GENOMICS 14, 63-69 (1992)

Mapping of the Regulatory Subunits RI/ and RII/ of cAMPDependent Protein Kinase Genes on Human Chromosome 7 RIGMOR SOLBERG,* PERTTI SISTONEN,1"ANN-LIZ TR.ASKELIN,1: DOMINIQUE BI~RUBF_,§ JACQUESSIMARD,§ PETER KRAJC:I,II TORE JAHNSEN,* AND ALBERT DE LA CHAPELLE:I: *Laboratory for Gene Technology, Institute of Pathology, Rikshospitalet, Oslo, and Institute of Medical Biochemistry, University of Oslo, Oslo, Norway; tFinnish Red Cross Blood Transfusion Service, Helsinki, Finland," SDepartment of Medical Genetics, University of Helsinki, and Folkhdlsan Institute of Genetics, Helsinki, Finland; §MRC Group in Molecular Endocrinology and Department of Human Genetics, CHUL Research Center and Lava/University, Quebec, Canada; and "Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, Rikshospitalet, Oslo, Norway Received March 26, 1992

T h e g e n e s e n c o d i n g the r e g u l a t o r y s u b u n i t s Rift (locus P R K A R 1 B ) and R I I f (locus P R K A R 2 B ) o f human cAMP-dependent protein kinase have been m a p p e d in the basic C E P H (Centre d'Etude du P o l y m o r p h i s m e H u m a i n ) f a m i l y p a n e l o f 4 0 f a m i l i e s to c h r o m o s o m e 7p and 7q, r e s p e c t i v e l y , u s i n g the enz y m e s H i n d I I I and B a n I I r e c o g n i z i n g the c o r r e s p o n d ing r e s t r i c t i o n f r a g m e n t length polymorphisms (RFLPs). P r e v i o u s data f r o m the C E P H d a t a b a s e and our p r e s e n t R F L P data w e r e u s e d to c o n s t r u c t a s i x point l o c a l f r a m e w o r k m a p i n c l u d i n g P R K A R 1 B and a seven-point framework map including PRKAR2B. The a n a l y s i s p l a c e d P R K A R I B as the m o s t distal o f the h i t h e r t o m a p p e d 7p m a r k e r loci and r e s u l t e d in an unequivocal order of pter-PRKAR 1B-D7S21-D7S 108D7S17-D7S149-D7S62-cen, with a significantly h i g h e r rate of m a l e than f e m a l e r e c o m b i n a t i o n b e t w e e n P R K A R 1 B and D 7 S 2 1 . T h e 7q r e g u l a t o r y g e n e locus, P R K A R 2 B , could also be p l a c e d in an u n a m b i g o u s o r d e r w i t h r e g a r d to the e x i s t i n g C E P H d a t a b a s e 7q m a r k e r loci, the resulting order being c e n - D 7 S 3 7 1 (COL 1 A 2 , D 7 S 7 9 ) - P R K A R 2 B - M E T - D 7 S 8 7 - T C R B qter. F u r t h e r m o r e , in s i t u h y b r i d i z a t i o n to m e t a p h a s e c h r o m o s o m e s p h y s i c a l l y m a p p e d P R K A R 2 B to band q 2 2 on c h r o m o s o m e 7. © 1992 AcademicPress,Inc.

number of human PKA subunits have been characterized at the cDNA level. They are called RIa (Sandberg et al., 1987, 1990), RIf~ (Solberg et al., 1991), RIIa 00yen et al., 1989), RIIfl (Levy et al., 1988), Ca (Maldonado and Hanks, 1988; Beebe et al., 1990), Cfl (Beebe et al., 1990), and C3~ (Beebe et al., 1990). In previous studies, we have mapped C~ to band p36 on chromosome 1 (Simard et al., 1992) and C~ to band q13 on chromosome 9 (Foss et al., 1992), using somatic cell hybrids and in situ hybridization. The human regulatory subunit RII/3 has been localized to the q31.1-qter region on chromosome 7 (Wainwright et al., 1987) by somatic cell hybrids and segregation analysis. The tissue-specific extinguisher 1 cDNA (locus TSE1), which was shown to encode the RIa subunit, has been localized to chromosome 17 (Boshart et al., 1991; Jones et al., 1991). In the present study we genetically mapped the genes encoding the regulatory subunits Rift (locus PRKAR1B) and RII/3 (locus PRKAR2B) on chromosome 7 by linkage analysis using the CEPH family panel (Dausset, 1986). In addition, in situ hybridization analysis was carried out to assign PRKAR2B to band q22 on chromosome 7. MATEP~IALS AND METHODS Probes. For RIB, a 0.9-kb EcoRI/PstI fragment of the human cDNA was used (So'berg et al., 1991). The RII/3 human cDNA probes used were a 1.6-kb BamHI fragment in the Southern blottings and a 3.3-kb EcoRI fragment in the in situ hybridization (Levy et al., 1988). RFLP analysis. For detection of an R F L P for RIB, genomic DNA (10 #g) from 16 unrelated healthy volunteers was digested with 14 restriction enzymes (BamHI, BglI, BgIII, EcoRI, HincII, HindIII, MboI, MspI, PstI, PvuII, RsaI, SstI, TaqI, and XbaI), subjected to electrophoresis in 0.8% agarose gels at 45 V for 16 h, and then transferred to nylon membranes (Schleicher & Schfiell, Dassel, Germany) by Southern blotting (Southern, 1975). The RI/3 probe was labeled~by either nick translation or random priming using Amersham kits (Amersham, Buckinghamshire, UK). I-[ybridization was performed at 42°C in 50% (v/v) formamide, 5x SSC (standard saline citrate; 1x = 0.15 M sodium chloride, 15 m M sodium citrate, pHT.0), 5X Denhardt's solution (1X = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 50 m M sodium phosphate buffer, pH

