Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constant region of kappa chain in the mouse

J. MoZ. Biol. (1974) 88, 43-63

Isolation of Messenger RNA for an Immunoglobulin Kappa Chain and Enumeration of the Genes for the Constant Region of Kappa Chain in the Mouse JANET STAVNEZER~,Ru CIDHC.HUANG~,EDWARD AND J. MICHAELBISHOP+-

STAVNEZER~

TDepartment of Microbiology University of California Medical X&o01 San Francisco, Calif. 94143, U.X.A. and t Biology Department Johns Hopkins University Baltimore, Md 21218, U.S.A. (Received

4 February

1974, and in revised

form

10 May

1974)

The messenger RNA for an immunoglobulin light (kappa) chain was isolated from the mouse myeloma MOPC41 and shown to be almost twofold longer than necessary to code for its protein product. DNA complementary to the mRNA was synthesized with the RNA-directed DNA polymerase of Rous sarcoma virus. Although the individual chains of the eDNAt contained an average of only 270 nucleotides, the cDNA was heterogeneous in molecular weight, allowing the isolation of a fraction of the cDNA 620 nucleotides long. This large GDNA would be long enough to code for nearly all (95%) of the constant region if all the untranslated region of the mRNA were between the 3’ terminal poly(A) and the constant region. On the other hand, if all the untranslated region of the mRNA were at the 5’ terminus, this cDNA would code for 93% of the entire kappa chain. The specificity of nucleotide sequences in the cDNA was documented by molecular hybridization with both template RNA and RNA from various myelomas. The amount of hybridization obtained with myeloma RNA was approximately proportional to the amount of kappa chain protein produced by the various myeloma cells. In addition, there was no hybridization with RNA isolated from either BALB/c mouse liver or Escherichia coli. The genes for the constant region of the kappa chain were enumerated in the mouse genome by annealing cDNA to DNA from mouse liver and MOPC41 myeloma. The haploid genome of both tissues contained three to four genes for the constant region of kappa chain even when tested under conditions that would detect distantly related nucleotide sequences. The fact that there are only a few genes coding for the constant region of kappa chains implies that specialized genetic mechanisms are required for the generation of antibody diversity.

1. Introduction There are two general theories molecules. The germ line theory t Abbreviations

used: cDNA,

to explain the origin of diversity among antibody (Hood & Prahl, 1972) postulates that each antibody

complementary

DNA; 43

SDS, sodium dodecyl

sulfate.

44

J. STAVNEZEB

ET

AL.

molecule is specified by a structural gene carried in the germ line. Alternatively, the somatic variation theory (Lennox & Cohn, 1967) postulates that a limited number of germ line genes generate a variety of antibody polypeptides by genetic processes that occur during somatic differentiation. The amino terminal portion of antibody molecules (variable region) varies greatly among antibodies and determines the antigenic specificity. The carboxy terminal portion (constant region) is virtually identical within each class of antibodies, except for a few amino acid interchanges, which segregate in a Mendelian fashion. The theories for the generation of antibody diversity apply specifically to the variable region, except Brown’s proposal of the presence of thousands of genes for the entire antibody molecule (Brown, 1972). But generally it is believed that there are two genes for each antibody polypeptide, which are joined during the development of each immunocyte (Dreyer & Bennett, 1965). The number of immunoglobulin genes can be determined by measuring the kinetics of reassociation of a highly labeled immunoglobulin-specific nucleic acid with an excess of unlabeled cellular DNA. This assay requires the isolation of a pure (or nearly pure) immunoglobulin messenger RNA, which can either be used in the hybridization experiment directly (if it is very highly labeled), or used as a template for the synthesis of highly labeled nucleic acid sequences in vitro. In the present experiments, single-stranded DNA complementary to immunoglobulin mRNA has been synthesized with tumor virus RNA-directed DNA polymerase and used as a probe for immunoglobulin sequences. We have chosen to use DNA-DNA annealing rather than DNA-RNA hybridization because the kinetics of DNA reassociation are almost completely dependent on the complexity of the DNA (Wetmur $ Davidson, 1968; Britten & Kohne, 1968). By contrast, the rate of RNA-DNA hybridization varies with the base composition and secondary structure of the RNA (Birnstiel et al., 1972; Bishop, 1969J972). We have previously isolated an immunoglobulin light chain mRNA from the mouse myeloma, MOPC41, and demonstrated that it can direct the synthesis of kappa chains in vitro (Stavnezer & Huang, 1971). The protein product was shown to be authentic MOPC41 kappa chain (except for the amino terminal peptide) by specific antibody-precipitation and analysis of tryptic peptides. We describe here the further purification of this light-chain mRNA and its transcription with RNAdirected DNA polymerase. We demonstrate that the cDNA is probably specific for immunoglobulin kappa chain sequences and describe the use of cDNAf- to determine the frequency of the kappa chain genes in MOPC41 and mouse liver DNAs.

2. Materials and Methods (a)

Mate&als

Oligo(dT)-cellulose was prepared by D. Givol (Gilham, 1964) or purchased from Searle Corp. [3H]deoxyribonucleoside triphosphates and [r*C]thymidine were obtained from Schwarz. RNAase and DNAase (electrophoretically pure) were purchased from Worthington. The DNAase was treated with iodoacetate to inactivate RNAase (Zimmerman & Sandeen, 1966). Pronase was obtained from Calbiochem. Hydroxyapatite was obtained from Bio-Rad. BioGel DNA-grade HTP was used for fractionation by batch and BioGel BTP for fractionation on columns. Frozen E. coli cells were given to us by H. Boyer. J . Taylor prepared and denoted the polio RNA and polio cDNA. A. Faras and J. Taylor t See footnote

on p. 43.

GENES

FOR

IMMUNOGLOBULIN

KAPPA

CHAIN

45

donated the RNA-directed DNA polymerase isolated from the Prague C strain of Rous sarcoma virus (Faras et a?., 1972). Unlabeled poly(dT) was obtained from Collaborative Research. All glassware was heated for 2 h at 160°C. Sterile glass-distilled water W&S used for preparing solutions. (b) Sources and maintenance

of nzyelomas

The solid tumors were obtained from M. Potter (MOPC41), M. Cohn (X5830), and M. Scharff (66.2) and were maintained in femade BALB/ c mice. XS63 cells were received as a suspension culture from M. Cohn. They were grown in Dulbecco’s modified Eagle’s medium plus 2 mm-glutamine and 10% heat-inactivated calf serum or fetal calf serum (Grand Island Biological). (c) Isolation

