Shuttle vectors to study somatic mutagenesis and regulation of gene expression in the immune system

147

Gene. 39 (1985) 147-153 Elsevier GENE

1417

Shuttle vectors to study somatic mutagenesis and regulation of gene expression in the immune system (Recombinant galactokinase)

DNA;

immunoglobulins;

B-cell; bovine papilloma virus; plasmids:

pV69, pCGBPV9;

Pedro A. Lazo

Institutefor Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 (U.S.A.) Tel. (215) 728-2445 (Received

March

(Revision

received

(Accepted

lst, 1985) May 20th, 1985)

June lOth, 1985)

SUMMARY

Somatic mutagenesis is one of the main mechanisms for generation of diversity in the immunoglobulin genes. A family of 16 shuttle vectors has been designed to identify the mutation mechanism. These vectors are based on bovine papilloma virus replicon and carry selective markers (NmR and ApR), a mutation marker (the Escherichia coli gaZK) as well as several segments from the immunoglobulin (Ig) heavy- and K light-chain genes in different orientations. They can also be used to study general mutagenesis and the relative contribution of different DNA sequences to the regulation of gene expression by competition with trans-acting factors. These vectors start replicating in mammalian cells two days after transfection. Stable transformants carry the plasmids in an extrachromosomal form for at least three months without evidence of structural alteration.

INTRODUCTION

Several mechanisms are implicated in the generation of antibody diversity: germline diversity (multiple V genes, and several D and J segments), flexibility of V-J and V-D-J joining, combinatorial diversity of heavy and light chains, and somatic mutagenesis (Tonegawa, 1983). Somatic mutagenesis was first identified by protein sequence of I chains (Weigert Abbreviations:

Ap, ampicillin;

bp, base pair(s);

papilloma

virus; Cm, chloramphenicol;

D, diversity;

globulin;

IVS, intervening

sequence;

GalK,

or 1000 bp;

Km,

joining;

kb, kilobase

lipopolysaccharide;

neo, gene coding

phosphotransferase

conferring

tibiotic

G418;

Nm, neomycin;

0378-l 119/85/$03.30

0

Ig, immuno-

galactokinase; kanamycin;

J, LPS,

for Tn5 aminoglycoside

resistance

to Km, Nm, and an-

R, resistance;

1985 Elsevier

BPV, bovine

Science

V, variable.

Publishers

et al., 1970) and confirmed later at the DNA level. The three types of Ig chain genes are able to mutate (Baltimore, 198 1; Tonegawa, 1983). The mutation mechanism seems to be a property of rearranged Ig genes but not of genes in germline configuration like the other allele of a V, gene productively rearranged or in germline J,. Somatic mutagenesis becomes active at the stage of pre-B cell (Ziegler et al., 1984) and seems to be continuous for several generations (Huppi et al., 1984; Clarke et al., 1985), but there is no evidence to link it to the replication mechanism or transcriptional activity. The Ig gene mutation rate is extremely high: about lo- 3 mutations/bp per division. This rate was determined measuring the reversion of antibody mutants to antigen binding (Cook and Scharff, 1977) and by analysis

148

of the accumulation of mutations in the response to influenza hemagglutinin (McKean et al., 1984; Clarke et al., 1985) and in an isogenic anti-idiotypic response (Sablitzky et al., 1985). The background mutation in mammalian cells is lop7 to lo- ” (Loeb and Kunkel, 1982). Some of the mutations have functional implications, the mutated antibody can have an increased affinity for its antigen (Grilliths et al., 1984) or acquire a new specificity, some of which might be against self (Diamond and ScharfT, 1984). The analysis of Ig genes in the response to influenza hemagglutinin (Clarke et al., 1985) has shown that the mechanism of mutation affects randomly both noncoding (flanks and IVS) and coding DNA (both silent and replacement mutations), in a 2-kb area around the rearranged Ig gene. This target area is less than one-third of the transcriptional unit. The initial studies located the mutations concentrated in the complementarity-determining regions, but that was the bias of the analysis since the role of antigen selection was unavoidable. Most mutations are not related to the gene function, but it seems that the antigen selects particular mutants (McKean et al., 1984; Clarke et al., 1985; Sablitzky et al., 1985). So far all the evidence suggests the existence of hypermutation mechanism but we do not really know what that mechanism is, whether it is developmentally regulated or cell-specific, or both. The sequencing approach to the problem has reached its limit and new and radically different ways need to be found to identify and characterize the mutator system. For this purpose I have developed a family of 16 shuttle vectors designed with the mutation mechanism in mind and their construction and other possible uses are reported here.