INTRODUCTION The cAMP-dependent protein kinase (PKA) (EC 2.7.1.37) is an essential enzyme in the signalling pathway of the second messenger cAMP (cyclic adenosine 3', 5'-monophosphate) (reviewed by Taylor et al., 1990; Scott, 1991). Through phosphorylation of target proteins, PKA controls many biochemical events in the cell, including regulation of metabolism (Krebs, 1972; Cohenl 1978), ion transport (Greengard and Browning, 1988), and gene transcription (Roesler et al., 1988; Huggenvik et al., 1991). The PKA holoenzyme is composed of two regulatory and two catalytic subunits and dissociates upon binding of cAMP to the regulatory subunits. A 63

0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form ~eserved.

64

SOLBERG ET AL.

6.5, 250 ~g/ml single-stranded salmon sperm DNA, and 0.1% SDS (sodium dodecyl sulfate). Washing was performed to a final stringency of 0.1X SSC/0.1% SDS at 50°C. The membranes were finally exposed to X-ray film (Hyperfilm-MP, Amersham) at -70°C for 5-8 days. ' We have previously shown RII~ to have a two-allele BanII polymorphism with alleles of 7.6 kb (allele 1) and 4.8 kb (allele 2) in size (Scambler et al., 1987). CEPH panel DNA (5 pg) was digested with HindIII for RIf~ and BanIIifor RII~, resioectively, and the resulting DNA fragments were separated in 0.8% agarose gels and blotted onto nylon filters (Zeta Probe, Bio-Rad, Richmond, CA). The filters were ultraviolet irradiated to crosslink DNA. Radi0active probes were prepared using the oligo labeling kit (Pharmacia LKB Biotechnology, Uppsala, Sweden). The filters were hybridized at 42°C in 50% formamide, 5X SSC, 1× Denhardt's, 20 mM sodium phosphate, pH 6.5, 50 t~g/ml single-stranded herring sperm DNA, and 10% (w/v) dextran sulfate. The membranes were washed in 3x SSC/0.1% SDS at 65°C and autioradiographed at -70°C for 2-10 days.

Linkage analysis.

The programs CLODSCORE and CILINK of the LINKAGE program package version 5.1 (Lathrop et al., 1984) were used in two-point and multipoint analysis of the informative CEPH families, respectively. Usually relatively well-spaced (Lathrop et al., 1989; Donis-Keller et al., 1989; Mishra et al., 1992) and/or informative markers, giving in pairwise analysis a lod score (Zmax)of >~3.0 against the test loci, were included in multipoint analysis. Multipoint analysis was started by calculating all four-point orders, includingthe test loci, with several different marker combinations. When an order with odds of 1000:1 or better over the second best order was found, a new marker locus was added and all permutations within the constraints of the best previous order were recursively computed. Information on two D7S62 polymorphisms (CRI-LIO20/TaqI, MspI), four MET (met protooncogene) polymorphisms (METD/TaqI, METH/ TaqI, METH/MspI, and pHOS6/TaqI), as well as four TCRB (T cell receptor, /3 polypeptide) polymorphisms (VB1/TaqI, VB15/MspI, VB7/BamHI, and VB7/EcoRV) in the CEPH database (revised version 4, 1990), were combined by haplotyping the relevant families. Also two separate data sets for D7S85 (CRI-L72/MspI) were recoded and combined. Using the described procedure, a six-point order was calculated for PRKARIB and a seven-point order was calculated for PRKAR2B. The CEPH reference families included in the analyses were 17, 23, 28, 35, 37, 102, 884, 13293, 13294, 1331, 1332, 1333, 1334, 1345, 1346, 1362, 1375, 1413, 1416, 1421, and 1424 for PRKARIB and 12, 35, 45, 66, 104, 13294, 1331, 1332, 1346, 1349, 1408, 1420, and 1421 for PRKAR2B. The recombination fractions were translated to map distances using the Kosambi mapping function. In the multipointanalysis, the variable sex difference option of the CILINK program was used. The 90% confidence limits were estimated for the sex-average recombination fractions by the ~-1 lod unit method from the output of the MLINK program. In situ hybridization of RII~. Preparation of metaphase chromosomes, hybridization, and chromosome staining were performed essentially as described previously (B~rub~ et al., 1989; B~rub~ and Gagn~, 1990). Briefly, the phytohemagglutinin-stimulated lymphocytes were treated with 5-bromodeoxyuridine (30 ttg/ml) for the last 7 h of culture to generate high quality chromosomal banding. Chromosome preparations were then treated as described by B~rub~ and Gagn~ (1990). The slides were treated with RNase (100 ttg/ml) for 60 rain, washed in 2× SSC and 0.1x SSC at room temperature, and digested with 50 U exonuclease III (BRL) in 10 ttl exonuclease buffer (50 mM Tris-HC1, pH 8.0, 10 mM MgC12, I mM mercaptoethanol) under a coverslip for 30-60 min at room temperature. The exonuclease was washed out with 2X SSC, and the preparations were dehydrated by washing with 50, 70, and finally 100% ethanol and air dried. The 3Hlabeled RII~ cDNA probe was hybridized to the metaphase preparations at 42°C for 18 h. The slides were then rinsed in 50% formamide, 2X SSC at 45°C, followed by washes in 2x SSC at 45°C and at room temperature. Finally, the slides were dipped in 0.1X SSC at room temperature before dehydration in ethanol and air drying. The hybridized slides were coated with nuclear track emulsion (Kodak NTB2), followed by exposure at 4°C for 7-19 days. Chromosomes were stained with buffered Giemsa solution and the chromosomes in metaphase

