Gene, 79 (1989) 139-150

Elsevier

139

GEN 02998

Variant chromosomal arrangement of adult j?-globin genes in rat

(Recombinant DNA; genomic phage 1 library; Southern-blot analysis; restriction maps; haplotypes differing in number of genes)

Milena Stevanovit, Tatjana Paunesku, Danica RadosavljeviE, Radoje Drmanac and Radomir Crkvenjakov

Genetic Engineering Center, V. Stepe 283. 11000 Belgrade (Yugoslavia)

Received by J.L. Slightom: 20 July 1988

Revised: 10 December 1988

Accepted: 14 January 1989

SUMMARY

The genomic organization of three haplotypes of /I-globin genes was determined to resolve the question of the number of those genes in rat. Haplotype a, found in inbred strain DA, has three genes or pseudogenes, while haplotypes b, found in AO, Y5 and Wistar strains, and c, found in Wistar strain, have five genes or pseudogenes each. In haplotypes b and c, the first gene is of j mGor type and the remaining four are of jIminor type. Partial sequencing of six out of 13 genes shows that duplications of /Iminor genes are causing polymorphism in a number of genes. Also, in haplotype b two bminor genes have a 6.5kb intron 2, while in haplotype c only

one /Iminor gene contains such a large intron 2. The three structurally different haplotypes described are not interconvertible by single recombination events. The results indicate that the rat has the highest number of adult /Sglobin genes found in mammals so far.

INTRODUCTION

The globin genes of vertebrates are contained in multigene families whose expression is both develop- mentally and temporally regulated. These families are attractive for molecular studies because of their extensive previous genetic and biochemical analysis. The genes encoding CI- and b-like chains are arranged in closely linked clusters which differ in complexity in different species (Collins and Weissman, 1984). Rat hemoglobins are unusually heterogeneous com-

pared with those of other mammals. The rat also possesses multiple tl- and /3-globin loci. The simple questions of the number of hemoglobin molecules present in adult erythrocytes and their chain com- position have not yet been answered with certainty, but not for lack of trying.

Various authors differ in their estimates of the number of hemoglobins that range from 4 to 7 (French and Roberts, 1965; Marinkovic et al., 1967; Bunn and Drysdale, 1971). Similarly, the number of 0: and bchains is estimated to be 4 to 7 (Garrick et al.,

Correspondence to: Dr. R. Crkvenjakov, Genetic Engineering

Center, P.O. Box 794, 11000 Belgrade (Yugoslavia)

Tel. 11-491391; Fax(3811)492397.

counts/min; cDNA, DNA complementary to RNA; kb, kilo-

base(s) or 1000 bp; nt, nucleotide(s); p, plasmid; PolIk, Klenow

(large) fragment of E. co/i DNA polymerase I; XGal, S-bromo-

4-chloro-3-indolyl-/I-D-galactopyranoside; SDS, sodium dodecyl

Abbreviations: aa, amino acid(s); bp, base pair(s); cpm, sulfate; wt, wild type.

0378-l 119/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

140

1978; Chu et al., 1978). The rat appears to be excep-

tional among mammals in expressing three or possi-

bly four /I chains in its adult hemoglobins. Garrick

et al. (1978) proposed that the adult hemoglobins of

rat are constructed by combining three a and four

fi chains.

Also for more than 20 years it has been known that

there are two haplotypes of adult hemoglobins

(French and Roberts, 1965; Marinkovic et al., 1967;

Stoic et al., 1982). The polymorphism has been

attributed to the adult P-chain locus.

We have been studying Belgrade anemia (b/b)

(Sladic-Simic et al., 1966). Belgrade laboratory rats

have an hypochromic, microcytic anemia inherited

as an autosomal recessive trait. Studies on iron

metabolism indicate that the transport of iron from

plasma into reticulocytes is markedly decreased

(Edwards et al., 1978). Globin mRNA levels

(Garrick et al., 1978; Crkvenjakov et al., 1982) as

well as hemoglobin synthesis in b/b rats are 50% of

the normal.

This convinced us that a deeper understanding of

the anemia requires answers about the genetic

organization of rat globin gene families. Studies of

the structure of rat globin genes may provide

important insights into the mechanism by which

globin gene expression is regulated and may lead to

new approaches to understanding b/b anemia.

MATERIALS AND METHODS

(a) Enzymes and chemicals

Restriction endonucleases, T4 DNA ligase, PolIk,

PolI and XGal were purchased either from

Boehringer-Mannheim or Amersham. Deoxy- and

dideoxynucleotides were from New England

Biolabs, ultra-pure acrylamide and bis-acrylamide

were from BRL, urea from Sigma; [35S]dATP,

[ 32P]dCTP and [ 32P]dATP were from Amersham.

Gene Screen was obtained from NEN and the com-

ponents for Escherichiu coli media from Difco.

(b) DNA probes

The 3’ probe was a 350-bp PstI fragment from

pBRrgX (Crkvenjakov et al., 1984). The 5’ probe

mp37, an 800-bp HindHI-BumHI fragment from

3 1-kb cluster, was subcloned into M13mp8. Its posi-

tion on the map is shown in Fig. 1.

(c) Construction and screening of rat genomic library

The Mb01 partial digestion library of b/b liver

DNA was constructed in AEMBL3 vector

(Frischauf et al., 1983). Screening of library with

32P-labeled pBRrgX was performed according to

standard procedures (Maniatis et al., 1982).

(d) Isolation of phage DNA and subcloning of

restriction fragments

Propagation of recombinant bacteriophage and

CsCl isolation of phage DNA was performed as

described by Davis et al. (1980). Agarose-gel electro-

phoresis and restriction mapping were performed as

described in Maniatis et al. (1982). Fragments for

subcloning were obtained by digestion of the DNA

with appropriate restriction enzymes followed by

electrophoresis on agarose and subsequent electro-

elution from gel slices (Maniatis et al., 1982). These

fragments were ligated to appropriate Ml3 vectors.

