16). Lane 1, 0.2 gg of total reticulyocyte RNA was hybridized
`to 20 ng of the 1.8-kbp Hha globin fragment in 5 Al. Lane 2, 18 ng of
`the 1.8-kbp Hha fragment was hybridized in 2.5 Ail in the absence of
`any added RNA. Lane 3, 30 Asg of poly(A)+ RNA purified from cell
`line 6 was hybridized to 75 ng of the 1.8-kbp Hha fragment in 10 ,ul.
`The 1800-base pair band is the renatured Hha fragment. (B) Both
`total rabbit reticulocyte RNA and poly(A)+ RNA purified from cell
`line 6 were hybridized to a 5.60-kbp Pst I fragment containing the
`genomic copy of the rabbit f3-globin gene. The Berk-Sharp analysis
`was carried out by a procedure to be described elsewhere. Only the
`bottom half of the autoradiogram is shown and therefore lane-specific
`background present in lanes 1 and 3, as well as in the RNA- control
`(lane 2) is not shown. We believe that this background results from
`the formation of DNA-DNA duplexes between a small number of
`nicked Pst fragments prior to S1 treatment. Lane 1, 0.35 gg of total
`rabbit reticulocyte RNA was hybridized to 0.1 gg of the 5.60-kbp Pst
`fragment in 10 Al. Lane 2,0.12 ,ug of the Pst fragment was hybridized
`in 10 Ml in the absence of any RNA. Lane 3, 30 Mg of poly(A)+ RNA
`purified from cell line 6 was hybridized to 0.12 Mg of the 5.60-kbp Pst
`fragment in 10AMl.
`
`about 48 i 5 nucleotides present in mature 9S RNA of rabbit
`erythroblasts.
`
`DISCUSSION
`In these studies, we have constructed mouse cell lines that
`contain the rabbit f3-globin gene and have analyzed the ability
`of the mouse fibroblast recipient to transcribe and process this
`heterologous gene. Solution hybridization experiments in
`concert with RNA blotting techniques indicate that, in at least
`one transformed cell line, rabbit globin sequences are expressed
`in the cytoplasm as a polyadenylylated 9S species. Correct
`processing of the rabbit 13-globin gene has also been observed
`in tk+ mouse cell transformants in which the globin and tk
`plasmids have been ligated prior to transformation (20). Similar
`results have been obtained by using a viral vector to introduce
`the rabbit globin gene into monkey cells (21, 22). Taken to-
`gether, these results suggest that nonerythroid cells from het-
`erologous species contain the enzymes necessary to correctly
`process the intervening sequences of a rabbit gene whose ex-
`pression usually is restricted to erythroid cells.
`The level of expression of rabbit globin sequences in our
`transformant is low: five copies of globin RNA are present in
`the cytoplasm of each cell. Our results indicate that the two
`intervening sequences present in the original globin transcript
`are processed and removed at loci indistinguishable from those
`observed in rabbit erythroid cells. Surprisingly, 45 nucleotides
`present at the 5' terminus of mature rabbit mRNA are absent
`
`Merck Ex. 1034, pg 1112
`
`

`
`5688
`
`Cell Biology: Wold et al.
`from the /3-globin RNA sequence detected in the cytoplasm of
`the transformant we have examined. It is possible that incorrect
`initiation of transcription occurs about the globin gene in this
`mouse cell line. Alternatively, the globin sequences we detect
`may result from transcription of a long precursor that ultimately
`must undergo 5' processing to generate the mature 9S species.
`Incorrect processing at the 5' terminus in the mouse fibroblast
`could be responsible for our results. At present, it is difficult to
`distinguish among these alternatives. Because we are restricted
`in our analysis to a single transformant, we do not know whether
`these observations are common to all transformants expressing
`the globin gene or reflect a rare but interesting abberation. It
`should be noted, however, that in similar experiments by
`Weissmann and his colleagues (20) at least a portion of the
`rabbit globin RNA molecules transcribed in transformed mouse
`fibroblasts retain the correct 5' terminus.
`Several alternative explanations can be offered for the ex-
`pression of globin sequences in transformed fibroblasts. It is
`possible that constitutive synthesis of globin RNA occurs in
`cultured fibroblasts (23) at levels five to six orders of magnitude
`below the level observed in erythroblasts. The introduction of
`20 additional globin DNA templates may simply increase this
`constitutive transcription to the: levels observed in our trans-
`formant. Alternatively, it is possible that the homologous globin
`gene is repressed by factors that are partially overcome by a
`gene dosage effect provided by the introduction of 20 additional
`globin genes. Finally, normal repression of the globin gene in
`a fibroblast may depend upon the position of these sequences
`in the chromosome. At least some of the newly introduced genes
`are likely to reside at loci distant from the resident mouse globin
`genes. Some of these ectopic sites may support low level tran-
`scription. Our data do not permit us to distinguish among these
`and other alternatives.
`Although the number of rabbit globin genes within a given
`transformant remains stable for over a hundred generations of
`culture in hypoxanthine/aminopterin/thymidine (unpublished
`studies), it has not been possible to prove that these sequences
`are covalently integrated into recipient cell DNA. In previous
`studies, however, we have demonstrated that cotransformation
`of either OX174 or plasmid pBR322 results in the stable inte-
`gration of these sequences into high molecular nuclear DNA.
`In the present study, the globin gene represents a small internal
`segment of the high molecular weight concatenated phage
`DNA used in the transformation (Fig. 1A). Analysis of inte-
`gration sites covalently linked to donor DNA is therefore dif-
`ficult. Preliminary studies using radioactive X sequences as a
`probe in DNA, blotting experiments indicate that, in some of
`our cell lines, we have introduced a contiguous stretch of re-
`combinant phage DNA, with a minimum length of 50 kbp.
`The presence of 9S globin RNA in the cytoplasm of trans-
`formants suggests that this RNA may be translated to give rabbit
`3-globin polypeptide. Attempts to detect this protein in cell
`lysates using a purified anti-rabbit f3-globin antibody (kindly
`provided by S. Boyer) have thus far been unsuccessful. It is
`possible that the globin RNAs in our transformant are not
`translated or are translated with very low efficiency due to the
`absence of a functional ribosomal binding site. The cytoplasmic
`globin transcripts in our transformant lack about 48 nucleotides
`of untranslated 5' sequence (Fig. 1B), which includes 13 nu-
`
`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`2.
`3.
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`4.
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`6.
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`cleotides known to interact with the 40S ribosomal subunit in
`nuclease protection studies (24, 25). Even if translation did
`occur with normal efficiency, it is probable that the protein
`would exist at' levels below the limits of detection of our im-
`munologic assay due to the low level of globin RNA, and the
`observation that the half-life of (3 globin in the absence of heme
`and a globin may be less than 30 min (22).
`These studies indicate the potential value of cotransformation
`systems in the analysis of eukaryotic gene expression. The in-
`troduction of wild-type genes along with native and in vitro-
`constructed mutant genes into cultured cells provides an assay
`for the functional significance of sequence organization. It is
`obvious from these studies that this analysis will be facilitated
`by the ability to extend the generality of cotransformation to
`recipient cell lines, such as murine erythroleukemia cells, that
`provide a more appropriate environment for the study of het-
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