Abstract
The structure of a human serum amyloid A (SAA) genomic clone (SAAg9) has been analyzed and the nucleotide sequence of the coding regions is compared with that of the cDNA for apoSAA1. The leader and coding sequences of exons 2 and 3 are identical to SAA1. However, there are 10 nucleotide and 7 derived amino acid substitutions in exon 4. These changes are identical to the amino acid sequence of the amyloid protein associated with familial Mediterranean fever. In particular, the amino acid substitution (Thr to Phe) at residue 69 of SAA1 may have an important role in this type of hereditary amyloidosis. The genomic clone SAAg9 has been transfected into mouse L cells, and constitutive expression of human specific mRNA and protein were observed in stable transfected clones. The expression of both SAA mRNA and protein were increased by incubation of the transfected cells with purified human interleukin-1 (IL-1), both human and mouse recombinant IL-1, and recombinant human tumor necrosis factor alpha. The induction of SAA is pretranslational and is likely to be mediated by protein factor(s) since incubation with cycloheximide diminished IL-1-dependent increase in SAA mRNA.
Highlights
The genomic clone SAAg9 has been transfected into appears to be a gene familyfor human SAA [14].In mice, three SAA genes and their corresponding mRNAshave been described [15]
These were hybridized up to a thousandfold or more in response to tissue injury or under standard conditions [17] with the human specific portion of inflammation. It was originally identified as a serum protein homologous to amyloid A (AAp)rotein, the subunoitf amyloid fibrils in reactive amyloidosis, and SAA is thought to be the precursor for AA [1].A product precursor relationship has the SAAl cDNA probe [12] which had been radiolabeled using oligonucleotides as primers [18].Following hybridization, the filters were washed in 30 mM NaCl, 3 mM sodium citrate, 0.1% sodium dodecyl sulfate at 65 “C for 1 h, dried, and visualized by autoradiography
Coincubation of the cells with 10 units of purified IL-lP and cycloheximide (10 pg/ml) markedly reduced the level of specific mRNA compared to that obtained with IL-1 alone as theinducer (Fig. 5B).This result was observed in duplicate experiments and using two transfected cell lines
Summary
A ified Eagle's medium,10%fetal bovine serum, 1.36 mg/ml hypoxanthine, 17.4 pg/ml aminopterin, 388 pg/ml thymidine (HAT)).HAT-. Resistant colonies were isolated and propagated in selective media at all times. Endonucleases PuuII and PstI, electrophoresed on 1%agarose gel, transferred onto nitrocellulose filter, and hybridized to 32P-labeled humanspecificfragment of SAAl cDNA, pAlb. endonucleases PuuII and PstI, electrophoresed on 1%agarose gel, transferred onto nitrocellulose filter, and hybridized to 32P-labeled humanspecificfragment of SAAl cDNA, pAlb This probewas radiolabeled by using oligonucleotides as primers [18].Hybridizing bands were identified by autoradiography. Hoffmann-LaRoche),with 6 X lo6units/mg specific activity [22] and a PI of 5, was stored in 5 M guanidine hydrochlorideat -20 "C
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