A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.
The tertiary structure of the macrophage migration inhibitory factor (MIF) from rat liver (12,300 Mr) is presented at 2.2 A resolution. Each monomer consists of two beta/alpha/beta motifs aligned in quasi two-fold symmetry, comprising a domain consisting of a four-stranded mixed beta-sheet and two antiparallel alpha-helices. The protein exists as a trimer in the crystal. An extra beta-strand that is almost perpendicular to the other beta-strands joins to the beta-sheet of the neighbouring monomer in the trimer. Unexpected similarities were detected between MIF and two kinds of isomerase.
DNA complementary to human a-fetoprotein (AFP) mRNA was cloned in the plasmid pBR322. Analysis of three overlapping cDNA clones revealed most of the nucleotide sequence of AFP mRNA, and the remaining nucleotides at the 5' end of the mRNA were elucidated from a cloned genomic DNA fragment. The amino acid sequence was deduced from the nucleotide sequence, which revealed 19 amino acids in the signal sequence and 590 amino acids in mature AFP. There are 15 regularly spaced disulfide bridges, which generate a folding structure having three repeating domains. There is one potential N-glycosylation site, Asn-Phe-Thr, in the amino acid sequence. In comparison with mouse AFP, 66% of the amino acid sequence was conserved, with the highest identity (72%) in domain 3, followed by domain 2 (67%) and domain 1 (59%). In comparison with human albumin, a 39% conservation ofprimary structure was found. Again, the similarity was the highest in domain 3 and the lowest in domain 1. Human AFP and human albumin are similar in overall structure, but certain parts of the molecules differ significantly in their predicted secondary structure. a-Fetoprotein (AFP) is a major serum protein (Mr, =70,000) synthesized during fetal life (1-4). Reappearance of AFP in adult serum often signals pathological conditions, particularly hepatocarcinomas and teratocarcinomas (1-4). In contrast, albumin increases steadily during fetal and neonatal growth and shows no apparent changes in concentration associated with development of liver and germ cell tumors.In the past several years, DNAs complementary to mouse (5, 6) and rat (7) AFP mRNAs and rat (8) and human (9, 10) albumin mRNAs have been cloned. Nucleotide sequences of these clones and the amino acid sequences deduced from them have provided valuable information on the structure of these proteins. However, the rat cDNA clones are not full-length and consequently, molecular comparisons have been limited to partial sequences of mouse and rat AFPs. Also, comparisons between AFP and albumin were possible only interspecially because neither mouse albumin nor human AFP mRNA sequences were known in their entirety.In this paper we report the complete nucleotide sequence of human AFP mRNA and the amino acid sequence of AFP. These data allow comparisons of the entire primary structures of AFPs interspecially and AFP and albumin intraspecially. MATERIALS AND METHODSThe production of the cDNA clone, pHAF2, containing 841 nucleotides or about 40% of the human AFP mRNA sequence at the 3' end was described (11). By using the nick-translated insert of pHAF2 as a probe, a second clone, pHAF6, with an additional 320 nucleotides at the 5' end was obtained (Fig. 1).
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