Copwight 8 1990 hv the Genetic5 Society of‘ America

Physical Characterizationof Genetic Rearrangements atthe Mouse Renin Loci Kenneth J. Abel and Kenneth W. Gross Department of Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263 Manuscript received June2 1, 1989 Accepted for publication December 20, 1989 ABSTRACT Many inbred strains ofmice have a single locus encoding renin,Ren-1, whereas other inbred strains have two tandemly linked loci, Ren-1 and Ren-2. Each of these renin genes in inbred mice exhibits a unique pattern of tissue-specific expression. As a prerequisite to understanding the structural basis for the expression differences, we have physically characterized the sequence organization of this chromosomal region in both types of strains. Pulsed field gel electrophoresis was initially used to compare the long-range structureof this region in C57BL/6 (Ren-1)and DBA/2 (Ren-l+Ren-2) mice. The structure in both inbred strains is extremely similar, except for an additional 30 kb containing Ren-2 in DBA/2 mice. T h e boundaries of the extra 30-kb segment were sequenced and compared to homologous sequences flanking the Ren-1 alleles. This analysis identified the precise recombination site, and also the presence of a large insertion, between the renin loci in DBA/2. T h e renin gene duplicationapparentlyresultedfromrecombinationbetweensequencessharinglittlehomology, suggestingthatnonhomologouschromosomalbreakageandrejoining may havebeen involved miLhanis&ally in the event.-

uplication is a process which often leads to diversity of both sequence and function. There exist numerous examples ofclosely linked homologous loci and multigene families whose members are thought to have arisen by gene duplication. In many of these instances duplication is associated with new patterns of gene expression,since selection may maintain only one locus to specify the original function. Duplicated copies may acquire unique tissue-specific expression profiles, e.g., amylase genes (HAGENBUCHLE, BOVEYand YOUNG 1980), or new patterns of developmentally regulatedexpression, e.g., globin genes (reviewed in MANIATISet al. 1980). Thus, gene duplication can have importantevolutionary consequences, yet the precise molecular details of these events remain poorly understood. Inbred strains of mice have been found to differ with respect to the number of genes encoding renin. Some strains,forexampleC57BL/6 and BALB/c, carry a single renin locus, and have fixed the Ren-IC allele. Other strains, such as DBA/2, have fixed the Ren-I allele, Ren-I”, plus a highly homologous locus, Ren-2. The renin structuralgenes map coincidentwith the Rnr (renin regulator) locus, originally mapped by WILSONet al. 1978, on mouse chromosome one (PICCINI, KNOPF and GROSS1982). It has been proposed that Ren-2 appeared as a result of a relatively recent gene duplication event(PICCINI, KNOPF and GROSS

GENE

The publication costs of this article were partly defrayed by the payment of page charges. This articlemust therefore be hereby marked “advertisemenl” i n accordance with 18 U.S.C. $1754 solely to indicate this fact. Genetics 1 2 4 997-947 (April, 1990)

1982; MULLINS et al. 1982;PANTHIER,HOLMand ROUGEON1982). The two renin lociin DBA/2 are transcribed in the same relative direction, with Ren-2 approximately 20 kb upstream of Ren-I” (ABELand GROSS1988). There exist markeddifferences intissue-specific expression which characterize the DBA/2 loci, and also alleles at the Ren-1 locus. All three inbred renin genes express equivalently in the kidney (FIELDet al. 1984a; FIELD and GROSS1985). In contrast, Ren-2derived mRNAs accumulate in the submandibular gland (SMG) to levels two orders of magnitude greater than those of Ren-I” (PICCINI,KNOPF and GROSS 1982), which in turn exceed those of Ren-1” (MILLER et al. 1989). Also, only Ren-I” and Ren-2 are expressed in theadultadrenal, yet Ren-I” is exclusively expressed in male sex accessory gland tissue (FABIAN et al. 1989). In an effort to understand the structural basis for the gene-specific expression patterns, we examined the overall structure of this region of chromosome one inmice bearing either one or two renin loci. Pulsed field gel electrophoresis (PFGE) was used to constructlong-range maps forboth DBA/2 and C57BL/6. Comparison of these maps revealed a stretch of additional DNA containing Ren-2 in DBA/ 2, relative to C57BL/6. Here we report the precise limits of the duplicated segment, the sequence of the recombination junction, and the identification of a largeinsertion between the two DBA/2 loci. The identification of flanking sequences rearranged by