TABLE 1 P a i r w i s e Lod S c o r e s b e t w e e n P R K A R I B and C h r o m o s o m e 7p M a r k e r s Marker locus (probe/enzyme) 0M D7S21 (MS31/HinfI) D7S108 (CRI-S202/PstI) D7S17 (p7-26/XmnI) D7S149 (TS93/PstI) D7S85 (CRI-S72/MspI) D7S55 (CRI-L390/BglII)

0.12 0.04 0 0.15 0.05 0.16

OF 0.02 0.1 0.24 0.1 0.08 0.02

Z~

0M=F Zm~

36.6* 11.38 5.07 5.95 5.66 3.13

0.07 0.08 0.18 0.12 0.06 0.14

34.66 11.19 4.61 5.9 5.63 3.09

090~ 0.03-0.11 0.03-0.16 0.10-0.30 0.05-0.24 0.00-0.19 0.04-0.32

* P < 0.01 for sex difference.

were photographed. R-banding was performed using the modified fluorochrome-photolysis-Giemsa (FPG) method of Perry and Wolff (1974), and metaphase chromosomes were rephotographed before analysis. RESULTS

RFLP Analysis T h e R F L P s for RIB a n d RIIB were t e s t e d i n t h e C E P H c o l l e c t i o n of 40 t h r e e - g e n e r a t i o n r e f e r e n c e p e d i g r e e s ( D a u s s e t , 1986) b y s c r e e n i n g first t h e p a r e n t D N A s a m ples for l i n k a g e i n f o r m a t i v e f a m i l i e s . T h e RIB p r o b e detected a multiallelic polymorphism with the restriction e n z y m e H i n d I I I . A t l e a s t e i g h t alleles w i t h i n t h e size r a n g e 4 - 5 k b c o u l d b e d e t e c t e d . H o w e v e r , it was n o t possible to i d e n t i f y i n d i v i d u a l allelic f r a g m e n t s i n d i f f e r e n t f a m i l i e s s e p a r a t e l y , d u e t o t h e n a r r o w l i m i t s of t h e size r a n g e . T h e allele f r e q u e n c i e s of t h e RIIB p r o b e i n t h e p a r e n t s of t h e C E P H f a m i l i e s were 0.24 for t h e 7.6-kb allele (allele 1) a n d 0.76 for t h e 4 . 8 - k b allele (allele 2),, respectively.

Genetic M a p p i n g of P R K A R I B O f t h e C E P H f a m i l i e s , 21 s e l e c t e d for t h e R I B / H i n d I I I ( P R K A R I B ) s e g r e g a t i o n were used. T h e m a t e r i a l c o n s i s t e d of a t o t a l of 113 m a l e m e i o s e s a n d 119 f e m a l e m e i o s e s , of w h i c h 82 a n d 66 were p h a s e k n o w n , r e s p e c tively. T h e o b s e r v e d h e t e r o z y g o s i t y i n 69 p a r e n t s t e s t e d was 0.46. P a i r w i s e lod scores b e t w e e n C E P H d a t a b a s e m a r k e r s w i t h Zm~ >/ 3.0 a n d P R K A R I B are s h o w n i n T a b l e 1. T h e h i g h e s t s e x - a v e r a g e lod scores, Z m ~ = 34.7 a n d Zmax = 11.2, were o b t a i n e d w i t h m a r k e r s h a v i n g t h e m o s t dist a l r e p o r t e d l o c a t i o n o n t h e s h o r t a r m ( D o n i s - K e l l e r et al., 1989; M i s h r a et al., 1992), D 7 S 2 1 a n d D 7 S 1 0 8 , r e s p e c tively. F u r t h e r m o r e , there was a conspicuous, statistic a l l y s i g n i f i c a n t ( T a b l e 1; x 2 = 8.93) d i f f e r e n c e of t h e sex-specific r e c o m b i n a t i o n r a t e s ( v a r i a b l e vs n o sex diff e r e n c e ) b e t w e e n P R K A R I B a n d D 7 S 2 1 . Six of t h e m a r k e r s i n t h e C E P H c h r o m o s o m e 7 d a t a b a s e gave l o d scores e x c e e d i n g 3. F i v e of t h e s e h a v e r e c e n t l y b e e n rep o r t e d (D7S21, D 7 S 1 0 8 , D 7 S 1 7 , D 7 S 1 4 9 , a n d D 7 S 8 5 ; M i s h r a et al., 1992) to reside o n t h e t e l o m e r i c p o r t i o n of t h e s h o r t a r m of c h r o m o s o m e 7.