(e) Southern blots

DNA was isolated from kidneys and spleens,

digested, electrophoresed, blotted and hybridized as

described by Herrman et al. (1986). Probes were

labeled according to the procedure described by

Feinberg (1983).

RESULTS AND DISCUSSION

(a) Isolation of clone clusters containing #!-globin

genes

To settle the question of the number and the origin

of rat adult /I-globin genes we have undertaken their

isolation and characterization. As an initial probe,

we have used the B cDNA covering the last 27 nt of

the second exon and the entire third exon

(Crkvenjakov et al., 1984). The rat genomic library

was constructed in the IEMBL3 vector using b/b

DNA. After screening the library with the probe, a

number of recombinant phages was isolated carrying

rat genomic fragments of lo-15 kb. The clone HindIII-BumHI (Fig. 1) containing the entire 5’ half of the gene up to the end of the second exon was obtained by subcloning from fir”“‘. In repeated searches with both new 5’ and 3’ (cDNA) probes, 31 overlapping phages were isolated. Southern analysis of inserts revealed conserved EcoRI and BamHI sites and distinct PstI and KpnI sites in different genomic clones (data not shown). These results indicate the presence of multiple gene loci of similar but not identical structure. The identification of overlapping clones by restriction mapping was

141

difficult due to this complex feature. Three uncon- nected maps were obtained containing 3, 2.5 and 1 globin genes, respectively (Fig. 1). They are 31, 16 and 12 kb in length. Two of the clusters show an unusual feature at one of their loci: the absence of a third exon at 1 kb or less after the 5’ end of intron 2. Since the third exon is found in the largest cluster 6.5 kb downstream from the second exon, it is reasonable to assume that a large intron is specific for rat /I-globin locus and is not a truncated gene as it might appear if only the second cluster was availa- ble.

Fig. 1. Restriction maps of three clusters of /Lglobin genes obtained from genomic library of a b/b rat strain. The position of the genes

is indicated by boxes; the exons within are darkened. The nomenclature of the genes is explained in Fig. 5. The position of the 5’ probe

(HindUI-BnmHI fragment) is indicated by the bar above the first gene in the large cluster. The lines below the maps indicate position

of inserts of recombinant phages. Only three out of 14 different phages from the large cluster are represented. From top to bottom the

clusters are 31, 16, and 12 kb in length. Restriction endonucleases are: B, BumHI; E, EcoRI; G, BglII; H, HindUI; K, KpnI; M, MspI;

P, PstI; and X, XbaI.

142

(b) Restriction maps of haplotypes b and c c/c b/c b/b

kb To verify the restriction maps of clusters and to try

to link them, we have undertaken extensive genomic

Southern analysis using single and double digestion

with eight enzymes. In all cases, the internal restric-

tion fragments from cloned inserts were found to be

identical to genomic fragments hybridizing with both

5’ and 3’ probes (data not shown). However, a

complicating factor was revealed in that the line

carrying b/b mutation contains two haplotypes b and

c of the adult fi-globin locus and our library reflects

this polymorphism as well. To see if the haplotypes

are inherited a number of crosses was performed. An

example of DNA analysis of an offspring of parents

homozygous for haplotypes b and c is shown in

Fig. 2. The results demonstrate that haplotypes

segregate as codominant traits according to

mendelian rules.

The evidence for assignment of cloned segments

to haplotypes is shown in Figs. 3 and 4. The lo-kb

BglII and 9.7-kb MspI fragments are unique to 3 1-kb

cluster and to haplotype b. On the other hand 6.6-kb

BgZII and 6.6-kb XbaI fragments are unique to 16-kb

cluster and haplotype c. The 2-kb A4spI (Fig. 3) and

0.9-kb XbaI (Fig. 4) bands which are 12-kb cluster

specific are common to both haplotypes. The latter

band is poorly visible due to reduced similarity with

3’ probe. On this basis the 31-kb cluster belongs to

haplotype b, the 16-kb cluster to haplotype c and the

12-kb cluster occurs in both.

We have started with the hypothesis that the dif-

ference between b and c haplotypes is due to restric-

tion-site polymorphisms. If this were the case, a con-

struction of a single physical map describing the two

haplotypes would have been possible. The alterna-

tive is to have separate maps, at least in part, for each

haplotype. The second possibility proved to be

correct. The restriction maps of the two haplotypes

are presented in Fig. 5. The maps were obtained by

genomic analysis of DNA from animals homozygous

for the particular haplotype. Mapping was facilitated

by maps of cloned segments and guided by principles

of parsimony and maximal relatedness of the two

haplotypes.

In mapping of haplotype b the 31-kb and 12-kb

clusters could not be overlapped. The evidence for

the existence of uncloned regions comes from

MspI + XbaI and EglII + XbaI digestions. Since an

-2

Fig. 2. Southern hybridization analysis of an offspring (b/c) of a

female homozygous for haplotype c (c/c) and a male homozygous

for haplotype b (b/b). The 3ZP-labeled HindHI-BumHI fragment

containing the 5’ half of the /Ima) gene (Fig. 1) was used as a

probe. Rat kidney DNA (10 pg) was digested with BglII restric-

tion endonuclease, subjected to electrophoresis through a 1%

agarose gel and transferred to nylon membrane (Herrman et al.,

1986). The filter was hybridized overnight at 65°C in hybridiza-

tion buffer containing 5 x lo6 cpm/ml of probe (specific activity

1 x 10’ cpm/pg). The film was exposed for three days at -70°C.

