region of immunoglobulin light-chain mrna transcribed into complementary dna by rna-dependent dna...

Region of Immunoglobulin Light-Chain mRNA Transcribed into Complementary DNA byRNA-Dependent DNA Polymerase of Avian Myeloblastosis VirusAuthor(s): Israel SchechterSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 72, No. 7 (Jul., 1975), pp. 2511-2514Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/64749 .

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Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp. 2511-2514, July 1975 Biochemistry

Region of immunoglobulin light-ch complementary DNA by RNA-depc myeloblastosis virus

(cross-hybridizations/immunoglobulin subgroups/immunoglobul

ISRAEL SCHECHTER

Department of Chemical Immunology, The Weizmann Institute of Science, Reho York, N.Y.

Communicated by S. Spiegelman, March 24, 1975

ABSTRACT The mRNA coding for a K-type immunoglobulin light (L)-chain and its complementary DNA (cDNA) hy- bridize with a Crtl/2 of 2.6 X 10-4 moles of ribonucleotide X liter-i X sec, forming well-matched duplexes (melting temperature Tm = 89?). The molecular weight of the cDNA is about 280,000 (840 nucleotides) as determined by alkaline sucrose gradient centrifugation and from the extent of protection of the mRNA by the cDNA from ribonuclease digestion. The cDNA anneals with K-type mRNAs of the same and different subgroups with comparable Crtl/2 values, but not with a X-type mRNA. Thus, one K-type cDNA can be used to quantify the mRNAs coding for all K-type L-chains. The values of cDNA hybridized at saturation with various K-type mRNAs indicate that: (1) the cDNA is complementary to the entire constant region and to about half of the variable (V)-region; (2) V-regions of similar amino-acid sequence are coded by a similar nucleotide sequence; (3) the nucleic acid probe to one V-region may anneal and quantify V-region genes of members of the same subgroup.

The distribution of genetic markers on immunoglobulin (Ig) chains (1) and amino-acid sequence data (2) raised the possi- bility that different genes code for the variable (V, N-terminal) and constant (C, C-terminal) parts of the intact poly- peptide chain. According to the somatic mutation hypothesis, the genome contains a few V-region genes, whereas the germ line hypothesis predicts a large number of V-region genes. To resolve this issue requires highly labeled nucleic acid probes that are specific to V- and C-region sequences. Furthermore, it is necessary to find out to what extent a probe of one V-region would detect and quantify nucleic acid sequences of other V-regions. It is possible that a given V-region probe would anneal with V-regions coding for light (L)-chains of the same subgroup (i.e., L-chains having similar amino-acid sequence at the V-region) but would not detect V-regions of L-chains belonging to different subgroups.

The isolation of Ig L-chain mRNA from myeloma polysomes specifically precipitated with antibodies to L-chain has been achieved. Over 95% of the cell-free product programmed by the mRNA was L-chain protein, as determined from two-dimensional tryptic fingerprints (3, 4). We here report the enzymic synthesis and characterization of the DNA complementary to this highly purified mRNA.

Abbreviations: AMV, avian myeloblastosis virus; cDNA, complementary DNA; Ig, immunoglobulin; L-chain, light chain; V-region, variable region of immunoglobulin; C-region, constant region; Crt, product of RNA concentration (moles of nucleotide per liter) and time (sec); Tmn, melting temperature.

251

ain mRNA transcribed into ndent DNA polymerase of avian

in variable-region genes)