MAPPING OF RI~ AND RIIf~ TABLE Multipoint

2

Analysis Results for PRKAR1B Four, Five, and Six Markers

Including

Support

(loglo[ L1/ L2] )~

Order

4.4 4.43

PRKARIB-D7S21-D7S108-D7S149-D7S62 D7S149-PRKARIB-D7S21-D7S108-D7S62 PRKAR1B-D7S2Z-D7S108-D7S62-D7S149 PRKAR1B-D7S21-D7S149-D7S108-D7S62

4.72 7.57

5.15

10.60

PRKAR1B-D7S21-D7S108-D7S17-D7S149-D7S62 PRKAR1B-D7S21-D7S108-D7S149-D7S17-D7S62 D7S17-PRKARIB-D7S21-D7S108-D7S149-D7S62 PRKAR1B-D7S21-D7S17-D7S108-D7S149-D7S62

3.4 5.15

12.49

Note. Markers added into the frame of the best four- or five-point order are underlined. The four best supported orders are shown in each case. L1 is for the uppermost order in the frame. a L1, likelihood of the best supported order; /,2, likelihood of the competing order.

The results of the multipoint analyses are shown in Table 2. D7S62 (CRI-L1020, not shown in Table 1) instead of D7S55 was included in the four-point analysis because it was placed closer to D7S108 on the published maps and also because it gave more unambiguous results t h a n D7S55. The best four-point order placed P R K A R I B as the most distal marker with the following order and orientation: pter-PRKAR1B-D7S21-D7S108D7S62-cen. This order, being 25000 times better than the second best order, was well supported• Therefore, it was selected as the basic frame for additional markers• The following five-point analysis including D7S149 resulted once again in an unambiguous order with odds of 52000:1 over the second best within the original constraint. The analysis placed the marker between D7S108 and D7S62. By repeating this procedure we were also able to place D7S17 between D7S108 and D7S149 with odds of 2500:1 over the second best supported order• Thus each of the markers in the final six-point order pter-PRKAR1B-D7S21-D7S108-D7S17-D7S149D7S62-cen (Table 2) conform to the criteria of framework markers (Keats et al., 1991) presently spanning the region of chromosome

could not be unequivocally placed either in relation to D7S149 or to D7S17. The genetic distances of the markers on male versus female maps are shown in Fig. 1. The suggested boundaries of physical map localization are indicated with lines (Tsui and Farrall, 1991).

Genetic Mapping of PRKAR2B

PRKARIB-D7S21-D7S108-D7S62 D7S62-D7S21-PRKAR1B-D7S108 D7S62-PRKAR1B-D7S21-D7S108 D7S21-PRKARIB-D7S108-D7S62

p-terminal

65

The 13 CEPH families informative for the RII~/BanII (PRKAR2B) polymorphism consisted of a total of 65 male meioses and 47 female meioses, of which 41 and 22 were phase known, respectively (heterozygosity was 0.36). Pairwise lod scores between PRKAR2B and 10 chromosome 7 CEPH database markers with lod scores >/3.0 •are shown in Table 3. The highest sex-average lod scores, Zmax ---- 12.10 and Z ~ = 7.05, were obtained for MET and D7S13, both indicating a relatively close linkage with sex-average recombination fractions of 0.04 and 0.06, respectively. For both markers only male recombination events were present (Table 3). However, comparison of the sex-specific with sex-average lod scores revealed that this difference was most likely due to chance attributed to fewer informative meioses in the female sex (46 vs 18 for MET, and 38 vs 10 for D7S13, respectively)• In fact the same was also the case for D7S87 and D7S8, i.e., all marker loci residing relatively close and probably distally to MET or between MALE

cM

m

m

22

-21 15.3 15.2 15.1 14 13

iiiiiii. . .f. DTs2110 s1719 11

11 11.2 11.1 1L1

11.21

e t al. ( 1 9 8 9 )

and Mishra

2 2

W'/S17 5

o,

7p

13

".

D7S62 35 D7S55

• " ' ' " • ' • , • • • .., ,

7. e t al. ( 1 9 9 2 ) ,

PRKARIB D7S21 D7S108

cM

D7S149

D7S149

D7S62

The order is in agreement with the maps of DonisKeller

FEMALE

PRKAR1B as a new most terminal polymorphic gene locus. Of the other marker loci shown in Table 1, the order of D7S55 in relation to D7S62 remained undetermined, a c e n t r o m e r i c p l a c e m e n t f a v o r e d w i t h o d d s o f 1.6:1 over a telomeric location. The previous location, on the other hand, is unequivocal in the map frame of Donis-Keller et al. (1989)• Two other loci on the aforementioned maps, D7S83 (CRI-S60/MspI) and D7S85 (CRI-S72/MspI), were each placed by five-point analysis between D7S108 and D7S62 with odds of 11000:1 and 910:1, r e s p e c t i v e l y , o v e r t h e s e c o n d b e s t p l a c e m e n t ,

but

11

-%

adding

D7S55 53 cM

76

FIG. 1. Locus PRKARIB on the male and female maps of the telomeric portion of human chromosome 7p. The genetic distances in centimorgans (cM) between marker loci are indicated. Approximate physical location boundaries on the cytogenetic presentation of the map are indicated with dotted lines. These are derived from published data on several markers, and do not intend to refine the physical map of these clusters of loci. The order and positions for six of the loci distal to D7S55 are based on the best order supported by the data, as explained in the text. The distance for D7S55 was calculated by sevenpoint analysis including all the loci, but the order shown was obtained from published data (Donis-Keller et al., 1989).