of mRNA

Messenger RNA was isolated from fresh or rapidly frozen tumors. All work was done at 0 to 4°C until SDS was added. About 6 g of myeloma were minced, and homogenized (about 6 strokes) in 30 ml of cold 4 mlvr-TriseHCl (pH 7.4), 15 mM-MgCl,, 2.5 mna-KCl, 250 mx-sucrose in a motor-driven Teflon pestle tissue grinder. The nuclei were pelleted by centrifugation at 3000 g for 10 min, and the supernatant solution was centrifuged at 105,000 g to pellet the microsomes (Kuff et al., 1962). The RNA was extracted from the microsomd pellets by rapid homogenization in 2% SDS, 5 mM-Tris=HCl (pH 7.4) (2.2 ml/g tumor). The suspension was incubated at 37°C for 5 to 15 min, and layered over six 5% to 20% sucrose gradients in 0.5% SDS, 0.1 M-NaCl, at 0.01 M-EDTA (pH 7), in the Spinco SW27 0.01 M-Tris.HCl (pH 7.4) (Vaughn et ah, 1967). After centrifugation rotor at 25,000 revs/mm for 16 to 20 h at 24”C, the gradients were fractionated, and the absorbance at 260 nm was determined. The material sedimenting at approximately 9 to 15 S was pooled and ethanol-precipitated overnight. The RNA was pelleted, dissolved in 1% SDS, 5 mM-Tris*HCl (pH 7.4), and centrifuged on three 10% to 70% sucrose gradients (Neal & Florini, 1972) (in the same buffer as above) in the SW27 rotor at 25,000 revs/min at 25°C for 19 to 21 h. The gradients were fractionated, the absorbance at 260 nm determined, and the small peak of RNA, indicated in Fig. l(a), which had the highest specific activity for the synthesis of light chain in vitro (Stavnezer & Huang, 1971), was ethanol-precipitated overnight. The RNA was pelleted, dissolved in 0.1 M-Tris*HCl (pH 9), 1% SDS, and extracted with cold phenol. The phenol pha.se was re-extracted and the combined aqueous phases were ethanol-precipitated. The RNA was dissolved in 0.5 M-KCl, 0.01 M-Tris*HCl (pH 7*6), and adsorbed to an oligo(dT)-cellulose column (Gilham, 1964) according to the procedure of Swan et al. (1972). The column was washed with the loading buffer, and then the mRNA was eluted with 0.01 ;M-Tris.HCl (pH 7.6). Appreciable quantities of rRNA contaminate the mRNA after one passage over oligo(dT)-cellulose (Swan et al., 1972; J. Stavnezer, unpublished data). Hence, in some cases the mRNA was adsorbed to oligo(dT)-cellulose twice. After precipitation with ethanol, the RNA was dissolved in 0.1 M-Tris.HCl (pH 8) or 1 mmEDTA (pH 7), an equal volume of dimethylsulfoxide, and 2 vol. dimethyl formamide. The RNA was centrifuged in a 5% to 50% or a 5% to 60% sucrose gradient in 99% dimethylsulfoxide, 1 mM-EDTA (pH 7), 10 DIM-Lic1, 10 mM-Tris*HCl (pH 7.4) in the SW27 rotor at 25,000 revs/min at 28°C for 5 days (Strauss et al., 1968) The concentration of RNA in the gradient fractions was measured by absorbance at 260 nm after the RNA was precipitated with ethanol, centrifuged, and redissolved in 0.1% SDS. The fractions comprising the main peak of the RNA were pooled and precipitated with ethanol. (d) Proteh

synthesis

in vitro

The RNA was assayed for the ability to direct specific protein synthesis in the rabbit reticulocyte lysate system as described by Stavnezer & Huang (1971) with the following modifications. The system contained 30 PM-hemin (prepared as described in Adamson et al., 1968). Reactions (100 ~1) were incubated at 24°C for 1 h. The products of the reaction were assayed by a modification of the method of Rhoades et al. (1973). The reactions were stopped by the addition of 10 ~1 of 10% Triton X100, 10% sodium deoxycholate, and 10 d of 0.1 M-leuoine, 1.5 M-NaCl, 0.01 M-Tris.HCl (pH 7.6), and MOPC41 kappa

46

ET AL.

J. STAVNEZER

chain (50 pg/m.l). A sample of 10 ~1 was removed for determination of the incorporation into hot trichloroacetic acid-precipitable material. The remainder was assayed for antibody-precipitable material by adding 2.5 ~1 of antiserum, incubating for 30 min at 37°C and then overnight at 4°C. The reaction mixture was layered over 100 ~1 of 1.0 M-sucrose in 1% Triton X100, 1% sodium deoxycholate, 0.15 M-NaC1, 0.01 M-l@UCine, 0.01 MTris.HCl (pH 7*6), and centrifuged in a Beckman microfuge tube in the Sorvall HB4 rotor at 16,300 g. The tubes were placed in dry ice, the tips cut off, and placed in scintillation vials. Protosol (0.5 ml) (New England Nuclear) was added, and the samples were incubated at 60°C until the pellets dissolved. Ten ml of 2 x Liquifluor (New England Nuclear) was added, and the samples were counted.

(e) Preparation

of MOPCW

Gght chain and antiserzLm

The light chain was isolated from mouse urine as described by Stavnezer & Huang (1971). Antiserumto the light chain was obtained from rabbits, which had been injected at multiple intradermal sites with light chain emulsified with Freund’s adjuvant, followed 6 weeks later by injection of light chain in phosphate-buffered saline. Serum was collected 10 to 14 days after the final injection. (f) Isolation

of DNA from

BALLBIG livers and MOPC41

tumors

DNA was isolated from nuclei or from whole cells. Nuclei were prepared from frozen tissue by a modification of the method of Marushige & Bonner (1966). The nuclei were suspended by hand homogenization in SSC (0.15 M-NaCl, O-015 m-sodium citrate), plus 25 mu-EDTA (pH 8), and then brought to 1% in SDS. When the DNA was extracted from whole cells, chopped fresh tissues were homogenized in a motor-driven Teflon homogenizer (1 g/20 ml of 0.1 M-NaCl, 0.05 M-TrisHCl (pH 7*6), O-01 M-EDTA (pH 7)). The DNA was extracted from either nuclei or whole cells by a modification of the method of Berns & Thomas (1965), followed sometimes by chloroform extraction in 1 M-sodium perchlorate (Marmur, 1961). After the DNA was ethanol-precipitated and redissolved in 20 mrvr-TrisaHCl (pH 7.4), 10 mu-EDTA (pH 7), RNA was removed either by incubation with boiled RNAase (100 pg/ml) overnight at 37”C, followed by incubation with predigested Pronase (100 w/ml) at 37°C for 0.5 to 1 h, or by incubation in 0.6 N-NaOH at 37°C for 1 h. The DNA was again extracted with phenol, and sometimes also with chloroform. Before use in reassociation experiments, the DNA was sheared at 50,000 lb/in2 to a length of approximately 220 nucleotide pairs (H. E. Varmus & J. Stavnezer, unpublished data). It was chloroform-extracted, ethanol-precipitated, and redissolved in 3 mM-EDTA (PH 7). (g) Isolation of DNA from E. coli DNA was isolated cations. (1) The cells EDTA (pH 7), 0.4% clumps disappeared. precipitated, dissolved G50 (coarse) column.

from frozen E. coli cells as above except for the following modifiwere homogenized in 0.1 ivr-NaCl, 0.05 M-Tris.HCl (pH 7.6), 0.01 MSDS with a Virtis homogenizer for approximately 30 s, until the (2) After the flnal chloroform-extraction, the DNA was ethanolin 0.1 x SSC, and passed through a 0.9 cm x 40 cm Sephadex It was ethanol-precipitated, and dissolved in 1 mM-EDTA (pH 7).

