Construction and Screening of a Genomic Library Specific for Mouse

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Proc. Nati. Acad. Sci. USA Vol. 86, pp. 8482-8486, November 1989 Genetics Construction and screening of a genomic library specific for mouse chromosome 16 (somatic cell hybrids/repetitive DNA/chromosome-specific libraries/cDNA screening/chromosome map) UTE HOCHGESCHWENDER*, J. GREGOR SUTCLIFFE, AND MILES B. BRENNAN*t Department of Molecular Biology, MB 10, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by George R. Stark, August 9, 1989

ABSTRACT We have established a protocol for producing source of anonymous RFLP markers, the library could be libraries of specific mouse chromosomes. The mouse DNA- used directly to screen for candidate clones ofspecific genes. containing clones from a genomic library of a hamster-mouse We applied this general strategy in assembling a repre- somatic cell hybrid containing only one mouse chromosome are sentative genomic library of mouse chromosome 16 consis- identified by screening with radiolabeled mouse repetitive ting of 14,200 individual clones, equal to about two "chro- sequences after specifically blocking hamster repetitive se- mosome equivalents." Each clone occupies an individual quences; 95% of the mouse DNA-containing clones are iden- well in a microtiter tray, allowing repeated plating and tified. We have applied this protocol in producing a library of hybridization analysis of the library. mouse chromosome 16, consisting of 14,200 clones or two To exploit fully this or any other chromosome-specific "chromosome equivalents." Each clone occupies an individual library, it is necessary to screen the library with cDNA well in a microtiter tray, allowing the entire library to be probes to identify clones containing expressed sequences. repeatedly and reproducibly plated and analyzed by hybrid- However, cDNA probes contain sequences that are highly ization. Further, we have established a protocol for making repeated in the genome, and the hybridization of these cDNA probes specifically depleted of highly repetitive se- sequences to their cognates in a large fraction of genomic quences for probing libraries of genomic clones. By screening clones obscures the hybridization signal from the high- the chromosome 16 library with cDNA probes from mouse liver complexity fraction of the cDNA. In screenings of the and brain, we demonstrate the feasibility of identifying ex- chromosome 16-specific library with cDNA probes of adult pressed sequences and characterizing their patterns of expres- brain and adult liver, we demonstrate the feasibility of sion. Such chromosome-specific libraries can facilitate the specifically depleting cDNA probes of sequences highly isolation of dermed genetic loci as well as form the basis for the repeated in the genome and of correctly identifying the production of integrated transcriptional, genetic, and physical expression patterns of sequences in genomic clones. maps of entire chromosomes. MATERIALS AND METHODS Two major reasons for obtaining a complete collection of Cells. Baby hamster kidney (BHK) and Chinese hamster genomic clones for a single mammalian chromosome are the ovary (CHO) cells, NIH 3T3 mouse cells, and 9-6Az2 ham- analysis ofthe arrangement ofgenes on the chromosome and ster-mouse hybrid cells (5) were grown in Dulbecco's mod- the isolation of specific genetic loci mapped to the particular ified Eagle's medium with 10% (vol/vol) fetal bovine serum chromosome. With these goals in mind, we devised protocols (BHK, CHO, 9-6Az2) or bovine serum (NIH 3T3). The for producing mouse chromosome-specific libraries and for 9-6Az2 line contains only mouse chromosome 16. The pres- screening these libraries for transcribed sequences. While ence ofmouse chromosome 16 in our cultures was verified by human chromosome-specific libraries can be made from the probing a Southern blot of restriction endonuclease-digested DNA of flow-sorted chromosomes, the similarity in DNA DNA from mouse, hamster, and the hybrid cell line with the content, base composition, and centric heterochromatin of plasmid pMx34 (6)-a probe for the Mx gene known to lie on mouse chromosomes