A physical map of the human PI and AACT genes

GENOMICS 7,382-388 (1990)

A Physical Map of the Human P/ and AACT Genes

LOUISE SEFTON,* GAVIN KELSEY, t PHILLIP KEARNEY,**’ SUSAN POVEY,$ AND JONATHAN WOLFE*

*Department of Genetics and Biometry, University College London, 4 Stephenson Way, London NW1 ZHE, United Kingdom; tlnstitute of Cell and Tumour Biology, German Cancer Research Centre, Im Neuenheimerfeld 280, D-6900 Heidelberg,

Federal Republic of Germany; and SMRC Human Biochemical Genetics Unit, Galton Laboratory, 4 Stephenson Way, London NW1 2HE, United Kingdom

Received December 18, 1989; revised March 12, 1990

We have used probes from the human genes PI, PIL, and AACT (aI-antitrypsin, cwl-antitrypsin-related sequence, and al-antichymotrypsin) to make a pulsed- field map of the surrounding region of 14q31-32. We have discovered that the PI-PIL gene cluster is only 220 kb away from the AACT gene and that it is orientated in the opposite direction. The comparatively short distance between the genes comes as a surprise given previous estimates of the level of genetic recombination between them. @I iseo Academic pm. IDC.

(Kelsey et al., 1987; Bao et crl., 1988, Hofker et cd., 1987). The PZL sequence is intriguing in that alteration in the active site suggests an altered function (Bao et al., 1988); there is however no firm evidence that this gene is expressed and it seems most likely to be a pseudogene (Kelsey et al., 1988b; Hofker et al., 1987).

INTRODUCIION

al-Antitrypsin (PI or AAT) and al-antichymotrypsin (AACT) are the most closely related members of a large family of serine protease inhibitors (“serpins”). Other serpins found in human plasma include antithrombin III, Cl-esterase inhibitor, plasminogen ac- tivator inhibitor, and angiotensinogen. These proteins have common features of tertiary structure, functional domains, and reactive sites. PI and AACT have 42% homology at the protein level (Chandra et al., 1983), and the genes determining these proteins have identical organization and intron-exon junctions (Bao et al., 1987). Both genes have been mapped to the region 14q32.1 in man (Rabin et aZ., 1986), and it seems likely that the divergence of these two loci is a relatively re- cent event (Bao et al., 1987). Although in man each protein is coded by a single gene, the situation in mice is more complex, with clusters of expressed genes both for PI and for contrapsin (the mouse equivalent of AACT) on mouse chromosome 12 (Hill et al, 1985). In man no other functional protease inhibitor genes have been identified in 14q32, although sequences showing extremely close homology to ai-antitrypsin, designated PZL, have been identified about 10 kb 3’ to the PI gene

Family studies have shown close genetic linkage between PI and AACT. We (Kelsey et al., 1988a) have observed only one recombinant in at least 35 infor- mative meioses (2 = 2.29 at 8 = 0 in males and 2 = 6.11 at 0 = 0.032 in females), and Cox et al. (1989) have also reported a maximum lod score of 12.17 at 0 = 0.03 (sex not recorded) in another study (in which one family, not showing recombination, was in common with our study). Very strong linkage disequilibrium between protein and DNA polymorphisms has been observed in a region of about 25 kb extending from 5’ of PI to the PZL locus, where there is a common 2-kb deletion (Cox et al., 1985; Povey, 1989).

We and others have previously attempted to make physical maps of the region using pulsed-field gel electrophoresis and these results were reported in abstracts (Kearney et al., 1987; Cox et al., 1987). We present here a much more complete investigation, based on inde- pendently obtained results from two different cell lines, which has allowed us to produce a definitive physical map of the PI and AACT genes.

METHODS

DNA Preparation

High-molecular-weight DNA in agarose blocks was prepared essentially by the method of Smith et al. (1987) from the human lymphoblastoid line OXEN (Bishop et aZ., 1983) and from the human hepatoma cell line Hep G2 (Knowles et al., 1980).

Restriction Digests

1 Present address: St. Vincent’s Hospital, Sydney, New South In the case of OXEN DNA, restriction digests on Wales, Australia. approximately 0.5 Kg of DNA (one-third of a block)

o&w-7543/90 $3.00 Copyright 0 1990 hy Academic Press, Inc. AI1 riehts of remoduction in anv form reserved.

