Mapping of murine Interferon-α genes to chromosome 4

Gene,26(1983) 181-188

181

Elsevier GENE 910

Mapping of murine interferon-cl genes to chromosome 4 (Recombinant DNA; cDNA clones; Northern analysis; Southern blotting; somatic cell hybrids; virus-induced cells)

K.A. Kelley *, C.A. Kozak b, F. Dandoy c, F. Sor e, D. Skup ‘, J.D. Windass d, J. DeMaeyer-Guiguard ‘, P.M. Pitha a* and E. DeMaeyer c a ~~c~~#~ Center, The Johns H~~~in~ University School of ~e~~~ii~e, 3~lt~~ore, MI) 21205 (U.S.A) Tel. (301) 955-8871; b Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Iiealth, Bethesda, MI? (U.S.A.) Tel. (301) 496-2613: ’ I~tit~t Curie-~iolo~‘e, Campus d’Orsay, B~tirne~lt 110, F-91405 Orsay (France) Tel. 6-907-64-67. and d ZCI/U~iversity Joint Laboratory, Biachemistr~ Department, University of Leicester, Leicester LEl7RZZ (U.K.) Tel. 92-8513724 (Received September 2nd, 1983) (Accepted September 14th, 1983)

SUMMARY

A cDNA library was constructed from polysomal poly(A) + RNA from Newcastle disease virus (NDV)induced mouse C243 cells, and screened with a human interferon-a (HuIFN-a) cDNA probe. A cDNA clone for one of the murine interferon-a (MuIFN-ot) genes was isolated, and sequencing analysis revealed that it was a partial copy which is almost identical to the published sequence for the MuIFN~a2 gene. This partial cDNA clone represents a virus-induced message as seen by Northern blot andysis of RNA from NDV-induced C243 cells, and Southern blot analysis of DNA from BALB/c mouse revealed the presence of a multiple IFN-a gene family. The MuIFN-a genes were mapped to chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrid DNAs. _“. ._. -.

There are three major structural types of interferon; the c1-and ~-interfero~s are acid-stable pro* To whom reprint requests should be sent at The Johns Hopkins University Oncology Center, 600, North Wolfe St., Room I-109, Baltimore, MD 21205 (U.S.A.). Abbreviations: bp, base pairs; HAT, selective medium for the HGPRT + hybrid cells; HGPRT, hypoxanthine-guanine phosphoribosyl transferase; HuIFN, human interferon; IFN, interferon; kb, kilobase pairs; MuIFN, murine interferon; NDV, NewcastIe disease virus; PGM, phosphoglucomutase; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl, 0.015 M Na,.citrate, pH 7.6. 0378-l 119/83/$03.00 Q 1983 Elsevier Science Publishers

teins which are produced predominantly as a result of virus infection, while y-interferon is an acid-labile protein which is produced by mitogen- or antigenactivated T-lymphocytes (Stewart, 1979). All three types of HuIFN have been cloned using recombinant DNA techniques (Goeddel et al., 198 1; Streuii et al., 1980; Derynck et al., 1980; Taniguchi et al., 1980; Gray et al., 1982). HuIFN-a’s are a multiple gene family consisting of at least 13 genes (Goeddel et al., 198 1; Brack et al., 198 1), whereas HuIFN-P appears to be a single-copy gene (Derynck et al., 1980; Taniguchi et al., 1980), although several reports indicate that there may be more than one @interferon gene (Weissenbach et al., 1981; Sehgal and Sagar,

182

1980). Human c(- and j?-interferons have been mapped to chromosome 9 by Southern blot analysis of somatic cell hybrids (Owerbach et al., 198 1; Meager et al., 1979; Pitha et al., 1982) and in situ hybridizations (Trent et al., 1982). The mouse provides an important model for studying the genetics and the biology of interferon. Murine interferons, like their human counterparts, exhibit size heterogeneity (Knight, 1975; Taira et al., 1980; DeMaeyer-Guignard et al., 1978) some of which may be due to glycosylation. The aminoterminal amino acids of three size classes of MuIFNs have been determined, and there is amino acid homology between the smallest size class of murine interferon (M, 20000) and HuIFN-cr (Taira et al., 1980). This sequence homology was recently used (Shaw et al., 1983) to obtain a MuIFN-LY genomic clone by screening a mouse genomic library with a HuIFN-c( probe. In the present study, we have screened a mouse cDNA library with a heterologous HuIFN-a cDNA probe, and a clone was obtained that cross-hybridizes with several restriction fragments in mouse DNA, and whose sequence is almost identical to MuIFN-a2 (Shaw et al., 1983). This MuIFN-cr cDNA clone was used to localize the IFN-cr genes to mouse chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrids.