vot, Israel; and the Institute of Cancer Research, Columbia University, New

MATERIALS AND METHODS

The mRNAs coding for M-321, M-63, M-41, and M-104E L-chains were prepared from the respective mouse myeloma polysomes specifically precipitated with antibodies to L-chains, followed by chromatography on oligo(dT)-cellulose (3, 4). Over 95% of the cell-free products programmed in vitro (3) by each mRNA corresponded to the appropriate L-chain. When analyzed on 3.2% polyacrylamide gels made in 98% formamide (5), all mRNAs migrated as 15.5 i 0.5S bands, and they contained variable amounts of rRNA (Table 1). The M-321 mRNA was further purified by sucrose gradient centrifugation to yield preparation M-321p, having only 5% rRNA contamination. The DNA complementary to M- 321p mRNA (cDNA) was synthesized with the aid of the avian myeloblastosis virus (AMV) DNA polymerase (6) in the presence of [3H]dCTP (26 Ci/mmol), actinomycin D (0.01%), and (dT)10o primer. Hybridizations were performed at 68? in 0.02 M Na-phosphate (pH 7.0), 0.3 M NaCl, 2 mM EDTA, and 0.1% sodium dodecyl sulfate. Hybrids were as- sayed after digestion by staphylococcal nuclease (7). A highly purified M-321 mRNA that contained less than 0.5% rRNA (prepared by rerun on a sucrose gradient and analyzed on formamide gels) was radioiodinated as described (8).

RESULTS AND DISCUSSION

When presented to the AMV DNA polymerase, 30% (by weight) of the M-321p mRNA was reverse transcribed into cDNA. The cDNA hybridized with the M-321p mRNA with Crti/2 of 6 X 10-4 mole of nucleotide X liter-1 X see; at saturation 65% of the cDNA was annealed (not shown); and the hybrids showed a biphasic thermal denaturation curve (Fig. 1A). This suggested that the total cDNA product consisted of a heterogeneous fraction capable of forming weak hybrids and of a homogeneous fraction forming strong hybrids. Ac- cordingly, the M-321 mRNA-cDNA hybrids were separated on a preparative scale using hydroxylapatite columns. Mate- rial eluted at 60? (18%) was discarded. The temperature was then raised to 86? and 95? to yield, respectively, low- (30%) and high- (52%) melting hybrids. After removal of the mRNA (alkaline treatment) and salts (9) from these frac- tions, the high- and low-Tm cDNAs were back hybridized to the M-321 mRNA. Hybrids formed with the low-Tm cDNA had low Tm (73?) and melted over a wide temperature range (Fig. IB). This finding and other experiments (to be reported elsewhere) indicate that the low-Tm cDNA is main-

1



2512 Biochemistry: Schechter

T T I 1 I I I i

100 LA B oo

840 / -

/Tm=72?

20-

60 70 80 90 100 60 70 80 90 K Temp

FIG. 1. Thermal elution hydroxylapatite chromatography of L-ch to 4 ml of 0.12 M Na-phosphate (pH 6.8)-0.4% sodium dodecyl sulfat was washed with the same solution at increasing temperatures and th, termined. In panels A, B, and C the M-321p mRNA (56 ng) was hybric cDNAs were: A, unfractionated cDNA; B, low-Tm cDNA (fraction elut C, high-Tm cDNA (fraction eluted from the preparative hydroxylapati (0.5 ng) was hybridized with various L-chain mRNAs (150 ng each) t M-104E.

ly composed of relatively lengthy poly(dT) stretches linked to short heteropolymeric DNA. Therefore, this material was not used in further studies.

The high-Tm cDNA and M-321 mRNA hybridized with a Crtl/2 of 2.6 X 10-4 and at saturation 97% of the cDNA was annealed (Fig. 2). The hybrids consisted of a fairly homogeneous population of well-matched duplexes, since they had a high Tm (89?) with sharp denaturation profile (72% of the hybrids melted between 88? and 90?, Fig. IC). The specific- ity of the cDNA probe was ascertained from annealing it with globin mRNA and rRNA of rabbit reticulocytes, poly(A)-rich RNA and rRNA of mouse livers, and with Escherichia coli RNA. In all cases hybrids were not detected at high Crt values (> 10,000-fold the Crtl/2 of the homologous reaction, Fig. 2).

Other laboratories reported the synthesis of cDNA to L- chain mRNA. In one report the Crtl/2 value for the annealing (at 0.3 M NaCi) of the cDNA and mRNA was 1.8 X 10-3 (10). In the other report the Crt1/2 was 4.2 X 10-4, but the hybridizations were performed at 0.6 M NaCl (11). In the present study hybridizations were performed at 0.3 M NaCl.

