GENOMICS
9, 247-2.56
(1991)
The Chinese Hamster HPRT Gene: Restriction Analysis, and Multiplex PCR Deletion BELINDA
Map, Sequence Screen
1. F. RossmR,**t,’ JAMES C. FUSCOE,~ DONNA M. MUZNY,~ MARGARET Fox,t AND C. THOMAS CASKEY*,§
*institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030; tBiochem/cal Genetics, Paterson institute for Cancer Research, Christie ffospitai & Hoit Radium Institute, Manchester M20 98X; +Center for Environmental Health, University of Connecticut, Storrs, Connecticut 06268; and §Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 Received
June
26,
1990;
The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping X bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform t,han Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene. li 1~1 Academic PESS. I~C. -
INTRODUCTION
Hypoxanthine guanine phosphoribosyltransferase (HPRT’; inosine monophosphate:pyrophosphatase ’ To whom correspondence should be addressed at Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030. ’ Abbreviations used: EDTA, ethylenediaminetetraacetic acid: HPRT, hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8.); HAT, growth medium supplemented with h-ypoxanthine. amethopterin (methotrexate), and thymidine; PCR, polymerase chain reaction; RNase, ribonuclease; SDS, sodium dodecyl sulfate: + (superscript), proficient, as in HPRT’, ~ (superscript). deficient. as in HPRT
revised
September4,
1990
phosphoribosyltransferase; EC 2.4.2.8) is one of the enzymes responsible for the salvage of preformed purine bases during normal nucleic acid turnover in the mammalian cell (Caskey and Kruh, 1979). The Chinese hamster HPRT locus has been favored for studies of somatic mut.ation because cultured celIs do not have an absolute requirement for HPRT activity and so impose no limit on the type of mutation which may occur at the HPRT locus. HPRT’ and HPRTpopulations are easily isolated using simple selection media and in addition, the hemizygous nature of the gene in cells derived from male animals facilitates analysis of mutabions at, this locus. The HPRT gene is located on the X-chromosome in man (Nyhan et al., 1967), mouse (Chapman and Shows, 1976), and Chinese hamster (Farrell and Worton, 1977). The cDNA sequences of mouse, Chinese hamster, and human HPRT have been published (Jolly et al., 1983; Konecki et al., 1982) and the structures of the mouse and human HPRT genes have been described (Melton et al., 1984; Kim et al., 1986; Pate1 et al., 1986); recently 57 kb of the human HPRT locus were sequenced (Edwards et al., 1990). The mouse HPRT gene covers about 34 kb and the human gene 40 kb but the overall arrangement is similar in both cases. The coding sequence is split into nine exons and the intron/exon junctions for both species occur in the same positions in the cDNA sequence. This report presents restriction map and sequence data from the Chinese hamster HPRT gene and comparisons with the known mouse and human sequences (Melton et al., 1984; Edwards et al., 1990). The information is relevant to studies of deletions at the Chinese hamster HPRT locus, such as those reported by Fuscoe et al. (1983), Vrieling et al. (1985), and Breimer et al. (1986). In cases where part of the gene is delet,ed, it is possible to define approximately the extents of such deletions by Southern analysis (Morgan et al., 1990; Thacker et al., 1990). Since one band often contains more than one exon, the location
248
KOSSITEK
of deletion endpoints can be ambiguous and these procedures also take up to several days to perform. The availability of sequence flanking the coding regions has enabled use of the polymerase chain reaction (PCR) (Mullis and Faloona, 1987) to amplify HPRT exons as an alternative to Southern analysis for investigation of Chinese hamster cell lines in mutation studies. Such analysis using simultaneous amplification of HPRT exons in a multiplex PCR reaction (Chamberlain et al., 1988) is described here, allowing the rapid screening of deletion mutations in only a few hours. MATERIALS Origin
of Cell
AND
METHODS
Lines
The wild-type Chinese hamster cell lines V79S and RJKO were originally obtained from the trypsinized lung of a male Chinese hamster (Ford and Yerganian, 1958; Elkind and Sutton, 1960). The RJKlO cell line was obtained after treatment of RJKO cells with Nmethyl-N’-nitro-N-nitrosoguanidine and selection in 8-azaguanine and has no detectable HPRT activity (Beaudet et al., 1973). The RJK159 cell line is a spontaneous HAT-resistant revertant of RJKlO (Fenwick et al., 1977), there being a lo- to 20-fold amplification of the HPRT gene which has a point mutation within the coding sequence (Fenwick et al., 1984; Rossiter et al., 1990). The RJK88 cell line is a spontaneous HPRT mutant derived from RJKO cells (Fuscoe et al., 1983) and Southern analysis has indicated that the entire HPRT gene is deleted, leaving only unlinked HPRT pseudogene sequences (Fuscoe et al., 1983). The HPRT cell lines UCC-15,16, and 19 are independent 6-thioguanine-resistant clones from Chinese hamster ovary (CHO-Kl-BH4) cells; UCC-15 and 16 were derived from X-ray treatment (300 rad), and UCC-19 is a spontaneous mutant. Generation
of Oligonucleotides
and Probes
Oligonucleotides were synthesized by phosphoramidite chemistry (Sinha et al., 1984) using an Applied Biosystems, Inc. (Foster City, CA), 380B DNA synthesizer, precipitated in 2.5 M ammonium acetate/70% ethanol, and redissolved in water. Table 1 lists the various probes used in this report with the conditions of their use. Double-stranded probes (at >lOg cpm/pg) were prepared by the random primer method of Feinberg and Vogelstein (1983, 1984) using the Multiprime labeling system (Amersham Corp., Arlington Heights, IL) and oligonucleotides were end-labeled (to >2 X lo8 cpm/pg) using T4 polynucleotide kinase (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). Labeled DNA and oligonucleotide probes were separated from unincorporated
E’1’ AI>
nucleotides using G50 and G25 Sephadex spin columns (Boehringer-Mannheim Biochemicals, Indianapolis, IN), respectively. Southern Analysis, Genomic Bacteriophage Mapping
Library
Screening,
and
Genomic DNA was prepared by lysis ofcells in SDS and proteinase K, followed by phenol/chloroform extractions and RNase A treatment, or prepared from cells encapsulated in agarose plugs as described by Fuscoe et al. (1989). For Southern analysis 10 pg DNA was digested, electrophoresed through 0.8% agarose, and transferred to Zetaprobe (Bio-Rad Chemical Division, Richmond, CA) in 10X SSC (1X is 150 mM NaCl, 15 mM sodium citrate). Prehybridization of the membrane was performed in 6X SSC, 0.25% nonfat dried milk (Carnation Co., Los Angeles, CA), 0.1% SDS at, the appropriate temperature (Table 1) and hybridization was accomplished under the same conditions with the addition of 106cpm/ml [tu-32P]dCTP-labeled probe. The RJK159 genomic library was generated from a partial Mb01 digest of genomic DNA cloned into the BamHI site of bacteriophage Charon 28; the library was propagated on LE392 cells. Bacteriophage plaques grown in agarose were transferred to Hybond N (Amersham Corp.) and the filters probed in the same way as Southern blots. Clones identified by HPRT probes were mapped for restriction endonuclease recognition sites using the method of Rackwitz et al. (1984). Inverse
PCR
The scheme for this approach is based on the technique used by Triglia et al. (1988). Genomic DNA (3 pg) from RJKO, RJK159, and RJK88 cells was digested to completion with different restriction enzymes (AluI, BglII, MaeI, MaeII, PstI, RsaI, SauSAI, or TuyI) in One-Phor-All PLUS buffer (Pharmacia LKB Biotechnology, Inc.), heated to 65°C for 15 minutes, diluted to 3 pg/ml in the same buffer (1X concentration), and then circularized overnight at 16°C with 60 U/ml T4 DNA ligase and 0.1 mM ATP. The DNA was precipitated in 0.3 M sodium acetate/70% ethanol. One microgram of the digested DNA was used in a loo-p1 PCR with oligonucleotides 1225 and 1226 derived from known exon 1 sequence but oriented such that their 5’ ends faced each other, and located on opposite strands; the 5’ ends of the primers included either an EcoRI or a Hind111 site. Amplification would only proceed if the two sites for oligonucleotide priming were present in a circular molecule. Before amplification the DNA was heated to 95°C for 30 min.