MATERIALS

AND METHODS

All plasmids were prepared using standard methods (Maniatis et al., 1982). Restriction enzymes were from BRL or New England Biolabs used according to the manufacturer’s protocol. The following cell lines were used for transfection experiments: 7OZ/3 and MPC-11 were grown according to Perry and Kelley (1979) and NFS-5.4lcil

was grown according to Hardy et al. (1984). The cells were transfected using the DEAE-dextran protocol adapted to lymphoid cells in suspension by Deans et al. (1984). Low M, DNA was prepared using the method developed by Hirt (1967).

RESULTS

(a) Experimental design

The basic idea is to clone fragments of Ig genes in such a way that they are located next to a bacterial gene marker that can be used to score mutations quickly. The mutation of the marker gene will occur during the passage of the shuttle vector through mammalian cell lines and be detected by an easy bacterial assay; this approach permits the screening of a very high number of gene copies bypassing the need to perform DNA sequence analysis. With that purpose in mind the mouse k-light- and heavy-chain Ig genes have been split into several fragments both in germline and rearranged configurations, and placed next to the galK gene from E. coli as mutation markers. BPV vectors were used because they have the ability to replicate in mammalian cells as a plasmid. For that particular work two shuttle vectors have been used: pV69 (Meneguzzi et al., 1984), that has the 69% of BPV genome and a single Hind111 site, and pCGBPV9d5 (Matthias et al., 1983) with the entire BPV genome and a single BamHI site. Both vectors have the neo gene to select with antibiotic G418 in mammalian cells and with Km in E. coli. (b) Cloning strategy

The general cloning strategy consisted in preparing all Ig DNA segments and galK as HindIII-BarnHI restriction fragments. In that way they could be first coligated and by appropriate single restriction enzyme digestion cloned next to each other in the BarnHI or Hind111 site of the shuttle vector of choice (Fig. 1). These restriction sites are located in the bacterial portion of the shuttle vector. Usually 1.5 pg of plasmid with Ig fragment and 1 pg of pUCgalK were digested with BamHI +HindIII followed by ligation to each other. The ligated population was

149

T4 DNA

from which gulK can be recovered with different restriction sites at its ends. The gulK mutations can be assayed using the E. co/i strain HBlOl (F-, hsdS20 (r, - , mn - ; recA 13, gaZK2) on MacConkey indicator plates with galactose (Miller, 1972). From the IClight chain the following Ig fragments were used: a germline and a productively rearranged VK2, gene, the germline J, cluster and the enhancer and C, sections. From the heavy chain a productively rearranged V gene from the 5558 family, several J genes and the enhancer were used to construct the vectors. The descriptions of the individual Ig fragments are presented in the legend of Fig. 2. In all cases where the Ig gene segment has a sequence of known function, this sequence is less than 300 bp from the gulK gene. All the vectors with their respective transcriptional orientations are shown in Fig. 2. The constructions of Ig and g&K can be isolated as single restriction fragments (BamHI or HindIII) that can be transferred to other vectors, like SV40, if required.

ligase

B

gal K

galK

Ig

pCGBPVSA5

galK

1s

+

+ -Barn

HI

pV69 Hind III

T4 DNA ligase Transfect

Screen for Fig. 1. General fragments

cloning

are pUCl2

I

red’ colonies Ig

strategy.

(d) State of shuttle vectors in transfected lymphoid cells

HE3101 (gal K-1

(gal K+)

inserts The plasmids

or pUC 13 unless otherwise

containing indicated

Ig

in the

legend of Fig. 2.

split into two portions, and digested with either Hind111 or BamHI for subcloning in shuttle vectors by ligation to 1 pg of either HindIII-digested pV69 or BumHI-digested pCGBPV9dS. This approach of double cloning simplified considerably the process of vector construction by eliminating the use of linkers and at the same time allowing us to dictate the orientation and location of the fragments in a single step. If the DNA fragment did not have these restriction sites, they were first subcloned in pUC12 (Vieira and Messing, 1982). (c) Gene fragments and shuttle vector construction

The galK gene was isolated from plasmid pGSCII (Razzaque et al., 1984) as a 2-kb HindIII-BarnHI fragment and subcloned in pUC12 to form pUCgulK

The possible uses of the vectors reported here for mutagenesis in the immune system, general mutagenesis, or characterization of minichromosome structure induced by specific sequences rely on the presence of these vectors in a nonintegrated or plasmidial form in transfected mammalian cells. Three lymphoid cell lines: 702/3, MPC-11 and NFS-5.4~2 were transfected with plasmids 5, p69BR, p69EC,-7, p69VJ,,-558 and ~69V,,,,p69E,. Digestion of Hirt extracts with DpnI to degrade plasmid of bacterial origin showed that approx. 10% of the plasmid DNA has replicated in the mammalian cell two days after transfection. DNA was isolated daily until day six after transfection; at this moment the antibiotic G418 was added to select for stable transformation. DNA from stable transformants was isolated after three months of growth in selective conditions. The result of such analysis using plasmid p69V,,,,-5 is shown in Fig. 3. The DNA remains in an extrachromosomal form in the three cell lines even after a very long period of culture. No evidence of integration in the host genome was detected by Southern blot analysis. The plasmid DNA isolated from mammalian cells mi-