Table 1. The cross hybridization of M-321 cDNA with mRNAs coding for other immunoglobulin light chains

Sample Crtl/2 (mole X Satura- composition liter-' x sec) tion

level mRNA mRNA rRNA Observed Correctedt (%)

M-321p (K) 95 5 2.7 x 10-4 2.6X 10-4 97 M-321 (K) 35 65 8.3 X 10-4 2.9 X 10- 4 96 M-63 (K) 33 67 1.0 X 10-3 3.3 X 10-4 95 M-41 (K) 37 63 1.2 X 10-3 4.4 X 10-4 75 M-104E (X) 39 61 2.5 x 10-2 9.7 X 10-3 75

*The amount of mRNA (15.5S band plus degraded mRNA that ranged in size from 15.5 to 9.5 S, ref. 3) was determined from the scanning of stained polyacrylamide gels made in 98% formamide.

tThe corrected Crtl/2 value was calculated by multiplication of the observed Crtl/2 by the fraction of mRNA in the sample (see text).

Proc. Nat. Acad. Sci. USA 72 (1975)

I - I I I I I I I I

c D 6W

- Tm=89? Tm=87?

\l^^ . , i | I.i. . 06070 80 90 100 60 70 80 90 100 roature?C

ain mRNA-cDNA hybrids. The annealing reaction (10 l1) was diluted e and loaded on an 0.5 g hydroxylapatite column at 60?. The column L acid-insoluble radioactivity eluted at different temperatures was de- lized with 0.4 ng [3H]cDNA (4.4 X 104 dpm/ng) to Crt of 9 X 10-2. The ed from the preparative hydroxylapatite column between 60 and 86?); te column between 86 and 95?). In panel D, the M-321 high-Tm cDNA ) Crt of 8 X 10-1. The mRNAs were: X, M-321; A, M-63; o, M-41; A,

An increase in salt concentration decreases the Crtl/2 value (12); therefore, it seems that our Crt/12 is again lower (when corrected to equal salt concentration), indicating a purer mRNA preparation. In both studies (10, 11) the mRNA used as template was prepared by repeated sucrose gradient centrifugation and chromatography on oligo(dT)-cellulose of the 12-14S RNA obtained from the total microsome population of myeloma tumors. It has been previously shown that this RNA fraction is contaminated by non-L-chain mRNAs, and that the above procedures do not lead to any significant biological purification of the L-chain mRNA (3, 13). On the other hand, the L-chain mRNA prepared from immune-precipitated polysomes is >95% pure (3, 13).

The cDNA was sized on an alkaline sucrose gradient with the 18S kX174 phage DNA as a reference standard. A value of 8 S was obtained (Fig. 3), corresponding to a molecular weight of about 280,000 (14). The size of the M-321 mRNA is about 440,000 daltons (15.5 S on formamide gels, ref. 3),

-5 -4 -3 -2 -I 0 2

20

80

Log C,t (mole x liter-' x sec)

FIG. 2. Kinetics of hybridization of M-321 cDNA (high-Tm fraction) with various mRNA preparations. The L-chain mRNAs were: O, M-321p; X, M-321; A, M-63; o, M-41; A, M-104E. The controls were: globin mRNA (v) and rRNA (v) of rabbit reticulocytes; poly(A)-rich RNA (e) and rRNA (@) of mouse liver; total RNA of E. coli (-).



Biochemistry: Schechter

I I I I I I

ox DNA 6

10 20 30 Fraction number

FIG. 3. Alkaline sucrose gradient centrifugation of the high- Tm cDNA fraction. The cDNA was layered onto a 15-30% sucrose gradient containing 0.9 M NaCI-O.1 M NaOH and spun (50,000 rpm/12 hr) at 0? in the Spinco SW-56 rotor. bX174 DNA was run on a parallel gradient as marker.