THE
CHINESE
HAMSTER
TABLE Description Probe Exon
1
Exon 1 Exons 2 and 8 Exon
4
Exon
5
Exon Exon
5 6
Exons 7-9 cDNA 7BE260 iHB80
Oligo
739
of Hybridization Description
Probes of DNA
of genomic
DNA
The PCR reaction mixture contained 50 mM KCl, 10 mM Tris, pH 8.3, 1.5 mM MgCl,, 0.01% gelatin, 200 PM each nucleotide, 1 &f each primer, and 2 U AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT), covered with 50 ~1 paraffin oil; 30 cycles of (94”C/30 s, 58”C/ 30 s, 72”C/2 min) were performed. One microliter of the PCR product was amplified further in an identical reaction to the first. The PCR was carried out in a Perkin-Elmer Cetus thermocycler using the “step cycle.” The products of the inverse PCR reactions were electrophoresed through 1% agarose and any products present in RJKO and RJK159, but not in RJK88, were isolated and digested with EcoRI and HindIII, then subcloned into EcoRI- and HindIII-digested pTZ18 and pTZ19 (Mead et al., 1986). Verification that the fragments contained exon 1 was obtained by the hybridization of oligonucleotide 739 derived from exon 1 sequence but not included in the priming molecules. Oligonucleotide sequences: 1225: 1226: 739:
249
Study
and Conditions
of Their
Hybridization temperature
fragment
?47-bp exon 5 PCR product 93bp MnlI-AluI cDNA fragment, includes 10 bp of exon 5 354.bp HindIII-Pat1 cDNA fragment 750.bp ps7stI fragment from pHPT20 (4) 260.bp BarnHI-EcoRI fragment which is the 3’ end of 7E0.9 marked in Fig. 1 80.bp BarnHI-Hind111 fragment from the middle of 7E0.9 marked in Fig. 1: it lies 5’ to 7EB260 20.mer oligodeoxynucleotide: 5’.dCACGACGCTGGGGCTGCGGG analysis
GENE
1
Used in This
107-bp NarI-BstNI cDNA fragment. includes 17 bp of exon 2 730-bp inverse PCR product 307.bp Rbul-HincII cDNA fragment, inclues 9 bp of exon 4 Xl-bp AluILMnlI cDNA fragment. includes 7 bp of exon 3 and 8 bp of exon 5 1%mer oligodeoxynucleotide: 5’.dTTCCACAATCAAGACATT-3’
Note. These probes were used in Southern Materials and Methods.
HPRT
5’-CGAAGCTTGGAGGAAGCCCGCAGAGGAG-3’ 5’-TCGAATTCGCCAGCCGACCGATTCCGTC-3’ 5’.CACGACGCTGGGGCTGCGGG-3’
Sequencing and Sequence Analysis Single-stranded template DNA was prepared from Ml3 (Messing, 1983) or pTZ (Mead et al., 1986) re-
Use Washing
48°C
2X SSC/63Y‘
5Fi”c 50°c
2X SSC/65”C 2 Y SSC/65”C
55°C
0.5~
SSC/SSY:
6~ SSC/43”C 6~ SK/room temperature 0.5x SSC/65”C 2X SSC/S!iT 55°C 55°C
2x SSC/Sfi’C 0.5~ SSC/SS”C 2x SSC/65”C
50°C
2x SSC/65”C
48°C
2x SSC/48”C
and for the screening
of the RJK159
genomic
conditions
library
(3 min)
as described
then
in
combinants containing regions from the Chinese hamster HPRT gene and sequenced manually using the dideoxynucleotide chain terminat,ion method as modified by Biggin et al. (1983) or using an Applied Biosystems, Inc., 370A DNA sequencer. To sequence through GC compressions the Sequenase Version 2 kit from U.S. Biochemical Corp. (Cleveland, OH) was used with dITP nucleotide analogs and sequencing reactions were performed at 50°C. The sequencing products were electrophoresed through 5 and 6% polyacrylamide gels in the presence of 8.5 M urea. Sequences were analyzed using the progressive sequence alignment method of Feng and Doolittle (1987), through the EuGene user interface of the Molecular Biology Information Resource (MBIR) at Baylor College of Medicine (C. Lawrence, Department of Cell Biology). Multiplex Pal-vmerase Chain Reaction and Gel Analysis In vitro amplification of 0.4 PLggenomic DNA was carried out in a 30-~1 reaction mixture of 6.7 mM MgCl,, 16.6 mM (NH&SO,, 5 mM ,0-mercaptoethanol, 6.8 PM EDTA, 67 mM Tris, pH 8.8, 10% dimethyl sulfoxide with 1.5 mM each deoxynucleotide, 133 nM to 1.67 PM each primer (Table 2), and 4 U AmpliTaq. The reaction mixture was covered with 25
250
ROSSITER
TABLE
2
Oligodeoxynucleotide Primers Used for PCR Amplification of the Chinese Hamster HPRT Gene Exon 1, 321 bp (66.7 nM)GTA CCTGGC (66.7 nM) TCCGCTCTG Exon 2. 166 (I..iOj&) (I.50 PM)
Exon
CCC AGGAGC CAC C CTGAAGAGTCCCG
hp AGC TTA TGC TCTGAT TTGAAA TCAGCT G ATTAAG ATC TTA CTT ACC TGT CCATAATC
3, 220 hp
(1.33 PM) II.33 ,uM) Exon~.19lhp (1.33 PM)
CCGTGATTT TAT TTT TGT AGG ACT GAAAG AAT GAA TTA TAC TTA CAC AGT AGC TCT TC GTGTAT
TCAAGAATATGC
ATG TAA ATGATG
(1.43 q’v4) CAAGTGAGTGAT TGAAAG CAC AGT TAC Exon F, 247 bp ( I.:&IM) AAC ATA TGG GTC AAA TAT TCT TTC TAA TAG (1.33
Exon (1.33 (1.33
Exons
&f) PM)
pM) 7 and nM) nM)
(133 (133 Exon 9, 744 (1.67&J
il.67
GGC TTA CCT ATA GTA TAC ACT AAG CTG
6. 145 hp
pM)
TTA CCA CTT ACC ATT AAA TAC CTC TTT TC CTA CTT TAA AAT GGC ATACAT ACC TTG C
8. 423 hp GTA ATA TTT TGT AAT TAA CAG CTT GCT GG TCA GTC TGG TCA AAT GAC GAG GTG C hp CAATTCTCTAATGTTGCT CTTACC TCTC CAT GCA GAG TTC TAT AAGAGA CAGTCC
Note. The sequence (5' to 3') of each primer with the final concentration in the PCR and resulting PCR product.
is shown the
length
together of the
~1 paraffin oil and subjected to 30 cycles of (94”C/l min, 58”C/l min, 7O”C/7 min), preceded by 5 min at 94°C and followed by 13 min at 7O”C, then incubation at 4°C. The reaction products (lo-15 ~1) were then electrophoresed through 4% agarose [3 parts NuSieve (FMC BioProducts, Rockland, ME), 1 part SeaKem (FMC BioProducts) ] for analysis. Reaction conditions for amplification of exon 1 alone were identical to the multiplex conditions except that the temperature cycle parameters were 30 cycles of (94’C/l min, 6O”C/l min, 7O”C/45 s), preceded by 5 min at 94°C and followed by 19 min at 70°C.
ET
AL.
