Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1681-1685, March 1990 Genetics Comparison of human ZFY and ZFX transcripts (sex determination/"zinc rmgers"/X chromosome/Y chromosome/polymerase chain reaction) MARK S. PALMER, PHILIPPE BERTA*, ANDREW H. SINCLAIR, BARBARA PYM, AND PETER N. GOODFELLOW Human Molecular Genetics, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom Communicated by M. F. Lyon, November 16, 1989 ABSTRACT ZFY is a candidate for the primary sex- Southern Blotting. Endonuclease-digested DNA was elec- determining gene (TDF, testis-determining factor) on the hu- trophoresed in 0.8% agarose gels and transferred and fixed to man Y chromosome. We have isolated cDNA clones ofZFY and Hybond-N+ filters (Amersham) (10). its homologue on the X chromosome, ZFX. The transcripts of Hybridization. (i) Oligonucleotide probes. Oligonucleotide these genes are very similar to each other and encode predicted 1818 was labeled with 32P at its 5' end with phage T4 proteins of equal size. The conceptual amino acid sequence of polynucleotide kinase. Library filters were hybridized in 5 x both proteins contains an acidic domain, similar to the acti- SSC/20 mM sodium phosphate, pH 7.0/lOx Denhardt's vation domain of transcription factors, and a potential nucleic solution/10%o dextran sulfate/7% SDS containing oligonu- acid-binding domain of 13 "zinc ringers."' We have used the cleotide (106 cpm/ml) and heterologous DNA (100,ug/ml). polymerase chain reaction to demonstrate the expression of (ii) Plasmidprobes. Plasmid inserts were radiolabeled by the ZFY and ZFX in a wide range of adult and fetal human tissues random primer method (11). Filters were hybridized in 5X and to show that ZFX is expressed from the inactive X SSPE/5x Denhardt's solution/0.5% SDS containing 0.5 x chromosome present in human-mouse hybrids. 106 cpm/ml. Final wash conditions were 0.2x SSC at 650C. (SSC is 150 mM NaCl/15 mM sodium citrate, pH 7.0; The primary signal for male sex determination in mammals is Denhardt's solution is 0.02% Ficoll/0.02% polyvinylpyrroli- a testis-determining factor (1) encoded by TDF, a gene on the done/0.02% bovine serum albumin; SSPE is 180 mM NaCl/ Y chromosome. Genetic analysis of sex-reversed individuals 10 mM sodium phosphate, pH 8.0/1 mM EDTA.) been used refine the TDF Sequencing of cDNA Clones. cDNAs derived from Agtll has to chromosomal location of to libraries or by PCR were subcloned into pBluescript vectors an interval of 140 kilobases (kb) on the short arm ofthe human (Stratagene). Plasmids were sequenced as double-stranded Y chromosome (2-4). Sequences within this interval were DNA by using synthetic oligonucleotide primers and Seque- found to be conserved on the Y chromosome in a variety of nase (United States Biochemical). eutherian mammals and also cross-hybridized to sequences Preparation of RNA. Total cellular RNA was extracted on the X chromosome. The nucleic acid sequence of a Y from cell lines and human tissues by the guanidinium thio- chromosome genomic fragment encoded an open reading cyanate method (12), and poly(A)+ mRNA was isolated by frame whose predicted amino acid sequence was a tandem oligo(dT)-cellulose column chromatography. repeat of 13 "zinc-finger" domains. The Y chromosome gene Northern Hybridization. mRNA (5 ,g) was electropho- encoding this zinc-finger protein was called ZFY, and the resed in a 0.8% agarose gel with Mops buffer and 2.2 M homologous sequence on the normal X chromosome was formaldehyde and then transferred to Hybond-N. The filter called ZFX. The zinc-finger motif has been associated with was hybridized at 46°C in Sx SSPE/50% (vol/vol) formam- nucleic acid-binding proteins, and if ZFY is TDF this is ide/5x Denhardt's solution/0.5% SDS with probe at 0.5 X consistent with the proposed regulatory function of TDF. 106 cpm/ml. The filter was washed 15 min in 2x SSPE/0.1% Support for the equivalence ofZFYand TDFwas obtained by SDS, 30 min in lx SSPE/0.1% SDS at 42°C, and 15 min in studying the sex-determining region of mice (5-7). However, 0.1x SSPE/0.1% SDS at room temperature. the finding of homologous genes in marsupials solely on PCR Amplification of cDNA. (i) Specificfirst-strand cDNA autosomes, and not on the sex chromosomes, suggests that synthesis. RNA (5 ,ug) was reverse-transcribed (13), using ZFY might not be the primary signal for sex determination avian myeloblastosis virus reverse transcriptase and syn- (8). Alternatively, eutherians and metatherians may have thetic oligonucleotide primers. (ii) PCR. This was done evolved different mechanisms for sex determination. essentially as described (14) for 35 