The size markers were restriction fragments derived from

1 EMBL3.

XbaI site is 3 kb from the right end of 3 1-kb cluster

and this region is devoid of either lMsp1 or BgZII sites,

in genomic analysis it must be represented in frag-

ments equal to or larger than 3 kb in XbaI + MspI

and XbaI + BgUI double digestions. Of the frag-

ments in each digestion satisfying this criterion, one

or two are found in cloned DNA indicating that

the remaining 6.9-kb XbaI-MspI and 7-kb XbuI- Bg/II fragments are coming from the right

end of the 31-kb cluster and contain 3.9 and 4 kb of

uncloned DNA, respectively. Since both 5’ and 3’

probes hybridize to these fragments and the cloned

part contains the third exon, the uncloned portion

must have the first and second exon. The same argu-

ment for the 9.7-kb XbuI fragment extends the

uncloned DNA to a total of 6.7 kb.

143

345678 kb

Fig. 3. Southern hybridization analysis of DNA of haplotypes b and c. The 32P-labeled HindHI-BumHI fragment containing the 5’ half

of the pmaJ gene (Fig. 1) was used as a probe. Lanes 1, 3, 5, 7 and 9 contain DNA from haplotype c and lanes 2,4,6,8 and 10 contain

DNA from haplotype b. The enzymes used were: lanes 1 and 2, MspI + XbaI; 3 and 4, Mspl; 5 and 6, XbaI; 7 and 8, XbaI + BglII;

9 and 10, BglII. The hybridization was performed as described in Fig. 2.

The existence of the 6.9-kb MspI-XbaI and 7- and

5.8-kb BglII-XbaI fragments of the same size in both

haplotypes indicates the identity of restriction sites in

this region. Also as mentioned before the 12-kb

cluster is common to both as well. Therefore, the

mapping information about haplotype c can be used

independently to derive the joint map of the two

haplotypes in this region. The XbaI digestion of

haplotype c reveals five fragments totaling 42.3 kb.

Their order (17.6,6.6,7.8 and 1 kb) is determined by

XbaI + BglII and BglII digestions, and a map of the

16-kb cluster which belongs to haplotype c. The 8-kb

XbaI fragment from c contains 4.5 kb of uncloned

DNA and 3.5 kb of the left end of the 12-kb cluster.

Since it hybridizes with both 5’ and 3’ probes, this

uncloned part contains the third exon. Due to the

identity of the two haplotypes in the region, haplotype

b must also contain 4.5 kb of uncloned DNA with

the third exon added to 6.7 kb preceding it with the

first and second exon. The perfect duplication thus

shown for haplotype b is the only solution accom-

modating the demonstration of 6.7 kb of uncloned

DNA and the absence of new bands in double diges-

tions withXbaI,MspI and BglII enzymes. Compared

with singlet bands the doublet bands overall appear

to give more intense signals (Figs. 3 and 4), which is

to be expected considering the variable homology of

the probes with different bands.

The left side of the haplotype c map is derived from

the finding that the 12.5-kb fragment of the 3’

portion of 24-kb MspI fragment is found in the 16-kb

‘cluster leaving a 11.5-kb 5’ portion containing a gene

which shows the same HindIII, BamHI and EcoRI

sites as the first gene of haplotype b.

144

3 4 56 7 8,,

Fig. 4. Southern hyb~dizatio~ analysis of DNA of haplot~es b and c. The 32P-labeled cDNA (Crkvenj~ov et al., 1984) containing the

3’ half of the fi was used as a probe. Lanes gene 1, 3, 5, 7 and 9 contain DNA from haplotype c and lanes 2, 4, 6, 8 and 10 contain

DNA from ~aploty~ 6. The enzymes used were: lanes: 1 and 2, Mq?I + Xbal; 3 and 4, Π 5 and 6, XbaI; 7 and 8, X&I + BglIf;

9 and 10, BglII. Hybridization was performed as described in Fig. 2.

Fig. 5. The org~ization of &globin gene family in h~plotypes b and c present in Wistar rats. The genes are represented by boxes. The dashed-line portions of the map represent uncloned DNA, the solid lines show the region of the three clusters which have been cloned. The 12-kb cluster is represented at the right-hand side ofboth maps. The vertical lines linking the maps of haplotypes enclose the regions different in the two haplotypes. For abbreviations of restriction endonucleases, see Fig. 1. Several restriction sites at the very left end of the haplotype c map, marked with dashed vertical lines, have not been expe~mentally confirmed. The genes are named according to haplotype (subscript), and type of chain and a lower-case letter (superscript) to differentiate them within the haplotypes, In case of duplicated genes, prime (‘) and double-prime 1”) symbols were used.

(c) The structure of three gene haplotype a

Although closely related the two haplotypes are

not easily interconvertible without the intermediate

haplotypes. Therefore we have looked for the addi-

tional haplotypes in locally available inbred strains.

One was found in DA strain, while A0 and Y5

strains have haplotype b. The new haplotype was

called haplotype ~1, which seems the least complex.

An example of the comparison of three haplotypes is

shown in Fig. 6A. We have constructed a provisional

restriction map of haplotype a by Southern genomic

analysis with four enzymes (Fig. ‘6C). XbaI digestion

shows that haplotype a is about 20 kb long (Fig. 6B).

Besides a 19-kb fragment, which hybridizes with

both probes, X&I generates one small 1.2-kb frag-

ment which hybridizes only with 3’ probe (not

shown). This suggests that XbaI has a site in the

second intron of the last gene. This is further

strengthened by inclusion of BgZII in the analysis

(Fig. 6B). When the 14- and 5.5kb BgZII fragments

were subjected to double digestion with XbaI, only

the 5.5-kb fragment stays intact, while the 14-kb

fragment is reduced to a 6.5-kb band which hybrid-

izes with both probes and a 1.2-kb fragment which

hybridizes with only the third exon probe. One gene

is contained within the 5.5-kb BgZII fragment, the

following one in the 6.5-kb BglII-XbaI fragment

which also contains the 5’ half of the third gene at

its very end. Moreover, the data obtained with PstI

(Fig. 6B) demonstrate that haplotype b contains

three genes and confirm the order of fragments. This

map is shown in Fig. 6C. The degree of conservation

of restriction sites is much less among haplotype a

and either b or c haplotypes, than among the later

haplotypes. Since only four enzymes were used in

this analysis and no similarities between haplotypes

were observed, the map should be considered as

tentative.