has thus far prevented their purification chromosome 16 (7). The hybrid line exhibited the mouse by this method (1, 2), although it may be possible to make use hybridization pattern superimposed upon the hamster pat- of Robertsonian translocation strains to purify fused chro- tern, thus proving that our hybrid contained mouse chromo- mosomes. The strategy for producing mouse chromosome- some 16 (data not shown). The intensity of hybridization to specific libraries is to screen genomic libraries from somatic the mouse band in the hybrid sample was submolar, consis- cell hybrids, which contain only a single chromosome from tent with the known mitotic instability of mouse chromo- one of the parent species, with repetitive DNA from that somes in hybrid lines. species. A similar approach has been used to identify human Purification of Nucleic Acids. High molecular weight DNA and mouse chromosome-specific restriction fragment length from tissue culture cells was isolated as described by Brison polymorphism (RFLP) markers from libraries of hamster- et al. (8). DNA from mice was prepared by removing the human and hamster-mouse somatic cell hybrids (3, 4). How- brain, washing it briefly in phosphate-buffered saline before ever, to extend this approach to the production of repre- homogenizing it gently in 10 mM Tris/1 mM EDTA, pH 7.5 sentative mouse chromosome-specific libraries, it is neces- (TE buffer) in a loose pestle glass homogenizer. The homog- sary that the vast majority, rather than only a fraction (4), of enate was lysed by addition of NaDodSO4 to 1% and then the mouse DNA-containing clones be identified. Such a further processed like DNA from tissue culture cells. When quantitative increase in the representation in the library the DNA was to be sheared, it was sonicated extensively at would allow a qualitative difference in its use: rather than a Abbreviation: RFLP, restriction fragment length polymorphism. *Present address: Brookdale Center for Molecular Biology, The The publication costs of this article were defrayed in part by page charge Mount Sinai School of Medicine, One Gustave L. Levy Place, New payment. This article must therefore be hereby marked "advertisement" York, NY 10029. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8482 Downloaded by guest on September 29, 2021 Genetics: Hochgeschwender et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8483 high power until the size of the DNA fragments was about 500 (9) (pH 7.0) at room temperature, heated to 680C in a large nucleotides as shown by electrophoresis in an agarose gel. volume of 99% formamide (16), washed several times with Phage DNA and plasmid DNA preparations were done TE buffer, and stored at 40C until next use. following standard procedures (9). Screening Somatic Cell Hybrid Libraries. Phage lifts were Total cellular RNA was prepared from fresh mouse tissues prepared by using Biotrans membranes (ICN) according to by the procedure of Chirgwin et al. (10). Enrichment for the supplier's recommendations. No more than five filters poly(A)+ RNA was performed by the procedure of Aviv and were hybridized in one plastic bag. For screening the somatic Leder (11). cell hybrid libraries, filters were first prehybridized at 680C Construction of Libraries. Phage libraries of genomic DNA overnight in the general hybridization solution [6x SSC were constructed by following standard procedures (9). DNA containing 0.5% NaDodSO4, 0.2% sodium pyrophosphate, from BHK cells and NIH 3T3 cells was digested to comple- Sx Denhardt's solution (9), and 10 mM EDTA; lx SSC = tion with BamHI and ligated into phage AEMBL3 (12). DNA 0.15 M NaCI/0.015 M sodium citrate, pH 7] supplemented from 9-6Az2 cells was digested partially with the enzyme with salmon sperm carrier DNA to a final concentration of Sau3AI, and the 9- to 23-kilobase (kb) fragments were 100 gg/ml. This first prehybridization was followed by a isolated on DE-81 paper (Whatman) and ligated into phage second overnight prehybridization in which the general hy- ADASH (Stratagene). DNA from NIH 3T3 cells was digested bridization solution was supplemented with sheared hamster to completion with EcoRI and ligated into phage AZAP