Expression of λ and K Genes Can Occur in all B Cells and is Initiated Around the Same Pre-B-Cell Developmental Stage

Transgenic mice that carry a λ2 transgene under the control of the Vλ2 promoter and the Eλ2-4 enhancer (λ2Eλ mice) are described. A high proportion of B cells in the spleen and the bone marrow express the λ transgene on the cell membrane. λ2 protein is synthesized by all λ2Eλ-derived spleen B-cell hybridomas that have retained the transgene, suggesting that all B cells have the ability to express λ genes. Feedback inhibition of endogenous K-gene rearrangement is significant, but not complete. The results are similar to those with transgenic mice expressing the same λ2 transgene under the control of the heavy-chain enhancer (λ2EH mice). Although the λ2EH transgene is expressed before the λ2Eλ transgene, feedback inhibition seems to occur at about the same stage of B-cell development, regardless of the timing of expression of the λ transgenes. Apparently, feedback is not necessarily coincident with the assembly of a heavy-chain/light-chain complex in pre-B cells. Expression of λ in the normal fetal liver coincides with the expression of K; thus, it appears that λ-gene transcription is not delayed. The differential rearrangement of K and λ genes is discussed in the light of these findings.


INTRODUCTION
Mammals produce both c and light chains but in vastly different proportions. At one end of the spectrum, mouse serum Igs are about 95% c+. At the other end, horse Igs are 95 % +, whereas human light chains are divided between 60% c + and 40% ,+ (Eisen and Reilly, 1985). It is not clear how these ratios are regulated. Selection of c + cells in the peripheral lymphoid organs only partially explains the skewing, because even in the bone marrow, c cells outnumber + cells by at least 5:1 (this paper; Rolink et al., 1993). Analysis of mouse and human Ig producing lymphomas and plasmacytomas has led to the hypothesis that c and , genes are sequentially activated (Hieter et al., 1981). It was found that most c-producing B cells have their , genes in germline configuration. In contrast, B cells with functional , genes have, with few exceptions, "Present address: Department of Biochemistry, Northwestern University, Evanston, Illinois. tCorresponding author. both c genes rearranged or even deleted. The sequential model postulated that developing B cells would first acquire the ability to rearrange c genes. , genes would be activated only after both c genes had been nonproductively rearranged. A modification of this model is one in which the induction of ,-gene rearrangement requires the inactivation of the c locus by rearrangement of the RS element located 3' of the Cc gene (Moore et al., 1985).
Recently, the sequential model, in which X rearrangement depends upon prior events at the c locus, has essentially been ruled out by the analysis of c knockout mice, which, despite the absence of any rearrangement in the c locus, produce large numbers of B cells with rearranged , genes (Takeda et al., 1993). The ordered rearrangement of Ig genes is controlled by a V(D)J recombinase that is present in pro/preB cells until correct heavy-and light-chain gene rearrangements have occurred (reviewed in Storb et al., 1989). It was shown with c transgenic mice, that B cells that express the c transgene together with an endogenous heavy-chain gene 14 LAMBDA TRANSGENIC MICE have their endogenous c genes mostly unrearranged (Ritchie et al., 1984). This was taken as evidence for the shutoff of the V(D)J recombinase after completion of correct rearrangements of both a heavychain and a light-chain gene (Ritchie et al., 1984). Similarly, it has been shown in mice that express a X transgene under the control of the heavy-chain enhancer that endogenous c-gene rearrangement is strongly inhibited Neuberger et al., 1989). Thus, it was clear that , , presumably the protein, can deliver a rearrangement feedback signal.
A puzzling observation was the finding of normal numbers of X-producing B cells in c transgenic mice, while endogenous c-producing B cells were greatly diminished (Gollahon et al., 1988). This led to the hypothesis of two distinct B-cell populations with different abilities to regulate c-and ,-gene expression. One, the c lineage, was thought to rearrange only c genes, whereas the other, the c/, lineage, was thought to be able to rearrange both c and , genes (Gollahon et al., 1988). To test this model, it had to be determined whether X genes can be expressed in all or in only a subset of B cells (Cuisinier et al., 1992). The enhancers driving -gene expression had for a long time been elusive, because it was found that, unlike heavy-chain and c genes, no enhancers were present in the JC intron of X genes (Picard and Schaffner 1984;Hagman et al. 1990). Recently, two strong B-cell-specific enhancers were found to be located 16 kb and 35 kb 3' of the C,2-4 and C,3-1 constant-region gene clusters, respectively (Hagman et al., 1990). It was then possible to make transgenic mice with transgenes driven by the transcriptional control elements. We report here the analysis of transgenic mice with a rearranged 2 transgene under the control of the V,2 promoter and the E,2-4 enhancer. They were generated to determine if all B cells can produce , and to obtain additional clues concerning the control of the differential expression of c and , genes.