(d) The partial sequence analysis of cloned genes

The evidence used to localize genes and their parts

was obtained by hybridization. To further charac-

terize the hybridizing fragments as containing globin

genes and to prove their identity we have partially

sequenced a majority of genes from haplotypes b and

c. A convenient BumHI site is situated at the end of

the second exon in all putative p genes. It was used

A.1 23 B

145

4 56789

kb

C. HAPLOTWEa

P r l~___LTI-_--“___~__--~~L___L_~~~__-_~

&I’? OF? l-j??

Fig. 6. The structure of the haplotype a present in DA inbred

strain. (Panel A) Southern hybridization analysis with the 5’

probe of MspI-digested homozygous DNA of different haplo-

types. Lanes: 1, A0 strain (haplotype b); 2, Wistar strain (haplo-

type c); 3, DA strain (haplotype a). (Panel B) Southern hybridi-

zation analysis of the haplotype a DNA with three genes. The

32P-labeled 5’ probe was used for hybridization. Lanes: 4, BglII;

5, BglII + XbaI; 6, BglII + PstI; 7, Pstl; 8, PstI f XbaI; 9, XbaI.

The faint 1.2 and 0.8-kb bands are poorly visible on the photo-

graph. Hybridization was performed as described in Fig. 2. (Map

C) The physical map of haplotype a. It was derived from (A) and

(B) and additional data for four enzymes. For abbreviations of

restriction endonucleases, see Fig. 1. A dashed line means that

no DNA from this haplotype was cloned.

as the starting point in sequencing six out of seven

cloned genes. The reading frame covering aa posi-

tions 30 to 100 of typical mammalian b chain was

found in all (Fig. 7). In one case, the partial sequence

was only long enough to cover positions 82 to 100

of a typical mammalian B chain (Fig. 7). The nu-

cleotide sequences share identity to AC1 strain

/I-globin cDNA (Ohshita and Hozumi, 1987),

146

100 RAT S maj/ L/111) ~VWPWrpRUPOSPOOLssA~~~~*_~~ KEVINAFNMiWMLDNW(~~ATFAHLSElliCDRllIMP

RAT 11 P ~3 -____________~K_-__________---- ___--~.______________~~-~-__~~_________~~~_~~~~

RAT D m’n ? ______________SK_____________--_o ______~______~______~~~~~~~~~_~~~~~~~_~~~~~~~~~~~

RAT #&in ? %ln X’

____________~K______________0-__------_______________________________________

RAT 6, I____________ __I_________ SK --a --_----_____________~~~~~~~__~~~~~__~~_~~~_~~~ RAT ,, minv’;W” __________-___________________OS_______-____-_

’ mlnZ ____.______-~K_______L ____-- -Q

RAT fl,,c _____________-fi~________________-p-~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

MOUSE RmaJ _________-__________----- ____- A-------------T-------N---S-_-_-S_____________

MOUSE Bmin ______________________~___~~~~~~~~~~~~ --------T---Q----N-------_-_S_______________

“Q,,&y a,, , ---~--_-___--,L-~--N mm__ L ____r__ RI&--------ITS,&- V-NM- ---E---________________

HOUSE I3h 0 ~~_~~~~~~~___~__~_~~__~~~~~~~~--R~----------~~S~~~~-~~--~~--~~-~~~~~_~~~~~~

MOUSE Y --~---~---~~~~~~~~~~~~~~~~-------R----------_~~-_G~,-~~~-~~--SAL_IL_______________

HUMAN D ____________. F-E _____ _nD-V __-___ -----------LG--S--A------------~---_ ___________

HUMAN a _______ ______ _F_E__--__p,,-!, ________- --------LG--S---A-----------~ ______ _________

HUflANYA-G ______________ F _ _-v-w N_-------------------LTSL~-~~----D-_---~~~~~~__~_______~_~

HUtIAN & ~~_~~~_______~_____~___p____~~~~~------~---~~S-~-~~--~~----~A_.~--_~---~-----_~

Fig. 7. The comparison of amino acid sequences covering globin-chain residues 31 to 100 in various #I-globin and P-like chains. The ’ derived translations of partial nucleotide sequences of five genes described in this work are presented. They are pbmaJ, BcmlnX”, fic:mi”W’:w”, and pblcminZ. The other sequences are taken from the literature: r’t’g and ‘I’/? (Garrick et al., 1978; Garrick and Garrick,

1983), Bacimin? (Ohshita and Hozumi, 1987), psd”““? (Wong et al., 1988); mouse jmaJ, mouse pm’“, mouse fil, mouse fi0, mouse Y

(Konkel et al., 1979; Hansen et al., 1982; Hill et al., 1984); human /?, human 6, human y”-” and human E (Lawn et al., 1980; Spritz et al.,

1980; Slightom et al., 1980; Baralle et al., 1980). The dashes indicate concordance with the sequence at the top at the given position.

0,rn’J JO AGG CTG CTG GTT GTC TAC CCT TGG /KC CAG AGG TAC TTT GAT AGC TTT GGG Ght CTG TCC TCT GCC TCT GCT 54

n-mlnw t

0 mmw c

%c minx

%c mlnZ

‘b minY

fi mh? .‘I

8,dm’“7

% mal

o minW t

G mlnW I-

Gbc minx

‘bc minZ

‘b l”i”Y

R ,mirll ,‘I

13Sdm’“7

‘b mai

G mloW c

G nvnw r

Gbc minx

‘bc minZ

ob minY

‘4 mh?

%d mln?

