The nucleotide sequence of the rabbit embryonic globin gene beta 4.

R C Hardison

doi:10.1016/s0021-9258(18)32118-5

Abstract

In the rabbit, two beta-like globin genes, beta 3 and beta 4, are expressed in embryonic erythrocytes and one, beta 1, is expressed in fetal and adult erythrocytes. The complete nucleotide sequence of embryonic gene beta 4 is presented, including the region from 582 base pairs before the cap site to 508 base pairs after the poly(A) addition site. beta 4 has the tripartite structure characteristic of animal globin genes, being interrupted by intervening sequences within codon 30 and between codons 104 and 105. Conserved sequences thought to play roles in transcription, RNA processing, and translation are found in beta 4. Rabbit gene beta 4 shows substantial homology to the human embryonic epsilon-globin gene, not only in message-coding blocks, but also in the 5' and 3' flanking regions and in the two intervening sequences. Comparison of all four rabbit beta-like genes with those of humans suggests that a primordial four gene family, 5'-epsilon-gamma-delta-beta-3', existed prior to the mammalian radiation. An alignment of the 5' flanking sequences of a collection of animal beta-like globin genes shows some conserved regions that differ between the adult genes and the embryonic/fetal genes. These could reflect a common evolutionary lineage rather than stage-specific control sequences.

Highlights

MRNA from both embryonic globin genes /33 and 8 4 /34 (Hardison et al, 1979) have been determined
RNA from this stage of gestation was hybridized with an nucleic acids were dissolved in10pl of 70%formamide in electropho- excess of the labeled DNA probes under conditions where resis buffer and analyzed on a sequencing gel
Following hybridization and digestion with the single strand specific nuclease SI, the Sequencing Strategy-The nucleotide sequence of the rab- size of the protected DNA fragment was determined by elecbit embryonic P-like globin genep3 was determined using the trophoresis on a denaturing polyacrylamide gel with a sestrategy outlined in the lower halfof Fig. 1.The sequence was quencing ladder included for sizemarkers

Summary

EXPERIMENTAL PROCEDURES

As it is in the human fetal y-globin genes. Small The recombinant plasmids with inserts of the 4.5-kb Bgl I1 frag(124 bp) and large (817 bp) intervening sequences are ment containing gene p3 (pBg1 4.5.p3), the 0.8-kb Barn HI-Eco RI located between codon3s0 and 31 and between104 and fragment containing intervening sequence I1of gene p3 End Labezing and Sequencing-Plasmid DNA (usually 20-30 pglp) was digested by restriction endonucleases in a standard buffer of. Plasmid DNA to be labeled by polynucleotide kinase was purified from RNA by passage over an A5M column (Bio-Rad) with 0.3 M NaC1,50 mM Tris-HCI, pH 8, and 1 mM EDTA before digestion with restriction endonucleases. 80% (v/v) deionized formamide, 0.04 M Hepes, pH 7.5, 0.40 M NaC1, gene p3 on the message complementary strand at the Msp I and 0.001 M EDTA (Favaloroet d.,1980).The hybridizationmix was site located 70 nucleotides past the ATG initiation codon in covered with paraffinoil, denatured at 95 "C for 5 min, and annealed the first message-coding block (Fig. 2C). RNA from this stage of gestation was hybridized with an nucleic acids were dissolved in10pl of 70%formamide in electropho- excess of the labeled DNA probes under conditions where resis buffer and analyzed on a sequencing gel

AND DISCUSSION

MlP I

SeK Ser

GGC AAG GAA F C

IVS X

Sequence GofloRbainbbit