382

PHYSICAL MAP OF THE HUMAN PI AND AACT GENES 383

were carried out in 150 ~1 of restriction buffer with 20 units of enzyme for 4 h. For double digests, blocks were rinsed in TE and incubated for a further 4 h with 20 units of the second enzyme in the appropriate restriction buffer. The incubation temperatures and buffer compositions were as recommended by the manufacturers, with the exception that 1 mM spermidine was included in buffers having an NaCl concentration of 50 mM or greater. Digests were terminated by the ad- dition of 50 ~1 of loading buffer (7 M urea, 50% (w/v) sucrose, 0.005% (w/v) bromphenol blue, and xylene cyanol).

In the case of Hep G2 DNA, whole blocks containing approximately 2 pg of DNA were used for digests which were performed in 120 ~1, using up to 20 units of enzyme for a minimum of 4 h. No spermidine was used. After digestion, EDTA was added to 20 mM and blocks were stored briefly on ice before loading. No loading buffer was used.

Electrophoresis and Blotting

Field-inversion gel electrophoresis (FIGE) was performed in a Pharmacia gel tank with platinum elec- trodes. The gels were 1% agarose with 0.5X TBE electrophoresis buffer (Maniatis et al., 1982) and were run for 16 h at 9°C and 10 V/cm. A linear time ramp, with forward times ranging from 3 to 60 s and a forward to reverse ratio of 3:1, was controlled by a BBC B+ mi- crocomputer (paper in preparation). Pulsed-field gel electrophoresis (PFGE) of OXEN DNA digests was performed in an LKB Pulsaphor apparatus using a hexagonal electrode array for 36 h at 170 V, with a pulse time of 50 s and at a temperature of 10°C. The DNA was separated in a 1% agarose gel in 0.1 M TBE buffer (Pulsaphor manual). PFGE of Hep G2 digests was carried out in a similar apparatus using 20 X 20- cm gels cast and run in 0.25X TBE. Several running conditions were used and these are detailed in the leg- ends to Table 2 and Fig. 5. On the OXEN gels size markers were yeast chromosomes from Saccharomyces cereuisiae (strain A364a) and concatemers of X gtWES.B DNA (monomer size 40.4 kb). On the Hep G2 gels, size markers were concatemers of X ~I857 (monomer size 48.5 kb). Because the relative electro- phoretic mobility of the size markers differed between the FIGE and PFGE, sizes for mapping the region around the PI family were taken only from the PFGE blots relative to the X concatemers. After electrophoresis, OXEN gels were blotted onto a GeneScreen plus nylon membrane in alkali, and Hep G2 gels were transferred to Pall Biodyne B membranes in 20X SSC according to the respective manufacturer’s instructions.

Probes

The restriction maps of the cloned regions around PZ, PZL, and AACT are shown in Fig. 1. There was no

pAT4.7 psP64wA - -

4kb c1

HH H

pACE3.4 -

FIG. 1. Outline restriction maps of the cosmid contigx containing the PZ,PZL, and AACT genes showing the positions of the subcloned fragments pAT4.7, pSP64RSA, and PACE used as probes in these experiments. E, EcoRI; H, HindIII; S, SalI. The upper line represents the PZ,PZL contig and the lower line represents the AACT contig. Exons I to V of PI are shown as solid blocks, exons II to V of PZL are shown as open boxes, and exons I to V of AACT are shown as solid blocks. All genes are oriented with their 5’ ends to the left.

overlap between the two groups of clones. The probe for the PI gene (pAT4.7) is a 4.7-kb EcoRI fragment including exon I (Kelsey et al., 1987). Approximately 25 kb downstream from the pAT4.7 probe is the PZL gene. A l.l-kb P&I/Hind111 fragment (SP64RSA) (Kelsey et al., 1988b) was used to identify this locus. However, this probe contains a short region (exon II) of homology to the PI gene. A 3.4-kb EcoRI fragment (pACE3.4) spanning exon 2 of the gene was used as a probe for AACT (Kelsey et al., 1988a). Probes were labeled with [(r-32P]dCTP using essentially the protocol of Feinberg and Vogelstein (1983). Hybridizations were carried out using the method of Church and Gilbert (1984) without the inclusion of bovine serum albumin. Blots were autoradiographed for up to 14 days with Amersham Hyperfine or Kodak X-Omat AR film at -70°C. Probes were stripped with NaOH according to the membrane manufacturers’ instructions and the fil- ters were autoradiographed again to ensure complete removal.

