107 GLWP. 40 (1985) 107-i 14

Elscvicr

GENE 1464

Structure and expression of K-chain genes in two IgE-producing rat immunocytomas

(Recombinant DNA; immunoglobulin; somatic mutation; C region; V region; J region)

Lars Hellman”*, Ake Engstram”, Hans Bennichb and Ulf Pettersso$

(Received April 3rd. 19X5)

(Revision received August 16th. 19X5)

(Accepted August IYth, 1985)

-- _-

The light chain expression in two IgE-producing rat ~mmunocytomas, IR2 and IR162, was studied. Both

immunocytomas produce Iight chains of the K type. The K chains were characterized at the protein level by

sodium dodecylsulfate (SDS)-polyacryl~ide gel eIectrophoresis (PAGE) and amino acid (aa) sequencing.

cDNA clones co~esponding to the k-chain mRNA were also prepared and sequenced. The results showed

that rat ti chains have the same structure as their mouse counte~arts with respect to signal sequence cleavage,

somatic mutations in the V-J region and invariance of all the aa positions which are strongly conserved in the

frame work regions of mouse V, chains (> 95 % conservation). Results from studies on k--chain transcription

lend support to the ahelic exclusion model with only one functionally expressed light chain in each immuno-

cytoma.

INTRODUCTION

Several characteristics of L- and H-chain expres-

sion have been clarified by studies of mouse myelo-

mas. The k-chain locus is activated through somatic

recombination where one V gene out of a large num-

---- * To whom correspondence and reprint requests should be

addrcsscd.

Abbreviations: aa, amino acid(s); Ad, adenovirus; AMV, avian

mvcloblastosis virus; bp, base pair(s); CDR, complementarity

determining region; DEAE, diethylaminoethyl; 1.1, heavy; kb.

kilobasc pairs; L, light; nt, nuclcotide(s); PA, polyacry(amide;

PAGE, polyacrylamidc gel electrophoresis; SDS. sodium dode-

c~lwlt,tc.

bcr of germ-line V genes is connected to one out of

four functional germ-line J segments (Max et al.,

1979; Sakano et al., 1979). In this way a functional

transcription unit is created where one V gene is

specifically activated to a high level ofexpression. An

enhancer sequence located in the intron between the

J and C exons plays a critical role in the activation

process (Emorine et al., 1983; Queen and Baltimore,

1983; Picard and Schaffner, 1984). When compari-

sons were made between germ-line and expressed

V-J regions no germ-line sequence was found that

perfectly matched the expressed VJ exons due to

point mutations. These are introduced in a limited

area in the VJ region of the rearranged K gene, by an

unknown mechanism. These somatic mutations are

centered around the VJ exon and no mutations are

1137X-I I I%,:X5:SO3.30 0 19X5 Elsevicr Science Publishers

found in the C exon, ~though it is located only a few

kb downstream in the transcription unit (Gearhardt

and Bogenhagen, 1983; for a review see Tonegawa,

1983).

So far only a few studies have been reported on rat

immunoglobulins and their genes (Sire et al., 1982;

Breiner et al., 1982; Hellman et al., 1982a,b; Steen

et al., 1984). Only one complete and a few partial rat

V-region sequences have to date been reported on

and so far none at the nt level. Rat germ-line J ~ and

C, regions have been characterized in detail by

cloning and sequencing the corresponding genomic

locus (Sheppard and Gutman, 198 1; 1982; Burstein

et al., 1982) but no expressed rat x chains have been

studied at the nt level.

In the present study we report the primary struc-

ture of two functionally expressed rat K chains and

some properties of ti-chain expression in the rat.

Our results show that k--chain expression in the rat

is very similar to that in the mouse. Somatic mu-

tations are found exclusively in the VJ region and

there are no nt differences or aa replacements in the

C regions of the two immunocytomas studied. All the

aa which are highly conserved (> 9.5 “/,) in the frame-

work regions of the mouse V, chains (Kabat et al.,

1983) are invariant in the four rat h- chains included

in the present comparison.

MATERIALS AND METHODS

(a) Tumors

The tumors were grown and prepared according

to already published procedures (Hellman et al.,

1982a; Steen et al., 1984).

(b) cDNA cloning

Methods for RNA preparation and cDNA cloning

have been described before (Hellman et al., 1982a;

Steen et al., 1984). Colony hybridization was per-

formed according to the method of Grunstein and

Hogness (1975).