AGC CTG CTG GTT GTC TAC CCT TGC ACC CAC ACG TAC TTT TCrkhA TTT CCC CAC CTC TCC TCT CCC TCT CCT

AC6 CTG CTG GTT 6TC TAC CCT TG6 ACC CAG A66 TAC TTT KUAA TTT GGG GAC CT6 TCC TCT CCC TCT 6CT

AGG CT6 m GTT 6TC TAC CCT TGG ACC CA6 A66 TACTTT ILIJAA TTT GG6 GAC CTG Tee rcr m rcr 6cr AW CTG CT6 GTT GTC TAC OCi ltiti ACC CA6 A(iti TACTTT TCT AAA TTT GM GAC Cl6 TCC lC1 liCC TCl 60

________________-___-_________I____ _-_____-__----_.--__-_____-___________________________________

A66 CTR CTG MT lilC TAC CCT TGG KC CA6 A6G TACTTT TCT TTT 13% GAC CTG TCC TCT &CC TCT KT

AGG CTG CTG GTT GTC TAC CCT T6G ACC CAG AGG TAC TTT CL%, TTf GGG GAC CTG TCC TCT &C TCT @CT

ATC ATG GGT hhC CCT AAG GTG AAG GCC CAT GGC AAG A~li GTG ATA AAC KC ?Tc AAI 6~1 (itic ~Tti AAA CAC ‘~3

ATC ATG GCT AAC CCC GTC AAC CCC CAT CCC AAG AAG GTG ATA &J CCC TTC AAT CAT c6c CT6 MA CAC

ATC ATG GGT AAC Ccc Gi6 AAG GCC CAT CCC AAG AAG GTfi ATA M KC TTC AAT kAT fiN: CTfi AAA CAC

AK ATG GGT AAC CCC 6TG AA6 GCC CAT G6C AfM AAG GTG ATA AAC GCC TTC AAT 6AT GGC CTG AAA CAC

AIC Al6 V.il AAL’ CC0 CAA GTG Ah(i CCC CAT GGC AAU AAG GTG ATA &lI GCC TTC AAT GAT GGC CTG Ahh CAC

______-_______-____-___ _____ -___-___----------------- -__._ --- -.____ ____ ____ _____________

ATC ATG GGT AAC CCC CAG GTG AAG 6CC CAT GGC AAG AAG 6TG ATA A&I GCC TTC AAT 6AT 6GC CT6 AAA CAC

ATC ATG GGT AAC CCC CAG GTG AAG GCC CAT GGC AAG AA6 6TG ATAAAI GCC TTC AAT 6AT 66C CT6 AAA CAC

TTG GAC AAC CTC AAG GGC ACC TTT GCT CAT CTG AGT GAA CTC CAC TGT AAG GGC CTG CAT GTG GA1 CC 100

TTG CAC ,\AC CTC AAC CCC ACC TTT CC1 CAT CTG ACT CAA CTC CAC TGT CAC AAG CTG CAT GTG CAT CC

TTG GAC AAC CTC AAG ijGC ACC iTT GCT CAT CTG AGT GAA CTC CAC TGT GAC AAG CTG CAT GTG 6AT CC

TT‘j GAC AAC CTC AA6 GGC ACC TiT m& CT8 A61 GAA CTC CAC TGT 6AC AAd Ci6 CAT GTG BAT CC

TTG i.,,,C AAC CTC AA‘i w,‘ ACC TTT GCT CAT CTti ACT GAA CIC CAC TtiT (iAC AAli CTG CAT tilti (iAT CC

___-____-_ CTC AAG GGC ACC TTT GCT CAT CTG AGT 6AA CTC CAC TGT GAC AA6 CT@ CAT CT6 6AT CC

TTG SAC AAC CTC AA,i MC ACT TTT I%-T CAT CTCI ACIT GAA CTC CAC TGT (iAC AA6 CTG CAT CITR G*T CC

TTO GAC AAC CTC AAG r,r,C KC TTT GCT CAT CTG AGT GAA CTC CAC TGT GAC AA6 CTG CAT GTG 6AT CC

Fig. 8. Comparison of partial nucleotide sequences of cloned rat genes from haplotypes b and c with published rat /? gene sequences.

The nomenclature ofFig. 6 was used. The numbers indicate the aa position in the /?chain of the reading frames presented. The additional

60-bp sequence from BmlnYb gene is included.

147

Sprague-Dawley rat fi-globin gene sequence (Wong

et al., 1988) and amino acid sequences of rat “ii/I and

“‘B-globin chains (Garrick et al., 1978; Garrick and

Garrick, 1983) (Fig. 8) allowing no doubt about the

identity of cloned genes.

This is valid, if all genes reported here are for b

instead of 6, y and E chains, which are part of a P-like

globin gene family as well (Wood and Weatherall,

1983). Two lines of evidence point to identification

of the genes we describe here as belonging to the

a-type. The first comes from partial sequencing of

second exons of cloned genes and conceptual trans-

lation of the coding regions obtained. As shown in

Fig. 7 protein sequences are all of rat /? type more

similar among themselves (three differences out of 70

residues) and to mouse fi”% (5) and Bmi” (4) than to

any of the embryonal and fetal chains from mouse or

man (at least eleven different residues out of 70). The

second line of evidence concerns the constant feature

of the organization of the /?-like globin gene family of

mammals in that the adult /?gene(s) are located at the

3’ end of the clusters (Collins and Weissman, 1984).

The exception is found in goat (Townes et al., 1984).

In mouse, the rodent most closely related to rat, /Pa’

and /Imin genes are the last two genes in the cluster.

The most conserved gene in haplotypes b and c,

compared to mouse /I genes is the 5’ -gene which we

have identified as 8”“‘. If the rule mentioned above

applies to the rat, all other genes which lie down-

stream from it would also be adult expressed p-type.