K. J. Abel and K. W. Gross

938

both the duplication event and the insertion may help to identify sequence elements which regulate renin gene expression. MATERIALSANDMETHODS

Mice: C57BL/6JRos and DBA/2JRos mice were obtained from West Seneca Laboratories, and fromstocks maintained at RPMI. GenomicSouthernblotanalyses: Restrictionenzyme digests of genomic DNAs were separated on 0.8% agarose gels, transferred onto nitrocellulose, and hybridized as described (ABELand GROSS1988). PFGE-Southern blot analyses: Pulsed field gel electrophoresis was performed using a CHEF apparatus (contourclamped homogenous electric fields) (CHU,VOLLRATH and DAVIS1986), built by Owl Scientific Plastics (Cambridge, Massachusetts). CHEF gels were 1% agarose in 0.5X TBE running buffer. Gels were run for 24 hr at 170 V using various switching intervals (indicated in figure legends). Digests of embedded spleen DNAs, transfers onto nitrocellulose or nylon membranes, and hybridizations were performed as described (ABELand GROSS1988). In Figure 2C, DNAs werefirstdigested to completion with NotI. T o generate partial digests, the DNA plugs were then dialyzed 30 min in T E buffer,equilibrated30 min in1X BssHII digestion buffer, and incubated for 1.5 h r with 0.1 unit of BssHII (80 pl total volume). Intact chromosomesof the yeast strain A364a, and ladders of lambda DNA oligomers (unit length, 48.5 kb) were included as size standards. DNA probes: pDD-1 D2 is a full-length renin cDNA isolated from a DBA/2 submandibular gland (SMG) cDNA library (FIELDet al. 1984a). pSM479is an incomplete DBA/ 2 SMG renin cDNA, containing only a portion of exon eight plus exon nine (PICCINI,KNOPF and GROSS1982). p3’Ren1 was isolated from the 3’ flanking region of Ren-1” (ABEL and GROSS1988).p5‘RenPXIR containsa 0.8kb XbaIEcoRI fragment derived from the 5’ flanking sequences of Ren-2-containing a cosmid clone(FIELD et al. 1984b). p5’34X/P contains a 0.35 kb XbaI-PstI fragment derived from a genomicsubclone containing Ren-1” 5‘ flanking sequences (pRn34, kindly provided by D. BURT and W. BRAMMAR). Genomic clones: Some of the presented sequences were derived from previously isolated genomic clones, including the Ren-2 cosmid clone (above) and a lambda clone containing Ren-1‘. 5’ flanking regions (lambdaBALB1) (FIELDet al. 1984b), andgenomic subclones ofRen-1” 5’ and 3’ flanking regions (pRn34, pRn26-, D. BURTand W. BRAMMAR). T o obtaina Ren-1‘’ 3’ flanking clone,approximately 600,000 plaques from a BALB/c library (generously prowere screened with the probe, p3’Renvided by s. WEAVER) 1 (above).Nitrocellulosefilter lifts were baked, and prewashed 2 hr at 65” in: 50 mM Tris (pH 8.0), 1 M NaC1, 1 nlM EDTA(Na)2,0.1 % SDS. Prehybridization and hybridization solutions were: 50% formamide, 5X Denhardt’s solution, 5X SSC, 20 mM NaP04 (pH 7.2), 150 rg/ml denatured salmon sperm DNA. Filters were prehybridized 2 h r at 42”. DNA probes were prepared to specific activities of 1 Og dpm/pg by the oligonucleotide random priming method (FEINBERG and VOGELSTEIN 1983). T h e hybridization incubation, 24 hr at 42“, contained 5 X lo5cpm/ml of denatured probe. Washes were done in 0. l x SSC, 0.1 % SDS at 65” for several hours. Filters were exposed to Kodak XAR-5 film for 1-2 days at -70”. Five positives were plaque purified. Of these, lambda3’ lc-lA was selected for subcloning and sequencing.