150

A

B p5VJH1 -558

H

Ri&

golK_

p69 ‘k2tc-5

M

p69JK17

H 4:

p69 IVS,31

H?&(B) 1

JK

(8) . I

z

H vJi+l B

BI

galK

Hr

H

z

H

x

P5JH2

H

EK

H

H

p69VJHf-558

u

g

*

B

golK

B5.H

H

P69JH2 v

HCB

vJHl

H-

HG

B

F

B I

*

P5h-j

(B)

p5 I”&

HGB 1

Fig. 2. Map of shuttle Ig fragments.

i%

H =i(B)

w

HBRB I J

H

B I

p5BR

H

I

vectors

E, enhancer;

with a common

BP”

derived

from pV69 (A) or pCGBPV9d5

C, constant

region;

5’ overhang.

et al., 1984) and subcloned rearranged

B, BarnHI;

H, HindHI;

were used. (1) V d,Xo: a 1.6-kb PsrI-BumHI productively

gene of myeloma

R, EcoRI;

fragment

into a HindIII-EcoRI

V rcz,c gene has two internal Hind111 fragment has an internal

from myeloma

BR, EarnHI-EcoRI

required

are erased.

K light chain

(4)The

HindIII-BamHI been published

fragment

PC3741

has the V,,,

PC2413 in ,l Charon

for the function enhancer

and 0.4 kb with the J,, as a 1.2-kb BamHI-XbaI

fragment

(7) The heavy chain enhancer

(PUCE,)

for further isolation was ligated

is converted

(C,)

fragment.

A detailed identified

used in this case because

PC8778

map comprising

to JH,. This clone, pVhJ558

as a 0.7-kb BnmHI-Hind111

to g&K and subcloned

fragment.

in the papilloma

in pUCl2

(8) For a control vectors

It

of IVS without

fragments

were

(pUCJ,)

subcloned

and Baltimore, (Brodeur

and Riblet,

J,,

and J,,

1984). gene

was isolated

in a BumHI-Hind111

of pJ11 and subcloned

shuttle vector the small HindIlI-BamHI

in the same way.

region has

1983). (5) The

of 0.8 kb with the VJ,,

to be converted

digestion

as a 3.7-kb

the germline J,-C,

was used as such for ligation to galK. (6) The J, cluster containing by XbaI-Hind111

the

as a 2.7-kb

ends but aRer the ligation these two sites

from myeloma

into two BamHI-Hind111

(Gillies et al., 1983) was isolated

in

in the Hind111 site of pUC12.

as 5’ of C, region (Queen

rearranged

the

(pUCV,,,c)

of IVS were subcloned

was subcloned

restriction

family

(2)V,,,,:

as a 9-kb Hind111 fragment

could not be conveniently

region

Ig fragments

gene of the V,,,

in the SmaI site ofpUC12

J, and beginning

of the

by two restriction

with the germline J, cluster and a I-kb fragment

from pJl1 (Marcu et al., 1980) and subcloned

fragment.

from pBR322

location

5558 productively

that by Hind111 digestion

gene. This plasmid

It was subcloned

of h’genes. Bg01 ends can be ligated to BamHI and the a-constant

heavy chain V gene is the gene from myeloma

into a BamHI-Hind111

to J,,.

orientation

of ends created

with agermline

with the gene was subcloned

fragment

the transcriptional

of pBR322. The following

fragment

27 by K. Huppi. This fragment

a 1.7-kb HindIII-BglII

in pUC12 (PC,) and used as indicated.

fragment

g ene joined

that was ligated to gulK. BamHI

(Van Ness et al., 1982) and the enhancer

is a 1.2-kb BamHI

small fragment

) to be converted

(pUCV,,i,.

indicate

as result of ligation

sites in the coding region. (3) The germline

Bg/II site that generates

any known sequence

erased

was isolated from the 6-kbEcoR1

in pUCl2

fragment

BumHI

(B). The arrows

(B), BamHI

27 by K. Huppi. From this new clone, a I-kb AccI fragment

and converted

1

L

BP”

I Charon

B .. 91 I

BBRH

golK

p69 BR

(Heindrich

B

..