(h) Preparation

of W-labeled

unique-sequence

mozcse DNA

BALB/c 3T3 cells, or 3T3 cells that had been infected with the B77 strain of Rous sarcoma virus (obtained from P. Vogt (Varmus et al., 1973)), were plated at one-fifth confluency in medium 199A (without nucleosides ; Grand Island Biological), 5% dialyzed calf serum, 1% Tryptose phosphate broth, and 2.5 or 1.25 PCi [W]thymidine/ml. They were grown until nearly confluent (3 days). The DNA was isolated by adsorption to, and elution from, hydroxyapatite in the presence of urea (Britten et al., 1970). The DNA was sheared, dialyzed against 1 mM-EDTA, and ethanol-precipitated. It was dissolved in 3 mM-EDTA (pH 7), denatured, incubated‘to Cot7 = 600 in 0.4 M-PO~ buffer (equimolar = Na2HP04 and NaHsPO,) at 68”C, and fractionated by the batch procedure on hydroxyapatite (as described below). The single-stranded DNA was passed through a t C,t is the product

of the initial

molar concentration

of DNA nucleotides

and time, in seoonds.

GENES

FOR

IMMUNOGLOBULIN

KAPPA

CHAIN

47

Sephadex G50 (coarse) column (0.9 cm x 40 cm), and precipitated with ethanol. This unique-sequence DNA had a specific activity of approximately 14,006 ots/min per pg. (i) I.solatiom

of total cell RNA

Method 1. Total cell RNA was isolated from minced fresh or frozen tissues, which were homogenized in a Virtis homogenizer in 60 ml cold phenol and 30 ml of 50 mMTris*HCl (pH 9), 10 mM-EDTA (pH 7), 0.5% SDS at room temperature. The buffer and phenol volume each were brought to 60 ml/g tissue, and the RNA was extracted twice at 6O”C, and once at room temperature. After ethanol precipitation and centrifugation, the RNA was dissolved in O-02 m-Tris*HCl (pH 7.4), 0.01 M-MgCl, adjusted to a coneentration of 500 pg/ml and digested with DNAase (20 pg/ml) for 2 h at room temperature. After adjustment to 10 mM-EDTA (pH 7), 50 mM-TrisHCl (pH 9-O), the RNA was phenolextracted twice, precipitated with ethanol, and dissolved in 1 mm-EDTA (pH 7). It was stored at -70°C. Method 2. Total cell RNA was isolated from frozen E. co& cells or fresh XX63 cells by method 1, except before the phenol extractions the cells were digested for 1 h at 37°C with 500 pg Pronase/pl in O-1 H-NaCI, 0.05 M-Tris.HCl (pH 7-41, 0.01 M-EDTA (pH 7), 0.5% SDS. The E. coli cells were suspended at 1 g/25 ml; the XS63 cells at 2 IO7 cells/ml. (j) Polyacrylamide

gel electrophoresis

of mRNA

Polyacrylamide gel electrophoresis was performed in 3% gels according to the method of Peacock & Dingman (1967). When the molecular weight of the mRNA was to be determined the RNA was dissolved in 50% formamide and 50% of 10 x electrophoresis chamber buffer. Electrophoresis was performed for 2 h at 2 n&/gel, either at 6°C or 26°C. The RNA was visualized with Stains-all (Dahlberg et al., 1969) and scanned in a Gilford spectrophotometer at 560 nm. (k) Preparation of cDNA DNA complementary to the light chain mRNA was synthesized in a reaction mixture containing 40 m&r-Tris*Ha (pH Sal), 9 mM-MgCl, 2% of 2-mercaptoethanol, 100 rg actinomycin D (Calbioehem)/ml, 0.2 yg oligo(dT),, - is (Collaborative Research)/ml, 10 PM each of r3H]dATP (5 Ci/mmol), [3H]dGTP (17.7 Ci/mmol), [3H]TTP (17.3 Ci/mmol), E3H]dCTP (30 Ci/mmol), O-75 pg light chain mRNA/ml, and 0.2 units of purified Rous sarcoma virus DNA polymerase/ml. The ethanol was evaporated from the labeled nucleoside triphosphates before they were used. After incubation at 37°C for 4 h, SDS was added (O*1o/o) and the incubation was continued for 5 min more. The DNA was precipitated with ethanol overnight and pelleted by centrifuging at 16,300 g for 20 min. It was dissolved in 3 mm-EDTA (pH 7), digested with RNAase (100 pg/ml) for 45 min at 37°C; and fractionated on hydroxyapatite by the batch procedure (Leong et al., 1972). The cDNA, which eluted in 0.16 M-PO, buffer, was passed through a Sephadex G50 column (0.9 cm x 40 cm) in 0.3 M-NaCl, 0.01 na-Tris * KC1 (pH 7.4), 0.001 M-EDTA (pH 7), 0.1% SDS. The material in the void volume was pooled, combined with 40 pg of E. coli DNA, and precipitated with ethanol overnight. The cDNA was dissolved in 1 mM-EDTA (pH 7), sheared at 50,000 lb/in2, chloroform-extracted, ethanol-precipitated, and redissolved in 1 mM-EDTA (pH 7). The specific activity of the cDNA was estimated to be 23,000 cts/min/ ng from the base composition of the cDNA, and the specific activities of the nucleoside triphosphates. (1) Isolation of large cDNA Complementary DNA synthesized using all 4 [3H]deoxynucleoside triphosphates to 750 pM) was isolated as above, and then centrifuged twice on a neutral 5% to sucrose gradient in either the SW41 or the SW65 rotor. The cRNA larger than was pooled from the first gradient, and the largest two-thirds of the cDNA (5 to was pooled from the second gradient. (m) Sedimentation The sedimentation coefficient in a 5% to 20% sucrose gradient

of cDNA

in alkaline

(120 20% 6.9 S 9 S)

sucrose

of unsheared oDNA was determined by sedimentation in 0.1 m-NaGH, 0.9 &r-NaCl, 0.01 M-EDTA in the SW50.1

48

J. STAVNEZER

E!Z’ AL.

rotor at 50,000 revs/min for 7.25 h or in the SW65 rotor at 64,000 revs/mm for 4.25 h at 20°C. Before centrifugation, the cDNA and a marker (6.1 S sea urchin DNA or 5.0 S i4C-labeled rat DNA) were incubated in 0.1 N-NaOH, 0.01 M-EDTA for 0.5 h at 37°C. The sea urchin DNA, kindly donated by D. Williams, had been sheared and sized on a Beckman model E centrifuge. The l*C-labeled rat DNA had been sheared and then sized in an alkaline sucrose gradient using the 6.1 S sea urchin DNA as a marker. The molecular weight of the cDNA was calculated according to Prune11 & Bernardi (1973). (n) Hydroxyapatite