(c) Sl analysis

Two x-chain cDNA clones IR2 : 13 and IR 162 : 6

were cleaved with the appropriate restriction en-

zymes and 5’-end-1abeIed with polynu~leotide ki-

nase. The fragments of interest were isolated from

5% PA gels by diffusion and were purified by DEAE

column chromatography. The end-labeled fragment

was dissolved in 0.01 M Tris HCl, 0.001 M EDTA

pH 7.9 (TE) and made RNase-free by phenol ex-

traction. It was then precipitated together with

carrier RNA and the specific mRNA preparation to

be analysed. Sl analysis was carried out according

to published procedures (Berk and Sharp, 1977;

Weaver and Weissmann, 1979).

(d) Northern blot analysis

Northern blot analysis was performed essentially

according to published procedures (Goldberg, 1980;

Thomas, 1980; Steen et al., 1984) with a slight modi-

fication. The sample was denatured in 5Of’,, for-

mamide, 20 mM morpholinopropane sulfonic acid,

2.2 M formaldehyde at 65 ‘C for 3 min prior to

loading on 20, agarose gels.

(e) Plasmid purification

Mini-scale preparations were made according to

the method of Birnboim and Doly (1979). Large-

scale plasmid puri~cation was done according to the

method of Bywater et al. (1983).

(f) Isolation and purification of rat immunoglo-

bulin li: (IgE)

Rat myeloma proteins were separated on a Se-

phadex G- 100 column in 1 M acetic acid-O. 1 M for-

mic acid. After lyophilisation the purity of the

isolated polypeptides was assessed by SDS-PAGE

in 4-20’1; gradient gels under reducing conditions.

(g) Amino acid sequencing

The purified L chains were reduced completely in

6 M guanidine-HCl, 0.5 M Tris . I-ICI pH 8.0 and

alkylated with iodoacetic acid. Cleavage of the pro-

teins, purification of peptides, and sequencing were

performed as described by Engstrom et al. (1984).

M lR162 lR2 M M (+) C-1 M

Fig. I. Analysis of in viva and in vitro synthesized H and L chains of immunoglobulin E from the IR2 and IR162 immunocytomas.

(Panel A) Analysis of in viva synthesized I&E from the IRI62 and IR2 immunocytomas by SDS-PAGE. Ad2 proteins wet-c used as

size markers (M). (Panel B) In vitro synthesis of IR162 K chains. mRNA was extracted from the IRI62 immunocytoma and translated

in vitro. The synthesis of ti chains was detected by immunoprecipitation using a K-specific antiserum. In vitro translation was carried

out both in the presence ( + ) and in the absence ( - ) of a microsomal membrane fraction. (Panel C) Identification of a cDNA clone

encoding the IR2 K chain. DNA was extracted from the cDNA clone and from the pBR322 vector and was immobilized on a

nitrncellulose filter. The filter was used for selection of RNA from the IR2 immunocytoma. The RNA was eluted from the filter and

translated in vitro. Ad2 proteins were used as size markers (M).

RESIILTS AND DISCUSSION

(a) Synthesis of light and heavy chains in the IR2 and

IR162 immunocytomas

For this study the two immunocytomas IR2 and

IR162 from the rat strain LouC/Wsl were chosen

(Bazin et al., 1974; Bazin and Pauwels, 1982). They

produce and secrete large amounts of IgE when pro-

pagated intraperitoneally in congenic Lou rats. The

myeloma proteins were purified from the serum of

Lou rats, carrying the appropriate immunocytoma

and analysed by SDS-PAGE under reducing condi-

tions. IgE from both immunocytomas contained E

chains which were identical in size, about

90-95 kDa1 (Fig. 1A). The L chains, on the other

hand, differed in size and were estimated to be

25 kDal and 21 kDa1 in the IR2 and IR162 tumors,

respectively (Fig. 1A).

(b) mRNA puri~cation and in vitro translation

mRNA was purified from both tumors and trans-

lated in vitro. The products were assayed by immu-

noprecipitation using monospecific antisera for I: and

ti chains (Karlsson et al., 1979). Translation was

performed in the presence or absence of microsomal

membranes to study the different processing steps

involved in the maturation of ti chains. The results

which are shown in Fig. 1B demonstrate that the

IR 162 k’ chain is processed to its mature form by the

addition of microsomal membranes and the size of

the in vitro processed product is identical with that

of its in vivo counterpart. This observation together

with an observed size reduction of about l-2 kDal

during maturation suggests that maturation only in-

cludes signal sequence cleavage and that no glycosyl

residues are added during processing. In the case of

IR2 the results from in vitro processing were not

I IO

completely conclusive, since the unprocessed 1~

chain had the same apparent M, as the mature chain

(results not shown). This might imply that cleavage

is followed by glycosylation. To determine whether

the IR2 K chains indeed were glycosylated, the con-

tents of amino sugars were determined in con-

junction with aa analysis and the contents of neutral

carbohydrate were estimated by the orcinol method

(Vasseur, 1948). The results revealed less than one

carbohydrate moiety per K chain in both IR2 and

IR162.