Subcloning/sequencing: Restriction fragments from genomic clones (see Figure 6)were subcloned into theplasmid vectors pTZ18R and pTZ19R (Pharmacia), which yielded single-stranded sequencing templates upon infection of host cells with the helper phage, M 13K07 (VIEIRAand MESSING 1987). Dideoxynucleotide sequencing was performed with Sequenase kits (United States Biochemical), using the M 13 reverse sequencing primer, and as necessary to complete internal sequences, custom oligonucleotide primers synthesized on an Applied Biosystems 380A DNA synthesizer. In each case (except for 3’ Ren-1‘:) regions to be sequenced were represented in at least two subclones, with genomic sequences in both orientations relative to the M 13 primer binding site. All sequences were determined in duplicate, once using dGTP in the labeling reaction and again using dITP, which eliminates compression artifacts observedwith dGTP (TABOR and RICHARDSON 1987). T h e sequenced DBA/2 subclones are: Region A (derived from cosmidRen2D)-plasmids p5‘R2-18.1 and -18.2 contain a 1.2-kb BamHI-PstI fragment in bothorientations in pTZl8R; and p5’R2B/X19 contains a 0.9-kb BamHI-XbaI fragment in pTZ19R. Region B (derivedfromtheRen2 cosmid)- p3’R2Xho19 containsa1-kb XhoI fragment in pTZ19R; and p3’R2X/B18contains a 0.85-kbXbaI-BamHI fragment in pTZ18R. Region C (derived from a genomic subclone ofRen-1” 5’ flanking regions, pRn34)-the plasmids p5’RlR/X-18 and -19 contain a 1.2-kb EcoRI-XbaI fragment in bothpTZ18RandpTZlSR,andp5’RlX/P18 contains a 0.35-kb XbaI-PstI fragment in pTZ18R. Region D (derived from pRn26)- p3’RlX/P-18 and -19 contain a 0.8-kb XbaI-PstI fragment in both pTZ 18R and pTZ 19R. T h e sequenced BALB/c subclones are: Region E (derived from lambdaBALBl)-p5’BALBl/l8 contains 1-kb a BamHI-PstI genomic fragment (plus flanking lambda vector sequences) in pTZ18R; and p5’BALB B/Pl8 contains the same fragment (minus vector sequences) in pTZ18R. Region F (derivedfrom lambda3’1‘:-1A)-p3’BALB19 containsa 1.5-kb XhoI-SstI fragment in pTZ19R. Each of these regions is shown diagrammatically in Figure 6. RESULTS

The physical linkage relationship of the Ren-1” and Ren-2 loci inDBA/2 mice was recently reported (ABEL and GROSS1988). The two loci are transcribed in the same relative direction, with Ren-2 roughly 20 kb upstream of Ren-1”. However, the precise duplication junction between the twolociwas not determined. The structure of this chromosomal region in C57BL/ 6 mice has now also been examined, and comparison of the regional structures in both inbred strains has helped to identify both the extent and the boundaries of the duplicated segment containingRen-2. PFGE mapping of this chromosomal region in C57BL/6 and DBA/2 mice: PFGE was performed using contour-clampedhomogeneouselectric fields (CHEF) (CHU, VOLLRATHand DAVIS1986) to separate digests of high molecular weight spleen DNA preparedfrombothinbredstrains. Digests were performed using enzymes which cleave mammalian DNA infrequentlyeither because cytosine methylation at CpG dinuleotides renders recognition sequences resistant to digestion, or because of lengthy recognition