of DNA

fractiomtion

Two procedures for separation of single and double-stranded DNA on hydroxyapatite were used. In both instances the results were corrected on the basis of the elution of linear denatured and native h phage DNA. Batch procedure. This procedure is described in Leong et al. (1972). Column procedure. The hydroxyapatite was boiled 10 min as a slurry in 0.14 M-PO, buffer. The columns were formed in Pasteur pipettes or plastic syringes using at least 1 ml packed hydroxyapatite/250 pg DNA. The columns were arranged in a Lucite box, which was filled with water maintained at the desired temperature by a Braun circulating water heater. The DNA (in cold 0.14 M-PO~ buffer) was loaded onto the column which was then washed with 0.14 M-PO~ buffer (at the column temperature) to elute singlestranded DNA, and 0.4 M-PO, buffer to elute the double-stranded DNA. In order to elute single-stranded DNA, the column procedure requires a lower concentration of phosphate than the batch procedure (J. Stavnezer, unpublished data). Therefore, 0.14 M-PO, buffer was used to elute the single-stranded DNA in the column procedure, and 0.16 M-PO, buffer in the batch procedure. The temperature used for hydroxyapatite fractionation was chosen to preserve the integrity of all duplexes formed during the annealing reaction. (0) Xl n&ease

assay for secondary

structure

Si single-strand specific nuclease from Aspergillus by L. Fanshier (Sutton, 1971). It was used to assay zation as described in Sullivan et al. (1973). (p) DNA-DNA

oryzae

of nucleic was

the extent

acids

prepared of nucleic

and donated acid hybridi-

annealing

DNA samples were denatured by boiling 2 to 10 min (depending on the volume) in 1 mM-EDTA (pH 7). Salt was added, the reaction mixtures were overlayered with mineral oil to prevent evaporation, and then incubated at the appropriate temperature, Samples were removed at various times, diluted at least 1:20 in cold 0.14 or 0.01 M-PO, buffer, depending on the fractionation procedure. They were stored at 4°C for 0 to 5 days before being assayed for nucleic acid secondary structure. The percentage annealed was plotted against the product of the initial molar concentration of DNA nucleotides and time in seconds (Cot), and multiplied by a constant to correct the incubation conditions to standard conditions when the factor was known (Britten & Smith, 1970). M-N&+, 68%; (2) non-stringent Conditions for reassociation : (1) stringent conditions-96 conditions-O.21 M-Na+, 51’C; and 1.5 iv-Na +, 59°C; reactions at 15 M-Na+, 59’C were more practical than reactions at 0.12 M-Na + 51°C because of the greater rate of reaction. Rice & Paul (1972) found that when annealing in 1.5 M-N~+ that 59°C was the lowest temperature that retained specificity in the reaction between the nucleic acids of rat and E. coli. We found that under these conditions there was no annealing of the cDNA and E. coli DNA at C,t = 510. (3) Least stringent conditions-l.0 M-PO, buffer, 54°C. (q) RNA-DNA

hybridization

To destroy any RNAase present (J. Stavnezer, unpublished data), the cDNA was boiled 3 min in 0.3 N-NaOH, 1 mM-EDTA, and then neutralized with HCl. The reaction mixture contained RNA, 0.3 M-NaCl, 0.02 M-Tris*HCl (pH 7*4), 0.001 M-EDTA (pH 7), and 1 mg E. coli RNA/ml, which was included to reduce the effect of trace amounts of RNAase. Hybridizations were performed at 68°C with varying concentrations of RNA. They were either overlayered with mineral oil (50 ~1 reactions), or sealed between mineral oil layers

GENES FOR IMMUNOGLOBULINKAPPA

49

CHAIN

in capillary tubes (5 or 10 ~1 reactions). Samples were analyzed by the S, nuclease assay. The percentage hybridization was plotted versus the product of the molar concentration of RNA nucleotides and time in set (Cot), multiplied by the rate correction factor of 2.3. (r) Thermal

denatwatkm

of RNA-DNA

hybrids

RNA-DNA hybrids were divided in equal portions and incubated separately in 0.01 %I-TriseHCl (pH 8.1) for 10 min at the indicated temperatures, and placed immediately in an ice/water bath. The samples were assayed with S1 nuclease for the percentage hybrid remaining at each temperature. Samples incubated at 0°C were assayed with and without Si nuclease to determine the total amount of hybridization. (s) Thermal

denaturation

of the annealed

DNA

The products of the reassociation reactions were loaded onto hydroxyapatite columns in 0.14 M-PO, buffer at 45°C or 50°C. The single-stranded DNA was eluted, the column flow was stopped, the temperature was raised 5”C, and held for 5 min before the singlestranded DNA was eluted at each increment of temperature. (t) Determination

of the base composition.

The base composition of the cDNA was determined micrococcal nuclease and splenic phosphodiesterase, electrophoresis (Taylor et al., 1972). (u) Measurement

of nucleic

of cDNA

by J. Taylor by digestion followed by high-voltage

with paper

acid concentration

The amount of nucleic acid was usually determined by measurement of the absorbance at 260 nm in a Gilford model 2000 speotrophotometer. The absorbance of RNA was assumed to be 1 Azeo unit/40 pg and that of sheared DNA to be 1 A2e0 unit/45 pg. The data in Table 1 on the yield of RNA in the mRNA isolation procedure were obtained with the oroinol test and corrected for interference by DNA with the diphenylamine test, as described by Schneider (1957).

3. Results (a) Purification

of the immunoglo6ulin

light

chain

messenger

The light chain mRNA was first released from the microsomal fraction of the MOPC41 cells by treatment with SDS, and then isolated by sedimentation through two successive sucrose gradients. At this stage 90 to 95% of the RNA migrated as a distinct species during polyacrylamide gel electrophoresis (Fig. l(b)). The RNA was TABLE 1 PuriJication

oj’ light chain messenger

Yield 1. 2. 3. 4. 5. 6. 7.

Homogenate Post-nuclear supernatant Microsomal extract Pool from 5% to 20% sucrose gradient Pool from 10% to 70% gradient Adsorbed to oligo(dT)-cellulose (one cycle) Pool from dimethylsulfoxide gradient

(%)

100 94 27 3.4 0.2 0.08 0.06

RNA Relative synthesis of kappa chain per Azso unit RNA?

17 185 455

i Relative synthesis : the amount of [14C]leucine incorporated into material precipitable wit,h antibody after synthesis in the presence of mRNA relative to the amount incorporated in the absence of mRNB. 4

J. STAVNEZER I

I

I

ET

AL.

I

(a)

01

Bottom

,

I

I

IO

20 Fraction

I 0

2

I

I

30 no.