i.e., about 63.5% of the mRNA was copied into cDNA. Since we used only one reference standard, and the effect of large poly(A) segment on the mobility of RNA in gels has not been defined, we estimated the extent of mRNA transcription by another method. We determined the fraction of ribonuclease-resistant RNA in the hybrids formed between the labeled [1251]mRNA and cDNA. As seen from Fig. 4, 56% of the mRNA was protected from ribonuclease digestion by the cDNA, implying that the cDNA is complementary in size to 56% of the mRNA. This is a minimum figure, since only the heteropolymeric portion of the cDNA was scored. The oligo(dT) segment in the cDNA is not scored because it anneals with the poly(A) moiety of the mRNA and the 125I labels mainly cytosine residues (8). The size of the poly(A) moiety in the L-chain mRNA is about 65,000 daltons (15). Assuming that on the average half of the poly(A) was transcribed, then the oligo(dT) segment (32,000 daltons) corresponds to 7.2% of the mRNA, and the corrected figure from the protection

50 - - 56 %

1 I

a 25- E

5 10 15 c DNA/mRNA (w/w)

FIG. 4. Protection of M-321 [125I]mRNA by M-321 cDNA from hydrolysis by ribonucleases. The [125I]mRNA (8 X 103cpm/ ng) was hybridized with increasing amounts of cDNA up to a Crt of 3.3 X 10-2. The annealing reaction (10 Ml) was diluted to 0.35 ml of 0.3 M NaCl-2 mM EDTA-50 mM Tris-HCl (pH 7.5), and divided into two aliquots. To one aliquot RNase A and RNase T1 were added (each to 34 Mg/ml), and incubation was at 36? for 0.5 hr. The extent of mRNA protection was calculated from the acid-insoluble 125I radioactivity in both aliquots. Over 99% of the [l25I]mRNA was acid-precipitable, and 99.6% was ribonuclease-sensitive in the absence of added cDNA.

Proc. Nat. Acad. Sci. USA 72 (1975) 2513

P V C P U A (a) , , ?? ? mRNA

(b) , U ,P, V , C P, A,

(c , U P, ,V , C ,P, U. A,

\ ____ __H T, cDNA

FIG. 5. Schematic representation of possible complementary regions between the cDNA and mRNA of immunoglobulin L- chain. The M-321 mRNA (440,000 daltons), cDNA (280,000 daltons) and their divisions are drawn to scale (see text). Nucleotide sequences in the mRNA are: P, for the amino- and carboxyl-terminal extra pieces of the L-chain precursor (17, 18); V, variable region; C, constant region; A, poly(A) at the 3' end; U, untranslated region. The location of the untranslated region is not yet known; therefore, it is represented in three arbitrary arrangements. Nucle- otide sequences in the cDNA are: H, heteropolymeric DNA; T, poly(dT).

experiment would be 63.2% (56 + 7.2) transcription. Thus, comparable values for the size of the cDNA (about 63.5% of the mRNA, or 280,000 daltons) were estimated by two different methods.

Of the nucleotide mass of the M-321 mRNA, 220,000 daltons are required to code for the V- and C-regions of the mature L-chain (16), 45,000 daltons to code for the extra pieces in the L-chain precursor (20 amino-acid residues at the N-terminus and about 25 residues at the C-terminus, refs. 17 and 18), about 65,000 daltons for the poly(A) at the 3' end of the mRNA (15), and a residual untranslated mass of about 110,000 daltons whose position has not yet been determined. In Fig. 5 the mRNA and cDNA are lined up to scale in three possible combinations. The untranslated mass was arbitrarily placed at the 3' (a) and 5' (b) ends of the translated region, and, when equally divided, between the two ends (c); it is evident that in all combinations the cDNA is complementary to the entire C-region of the L-chain. The data quoted below suggest that the cDNA may contain sub- stantial amount (about 50%) of V-region sequences, vide infra, and that scheme (c) in Fig. 5 is the best approximation for the structure of the L-chain mRNA.

The M-321, M-63, and M-41 mouse myeloma L-chains are of the K-type. They share an identical C-region, which is the C-terminal half of the molecule, but differ at the V-region, which is the N-terminal half. The M-321 and M-63 are of the same subgroup and differ at the V-region only in eight out of 111 amino-acid residues. The M-41 belongs to a different subgroup and differs from M-321 V-region in 53 positions (16). The X-type M-104E L-chain differs extensive- ly in sequence from M-321 both in the C- and V-regions (19). The M-321 cDNA was annealed with the mRNAs coding for these L-chains. The observed Crti/2 values (Fig. 2) were corrected for rRNA in the mRNA preparations (Table 1). This is justified since we found that myeloma rRNA was a poor template (0.8% when compared to the mRNA), and the cDNA did not anneal with the mouse liver rRNA (Fig. 2). Indeed, the corrected Crtl/2 values of M-321p (5% rRNA contaminant) and M-321 (65% rRNA contaminant) were very similar (Table 1).