MATERIALS

AND METHODS

(a) Preparation

and cloning of MuIFN-IX cDNA

IFN mRNA was obtained by preparing polysomal poly(A) + RNA from NDV-induced mouse C243 cells as described previously (Skup et al., 1982). Double-stranded cDNA was prepared and annealed with pAT153 DNA at the Pst I site, and annealed mixtures were used to transform Escherichia coli strain JA221, also as previously described (Skup et al., 1982). Clone pMF1204 was selected by the hybridization method of Grunstein and Hogness (1975), using a nick-translated HuIFN-cr probe derived from a cloned Namalva HuIFN-cr cDNA (Skup et al., 1981). Nick translations were according to the method of Rigby et al. (1977). Sequencing was performed by the method of Maxam and Gilbert (1977).

(b) Somatic cell hybrid construction

Somatic cell hybrids were constructed between HGPRT- cells of the Chinese hamster line E36 and peritoneal cells of three different mouse strains (Kozak et al., 1975; Kozak and Rowe, 1979; 1980a) using HAT selection (Szybalska and Szybalski, 1962). The mouse chromosomal content of each line was determined using Giemsa-trypsin banding followed by staining with Hoechst 32258 (Kozak et al., 1977). Hybrids were also typed for the expression of mouse isoenzyme markers on 13 chromosomes (Kozak and Rowe, 1980b). Somatic cell hybrids HM6, HM7, HM12, HM15, HM16, HM18, HM20, HM22, HM23, HM27, HM32, HM33, HM35, HM36, VEM12-4K3 and BM34 were analyzed for the presence of the MuIFN-a sequences. (c) Southern blot analysis of somatic cell hybrids

Somatic cell hybrid DNAs were extracted as described by Heisterkamp et al. (1982); these DNAs were digested with BarnHI, electrophoresed through 0.5 % agarose gels, and transferred to nitrocellulose membranes in 20 x SSC, essentially as described by Southern (1975). The 392-bp PstI-HincII fragment from pMF1204 which was used as a probe was purified by phenol extraction from low melting agarose followed by chromatography on DEAE cellulose (DE52), and nick-translated according to Rigby et al. (1977). Specific activities of l-2 x 10’ dpm/pg of fragment were routinely obtained. The restricted DNAs were transferred to nitrocellulose membranes and hybridized to 32P-labeled probe [in 50% formamide, 5 x SSC, 50 mM K. phosphate, pH 6.5, 250 pgg/mldenatured herring sperm DNA, 10 pg/ml poly(A), 0.1% SDS] at 37°C for 48 h. Membranes were washed two times in 2 x SSC, 0.1 y0 SDS at 55°C followed by two 15-min washes in 0.5 x SSC, 0.1% SDS at 55°C and exposed to Kodak XAR-5 film for 3-10 days.

RESULTS

A cDNA library of polysomal poly(A)+ RNA from induced mouse C243 cells was constructed and

183

screened with a HuIFN-or cDNA

was cloned from Namalva

1981) and

the mature

cDNA probe. This human

corresponds

cells (Skup et al.,

to HuIFN-CXA

protein.

MuIFN-crl

and all of the HuIFN-u

(Shaw

et al., 1983)

genes code for mature

pro-

teins that are 166 amino acids long, but MuIFN-cr2

(Goeddel

et al., 1981). The mouse cDNA clone pMF1204 was selected by this screening and subjected to restriction

codes for a protein

that has an extra amino acid at

tailing used

the carboxyl terminus. The cDNA clone pMF1204 also codes for this extra amino acid, indicating that

to construct the cDNA library should regenerate PstI sites flanking the cDNA insert, and PstI digests

it is the same as MuIFN-~2. There are, however, several differences between the published MuIFN

of pMF1204

and

enzyme analysis. The poly(dG)-poly(dC)

revealed that the cDNA insert is 820 bp

long (Fig. 1). To determine partial or complete sequence

whether

pMF1204

copy of an mRNA,

was determined

according

is a

its nucleotide to the strategy

(Goeddel et al., 1981), an 407 bp was found which nucleotide and amino acid human a-interferon genes.

message for MuIFN-a’s is approx. 1200 bp long, and together with the sequence comparison to the human gene it can be seen that pMF1204 is a partial copy of a MuIFN-IX mRNA, and that it is missing sequences at the 5’ end of the message. While this work was in progress, the sequences of two MuIFN-X’S have been published, and pMF1204 is almost identical to MuIFN-ct2 (Shaw et al., 1983). Comparison of pMF1204 to MuIFN-cr2 showed that acid signal sequence

0

that code for the 23 amino

and the first 31 amino acids of

200

L

800 I

600 I

400 I

er

1

0

P HlrlfI

P

HlrlClI

T

P

P

Ps.fI

Fig. 1. Restriction is the

sequencing.

