Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x

Gene, 31 (1984) 241-245

241

Elsevier GENE

1092

Short Communications Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x (Recombinant DNA; in vitro translation; gene complexes; evolution)

Maria-Giulia Farace*, Alison Hill, Marco Tripodi*, Richard W. Padgett, Ciweppe Raschella*, Roberto Gambari*, Antonio Fantoni*, Clyde A. Hutchison, III** and Marshall H. Edgell Department of Microbiology and Immunology, Curriculum in Genetics, Program in Molecular Biology and Biotechnology, The Universityof North Carolina, Chapel Hill, NC 27514 (U.S.A.) Tel. (919) 966-5396, and *D@artimento di Biopatologia Umana, Universityof Rome, Rome 00100 (Italy) Tel. 01 l-39-6-490047 (Received

December

(Revision

received

(Accepted

8th, 1983) May 2nd, 1984)

June lst, 1984)

SUMMARY

Cytoplasmic poly(A)+mRNA from 1Zday mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the a-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic a-like globin chain. The insert sequence is 3 16 nucleotides long, contains the codons corresponding to amino acid residues 43-141, and extends into the 3’ untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known a globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the a- and p-globin gene complexes.

INTRODUCTION

The globin gene complex represents an appealing system to study the evolution and regulation of gene families since there is temporal and spatial expression of members of the gene family during development. Early in mouse fetal development four globin chains are produced, the embryonic x (x-like chain), the embryonic y and z (B-like chains) and the adult ** To whom correspondence Abbreviations: base pairs;

should be sent at the first address.

AM virus, avian myeloblastosis MY, million years;

SDS,

SSC, 0.15 M NaCl, 0.015 M Na,.citrate,

0378-l 119/84/$3.00

0

1984 Elsevier

sodium

virus; kb, kilododecyl

pH 7.6.

Science

Publishers

sulfate;

achain (Fantoni et al., 1967). The yolk-sac-erythroid cells produce the x and z globin chains early in ontogeny of the mouse and by day 14 the fetal liver replaces the yolk-sac-erythroid cells as the predominant site of erythropoiesis and synthesizes only the adult chains. The developmental signals producing the shift from the synthesis of embryonic globins to the synthesis of fetal and adult globins are so far unknown but may act on specific tissues rather than altering the expression within a single cell type. The nucleotide sequence of the coding and noncoding regions of the loci will presumably be essential in understanding globin gene controls. Data are already available on

242

the organization and nucleotide structure of the pglobin locus of the mouse (Jahn et al., 1980). Seven P-like globin structures are closely linked within 65 kb of DNA. The order of these genes is 5’-Hbb-y, Hbb-j?hO,Hbb-fihl, Hbb$h2, Hbb$h3, Hbb$I,

Hbb-

/l2-3’ (Jahn et al., 1980); Brown et al., 1982). Leder et al. (198 1) have cloned and restriction mapped the functional c( and cc-likegenes of BALB/c mice. The order of the genes is 5’-r-like embryonic x, x 1, and x 2-3’. Here we report the cloning and sequencing of the embryonic x globin cDNA from the C57BL mouse. The inserted sequence is 316 nucleotides long and provides the amino acid sequence from residue 43 to the C-terminus at position 141. The isolation of a cDNA clone constitutes formal identification of the gene coding for the x protein.

MATERIALS

AND METHODS

Yolk-sac-erythroid cells were isolated from circulating blood of C57 BL/cas fetuses at the 12th day of development and mRNA was purified. Doublestranded DNA was synthesized (Fantoni et al., 1979) and cloned into pBR322. Colonies were screened by hybridization to 32Plabeled cDNA from peripheral blood of 1Zday mouse embryos. 86 recombinant cDNA clones were selected that represented the major transcription products from embryonic blood cells. Of these clones, 24 also hybridized strongly to 32P-labeled globin cDNA from adult mice and were presumed to be specific for the adult a-globin since they are the only major adult sequences present at day 12. Representative clones were used to hybrid-select and in vitro-translate mRNA selected from embryonic cytoplasmic poly(A) ‘RNA. The resulting protein was electrophoresed with lysates of embryonic globin chains to identify the clone.