DNA (to a final concentration of 100 in addition to the (Stratagene). Ligated DNAs were packaged in vitro (Giga- ,ug/ml) pack; Stratagene) and plated on strain LE 392/P2 (AEMBL3 salmon sperm carrier DNA. This second prehybridization and ADASH libraries) or BB4 (ref. 13; AZAP library). Li- step was followed by an overnight hybridization step, for braries were plated and screened as primary libraries, except which the general hybridization solution was supplemented for the AEMBL 3-NIH 3T3 and -BHK libraries, which were with 100 ,ug ofsalmon sperm carrier DNA and ofyeast carrier amplified. Phage clones from the AZAP-NIH 3T3 library were RNA per ml and with 32P-labeled mouse genomic DNA at 1 converted into plasmid clones by the supplier's automatic x 107 cpm/ml. Filters were first washed at room temperature excision protocol. in 2x SSC/0.1% NaDodSO4, followed by two stringent Preparation of Radioactive Probes. High molecular weight washes in 0.5x SSC/0.1% NaDodSO4 at 680C. genomic DNA was labeled with 32P by nick translation (14). Positive plaques were picked into individual wells of mi- DNAs of phage and plasmid clones were labeled by random crotiter plates containing 0.1 ml of SM medium (9). Initially, hexamer oligonucleotide priming (15). Radioactive cDNA the library was stored at 40C. Later, dimethyl sulfoxide was probes were constructed by first synthesizing unlabeled added to 7% (vol/vol), and the plates were stored at -70°C cDNA from 2 ,ug of poly(A)+ RNA by using an oligo(dT) sealed in plastic bags. For plating, trays were thawed, primer (9). The RNA template was removed by treating for aliquots were plated, and trays were refrozen and stored at 20 min at 68°C with 0.1 M sodium hydroxide, the cDNA was -70°C. Several cycles of freezing/thawing have not dimin- precipitated twice with ethanol and then used as template for ished phage titers noticeably. a labeling reaction using random hexamer oligonucleotide Screening the Chromosome 16 Library. Filters were pre- priming. For all labeling reactions [a-32P]dATP (3000 Ci/ hybridized and hybridized in the general hybridization solu- mmol; 1 Ci = 37 GBq) was used. tion supplemented with 100 ,ug of salmon sperm carrier DNA Removal of Highly Repeated Sequences from DNA Probes. and of yeast carrier RNA per ml. Radioactive probe was High molecular weight denatured DNA from mice was bound added to 1 x 107 cpm/ml. When probes that were depleted to diazonium cellulose as described by Goldberg et al. (16) of repetitive elements by hybridization with DNA-cellulose and Brison et al. (8). Diazotization of m-aminobenzyloxy- were used, the filter hybridization was carried out in the methyl cellulose (ICN) was carried out by following the presence of 50% formamide at 42°C. Filters were washed in protocol of Goldberg et al. (16) except that pure sulfuric acid 2x SSC/0.1% NaDodSO4 at 68°C. was used to reprecipitate the cellulose. At the end of the diazotization reaction, excess HNO2 was destroyed by add- ing solid urea (17). The buffer for washing the cellulose was RESULTS 250 mM sodium acetate (pH 4.0). Construction of Chromosome-Specific Libraries. In our For coupling, genomic DNA (1 mg in 0.9 ml of H20) was approach to making chromosome-specific libraries, genomic boiled for 2 min, and 0.1 ml of 2.5 M sodium acetate (pH 4.0) libraries are constructed from DNA of hamster-mouse so- was added. The DNA was diluted with 4 volumes ofdimethyl matic cell hybrids that contain only a single mouse chromo- sulfoxide, added to freshly prepared pelleted diazonium some. The mouse DNA-containing clones are identified by cellulose (100 mg/mg of DNA), and the mixture was incu- hybridizing plaque lifts with highly repetitive mouse se- bated for 20 hr at room temperature (8) while continuously quences. To establish conditions under which mouse highly agitated on a rocking platform. The DNA-cellulose was repetitive sequences would hybridize with most mouse ge- washed as described by Goldberg et al. (ref. 16; except that nomic clones, we prepared plates with 100 mouse clones from TE buffer was used in the final washing step) and stored in TE the NIH 3T3 library. Filters lifted from these plates were buffer at 4°C. hybridized