RESULTS

Linkage of PI and AACT by FZGE

Linkage of PZ and AACT was demonstrated by the cohybridization of their respective probes to a number of fragments of differing sizes in single digests of OXEN DNA (Fig. 2). Both AACT and PI probes hybridized to a 600-kb Not1 fragment and to 600- and 490-kb NruI fragments. Smaller NruI fragments were not shared. EC& and MluI had partial digest fragments of 490 and 330 kb, respectively, common to both probes. In ad- dition, for these two enzymes the sum of the complete digestion fragments for PI and AACT was equal to the length of the first partial digest common to both probes. This indicates that there are single partially digesting

384

b) C) = PI AACT

j i

SEFTON ET AL.

PIL PI AACl

FIG. 2. A field-inversion gel (a) of digests of OXEN DNA, blotted and probed with pAT4.7 (b) andpACE3.4 (c). Size markers were yeast (Sacchuromyces cereuisioe) chromosomes, X gtWES.B oligomers, and X ~I857 monomer plus its digest with Hind111 (only the 48.5- and 23.1-kb bands show up clearly on the photograph of this lane).

sites for each of these enzymes that separate the two loci. The 330-kb MZuI fragment was the smallest band cohybridizing to PI and AACT probes, and this enzyme was therefore used in double digests to map the region common to the two genes.

Mapping AACT, PI, and PIL within the Common MluI Fragment

The physical map of the region including PI and AACT was constructed using double digests of BssHII, MU, SalI, and SfiI (Figs. 3 and 4a and Table 1). The approximately 350-kb MZuI fragment common to AACT and PI consisted of two complete digest fragments: a 260-kb PI fragment and an 80-kb AACT fragment. The 260-kb fragment recognized by pAT4.7 was not further reduced by digestion with SalI, which also gave a fragment of very similar size with this probe. The MZuI and Sal1 fragments are reduced by BssHII to 130 and 125 kb, respectively, supporting the separation of one pair of MluI and Sal1 sites by 5 kb and, in combination with the Sal1 and MluI double digest result above, suggesting that the other pair of sites is coincident. pAT4.7 hybridizes to a number of SfiI fragments including lo-, 65-, and 80-kb fragments. Se- quence data (Long et al., 1984) predict that an SfiI site exists within the pAT4.7 probe. However, this site was not cleaved in the OXEN genomic DNA as shown by double digestion with EcoRI and SfiI followed by con-

FIG. 3. A pulsed-field gel of digests of OXEN DNA, blotted and probed with pSP64RSA (PIL), pAT4.7 (PI), and pACE3.4 (AACT). Size markers were as for Fig. 1 and are not shown; however, fragment sizes are given in Table 1.

ventional gel electrophoresis and Southern blotting (data not shown). After double digestion with BssHII, the 65- and 80-kb SfiI fragments were reduced to 50 kb. This locates the PI probe to a lo-kb region within the 260-kb MluI fragment. The orientation of PI was determined by probing with the PIL probe SP64RSA, which is downstream of PI. This probe reveals fragment sizes identical to those given by pAT4.7 except for digests involving SfiI. A 165-kb SfiI fragment, which is recognized by SP64RSA but not by pAT4.7 and which is not cleaved by BssHII, positions PIL to the left of

pSP64RSA PAT., 7 1Whb

b)

pAT4 7 p*cE,3 4

b IB) w:: i%

M SNOS Sf su.4 ai? es‘s‘ S‘Ss2 s T,i Sf8 “e” M M

FIG. 4. Restriction maps drawn (a) from the data of Fig. 3 and Table 1 and (b) from the data of Fig. 5 and Table 2. B, BssHII; M, Mluk No, NotI; Nr, NruI; S, SalI; SC, S&I; Sf, SfiI. Sites in parentheses cut only partially. Also shown above map (a) are the extents of the contigs drawn in Fig. 1, the approximate extents of the genes, and the positions of the probes. The positions of the probes used to make map (b) are repeated above it. While the maps show every restriction site that we have deduced from the data, they are of course incomplete in the sense that sites beyond those that cut to completion are invisible to this analysis. These may include some BssHII and Sal1 sites between PI and AACT as well as many sites outside them.