66

SOLBERG ET AL. TABLE Pairwise

Lod Scores between

Marker locus (probe/enzyme) M E T (haplotyped) D7S13 (B79a/MspI) D7S87 (CRI-S94/TaqI) D7S8 (pJ3.11/MspI) D7S64 (CRI-L1238/EcoRI) D7S107 (CRI-S25/MspI) D7S371 (pTHH28/MspI) COL1A2 (NJ3/EcoRI) D7S80 (CRI-S29/TaqI) T C R B (haplotyped)

3

PRKAR2B

and Chromosome

7q Markers

0M

OF

Zmax

0M_F

Zmax

090%

0.06 0.09 0.05 0.08 0.04 0 0.06 0.05 0 0.2

0 0 0 0 0.5 0.23 0.5 0.44 -0.29

12.55 7.38 5.23 5.23 5.16" 5.12 3.19 3.1" 3.59 3.73

0.04 0.07 0.04 0.05 0.12 0.06 0.06 0.18 0 0.23

12.1 7.05 5.18 4.79 3.55 4.51 3.16 1.79 3.59 3.58

0.01-0.12 0.02-0.18 0.00-0.17 0.00-0.17 0.03-0.28 0.01-0.19 0.00-0.27 -0.00-0.28 0.14-0.35

*P < 0.05 for sex difference.

PRKAR2B and MET (no equivocal placement indicated for D7S13 on the map of Donis-Keller et al., 1989, and a proximal placement on the map of Lathrop et al., 1989). In contrast to previously mentioned loci, D7S64, D7S107, D7S371, and COLIA2 (collagen type I, a 2) exhibited a reverse sex-specific linkage relationship to PRKAR2B, which was significant for COLIA2 (x 2 = 6.03) and for D7S64 (~(2 = 7.41) but not for D7S371. For this marker, the high apparent recombination rate in females was due to insignificant negative lod scores and was therefore due to paucity of information. On the other hand, the recombination rate seemed similar for the two sexes between PRKAR2B and TRCB, which has been placed most distal (Lathrop et al., 1989; DonisKeller et al., 1989) of the markers included in this analysis. For D7S80, only 11 male meioses in two of the families were informative. The results of the multipoint analyses are shown in Table 4 and Fig. 2. The best orders for four- and fiveTABLE

point maps were well supported, with odds exceeding 1000:1 over the second best order. However, the placement for D7S371 incorporated in the best five-point order was less firmly supported (odds 120:1). Therefore, both the best and second best orders were included in the calculation of the final seven-point order with the addition of COL1A2. This resulted in an order cen-D7S371(COL1A2, D 7 S 7 9 ) - P R K A R 2 B - M E T - D 7 S 8 7 - T C R B qter, with orientation as reported previously (Lathrop et al., 1989; Donis-Keller et al., 1989). The order was supported by odds of 72000:1 over the second best order and is in good agreement with aforementioned published MALE

cM

FEMALE

cM

m D7S371

25

B

C O L 1 A 2 , D7S79

4 28

IL2

Multipoint Analysis Results for PRKAR2B Including Four, Five, Six, and Seven Markers Support Order

D7S79-PRKAR2B-MET-TCRB PRKAR2B-D7S79 MET-TCRB TCRB PRKAR2B-MET-D7S79 PRKAR2B-MET-D7S79-TCRB D7S79-PRKAR2B-MET-D7S87-TCRB D7S79-PRKAR2B D7S87-MET-TCRB D7S79 D 7 S 8 7 - P R K A R 2 B - M E T - T C R B D7S87-D7S79-PRKAR2B-MET-TCRB D 7 S 3 7 1 - D 7 S 7 9 - P R K A R 2 B - M E T D7S87 T C R B D7S79-D7S371-PRKAR2B-MET-D7S87-TCRB D7S79-PRKAR2B-MET-D7S87-TCRB-D7S371 D7S79-PRKAR2B-MET-D7S87-D7S371-TCRB D7S371 ( C O L 1 A 2 , D 7 S 7 9 ) - P R K A R 2 B - M E T - D 7 S 8 7 T C R B D7S79-D7S371-COL1A2-PRKAR2B-MET D7S87-TCRB COLIA2-D7S79-D7S371 PRKAR2B-MET-D7S87-TCRB D7S79-C_OL1A2 D 7 S 3 7 1 - P R K A R 2 B M E T - D 7 S 8 7 - T C R B

(lOg~o[L1/L2]) ~

11.1 1L1 11.21 11.22 11,23 D

21.1 21.2 21.3

3.89

22

4.41 6.41

31.1

D7S371

-- PRKAR2B

4

31.2 --MET - - D7S87

6 6

13

- - TCRB

--TCRB 48

2.08

Note. Markers added into the frame of the best four-, five-, or sixpoint order are underlined. The four best supported orders are shown in each.case. L1 is for the uppermost order in the frame. a L1, likelihood of the best supported order; L2, likelihood of the competing order.