with 32P-labeled mouse (NIH 3T3) genomic DNA Hybridization of labeled DNA probes with DNA-cellulose at 68°C for 18 hr and then washed over a range of stringencies was performed as described by Brison et al. (8). After (i.e.,0.lx SSCat730C,0.lx SSCat680C,0.5x SSCat680C, prehybridization of the DNA-cellulose, 32P-labeled dena- 2x SSC at 68°C). We used genomic mouse DNA as a source tured probe was added to a 500-fold excess of immobilized of highly repeated elements rather than cloned examples of DNA in a hybridization volume adjusted to 125 ,ug of immo- such elements to insure the widest representation ofdifferent bilized DNA per ml. The mixture was heated to 80°C for 2 highly repetitive elements in the probe. At a washing strin- min, cooled to 37°C, and incubated for 48 hr with constant gency of 0.5x SSC at 68°C, most of the mouse clones were agitation on a rocking platform. Every 12 hr the DNA- still identified, whereas at higher stringencies, the signal was cellulose was pelleted by centrifugation, and the probe in the diminished. To determine the efficiency of isolating mouse supernatant solution was melted by heating for 10 min at clones with these conditions, we labeled mouse (NIH 3T3) 80'C, cooled to 370C, and then added back to the same genomic DNA and A phage vector (EMBL3) DNA by nick- DNA-cellulose for continued incubation at 370C. To recover translation and used these in parallel to probe duplicate lifts the DNA-cellulose for reuse, it was washed twice in 2 x SSPE from plates of the NIH 3T3 library (Fig. LA). Comparison of Downloaded by guest on September 29, 2021 8484 Genetics: Hochgeschwender et al. Proc. Nati. Acad. Sci. USA 86 (1989)

A a:'* b .C 8..xA clones could be quantitatively identified from the somatic cell hybrid library (f in Fig. 1B). The optimal screening density is low enough that the

F . *. . mouse DNA-containing clones can be identified and picked D. * i relatively uncontaminated by surrounding hamster clones, while the total number of plates is kept within a reasonable limit. A concentration of 1000 clones per 10-cm diameter plate (or 3000 clones per 15-cm diameter plate) was established empirically as the upper limit to allow the isolation of relatively pure mouse plaques with precision and complete- ness. Production of a Murine Chromosome 16 Library. We applied the general strategy for construction of chromosome- specific libraries to mouse chromosome 16. Genomic libraries from DNA of the chromosome 16-only hybrid cell line Films* * ar* ea a i 9-6Az2 were prepared and screened for mouse DNA- Filcontaining clones according to the protocol described above. * . conFilters with mouse clones alone and hamster clones alone were included as controls in all steps. Exposure time was adjusted to be short enough so that the filter with hamster clones alone gave no background signal and long enough so w .i,e. that the filter with mouse clones alone showed signals for >95% of clones FIG. 1. Identifying mouse DNA-containing ci[ones among ham- plated. ster DNA-containingoftetoflerssosta2s clones. (A) Duplicate liftsoffrom a plate of the In total, 375 plates were screened. Libraries were prepared NIHplaque3T3 library were hybridized with NIH 3T3 DINA (a) or A phage as needed during the plating process. From a total number of DNA (b). (c) Films are overlaid at an offset to faciilitate comparison. 1 x 106 clones plated, 14,200 mouse DNA-containing clones (B) Duplicate lifts from plates of the NIH 3T3 libirary (a and d), the were identified and picked into microtiter wells; 71 wells per BHK library (b and e), and the 9-6Az2 librarf y (c and f) were microtiter plate were used; therefore, the collection, called prehybridized with carrier DNA alone (a-c) or wit) 100 ig ofsheared hereafter the chromosome 16 library, is contained in 200 hamster DNA per ml in addition (d-f) before iybridization with microtiter plates. The chromosome 16 library is plated by 32P-labeled genomic mouse DNA. All films were exposed for 36 hr transferring a small amount of liquid from each well to a with intensifying screens. bacterial lawn by means ofa multiprong device. When plated, plaques of the two filters shows that 128 of 1.30 of the clones the