XCH-HPT 12 using both exon 5 and exon 6 probes against the genomic library. The first exon and a large part of the first intron was not successfully isolated from two different RJK159 libraries, using a variety of exon l-specific probes, but the regions flanking exon 1 were eventually isolated by the alternative method of inverse PCR (see below). The bacteriophage clones were digested to completion with the restriction endonucleases BamHI, BglII, EcoRI, HindIII, and PstI and the products carefully compared with standard markers to determine their size. Fragments containing coding regions were identified by Southern analysis using exon-specific probes as described in Table 1. Restriction enzyme recognit.ion sites for the five selected enzymes were determined by the mapping procedure described under Materials and Methods. If there was any discrepancy bet,ween the two methods in determining the distance between restriction sites, the size obtained by comp1et.e digestion was taken to be more accurate. The data are collated in Fig. 1. The location of each of the exons was determined either by their position relative to one of the restriction endonuclease sit.esin the map or by their position relative to a different restriction endonuclease recognition site and determination of the location of that other site relative to one of the mapping sites by means of double digestions and Southern analysis. Exons 1,4, and 6 were located by the latter procedure; exon 1 lies 444 bp 5’ of a Sac1 site, exon 4 lies 25 bp 3’ of an NsiI site, and exon 6 lies 2 bp 3’ of a nra1 site. Restriction fragment sizes predict,ed by the map shown in Fig. 1 were confirmed by Southern analysis of genomic DNA from RJKO and RJK159 cells, probed with HPRT cDNA or exon-specific probes (Table 1). Figure 2B shows a schematic of the exons contained within the bands observed in a Southern (Fig. 2A) when HPRT cDNA is used as a probe; exons 1 and 5 are not usually seen under these conditions because of the short length of homology with the probe. Exon
RESULTS
Restriction Chinese
Map and Southern Analysis Hamster HPRT Gene
of
the
HPRT cDNA was used initially to screen the RJK159 genomic library, resulting in the isolation of the clones XCH-HPT 1,2,5, and 7 (Fig. 1). On hybridization with exon-specific probes (Table 1) these clones were found to contain exons 2-9, but restriction mapping revealed that a port,ion of the gene between exons 5 and 6 was not represented. The gap between exons 5 and 6 was closed by isolation of clone
1 Cloned
hy Inverse
PCR
The first exons from the RJKO and RJK159 HPRT genes were isolated by the inverse PCR method described under Materials and Methods. Eight, restriction endonucleases were used in the attempt to clone the Chinese hamster HPRT exon 1 (AU, BglII, MaeI, MaeII, PstI, RsaI, SauSAI, and TayI), the majority having 4-base recognition sequences, and none having a (X-rich recognition sequence (since the promoter region was expected to be (X-rich and would therefore be digested by such enzymes). The inverse PCR was deemed to be successful if a fragment was generated from R?JKO and RJK159 cells but was ab-
THE
I
I I
I
I
HAMSTER
I 1
1
L
1
I
II
I, 1
II
2
I I 3
I
I
I
1
I
BglTI
I
I EcoKI
I
,
4
I I
I
I
1
251
(;ENE J BumHI
I I
/;;\
I
HI’RT
I
,*,
I
12
CHINESE
I
II 5
I
HmdIIl
I I II 1 6
78
9
PstI
EXONS
KH-HFTU2 KH-HFT#I2 ICH-HPT#I ICH-HFT#S XCH-HIT-#1 FIG. 1. Restriction map of the Chinese hamster HPRT gene. The location of the enzymes BarnHI, J&$11, EcoRI, HindIII. and PstI are shown. together with the positions clones from which exons 2-9 were isolated. The positions of the restriction sites around known whether there are additional sites within the dotted regions of the B&II and P&I two sites close together or a single site. The 900.bp EcoRI fragment indicated by an analysis descrihedin Fig. 2 and elsewhere. .
sent in RJK88 cells (where the HPRT gene is deleted). Only the enzymes AluI and RsaI yielded exon 1 fragments from this procedure and since the AluI fragment was contained within the RsaI fragment it was not investigated further. The 730-bp inverse PCR fragment subcloned into pTZ18 and pTZ19 vectors contained a unique RsaI site which marked the joining of the 5’ and 3’ flanking sequences. Since the fragment was inverted with respect to the gene organization, sequencing in from the ends actually corresponded to sequencing outward from exon 1 in both directions. Sequencing of the region 3’ to exon 1, in the first intron, proved particularly difficult because of strong stop signals and GC compressions. These problems were eventually overcome by the technical modifications described under Materials and Methods. The entire fragment was sequenced with the use of additional oligonucleotides as sequencing primers. Figure 1 indicates the genomic fragments t.o which the exon 1 inverse PCR product hybridizes. These Southerns and those performed with probes from within the 900-bp E’coRI fragment allowed the posit,ioning of the first exon with respect to the rest of the gene, since a large BumHI fragment (13.5 kb) includes both exon 1 and the 5’ portion of the 900-bp EcoRI fragment from intron 1. Sequences from the Chinese Hamster
HPRT
Gene
Small fragments (a few hundred base pairs) containing HPRT exons 2-9 were isolated from the bacteriophage clones shown in Fig. 1, subcloned into Ml3 or pTZ vectors, and used as templates for sequencing the regions flanking the exons. The sequence deter-
restriction endonuclease recognition sites lor the of the exons and the extent of the bacteriophage exon 1 are deduced from Southern analysis. It is not maps. The two f’stl sites enclosed by an oval may be asterisk was used to generate probes for Southern
mined, deposited with the EMBL Data Library (ACcession Nos. X53073-80), is from the RJK159 cell line which contains one point mutation within exon 6; the wild-type sequence at this position was identified by sequencing an HPRT mutant with a different point mutation (Rossiter et al., 1990). Since the fragment containing exon 1 was generated via the PCR, it was possible that the sequence might contain polymerase errors. For each cell line (RJKO and RJK159) three clones were sequenced in each direction (13 in total) and whenever there was a difference between clones, the sequence found in two of the three was taken as being correct. In all, there were three PCR errors found in the i’30-bp fragment and no differences found between the two cell lines. Comparison of Chinese Hamster, HPRT Sequences
Mouse, and Human
The complete human HPRT locus has been sequenced (Edwards et al., 1990) and some mouse HPRT sequence has been reported (Melton et al., 1984); this information provided the possibility of comparing three mammalian species in terms of their sequence conservation within the HPRT gene. Figure 3 shows the alignment of human, Chinese hamster, and mouse sequences around exon 1 and the promoter region. Although the coding region (marked within the large box) and the few bases on either side are quite homologous, the degree of identity between the human and the Chinese hamster sequences and between the human and the mouse sequences over the whole region is less than 40%. Very little flanking sequence from the mouse HPRT gene has been published except for the exon 1
252
ROSSITER
A.
B
Bg E
H
4.31
probe: cDNA
B. E 16.X-6-9 13.6-2-4
6.6-y 5.093
4.6-
of the Chinese
Hamster
9.0 2 8.2 =6-g 6.6-d 5.2-y
3
3.5-v
3.3-3 2.1~w
3.0-2 2.2-9 1.9-v 1.6-7-S 1.2-3
Anal.ysis
P
H
;;I;=;;
PCR Gene
PCR primers for the multiplex amplification of Chinese hamster HPRT exons were originally selected to generate fragments of different sizes which could be resolved by agarose gel electrophoresis (Table 2). Exons 7 and 8 are amplified on a single fragment since they are only 165 bp apart. When possible the primers were located beyond t.he immediate splice sites so that these regions would be amplified together with the exons. The oligonucleotides were designed to share similar melting temperatures, therefore those with a higher AT content are longer than the others. Although all primers designed were able to amplify individual exons, not all were able to participate in the multiplex reaction successfully; the set of 16 described in Table 2 derives from a larger set of 38 oligonucleotides actually synthesized. It was never possi-
23.lkb 9.42 6.56
Bt?
AL,
Multiplex HPRT
P origin
B
ET
1.2-y
FIG. 2. Southern analysis of the Chinese hamster HPRT gene using cDNA and exon-specific probes. (A) Sout,hern analysis of V79S DNA digested with BarnHI (B), R~111 (Bg). EcoRI (E), Hind111 (H). and PstI (P) and probed with HPRT cDNA (Table 1). (B) The schematic illustrates the hands identified by a cDNA probe on a Southern blot containing wild-type genomic DNA digested as in (A). The size of each hand in kilobases is shown on its left and the exons represented within it on the right. The symbol $ represents pseudogene fragments. The second largest RamHI fragment is a doublet and the 4.6-kh &$I1 fragment is a triplet. The sizes of the larger fragment.s were determined from Southern gels which were run longer than those shown here, for bett,er resolution. These exon assignments were predicted from the restriction map in Fig. 1 and confirmed by Southern analysis using exon-specitic probes (data not shown).
region so sequences surrounding the remaining eight exons were only compared between human and Chinese hamster. Such analysis of the sequences around exons 7 and 8 is shown in Fig. 4 and in contrast to the region around the first exon, the degree of identity over the whole region shown here is greater than 80%. Comparison of human and Chinese hamster sequences from around the other coding regions of the HPRT gene also showed considerable intron homology (analysis not shown).
FIG. 3. Comparison of human, Chinese hamster, and mouse HPRT sequences around exon 1. Sequences from the 5’ regions of the human, Chinese hamster, and mouse HPRT genes are aligned using the computer program described under Materials and Methods. The large box is drawn around the coding region of exon 1 and smaller hoxes are drawn around the sequences .5’-CGCGGG-:I’.
THE
CHINESE
HAMSTER
FIG. 4. Comparison of human and Chinese hamster HPRT sequences around exons 7 and 8. Sequences from exons 7 and 8 ofthe human and Chinese hamster HPRT genes are aligned as in Fig. 3: boxes are drawn around the coding regions.
ble to combine several combinations of exon 1 primers with the remaining set of primers and so the multiplex PCR includes exons 2-9; exon 1 is amplified in a separate reaction. Figure 5 shows the multiplex seven-component reaction and the exon 1 amplification, used to screen a series of HPRT cell lines including two HPRT partial deletion mutants. In one of the mutants (UCC-15, lane 3) exon 4 is missing; in the other (UCC-19, lane 4) exons 4,5, and 6 are missing. Included in the analysis are wild-t,ype cell lines (RJKO and UCC-1, lanes 1 and 2) and cell lines with complet,e deletions of the HPRT gene (RJK88 and UCC-16, lanes 5 and 6).