BglII

map and sequencing

The wavy lines indicate box

* .

T HPau DdeI

coding

vector

region

Restriction

strategy

(pAT153)

of pMF1204

for pMF1204.

sequences. as

determinations.

indicate

The open

determined

sites labeled by 5’-end labeling

cated by tilled circles (O), and arrows extent of sequence

.

‘L

1 ,_l -

by

are indi-

the direction

The top scale is in bp.

pMF1204

has

When six base

compared

to

substitutions region.

Of the five that are found in the coding region, two

Northern blot analysis of poly(A)+ RNA from NDV-induced and uninduced mouse C243 cells revealed that pMF1204 hybridizes strongly with a 12s mRNA (not shown) which is similar in size to the message for HuIFN-a’s. This result suggests that the

it lacks the nucleotides

MuIFN-~2,

sequences.

(Fig. 2), one of which is in the 3’ untranslated

shown in Fig. 1. The nucleotide and predicted amino acid sequences of pMF1204 are shown in Fig. 2. By analogy with HuIFN-c( open reading frame of showed a high degree of sequence homology with

pMF1204

and

(at positions 262 and 288 of Fig. 2) do not cause a change in the amino acid coded for at these positions. The other three substitutions cause changes from leu to pro, gln to lys, and ala to thr at positions 206,244, and 262, respectively, in Fig. 2. Furthermore, in the 3’ untranslated region of pMF1204, there is an additional single base deletion and a 8-bp insertion when compared to MuIFN-~2. The chromosomal localization of the MuIFN-cr genes was established by Southern blot analysis of DNAs from hamster/mouse Hybrids were constructed

somatic between

cell hybrids. cells of the

Chinese hamster line E36 and peritoneal cells or spleen cells of three different mouse strains (Kozak et al., 1975; Kozak and Rowe, 1979; 1980a). The mouse chromosomal content of each line was determined using Giemsa-trypsin banding followed by staining with Hoechst 33258 (Kozak et al., 1977). Each hybrid was also typed for various genetic markers present on different mouse chromosomes (Kozak and Rowe, 1980a). Both hamster and mouse genomic DNAs have sequences homologous to the 392 bp PstI-HincII fragment (which contains only the coding region) of pMF1204 that was used to screen the mouse/hamster hybrids (Fig. 3). Analysis of 16 somatic cell hybrids with different complements of mouse chromosomes showed that all 16 hybrids had the hamster BumHI interferon-u fragments; three hybrids also contained all of the mouse BamHI interferon-a fragments. Correlations between mouse a-interferon sequences and mouse chromosomes in these hybrids showed that all three positive clones contain mouse chromosome 4, and all of the clones lacking mouse interferon-cc sequences also lack chromosome 4 (Table I). All other chromosomes showed discordant segregation with mouse a-interferon bands. One highly

IX4

1 10 20 30 40 50 60 Asp Arg Gin Asp Phe Gly Phe Pro Leu Glu Lys Val Asp Asn Gln Gln Ile Gin Lys Ala GAC AGG CAG GAC l-l-iGGA TTC CCC TTG GAG AAG GTG GAT AAC CAG CAG ATC CAG AAG GCT z 110 120 100 70 90 80 Gin Ala Ile Pro Val Leu Arg Asp Leu Thr Gin Gln Thr Leu Asn Leu Phe Thr Ser Lys CAA GCC ATC CCT GTG CTG CGA GAT CTT ACT CAG CAG ACC TTG AAC CTC TTC ACA TCA AAG 140 170 180 150 160 130 Ala Ser Ser Ala Ala Trp Asn Ala Thr Leu Leu Asp Ser Phe Cys Asn Asp Leu His Gin GCT TCA

TCT GCT GCT

TGG AAT GCA ACC CTC CTA GAC TCA TTC TGC AAT GAC CTC CAC CAG

230 240 190 220 210 200 Gln Leu Asn Asp Leu Gln Thr Cys Pro Met Gln Gln Val Gly Val Gln Glu Pro Pro Leu CAG CTC AAT GAC CTG CAA ACC TGT' CCG ATG CAG CAG GTG GGG GTG CAG GAA CCT &T