contents of four microtiter plates are placed on a single are identified by the mouse DNA probe. agar plate by offsetting the multiprong device. Therefore, the complete library can be represented on 50 filters. Next, we defined conditions under whice labeled mouse Mouse chromosome 16 has a length of approximately 64 DNA does not hybridize to hamster DNA-cc)ntainingntaining clones. centimorgans or 1.2 x 108 base pairs (bp). Since the average It was necessary to avoid any backgrourid signals from insert size is -17 kb, 14,000 clones equal 2.4 x 108 bp or two hamster DNA-containing clones because we planned to iden- "chromosome equivalents." The discrepancy in the number tify a small number of mouse clones among a large number of of mouse DNA-containing clones expected from the four hamster clones and because the signal fromIl various mouse genome-equivalent somatic cell hybrid library and the two DNA-containing clones ranged considerably in intensity (see chromosome equivalents isolated here is a reflection of the Fig. 1A). Accordingly, we prepared plates wiith 1000 hamster mitotic instability of chromosome 16 in the somatic cell clones from the BHK library. Filters lifted firom these plates hybrids (see Materials and Methods). were prehybridized with either carrier DNAA alone or with To check the representation ofthe chromosome 16 library, various amounts of sheared BHK DNA (in a range of 20-200 the library was initially screened with a probe known to lbg/ml) before hybridization with 32P-labeled genomic mouse identify a chromosome 16 gene. Screening with a cDNA DNA by using the conditions established for Fig. lA. We clone representing the full-length mRNA of one ofthe mouse found that a prehybridization step with 10(org of sheared Mx genes identified four strongly hybridizing clones and hamster DNA per ml lowers the backgrounDd from hamster several more weakly hybridizing clones. The clones were DNA-containing clones to negligible levels (Frig. 1B, compare shown to contain the mouse Mx gene by Southern blots to b and e). To insure that the hamster DNA irehybridization restriction digests of the clone DNA (data not shown). Since step did not interfere with the signal from lhybridization of the mouse Mx gene is represented in the haploid genome by two copies per chromosome 16 (18), the result is within the labeled mouse genomic DNA to mouse Ihybridizataining)NA-containing expected range for a library consisting of two "chromosome clones, we made duplicate lifts from plate s of 300 mouse euvlns clones from the NIH 3T3 library, prehybMridized with or To insure that the clones in the chromosome 16 library are without sheared hamster DNA and then lhybridized with derived from mouse chromosome 16, five random clones labeled mouse genomic DNA. We did not det:ect a substantial were used to probe Southern blots containing DNA of mouse decrease in signal from mouse DNA-containiing clones after (C57BL/6), hamster (cell line E36), and two mouse-hamster prehybridization with sheared hamster genc)mic DNA (Fig. somatic cell hybrids, which contain a common set of mouse 1B, compare a and d). chromosomes (chromosomes 2, 7, 12, 15, and 17) but differ Finally, duplicate lifts of the hamster-mou se chromosome in one other chromosome: chromosome 1 for cell line HM57 16 library (see below) were probed with labe]led mouse DNA and chromosome 16 for cell line HM96 (Christine Kozak, after prehybridization with or without shearesd hamster DNA. personal communication). Each clone exhibited a hybridiza- In the absence of prehybridization with slheared hamster tion pattern consistent only with a location on chromosome DNA, the background signal from the Ihamster DNA- 16; two examples of these are shown in Fig 2. containing clones precludes identification of the mouse Screening of Chromosome-Specific Libraries with cDNA DNA-containing clones (c in Fig. 1B). But,,after prehybrid- Probes. The use of cDNA probes to screen genomic libraries ization with hamster DNA, the mouse I)NA-containing is not widespread, owing in part to the difficulties of highly Downloaded by guest on September 29, 2021 Genetics: Hochgeschwender et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8485 