HPRT
253
(:ENE
likely to be several hundred kilobases (Stark and Wahl, 1984). There is considerable evidence that the amplified HPRT gene characterized from the RJK159 cell line is ident,ical (apart from the exon 6 point. mutation) to that in wild-type RJKO and V79S cells and other Chinese hamster cell lines. First, Southern analysis indicates that the gene structure is identical at least as far as the extent of the largest fragment recognized by the cDNA probe (Fig. 2). Second, the region surrounding exon 1 was sequenced from both RJKO and RJK159 cells and was found to be identical in the two cell lines. Third, primers for PCR amplification of individual exons based on sequence from the RJK159 HPRT gene are able to amplify exons from RJKO and Chinese hamster ovary (CHO) cells (Fig. 5; Xu et al., 1989). Comparison Hamster,
of HPRT Genes between Human, and Mouse
Chinese
The overall structure of the HPRT gene in Chinese hamster cells (Fig. 1) is similar to that in human and mouse cells; the first int,ron is the largest, exons 7, 8, and 9 lie very close together, and the positions of the
DISCUSSION Development of a restriction map for the Chinese hamster HPRT gene in RJK159 cells has led to the isolation and sequencing of all nine exons and their flanking regions, and the development of a multiplex PCR deletion screen. This information allows a more accurate assessment, of Southern experiments since the content of each HPRT band is defined; the multiplex PCR screen permits a rapid analysis of HPRT deletion mutants and the generation of specific fragments of the HPRT gene for further analysis. HPRT Gene Structure Is the Same in Different Chinese Hamster Cell Lines Since the RJK159 cell line contains an amplified HPRT locus, it is important to demonstrate that the gene structure has not been altered as a result of the amplification process. Alteration of the gene structure is not expected since the amplification unit is
I?.
-9 -7&8 l-5 -3 -4 -2 -6
FIG. 5. Screen of wild-t?ipe cells and HPRT-deficient mutants hy multiplex PCR analysis. (A) The left-hand group of reactions was performed with the primers for amplification of exon 1 of the Chinese hamster HPRT gene (Table 2). The right-hand group of reactions was performed with the multiplex primers for the simultaneous amplification of exons 2-9 of the Chinese hamster HPRT gene (Table %). Lanes: (M) WarIII-digested 4X171 markers; (1) RJKO; (2) [ICC-l; (3) UCC-15; (4) IICC-19; (5) (JCC-16; (6) R.JKSX; (7) no DNA. (B) Schematic indicating the location of the individual exon hands wit,hin the multiplex amplification reaction.
254
ROSSITER
intron/exon boundaries are conserved exactly between all three species, The size of 36 kb is intermediate between the mouse gene (about 34 kb) (Melton et al., 1984) and the human gene (39.8 kb) (Edwards et al., 1990). The human, mouse, and Chinese hamster HPRT genes lack consensus CAAT and TATA sequencesimmediately 5’ to the initiation site and the region around the first exon contains several 5’-GGCGGG-3’ promoter elements. These are both features of ubiquitously expressed housekeeping genes, such as phosphoglycerate kinase (Singer-Sam et al., 1984), adenosine deaminase (Wiginton et al.. 1986), adenosine phosphoribosyltransferase (Broderick et al., 1987), lysosomal acid phosphatase (Geier et al., 1989), 5-lipoxygenase (Funk et al., 19891, and dihydrofolate reductase (Means and Farnham, 1990). Figure 3 shows that, apart from the coding region of exon 1 and the immediate flanking sequences,there is not a great deal of homology between the human, the mouse, and the Chinese hamster 5’ HPRT sequences. Despite this fact, the locations of the GC element,s appear to be conserved in two clusters, one about 200 bp 5’ to the initiation codon and the other about 150 bp 3’ to the first exon. This would suggest that these GC “boxes” indeed have some functional importance and raise the possibility that the “promoter region” of the mammalian HPRT gene extends both 5’ and 3’ of the first exon. Coding regions between the three speciesare highly conserved, as are the immediate flanking regions which form the splice signals (Fig. 4). The intron homology around exons 2-9 is greater than that observed around exon 1 but it is not known whether such homology may reflect functional importance, perhaps for splicing, or merely similarity because of a common ancestry. A similar phenomenon of intron homology between mammalian species has been observed in N- and o-globin genes (Hardison and Gelinas, 1986; Hardies et al.. 1984; Hardison, 1984) and y-crystallin genes (den Dunnen et al., 1989). It is also not known in these cases if similarity reflects a possible functional importance.
Multiplex
L3eletion Screen
The multiplex PCR deletion screen for the Chinese hamster HPRT gene can yield more precise information about the endpoints of a deletion than Southern analysis since the presence of each exon can be determined individually (apart from exons 7 and 8, which lie very close together). In contrast, a band on a genomic Southern blot often represents several exons (Fig. 2). The multiplex PCR deletion screen (Fig. 5) is quicker to perform than Southern analysis, taking
ET
AI>
only a few hours for both the reaction and the gel analysis, and avoids the use of radioisotopes. The PCR analysis does not yield any amplification products when RJK88 DNA is used as a template; this is expected since the entire HPRT functional gene is absent in RJK88 cells (Fuscoe et al., 1983). There are, however, HPRT pseudogene sequences in these cells (the genome location is unknown) and the fact that no amplification products are seen would suggest that the pseudogene in these cells is in a processed form, lacking intron sequences. This is in agreement with the studies of Isamat et al. (1988) which suggest,that the processed mouse HPRT pseudogene shares a common origin with the Chinese hamster HPRT pseudogene. There is also evidence to suggest that the four human HPRT pseudogenes (with autosomal locations) are in the processed form (Pate1 et al., 1984). The multiplex oligonucleotide primers used in this study are made partly or completely from intron sequences so they would not be expected to hybridize t.o cDNA or processed pseudogene sequences. It is possible to sequence PCR products directly without the need for subcloning and thus detect point mutations in addition to deletions (Gibbs et al., 1990). Ideally the PCR primers should be located several nucleotides from the exon/intron boundary to permit sequencing of the entire exon and its flanking region. It should be not.ed however that because of the limited sequence available the oligonucleotide primers used for amplification of the Chinese hamster HPRT gene were designed primarily to generate fragments of different sizes to permit resolution of the products by agarose gel electrophoresis and 4 of 16 of the primers contain some exon sequence. This raises the possibility that the absence of a band in the multiplex PCR could result from a mismatch within the primer binding site rather than an exon deletion although if more than one band is missing from the multiplex reaction, a deletion including the appropriat,e exons is the most likely explanation. If only one band is missing, an additional PCR could be performed with different priming oligonucleotides further from the exon boundaries; if amplification were observed a point mutation would be the most likely explanation and the PCR product could be sequenced; lack of amplification would suggest a deletion affecting that particular exon. The region around exon 1 is much more CC-rich than the rest of the gene and may be a reason why exon 1 primers were incompatible with the rest of the multiplex reaction. The addition of even small amounts of exon 1 primers to the seven-component multiplex resulted in either spurious additional products or loss of one or more of the other products. The ability to amplify individual exons of the Chinese hamster HPRT gene provides a means of gener-
THE
CHINESE
HAMSTER
ating exon-specific fragments larger than those engineered from cDNA (Table 1) and yet small enough not to include repetitive intron sequences which can interfere with hybridization. Such fragments have t.he potential to be useful as probes in Southern analysis, particularly in cases of’ gene rearrangements which may elude detection by multiplex PCR.
HPRT 11.
ELKIND, M. M.. AND SUTTON, H. (1960). Radiation response of mammalian cells grown in culture. I. Repair of X-ray damage in surviving Chinese hamster cells. Radiat. Res. 13: 556593.
12.
FARRELL, S. A., AND WORTON, R. G. (1977). Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids. Somatic Cell Genet. 3: 539-551.
13.
FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique radiolabeling DNA restriction endonuclease fragments high specific activity. Anal. Biochem. 132: 6-13.
14.
FEINBERG, A. P., AND VOGELSTEIN, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Hiochem. 137: 266267.
15.
