In
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`EP 0 314161 A1
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`it was difficult to determine minimum size restriction fragments necessary to contain promoter and
`genes,
`L-V sequences. The rearranged k genes were therefore cloned as both EcoR l and Hind Ill fragments.
`Briefly. bacteriophage libraries were made from Hind III and from EcoFi I digested and size fractionated
`genomic DNA from LCL 901. The libraries were screened with the 0x3 probe, prepared as described in the
`following section. The variable region genes were then subcloned into pUC18 to facilitate mapping of
`restriction sites and identification of duplicate clones. The rearranged k genes were then inserted into the
`pUCG-MCSll-MHE cassette, described above, to form expressible lambda light chain genes. The details are
`set forth below.
`
`An oligonucleotide probe was used to clone a germline lambda constant region gene, and a portion of
`this clone was used as a probe for detecting rearranged lambda genes in human lymphoblastoid cell lines.
`Briefly, a human germiine DNA library containing Hind lll fragments of approximately 9 kb was prepared. A
`synthetic oligonucleotide probe homologous to a region of identity among Ck 1-3 was prepared and used to
`screen the library. Hybridization and washing conditions were utilized such that only a few plaques
`hybridized. These plaques were purified and the DNA was prepared and analyzed to determine whether any
`of them corresponded to the published map of the lambda locus. Clone HP2-Cx 5 corresponded to the Hind
`lll fragment containing the Ck 3 and 4 genes. An EcoFi I-Hlnd lll fragment containing me was subcloned
`into pUC18 and a 0.8 kb EcoFi l-Bgl
`|l fragment derived from it, referred to as the 0x3 probe, was used to
`screen for rearranged lambda genes in a library derived from cell line 901.
`The details are as follows: Human placenta was used as a source of germline DNA. Genomic DNA was
`prepared as described above. According to Hieter et al.,1981,s__upra, the human k 1 and x 2 genes are
`found on an 8.8 kb Hind lll fragment and the human A 3 and x 4 genes are found on a contiguous 9.2 kb
`Hind I" fragment. Therefore, to prepare a library containing the lambda genes human placental DNA was
`digested to completion with Hind lll, phenol and chloroform extracted, ethanol precipitated and resuspended
`in TE buffer. Fifty micrograms of the digested DNA was fractionated on a 10-40% sucrose gradient as
`described above and fractions containing DNA of 8.2 to 11.5 kb were each ligated with XL47.1-Hind Ill
`treated phage arms (prepared as described above; approximately 5% of each fraction was ligated with 100
`ng of phage arms).
`.
`To screen the bacteriophage library, a 26 base oligonucleotide of
`the following sequence was
`synthesized on an Applied Biosystems 380A DNA Synthesizer. 5 GCTGC CAGGT CACGC ATGAA
`GGGAG C 3. This sequence corresponds precisely to a region of identical nucleotide sequence among CR
`1
`through 3 (nucleotides 251-276, according to the numbering of Hieter et al., i__bid). The lambda probe was
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`radiolabeled with 32FuyATP by a T4 DNA kinase reaction (Maniatis et al.. supra, p.—125) to a specific activity
`of approximately 108 cpm/LLg.
`The library was plated and phage DNA lifted onto nitrocellulose paper (in duplicate) as described by
`Maniatis et al., supra, p. 320. The filters were prehybridized at 37° C for 4 hours in a solution containing 8X
`SSC, 5X Denhardt's solution, 0.15% pyrophosphate, and 0.1 mg/ml tRNA. To decrease the non-specific
`binding of the probe to nitrocellulose. the probe was prehybridized in the above solution to a piece of
`nitrocellulose. The filters containing the phage were then hybridized with the "prehybridized" probe at 106
`cpm/ml overnight at 37°C. Following hybridization. the fluid was stored at -20° C and used again for the
`next round of hybridization. The filters were washed under progressively more stringent conditions until only
`a few positive plaques remained. Washing one of the sets of filters in 0.25X SSC at 45 C produced only
`two positive hybridization signals. Both of these signals. in addition to many more were also present in the
`duplicate set of filters which had been washed in 1X SSC at 45° C.
`A single plaque (HPZ-st) was picked, diluted and plated out again. Filters were prehybridized as
`described above. and then hybridized in the fluid remaining from the first
`library screen. Filters were’
`washed in 1X SSC at 450. Many dark hybridization signals were seen against a background of light
`signals, suggesting that there had been some enrichment for the positive plaque and that most of the
`hybridization seen after washing in 1X SSC at 45 C was non-specific. Two of the plaques hybridizing most
`strongly were picked and replated This process was continued until the plaques were pure.
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`Phage DNA was then prepared as described in Maniatis et al., supra. p. 77. Hind Ill and EcoR l digests
`of the phage DNA gave fragment sizes suggesting that clone HP2-Cx5 contained the human 0x3 and 0x4
`genes: the total Hind I" insert size was 95-10 kb, including two 4.5 kb EcoR I-Hind l|| fragments and a 1 kb
`EcoFi I fragment. The oligonucleotide probe hybridized with the 4.5 kb EcoR l-Hind lll bands.
`The 10 kb Hind lll insert from clone HP2-Cx5 was subcloned into pUCtB to form prS/4. The 4.5 kb
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`35
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`10
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`75
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`20
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`25
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`3D
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`35
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`40
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`45
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`50
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`55
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