A B While treatment by this method depletes cDNA probes 1 2 3 4 1 2 3 4 from central nervous system tissue of most classes of repetitive elements, there are some classes of repetitive elements that are present at relatively higher levels in the mRNA population than in the genome. Because of their relative underrepresentation in genomic DNA, these particular repetitive elements are not sufficiently eliminated from the cDNA probe by hybridization with immobilized high molecular weight genomic DNA. Therefore, we "spiked" the DNA-cellulose with DNA clones carrying these classes of repetitive elements. We constructed a library with EcoRI- digested NIH 3T3 DNA in the A phage vector ZAP. This library was plated, and 355 single clones were immediately picked into microtiter arrays. Filters lifted from this collection of clones were hybridized with a cDNA probe from mouse cerebellum depleted of highly repetitive elements by prehybridization with DNA-cellulose. Of 355 clones, 30 gave a signal and were therefore selected as clones probably carrying repetitive sequences relatively overrepresented in central nervous system mRNA compared with their distri- bution in the genome. These phage clones were converted into plasmids, the individual plasmid cultures were pooled, and plasmid DNA was prepared from this pool. The DNA FIG. 2. Chromosomal assignment of clones from the chromo- was cut with Xho I to linearize the plasmids and coupled to some 16 library. Southern blots were prepared (9) with EcoRI- diazotized cellulose. When a mixture of genomic mouse digested (A) or HindIlI-digested (B) DNA (10 Ag per lane) from DNA-cellulose and plasmid DNA-cellulose was used for C57BL/6 mice (lane 1), HM96 cells (lane 2), E36 cells (lane 3), and prehybridization with cDNA probes, depletion of highly HM 57 cells (lane 4) and were hybridized with chromosome 16 clones repetitive elements was complete, leaving no background of 2368 (A) and 55E8 (B). To avoid hybridization with highly repetitive false positive signals from hybridization with repetitive ele- sequences, prehybridization of blots was followed by hybridization ments. The signals from unique sequences were not dimin- with 100 p~g of sheared mouse genomic DNA per ml before hybridization with radioactive probes. Blots were washed in 0.2x SSC/ ished by this procedure (Fig. 3). 0.1% NaDodSO4 at 680C and exposed for 2 weeks with intensifying Screening the Chromosome 16 Library for Sequences Ex- screens. DNA size standards are (from top to bottom) 23, 9.4, 6.5, pressed in Brain and Liver. To identify genomic clones 4.3, 2.3, 2.0, and 0.5 kb. carrying sequences expressed in different tissues, the entire chromosome 16 library was screened with cDNA probes repetitive elements present both in genomic sequences and in derived from mRNA of adult mouse brain and liver. Reverse- mRNAs. The concentration of RNA transcripts from highly transcribed cDNAs were radioactively labeled by random repetitive elements is especially high in the central nervous hexamer priming and the labeled probes were depleted of system. In cDNA probes produced by reverse transcription repetitive sequences as described above. These screenings of oligo(dT)-primed RNA, there is a large amount of material identified 208 clones that hybridized with the brain probe and that hybridizes to highly repetitive elements. This material presumably has three sources: priming of polymerase III ~b < ,, transcripts from internal stretches, the highly repetitive A, t elements in the 3' and 5' untranslated v. o #' regions of polymerase * * .. II mRNA transcripts, and the introns of nuclear RNA. The