RESULTS

AND DISCUSSION

(a) Identification pHE52

and by hybrid selection and translation of complementary mRNA. The cDNA insert from pHE52 was sequenced using Maxam and Gilbert (1980) protocols. Each nucleotide of the sequence was confirmed by sequencing from at least two different restriction sites. The cDNA insert is 316 nucleotides long and covers 70% of the coding sequence and 50% of the whole mRNA molecule. The insert begins with its 5’ end at codon corresponding to the amino acid residue 43, continues to the carboxy terminus at position 141 and includes 15 nucleotides of the 3’ untranslated region (Fig. 1). An examination of the amino acid sequence encoded by the globin cDNA (Fig. 1) shows that the globin cDNA specifies the characteristic Lys-TyrArg carboxy terminus of cc-likeglobins. Nick-translated pHE52 DNA hybridized to transfers of EcoRIdigested BALB/c DNA shows the most intense hybridization to a 4.2-kb band reported to contain the embryonic r-like gene x by Leder et al. (1981) (not shown). An mRNA was isolated from the 1l-day embryonic mRNA population by hybrid selection to DNA prepared from pHE52 and was translated in a reticulocyte lysate protein-synthesizing system. This mRNA gave rise to a protein chain which co-electrophoresed with one of the three embryonic globin chains. The other two chains have been identified as the B-like embryonic chains y and z (Fig. 2). It is, therefore, concluded that this cloned cDNA codes for the a-like embryonic globin chain x. Melderis et al. (1974) have determined the amino acid composition of tryptic peptides from the x protein. Their amino acid composition data matches the deduced amino acid sequence encoded by pHE52 except for three residues. Two of these three differences can be explained by a change of only one base pair each. These discrepancies could be due to strain differences between BALB/c, in which the tryptic peptide compositions were determined, and C57BL/Cas, from which the cDNA was prepared. We cannot exclude the possibility of errors introduced by reverse transcriptase and DNA polymerase I (Browne et al., 1977; Vanin et al., 1981).

of the globin chain encoded by

(b) Comparison between the nucleotide sequence of x and other a globins

The plasmid pHE52 was grown and purified for further characterization by nucleotide sequencing

Analysis of the P-like genes of vertebrates has shown that evolutionary divergence between em-

243 50 Leu Pro His Phe Asp Leu His His Sly Ser Gln Gln Leu Arg Ala His Gly Phe Lys GGCCTCCCCCACT'rCGACCTGCACCATGGGTCTCAGCAGTTGCGGGCCCACGGCTTCAAG I I II IIIII II I III II III III I II I I I I I I I I I CTTTCCTCACTTTGATGTAAGCCACGGCTCTGCCCAGGTCAAGGGTCACGGCAAGAAG Phe Val Ser Ala Val Lys Gly LYS I

1

I

1

1

Embryonic Adult

8 8 , 100 Ile Met Thr Ala Val Gly Asp Ala Val Lys Ser Ile Asp %n Leu Ser Ser Ala Leu Thr ATCATGACCGCCGTAGGGGATGCGGTTAAGAGAGCATCGACAACCTCTCTAGTGCTTTGACT I I II I I III I I IIII IIII I III1 III II GTCGCCGATGCGCTGGCCAGTGCTGCAGGCCACC'rCGATGACCTGCCCGGTGCCT'rG'rCT Val Ala Asp Leu Ala Ser Ala Gly His Leu Pro Gly Ser ASP I

Alpha

I

Embrvonic x Adult Alpha

I I I 150 Lys Leu Ser Glu Leu His Ala Tyr Ile Leu Arg Val Asp Pro Val Asn Phe Lys Leu Leu AAGCTGAGCGAGCTGCATGCCTACATCCTGCGTGTGTG~ATCCGGTCAACT'rCAAGCTCCTG IIIIlIII IIIIIIIII III IIIIIIIIIIIIII IIIIIIIII/IIIIIIII GCTCTGAGCGACCTGCATGCCCACAAGCTGCGTGTGSATCCCG'rCAACT'rCAAGCTCCTG Ala His Lys ASP I

x

I

Embryonic Adult

I I 1 I 200 Ser His Cys Leu Leu Val Thr Met Ala Ala Ar’J Phe Pro Ala Asp Phe Thr Pro Glu Val TCCCACTGTCTGCTGGTCACAA,rGGCCGCACGCTT'rCCCGCCGACTTCACCCCTGAGGTC IIIIII IIIIIIII II III1 II II IIIII IIIIIIII I III AGCCACTGCCTGCTGGTGACCT'rGGCTAGCCACCACCCTGCCGATTTCACCCCCGCGG'rA Leu Ser His His Ala

x

Alpha

I

I I I 1 250 300 His Glu Ala Trp Asp Lys Phe Met Ser Ile Leu Ser Ser Ile Leu Thr Glu Lys Tyr Arg CACGAAGCCTGGGACAAGTTCATGTCTATCCTGTCTTCTA'rCC'rGACTGAGAAGTACCGC III I IIIIII~lII I I I I I I IIIII I I I I I I I I CATGCCTCTCTGGACAAATTCCTTGCCTCTGTGAGCACCGTGCTGACCTCCAAGTACCGT Ala Ser Leu Leu Ala Ser Val Thr Val Ser 1

