Methionine Lab Report

A human heart cDNA library was screened with a probe corresponding to the mouse PPARg (7). Three overlapping clones were identified, purified, and sequenced. The nucleotide sequence is shown in Fig. 1. The longest open reading frame starting from the nucleotide at position 91 coded for a polypeptide of 505 amino acids. There was an in-frame stop codon upstream of this methionine suggesting the translation initiation occurred from this codon. The second and third methionine codons were at positions 29 and 31 in the amino acid sequence.
The first and third methionine codons in hPPARg2 were in a context appropriate for translation initiation, i.e. the Kozak sequence (25), and were conserved between mice and man. The second methionine codon was

The DNA binding domains were 83% conserved between hPPARg2 and hPPARa or hPPARb. Further, three amino acids were present between

the two cysteines in the D-box (amino acids 177–179), a characteristic feature of all PPARs known to date. Based on these observations we believe this human isoform is hPPARg2.
To determine if there are multiple translation start sites for hPPARg2, as in mPPARg2, coupled in vitro transcription/ translation reactions were performed in the presence of
[35S]methionine and pCMVhPPARg2 as template. Two bands were observed by PAGE (Fig. 2). The upper band (57 kDa) corresponded to translation initiation from the methionine at position 1. The lower band (53 kDa) probably corresponded to translation initiation from the methionine at position 31. We cannot rigorously discount translation initiation from the methionine at position 29. However, since this methionine was not within a good Kozak sequence and was also absent in mPPARg2, we think it is unlikely. Indeed, in vitro transcription/ translation of pCMXmPPARg1 (10) and pCMVhPPARg1 also gives rise to bands that comigrate with the lower band observed with pCMVPPARg2. Hence, in analogy with mPPARg1, we called the smaller polypeptide

The transcriptional response of hPPARg2 to PPARg activators was determined in a cotransfection assay (Fig. 4A) and compared with hPPARg1 (Fig. 4B). PPARg1 and PPARg2 are activated by BRL 49653 with an EC50 of approximately 100 nM and by 15-deoxy-D12,14-prostaglandin J2 (an endogenous
PPARg ligand) (26, 27) with an EC50 around 3 mM. They are also activated by 5,8,11,14-eicosatetraenoic acid and 2-bromopalmitate.
The response of hPPARg2 to these four activators is very similar to that of hPPARg1. We conclude that both hPPARg1 and hPPARg2 are similarly activated by known PPARg activators. Since PPARg2 binds to PPREs as a heterodimer with RXR, we next determined the transcriptional response of the
PPARg2/RXR heterodimer to an RXR ligand. LG100268 (28) is a highly selective RXR ligand (Kd ;3 nM). Both BRL 49653 and
LG100268 transcriptionally activated the PPARg2/RXR heterodimer
(Fig. 5A), and the transcriptional response observed with both ligands was greater than that observed individually.
RXR agonists activated a reporter containing the hydratase
(bifunctional enzyme) PPRE. They also induced expression of the hydratase gene in vivo, and increased induction is seen with a combination of RXR and PPAR