40

Bottom

I 4

I 6

Distance migrated

20

40 60 Fraction no

I 8

IO

(cm )

FIG. 1. Purification of MOPC41 light chain eRNA. (a) Centrifugation of MOPC41 mRNA in a 10% to 70% sucrose gradient. The large peak (fractions 11 to 18) was due to SDS, which formed a precipitate at this point in the gradient. The RNA under the brrtcket was pooled and precipitated with ethanol. (b) The RNA obtained from the sucrose gradient shown in (a) analyzed by electrophoresis in a polyscrylamide gel, which was scanned as described in Materials and Methods. (c) The RNA adsorbed to, and eluted from, oligo(dT)-oellulose centrifuged in a 5% to 60% sucrose gradient in 99 y0 dimethylsulfoxide.

further purified by adsorption to, and elution from, oligo(dT)-cellulose, and finally by sedimentation through a sucrose gradient containing the denaturant, dimethylsulfoxide (Fig. l(c)). The RNA under the brackets was pooled and ethanol-precipitated. The extent of purification and yield of RNA at each step in the procedure is summarized in Table 1. The final yield of the purified mRNA was O*O6o/oof the total cellular RNA. The relative activity of the RNA as messenger for kappa chain synthesis in vitro increased 27-fold between steps 4 and 7. The total purification was probably much greater than this, but before step 4 the RNA contained SDS and was not used in the translation assay. (b) Xize of the messerqer RNA The molecular weight of the mRNA was determined by electrophoresis in 3% polyacrylamide gels at two temperatures, 6°C and 26°C (Groot et al., 1970; David & Chase, 1972), using mouse 28 S, 18 S, 5 S and 4 S RNAs as molecular weight standards. Electrophoresis at both temperatures gave identical results : 4.0 x IO5 M,, which is equivalent to 1180 nucleotides. This result is identical to that of Brownlee et al. (1973), who used polyaorylamide gel electrophoresis in formamide; by contrast

GENES

FOR IMMUNOGLOBULIN

KAPPA

51

CHAIN

Swan et al. (1972) and Delovitch & Baglioni (1973) reported a length of 800 to 900 nucleotides determined by rate zonal centrifugation. (c) Synthesis and characterization

of complementary

DNA

Complementary DNA was transcribed from the purified mRNA using DNA polymerase of Rous sarcoma virus. The reaction was dependent on the presence of oligo(dT),, _ 18 primer (20-fold stimulation), and inhibited by RNAase (pretreatment for 20 min at 37°C resulted in 88% inhibition). In the reaction using all four [3H] deoxynucleoside triphosphates, each at a concentration of 10 PM, 180 ng of cDNA was synthesized when 750 ng of mRNA was used as template.

$00

I1: 300

I i

20

IO (a)

2

100

20

IU

Fraction no.

200

.5 5 v) 5

(b)

FIG. 2. Rate zonal centrifugation of oDNA in an alkaline sucrose gradient. (a) Unsheared total cDNA was centrifuged in a 5% to 20% sucrose gradient in 0.1 M-NaOH, 0.9 M-NaCI, 0.01 M-EDTA at 60,000 revs/min in the SW50.1 rotor for 7.25 h at 2O’C. The position of the mean S-value of the cDNA (5.4 S) is indicated. The S-value was determined by reference to the sedimentation of sheared sea urchin DNA (6.1 8). (b) Purified, large cDNA was centrifuged as in (a) in the SW65 rotor for 4.25 h. The position of the mean S-value of the oDNA (7.8 S) is indicated. The S-value was determined by reference to au internal marker of sheared 14C-labeled rat DNA (5.0 S). -O-O-, cDNA; -e-e--, sheared rat DNA.

The cDNA was heterogeneous in size with a mean sedimentation coefficient of 5.4 S when analyzed by sedimentation through an alkaline sucrose gradient (Fig. 2(a)). DNA of t,his molecular weight (89,200) contains 271 nucleotides. Large cDNA was isolated from cDNA synthesized using all four [3H]deoxynucleotide triphosphate precursors at concentrations of 120 to 750 PM. Before selection by size on sucrose gradients, the mean sedimentation value of this cDNA was 5.6 S. The largest @DNA was pooled from two successive neutral sucrose gradients, yielding cDNA with a mean S-value of 7.8 on an alkaline sucrose gradient (Fig. 2(b)). DNA of this molecular weight (204,000) contains 620 nucleotides. Most of the following experiments were performed with total cDNA; when the large cDNA was used it is so indicated. The base composition of the cDNA was 21.8% cytidine, 22=90/, adenine, 21.9% guanine, and 33.4% thymidine. Although the base composition of the light chain mRNA is unknown, the proportion of thymidine in the cDNA is relatively high, suggesting that the cDNA contains oligo(dT). The presence of oligo(dT) was

62

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confirmed by incubating the cDNA with unlabeled poly(A) followed by digestion with S1 nuclease. There was 6% hybridization, indicating that 3% of the cDNA is labeled oligo(dT) (the specific activity of the [3H]TTP was l-9 times the specific activity of the average of the four [3H]deoxynucleoside triphosphate precursors). Only 1% of the large cDNA hybridized with poly(A). These data are corrected for the amount of S1 nuclease-resistant material obtained (80%) when 3H-labeled poly(dT) was incubated with poly(A) under identical conditions. Since initiation of DNA synthesis on light chain mRNA requires an ol.igo(dT) primer, all the oDNA chains are presumably initiated at the 3’ end of the structural gene, where the poly(A) is located (Mendecki et al., 1972). If one subtracts the contribution to the molecular weight of the labeled oligo(dT) (7 nucleotides), and of the unlabeled oligo(dT) primer (12 to 18 nucleotides), which should be covalently linked to the cDNA, the cDNA probably corresponds to 82 to 84 amino acids. The large cDNA corresponds to 198 to 200 amino acids.

log Cot (corrected) FIG. 3. Hybridization of cDNA with purified light chain mRNA. Varying amount of purified mRNA (adsorbed to oligo(dT)-cellulose twice) were incubated with O-025 ng of cDNA (both sheared and unsheared) at 68°C in 100 ~1 of 0.3 ~-N&cl, 0.02 M-Tris*HCl (pH 7.4), 0.001 M-EDTA (pH 7). After 18 to 20 h, samples were removed and assayed for secondary struoture with S1 nucleate. The C,t, of the light chain mRNA hybridized with light chain cDNA is indicated by the horizontal line on the curve. The Grtt of the hybridization of polio RNA and polio oDNA, which was performed and assayed in a similar manner, w&s 4-7 x 10e3.

(d) Hybridization of complementary DNA with template RNA The kinetics of hybridization between the cDNA and the isolated kappa chain mRNA (adsorbed to oligo(dT)-cellulose twice) were measured to investigate the purity of the template RNA and cDNA (Fig. 3). The reaction occurred in a IOO-fold range of C,t values, and attained a fmal value of 100% hybridization, indicating that the cDNA is transcribed from a population of RNA molecules of similar complexityt. i Complexity is defined as the number of nucleotides in unique sequenoes, and is inversely proportional to the rate of annealing of the nucleic acid (Britten, 1970). To calculate the oomplexity of & nucleic acid relative to another of known complexity the Co6t values (or C,t, values) are compared (Birnstiel et al., 1972).