cycles, with annealing at We have isolated cDNA clones derived from transcripts of 600C. both ZFYand ZFX and we report their sequences here.t We Analysis ofPCR-Amplified cDNA. Amplified products were used the nucleotide sequences to design primers for the extracted in chloroform and then precipitated with spermi- polymerase chain reaction (PCR), which enabled us to look dine (15). Washed precipitates were digested with BamHI at their expression in different tissues and cell types. and run in a 1% agarose gel containing ethidium bromide. Oligodeoxynucleotides. The sequence of oligonucleotide 1818 (5'-AACAAGATGCATAAATGCAAATTCTGCGAA- MATERIALS AND METHODS TATGAGAC-3') is from a region of the published genomic cDNA Libraries. Adult testis cDNA and HeLa cell cDNA sequence ofthe ZFYzinc-finger domain (4) predicted to have libraries, both in bacteriophage Agtll, were obtained from minimum codon degeneracy; the sequence corresponds to Clontech. A cDNA library (pCD2Bassing) derived from a nucleotides 1560-1597 of the ZFY transcript reported here. human foreskin fibroblast line was kindly provided by H. Okayama in the vector pCD2 (9). Abbreviation: PCR, polymerase chain reaction. Cell Lines. These are described in Figs. 2 and 6. *Permanent address: Centre de Recherches de Biochimie Macro- moleculaire, Centre National de la Recherche Scientifique LP 8402/Institut National de la Santd et de la Recherche Medicale The publication costs of this article were defrayed in part by page charge U.249, rte de Mende, 34033 Montpellier Cedex, France. payment. This article must therefore be hereby marked "advertisement" tThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M30607 and M30608). 1681 Downloaded by guest on October 1, 2021 1682 Genetics: Palmer et al. Proc. Natl. Acad. Sci. USA 87 (1990) Oligonucleotide 5242 (5'-CCCGAATTCGCTGTGACTGAT- A B 8'9 GAGAATTAAAGGC-3') is identical with nucleotides 233- d9 X Y X Y H 9 X 4 X Y H 257 of ZFX plus an EcoRI linker at the 5' end. Oligonucle- 9.4 _ _ otide 3039 (5'-CCTCGACTTAAACTTCTTCC-3') is comple- 9.4 s mentary to nucleotides 1306-1325 of ZFY and 1550-1569 of ZFX. Oligonucleotide 3043 has the same sequence as oligo- nucleotide 3039 but has an EcoRI linker at the 5' end. 4A 4A Oligonucleotide 5245 (5'-CATGATAGTGTAGTGGAAG- Q 0 CAGAAA-3') is identical with nucleotides 486-510 of ZFY and 730-754 of ZFX. Oligonucleotide 5244 (5'-CCTC- is complementary to TCCTACGATCACTTCCATATA-3') 2.0 - 4S 2.0...- nucleotides 981-1005 of ZFY and 1225-1249 of ZFX. 40 -w_0,,0W. RESULTS 1.1 1.1 Identification of Partial Transcripts of ZFY and ZFX. An adult testis cDNA library was screened with oligonucleotide 06 Q68 1818. A positive phage plaque was isolated and found to contain a 2.3-kb EcoRI insert. This was subcloned into pBluescript and called pMF-1 (Fig. 1). pMF-1 includes at its 3' end a sequence that is identical (except for one base FIG. 2. Southern blot hybridizations. DNA was prepared from difference) with the nucleotide sequence of the genomic the following sources. Lanes 6, male cell line PGF (16); lanes 9, domain entered into the EMBL data base (acces- female cell line WT49 (17); lanes 4X, a 48XXXX cell line, GM1416B zinc-finger (Coriell Institute for Medical Research, Camden, NJ); lanes 4Y, a sion no. J03134) from nucleotide 73 of the genomic sequence 49XYYYY cell line, Oxen (18); lanes X, a hamster-human hybrid cell through to its 3' end. line containing the human X chromosome, C12D (19); lanes Y, a pMF-1 was used to screen a HeLa cDNA library (HeLa hamster-human hybrid containing the human Y chromosome, 853 cells carry no Y chromosome). The 1.3-kb EcoRI insert of a (20); lanes H, the hamster parent cell line, W3GH (19); lane 9, positive phage was subcloned into plasmid (pPB, Fig. 1). pPB hamster-human hybrid containing human chromosome 9p, CF11-4 must contain a transcript from ZFX or a related transcript (21). Filters were screened with pMF-1 (A) or pPB (B). Numbers at from an autosome. pPB lacks the zinc-finger domain but is left of autoradiographs are size markers (kb). Lanes do not all contain homologous to the 5' end of pMF-1. It also extends further 5' the same amount of DNA. than pMF-1 by about 400 bp. There is one continuous open reading frame in pPB with no evidence of a 5' untranslated Affara et al. (22) isolated a transcript that contained the region. Comparison of these sequences suggested that the zinc-finger region but whose 5' sequence was shown, by in first 52 bp at the 5' end of pMF-1 are inverted and probably situ hybridization, to be derived from the short arm of arose as an artifact during the construction of the library. chromosome 9 (9p).