XH

H

-golK

)

p=EH

B

~

Hj;;B

JH-7

enzymes

H2

p5 JH-7

p69EC,-7

p69

B -

in pUCl2 fragment

151

5

tom \’ gz x-

days after transfection R2345 6

Rbr-r=

A

B

Fig. 3. Southern

blot analysis

from

lymphoid

transfected

transfected

with p69V,,,-

of low-M,

5 was prepared

splitting the culture in two: one halfto to cominue

of different

(Hirt

from

extract)

7OZ/3 cells

on a daily basis by

isolate DNA and the other

the culture. At day six the antibiotic

to select for stable transformants. mants

DNA

cells. (A) DNA

G4l8 was added

(B) DNA from stable transfor-

cell lines transfected

with p69V,,,,-5

that

have been growing in G418 (400 pg/ml) for at least three months. The different supercoil

bands

and relaxed

pg of p69V,,,,-5. used as a probe

are monomeric

and

forms. The reference

32P-labeled (Maniatis

nick-translated

plasmid

in

lane (R) contains

dimeric

50

p69V,,,,-5

was

et al., 1982).

grates in gels like the control (Fig. 3), suggesting that no major alteration of the plasmid DNA like large deletions or recombinations have taken place.

DISCUSSION

Though these shuttle plasmids have been designed with the mechanism of somatic mutagenesis in the immune system in mind there are also other aspects of the biology of the system for which these vectors can be of use. Vectors p69BR and p5BR can be used to study the mutation background in normal cells by using the gulK gene, and for general mutagenesis. Vectors p69IVS,-31 and pSlVS, can also be used for this

purpose if nothing is found in the intervening sequence. Studying mutagenesis with shuttle vectors one must be aware of the fact that the transfection of mammalian cells usually results in significant damage to the plasmid molecules. Shuttle vectors with the 1acZ (Lebkowski et al., 1984), gulK (Razzaque et al., 1984) or@ (Ashman et al., 1984) genes have a spontaneous mutation frequency of 2%. This vector damage seems to happen in the very early stages of transfection before replication in mammalian cells has taken place. The background mutation frequency does not increase with time (Lebkowski et al., 1984; Razzaque et al., 1984) even if one waits for the appearance of stable transformants and follows them (Ashman et al., 1984). This damage seems to be vector-independent. The mutation rate of Ig genes is about 10 - 3 mutations/bp per generation (Cook et al., 1977; McKean et al., 1984; Sablitzky et al., 1985). Based on this rate, in the first live generations between five to ten mutations will be accumulated per gene copy in the target area. This number of mutations should result in a mutation frequency near loo%, that is well above the 2% background. In some pre-B cells the mutation background seems to be less than 0.3% (P.A.L., unpublished data). Regarding the existence of specific mutator systems, a similar approach has been successful in the detection of an inducible error-prone mutation mechanism (Sarkar et al., 1984), even though the discrimination level was at least ten-fold lower than that expected for the Ig genes. These vectors not only should allow the identification of the sequences involved in the mutator mechanism, but also whether this mechanism is developmentally regulated and/or is tissue-specific by transformation of different cell lines. A similar approach can be used with T-cell receptor genes for which the occurrence of somatic mutation in the P-chain has already been reported (Augustin and Kim, 1984). The other main use of these plasmids is a consequence of the dissection of Ig genes. They can be used in competition experiments to determine the relative contribution of specific DNA sequences to the regulation of Ig gene expression. This kind of approach has already proven successful for enhancer sequences linked to the CmR gene (Mercola et al., 1985). For that purpose, several plasmids can be used to study promoter regions in rearranged (~69V,2,,- 5, p69VJ,,558 and p5VJ,,558) or germ-

line configuration (p69V,,,,0). This p69V,,iX0 has also a recombination signal at its 3’ side. Germline recombination signals in a 5’ location are found in plasmids p69J,-17, p69J,-7, p5J,-7 or as a single signal in p69J,, and p5J,,. Enhancer sequences from the K light chain are found in p69EC,-7 and from the heavy chain in p69E, and p5E,. As a result of the extrachromosomal nature of BPV virus, the vectors can be retrieved as minichromosomes (Reeves et al., 1985) to detect the transacting factors that recognize specific DNA sequences. The results shown here support this possible use; the cloning of other genes in BPV vectors sometimes results in their integration in the host genome. It is expected that this family of shuttle vectors should give significant information on several aspects of Ig gene expression and mutagenesis either immune-specific or general.

Cook,

W.D.

and

Scharff,

mouse myeloma

mutants

of

5687-5691. an immunoglobulin phocytes. Diamond,

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G.M.,

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I am grateful to M. Weigert for encouragement and support, V. Hay for technical assistance, D. Dimitri for typing and P. Brodeur, F. Cuzin, M. Seidman and P. Matthias for providing several plasmids. I have been supported by fellowships from the NIH Fogarty International Center and the Cancer Research Institute, Inc., New York.

Antigen-binding

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Hirt,

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