GENES

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CHAIN

The complexity of the mRNA was computed by comparing its C,t+ (3.3 x 10e3) to that of a standard (polio RNA), and found to be 5250 nucleotides. This is 4.4 times greater than that predicted from the molecular weight of the mRNA. If rabbit hemoglobin a-chain mRNA is used as the standard (CJ* = 8.8 x 10m4; N. D. Hastie, M. G. Farace, K. B. Freeman t J. 0. Bishop, personal communication), the complexity of the kappa chain mRNA is 2.2 times too great (see Discussion). (e) Hybridization

of the complementary

DNA with cellular

RNAs

The specificity of the cDNA was investigated by hybridization with total cell RNA from four myelomas (Fig. 4), which produce different amounts of kappa chain protein: (1) MOPC41-souroe of the kappa chain mRNA; (2) 66.2-a myeloma that produces only kappa chains, in one-third of the amount synthesized by MOPC41 (R. Laskov & M. Scharff, personal communication); (3) h5830-a myeloma that primarily produces lambda chains, although a small amount of kappa chain, approximately 5% of the amount of lambda chain, was detected in the urine of a mouse bearing a small h5830 tumor (R. Riblet, personal communication) ; and (4) XS63-a “non-producer” cell line derived from myeloma 563, which in reality synthesizes 1% of the amount of IgA that the parent 563 cells synthesize (Schubert & Horibata, 1968). The relative amount of kappa chain RNA in each myeloma, computed from the Crtt of the hybridization (see Materials and Methods), was approximately proportional to the amount of kappa chain produced by each myeloma (see inset to Fig. 4). The fact that the hybridization between XS63 RNA and cDNA only reaches 58% probably results from an insufficient RNA excess, since the ratio of cellular kappa chain sequences to cDNA in the experimental points taken at Crt = 96 is 2.1 and at C$ = 960 is 21 (assuming that O*O02°/0of the cell RNA codes for the kappa

80

t

MOPC41 66 2

32

01

13

h5a30

63

XS63

204

0 025 0005 0002

I

L

-3

-2

-L

-I

0 log C, t

I (corrected

2

3

4

)

FIG. 4. Hybridization of light chain cDNA with cellular RNAs. Varying amounts of cellular RNA were incubated with 0.034 ng of light chain cDNA in 50 ~1 of 0.3 M-N&~, 0.02 M-Tris.HCl (pH 7.4), 0.001 M-EDTA (pH 7) at 68°C for approximately 48 h. The hybrids were assayed with S, nuclease. -O-O--, MOPC41 RNA; -O-•-, 66.2; -u--O-, X5830 RNA; -A-A-, XS63 RNA; -A-A---, BALR/c liver RNA; -m-m--, E. co& RNA. The C,t: values s,re indicated by the horizontal line on each curve.

54

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chain constant region). RNA from BALB/c mouse livers and E. coli contained no detectable nucleotide sequences (less than 1 part in 106) complementary to the kappa chain cDNA. These results suggest that the cDNA is specific for the immunoglobulin kappa chain. The large cDNA was also tested for specificity by hybridization with cell RNAs. At a C,t = lo2 it hybridized with RNA from the myeloma cells MOPC41 (loo%), 66.2 (84%), h5830 (88%), but not significantly with RNA from mouse liver (15%) or E. coli (5%). It could be argued that the lower rate of hybridization obtained with myelomas 66.2 and h5830 was due to the formation of poorly matched hybrids between the cDNA and a different kappa chain mRNA (in the case of 66*2), or with lambda chain mRNA (/\5830) (Sutton & McCallum, 1971). To clarify this point the fidelity of basepairing was ascertained by examining the thermal denaturation of the hybrids formed with MOPC41,66*2 and h5830 RNA. The T, values (the mid-point of the temperature melting curve) of all three hybrids were identical (Fig. 5), suggesting that the lower rate of hybridization was due to the fact that myelomas 66.2 and h5830 contained Iess kappa chain RNA than MOPC41, rather than to a reduction in rate caused by mismatching.

Temperature

(“C 1

FIG. 5. Thermal denaturation of the hybrids between cDNA and myeloma cell RNAs. The dissolved hybrids were formed by incubation to C,r = 1.5 X 10 4. They were ethanol-precipitated, in 0.01 na-Tris.HCl (pH 8*1), incubated at the indicated temperatures, and quickly chilled to 0°C. The seoondary structure was measured with S1 nuclease. The horizontal line on the curves at 50% dissociation indicates that all three hybrids had the identical T, = 65.6’C (65.3 to GS”C).

(f) Enumeration of kappa chain sequencesin the mouse genome The number of genes for the constant region immunoglobulin kappa chains in the mouse genome was determined from the kinetics of nucleic acid reassociation. A relatively large amount of DNA isolated from mouse liver or MOPC41 was melted and reassociated in the presence of 3H-labeled cDNA and 14C-labeled unique-sequence mouse DNA. The rate of annealing of the labeled nucleic acids will be determined by the concentration of unlabeled sequences, if the ratio of sequences in the unlabeled DNA to identical sequences in the labeled DNA is large and if self-reassociation of the labeled unique-sequence DNA is avoided by keeping its concentration low. By

GENES

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55

comparing the CotI of the labeled light chain-specific DNA to that of the labeled unique-sequence DNA, the frequency of the light chain sequences in the genome can be determined. In an effort to detect both closely and distantly related kappa chain genes, the reassociations were carried out under three different sets of conditions, which permit varying degrees of mismatching in duplex DNA (McCarthy & Church, 1970). The cDNA was annealed with DNA isolated from both mouse liver and MOPC41 in an effort to detect amplification of the genes for the constant region of kappa chains in the myeloma DNA. (g) Annealing of the com$ementary DNA under three different sets of conditions When the unique-sequence DNA and cDNA were annealed with liver or MOPC41 DNA under our most stringent conditions (0.6 M-Na+ at 68°C; Fig. B), we found

60 Unique sequence Cotl12= 1050 P

RI-l t

cDNA

G-J,,7

Unique sequence

0

I

Cot,,r = 1500

2

3

4

log Cot (corrected)

Fm. 6. Annealing of light chain cDNA with liver and MOPC41 DNA in 06 M-Na+ at 68°C. Complementary DNA (O-75 ng) and 1.57 pg of 14C-labeled unique-sequence DNA were incubated either with (a) 561 pg of liver DNA or (b) 576 pg of MOPC41 DNA in a total volume of 100 t;l. Ten ~1 portions were removed at vsrying times (up to 49 h) and the amount of annealing was measured on hydroxyapatite using the batch procedure. The results of the elution of oDNA were corrected for the elution of cDNA from hydroxyapatite after incubation in the absence of cell DNA (16% double-stranded). This correction w&s only necessary when the batch procedure was used. The horizontal lines on the curves indicate the Cot+ values. -O-O--, cDNA; -a-+--, unique-sequence DNA.

56

ET AL.

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TABLET The number of genesfor the constant region of kappa chain in the mouse Conditions

for reassociation

DNA

Unique

cot* sequence

Kappa

chain

No. of kappa genes

(1) 0.6 M-N&+, 68°C (No unlabeled poly(dT))

Liver MOPC41

1050 1500

750 830

(2) 0.21 M-N&+, (No unlabeled

MOPC41

1700

t(a) 0.27 (b) 650

MOPC41 Liver

430 680

103 340

2.1 1.7

(4) As in (3) except with unsheared cDNA (270 nucleotides long)

MOPC41

530

180

2.9

(5) 1.5 M-NB+, 59T, &S in (3) except with large cDNA (620 nuoleotides long)

MOPC41

1100

290

3.6

54°C (6) 1.5 M-N&+, (Including unlabeled poly(dT))

MOPC41

580

610

1

(3)

1.5

M-N&+,

(Including PWdT))

61’C

poly(dT))

59°C

unlabeled

t (a) C,,l+ of the fast reaction

of the oDNA;

(b)

1.4 1.8 6300 2.6

Cot&of the slow reaction of the cDNA.

