Correct transcription of a cloned mouse immunoglobulin - Europe PMC

Proc. Natt Acad. Sci. USA Vol. 80, pp. 417-421, January 1983

Biochemistry

Correct transcription of a cloned mouse immunoglobulin gene in vivo (promoter/simian virus 40/enhancer/transient-expression assay)

DIDIER PICARD AND WALTER SCHAFFNER Institut fur Molekularbiologie II der Universitit Zurich, HMnggerberg, 8093 Zurich, Switzerland

Communicated by A. Frey-Wyssling, October 27, 1982

ABSTRACT We have obtained correct transcripts from the mouse immunoglobulin AI light chain gene on transfection into human HeLa cells. Linkage to simian virus 40 (SV40) DNA containing a transcriptional "enhancer" element was required to raise A-chain gene transcription to a detectable level in our transientexpression assay. The transcripts had the same 5' end as authentic Al mRNA when the SV40 enhancer element was 150 base pairs upstream from the cap site. The situation was different when the A-chain gene promoter was separated from the SV40 sequences by more than 1 kilobase pair of spacer DNA; then, A-chain gene transcripts were not correctly initiated in human HeLa, monkey CV-1, and mouse 3T6 cells. In this respect, the A-chain gene behaves differently from the rabbit 3-globin gene, which can be activated by the SV40 enhancer over distances of several kilobases [Banerji, J., Rusconi, S. & Schaffner, W. (1981) Cell 27, 299-308].

To date, most of the DNA sequences necessary for efficient transcription of eukaryotic genes in vivo have been located within some 100 base pairs (bp) upstream from the transcription initiation site (1). However, a bidirectional "modulator" element located more than 110 bp upstream of a sea urchin H2A histone gene has been found (2) that is required for maximal expression of this gene. Furthermore, it has been found that both a small DNA fragment of simian virus 40 (SV40) and one of polyoma virus enhance the transcription of a linked rabbit ,globin gene in vivo by two orders of magnitude. The enhancing DNA segment ("enhancer"), which is located between the viral origin of replication and the late genes, acts in either orientation and over distances of at least 1,400 bp (refs. 3, 4; cf. also ref. 5). These properties may be exploited to investigate the expression of a wide range of cloned genes. In the present study, we have linked a somatically rearranged gene coding for a mouse immunoglobulin Al light chain (6, 7) to SV40 DNA and transfected it into mammalian cells. We were interested in developing an in vivo system that could be used to study immunoglobulin gene expression. It has been shown that a human K variable region gene gives similar patterns of initiation of transcription whether injected into Xenopus oocytes or assayed in vitro (8). However, it is unknown whether correct initiation occurs in these systems because the natural cap site has not been mapped. As recently reported, no specific transcripts from a mouse K light chain gene could be detected in mammalian cells unless the K-gene promoter was replaced by the SV40 late promoter (9). Here, we show that linking the A-chain gene with its own promoter to SV40 DNA leads to the accumulation of high levels of A-chain gene-specific transcripts in a transient-expression assay. These RNAs are not faithfully initiated when the SV40 sequences containing the enhancer are located more than 1 The publication costs ofthis 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.

kilobase pair (kb) from the A-chain gene cap site. However, correct initiation is observed when the enhancer element is inserted 150 bp upstream from the transcription initiation site. MATERIALS AND METHODS Plasmid Construction. The genomic mouse AI light chain gene clone AgtWES Ig 303A, isolated from the myeloma line HOPC2020, was a gift from S. Tonegawa (Cambridge, MA) (6, 7). The A-chain gene was excised from AgtWES Ig 303A and recloned (Fig. 1) as follows. (i) Clone B1, the 7.4-kb EcoRI Achain gene fragment, was inserted into the EcoRI site of the previously constructed recombinant plasmid pBSV-early (10). (ii) pIS+, the 7.4-kb A-chain gene fragment, was cloned by means of synthetic EcoRI linkers into the Kpn I site of a SV40 recombinant plasmid. The SV40 recombinant was constructed by ligating the BamHI/EcoRI fragment from pBR327 (11) to the large BamHI/Hae II fragment from SV40, thereby fusing the EcoRI and Hae II sites. (iii) JSIB+, the 4.4-kb A-chain gene fragment generated by partial Xba I digestion, was inserted into the Xba I site about 1,100 bp downstream from a rabbit ,globin gene in a globin gene-SV40-pJC-1 recombinant plasmid. The A-chain gene was in the same orientation as the 3globin gene in JSI+ but in the opposite orientation in clone JSIB-. Both clones retain about 290 bp upstream from the AUG of the Achain gene and more than 1,000 bp downstream from the gene. (iv) pIX, the 7.4-kb EcoRI A-chain gene fragment, was first inserted into the EcoRI site of pBR322. In this recombinant, the Sac I site upstream from the A-chain gene was changed into a Xho I site by using synthetic linkers. pIXE, the subcloned SV40 DNA fragment spanning map positions 100-272 (12), which includes the 72-bp repeats (13), was inserted into the newly created Xho I site ofclone pIX. Standard cloning procedures were used. All work involving recombinant plasmids was done under conditions conforming to the standards outlined in the National Institutes of Health guidelines for recombinant DNA research. Cell Growth and Transfection. The BALB/c myeloma line HOPC2020 was maintained by injection into female BALB/c mice (14). Tissue culture cell lines were grown and transfected by the calcium phosphate coprecipitation technique (15) as described (3). RNA Analysis. RNA extracted from transfected cells was incubated with RNase-free DNase to remove residual input plasmid DNA. To analyze the transcripts by nuclease Si mapping (16, 17), we used either a double-stranded probe with a homogeneously labeled minus strand (B. Wieringa, P. Dierks, and C. Weissmann, personal communication) or an end-labeled single-stranded DNA probe. Using the former probe, we incubated the RNADNA hybrids with RNase-free DNase prior to nuclease S1 digestion to specifically cleave renatured DNA Abbreviations: bp, base pair(s); kb, kilobase pair(s); SV40, simian virus 40; T antigen, SV40 large tumor antigen.