FENG, D. F., AND DOOLITTLE, R. F. (1987). Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25: 351-360. FENWICK, R. G.,
9, 247-2.56
(1991)
The Chinese Hamster HPRT Gene: Restriction Analysis, and Multiplex PCR Deletion BELINDA
Map, Sequence Screen
1. F. RossmR,**t,’ JAMES C. FUSCOE,~ DONNA M. MUZNY,~ MARGARET Fox,t AND C. THOMAS CASKEY*,§
*institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030; tBiochem/cal Genetics, Paterson institute for Cancer Research, Christie ffospitai & Hoit Radium Institute, Manchester M20 98X; +Center for Environmental Health, University of Connecticut, Storrs, Connecticut 06268; and §Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 Received
June
26,
1990;
The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping X bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform t,han Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene. li 1~1 Academic PESS. I~C. -
INTRODUCTION
Hypoxanthine guanine phosphoribosyltransferase (HPRT’; inosine monophosphate:pyrophosphatase ’ To whom correspondence should be addressed at Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030. ’ Abbreviations used: EDTA, ethylenediaminetetraacetic acid: HPRT, hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8.); HAT, growth medium supplemented with h-ypoxanthine. amethopterin (methotrexate), and thymidine; PCR, polymerase chain reaction; RNase, ribonuclease; SDS, sodium dodecyl sulfate: + (superscript), proficient, as in HPRT’, ~ (superscript). deficient. as in HPRT
revised
September4,
1990
phosphoribosyltransferase; EC 2.4.2.8) is one of the enzymes responsible for the salvage of preformed purine bases during normal nucleic acid turnover in the mammalian cell (Caskey and Kruh, 1979). The Chinese hamster HPRT locus has been favored for studies of somatic mut.ation because cultured celIs do not have an absolute requirement for HPRT activity and so impose no limit on the type of mutation which may occur at the HPRT locus. HPRT’ and HPRTpopulations are easily isolated using simple selection media and in addition, the hemizygous nature of the gene in cells derived from male animals facilitates analysis of mutabions at, this locus. The HPRT gene is located on the X-chromosome in man (Nyhan et al., 1967), mouse (Chapman and Shows, 1976), and Chinese hamster (Farrell and Worton, 1977). The cDNA sequences of mouse, Chinese hamster, and human HPRT have been published (Jolly et al., 1983; Konecki et al., 1982) and the structures of the mouse and human HPRT genes have been described (Melton et al., 1984; Kim et al., 1986; Pate1 et al., 1986); recently 57 kb of the human HPRT locus were sequenced (Edwards et al., 1990). The mouse HPRT gene covers about 34 kb and the human gene 40 kb but the overall arrangement is similar in both cases. The coding sequence is split into nine exons and the intron/exon junctions for both species occur in the same positions in the cDNA sequence. This report presents restriction map and sequence data from the Chinese hamster HPRT gene and comparisons with the known mouse and human sequences (Melton et al., 1984; Edwards et al., 1990). The information is relevant to studies of deletions at the Chinese hamster HPRT locus, such as those reported by Fuscoe et al. (1983), Vrieling et al. (1985), and Breimer et al. (1986). In cases where part of the gene is delet,ed, it is possible to define approximately the extents of such deletions by Southern analysis (Morgan et al., 1990; Thacker et al., 1990). Since one band often contains more than one exon, the location
248
KOSSITEK
of deletion endpoints can be ambiguous and these procedures also take up to several days to perform. The availability of sequence flanking the coding regions has enabled use of the polymerase chain reaction (PCR) (Mullis and Faloona, 1987) to amplify HPRT exons as an alternative to Southern analysis for investigation of Chinese hamster cell lines in mutation studies. Such analysis using simultaneous amplification of HPRT exons in a multiplex PCR reaction (Chamberlain et al., 1988) is described here, allowing the rapid screening of deletion mutations in only a few hours. MATERIALS Origin
of Cell
AND
METHODS
Lines
The wild-type Chinese hamster cell lines V79S and RJKO were originally obtained from the trypsinized lung of a male Chinese hamster (Ford and Yerganian, 1958; Elkind and Sutton, 1960). The RJKlO cell line was obtained after treatment of RJKO cells with Nmethyl-N’-nitro-N-nitrosoguanidine and selection in 8-azaguanine and has no detectable HPRT activity (Beaudet et al., 1973). The RJK159 cell line is a spontaneous HAT-resistant revertant of RJKlO (Fenwick et al., 1977), there being a lo- to 20-fold amplification of the HPRT gene which has a point mutation within the coding sequence (Fenwick et al., 1984; Rossiter et al., 1990). The RJK88 cell line is a spontaneous HPRT mutant derived from RJKO cells (Fuscoe et al., 1983) and Southern analysis has indicated that the entire HPRT gene is deleted, leaving only unlinked HPRT pseudogene sequences (Fuscoe et al., 1983). The HPRT cell lines UCC-15,16, and 19 are independent 6-thioguanine-resistant clones from Chinese hamster ovary (CHO-Kl-BH4) cells; UCC-15 and 16 were derived from X-ray treatment (300 rad), and UCC-19 is a spontaneous mutant. Generation
of Oligonucleotides
and Probes
Oligonucleotides were synthesized by phosphoramidite chemistry (Sinha et al., 1984) using an Applied Biosystems, Inc. (Foster City, CA), 380B DNA synthesizer, precipitated in 2.5 M ammonium acetate/70% ethanol, and redissolved in water. Table 1 lists the various probes used in this report with the conditions of their use. Double-stranded probes (at >lOg cpm/pg) were prepared by the random primer method of Feinberg and Vogelstein (1983, 1984) using the Multiprime labeling system (Amersham Corp., Arlington Heights, IL) and oligonucleotides were end-labeled (to >2 X lo8 cpm/pg) using T4 polynucleotide kinase (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). Labeled DNA and oligonucleotide probes were separated from unincorporated
E’1’ AI>
nucleotides using G50 and G25 Sephadex spin columns (Boehringer-Mannheim Biochemicals, Indianapolis, IN), respectively. Southern Analysis, Genomic Bacteriophage Mapping
Library
Screening,
and
Genomic DNA was prepared by lysis ofcells in SDS and proteinase K, followed by phenol/chloroform extractions and RNase A treatment, or prepared from cells encapsulated in agarose plugs as described by Fuscoe et al. (1989). For Southern analysis 10 pg DNA was digested, electrophoresed through 0.8% agarose, and transferred to Zetaprobe (Bio-Rad Chemical Division, Richmond, CA) in 10X SSC (1X is 150 mM NaCl, 15 mM sodium citrate). Prehybridization of the membrane was performed in 6X SSC, 0.25% nonfat dried milk (Carnation Co., Los Angeles, CA), 0.1% SDS at, the appropriate temperature (Table 1) and hybridization was accomplished under the same conditions with the addition of 106cpm/ml [tu-32P]dCTP-labeled probe. The RJK159 genomic library was generated from a partial Mb01 digest of genomic DNA cloned into the BamHI site of bacteriophage Charon 28; the library was propagated on LE392 cells. Bacteriophage plaques grown in agarose were transferred to Hybond N (Amersham Corp.) and the filters probed in the same way as Southern blots. Clones identified by HPRT probes were mapped for restriction endonuclease recognition sites using the method of Rackwitz et al. (1984). Inverse
PCR
The scheme for this approach is based on the technique used by Triglia et al. (1988). Genomic DNA (3 pg) from RJKO, RJK159, and RJK88 cells was digested to completion with different restriction enzymes (AluI, BglII, MaeI, MaeII, PstI, RsaI, SauSAI, or TuyI) in One-Phor-All PLUS buffer (Pharmacia LKB Biotechnology, Inc.), heated to 65°C for 15 minutes, diluted to 3 pg/ml in the same buffer (1X concentration), and then circularized overnight at 16°C with 60 U/ml T4 DNA ligase and 0.1 mM ATP. The DNA was precipitated in 0.3 M sodium acetate/70% ethanol. One microgram of the digested DNA was used in a loo-p1 PCR with oligonucleotides 1225 and 1226 derived from known exon 1 sequence but oriented such that their 5’ ends faced each other, and located on opposite strands; the 5’ ends of the primers included either an EcoRI or a Hind111 site. Amplification would only proceed if the two sites for oligonucleotide priming were present in a circular molecule. Before amplification the DNA was heated to 95°C for 30 min.