CTG

i

290 300 270 280 Val Arg Lys Tyr Phe His Arg Ile Thr Val Tyr Leu

250 260 Thr &s_ Glu Asp Ala Leu Leu

ACC AAG GAA GAC GCC CTG CTG ACT GTG AGG AAA TAT TTC CAC AGG ATT ACT GTG TAC CTG z "G 360 350 340 330 310 320 Arg Glu Lys Lys His Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Val Trp Arg Ala ;

AGA GAG AAG AAA CAC AGC CCC TGT GCC TGG GAG GTG GTC AGA GCA GAA GTC TGG AGA GCC 420

410 400 370 390 380 Leu Ser Ser Ser Val Asn Leu Leu Pro Arg Leu Ser Glu Glu Lys Glu

CTG TCT TCC TCA GTC AAC TTG CTG CCA AGA CTG AGT GAA GAG AAG GAG[TGBj GTGT~~C -r 430 440 450 460 470 480 & 490 AAAGTGTGGAGAGACCTCCCTTGG;;CTAGAAACTGCATCTCArrrrATAAGCTCTCC~AAAACTCTCArrACCTT E 500 510 520 530 540 550 560 570 CAGTATGAATACAaTCAaCCTGCCTArmrrCATAATAnGAGCAATTATTTTCAGTATGTACATCCATGCCTGTA 630 620 610 580 590 600 TCTGCACCCATTTGTTCTTATTTATTTATlTATTTATTTATTTATTTATTTATTTATTTA,ATG%AATAT% 670 660 TAAGGTAmATGrrAAAiArrTCAA~~CATA~CATAAAA 750 740 TTGCllTAGAAGA

730 TATTC

Fig. 2. DNA and amino acid sequence are boxed.

Nucleotide

asterisk

indicates

changed

by the indicated

in the MulFN-~2

differences

base substitutions,

ofthe cDNA

between

with the base found

base substitutions

sequence.

insert of~~~l2~4.

the sequences

The underlined

arc underlined. nuclcotide

The stop codon (WA)

of pMF1204 at that position

and MuIFN-a2 in MulFN-x2

The arrow between

sequence

positions

and ~iyadenyla~ion

(Shaw indicated

below. The amino

485 and 486 indicates

is not found in MulFN-x.2.

signal (AATA.AA)

et al., 19X3) are shown

as follows:

acids that are

where a T is inserted

185

hybrids

kb

were positive

mouse PGM-2

for MuIFN-a

feron is present

and

activity and one hybrid lacked both.

These data demonstrate

23.7 -

sequences

that the locus for cl-inter-

on mouse chromosome

4.

DISCUSSION

Southern

4.3-

analysis

of mouse DNA restricted

several different enzymes least

ten

fragments

MuIFN-r cDNA published results). results, as well as 1980; Shaw et al.,

2.32.0-

revealed the presence that

hybridized

with

with of at the

clone pMF 1204 (Fig. 3 and unIt can be concluded from these the work of others (Taira et al., 1983), that like the HuIFN-r’s,

the MuIFN-LX’S exist as a multiple gene family. Southern blot analysis of hamster/mouse somatic cell hybrid DNAs revealed that the MuIFN-r genes are located on chromosome 4. In the hamster/mouse hybrids that were positive for MuIFN-r (Fig. 3), all of the mouse fragments that hybridize to pMF1204 were always seen after longer exposures, indicating that the c(-IFN genes are located on one chromosome. Several studies have shown that HuIFN-r

12345678 Fig. 3. Hybridization region fragment somatic

cell hybrids.

phoresed blotting section

through

nick-translated

in MATERIALS

lane 3, somatic

HM15;

cell hybrid

and lane 8, somatic

with BarnHI,

gels and analyzed DNA;

cell hybrid

lane 5, somatic HM23;

coding

from mouse/hamster

DNAs were digested

0.5% agarose

as described

spleen DNA;

Pst I-Hi&I

to DNAs

c. Lane 1, E36 hamster

cell hybrid somatic

of

from pMF1204

AND

electro-

by Southern METHODS,

lane 2, BALB/c

mouse

HM6; lane 4, somatic

cell hybrid

lane 7, somatic

HM18;

cell hybrid

lane 6, HM32;

cell hybrid HM33. Sizes ofHindIII-restricted

1 DNA and HueHI-digested

@Xl74 DNA markers

are indicated

in kb.