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Biochemistry: Picard and Schaffner

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Proc. Nad Acad. Sci. USA 80 (1983)

caD-n

c

B

EcoRI

(Kpn 1)

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C

pi S+ BamHI

Amp EcoRI

EcoR/ (KpnI)

D

E

EcoRI plasmid DNA

SV40 DNA

mouse

DNA

rabbit DNA

FIG. 1. Schematic representation of recombinant DNAs used. (A) The mouse EcoRI DNA fragment containing the immunoglobulin Al light chain gene (6, 7). The black line, the black bars, the hatched bars, and the open bars are noncoding DNA, translated regions, untranslated regions, and intervening sequences (IVS1 and IVS2), respectively. (B) Map of clone B1. (C) Map of pIS+. (D) Map of JSI,8+; in JSI(-, the A-chain gene is in the opposite orientation. (E) Map of pLX; pIXE contains the SV40 enhancer element at the Sac I site upstream from the A-chain gene. Amp, (3-lactamase gene; ori, SV40 origin of replication; e, SV40 early region; 1, late region; V, J, and C, variable, joining, and constant regions of the A-chain gene, respectively.

probe (B. Wieringa, P. Dierks, and C. Weissmann, personal communication). For "primer extension" with reverse transcriptase (18), a single-stranded DNA primer was prepared by exonuclease III digestion of an end-labeled DNA fragment (19). RNase-Free DNase. Macaloid. The mineral macaloid, a clay commonly used as a lubricant in oil well drilling, has been known for many years to adsorb RNase, and it has been used to remove RNase from preparations of RNA (ref. 20, cited in ref. 21) and bovine serum albumin (M. A. Billeter, personal communication). One-half gram of macaloid powder (a gift from the Georg Langer Company, Ritterhude/Bremen, Federal Republic of Germany) was suspended in 50 ml of 50 mM Tris HCl (pH 7.6) and the suspension was boiled for 5 min with constant agitation and then centrifuged at room temperature for 5 min at 4,000 rpm (= 2,500 g). The supernatant was discarded, and the sticky pellet was completely suspended in 40 ml of 50 mM Tris HCl (pH 7.6). Centrifugation and resuspension of the pellet were repeated twice more. Particularly in the later centrifugations, the pellet tended to consist of an upper jelly-like part and a more solid and sticky lower part. A fourth centrifugation

step was done for 15 min at 5,000 rpm. Then, the pellet was suspended in 30 ml of Tris'HCl (pH 7.6) to give a final macaloid concentration of 16 mg/ml. Such suspensions have been stored for more than 20 months in the refrigerator without detectable loss of activity. Macaloid from other sources (two lots from NL Chemicals, Hightstown, NJ, and one lot from National Lead, Houston, TX) were also suitable for DNase I purification. Macaloid treatment of DNase. The content (100 mg) of one ampoule of lyphilized DNase I (Worthington 2139 DP; 1,588 Kunitz units/mg) was dissolved in 5 ml of enzyme dilution buffer [20 mM Tris HCI, pH 7.6/50 mM NaCI/1 mM dithiothreitol/50% (vol/vol) glycerol/bovine serum albumin (100 tkg/ ml); ref. 10]. The actual protein concentration of the resulting solution as measured by the method of Schaffner and Weissmann (22) was 12.9 mg/ml. Two milliliters of concentrated DNase solution was diluted with 6 ml of cold 50 mM Tris HCl (pH 7.6) at 00C in a 13-ml polyethylene tube. Macaloid (16 mg/ ml) was resuspended by quick shaking, and 2.8 ml of the suspension was added to the DNase. The content of the tube was mixed on a rotating wheel (30 rpm) at 40C for 30 min. The tube was centrifuged for 10 min at 8,000 rpm. The supernatant was decanted into a tube that contained 2.8 ml of macaloid suspension and this was mixed for 30 min at 4°C. The tube was centrifuged at 10,000 rpm for 15 min. The supernatant was removed without taking any macaloid along, mixed with an equal volume of cold autoclaved anhydrous glycerol, and stored in aliquots at -20°C. Such preparations (activity, 0.9 unit/,ul) were stable for at least 6 months. Fifty percent of the DNase activity was recovered, and its specific activity had tripled to 4,900 units/ mg. Three further batches of DNase (Worthington DP 36B844X, 1,850 units/mg; Worthington 2006 D, 2,722 units/mg; and Sigma DN-CL, 2,500 units/mg) also gave highly active RNasefree DNase preparations. Analysis of RNA that had been incubated with either DNase I or sterile 50% glycerol showed that 1 unit of DNase cleaved