approximately 1.6 copies per haploid genome (Table 2). When the same experiments were performed with non-stringent conditions (O-21 M-Na+ at 51”C), the cDNA annealed with MOPC41 DNA in a two-step reaction (Fig. 7). Thirty percent of the cDNA annealed at a C,t + indicating 6300 copies per haploid genome, and 60% of the cDNA annealed at a Cot+ indicating 2.6 copies of this portion of the sequence per haploid genome. Approximately the same results were obtained with mouse liver DNA. The addition of unlabeled poly(dT) to the reaction mixture completely eliminated the more rapid component of the reaction (Fig. 7). Therefore, we conclude that this component is due to a reaction between poly(dT) on the cDNA and poly(dA) in the mouse genome, and thereafter eliminated it by the addition of unlabeled poly(dT) to all reassociations. The proportion of the cDNA that annealed in the more rapid component of the reaction is greater than the amount of poly(dT) in the cDNA because the reassociation product was assayed by binding to hydroxyapatite. This procedure scores as duplex the single-stranded DNA that is covalently linked to duplex DNA. From these experiments, and from one in which 3H-labeled poly(dT) was annealed with MOPC41 DNA (J. Stavnezer, unpublished data), we conclude that 7x 10m5 to 16x low5 part of the mouse genome is poly(A)*poly(dT). The standard for this calculation was the Cot; of poly(dA) .poly(dT) determined by Taylor et aZ. (1973). When the cDNA was annealed with MOPC41 and liver DNA in the presence of competing poly(dT) under non-stringent conditions (1.5 M-N%+, 59”C), 1.9 copies of the kappa chain constant region were detected (Table 2).

GENES

100 L -2

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-I

0

I log Co’

KAPPA

2

67

CHAIN

3

4

FIG. 7. Annealing of light chain cDNA with MOPC41 DNA in 0.14 or-PO4 buffer at 51°C in the absence and in the presence of unlabeled poly(dT). Complementary DNA (0.47 ng) and 0.5 pg of 14C-labeled unique-sequence DNA were incubated either with 18 pg of MOPE41 DNA in 500 p1 (Cot = IO-% to 1) or 2.12 mg of MOPC41 DNA in 130 ~1 (c,t = 1 to 1.3 x lo*) for varying time (up to 70 h). The secondary structure was measured on hydroxyapatite columns, eluting single-stranded DNA with 0.14 M-PO, buffer at 45°C and double-stranded DNA with 0.4 M-PO, buffer. The horizontal lines indicate the Cat+ values. --O-O-, cDNA; -e-e-, uniquesequence DNA; -m-•--, cDNA in the presence of 1 pg unlabeled poly(dT).

When the cDNA was annealed with MOPC41 DNA under still less stringent conditions, 15 M-N~+ at 54”C, we detected approximately one copy per haploid genome (Table 2). Under these conditions 8% of the cDNA reacted with E. coli DNA at C,t = lo3 in the presence of excess unlabeled poly(dT). All the experiments described above were performed with cDNA that was sheared to a length of 125 nucleotides (J. Stavnezer, unpublished data). When unsheared cDNA was reassociated with MOPC41 DNA under non-stringent conditions (1.5 M-Na*, 59”C), slightly more copies were detected (2.9) (Table 2), in agreement with the effect of length on the rate of annealing (Wetmur & Davidson, 1968) (see below). (h) Annealing

of the large complementary

DNA

The kappa chain mRNA contains 300 more nucleotides than needed to code for the kappa chain. It is possible that these untranslated nucleotides are between the constant region sequence and the 3’ terminal poly(A). The hybridization studies of Firtel & Lodish (1973) and Molloy et al. (1974) suggest that the structural gene sequences of mRNAs are adjacent to the poly(A), whereas the sequencing data of Proudfoot & Brownlee (1974) show that there are at least five untranslated nucleotides between the poly(A) and the globin structural gene. Therefore, we isolated cDNA large enough to code for 100 amino acids of the constant region, even if all 300 untranslated nucleotides were between the poly(A) and the constant region sequence. If there were no untranslated nucleotides between the poly(A) and the constant region sequence this large eDNA would code for 93% of the entire kappa chain,

58

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0

ET

I log cot

AL.

2

3

4

FIG. 8. Annealing of large cDNA with MOPC41 DNA in 1.0 M-PO, buffer at 59°C in the presence of unlabeled poly(dT). Unsheared large oDNA (0.2 ng) and 0.12 pg of 14C-labeled unique-sequence DNA were incubated with 265 pg of MOPC41 DNA in 500 ~1 (Cot = 0.1 to 10*3), or 2.61 pg of MOPGIl DNA in 300 ~1 (C,t = 7 x 1O3) for varying times up to 75 h. The amounts of unlabeled poly(dT) included in the above reactions were 0.6 pg and 7.6 pg, respectively. -O-O--, cDNA; --a--e-, unique-sequence DNA.

When unsheared stringent conditions agreement with the the effect of length

large cDNA was annealed with MOPC41 DNA under non(1.5 M-Na +, 59”C), 3.8 copies were detected (Fig. 8). This is in number of copies detected using total cDNA, after correction for on the rate of annealing (see below).

(i) Thermal denaturation

of the annealed

complementary

DNA

The stability of the duplexes between cDNA and MOPC41 was investigated by thermal denaturation on hydroxyapatite (Fig. 9). The l%labeled unique-sequence DNA was also reassociated and melted as an internal standard. The differences in the T, values (AT,) of the unique and cDNA were 5°C in the hybrids formed at stringent conditions and 8°C in those formed at non-stringent conditions. These data could be corrected for the difference in size and G+C content of the cDNA and unique-sequence DNA (Thomas & Dan&, 1973; Mandel $ Marmur, 1968). However, the resulting correction is smaller than the variability in the data. The AT, values indicate that the duplex formed with stringent conditions is approximately 2*5”,b mismatched, and that formed with the non-stringent conditions is approximately 45% mismatched (Ullman & McCarthy, 1973). The presence of mismatching supports our conclusion that the cDNA is hybridized with a few kappa chain genes with slightly different nucleotide sequences. (j) The number of genes for the constant region of kappa ckrin

in the mouse

The copy number estimations should be corrected for: (1) the reduction in the rate of annealing caused by the small size of the sheared cDNA relative to the labeled unique-sequence DNA (correction factor = 1.33) (correction factor for the large cDNA = 056) (Wetmur & Davidson, 1968) ; (2) the reduction in annealing rate caused by t.he amount of mismatching between the cDNA and the cellular kappa

GENES

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-

5 Temperature

55

65

I 75

Tm=765T

I 85

I 95

PC)

(a)

(b)

FIG. 9. Thermal denaturation of the light chain cDNA-MOPC41 DNA hybrid. (a) Complementary DNA (0.17 ng) and 0.26 pg of 14C-lrtbeled unique-sequence DNA were annealed with 2 mg of MOPC41 DNA and 5 pg of unlabeled poly(dT) in 200 ~1 of 0.6 M-Na+ at 68°C to a C,t = 2 x 104. The hybrid was bound to hydroxyapatite in 0.14 M-PO, buffer at 5O”C, the temperature was raised to 6O”C, and the amount of hybrid melted above 60°C was determined. (b) Complementary DNA (0.36 ng) and 0.26 pg of l*C-labeled unique-sequence DNA were annealed with 4 mg of MOPC41 DNA in the presence of 10 pg of unlabeled poly(dT) in 400 ~1 of 1.0 M-PO, buffer at 69°C to C,t = 4.7 x 10s. The hybrid was bound to hydroxyapatite in 0.14 X-PO, buffer at 45”C, and the amount of hybrid melted in 5 deg. C increments was determined. -O--O--, cDNA; -@-a--, unique-sequence DNA.

chain sequences (correction factor = 1.7) (McCarthy & Farquhar, 1972; Bonner et al., 1972) and (3) the increase in the rate of hybridization of the cDNA caused by the contribution of the cDNA to the concentration of the kappa chain sequences (correction factor = 0.8) (Sullivan et al., 1973). Applying these corrections to the reassociation data obtained with both the sheared and the large cDNAs we estimate that each haploid mouse genome contains three to four genes for the kappa chain constant region.