Tissue-Specific Expression
Expression of the ,2E, transgenes was determined by Northern blots of total RNAs from a variety of tissues. It was found that the ,2E, transgene was transcribed in the spleen, but not in thymus T cells (some , mRNA was seen in thymus, but it was matched by c mRNA from B-cell contamination), liver, kidney and heart, suggesting that expression is restricted to B cells (data not shown).

FACS Analysis of Spleen Cells
To determine the distribution of ,+ and c + cells in the peripheral lymphoid organs, spleen cells of ,2 transgenic mice and normal littermates were analyzed by fluorescence-activated cell sorter (FACS).  (,96) of the B cells are /. The majority of these coexpress c (46% or 56%). Only 22% or 15% of the total B cells are X-only cells, 32% or 29% are c-only. Clearly, as has been found previously with ,2EH-gene transgenic mice Neuberger et al., 1989;Rudin et al., 1991), there is feedback on endogenous c-gene rearrangement, but it is incomplete. No significant difference is seen between 94 and 96 ,2E, transgenic mice. The degree of feedback in the ,2E mice seems very similar to that found in the ,2EH mice: Both show a decrease in total c + cells and a substantial population of ,-only cells. However, in the mice with the ,2EH transgene more of the c cells coexpress , (32% versus 16%).
Perhaps this is a reflection of the somewhat greater amounts of protein on mature B cells of these mice due to greater efficiency of the heavychain enhancer at this stage. The total ,+ B cells can be roughly divided into two populations based on staining intensity with anti-(not shown). In the ,2EH transgenic mice, 42% of the total ,+ B cells express high levels of ,, however, only 31% of the + cells in ,2E, mice fall in this category.
Why are not 100% of the B cells ,+? We assume that this is due to our inability to detect low levels of , in many double-positive cells. This assumption is supported by the analysis of hybridomas.   (Table  2). It was found that all the c-only hybridomas had lost the transgenes. Similar results were found with hybridomas of 2EH mice (Hagman el al., 1989). Based on these findings, it appears that the ,2 transgene under the control of its own enhancer can be expressed in all B cells. It is not clear whether most of the :+ cells in the spleen (as determined by FACS analysis) have also lost the transgene. The fact that the percentages of :-only cells in spleen and hybridomas agree so well may suggest that this is the case. However, hybridomas are notorious for the loss of chromosomes due to their polyploidy. It appears more likely that the "K-only" B cells in the spleen express the transgene, but that K has a higher affinity for many heavy chains. It has been shown that in a competitive situation, even a slightly higher affinity of one type of light chain with the heavy chain results in its preferential association with the heavy chain (Grey and Mannik, 1965;Margulies et al., 1976;Klein et al., 1979).
All ,-only hybridomas show only germline : genes. Of course, it cannot be ruled out that some of them have lost a chromosome carrying a rearranged c gene. However, in normal mice, only 60% of the + cells have retained one germline allele and essentially none of the ,+ cells retain a germline c allele (Coleclough et al., 1981). Thus, the , hybridomas from these 2E, mice support the conclusion from the FACS analysis that the X2E transgene causes feedback inhibition of c-gene rearrangement.
On the other hand, only 30% of the double ://,+ hybridomas and none of the c hybridomas show a germline c allele. Presumably, the c cells that have a germline allele had produced a functional c allele by rearrangement before expression of the  dH genes analyzed by digestion with Barn HI and probing with JH 3-4 probe (Manz et al., 1988). G=germline band; R--rearranged band; band detected eDigestion with EcoRI and probing with 5' DH probe (Manz et al., 1988) shows five bands if rearrangement has occurred (=G). DJ rearrangement generally leads to reduction of the number of bands (1B, 3B=1 bands). ---VD rearrangement deletes all bands with this probe. fDigestion with EcoRI and probing with 5' JH (Manz et al., 1988)  transgene led to feedback inhibition of the V(D)J recombinase. Because the c-only and double c/ hybridomas have fewer germline c alleles than c cells from normal mice, the parent B cells must have been selectively enriched, presumably by antigen. The presence of a band in Southern blots of DNA hybridizing with a probe derived from a region 5' of JH1 indicates the presence of an unrearranged heavy-chain allele (Alt et al., 1984). Many of the hybridomas showed evidence of such a germline H allele (Table 2). This was confirmed by the presence of germline bands when the blots were probed with a sequence from 5' of DH ( Table 2). The proportion of hybridomas with germline heavy-chain alleles is similar to that of hybridomas from ,2EH transgenic mice . Germline heavy-chain alleles are very rare in hybridomas from normal mice (Roth et al., 1993;Weaver et al., 1985). In transgenic hybridomas that produce a heavy chain, the presence of a germline H locus presumably indicates that the productive heavy-chain gene rearrangement occurred in a pre-B cell already expressing the light-chain transgene and that the V(D)J recombinase was inactivated before the second heavy-chain allele could begin to rearrange.