From the comparison of the published and obtained

sequences, it appears that the first gene in both haplo-

types codes for pm”’ or ““fl chain (Garrick et al.,

1978). The remaining sequences are similar among

themselves and to “‘bchain and we propose to group

them in the Brni” class.

The rat genes with the normally sized introns have

the characteristic EcoRI 180-bp fragment which

starts in the second intron and ends at aa position 17

in the third exon or position 121 of /? chain and the

neighboring 1.4-kb EcoRI fragment containing the

rest of the third exon. The sequences of 180-bp frag-

ment from the /I”“’ gene and 1.4-kb fragment from

the /IminZ gene show the expected reading frames for

17 and 24 aa, respectively (not shown, and Fig. 9).

The third exon and 3’-untranslated sequence of the

fifth gene in haplotype b shows a high degree of

identity with a rat globin gene from Sprague-Dawley

strain which is active (Wong et al., 1988). Since the

III EXON FROM Eco RI site /I21 %a.--146 a.a.

121 T P/S C A OAAFO KVV

Bbc mln 2

%d mln?

Oaci mln?

gmln?

“bc mln 2

‘sd mln?

B aci mln?

gmln?

%c mln 2

Bsdm’n?

%I mln?

Dmln?

‘bc mln 2

LJSdmln7

Oacl mln?

,,mln?

%C mln 2

%d mln?

Dacl mln’t

Umlrl?

ACC CCC TGT GCA CAC GCT GCC rrc CAG AAG GTG GTG __________ .-._ ___-- _-.--_.- ------___-_A

_-___C___--__-----_--_---._----.._----___--____

____--__A__-__----___-------_____--___---__._

A ti v AS ALA HK Y Ii 146

GCT CGA GTG GCC AGT GAC ClG GCT CAC AAG TAC CAC

________-____________C_.__-__________________

____ ___ -__----__C-___-_-__---___--___

________---___--_C___C--T__________.._____.______

TAAGCCCCCTTTCCTGCTGTGTTATGCACAAAGGTTATGTGTC

_____.._______ _____._ TGTCT_________ _.__ ___ ____ __.

TAAACCTCTTTTCCTGCTCTTGTCTTTGTGCAATGGTCAATTG

TAAACCTCTTTTCCTGCTCTGGTCTTTGTGCAATGGTCAATTG

CCCTAGAGAACAACTGTCAACTGTGGGGGGAAATGATGAAGG

__G___~____ __ ..-_ __ ---__. __ _ __~_ .__. _-__-___.

TTCCCAAGAGAGCATCTGTCAGTTGTTGTCAAAATGACAAAG

TTCCCAGAGAGCATCTGTCAGTTGTTGTCAAAATGACATAAG

CCTTTGGGCAGCTAGCTTATCTAATAAATCATATTTACTTTA

_____.___ ____ __ ____. C_A-CTA-T- AATC_TA-TTAC-T

ACCTTTGAAAATCTGTCCTACTAATTAAAGGCATTTACTTTC

ACCTTGAAATCGTCCACAATAAAGGCATTACTTCACG

Fig. 9. Comparison ofthird-exon sequences of BbicminZ gene and

published rat /?-globin gene sequences. Nomenclature as in

Fig. 6, except that the sequence of 3’ probe (Crkvenjakov et al.,

1984) is included as bmin?. The dashes indicate concordance with

the sequence at the top for the given position.

3’ genes of haplotype b and c are indistinguishable

by restriction mapping, this argument applies to

both.

The sequences of the two cDNAs are known

(Crkvenjakov et al., 1984; Ohshita and Hozumi,

1987). The cDNA from AC1 strain shows overall

similarity to sequenced parts of three minor genes

from haplotype c and to the “‘p chain (Garrick and

Garrick, 1983). The 3’-untranslated parts of third

exons of the cDNAs (Fig. 9) clearly share a higher

degree of identity among themselves than with the

3’-untranslated sequence of /IminZ gene. Hence, at

least one additional /Pin gene is active in one of the

haplotypes. The cDNA we have used as a probe

belongs to either haplotypes b or c since the mRNA

148

was obtained from animals of these haplotypes

(Crkvenjakov et al., 1984). It has the AvuI site in the

third exon. This site is present only in putative third

exons of genes with large introns. Since the 8”“’ gene

lacks the AvaI site, the cDNA cannot be derived

from it. Then, there appear to be four active globin

genes if all three active Bmi” genes are in the same

haplotype or three if the two cDNAs are derived

from different haplotypes. From this argument there

is a near certainty that all three genes of haplotype

a are active. There is no sequencing information

about the third exons from P-globin genes which

contain the 6-kb intron 2. Their identification

presently rests on hybridization and the existence of

an internal AvaI site.

Our results can be summarized as showing that

presumptive genes or 5’ halves of genes with large

introns detected by hybridization, are the /?-globin

genes or their pseudogenes of nearly intact structure

by the combined criteria of conserved restriction

sites and partial sequencing.

(e) Haplotypes organization of /?-globin gene family

in mammals

The existence of several haplotypes of/&like globin

gene family seems to be common among mammals

studied. Such haplotypes have been found in mouse,

sheep, and horse (Garrick and Garrick, 1983).

Generally, allelism is based on existence of sequence-

variant genes at the same locus in two or more haplo-

types. In case of rat, the additional feature is present.

The haplotypes differ in the number and the spacing

of genes as well in sequences of genes. The borderline

example in this respect is mouse haplotype s in which

a greater part of fl0 gene has been deleted without

apparent consequences (Holdener-Kenny and

Weaver, 1986). It can fall in either group depending

on whether the gene remnant is counted as a gene or

not.

It is axiomatic that allelic variations existing in

nature are fixed in inbred strains. The mouse inbred

strains show three haplotypes with two pgenes each.