The complete aa sequence was also determined for

the IR2 K chain by sequence analysis of chemically

and enzymatically derived peptides. All aa residues

were accounted for by the sequence analysis giving

further indication that the IR2 ti chain is unglycosy-

lated. In addition no putative N glycosyl sites (Asn-

X-Thr/Ser) are present in the sequence of the V

region from the IR162 K chain.

(c) Construction of cDNA clones cotrespondin~ to

the x-chain mRNA

mRN.4 from the two immunocytomas was f’rac-

tionated in a 15-30% sucrose gradient and the

fractions containing the highest amount of ti-chain

mRNA were used as substrate for cDNA synthesis.

The resulting cDNA was cloned into the pBR322

vector and DNA from selected clones was extracted

and used for hybridization selection followed by in

vitro translation. One clone out of the first 20

selected mRNAs after in vitro translation yielded a

prominent poIypeptide band of the same size as the

unprocessed K chains (Fig. 1C). The clone was par-

tially sequenced, and the deduced aa sequence was

found to match the aa sequence of rat K chains (see

below). This clone was subsequently used to screen

cDNA libraries containing IR2 and IR162 mRNA

sequences. The cDNA inserts of about 30 positive

clones from each of the immunocytomas were

analysed by PAGE after restriction enzyme cleavage

and the clone from each immunocytoma which

contained the longest insert was mapped and se-

quenced according to the strategy that is outlined in

Fig. 2. The two sequenced clones were found to

cover the entire coding region of the IR2 and

IR162 K mRNAs. The entire insert in clone IR 162: 6

was sequenced to determine the complete structure

of a rat x-chain mRNA. To study the structure of the

L >

1 0 0 h ,,

I:ig. 2. Strategy fix sequencing of K chains. A schcniatic illtk

tration ofthc structure ofthe K chain is indlcated. The horizontal

lines in the upper part of the figurc indicate regions of the IRL

and the IR162 I chains which were studied by aa seqwncc

xnalysis. The cleavage sites for selcctcd restriction enzymes xre

rndicated in the lowr part of the figure. The arrows indicate the

;m1ount of seyuencc iIlf~rrn~ti~)n that was obtained from individ-

very 5’ end of the IR162 ti mRNA primer extension

was used; a HindIII-PvuII fragment from the 5’

region of the IR162:6 clone was isolated, 5’-cnd-

labeled, and hybridized to IRl62 mRNA before

being extended with AMV polymerase. The extended

material was fractionated on a 6’1;, sequencing gel

and the longest extension product was eluted from

the gel and scqucnced. Fig. 3 shows the scquencc.

For the cDNA clone IR2: 13, only the 5’ part,

covering the 5’-noncoding region together with the

signal sequence and the V and J segments, was

sequenced (Fig. 3). The C region of the IR2 : 13 clone

was studied by extensive restriction mapping and the

results revealed no differences as compared with the

constant region of IR162.

(d) Amino acid sequence determination

The purified IgE L chains were sequenced to lo-

cate the exact points of signal sequence cleavage and

to confirm the aa sequence that was predicted from

the cDNA clones. The IR2 ti chain was sequenced

through its entire length. For IR162 only selected

r-egions were studied. Its sequence is, however,

known from the sequence of cDNA clone IR 162 : 6.

III

Fig. 3. The nuclcotide sequences of K chains from the IR2 and

IRl62 immunocytomas. The position ofthe poly(.4) addition site

is indicated as well as the junction between the J and the V

segments. Nucleotides which differ between the IR2 and IRl62

sequencec are indicated. The arrows indicate the positions where

signal sequence cleavage takes place. The broken lines indicate

the 5’ ends of the cDNA clones.

The results made it possible to compare the IR2 and

IR 162 I;-chain sequences with the published Lou rat

germ-line sequence for the constant region (Shep-

pard and Gutman, 1981). The comparison showed

no aa replacements in either of the immunocytomas.