Most of the ,-only hybridomas show only germline heavy-chain genes with no evidence of any heavy-chain gene rearrangement. Although many of these may have lost one chromosome 12 carrying the rearranged heavy-chain allele, it seems unlikely that all of them did, particularly, given that all the c + and double-producing hybridomas show at least one rearranegd heavy-chain allele. It appears then that these mice may have some pre-B cells in the spleen and that these cells can be readily fused with myeloma cells. To a lesser degree, a similar finding was made with hybridomas from 2EH mice . It is puzzling that in FACS analysis, many more ,-only B cells were found than + hybridomas that have heavy chains. This has not been further explored.
FACS Analysis of Bone Marrow B Cells Because spleen B cells represent a population of antigen-selected cells, we analyzed bone marrow cells in order to evaluate the distribution of c and , cells that are largely unselected (Table 3). 33.9 to 51% of the total lymphoid cells in the bone marrow were Ig /. The averages were 43.5 +4.5 for normal littermates, 47.2+4.5 (,94) and 34.3 +0.6 (,96) for ,2EK transgenics, and 40.6 + 1.5 for 2EH transgenic mice. 17% of Ig bone marrow B cells of normal littermates express X. This is slightly higher than in spleen and persumably more closely reflects the actual rate of , rearrangements in the bone marrow as described before (Kim et al., 1994).
In the , transgenic mice, an average of 51 to 69% of the bone marrow B cells are ,+ (Table 3). In contrast to the spleen (Table 1), in the bone marrow of transgenic mice, more than 50% of ,+ cells express only. However, the Z, transgenic mice also have a higher percentage of c-only versus c/ B cells. Does this mean that the endogenous c enhancer is active before the transgenic X enhancer? This is unlikely given findings with fetal liver (see what follows). Also in ,2EH transgenic mice, c-only Compared with normal littermates, in , transgenic mice, regardless 6f the type of enhancer present in the transgenes, the strongest" transgeneinduced effect in bone marrow cells is a reduction by over 50% of the number of B220high/l + lymphoid cells (Fig. 1). Thus, both types of transgenes induce feedback inhibition of endogenous c-gene rearrangement, although the inhibition seems stronger with the K2EH transgene (Fig. 1B). However, B220w/c cells are present in very similar numbers in normal and both types of , transgenic mice. The  reduction in :+ cells occurs, therefore, at the transition from B220 w to B220 high B cells.

Kappa and Lambda and mRNAs in Fetal Livers
In order to more directly determine the timing of expression of the transgenes, fetal liver RNAs were analyzed (Fig. 2). Expression of the X2EH transgene is detectable in Northern blots in day 14 fetal livers and reaches a peak at day 15. Expression of the X2EZ transgenes is seen at day 15 and reaches a plateau at day 16 in Northern blots of fetal liver RNA of 96 mice ( Fig. 2A). In Northern blots of RNA from 94 mice, the mature 2 mRNA signal is not seen until day 16, at which time peak levels are present also in this mouse ( Fig. 2A). This expression pattern is confirmed by PCR of fetal liver RNAs from X94 mice (data not shown). Presumably, the overall levels are lower in ,94 compared with X96 due to the lower-transgene copy number. However, the time course seems to be the same in both ,2E, transgenic lines with peak levels being delayed by about 1 day compared with the ,2EH transgenic line. There seems to be a decline of , RNA levels for both ,2E, transgenic lines at day 18, but not for the ,2EH line. Perhaps this is due to limiting amounts of transcription factors shared between : and genes, such as PU.1 (Eisenbeis et  reverse transcriptase are shown on the left, and without reverse transcriptase on the right for the three assays 0c , V, , and C,1) in which the amplified RNA transcripts are of the same size as amplified DNA. There is a background level of CM amplification without reverse transcriptase, but the level with reverse transcriptase well exceeds it. The days of gestation are indicated (17 E, L day 17 early, late). 21 petition may exist between endogenous K and/or gene expression and the expression of the 2EX transgenes. Because this seems not the case for the heavy-chain enhancer, the latter factors may not be limiting in the developing B cells. To compare the timing of expression of endogenous : and , genes, fetal liver RNA from normal mice was analyzed by PCR, because Northern blots were found not to be sensitive enough. No evidence for the expression of rearranged : and genes was obtained at day 14, but the mature forms of both K and , mRNAs were clearly present at day 15 (Fig.   2B). The mRNAs were.determined using V and C primers, which detect all three forms of ,mRNA (V2JC2, V1JC3, and VIJC1; see Materials and Methods). A similar profile of expression was seen with VIJC1 specific primers (Fig. 2B). These data suggest that competency for rearrangement and expression of : and genes arise in parallel.