In s haplotype both genes are of major type and

identical (Erhart et al., 1985) in d and p haplotypes

major and minor genes are apparent (Konkel et al.,

1979). All three haplotypes have been found in nature

(Berry and Peters, 1977). The fact that changes deal-

ing with regulation of activity as profound as those

concerning rat gene organization are also found in

mouse haplotypes, but keeping the number of genes

constant, seems to favor the possibility of the pres-

ence of these rat haplotypes in nature. An alternative

is the introgression mechanism similar to the one

demonstrated in mouse for Y chromosome on the

level of partially hybridizing subspecies (Bishop

et al., 1985). Since a closely related species, R. rams, has several subspecies, a source of variation may

exist. The difficulty is the absence of crossbreeding

between R. norvegicus and R. rattus (Robinson,

1965), and the observed variation may be the result

of inbreeding. The C57BL/lO mouse inbred strain

has one MHC class-I gene in D region (Weiss et al.,

1984), while BALB/c inbred strain has live genes

(Steinmetz et al., 1986). It would be interesting to

know if this difference in the number of genes is

found in MHC haplotypes from wt. If not, like the

case described here, it might be an illustration of

changes in the number of members of a gene family

upon inbreeding.

(f) Conclusions

We have presented the restriction maps showing

differences in organization of B-globin genes in three

rat strains. Cloned DNA covers 80% of the haplo-

type b map, 60% of haplotype c map, whereas no

DNA was cloned from haplotype a. Here we show

evidence for the existence of 13 genes for adult

b-globin chains alone. The limited cloning of 6.5

genes together with extensive genomic restriction

mapping has provided sufficient evidence for the

genetic organization of haplotypes b and c and the

general outline of genetic organization of haplotype

a. The reported numbers of B-chain genes, live for

the haplotypes b and c and three for the haplotype a,

are the highest for the /? genes of mammals. The

similarities of genes within b and c haplotypes

exclude the possibility of the occurrence of a tripli-

cation event as in the goat (Townes et al., 1984).

Although final identification must await complete

sequencing, it appears reasonable to consider the

described genes of all three haplotypes as belonging

to the p type.

We cannot offer any direct evidence on the

question of which genes are active and which are

pseudogenes. Since complete identity exists among

149

the sequence of r’rrJ9 (G at-rick et al., 1978), the most abundant adult b-globin chain, and our 8”“’ second exon sequence, it is reasonable to assume that this is an active gene. Which of jImin genes are active remains to be determined. Our results demonstrate the extent of process of duplication possible in evolu- tion of /I-globin genes of rodents. The described haplotypes represent some ofthe steps in the process. Although we do not yet propose a detailed scheme for the evolution of described haplotypes, the main events are indicated by our results about multiplicity of flmin genes in three haplotypes. The rat ancestor which had a fi-globin family organization similar to mouse, underwent a duplication of pmin locus pro- ducing two jImin genes, as found today in haplotype a. Further duplication(s) gave rise to five gene loci as found today in haplotypes b and c. It is tempting to speculate that the expansion of /I genes was accom- panied by the elimination of some preadult genes. The absence of specific fetal globin chains during rat development (Hunter and Paul, 1969) seems to lend some support to this possibility. One can conclude that rat P-globin gene haplotypes form a separate group in terms of genetic organization. Interestingly, rare haplotypes in humans carry three instead of two y genes (Trent et al., 1981). The existence of such variation indicates the absence of constraints of con- stancy of the gene numbers in evolution of P-like- globin gene family.

The variation we found among inbred strains was unexpected. It could be explained in several ways. One, that haplotypes similar to b and c exist in wt populations of R. norvegicus. Second, that by some unknown mechanism of horizontal transfer part of the fi-globin-gene family from a related species has been introduced into progenitors of inbred strains. Third, that the increase in the number of genes is due to inbreeding rearrangement. Further experimen- tation is needed to elucidate which of the three possi- bilities is the correct one.

ACKNOWLEDGEMENTS

This research was supported in part by SIZ for Science, Serbia and U.S.-Yugoslav Joint Board on Scientific and Technological Cooperation, Grant JF 789.

REFERENCES

Baralle, F.E., Shoulders, C.C. and Proudfoot, N.J.: The primary

structure of the human s-globin gene. Cell 21(1980) 621-626.

Berry, R.J. and Peters, J.: Heterogeneous heterozygosities in Mus

musculus populations. Proc. Roy. Sot. Lond. 197 (1977)

485-503.

Bishop, C.E., Boursot, P., Baron, B., Bonhomme, F. and Hatat,

D.: Most classical Mus musculus domesticus laboratory mouse

strains carry a Mus musculus musculus Y chromosome. Nature

325 (1985) 70-72.

Bunn, H.F. and Drysdale, J.W.: The separation of human and

animal hemoglobins by isoelectric focusing in polyacryle-

amide gel. Biochim. Biophys. Acta 229 (1971) 42-56.

Chu, M.L., Garrick, L.M. and Garrick, M.D.: Deficiency of

globin messenger RNA in reticulocytes of the Belgrade rat.

Biochemistry 17 (1978) 5128-5133.

Collins, F.S. and Weissman, S.M.: The molecular genetics of

human hemoglobin. Progr. Nucleic Acids Res. Mol. Biol. 3 1

(1984) 315-437.

Crkvenjakov, R., Maksimovic, V. and GliSin, V.: A pool of non-

polysomal globin mRNAs in globin deficient reticulocytes of

the anemic Belgrade rat. Biochem. Biophys. Res. Commun.

105 (1982) 1524-1531.

Crkvenjakov, R., Bucan, M., Konstantinovic, M., Fogel, M.,

Savic, A. and GliSin, V.: Characterization of two rat globin

cDNA clones. Hemoglobin 8 (1984) 597-611.

Davis, R.W., Botstein, D. and Roth, J.R.: Advanced Bacterial

Genetics (A Manual for Genetic Engineering). Cold Spring

Harbor Laboratory, Cold Spring Harbor, NY, 1980.