problem of screening genomic libraries with cDNA probes *.* ****o -* 4*W 'S*.'* .. * .4 ' can be overcome by depleting the probes of highly repetitive *1 4¶9*i** t * f sequences by prehybridization with a large excess of such sequences. Our strategy to remove these elements was .b *~ adopted from Brison et al. (8), who developed the method to * . **t ; deplete genomic DNA probes of repetitive elements. Dena- tured high molecular weight mouse DNA is covalently attached to diazotized cellulose, and the labeled probe is kc. d hybridized to this cellulose-bound DNA for 48 hr. Although exact calculations are difficult, we estimate that this represents a Cot value of --50. The probe is labeled by random hexamer priming of unlabeled first-strand cDNA, rather than by incorporation of radioactive nucleotides during the reverse transcription step, to avoid selective loss of cDNA populations containing repetitive elements in their 3' untranslated regions. The DNA-cellulose with the attached repetitive elements from the probe is pelleted, and the supernatant cDNA, depleted of highly repetitive elements, is then used as FIG. 3. Screening of the chromosome 16 library. Duplicate lifts probe. (Attempts to remove highly repetitive sequences from a plate with 284 clones of the chromosome 16 library were with excess mouse DNA hybridized with cDNA probes prepared with RNA from adult mouse during hybridization by competition brain (a and c) or liver (b and d). The cDNA probes were either or prior to hybridization by annealing to an excess of mouse untreated (a and b) or depleted of highly repetitive elements by DNA and using the single-stranded fraction after passing over prehybridization with DNA-cellulose (c and d). All films were a hydroxyapatite column gave poor results.) exposed for 6 days with intensifying screens. Downloaded by guest on September 29, 2021 8486 Genetics: Hochgeschwender et al. Proc. Natl. Acad. Sci. USA 86 (1989) A B liminary characterization indicates that clones of the Mx at This B L B L B L 1 1 2 3 genes are present the expected frequency. approach 23 allows the production of representative libraries specific for other mouse chromosomes. To maximize the usefulness of

~~~~InH chromosome-specific libraries, it is necessary to screen these

... for expressed sequences. Our protocol for making cDNA -i-;L. W ..-.. ace. probes depleted of sequences highly repeated in the genome makes this possible. To permit information from such screen- __ h __ ings to be integrated, our library was constituted as a per- a.:i manent collection ofclones, each with a unique address, and ...... :. SL the whole available for repeated and .. w# plating hybridization *::: ...... analysis. an. There are few direct studies that compare patterns of gene ...... t expression in different mammalian tissues. RNA complexity ...... -. studies (20-23) suggest that between 10,000 and 100,000 .. genes are expressed in the rodent brain. The detected overlap a b in expressed mRNA between brain and other tissues has ranged from 90% to 35% (20, 21, 23). At the level ofdetection FIG. 4...... Northern X and Southern blot analyses of chromosome 16 of our about 84% of the on chromo- screenings, sequences clones. (A) Northern blots were prepared (19) with poly(A)+ RNA (2 some 16 expressed in brain are also expressed in liver. Such tug per lane) of adult mouse brain (lanes B) and liver (lanes L) and hybridized with chromosome 16 clones 1%H4 (lanes a), 195F5 (lanes analyses of collections of genomic clones, representative of b), and 165B9 (lanes c). Blots were washed in 2x SSC/0.1% single chromosomes or, ultimately, the entire genome, with NaDodSO4 at 680C. RNA size standards are (from top to bottom) 9.5, cDNA probes made from a variety of tissues at different 7.5, 4.4, 2.4, 1.3, and 0.24 kb. (B) Southern blots were prepared (9) developmental stages will provide specific information about with Hindi.1-digested DNA (10 tLg per lane) from NIH 3T3 cells gene expression unavailable at present by any other means. (lanes 1), 9-6Az2 cells (lanes 2), and CHO cells (lanes 3) and hybridized with chromosome 16 clones 1%H4 (lanes a), 195F5 (lanes We thank Dr. Christine Kozak for supplying us with the hamster- b), and 165B9 (lanes c). Blots were washed in 0.2x SSC/0.1% mouse hybrid cell line 9-6Az2 and DNA ofcell lines E36, HM57, and NaDodSO4 at 680C. DNA size standards are (from top to bottom) 23, HM96; Dr. Peter Staeheli for providing plasmid pMx34; and Andrea 9.4, 6.5, 4.3, 2.3, 2.0, and 0.5 kb. To avoid hybridization with highly Becker for technical assistance. This work was supported by grants repetitive sequences in both Northern and Southern blot analyses, GM 32355, NS 22347, and NS 22111 from the National Institutes of probes were depleted of these sequences by treatment with DNA- Health to J.G.S. and by a fellowship from Deutsche Forschungsge- cellulose, and prehybridization of blots was followed by hybridiza- meinschaft to U.H. This is publication no. 5745-MB from The tion with 100 ,ug of sheared mouse genomic DNA per ml before Research Institute of Scripps Clinic. hybridization with radioactive probes. 