To further compare the activation of the K and , loci in developing B cells, sterile transcripts from the two loci were analyzed (Fig 2C). Both :0 and C transcripts were seen at day 14. The C transcripts could be either germline or mature. However, because no mature , transcripts were found at day 14, these must be sterile. Sterile C, transcripts have also been seen in rat (Hellman et al., 1985). A spliced :0 transcript was also detectable at day 14. The latter had previously been reported only in Abelson murine leukemia virus transformed pre-B cell lines (Leclercq et al., 1989), but, as shown here, is also present in normal pre-B cells. A sterile V, transcript was not detectable until day 15 (Fig. 2C). Thus, it seems that the appearance of mature , mRNA and presumably rearrangement of genes does not occur until the VX promoter is active. This cannot be tested for : because no consensus sequence exists of the region 3' of V: genes.

DISCUSSION
The 2EK transgenes are expressed tissue specifically, namely, only in B cells. They are thus similar in expression to ,1 transgenes that contained a similar length of sequence upstream of the V, promoter, but included the complete sequences between C,1 and the EX3-1 enhancer (about 35 kb) and additional 10 kb 3' of the enhancer (Eccles et al., 1990). It appears from the findings with the 2E transgenic mice that , genes can be expressed in most, if not all B cells. Between 65% and 74% of spleen B lymphocytes in these mice are ,+. This proportion must be higher, considering that :/ coproducing B cells may often not express the transgenic chain in membrane Ig because of the competitive binding of : with the heavy chain (Grey and Mannik, 1965;Margulies et al., 1976;Klein et al., 1979). This is confirmed by spleen B-cell hybridomas containing the , transgene all of which secrete , light chains (24/24). These results suggest that all B cells are capable of expression. The data do not support a model of a separate : B-cell lineage (Gollahon et al., 1988). The implications for isotypic exclusion of : and genes will be discussed below.
The transgenes containing the , enhancer lead to significant feedback inhibition of :-gene rearrangement, very similar to that observed with the 2EH transgene (Tables 1 and 3 (Ma et al., 1992). A similar conclusion was drawn from studies with cultured pre-B cells (Rolink et al., 1993) and with pre-T cells (Turka et al., 1991). The suggestion of delayed feedback is further supported by the analysis of bone marrow B cells expressing : and B220 (Fig. 1). In transgenic mice with either the or heavy-chain enhancer, the percentages of B2201w/: + cells are about the same as in normal mice. However, the B220high/l + cells are reduced by 50% or more. This may indicate that : rearrangement continues during the B220 lw stage and is only inhibited at the time when B cells 22 LAMBDA TRANSGENIC MICE that have productively rearranged a c gene transit into the B220 high stage. In that way, a large proportion of newly arising B cells would coexpress endogenous c and transgenic X. Such a delayed feedback may explain the production of endogenous light chains replacing anti-self light chains in what has been termed "receptor editing" (Tiegs et al., 1993).
It would also explain the escape of endogenous L chain producing B cells from feedback inhibition by rearranged c or transgenes (Manz et al., 1988;Neuberger et al., 1989;Bogen and Weiss, 1991;Rudin et al., 1991).