Edwards, J., Huebers, H., Kunzler, C. and Finch, C.: Iron

metabolism in Belgrade rat. Blood 67 (1978) 623-630.

Erhart, M.A., Simons, K.S. and Weaver, S.: Evolution of the

mouse globin genes: a recent gene conversion in the HbbS

haplotype. Mol. Biol. Evol. 2 (1985) 304-320.

Feinberg, A.P. and Vogelstein, B.: A method for rapid labelling

DNA. Anal. Biochem. 132 (1983) 6-l 1.

French, E.A. and Roberts, K.B.: The multiple hemoglobins ofthe

rat. Proc. Physiol. Sot. 22 (1965) l-9.

Frischauf, A.-M., Lehrach, H., Poustka, A. and Murray, N.:

Lambda replacement vectors carrying polylinker sequences.

J. Mol. Biol. 170 (1983) 827-842.

Garrick, L.M., Sloan, R.L., Ryan, T.W., Klonowski, T.J. and

Garrick, M.D.: Primary structure of the fimaJ chain of rat

haemoglobins. Biochem. J. 173 (1978) 321-330.

Garrick, M.D. and Garrick, L.M.: Hemoglobins and globin

genes. In Agar, N.S. and Board, P.G. (Eds.), Red Blood Cells

of Domestic Mammals. Elsevier, Amsterdam, 1,983.

Hansen, J.N., Konkel, D.A. and Leder, P.: The sequence of a

mouse embryonic fl-globin gene. J. Biol. Chem. 257 (1982)

1048-1052.

Herrman, B., Bucan, M., Mains, P.E., Frischauf, A.-M., Silver,

L.M. and Lehrach, H.: Genetic analysis of the proximal

portion of the mouse t-complex: evidence for the second

inversion withill t haplotypes. Cell 44 (1986) 469-476.

Hill, A., Hardies, S.C., Phillips, S.J., Davis, M.G., Hutchison III,

C.A. and Edgell, M.H.: Two mouse early embryonic /I-globin

gene sequences. J. Biol. Chem. 259 (1984) 3739-3747.

150

Holdener-Kenny, B. and Weaver, S.: A naturally occurring dele-

tion in the mouse Hbb3 globin gene cluster. Proc. Nat]. Acad.

Sci. USA 83 (1986) 4374-4378.

Hunter, J.A. and Paul, J.: Hemoglobins to the foetal and adult

rat: sites of synthesis and the effects of erythropoietin. J.

Embryo]. Exp. Morphol. 21 (1969) 647-651.

Konkel, D.A., Maizel, J.V. and Leder, P.: The evolution and

sequence comparison of two recently diverged mouse chro-

mosomal /I-globin genes. Cell 18 (1979) 865-873.

Lawn, R.M., Efstratiadis, A., O’Connell, C. and Maniatis, T.: The

nucleotide sequence of the human /?-globin gene. Cell 21

(1980) 647-651.

Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning.

A Laboratory Manual. Cold Spring Harbor Laboratory, Cold

Spring Harbor, NY, 1982.

Marinkovic, D., Martinovic, J. and Kanazir, D.T.: Electro-

phoretic hemoglobin patterns in one random bred and two

inbred strains oflaboratory rats. Nature 213 (1967) 819-821.

Ohshita, Y. and Hozumi, T.: Sequence of rat /?-globin mRNA.

Nucleic Acids Res. 15 (1987) 1336.

Robinson, R.: Genetics of Norway Rat. Pergamon, London,

1965.

Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with

chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74

(1977) 5463-5467.

Sladic-Simic, D., Zivkovic, N., Pavic, D., Marinkovic, D.,

Martinovic, J. and Martinovic, P.N.: Hereditary, hypo-

chromic anemia in the laboratory rat. Genetics 53 (1966)

1079-1089.

Slightnm, J.L., Blechl, A.E. and Smithies, 0.: Human foetal Gy-

anti \ A y-globin genes: complete nucleotide sequences suggest

that DNA can be exchanged between these duplicated genes.

Cell 21 (1980) 627-638.

Spritz, R.A., DeRiel, J.K., Forget, B.G. and Weissman, S.M.:

Complete nucleotide sequence of the human b-globin gene.

Cell 21 (1980) 639-646.

Steinmetz, M., Stephan, D. and Fisher-Lindahl, K.: Gene organi-

zation and recombinational hotspots in the murine major

histocompatibility complex. Cell 44 (1986) 895-904.

Stoic, V., Kunz, H.W. and Gill III, T.J.: Hemoglobin poly-

morphism in inbred strains of rats (Ratfus norvegicus).

Biochem. Genet. 20 (1982) 395-403.

Townes, T.M., Fitzgerald, M.C. and Lingrel, J.B.: Triplication of

a four-gene set during evolution of the goat /?-globin locus

produced three genes now expressed differentially during

development. Proc. Natl. Acad. Sci. USA 81 (1984)

6589-6593.

Trent, R.J., Bowden, D.K., Old, J.M., Wainscoat, J.S., Clegg, J.B.

and Weatherall, D.J.: A novel rearrangement of the human

B-like globin gene cluster. Nucleic Acids Res. 9 (1981)

6723-6730.

Weiss, E.H., Golden, L., Fahner, K., Mellor, A.L., Devlins, J.J.,

Bullman, H., Tiddens, H., Bud, H. and Flavell, R.A.: Organi-

zation and evolution of the class I gene family in the major

histocompatibility complex of the C57BL/lO mouse. Nature

310 (1984) 650-655.

Wong, W.M., Lam, V.M.S., Cheng, L.Y.L. and Tam, J.W.O.:

Genomic sequence of a Sprague-Dawley rat p-globin gene.

Nucleic Acids Res. 16 (1988) 2342.

Wood, W.G. and Weatherall, D.J.: Developmental genetics of

the human haemoglobins. Biochem. J. 215 (1983) I-10.