The annealing of the M-321 cDNA with the X-type M- 104E mRNA was 37-fold slower than the homologous reaction (Table 1). Decreased annealing rate can be due to the formation of poorly matched hybrids (20), yet the hybrids formed here were well-matched. They had a high Tm and sharp melting curve like M-321 mRNA hybrids (Fig. 1D).



2514 Biochemistry: Schechter

The formation of well-matched hybrid with slow annealing rate and high saturation level (75%) rules out two possibili- ties: (1) that the X-type mRNA (which was present in the same amount as the K-type mRNAs) annealed with the K-type cDNA; (2) that the observed annealing is due to a common contaminant in the M-321 and M-104E mRNA preparations. Our interpretation is that the M-104E mRNA (X-type) contains about 3% (calculated from Crtl/2 values) of K-type mRNA, which hybridized with the M-321 cDNA. The origin of the K-type mRNA is either from the M-104E tumor (myelomas producing two Ig types were reported, ref. 2), or from lymphocytes spread in the tumor tissue.

The cDNA annealed with the K-type mRNAs of the same (M-321, M-63) and different (M-41) subgroups with comparable Crtl/2 values (Table 1), most probably due to a common C-region. Thus, one K-type cDNA would be a reliable probe to quantify, by hybridization kinetic experiments, the mRNA species coding for all K-type L-chains.

The identical Tm values and the sharp melting profiles obtained with all K-type mRNA hybrids (Fig. 1D) show that a large segment of these mRNAs form a well-matched du- plex with the cDNA. The mRNA segment complementary to the cDNA is larger in M-63 than in M-41 because the saturation level achieved with M-63 (95%) is larger than that achieved with M-41 (75%) (Table 1 and Fig. 2). Considering the sizes of the cDNA (280,000 daltons) and mRNAs (440,000 daltons), that reverse transcription starts from the 3' end of the mRNA (i.e., from the end coding for the C-region), and that M-321 and M-63 have very similar V-regions that are quite different from the V-region of M-41, the results suggest that the M-321 cDNA contains the entire C- region and presumably a portion of the V-region. It is un- likely that the difference at saturation between M-321 and M-63 to M-41 is due to the putative untranslated region at the 3' end of the mRNA, because it has been shown that the mouse genome contains only a few genes for the C-region (10, 11, 21). The difference of 21% (96-75) corresponds to about 59,000 daltons (0.21 X 280,000). Thus, the M-321 cDNA might contain about half of the V-region as shown schematically in Fig. 5c. If this is indeed the situation, then the similarity in Crtl/2 and saturation levels obtained with M-321 and M-63 indicates that V-regions with similar amino-acid sequence (as M-321 and M-63) are coded by similar nucleotide sequences. That is, the nucleic acid probe to one V-region might anneal and quantify the V-region genes of members of the same subgroup. It is important to confirm this conclusion with cDNAs and mRNAs from other

Proc. Nat. Acad. Sci. USA 72 (1975)

subgroups. To discriminate between the somatic mutation and germ line hypotheses, it is necessary to determine the number of V-region genes. For this purpose we have to know the range of structural variation in V-region sequences that can still be quantified by a nucleic-acid probe to a unique V-region.

I thank Dr. S. Spiegelman for his hospitality and encouragement during the course of these investigations. This work was supported in part by the Israel National Commission for Basic Research, and by the U.S. National Institutes of Health, National Cancer Institute Research Grant CA-02332 and Virus Cancer Program Contract NO1-CP-3-3258.

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235, 108-110. 6. Kacian, D. L. & Spiegelman, S. (1973) in Methods in Enzy-

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region of immunoglobulin light-chain mrna transcribed into complementary dna by rna-dependent dna...

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