The development of expression of Ig genes in fetal liver roughly parallels that of pre-B cells in the bone marrow, but represents a synchronized population of pre-B cells (Strasser et al., 1989). It is clear from the Northern analysis that the expression of the X2EH transgene precedes that of the ,2E, transgene (Fig 2). Expression of the former, being regulated by the heavy-chain enhancer, presumably is simultaneous with t-gene expression in fetal mice (Alt et al., 1981). Endogenous c-andgene expression is seen as sterile transcripts as early as day 14. Furthermore, mature c and , transcripts indicative of gene rearrangement are seen by day 15 of gestation. There is therefore no indication that the expression of c precedes that of ,.
If all B cells have the ability to rearrange both classes of light-chain genes at the same time, why then are less than 20% of the B cells arising in the normal bone marrow expressing a gene? Because the findings in K-knockout mice essentially refute a model that explains c/X isotypic exclusion by making , rearrangement dependent on a signal from the c locus, it appears that the chance of rearrangement for , is simply lower than for c. Stochastic models have been considered a number of times (Coleclough, 1983); the question remains why has a lower probability for rearrangement. The timing of the expression of c and , genes seems to be similar as shown by the findings with fetal liver RNAs (Fig   2). Additionally, pre-B cell lines have been found to express reporter genes under the control of the , enhancer (C. Rudin, B. Kurtz and U.S., unpublished). However, although , appears to be initially expressed at the same time as c, the strength of activation of the c and enhancers may be different. The c gene has two enhancers, whereas, due to spatial constraints (Sto;b et al., 1989;Hagman et al., 1990), each gene may be governed by only one of the two , enhancers. Unlike the X enhancers, the two c enhancers are controlled by different trans-activating factors. Only the c intron enhancer is activated by NFcB. Interestingly, the enhancers have a certain homology with the 3' c enhancer (Pongubala et al., 1992;Eisenbeis et al., 1993). Thus, the enhancers may compete with the 3' c enhancer for the binding of the same transactivating factors and the 3' c enhancer may have a higher binding affinity. To completely unravel the isotypic exclusion of c and , , the activation and relative strengths of the c and , enhancers must be more systematically analyzed once the transactivating proteins are fully understood.
Another possibility to explain the c/X imbalance is that the c and genes represent unequal targets for the V(D)J recombinase (Miller et al., 1982). It has been reported that in an in vitro system using rearrangement constructs containing the c and rearrangement signal sequences (RSS), the frequency of c rearrangements is higher (Ramsden and Wu, 1991). However, because rearrangement via the X RSSs in this report was unphysiologically low (two to three orders of magnitude lower than that via the c RSSs), this claim must be evaluated by additional experiments.
An important difference between the two loci are the numbers of RSSs in V and J genes that can serve as targets for rearrangement. The c locus has at least 50 V genes and 4 functional J genes (Tonegawa, 1983). The locus has only three V genes (V1, V2, and Vx) and three functional J genes (J1, J2, and J3) (Miller et al., 1988;Carson and Wu, 1989;. Thus, if the levels of V(D)J recombinase in pre-B cells are limiting, c genes will have a much greater chance for encounter with recombinase and therefore rearrangement. Recent data with transgenic c-rearrangement substrates have ruled out a mechanism in which the V(D)J recombinase would bind to a single RSS as a holo-enzyme complex with binding sites for both the 12-spacer RSS and the 23-spacer RSS and track to the nearest complementary RSS (Engler et al., 1993). The data are most compatible with binding and random encounter of at least two independent subunits, one with specificity for a 12-spacer RSS and the other for a 23-spacer RSS in a situation where the recombinase is present in a limiting quantity. Nonsaturating levels of the recombinase, at least at the stage of L-gene rearrangement, are also supported by the finding that about 60% of c B cells have one c allele in germline conformation (Coleclough et al., 1981). In this way, the problem of unequal levels of c and , expression would be reduced to unequal 23 numbers of recombinase targets. The suggestion of limiting recombinase levels will have to be tested.
Finally, the possibility of and c locus-specific rearrangement coactivators (or silencers; Lauster et al., 1993) that may exist in unequal concentrations must be considered in future experiments.