THE
CHINESE
HAMSTER
TABLE Description Probe Exon
1
Exon 1 Exons 2 and 8 Exon
4
Exon
5
Exon Exon
5 6
Exons 7-9 cDNA 7BE260 iHB80
Oligo
739
of Hybridization Description
Probes of DNA
of genomic
DNA
The PCR reaction mixture contained 50 mM KCl, 10 mM Tris, pH 8.3, 1.5 mM MgCl,, 0.01% gelatin, 200 PM each nucleotide, 1 &f each primer, and 2 U AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT), covered with 50 ~1 paraffin oil; 30 cycles of (94”C/30 s, 58”C/ 30 s, 72”C/2 min) were performed. One microliter of the PCR product was amplified further in an identical reaction to the first. The PCR was carried out in a Perkin-Elmer Cetus thermocycler using the “step cycle.” The products of the inverse PCR reactions were electrophoresed through 1% agarose and any products present in RJKO and RJK159, but not in RJK88, were isolated and digested with EcoRI and HindIII, then subcloned into EcoRI- and HindIII-digested pTZ18 and pTZ19 (Mead et al., 1986). Verification that the fragments contained exon 1 was obtained by the hybridization of oligonucleotide 739 derived from exon 1 sequence but not included in the priming molecules. Oligonucleotide sequences: 1225: 1226: 739:
249
Study
and Conditions
of Their
Hybridization temperature
fragment
?47-bp exon 5 PCR product 93bp MnlI-AluI cDNA fragment, includes 10 bp of exon 5 354.bp HindIII-Pat1 cDNA fragment 750.bp ps7stI fragment from pHPT20 (4) 260.bp BarnHI-EcoRI fragment which is the 3’ end of 7E0.9 marked in Fig. 1 80.bp BarnHI-Hind111 fragment from the middle of 7E0.9 marked in Fig. 1: it lies 5’ to 7EB260 20.mer oligodeoxynucleotide: 5’.dCACGACGCTGGGGCTGCGGG analysis
GENE
1
Used in This
107-bp NarI-BstNI cDNA fragment. includes 17 bp of exon 2 730-bp inverse PCR product 307.bp Rbul-HincII cDNA fragment, inclues 9 bp of exon 4 Xl-bp AluILMnlI cDNA fragment. includes 7 bp of exon 3 and 8 bp of exon 5 1%mer oligodeoxynucleotide: 5’.dTTCCACAATCAAGACATT-3’
Note. These probes were used in Southern Materials and Methods.
HPRT
5’-CGAAGCTTGGAGGAAGCCCGCAGAGGAG-3’ 5’-TCGAATTCGCCAGCCGACCGATTCCGTC-3’ 5’.CACGACGCTGGGGCTGCGGG-3’
Sequencing and Sequence Analysis Single-stranded template DNA was prepared from Ml3 (Messing, 1983) or pTZ (Mead et al., 1986) re-
Use Washing
48°C
2X SSC/63Y‘
5Fi”c 50°c
2X SSC/65”C 2 Y SSC/65”C
55°C
0.5~
SSC/SSY:
6~ SSC/43”C 6~ SK/room temperature 0.5x SSC/65”C 2X SSC/S!iT 55°C 55°C
2x SSC/Sfi’C 0.5~ SSC/SS”C 2x SSC/65”C
50°C
2x SSC/65”C
48°C
2x SSC/48”C
and for the screening
of the RJK159
genomic
conditions
library
(3 min)
as described
then
in
combinants containing regions from the Chinese hamster HPRT gene and sequenced manually using the dideoxynucleotide chain terminat,ion method as modified by Biggin et al. (1983) or using an Applied Biosystems, Inc., 370A DNA sequencer. To sequence through GC compressions the Sequenase Version 2 kit from U.S. Biochemical Corp. (Cleveland, OH) was used with dITP nucleotide analogs and sequencing reactions were performed at 50°C. The sequencing products were electrophoresed through 5 and 6% polyacrylamide gels in the presence of 8.5 M urea. Sequences were analyzed using the progressive sequence alignment method of Feng and Doolittle (1987), through the EuGene user interface of the Molecular Biology Information Resource (MBIR) at Baylor College of Medicine (C. Lawrence, Department of Cell Biology). Multiplex Pal-vmerase Chain Reaction and Gel Analysis In vitro amplification of 0.4 PLggenomic DNA was carried out in a 30-~1 reaction mixture of 6.7 mM MgCl,, 16.6 mM (NH&SO,, 5 mM ,0-mercaptoethanol, 6.8 PM EDTA, 67 mM Tris, pH 8.8, 10% dimethyl sulfoxide with 1.5 mM each deoxynucleotide, 133 nM to 1.67 PM each primer (Table 2), and 4 U AmpliTaq. The reaction mixture was covered with 25
250
ROSSITER
TABLE
2
Oligodeoxynucleotide Primers Used for PCR Amplification of the Chinese Hamster HPRT Gene Exon 1, 321 bp (66.7 nM)GTA CCTGGC (66.7 nM) TCCGCTCTG Exon 2. 166 (I..iOj&) (I.50 PM)
Exon
CCC AGGAGC CAC C CTGAAGAGTCCCG
hp AGC TTA TGC TCTGAT TTGAAA TCAGCT G ATTAAG ATC TTA CTT ACC TGT CCATAATC
3, 220 hp
(1.33 PM) II.33 ,uM) Exon~.19lhp (1.33 PM)
CCGTGATTT TAT TTT TGT AGG ACT GAAAG AAT GAA TTA TAC TTA CAC AGT AGC TCT TC GTGTAT
TCAAGAATATGC
ATG TAA ATGATG
(1.43 q’v4) CAAGTGAGTGAT TGAAAG CAC AGT TAC Exon F, 247 bp ( I.:&IM) AAC ATA TGG GTC AAA TAT TCT TTC TAA TAG (1.33
Exon (1.33 (1.33
Exons
&f) PM)
pM) 7 and nM) nM)
(133 (133 Exon 9, 744 (1.67&J
il.67
GGC TTA CCT ATA GTA TAC ACT AAG CTG
6. 145 hp
pM)
TTA CCA CTT ACC ATT AAA TAC CTC TTT TC CTA CTT TAA AAT GGC ATACAT ACC TTG C
8. 423 hp GTA ATA TTT TGT AAT TAA CAG CTT GCT GG TCA GTC TGG TCA AAT GAC GAG GTG C hp CAATTCTCTAATGTTGCT CTTACC TCTC CAT GCA GAG TTC TAT AAGAGA CAGTCC
Note. The sequence (5' to 3') of each primer with the final concentration in the PCR and resulting PCR product.
is shown the
length
together of the
~1 paraffin oil and subjected to 30 cycles of (94”C/l min, 58”C/l min, 7O”C/7 min), preceded by 5 min at 94°C and followed by 13 min at 7O”C, then incubation at 4°C. The reaction products (lo-15 ~1) were then electrophoresed through 4% agarose [3 parts NuSieve (FMC BioProducts, Rockland, ME), 1 part SeaKem (FMC BioProducts) ] for analysis. Reaction conditions for amplification of exon 1 alone were identical to the multiplex conditions except that the temperature cycle parameters were 30 cycles of (94’C/l min, 6O”C/l min, 7O”C/45 s), preceded by 5 min at 94°C and followed by 19 min at 70°C.
ET
AL.