D7S87

- - COLIA2, D7S79

3.34 5.53 13.54

5.86 7.26 7.26

PRKAR2B, M E T

m 19

~2

31.3

3.45 4.54

D

102

7q F I G . 2. Locus P R K A R 2 B on the seven-point map of markers spanning the middle portion of chromosome 7q. Male and female maps are shown with marker distances in centimorgans (cM). As the published physical location for the most proximal marker locus D7S371 on chromosome 7 (7p13-q22.1; Tsui and Farrall, 1991) is rather broad compared with the present genetic mapping data, no physical boundaries for the map are indicated. The published locations in the cytogenetic presentations are shown as physical anchor points for the map.

MAPPING OF Rift AND RIIf

67

FIG. 3. In situ hybridizationof RIIfi to metaphase chromosomes. (A) Giemsa-stainedmetaphase chromosomeswith silver grains. The specificsites of hybridizationon chromosome7 are indicatedby arrows. (B) The same metaphasechromosomesafter subsequentR-bandingby the fluerochrome-proteolysis-Giemsamethod. data. As no recombinations were found for either sex in the CEPH database, order could not be established between COLIA2 and D7S79 within the map frame shown. In Situ Hybridization The RII~ cDNA probe was physically assigned by in situ hybridization to metaphase chromosomes, which is illustrated in Fig. 3. A total of 137 metaphases were analyzed, and the distribution of silver grains on different chromosomes was determined (Fig. 4A). Of the total number of 386 grains counted, 35 (9.1%) occurred on chromosome 7. Of these grains, 46% (16/35) was located on the q22 band (Fig. 4B). This suggested a physical localization of PRKAR2B on 7q22. DISCUSSION The present data show that the loci of the genes encoding the regulatory subunits RIB (PRKAR1B) and RIIB (PRKAR2B) of cAMP-dependent protein kinase were located on chromosome 7p and 7q, respectively. Boshart et al. (1991) and Jones et al. (1991) have localized RIa as the gene product of the tissue-specific extinguisher 1 (TSE1) on human chromosome 17. In hybridization experiments, Boshart et al. (1991) used a specific 40~mer oligonucleotide complementary to nucleotides 23-62 of the human RIa coding region (Sandberg et al., 1987). Scambler et al. (1987) located a human RI subunit on chromosome 7, region pl-qter, when hybridizing somatic cell hybrids with a bovine RIa cDNA probe (Lee et al., 1983). This location was most likely due to cross-hybridization to RIB, based on the results in the present

paper and the data from Boshart et al. (1991) and Jones et aI. (1991). We have previously localized the human RIIB gene to the region 7q31.1-qter (Scambler et al., 1987; Wainwright et al., 1987), but the sublocalization has never been accurately determined. The more informative of the two regulatory subunit loci, P R K A R I B , was firmly placed as the most distal marker locus by our and existing C E P H family data in the map of marker loci of the telomeric portion of chromosome 7p. Furthermore, the inclusion of this marker locus in the analysis resulted in a more detailed framework map for this subregion, well in agreement with the recently published 2-cM map of 7p (Mishra et al., 1992), constructed by a slightly different method. Six useful framework loci, P R K A R I B , D7S21, D7S108, D7S17, D7S149, and D7S62, were included with well-supported order and locations. P R K A R I B also mapped more distally than any of the loci in the above-mentioned map, thus extending the map length in the telomeric direction. Two of the loci included here within the framework, D7S21 and D7S108, were also placed on the map of Mishra and colleagues with a very similar sex-specific recombination rate, while D7S17, D7S149, and D7S55 could not be equivocally placed. As D7S21 has been physically mapped to band p22 (Royle et al., 1988), our genetic mapping data would suggest a physical location of P R K A R I B in region pter-p22. It is interesting that the addition of this locus also brings to notion a new finding suggesting a similar sexspecific relationship between the most terminal chro-

68

SOLBERG ET AL. 20. 16. 12.

8. 4.

22

0-

I C "~

2

3

4

21

5

000

20. 16-

(3

13 12 11,2

A Z

00000

0

11'.1

..ll p

I

q

I p

6

B

I

7

11.21 11.22 11.23

8

9

10

II

O0

21.1 21.2 21,3

12

2O

q

22

0000000000000000

31.1 31.2 31.3

1

O0

32

I

/o 13

i

,

[

17

18

, ,I o /

I

15

16

19

20

21 22

Chromosomes

Y

IN 35

O0

X

7

F I G . 4. (A) Histogram distribution of 386 silver grains in 137 m e t a p h a s e cells hybridized with the 3H-labeled RII~ cDNA. A concentration of labeling is seen on chromosome 7. (B) Idiogram of chromosome 7 displaying 46% of the silver grains over the q22 band.