1

t

I

Embrvonic A Adult

Alpha

Embryonic Adult

x

x

Alpha

1

350

***

TAAGCTCCACCGCCACGACCCCC IIIIII I I TAAGCTGCCTTCTGCGGG

Embryonic Adult

Fig. 1.Nucleotide sequence ofpHE52 andthemouse adult a globin (Nishioka shown above the nucleotide are indicated

below the nucleotide

and 3’ C’s in the embryonic sequence

of pHE52,

sequence

the pBR322-cDNA 301-303

Positions

sequence.

sequence

labeled “Embryonic

residues in this run, including of the pBR322

sequence.

The short vertical lines indicate

show the termination

homology

are the result of the GC tailing during x” in the figure, is preceded

the two shown in the figure). The sequence to allow reconstruction

of the complete

cloning,

1979). The corresponding

between

r-like sequences

the two DNA sequences.

and indicate

the endpoints

sequence

This provides

of the pHE52

plasmid.

The 5’ G’s

of the cDNA.

The

(there are a total of 18 G

shown is followed at the 3’ end by GGCATCGTGG. of ATTGCTGCA.

Alpha

amino acids are

differs from the embryonic

at the 5’ end by AACGTTGTTGCCG,,

in the region of the PstI site shows a deletion

junctions

and Leder,

at which the adult amino acid sequence

x

sufficient The asterisks

Inspection

information

about

above positions

codon for the protein.

bryonic and adult globin genes occurred before the mammalian radiation. Consequently, the embryonic globin genes of vertebrates differ among themselves much less than embryonic and adult globin genes within the same species. Similar results are found when the analysis is done

on the z-like genes. At the nucleotide level, the divergence between the coding regions of the embryonic x cDNA sequence and the adult r-globin gene is 40% (Fig. 1). This value is very similar to the 38% divergence as seen in the comparison between z’and chicken U-, human i- and human r-, chicken n’-

244

b

a

this same time. Presumably, we are seeing that same history in the mouse sequences, that is, the embryonic-adult split occurred in an ancestor common to human, chicken and mouse. In contrast with the x genes, the embryonic-adult duplication for the mammalian b-globins occurred approx. 200 MY ago (Vanin et al., 198 1; Efstratiadis et al., 1977; 1980; Konkel et al., 1979; Richards et al., 1979; Baralle et al., 1980; Perler et al., 1980). The non-adult j3genes underwent an additional split to embryonic and fetal genes at about 100 MY ago (Vanin et al., 1981). Clearly, the response of the rand fi-globin complexes to adaptive pressures was different even though the genes are coordinately regulated during ontogeny.

c

ACKNOWLEDGEMENTS

Fig. 2. Autoradiogram vitro translation 13-day embryos;

(b) hybrid

bryos complementary rabbit

reticulocyte

translated described lation

of a polyacrylamide

products

selected

to pHE52;

mRNA

system

by Pelham and Jackson

were identified

from

(c) endogenous

in vitro translation

in a reticulocyte

gel used to analyze in

coded by: (a) poly(A)‘mRNA

from

11-day em-

mRNA of the

system. The mRNA (New England

Nuclear)

(1976). The products

by electrophoresis

was as

We thank Dr. T. Maniatis for providing sequence information prior to publication. We are also grateful to Dr. S.C. Hardies for helpful discussions. This work was partially supported by the Foundation “Istituto Pasteur-Fondazione Cenci Bolognetti”. C.A.H. and M.H.E. are supported by National Institutes of Health grants GM21313, GM30180 and A108998.

of trans-

in 12.5% acrylamide

gels with 6 M urea, 4% acetic acid and 0.4% Triton X-100 using 5% acetic acid, 0.1% P-mercaptoethanol, the electrophoretic

buffer.

volts) the gels were stained salicylate

After

0.1% Triton X-100 as

electrophoresis

and fluorographed

(18 h at 150

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