XCH-HPT 12 using both exon 5 and exon 6 probes against the genomic library. The first exon and a large part of the first intron was not successfully isolated from two different RJK159 libraries, using a variety of exon l-specific probes, but the regions flanking exon 1 were eventually isolated by the alternative method of inverse PCR (see below). The bacteriophage clones were digested to completion with the restriction endonucleases BamHI, BglII, EcoRI, HindIII, and PstI and the products carefully compared with standard markers to determine their size. Fragments containing coding regions were identified by Southern analysis using exon-specific probes as described in Table 1. Restriction enzyme recognit.ion sites for the five selected enzymes were determined by the mapping procedure described under Materials and Methods. If there was any discrepancy bet,ween the two methods in determining the distance between restriction sites, the size obtained by comp1et.e digestion was taken to be more accurate. The data are collated in Fig. 1. The location of each of the exons was determined either by their position relative to one of the restriction endonuclease sit.esin the map or by their position relative to a different restriction endonuclease recognition site and determination of the location of that other site relative to one of the mapping sites by means of double digestions and Southern analysis. Exons 1,4, and 6 were located by the latter procedure; exon 1 lies 444 bp 5’ of a Sac1 site, exon 4 lies 25 bp 3’ of an NsiI site, and exon 6 lies 2 bp 3’ of a nra1 site. Restriction fragment sizes predict,ed by the map shown in Fig. 1 were confirmed by Southern analysis of genomic DNA from RJKO and RJK159 cells, probed with HPRT cDNA or exon-specific probes (Table 1). Figure 2B shows a schematic of the exons contained within the bands observed in a Southern (Fig. 2A) when HPRT cDNA is used as a probe; exons 1 and 5 are not usually seen under these conditions because of the short length of homology with the probe. Exon
RESULTS
Restriction Chinese
Map and Southern Analysis Hamster HPRT Gene
of
the
HPRT cDNA was used initially to screen the RJK159 genomic library, resulting in the isolation of the clones XCH-HPT 1,2,5, and 7 (Fig. 1). On hybridization with exon-specific probes (Table 1) these clones were found to contain exons 2-9, but restriction mapping revealed that a port,ion of the gene between exons 5 and 6 was not represented. The gap between exons 5 and 6 was closed by isolation of clone
1 Cloned
hy Inverse
PCR
The first exons from the RJKO and RJK159 HPRT genes were isolated by the inverse PCR method described under Materials and Methods. Eight, restriction endonucleases were used in the attempt to clone the Chinese hamster HPRT exon 1 (AU, BglII, MaeI, MaeII, PstI, RsaI, SauSAI, and TayI), the majority having 4-base recognition sequences, and none having a (X-rich recognition sequence (since the promoter region was expected to be (X-rich and would therefore be digested by such enzymes). The inverse PCR was deemed to be successful if a fragment was generated from R?JKO and RJK159 cells but was ab-
THE
I
I I
I
I
HAMSTER
I 1
1
L
1
I
II
I, 1
II
2
I I 3
I
I
I
1
I
BglTI
I
I EcoKI
I
,
4
I I
I
I
1
251
(;ENE J BumHI
I I
/;;\
I
HI’RT
I
,*,
I
12
CHINESE
I
II 5
I
HmdIIl
I I II 1 6
78
9
PstI
EXONS
KH-HFTU2 KH-HFT#I2 ICH-HPT#I ICH-HFT#S XCH-HIT-#1 FIG. 1. Restriction map of the Chinese hamster HPRT gene. The location of the enzymes BarnHI, J&$11, EcoRI, HindIII. and PstI are shown. together with the positions clones from which exons 2-9 were isolated. The positions of the restriction sites around known whether there are additional sites within the dotted regions of the B&II and P&I two sites close together or a single site. The 900.bp EcoRI fragment indicated by an analysis descrihedin Fig. 2 and elsewhere. .
sent in RJK88 cells (where the HPRT gene is deleted). Only the enzymes AluI and RsaI yielded exon 1 fragments from this procedure and since the AluI fragment was contained within the RsaI fragment it was not investigated further. The 730-bp inverse PCR fragment subcloned into pTZ18 and pTZ19 vectors contained a unique RsaI site which marked the joining of the 5’ and 3’ flanking sequences. Since the fragment was inverted with respect to the gene organization, sequencing in from the ends actually corresponded to sequencing outward from exon 1 in both directions. Sequencing of the region 3’ to exon 1, in the first intron, proved particularly difficult because of strong stop signals and GC compressions. These problems were eventually overcome by the technical modifications described under Materials and Methods. The entire fragment was sequenced with the use of additional oligonucleotides as sequencing primers. Figure 1 indicates the genomic fragments t.o which the exon 1 inverse PCR product hybridizes. These Southerns and those performed with probes from within the 900-bp E’coRI fragment allowed the posit,ioning of the first exon with respect to the rest of the gene, since a large BumHI fragment (13.5 kb) includes both exon 1 and the 5’ portion of the 900-bp EcoRI fragment from intron 1. Sequences from the Chinese Hamster
HPRT
Gene
Small fragments (a few hundred base pairs) containing HPRT exons 2-9 were isolated from the bacteriophage clones shown in Fig. 1, subcloned into Ml3 or pTZ vectors, and used as templates for sequencing the regions flanking the exons. The sequence deter-
restriction endonuclease recognition sites lor the of the exons and the extent of the bacteriophage exon 1 are deduced from Southern analysis. It is not maps. The two f’stl sites enclosed by an oval may be asterisk was used to generate probes for Southern
mined, deposited with the EMBL Data Library (ACcession Nos. X53073-80), is from the RJK159 cell line which contains one point mutation within exon 6; the wild-type sequence at this position was identified by sequencing an HPRT mutant with a different point mutation (Rossiter et al., 1990). Since the fragment containing exon 1 was generated via the PCR, it was possible that the sequence might contain polymerase errors. For each cell line (RJKO and RJK159) three clones were sequenced in each direction (13 in total) and whenever there was a difference between clones, the sequence found in two of the three was taken as being correct. In all, there were three PCR errors found in the i’30-bp fragment and no differences found between the two cell lines. Comparison of Chinese Hamster, HPRT Sequences
Mouse, and Human
The complete human HPRT locus has been sequenced (Edwards et al., 1990) and some mouse HPRT sequence has been reported (Melton et al., 1984); this information provided the possibility of comparing three mammalian species in terms of their sequence conservation within the HPRT gene. Figure 3 shows the alignment of human, Chinese hamster, and mouse sequences around exon 1 and the promoter region. Although the coding region (marked within the large box) and the few bases on either side are quite homologous, the degree of identity between the human and the Chinese hamster sequences and between the human and the mouse sequences over the whole region is less than 40%. Very little flanking sequence from the mouse HPRT gene has been published except for the exon 1
252
ROSSITER
A.
B
Bg E
H
4.31
probe: cDNA
B. E 16.X-6-9 13.6-2-4
6.6-y 5.093
4.6-
of the Chinese
Hamster
9.0 2 8.2 =6-g 6.6-d 5.2-y
3
3.5-v
3.3-3 2.1~w
3.0-2 2.2-9 1.9-v 1.6-7-S 1.2-3
Anal.ysis
P
H
;;I;=;;
PCR Gene
PCR primers for the multiplex amplification of Chinese hamster HPRT exons were originally selected to generate fragments of different sizes which could be resolved by agarose gel electrophoresis (Table 2). Exons 7 and 8 are amplified on a single fragment since they are only 165 bp apart. When possible the primers were located beyond t.he immediate splice sites so that these regions would be amplified together with the exons. The oligonucleotides were designed to share similar melting temperatures, therefore those with a higher AT content are longer than the others. Although all primers designed were able to amplify individual exons, not all were able to participate in the multiplex reaction successfully; the set of 16 described in Table 2 derives from a larger set of 38 oligonucleotides actually synthesized. It was never possi-
23.lkb 9.42 6.56
Bt?
AL,
Multiplex HPRT
P origin
B
ET
1.2-y
FIG. 2. Southern analysis of the Chinese hamster HPRT gene using cDNA and exon-specific probes. (A) Sout,hern analysis of V79S DNA digested with BarnHI (B), R~111 (Bg). EcoRI (E), Hind111 (H). and PstI (P) and probed with HPRT cDNA (Table 1). (B) The schematic illustrates the hands identified by a cDNA probe on a Southern blot containing wild-type genomic DNA digested as in (A). The size of each hand in kilobases is shown on its left and the exons represented within it on the right. The symbol $ represents pseudogene fragments. The second largest RamHI fragment is a doublet and the 4.6-kh &$I1 fragment is a triplet. The sizes of the larger fragment.s were determined from Southern gels which were run longer than those shown here, for bett,er resolution. These exon assignments were predicted from the restriction map in Fig. 1 and confirmed by Southern analysis using exon-specitic probes (data not shown).
region so sequences surrounding the remaining eight exons were only compared between human and Chinese hamster. Such analysis of the sequences around exons 7 and 8 is shown in Fig. 4 and in contrast to the region around the first exon, the degree of identity over the whole region shown here is greater than 80%. Comparison of human and Chinese hamster sequences from around the other coding regions of the HPRT gene also showed considerable intron homology (analysis not shown).
FIG. 3. Comparison of human, Chinese hamster, and mouse HPRT sequences around exon 1. Sequences from the 5’ regions of the human, Chinese hamster, and mouse HPRT genes are aligned using the computer program described under Materials and Methods. The large box is drawn around the coding region of exon 1 and smaller hoxes are drawn around the sequences .5’-CGCGGG-:I’.