TABLE 1

The Restriction Fragment Sizes Measured from Fig. 3

PIL

250 225

BssHII MluI Sal1 w

PI AACT PIL PI AACT PIL PI AACT PIL PI AACT

250 355 350 350 350 255 255 70 165 (190) (195) 225 215 260 260 80 (10) (135) (175) 6-W 230 (18’3 80 135

65 10

BssHII + MluI BssHII + Sal1 BssHII + SfiI MluI + Sal1

PIL PI AACT PIL PI AACT PIL PI AACT PIL PI AACT

250 250 125 125 165 (190) 135 255 255 70 225 225 230 (6% 70 50 105 (180) 30 130 130 105 (10) 10

(65)

MluI + SfI Sal1 + SfiI

PIL PI AACT PIL PI AACT

135 030) 85 80 85 80

65 65 30 (10) 10 10

Note. PIL, probe pSP64RSA, PI, probe pAT4.7; AACT, probe pACE3.4. Fragments in parentheses ( ) were very weak.

PI (as the map is drawn in Fig. 4). This conclusion is verified by the MluI plus SfiI and Sal I plus SfiI double digests which bisect this SfiI fragment. Furthermore, these digests confirm theseparation of this pair of Sal I and MluI sites by 5 kb.

The smaller MluI fragment hybridizing to the AACT probe, pACE3.4, could be positioned at either end of the 260-kb MluI fragment hybridizing to pAT4.7 and SP64RSA. However, if it were on the left, then pACE3.4 would necessarily hybridize to the same BssHII fragment as pAT4.7 and SP64RSA and to the same SfiI fragment as SP64RSA. It does not. Therefore it must be on the right of the map. pACE3.4 also hybridizes to a 70-kb Sal1 fragment which is reduced to 30 kb on double digestion with MluI. The Sal1 fragment is also reduced to 30 kb by double digestion with SfiI but is not cleaved by BssHII. A pair of Sal1 sites is located just upstream of pACE3.4 (Fig. l), and at least the nearer of the two sites is cleaved in OXEN genomic DNA (data not shown). Therefore this probe can be positioned exactly and the orientation of AACT can be deduced.

Thus the two genes PI and AACT are separated by 220 f 5 kb and are orientated in opposite directions with their 5’ ends pointing toward each other.

A Restriction Map from a Second Cell Line

We have also prepared a map from a second cell line (the human hepatoma derived cell line Hep G2). In this case we have used three additional restriction enzymes, SacII, NruI, and Not1 (Table 2). The Hep G2 blots were probed with pACE3.4 and pAT4.7. Figure 5 shows the results obtained from one gel, and Table 2 gives the estimated band sizes of all the fragments deduced from measurements of five gels. Many restriction fragments were common to both probes, some enzymes (e.g., MluI and NotI) gave only common fragments, and other enzymes (e.g., SacII) gave some common and some probe-specific fragments. This result strengthens the conclusion of the close physical linkage inferred from the OXEN data. However, as with OXEN DNA, Sal1 and BssHII gave only probe-specific fragments. In general, the digestions were less complete than those for OXEN, which may be a consequence of more DNA being digested with less enzyme. Whatever the reason, one benefit is that the map extends to a greater distance from the probes, and a second benefit is that the use of additional enzymes leads to the iden- tification of the nearest putative CpG islands. This map and the OXEN map were produced completely inde- pendently and agree remarkably well although there are several restriction site changes between the lines.

386

BssHII

AT ACE

SEFTON ET AL.