To study the structure of the signal sequence at the

protein level, aa sequencing was performed on x

chains synthesized in vitro in the presence of

radioactively labeled Met, Leu and Pro. The results

obtained were in complete agreement with the pre-

dicted sequence of the signal peptide in IR162 and,

moreover, they made it possible to determine the

exact start point that is used for translation; it is the

lirst of two adjacent Met residues (see Fig. 3 for

details).

(e) Size of light- and heavy-chain polypeptides after

different processing stages

The IR2 E chain is made as a primary translation

product of M, 64670 as determined from the nt se-

quence ~~eliman et al., 1982b). After signal se-

quence cIeavage it is reduced to an M, of 62521

(Hellm~ et al., 1982b). Analysis by SDS-PAGE

yielded a considerably higher estimate for the mature

F. chains which apparently is due to extensive glyco-

sylation (Hellman et al., 1982b).

The apparent discrepancy in size between IR2 and

IR162 ti chains as determined by SDS-PAGE is

difficult to explain. The unprocessed polypeptides

differed by only three aa and their predicted molecu-

lar M,.s are 25954 and 25390, respectively. After

signal-sequence cleavage their M,s are reduced to

23 517 and 23 138. Since no glycosyl residues appear

to be present in the mature x chains the observed

mobility difference is most likely caused by differ-

ences in aa composition and charge (Sheng and

Knight, 197 1).

(f) Features of rat IC chains

A comparison between the established L-chain

sequences and published germ-line J- and C-region

sequences (Sheppard and Gutman, 1981) revealed

no nt differences in the C regions ofthe two expressed

ti chains. The J segments of IR2 and IR162, in

contrast, have no identical homologue among the

germ-line .I segments (Breiner et al., 1982; Sheppard

and Gutman, 1982). Therefore, we conclude that

somatic mutations have occurred exclusively in the

V-J region of rat F; chains as is the case for mouse

h’ chains (Gearhardt and Bogenhagen, 1983; Tone-

gawa, 1983) (Fig. 4).

The signal sequences are 20 and 22 aa long and

both are cleaved after a cysteine residue which is a

common property of many K-chain signal sequences

(Kabat et al., 1983). The hydrophobicity patterns are

the same as for other signal sequences with an inter-

nal stretch of strongly hydrophobic aa.

The V regions are 94 and 95 aa long for IR 162 and

IR2, respectively. The extra aa in V,nz is located at

the junction with the J segment and is probably due

to variability in the site of recombination (Early and

Hood, 1981). This is apparently a common phe-

nomenon and provides an extra source of variation

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lyr Arq IGG ACG TTC GGT GGA GGC ACC AAG CTG GAA TTG AAA CGT

As" Thr Phe Gly Ala G,y Thr lyr, Lee G," Le" Lys Arq AAC ACG TTT GGA GCT GGG ACC AA6 CTG GAA CTG AA.4 CGT

Asp Thr Phe Gly Ala Gly Thr Lys LPu Glu Leu Lvr A19 GAC ACG TTT GGA GCT GGG ACC AAG CT6 GAA CTG AAb CGT

Tyr Thr Phr Gly Ala Gly Thr Lys Leu Glu Leu Lys Arq TAC AC6 TTT GGA GCT GGG ACC ,%A6 CTG GA& CTG AAA CGT

Fhe Thr Phe Giy Ser Gly Thr LYI Lear Glu I!e Lye Arq TTC ACG TTC GGC TCA GGG ACG AAG TTR GAA kTA AA& CGT

ieu Thr me Gly 5ei Giy mr iys Leu Gl" IlP iys Arq CTC ACT, TX GGT TCT GGG ACC ARC, CTG GAG ATC AA4 CGT

Tyr Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys Arq TAC ACG TTT GGA LCC' GGG ACC AAG CTG GkA CTG AAA CGG (JZi

Thr Thr Phe Gly 61~ Gly Thr Lvs LFU Glu Lelr Lys Arq AQ ACG TTT GGA G$T GGG ACC AAG CTA GR4 CTI; AAA CGG (JZh)

Fig. 4. A comparison between the sequences of the J segments

pl-esent in the IR2 and the IR162 K chains and germ-lint se-

yucnces for rat 3 segments (Breiner et al., 1982; Sheppard and

Gutman, 1982). The black dots indicate nt which must have been

mutated in order to convert the corresponding germ-line se-

quence to the sequence found in the rearranged k chain.

in the CDR3 region of the L chain (Tonegawa, 1983).