4. Discussion (a) Specijcity of the complementary DNA for immunoglobulin kappa chain The validity of our conclusions depends on the specificity of the cDNA for immunoglobulin kappa chains. Hybridization of the purified mRNA with cDNA

60

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transcribed from this RNA suggests that the cDNA is transcribed from a single species of RNA, although the shape of hybridization curves is insensitive to moderate inhomogeneity. It is possible that this RNA preparation could contain a number of RNAs serving as templates for DNA synthesis if the concentration and complexity of the RNAs were to vary proportionally so that they all had similar C,t, values. However, this is unlikely because the cDNA hybridizes specifically with RNA from cells producing immunoglobulin kappa chain. The C,t, of the hybridization between mRNA and cDNA is 3.3 x 10w3, which is two to four times greater than that predicted from the molecular weight of the mRNA. Although the complexity is higher than expected it is within the expected range of variation found for the rate of RNA-DNA hybridization (Birnstiel et al., 1972; Strauss & Bonner, 1972; Bishop, 1969,1972). Hybridization rates commonly vary two to threefold among different RNAs even when the temperature and salt. concentration are held constant. Base composition has been found to affect the optimum temperature, and hence the rate, of RNA-DNA hybridization more than it affects the rate of DNA reassociation (Birnstiel et al., 1972), so that it is quite possible that we did not perform the hybridization at the optimum temperature for this particular RNA. It is also possible that our mRNA preparation contains RNAs not transcribed by the DNA polymerase. However, it is unlikely that an mRNA present as a small contaminant in the preparation of mRNA for kappa chain is the template for cDNA, since the mass ratio of cDNA synthesized to template RNA was 1:4, and RNA-directed DNA polymerases do not synthesize an excess of DNA product relative to the amount of template present (Verma et al., 1972; Faras et al., 1972; Leis & Hurwitz, 1972). (b) Enumeration of kappa chain constant region genes Since initiation of DNA synthesis on light chain mRNA requires an oligo(dT) primer, all the cDNA chains are presumably initiated at the 3’ end of the structural gene, where the poly(A) is located. If one subtracts the contribution to the molecular weight of the labeled oligo(dT) (3% of the cDNA, or 7 nucleotides), and of the unlabeled oligo(dT) primer (12 to 18 nucleotides), which should be covalently linked to the cDNA, the average cDNA would correspond to 82 to 84 amino acids, or 75% of the light chain constant region, and the large cDNA to 199 amino acids or 93% of the entire kappa chain. This assumes the untranslated nucleotides in the mRNA are at the 5’ terminus. However, if there are 300 untranslated nucleotides between the 3’ terminal poly(A) and the constant region, the average cDNA would contain no translated sequences and the large cDNA would code for 95% of the constant region. Two groups have measured the rate of hybridization between mouse cell DNA and labeled RNA prepared from mouse myelomas producing kappa chains (Delovitch & Baglioni, 1973; Storb, 1974). Using the reassociation of cell DNA as the standard for their RNA-DNA hybridization, Delovitch & Baglioni measured 40 to 1000 copies, and Storb measured 2500 copies of the kappa chain gene per mouse genome. We believe the discrepancy between their results and ours may possibly be due to one or more of the following factors. (1) The final amount of hybridization of their mRNA was less than or equal to 50%, so they may have determined the reiteration of the variable region half of the light chain. (2) It is possible they were hybridizing ot,her species of RNA, in addition to light chain mRNA, since the purity of the

GENES

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61

RNA was not determined. (3) They used a DNA standard for their hybridization, which may be inaccurate since the relative rates of RNA-DNA hybridization and DNA reassociation vary with different reaction conditions and. species of RNA (Straus & Bonner, 1972; Birnstiel et al., 1972). Premkumar et al. (1974) have hybridized labeled mRNA for immunoglobulin heavy chains with cell DNA. They obtained a biphasic hybridization reaction, suggesting the presence of 5900 variabie region genes and eight constant region genes. Provided that their mRNA is pure and that the hybridization obtained at low C,t values is actually that of the variable region, their data suggests that our cDNA only codes for the constant region of kappa chain. This assumes that the reiteration of the variable region of the heavy and light chains is comparable. Hybrids formed between the total cDNA and three different myeloma tumors all had the same T, when denatured with heat. This similarity substantiates our conclusion that the cDNA is hybridizing only with RNA representing the kappa chain constant region. The high thermal stability of the hybrids with the myeloma 66.2 RNA also rules out the hypothesis that the small amount of hybridization obtained with 66.2 is due to the reduction in rate caused by a divergence in the sequences of MOPC41 and 66.2 constant regions. Instead, it appears that 66.2 synthesizes 23% of the amount of kappa chain mRNA that MOPC41 synthesizes. This is in accord with the relative amounts of kappa chain produced by these myelomas. We have concluded that the nucleic acid sequences for the constant region of kappa chain differ by 2.5 to 4.5%. This does not agree with the available amino acid sequence data, which indicates a smaller amount of divergence. The constant regions of the three BALB/c myeloma kappa chains that have been sequenced (MOPC41 and MOPC70 by Gray et al. (1967); MOPC21 by Milstein & Svasti (1971) differ in only four amino acids, whose codons could differ by only one base each. If this were true the differences among the DNA sequences would be only l-2%, although they could easily diverge further if there were wobble in the third base. In addition, the three kappa chains that have been sequenced to date may not represent the entire range of variation among kappa chains. (c) Theoretical

implications

The cDNA used in these studies may be complementary to only the constant region of the kappa chain. Consequently, we cannot distinguish between the germ line and the somatic theories of antibody variability. Since we find only a few genes coding for the constant region of kappa chains, the entirety of every kappa chain polypeptide is not represented by a distinct gene. Therefore, the generation of antibody diversity requires specialized genetic mechanisms. There are three possibilities. (1) If there are numerous genes coding for the variable region of the kappa chain, the variable and constant region genes must somehow join during the development of the immunocyte. This would require a specific mechanism for excision and integration. (2) If there are a few variable region genes, and they are separate from the constant region genes, somatic mutation of the variable region would be necessary in addition to excision and integration. (3) If there are a few genes coding for the entire kappa chain, the variable half of the gene must undergo somatic mutation, while the constant half is maintained invariant. Although there is no precedent for any of

62

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ET

AL.

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