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FIG. 4. Comparison of human and Chinese hamster HPRT sequences around exons 7 and 8. Sequences from exons 7 and 8 ofthe human and Chinese hamster HPRT genes are aligned as in Fig. 3: boxes are drawn around the coding regions.
ble to combine several combinations of exon 1 primers with the remaining set of primers and so the multiplex PCR includes exons 2-9; exon 1 is amplified in a separate reaction. Figure 5 shows the multiplex seven-component reaction and the exon 1 amplification, used to screen a series of HPRT cell lines including two HPRT partial deletion mutants. In one of the mutants (UCC-15, lane 3) exon 4 is missing; in the other (UCC-19, lane 4) exons 4,5, and 6 are missing. Included in the analysis are wild-t,ype cell lines (RJKO and UCC-1, lanes 1 and 2) and cell lines with complet,e deletions of the HPRT gene (RJK88 and UCC-16, lanes 5 and 6).
HPRT
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likely to be several hundred kilobases (Stark and Wahl, 1984). There is considerable evidence that the amplified HPRT gene characterized from the RJK159 cell line is ident,ical (apart from the exon 6 point. mutation) to that in wild-type RJKO and V79S cells and other Chinese hamster cell lines. First, Southern analysis indicates that the gene structure is identical at least as far as the extent of the largest fragment recognized by the cDNA probe (Fig. 2). Second, the region surrounding exon 1 was sequenced from both RJKO and RJK159 cells and was found to be identical in the two cell lines. Third, primers for PCR amplification of individual exons based on sequence from the RJK159 HPRT gene are able to amplify exons from RJKO and Chinese hamster ovary (CHO) cells (Fig. 5; Xu et al., 1989). Comparison Hamster,
of HPRT Genes between Human, and Mouse
Chinese
The overall structure of the HPRT gene in Chinese hamster cells (Fig. 1) is similar to that in human and mouse cells; the first int,ron is the largest, exons 7, 8, and 9 lie very close together, and the positions of the
DISCUSSION Development of a restriction map for the Chinese hamster HPRT gene in RJK159 cells has led to the isolation and sequencing of all nine exons and their flanking regions, and the development of a multiplex PCR deletion screen. This information allows a more accurate assessment, of Southern experiments since the content of each HPRT band is defined; the multiplex PCR screen permits a rapid analysis of HPRT deletion mutants and the generation of specific fragments of the HPRT gene for further analysis. HPRT Gene Structure Is the Same in Different Chinese Hamster Cell Lines Since the RJK159 cell line contains an amplified HPRT locus, it is important to demonstrate that the gene structure has not been altered as a result of the amplification process. Alteration of the gene structure is not expected since the amplification unit is
I?.
-9 -7&8 l-5 -3 -4 -2 -6
FIG. 5. Screen of wild-t?ipe cells and HPRT-deficient mutants hy multiplex PCR analysis. (A) The left-hand group of reactions was performed with the primers for amplification of exon 1 of the Chinese hamster HPRT gene (Table 2). The right-hand group of reactions was performed with the multiplex primers for the simultaneous amplification of exons 2-9 of the Chinese hamster HPRT gene (Table %). Lanes: (M) WarIII-digested 4X171 markers; (1) RJKO; (2) [ICC-l; (3) UCC-15; (4) IICC-19; (5) (JCC-16; (6) R.JKSX; (7) no DNA. (B) Schematic indicating the location of the individual exon hands wit,hin the multiplex amplification reaction.
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intron/exon boundaries are conserved exactly between all three species, The size of 36 kb is intermediate between the mouse gene (about 34 kb) (Melton et al., 1984) and the human gene (39.8 kb) (Edwards et al., 1990). The human, mouse, and Chinese hamster HPRT genes lack consensus CAAT and TATA sequencesimmediately 5’ to the initiation site and the region around the first exon contains several 5’-GGCGGG-3’ promoter elements. These are both features of ubiquitously expressed housekeeping genes, such as phosphoglycerate kinase (Singer-Sam et al., 1984), adenosine deaminase (Wiginton et al.. 1986), adenosine phosphoribosyltransferase (Broderick et al., 1987), lysosomal acid phosphatase (Geier et al., 1989), 5-lipoxygenase (Funk et al., 19891, and dihydrofolate reductase (Means and Farnham, 1990). Figure 3 shows that, apart from the coding region of exon 1 and the immediate flanking sequences,there is not a great deal of homology between the human, the mouse, and the Chinese hamster 5’ HPRT sequences. Despite this fact, the locations of the GC element,s appear to be conserved in two clusters, one about 200 bp 5’ to the initiation codon and the other about 150 bp 3’ to the first exon. This would suggest that these GC “boxes” indeed have some functional importance and raise the possibility that the “promoter region” of the mammalian HPRT gene extends both 5’ and 3’ of the first exon. Coding regions between the three speciesare highly conserved, as are the immediate flanking regions which form the splice signals (Fig. 4). The intron homology around exons 2-9 is greater than that observed around exon 1 but it is not known whether such homology may reflect functional importance, perhaps for splicing, or merely similarity because of a common ancestry. A similar phenomenon of intron homology between mammalian species has been observed in N- and o-globin genes (Hardison and Gelinas, 1986; Hardies et al.. 1984; Hardison, 1984) and y-crystallin genes (den Dunnen et al., 1989). It is also not known in these cases if similarity reflects a possible functional importance.
Multiplex
L3eletion Screen
The multiplex PCR deletion screen for the Chinese hamster HPRT gene can yield more precise information about the endpoints of a deletion than Southern analysis since the presence of each exon can be determined individually (apart from exons 7 and 8, which lie very close together). In contrast, a band on a genomic Southern blot often represents several exons (Fig. 2). The multiplex PCR deletion screen (Fig. 5) is quicker to perform than Southern analysis, taking
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only a few hours for both the reaction and the gel analysis, and avoids the use of radioisotopes. The PCR analysis does not yield any amplification products when RJK88 DNA is used as a template; this is expected since the entire HPRT functional gene is absent in RJK88 cells (Fuscoe et al., 1983). There are, however, HPRT pseudogene sequences in these cells (the genome location is unknown) and the fact that no amplification products are seen would suggest that the pseudogene in these cells is in a processed form, lacking intron sequences. This is in agreement with the studies of Isamat et al. (1988) which suggest,that the processed mouse HPRT pseudogene shares a common origin with the Chinese hamster HPRT pseudogene. There is also evidence to suggest that the four human HPRT pseudogenes (with autosomal locations) are in the processed form (Pate1 et al., 1984). The multiplex oligonucleotide primers used in this study are made partly or completely from intron sequences so they would not be expected to hybridize t.o cDNA or processed pseudogene sequences. It is possible to sequence PCR products directly without the need for subcloning and thus detect point mutations in addition to deletions (Gibbs et al., 1990). Ideally the PCR primers should be located several nucleotides from the exon/intron boundary to permit sequencing of the entire exon and its flanking region. It should be not.ed however that because of the limited sequence available the oligonucleotide primers used for amplification of the Chinese hamster HPRT gene were designed primarily to generate fragments of different sizes to permit resolution of the products by agarose gel electrophoresis and 4 of 16 of the primers contain some exon sequence. This raises the possibility that the absence of a band in the multiplex PCR could result from a mismatch within the primer binding site rather than an exon deletion although if more than one band is missing from the multiplex reaction, a deletion including the appropriat,e exons is the most likely explanation. If only one band is missing, an additional PCR could be performed with different priming oligonucleotides further from the exon boundaries; if amplification were observed a point mutation would be the most likely explanation and the PCR product could be sequenced; lack of amplification would suggest a deletion affecting that particular exon. The region around exon 1 is much more CC-rich than the rest of the gene and may be a reason why exon 1 primers were incompatible with the rest of the multiplex reaction. The addition of even small amounts of exon 1 primers to the seven-component multiplex resulted in either spurious additional products or loss of one or more of the other products. The ability to amplify individual exons of the Chinese hamster HPRT gene provides a means of gener-
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ating exon-specific fragments larger than those engineered from cDNA (Table 1) and yet small enough not to include repetitive intron sequences which can interfere with hybridization. Such fragments have t.he potential to be useful as probes in Southern analysis, particularly in cases of’ gene rearrangements which may elude detection by multiplex PCR.
HPRT 11.
ELKIND, M. M.. AND SUTTON, H. (1960). Radiation response of mammalian cells grown in culture. I. Repair of X-ray damage in surviving Chinese hamster cells. Radiat. Res. 13: 556593.
12.
FARRELL, S. A., AND WORTON, R. G. (1977). Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids. Somatic Cell Genet. 3: 539-551.
13.
FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique radiolabeling DNA restriction endonuclease fragments high specific activity. Anal. Biochem. 132: 6-13.
14.
FEINBERG, A. P., AND VOGELSTEIN, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Hiochem. 137: 266267.
15.
FENG, D. F., AND DOOLITTLE, R. F. (1987). Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25: 351-360. FENWICK, R. G.,