X2 Transgenes
Construction of the 2 transgene containing the H-chain enhancer (,2EH) and the transgenic line carrying this transgene (2-1275) have been described . A ,2 transgene driven by the E2-4 enhancer was constructed as follows ( Fig. 3): The 1.6-kb Xhol-Sall fragment containing the ,2 enhancer was isolated from pA16-N16 (Hagman et al., 1990). This A16XS1.6 fragment was then subcloned into the Sall site of X2-4.4X, which contains the functionary rearranged ,2 gene of the plamacytoma MOPC 315 (Wu et al., 1982;Hagman, 1989;Hagman et al., 1989). The resulting plasmid, 2EK, was digested with Sall and Pvul to remove most of the vector, the insert was isolated from Sea Plaque (FMC Corp., Rockland, ME) lowmelting agarose gels, and microinjected into the pronucleus of C57BL/6J zygotes. Transgenic Mice Two X2EX transgenic lines were chosen for analysis. One line, 2E-94, has 8-10 copies of the transgene, and the other line, 2E-96, has approximately 50 copies. Hybridomas 2E-94 mice were injected with 20 tg of LPS intraperitoneally 3 days prior to fusion. Hybridomas were generated as previously described (Manz et al., 1988). Supernatants were screened for Ig L chains in an ELISA assay, as described in detail elsewhere . Briefly, for kappa production, goat anti-mouse (GAM)Ig (1:400; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was the solid-phase antibody followed by peroxidase-conjugated GAMK (1:1000; Southern Biotechnology Assoc., Birmingham, AL). Lambda L-chains were detected using a combination of GAMX and peroxidase-conjugated GAM (1/200 and 1/1000, respectively; Southern Biotechnology Assoc.). In both assays, the TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories) was used as the substrate. The reaction was stopped with 1 M phosphoric acid and the absorbance was read at 450 nm.
Nucleic Acid Procedures and Probes DNA isolated from hybridomas or fetal tissue was phenol/chloroform extracted using standard techniques. Thirty micrograms of hybridoma DNA was digested with Barn HI or Eco RI (New England BioLabs, Beverly, MA), electrophoresed and blotted to Gene Screen Plus (New England Nuclear, Boston). Hydridization and reprobing procedures have been described (Manz et al., 1988).
RNA was isolated from the fetal liver of individual embryos at 14 to 18 days gestation according to the method of Chomczynski and Sacchi (1987). Body DNA was also isolated to detemine which embryos were transgenic.

RNA Analysis by Northern Blots
Twenty micrograms of fetal liver RNA or 5 tg of normal spleen RNA, as control, were analyzed by Northern blot hybridization as described . 2 mRNA was detected by a 32p_ labeled C2 probe . Under the same condition, there was no detectable signal of endogenous ,2/3 transcripts from nontransgenic fetal liver RNA (not shown). The blots were reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Fort et al., 1985). 24 LAMBDA TRANSGENIC MICE
The reaction was carried out at 42C for 2 hours.
After heat denaturation at 65 C for 15 min, 30 1 of TE was added to the reaction mixture. Ten microliters of the first-strand cDNA was amplified for mature c or , transcripts, and 1 tl of the cDNA for the control transcript (ribosomal protein-17 mRNA) using the following primers: Vc degenerate primer and Cc3 primer for c transcripts (product size of 695 bp), V,I and C2 primers for , transcripts (218 bp), V,I and C13 for ,1 transcripts (638 bp), RP17 primers for control transcripts (170 bp). For the sterile transcripts that do not span introns, total RNA was treated with RNase-free DNase (Promega). Five micrograms of DNase-treated RNA was used for the the cDNA synthesis as described earlier, with and without reverse transcriptase in parallel to make sure that the amplification occurs from the cDNA templates. The following primer pairs were used: Ko and Jc2 primers for the sterile c transcripts (product size of 500 bp), Ko and Cc3 for the spliced c transcripts (681 bp), VI and 3VM primers for the V serile transcripts (262 bp), CM5 and CM3 for C1 transcripts (466 bp).
The PCR reaction mixture contained Taq buffer (50 mM KC1, 10mM Tris, pH 8.3, 1.5 mM MgCI2, 0.01% (w/v) gelatin), 0.2 mM dNTPs, 1 U Taq polymerase, 25 pmoles of primers, and cDNA template in a volume of 25 1. Amplification was performed using a Perkin Elmer Cetus GenesAmp PCR System 9600 programmed for 30 cycles (28 cycles for the control transcript within the linear range of amplification) of thermal denaturation (at 95 C for 30 s), primer annealing (at 65 C for 30 s), and template extension (at 72C for 30 s). Ten microliters of the amplified products were analyzed by Southern blot hybridization using specific probes: C probe for mature c transcripts, Jc2 probe (Lewis et al., 1982) for sterile c transcripts, VM probe for mature and sterile V, transcripts, and C1 probe for C,1 transcripts.