Rearrangements at Mouse Renin Loci

939 FIGURE I .--L.oc;lliz;ltion of 2Yotl and .!fit sitrs flanking / h - l " . ('4) I)igests of 1113.412 ;mtl C.57131./6 spleen 1)NAs ~ w r esepmxtetl I)! colwention;ll clectrol~lloresis. ttxnsferred. and h~lwitlisrtlw i t 1 1 ;I 3' rntlrenin cDNA prohe,pSlf479. The probe deterts K p n l fragments extending intothe S' flanking regions. l h e lengths of ronlbinctl Hind1 I I- and Ilindlll BroKI-digested 1an~I)cla D N A . inclutletl ;IS size mlrkers. are indicated. The homologous 'Votl site cIow1stream of R P ~ - 2does notlie within the 2.5-kb K p n l fragtnent detected hv this probe. I) = I)RA/2. C = C57RI./(i. (u) Digests of C57RL/fi D N A were electrophoresed ;IS in (A) and hyhritlizetl w i t h ;I full-length renin cDNA prol)e. pDD-1D2. (C) Not1 digests of enlbedded spleen DNAs were sep;lr;ltetl b y CHEF. using $1 switching interval of 70 sec. The digests were transferred andhybridized with the full-length cDNA probe. Embedded. intact chromosomes of the yeast strain A364a were included as size markers.The lengths of the yeast chroI11osonIes were previouslv estimated by comparing their mobilities relativeto oligomers of intact1;lmhtl;l D N A ( u n i t length, 48..5

+

kb).

sequences, or both. We previously identified coincident, cleavable NotI and SmaI sites at positions 4 kb downstream of Ren-1" and 7 kb downstream of Ren2 (ARELand GROSS1988; see Figure 4). These sites lie at homologous positions downstream of each locus, the difference reflecting the insertion of a partial IAP genome immediately 3' of Ren-2 (BURT,REITHand BRAMMAR 1984). A cleavable BssHII site has also been found at this position, but downstream of Ren-2 only (K. ARELand K. GROSS,unpublished results). These sites demarcate a segment of 28 kb that encompasses Ren-1" (Figure 4). T o obtain a starting point for long-range mapping around the Ren-I" locus, NotI and S j I were tested for their abilities to cleave within a 23-kb KpnI fragment which extends downstream from an exon-3 KpnI site present in both Ren-li.' and Ren-I" (MULLINS et al. 1982; CHIRGWIN et al. 1984). As shown in Figure 1A, a renin cDNA probedetected 10-kb fragments in DBA/2 and C57BL/6 DNAs digested with KpnI+NotI, indicating that both the NotI site 3' of Ren-ID (above) and a Not1 site downstream of Ren-1" lie at homologous positions. The 2.5-kb fragment is attributed to Ren-2 (MULLINSet al. 1982). Figure 1B shows that an SjI site lies roughly 5 kb downstream of the Ren-1': NotI site. An S j I site also resides at an equivalent position downstream of Ren-I" (ABELand

GROSS1988). Both Ren-IC and Ren-2 lie within NotI fragments of approximately 550 kb (Figure lC), indicating that upstream NotI sites are similarly placed for Ren-1': and Ren-2. The regional structure upstream of the renin genes was examined by mapping additional sites relative to the 3' flanking NotI sites. Figure 2 shows Southern blot hybridization results for several enzymes, alone and in double digests with NotI. Using a renin cDNA probe,Figure 2Ashows that Sac11 sites reside at equivalent positions upstream of both Ren-2 and RenI", while upstream sites for Sal1 occupy different positions (see Figure 4). T h e smaller fragment in each of the DBA/2 double digests represents the 28 kb NotI fragmentcontaining Ren-I". Figure 2B shows hybridization results forbothinbredstrains using BssHII alone. In Figure 2C, NotI complete plus BssHII partial digests, in combination with a 5' flanking probe specific for Ren-2 and Ren-I", show that the first BssHII sites upstream of these two loci are also similarly placed. Figure3 shows hybridization results, using a 3' flanking probe specific for the Ren-I alleles, which were useful for mappingHpaI and S j I sites in C57BL/ 6, andfordownstream NotI sites in bothinbred strains. The results shown in Figures 1-3, together with the mapping results from our previous report,

940

K. J. Abel a n d K. W . Gross FIGL~RE 2.-l’F(:F. twpping of sitcs upstreant o f ‘ /