1. Gray, J. W. & Langlois, R. G. (1986) Annu. Rev. Biophys. Biophys. 226 clones that hybridized with the liver probe. Of these, 175 Chem. 15, 195-235. clones (84% of the brain-positive clones and 77% of the 2. Gray, J. W., Dean, P. N., Fuscoe, J. C., Peters, D. C., Trask, liver-positive clones) hybridized with both probes, 33 clones B. J., van den Engh, G. J. & Van Dilla, M. A. (1987) Science 238, (16%) hybridized with the brain probe only, and 51 clones 323-329. 3. Gusella, J. F., Keys, C., Varsanyi-Breiner, A., Kao, F.-T., Jones, (23%) hybridized with the liver probe only. Representative C., Puck, T. T. & Housman, D. (1980) Proc. Natl. Acad. Sci. USA filters of these screenings are shown in Fig. 3. 77, 2829-2833. From a selection of these clones, phage DNA was pre- 4. Kasahara, M., Figueroa, F. & Klein, J. (1987) Proc. Natl. Acad. Sci. pared, labeled by random hexamer priming, and used to USA 84, 3325-3328. of brain and liver RNA. the 5. Kozak, C. A. & Rowe, W. P. (1980) J. Exp. Med. 152, 1419-1423. probe Northern blots Reflecting 6. Staeheli, P., Haller, O., Boll, W., Lindenmann, J. & Weissman, C. primary screening, the clones fall in one of three classes: (1986) Cell 44, 147-158. expressed in brain and liver or expressed exclusively, or at 7. Staeheli, P., Pravtcheva, D., Lundin, L.-G., Acklin, M., Ruddle, F., significantly higher levels, in one tissue versus the other. Lindenmann, J. & Haller, 0. (1986) J. Virol. 58, 967-969. Examples of one clone from each class are shown in Fig. 4A. 8. Brison, O., Ardeshir, F. & Stark, G. R. (1982) Mol. Cell. Biol. 2, To insure that these clones are mouse 578-587. derived from the 9. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular chromosome, we have used them as probes of Southern blots Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold of mouse, hamster, and somatic cell hybrid genomic DNAs. Spring Harbor, NY). Representative examples are shown in Fig. 4B. Band patterns 10. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, are detected by the genomic clones in mouse DNA and mouse W. J. (1979) Biochemistry 18, 5294-5299. chromosome 16-hamster hybrid DNA. 11. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. 12. Frischauf, A.-M., Lehrach, H., Poustka, A. & Murray, N. (1983) J. DISCUSSION Mol. Biol. 170, 827-842. 13. Short, J. M., Fernandez, J. M., Sorge, J. A. & Huse, W. D. (1988) We have developed a method for constructing mouse chro- Nucleic Acids Res. 16, 7583-7600. mosome-specific libraries and have applied it to make a 14. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) J. library of mouse chromosome 16. This library consists of Mol. Biol. 113, 237-251. 15. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 14,200 clones with an average insert size ofapproximately 17 16. Goldberg, M. L., Lifton, R. P., Stark, G. R. & Williams, J. G. kb and represents roughly two "chromosome equivalents." (1979) Methods Enzymol. 68, 206-220. If the cloned fragments were generated randomly, the prob- 17. Noyes, B. E. & Stark, G. R. (1975) Cell 5, 301-310. ability of a given sequence being in this library would be 85%. 18. Staeheli, P. & Sutcliffe, J. G. (1988) Mol. Cell. Biol. 8, 4524-4528. As many genes contain several exons spread over tens of 19. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205. the at one exon 20. Hastie, N. D. & Bishop, J. 0. (1976) Cell 9, 761-774. kilobases, probability of having least of such 21. Bantle, J. A. & Hahn, W. E. (1976) Cell 8, 139-150. genes in the library is greater than 85%. Although our method 22. Chikaraishi, D. M. (1979) Biochemistry 18, 3249-3256. for generating insert fragments (partial digestion with the 23. Milner, R. J. & Sutcliffe, J. G. (1983) Nucleic Acids Res. 11, restriction enzyme Sau3A) is not absolutely random, a pre- 5497-5520. Downloaded by guest on September 29, 2021