mosome 7p markers as previously found for several other terminal c h r o m o s o m e regions (e.g., N a k a m u r a et al., 1988, 1989). In c o n t r a s t to the general linkage relationship between markers of o t h e r c h r o m o s o m a l regions, the r e c o m b i n a t i o n rate between the most telomeric markers tends to increase in the male sex versus t h a t observed in the female sex. T h i s " t e r m i n a l i z a t i o n " of the crossing over rate in male was most obvious between P R K A R 1 B and D7S21 and was e x t e n d e d to D7S108. Conversely, this could be considered to imply indirect support for the most t e r m i n a l p l a c e m e n t of P R K A R 1 B on the p r e s e n t map. F u r t h e r m o r e , the findings also lend some additional support for the idea t h a t this p h e n o m e n o n of terminalization might be a general one and t h a t the failure to observe it at least for some of the c h r o m o s o m e s could be due merely to the lack of markers with a location sufficiently telomeric. T h e fact t h a t t h e r e were relatively few informative meioses for P R K A R 2 B was obvious in the selection of the m a r k e r loci for the m u l t i p o i n t analysis. Although the middle c h r o m o s o m a l region of 7q is quite well populated with m a r k e r loci in the C E P H database, only a few of t h e m gave an unequivocal map order when P R K A R 2 B was included. However, for this marker, too, a well-supp o r t e d map consisting of six other m a r k e r loci (Table 4) could be constructed. Since the data generally consisted of more informative and particularly of more phasek n o w n male t h a n female meioses, the genetic distances on the constructed male map for P R K A R 2 B are consequently b e t t e r supported t h a n on the female map. T h e

c o m p a r i s o n of the sex-specific maps (Fig. 2) shows t h a t the length of the female map is more t h a n twice t h a t of the male map. Indeed, since the female map was based on relatively few informative meioses between P R K A R 2 B and M E T or D7S87, the length of this subregion m a y be an underestimate. T h e highest pairwise lod score of 12.55 was o b t a i n e d between M E T and P R K A R 2 B at a r e c o m b i n a t i o n fraction of 0.06 for male and at 0 for female based on 46 and 18 informative meioses, respectively. It is quite probable t h a t the lack of recombinations was due to sampling effect. T h e physical mapping of P R K A R 2 B to b a n d 7q22 agrees therefore relatively well with its genetic map 6 c M proximal (on the male map) to M E T , which has b e e n physically m a p p e d to the n e x t b a n d interval q31 (Tsui and Farrall, 1991). However, as this cytogenetic interval is r a t h e r broad, these results would suggest a r a t h e r proximal location for M E T within q31.1, as well as a r a t h e r distal location for P R K A R 2 B within the wide light b a n d q22. In an effort to create a b e t t e r chromosome 7 map, the p r e s e n t genetic maps, particularly the locus order, should prove to be valuable. ACKNOWLEDGMENTS We thank Hanne K/ihler for excellent technical assistance. This work was supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities (NAVF), Astri and Birger Torsteds Grant, Anders Jahres Foundation for the Promotion of Science, Nordic Insulin Foundation, The Sigrid Juselius Foundation, The Academy of Finland, and the Medical Research Council of Canada.

MAPPING OF RI~ AND RII~ REFERENCES Beebe, S. J., Oyen, 0., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990). Molecular cloning of a tissue specific protein kinase (C7) from human testis--Representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase. Mol. Endocrinol. 4: 465-475. B~rub~, D., The, V. L., Lachance, Y., Gagn~, R., and Labrie, F. (1989). Assignment of the human 3~-hydroxysteroid dehydrogenase gene (HSDB3) to the p13 band of chromosome 13. Cytogenet. Cell Genet. 52: 199-200. B~rub6, D., and Gagn~, R. (1990). Exonuclease digestion of human chromosomes for in situ hybridization and R-banding. Nucleic Acids Res. 18: 2831. Boshart, M., Weih, F., Nichols, M., and Schutz, G. (1991). The tissuespecific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase. Cell 66: 849-859. Cohen, P. (1978). The role of cAMP-dependent protein kinases in regulation of glycogen metabolism in mammalian skeletal muscle. Curr. Top. Cell Reg. 14: 117-196. Dausset, J. (1986). Le Centre d'Etude du Polymorphisme Humain. Presse Med. 15: 1801-1802. Donis-Keller, H., Helms, C., Green, P., Riethman, H., Ramachandra, S., Falls, K., Bowden, D. W., Weiffenbach, B., Keith, T., Stephens, K., Cannizzaro, L. A., Shows, T. B., Stewart, G. D., and van Keuren, M. (1989). A human genome linkage map with more than 500 RFLP loci and average marker spacing of 6 centiMorgans. Cytogenet. Cell Genet. 51: 991. Foss, K. B., Simard, J., B~rub~, D., Beebe, S. J., Sandberg, M., Grzeshik, K.-H., Hansson, V., and Jahnsen, T. (1992). Localization of the catalytic subunit C7 of the cAMP-dependent protein kinase gene (PRKACG) to human chromosome region 9q13. Cytogenet. Cell Genet. 60: 22-25. Greengard, P., and Browning, M. D. (1988). Studies of the physiological role of specific neuronal phosphoproteins. Adv. Second Messenger Phosphorylation Res. 21: 133-146. Huggenvik, J. I., Collard, M. W., Stofko, R. S., Seasholtz, A. F., and Uhler, M. D. (1991). Regulation of the human enkephalin promoter by two isoforms of the catalytic subunit of cyclic adenosine 3',5'monophosphate-dependent protein kinase. Mol. Endocrinol. 5: 921-930. Jones, K. W., Shapero, M. H., Chevrette, M., and Fournier, R. E. K. (1991). Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A. Cell 66: 861-872. Keats, B. J. B., Sherman, S. L., Morton, N. E., Robson, E. B., Buetow, K. H., Cartwright, P. E., Chakravarti, A., Francke, U., Green, P. P., and Ott, J. (1991). Guidelines for human linkage maps--An international system for human linkage maps (ISLM, 1990). Ann. Hum. Genet. 55: 1-6. Krebs, E. G. (1972). Protein kinases. Curt. Top. Cell Reg. 5: 99-133. Lathrop, G. M., Laloul, J.-M., Juliet, C., and Ott, J. (1984). Strategies for multilocus linkage analysis in human. Proc. Natl. Acad. Sci. USA 81: 3443-3446. Lathrop, G. M., O'Connell, P., Leppert, M., Nakamura, Y., Farrall, M., Tsui, L.-C., Lalouel, J.-M., and White, R. (1989). Twenty-five loci form a continuous linkage map of markers for human chromosome 7. Genomics 5: 866-873. Lee, D. C., Carmichael, D. F., Krebs, E. G., and McKnight, S. M. (1983). Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 80: 3608-3612. Levy, F. 0., Oyen, O., Sandberg, M., Task~n, K., Eskild, W., Hansson, V., and Jahnsen, T. {1988). Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic

69

adenosine monophosphate-dependent protein kinase from human testis. Mol. Endocrinol. 2: 1364-1373. Maldonado, F., and Hanks, S. K. (1988). A cDNA clone encoding human cAMP-dependent protein kinase catalytic subunit Ca. Nucleic Acids Res. 16: 8189-8190. Mishra, S. K., Helms, C., Dorsey, D., Permutt, M. A., and DonisKeller, H. (1992). A 2-cM genetic linkage map of human chromosome 7p that includes 47 loci. Genomics 12" 326-334. Nakamura, Y., Lathrop, M., O'Connell, P., Leppert, M., Lalouel, J.-M., and White, R. (1988). A primary map of ten DNA markers and two serological markers for human chromosome 19. Genomics 3" 67-71. Nakamura, Y., Lathrop, M., O'Connell, P., Leppert, M., Kambon, M. I., Lalouel, J.-M., and White, R. (1989). Frequent recombination is observed in the distal end of the long arm of chromosome 14. Genomics 4: 76-81. Oyen, O., Myklebust, F., Scott, J. D., Hansson, V., and Jahnsen, T. (1989). Human testis cDNA for the regulatory subunit RIIa of cAMP-dependent protein kinase encodes an alternate amino-terminal region. F E B S Lett. 246: 57-64. Perry, P., and Wolff, S. (1974). New Giemsa method for the differential staining of sister chromatids. Nature 251: 156-158. Roesler, W. J., Vandenbark, G. R., and Hanson, R. W. (1988). Cyclic AMP and the induction of eukaryotic gene transcription. J. Biol. Chem. 263: 9063-9066. Royle, N. J., Clarkson, R. E., Wong, Z., and Jeffreys, A. L. (1988). Clustering of hypervariable minisatellites in the proterminal regions of human autosomes. Genomics 3: 352-360. Sandberg, M., Task~n, K., Oyen, O., Hansson, V., and Jahnsen, T. (1987). Molecular cloning, cDNA structure and deduced amino acid sequence for a type I regulatory subunit of cAMP-dependent protein kinase from human testis. Biochem. Biophys. Res. Commun. 149: 939-945. Sandberg, M., Sk~lhegg, B., and Jahnsen, T. (1990). The two mRNA forms for the type Ia regulatory subunit of cAMP-dependent protein kinase from human testis are due to the use of different polyadenylation site signals. Biochem. Biophys. Res. Commun. 167: 323-330. Scambler, P., Oyen, O., Wainwright, B., Farrall, M., Law, H.-Y., Estivill, X., Sandberg, M., Williamson, R., and Jahnsen, T. (1987). Exclusion of catalytic and regulatory subunits of cAMP-dependent protein kinase as candidate genes for the defect causing cystic fibrosis. Am. J. Hum. Genet. 41: 925-932. Scott, J. D. (1991). Cyclic nucleotide-dependent protein kinases. Pharmacol. Ther. 50: 123-145. Simard, J., B6rub6, D., Sandberg, M., Grzeshik, K.-H., Gagn6, R., Hansson, V., and Jahnsen, T. (1992). Assignment of the gene encoding the catalytic subunit C~ of cAMP-dependent protein kinase to the p36 band on chromosome 1. Hum. Genet. 88: 653-657. Solberg, R., Task~n, K., Keiserud, A., and Jahnsen, T. (1991). Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI~ of cAMP-dependent protein kinases. Biochem. Biophys. Res. Commun. 176: 166-172. Southern, E. M. (1975). Detection of specific sequence among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503517. Taylor, S. S., Buechler~ J. A., and Yonemoto, W. (1990). cAMP-dependent protein kinase: Framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem. 59: 971-1005. Tsui, L.-C., and Farrall, M. (1991). Human Gene Mapping 11: Report of the committee on the genetic constitution of chromosome 7. Cytogenet. Cell Genet. 58: 337-381. Wainwright, B., Lench, N., Davies, K., Scambler, P., Kruyer, H., Williamson, R., Jahnsen, T., and Farrall, M. (1987). A human regulatory subunit of type II cAMP-dependent protein kinase localized by its linkage relationship to several cloned chromosome 7q markers. Cytogenet. Cell Genet. 45: 237-239.