The overall homology was only 590,; for the V and

J regions when IR2 and JR162 K chains were com-

pared (Figs. 5 and 6). Most of the aa replacements

are conservative although a high percentage of the

residues have been changed by mutation. Certain

properties seem nevertheless important for V regions

of both ti and A chains. The hydrophobicity patterns

are particularly well conserved (Fig. 6), and there are

Fig. 6. H~drophiiicit~ patterns of variable regions from different

imniuno~lobulin L chains. The hydrophilicity piots for the JR?.

and the JR162 K chains xc compared wrth corresponding

regions ofa mouse Vi.,, chain from MOPC 315 (Wu et al., 1983).

Hydrophilicity plots were made according to the method of Hopp

and Woods ( 1983).

IR162 IR2 s 2l1 IR102

lRl62 IR2 MouseVhiI

Fig. 5. An aa sequence comparison between varrable region sequences from four different rat V, chains and one mouse V,,, chain The

black boxes above the sequence indicate aa which are conserved in all four rat ti chains and the aa which are conserved between IR2.

I R 162 V regions, and a mouse V,,, region are shown in the lower panel. The black circles indicate aa which are conserved in more than

95 “<, ofall mouse V, chains studied (Kabat et al., 1983) and also in the four rat K chains. The open circles Indicate aa which arc conserved

in the mouse but not in the rat k’ chains. It is noteworthy that both these aa are located in the CDR regions. The open circles m the

lower panel indicate those aa which are not conserved when comparing mouse V, chains with V, chains expressed in IRZ and 1 R I62

and with B n~oust: V,,, chain.

113

also certain aa that seem to be under a very strong

evolutionary constraint. A comparison between the

established V, sequences and published sequences

for mouseV,regions (Kabat et al., 1983) reveals that

the aa which show a high degree of conservation in

the frame work regions of the mouse (> 95 74) (Kabat

et al., 1983) are all invariant in the four rat V, chains

included in our comparison (Fig. 5). The four V,

regions, which are shown in Fig. 5, probably repre-

sent four different V-gene families which diverged

early in evolution.

When analysing the sequence homology between

the constant regions of mouse and rat x chains an

exceptionally high percentage of aa substitutions

were found as opposed to silent mutations (Sheppard

and Gutman, 1981). A relatively high degree of mu-

tations are apparently allowed without changing the

functional properties of the L chain.

Over larger periods of time the selection against

unfavourable mutations becomes more pronounced,

as is seen when the mouse and human C, loci are

compared. Hieter et al. (1980) found that the most

conserved regions are centered around structural

and regulatory sequences such as the J and C exons,

and the enhancer element localized in the intron

between the J and C exons (Hieter et al., 1980).

(g) Analysis of light-chain transcripts

Cytoplasmic RNA from both immunocytomas

was studied by Northern blot analysis using a C-

region specific probe from clone IR162 : 6 (BstNI-

P.stI fragment; Fig. 2). Analysis of mRNA from the

I R2 tumor revealed only one mRNA species with an

approximate length of 1200 nt. A 5’-end-labeled

fragment from clone IR2: 13 was also used as a

probe for Sl analysis to confirm that only one X-

chain mRNA exists in this tumor. The analysis

revealed only one band, indicating one single

functionally rearranged k--chain gene in the IR2

tumor.

Analysis of mRNA from the IR162 tumor, in con-

trast, showed three different mRNA species which

were approximately 1200-, 850-, and 750-nt long.

Probes containing exclusively V-region specific se-

quences hybridized only to the full-length transcripts

in both tumors indicating that the two short RNA

species lack V-region sequences. This is due to an

aberrantly rearranged allele as is shown in Hellman

et al. ( 1985).

No transcripts corresponding to full-length i,-

chain mRNA (1200 bp) were detected in either

tumor using C,,- and C,,,-specific probes (not

shown) (Bothwell et al., 1981). We therefore con-

clude that only one L-chain gene is functionally

rearranged in each tumor, represented by cDNA

clones IR2 : 13 and IR162 : 6. This result supports the

allelic exclusion model (Early and Hood, 198 1; Wall

and Kuehl, 1983; Honjo, 1983).

ACKNOWLEDGEMENTS

The authors are indebted to Agneta Lissmats for

excellent technical assistance and to Jeanette Back-

man, Christina Pellettieri, and Linda Baltell for

typing several versions of this manuscript. We thank

Dr. Ursula Storb and Alfred Bothwell for mouse

i.-chain probes and Dr. Torbjdrn Karlsson for pro-

viding the immunocytomas. Financial support was

obtained from the Swedish Medical Research

Council, the Swedish National Board for Technical

Development, and the Marcus Borgstrom

Foundation.

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