TABLE 2

The Restriction Fragment Sizes Measured from Hep G2 Digests

MluI Not1 NTUI Sac11 Sill1 SfiI

AT ACE AT ACE AT ACE AT ACE AT ACE AT ACE

260 225 limit limit 65 115 600 600

535 535 355 355 310 310

BssHII 260 225 100 150 65 100

620 620

260 225 65 115

700 700 limit limit 620 620 620 620 535 535 535 535 490 490 490 490 430 430 430 430 355 355 355 355 310 310 310 310

Not1

620 620 540 540

(375) (375)

(620) (540) (455) 410 375 245

(620) (540) (455)

465 425 310 275

375 210 130

260 225 260 (210) 260 (120) 145 (120) 150 140

65 120 65 125 65

(540) (540) (490) (490) (450) (450) 375 375 355 355

(330) (330) (310) (310) 255 130 235 80

465 425

(310) 275 235

620 620 540 540

(375) (375) '540' 455 375 245

(620) 540 455 375 (185) 130

465 425 310 275

NruI (620) 540

(455) (410) 375 245

Sac11

(620) 540

(455)

375 130

465 425 310 275

465 425 300 260

Sail

(125) 75

(125)

(Ii,

(125) 75 30

125 75

(125) 75

75

285 260

170 130 85 60 10

(170) 65 50 20 10

(285) 260

(170)

130 85 60 11

(285) (260) 170 130 80 60 11

285 260 170 130 85 60 11

m5) (26’3 170 130 85 60 11

070) 130 85 60 10

285 260 200 170 145 125

125 100

(285) 260 200 170 145 125

(285) (260) 170 145 125

285 260 200 170 145 125

(285) (260) 200 170 145 125

70 35

Note. Fragment sixes in kb measured from five pulsed-field gels of Hep G2 digests. Gels were run as described in the text. Several pulse time ramps were used ranging from a 5- to 36-s linear ramp of reorientation frequency over 36 h (resolving 10-300 kb) to a 15- to 90-s linear ramp over 46 h (resolving 50-700 kb). Most digests were run under at least two sets of conditions. These figures are the consensus values over the whole set of gels only one of which is shown in Fig. 5. AT, probe pAT4.7; ACE, probe PACE; limit, band unresolved at the limiting mobility of the gel; ( ), very faint band.


a) PI b) AACT

FIG. 5. A pulsed-field gel of digests of HepG2 DNA, blotted and probed with pAT4.7 (a) and with pACE3.4 (b). Size markers were Xc1857 oligomers which were run in the two outside lanes. Pmbe pAT4.7 hybridizes weakly to XcI857, which can therefore be seen in

(a).

DISCUSSION

We have physically mapped a region of almost 1000 kb from bands 14q31-32 and have shown that within this region the AACT gene is 220 kb upstream of the PI,PIL gene cluster and oriented in the opposite direction. We, and others (Kearney et al., 1987; Cox et aL, 1987), have previously published incorrect maps of this region because we were misled by some very similar fragment sizes for the enzyme BssHII when probed with both PI and AACT probes (see Fig. 3). Two factors have helped us to improve our analysis. First, the change from FIGE to PFGE has sharpened the bands on the gels and has improved the resolution. Also the use of a second cell line has allowed us to construct a “consensus” map of sites in common between the two lines, which has confirmed the separation between the two loci. Equally important are the restriction site differences between the two cell lines that do not lead to any inconsistency between the two maps despite lead- ing to a number of different restriction fragment sizes between the two lines. The restriction site differences observed might represent true polymorphisms but are more likely to be caused by differential methylation. For example, the additional A&I and BssHII sites in Hep G2 DNA might reflect hypomethylation in a liver- derived cell line of the region around liver-specific genes. It is interesting that although there are CpG islands within the compass of our map (obvious as clusters of restriction sites) there are no obvious islands at the 5’ ends of any of the genes or in the interval between the genes and for 150 kb downstream of PIL

and 20 kb downstream of AACT. Possibly if more restriction enzymes were included in the map, this situation would change. However, the result is consistent with the observation that tissue-specific genes are less likely to be associated with CpG islands, whereas housekeeping genes are invariably so associated (Bird, 1987; Gardiner-Gander and Frommer, 1987).

In family studies the PI and AACT loci have been observed to recombine relatively frequently (ff = 0.03) (Kelsey et al., 1988a; Cox et al., 1989) considering their distance apart measured in this study. This corresponds roughly to 1 CM per 70 kb and if this represents the true recombination rate between these two loci, then the region is recombining at a frequency greatly in ex- cess of the genome average (usually estimated at 1 CM per 1000 kb). This is consistent with the very high figure for the general region of 14q32-qter discussed in Cox and Donlon (1989).

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A physical map of the human PI and AACT genes