segregated positive clone, BM14F, contains chromosome 4 and only three other chromosomes: 1,3, and 15. Conversely, one negative clone, 6BD, contains all of the mouse chromosomes except 4, 10, and 11. Finally, a comparison of mouse isozyme expression and a-interferon sequences in several additional hybrids showed that a-interferon correlated with PGM-2, an isozyme marker on chromosome 4. Two

genes are closely linked (Brack et al., 1981; Ullrich et al., 1982) on chromosome 9, and thus the mouse genes may be clustered in a similar manner on mouse chromosome 4. A number of genes involved in the production and sensitivity to interferon have been chromosomally mapped in both man and mouse. Sensitivity to interferon is under the influence of a locus that has been mapped to human chromosome 21 and mouse chromosome 16; both of these chromosomes also carry the locus for the enzyme superoxide dismutase. suggesting a shared region of homology. The genes for a-interferon have been shown to be clustered on chromosome 9 in man, but as yet no other common loci are present on both human chromosome 9 and mouse chromosome 4, suggesting that the region of homology may be very small. The localization of the structural genes for interferon on chromosome 4 is interesting in the light of the number of genes on this chromosome involved in the regulation of RNA tumor virus expression or in oncogenesis. The mouse homologue for one oncogene, c-mos, has been mapped to this chromosome using similar cell hybrids (Swan et al.. 1982). Chro-

186

TABLE

I

Correlation

between

specific mouse chromosomes

Mouse

Number

and mouse of hybrid

G(-IFN genes in 16 somatic

cell hybrids

a 0 %I discordant

clones

chromosome designation

a-interferon/chromosome

retention

-I-

+/+

+I-

-!+

1

1

9

2

4

38

2 3

1 2

8 9

2

I

5 4

44 31

4

3

13

0

0

0

5

1

12

2

1

19

6

0

9

3

4

44

I

2

4

1

9

63

8

0

12

3

0

19

9

1

11

2

2

25

10

0

11

3

1

25

11

0

12

3

0

19

12

1

5

2

8

63

13

1

9

2

4

38

14

1

12

2

0

13

15

3

3

0

10

63

16

1

8

2

5

44

17

1

6

2

7

56

18

1

8

2

5

44

19

0

9

3

4

44

x

1

8

2

5

44

a The mouse

chromosomes

’ The number ofhybrids

were identified

which contain

by Giemsa-trypsin

both the MuIFN-ccgenes

banding

followed

and the designated

by staining

The number of hybrids which contain the MuIFN-a

is indicated

the number

whereas

with Hoechst

mouse chromosome,

in the + / + or - / - column, respectively. in the + / - column,

c

b

33258 dye.

or which lack both, is indicated

genes but lack the designated

which lack the c+IFN genes but contain

the chromosome

chromosome

is indicated

in the -

I’+

column. ’ The “/, discordance of hybrids

for each mouse chromosome

is calculated

by dividing the sum of the + / - and - ,’+ columns

by the total number

examined.

mosome 4 also carries structural genes related to the mouse mammary tumor virus genome in at least one inbred strain (Morris et al., 1979), the Fv-1 locus, which restricts replication of N- or B-tropic leukemia viruses (Rowe et al., 1973), and a locus near Fv-1 which governs the expression of cell surface antigens related to the xenotropic retrovirus (Morse et al., 1979). It will be of interest to determine the relationship, if any, of the a-interferon locus with these genes on chromosome 4. The availability of clones for mouse interferon genes will allow a number of studies on the inducibility of MuIFN genes in vitro and in vivo to be conducted. The induction of interferon in vivo is affected by the genotype of the mouse (DeMaeyer et al., 1974), and the availability of cloned MuIFN genes will allow examination of whether the differ-

ences between these high- and low-producer strains of mice are due to the polymorphism of MuIFN genes or at the level of transcription of these genes. Differences in the expression of MuIFN-cr genes have also been seen in NDV-induced Ehrlich ascites tumor cells (Shaw et al., 1983) and the effects of various inducers on the levels of expression genes can be examined with the appropriate

of these probes.

ACKNOWLEDGEMENTS

We thank C. Corey, J. Sears, A. Cachard and L. Eusebe for technical assistance, and B. Schneider for typing the manuscript. This work was supported by NIH grant

187

AI-19737A and by CNRS ATP Immunopharmacologie, and is a partial fulfillment of doctoral requirements for K.A.Kelley. F. Dandoy received support from the Fondation pour la Recherche MCdicale Franwise.

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