http://repub.eur.nl/res/aut/3247/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/8612/ 1996-01-01T00:00:00Z Transglutaminases (TGases) are calcium-dependent enzymes catalysing the post-translational cross-linking of proteins. In the prostate at least two TGases are present, the ubiquitously expressed tissue-type TGase (TGC), and a prostate-restricted TGase (TGP). This paper deals with the molecular cloning and characterization of the cDNA encoding the human prostate TGase (hTGP). For this purpose we have screened a human prostate cDNA library with a probe from the active-site region of TGC. The largest isolated cDNA contained an open reading frame encoding a protein of 684 amino acids with a predicted molecular mass of 77 kDa as confirmed by in vitro transcription-translation and subsequent SDS/PAGE. The hTGP gene was tissue-specifically expressed in the prostate, yielding an mRNA of approx. 3.5 kb. Furthermore, a 3-fold androgen-induced upregulation of hTGP mRNA expression has been demonstrated in the recently developed human prostate cancer cell line, PC346C. Other well established human prostate cancer cell lines, LNCaP and PC-3, showed no detectable hTGP mRNA expression on a Northern bolt. The gene coding for prostate TGase was assigned to chromosome 3. http://repub.eur.nl/res/pub/39449/ 1993-05-26T00:00:00Z ln this study the androgen receptor (AR) transcription unit is presented. Chapter II describes the isolation and characterization of one genomic clone, from which the amino acid sequence of the N-terminai domain of the hAR was deduced. This amino acid sequence was characterized by the presence of several homopolymeric stretches, of which a long a-stretch {20 residues) and a long G-stretch (16 residues) are most conspicuous. In combination with a previously described eDNA clone, which encoded the hAR DBD and LSD, an open reading frame of 2730 bp was deduced, encoding a protein of 910 amino acids. Next, the complete coding region of the hAR gene was isolated from a genomic library, as described in Chapter Ill. The information for the hAR was found to be separated over eight exons. The total lenght of the single copy hAR gene, which is located on the X-chromosome, exceeds 90 kb. As described in Chapter ll, the N-terminal domain is present in one single exon. The DBD is encoded in the exons 2 and 3, with each exon containing the information for one "zinc-finger". The LBD is encoded by the exons 4 to 8. Interestingly, the positions of the introns were found to be conserved between the hAR gene and the cPR and hER genes. The experiments described in Chapter IV extend the work of the Chapters II and Ill and deal with the characterization of the complete hAR eDNA and gene. Northern blot analysis of hAR mRNA identified two hAR mRNA species of 11 and 8 kb, respectively, in the human prostatic tumor cell line LNCaP. A full-length hAR eDNA was constructed from eDNA and genomic clones. Structurally, the 11 kb eDNA consists of a long 5'-UTR (1.1 kb), a 2.7 kb ORF, and a very long 3'·UTR (6.8 kb). The complete 5'-UTR and 3'-UTR were found to be encoded in the exons 1 and 8 of the hAR gene, respectively, fixing the number of exons in this gene at 8. The promoter of the hAR gene, located 1.1 kb upstream from the ATG translation initiation codon in exon 1, was structurally and functionally characterized. Two major sites of transcription initiation in a 13 bp region were identified by 51-nuclease protection experiments. DNA fragments, spanning these sites of initiation, conferred promoter activity upon a promoterless reporter gene construct. 51-nuclease protection experiments with RNA from COS cells, transiently transfected with these constructs, showed usage of the correct initiation sites. Structurally, the promoter lacks TATA/CCAAT boxes and potential regulatory elements consist of a short GC-box (-59/-321. which includes an Sp1 binding sequence (-46/-37), and a long homopurine stretch (-60/-117). The 3'-UTR contains two equally effective polyadenylation signals at a mutual distance of 221 bp. In addition, it was shown that the 8 kb hAR mRNA results from an alternative splice in the 3'-UTR, which does not affect the hAR ORF http://repub.eur.nl/res/pub/8890/ 1990-01-01T00:00:00Z The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcription