Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Zhu et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0336133100

Supporting Text
Cloning Results for Vertebrate cDNAs Similar to the Spotted Seatrout mPR
Human mPRα.
Four similar EST clones from tissues of testis (dbEST ID: 1780349, 1731306), adrenal gland (dbEST ID: 990542), and ovary (dbEST ID: 3680423) were identified in human ESTs database by BLAST search using human genome sequence. An IMAGE clone (dbEST 1731306, IMAGE: 1621612) from testis was selected and purchased from Research Genetics (Huntsville, AL). The clone was fully sequenced in both directions and deposited into GenBank (accession no. AF313620). The full length of the clone has 1,262 nt, including a 22-bp poly(A) tail. The DNA sequence shows an open reading frame of 1,041 nucleotides, starting at the 97 nt and ending at the 1,137-nt position. The open reading frame encodes a polypeptide of 346 amino acid residues, including a 19-aa signal peptide. Human mPRβ.
Three similar EST clones from fetal brain (dbEST ID: 448468, 448148) and neuroepithelial cells (dbEST ID: 740252) were identified in human ESTs database by BLAST search using human genome sequence. All three clones were truncated at 5' end. An IMAGE clone (IMAGE: 563998; dbEST ID: 740252) was purchased from Research Genetics and fully sequenced. The DNA sequence of the clone has 2,221 bp, with 671 nt encoding a 219-aa partial amino acid sequence with incomplete 5' end ( GenBank accession no. AF313619). The sequence contains a large 3'-untranslated region, with four poly(A) signals at the 842, 1,017, 1,102 and 2,182 nt positions and an 18-bp poly(A) tail. Mouse mPRα.
Six similar mouse EST clones from mouse brain (dbEST ID: 3262253, 3262254, 2336487), lung (dbEST ID: 1609269), heart (dbEST ID: 779001), and embryo (dbEST ID: 735621) were identified by BLAST search by using human genomic sequence and mouse ests database in GenBank. A clone (dbEST ID: 3262253) was obtained from Dr. Katsuyuki Hashimoto (National Institute of Infectious Diseases, Japan). The clone (mb) was fully sequenced in both directions and the sequence was deposited into GenBank (accession no. AF3136180). The full length clone has 2,053 nt with a poly(A) signal at the 2,017-nt position and a 17-bp poly(A) tail. The mouse mPRα sequence shows an open reading frame of 1,038 nt, starting at the 62 nt and ending at 1,099 nt position. The open reading frame encodes a polypeptide of 345 amino acid residues.
Mouse mPRβ.
One mouse EST clone (dbEST ID: 2469454) from mouse testis was found by BLAST search using human genomic sequence and mouse ESTs database. An IMAGE (IMAGE Id: 567489) clone was purchased from Research Genetics and fully sequenced. The full length of the clone (mt) has 1,496 bp nucleotides with a poly(A) signal at 1,476 and a 16-bp poly(A) tail. The mt sequence shows an open reading frame of 1,065 nt starting at the 349 and ending at the 1,411 nt positions. The open reading frame encodes a polypeptide of 354 amino acid residues. The cDNA sequence was deposited in GenBank (accession no. AF 313617).
Pig mPRα.
One pig EST clone (dbEST ID no. 3819745) was obtained from pig embryo by using human genomic sequence and non-human and non-mouse ESTs database in GenBank. The clone was purchased from BACPAC Resource Center at the Children's Hospital Oakland Research Institute (Oakland, CA). The cDNA sequence was deposited into GenBank (accession no. AF313616). The full length of the clone has 2,254 bp nucleotides, with a poly(A) signal at the 2,204 nt position and a 34-bp poly(A) tail. The pig mPRα sequence shows an open reading frame of 1,053 nt starting at 156 and ending at the1,208 nt positions. The open reading frame encodes a polypeptide of 350 amino acid residues). Pig mPRβ.
One pig EST clone (dbEST ID no.1616528) obtained from pig intestine showed high homology to human genomic sequence by using BLAST search of non-human and non-mouse ESTs database in GenBank. The clone was obtained from Dr. Merete Fredholm (The Royal Veterinary and Agricultural University, Denmark). The cDNA was fully sequenced and deposited into GenBank (accession no. AF313615). The full length clone has 2,722 bp nucleotides with a 42-bp poly(A) tail. The pig mPRβ sequence shows an open reading frame of 1,064 nt, starting at 73 and ending at the 1,137 nt positions. The open reading frame encodes a polypeptide of 354 amino acid residues. Human, mouse, and Japanese pufferfish mPRg.
The full-length human mPRg (AK000197) has 2,398 nt and contains 330 aa. The mouse kidney mPRg (AK002481) has 1,179 nt and contains 330 aa. The partial sequence of pufferfish mPRg (302aa) was retrieved from Fugu genome database. Zebrafish mPRα and mPRβ.
One major fragment was amplified from zebrafish ovary cDNA by RT-PCR by using a set of paired primers, mPRmixF1/mPRmixR1 (Table1). The 450-bp PCR products were cloned and seven randomly selected clones were sequenced. The DNA sequences of four clones were identical (mPRα) and shared 72% identity to seatrout mPR. The DNA sequences of other three clones were also identical (mPRβ) but shared 48% identity to seatrout mPR. The longest cDNA of zebrafish mPRα obtained from RACE and RT-PCR had 1,478 nt and a 21-bp poly(A) tail. The zebrafish mPRα has an open reading frame encoding a polypeptide of 354 amino acid residues. The zebrafish mPRα shares 80% amino acid identity compared to seatrout mPR. The longest cDNA of zebrafish mPRβ obtained from RACE and RT-PCR had 1,452 bp nucleotides and a 22-bp poly(A) tail. The zebrafish mPRβ shows an open reading frame encoding 352 amino acid residues. Xenopus
mPR. One 450-bp fragment was amplified from Xenopus ovary cDNA by using nested RT-PCR and two sets of primers (Table1). The sequence of four randomly selected clones were identical and shared high homology (52% amino acid identity) to seatrout mPRα. The longest cDNA of Xenopus mPR obtained from RACE and RT-PCR had 1,721-bp nucleotide with a 33-bp poly(A) tail. The Xenopus mPR had an open reading frame that encodes 353 amino acid residues.