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Mouse Sebox Homeobox Gene Expression in Skin, Brain, Oocytes, and Two-Cell Embryos

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Mouse Sebox Homeobox Gene Expression in Skin, Brain, Oocytes, and Two-Cell Embryos Mouse Sebox homeobox gene expression in skin, brain, oocytes, and two-cell embryos Mario Cinquanta*†, Alessandra C. Rovescalli*, Christine A. Kozak‡, and Marshall Nirenberg*§ *Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, and ‡Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Contributed by Marshall Nirenberg, June 12, 2000 Sebox is a mouse paired-like homeobox gene, previously named CTAGTAC-3; in the second PCR, ϩP was 5Ј-GCGGGATCC- OG-9. Sebox genomic DNA and cDNA were cloned and sequenced. TGGAGCTGGAGCGGGTATT-3Ј, and ϪP was as above). The In addition, rat and human Sebox genomic DNAs were cloned and resulting PCR product was subcloned in pBluescriptII-KS(ϩ) sequenced, and the predicted amino acid sequences were com- (Stratagene) and sequenced. The 5Ј termini of Sebox mRNAs were pared. The mouse Sebox gene was mapped to chromosome 11 near identified by RNA ligase-mediated 5Ј-RACE, according to ϩ the Evi 2 locus. The mouse Sebox gene is expressed in brain, skin, Schaefer (5). Decapped poly(A) RNA was ligated to the 3Ј end ovary, and liver of mice. In the brain, the Sebox gene is expressed of 5Ј-UAAUACGACUCACUAUAGGGAGGAGGAAGCU- in the cerebral cortex and CA areas of the hippocampus, pontine CUAUUGCACUAGUCUCGAGGAUCCAAUAAA-3Ј, and nuclei, choroid plexus, and the cerebellum. Northern analysis and 1 ␮g of the ligated RNA was used for reverse transcription with a RNase protection assays revealed low levels of Sebox RNA in gene-specific primer (GSP1: 5Ј-GCTTCAGGCAGGTGAGTTA- Ј 12-day mouse embryos and higher levels in 18- and 19-day em- 3 ) catalyzed by SuperScript II (Life Technologies). The first-strand bryos. In late embryos and newborn mice, Sebox expression is cDNA then was used as a template for PCR amplification by using Ј localized in the epidermis. In adult mice, Sebox RNA was found in a reverse primer nested to GSP1 (GSP2: 5 -AGGATAGGGC- Ј maturing oocytes and in fertilized eggs; however, the abundance CTAGCTGCAAATAC-3 ) and a forward primer specific for the anchor oligonucleotide (anchor-1: 5Ј-GGAGGAGGAAGCTC- of Sebox RNA is decreased in the two-cell embryo, and little or Ј none was detected in the four-cell embryo. Hence, Sebox is a TATTGCACTA-3 ). An additional amplification then was per- maternally expressed homeobox gene. formed by using an aliquot from the previous PCR and primer pair: GSP3 (5Ј-CACCAACTGCCCGACACTGAAAG-3Ј; nested to Ј transcription factors ͉ OG-9 ͉ rat Sebox ͉ human Sebox ͉ epidermis GSP2), and anchor-2 primer (5 -TTGCACTAGTCTCGAG- GATCCAAT-3Ј; nested to anchor-1). The PCR products were cloned into pCRII (TA Cloning System, Invitrogen) and sequenced omeodomain proteins play a key role in coordination of in both directions. Hgene activity and determination of cell fate in the devel- Human genomic DNA from placentas of multiple individuals opment of organisms as diverse as plants, yeast, insects, and was obtained from CLONTECH and amplified by PCR by using mammals (1). The homeodomain region of the proteins, and Platinum Taq DNA Polymerase High Fidelity (Life Technolo- sometimes the functions of the proteins, have been conserved gies), using two primer pairs: ϩP: 5Ј-GAAGTATAGGATT- during evolution. The prd-like class of homeobox genes in- TCAGGCACAGA-3Ј; ϪP: 5Ј-TGGAGCTGGCAAAGGGTA- cludes several genes that encode proteins with a homeodomain AGGT-3Ј; and ϩP: 5Ј-GCCAGCCACCCTGAGCATGATA-3Ј; similar to that of prd but lack a paired domain, which is present ϪP: 5Ј-CAGGGAATCCACTTCTGATGTGTT-3Ј. Deoxyade- in homeodomain proteins of the paired class (2). Most of the nylic acid residues then were added to the 3Ј termini of amplified prd-like homeodomains have a glutamine residue at position DNA products catalyzed by Taq DNA polymerase, and DNA 50 of the homeodomain, whereas a serine residue is found at the fragments were ligated into pCRII. Three to five recombinant same position in the paired homeodomain (2). In addition, most clones from each PCR reaction mixture were isolated, and DNA of the prd-like homeobox genes have an intron between codons inserts were sequenced in both directions by using M13(-20) and for amino acid residues 46 and 47 of the homeodomain (2). reverse primers. Sebox,askin-embryo-brain-oocyte-specific homeobox gene Similarly, rat genomic DNA from kidneys of 200 outbred (formerly termed OG-9), is a prd-like class gene that was cloned Sprague–Dawley male rats was purchased from CLONTECH and previously in this laboratory (3). The amino acid sequence of the amplified with primers based on the mouse Sebox genomic ϩ Ј Sebox homeodomain is not closely related to any known home- sequence (primer pair 1: P, 5 -GGAACCTTCCCTGGCTCTAG- GATG-3Ј; ϪP, 5Ј-ATAGGATCCCCCAACCCACTGGCACAG- odomain. The murine homeodomain with the highest similarity Ј ϩ Ј to Sebox is Phox2 (65% identical amino acid residues) (4). 3 ; pair 2: P, 5 -GCCGGATCCCAGTCCTGTGGAGGCATCT- 3Ј; ϪP, 5Ј-CTTGGATCCGATGGCTGAGGTATAGATA- Here we describe the predicted amino acid sequence of the Ј ϩ Ј Ј Ϫ Sebox protein, the structure and chromosomal location of the AGGTC-3 ; pair 3: P, 5 -TGGGTCCAGGGCAGAGTT-3 ; P, 5Ј-TTGGGATCCATCACTCTCTCTTCAGATCCACAG-3Ј). Sebox gene, and the pattern of expression of the Sebox gene, and demonstrate that Sebox is a maternally expressed gene. We also identified the human SEBOX gene and cloned and sequenced rat Abbreviation: RACE, rapid amplification of cDNA ends. Sebox genomic DNA. Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos., mouse Sebox genomic DNA (AF284336); rat Sebox genomic DNA Materials and Methods (AF284338); and human SEBOX genomic DNA (AF284337)]. Cloning. The 3Ј-end of the mouse Sebox gene was cloned by rapid †Present address: Department of Experimental Oncology, European Institute of Oncology, amplification of cDNA ends (RACE) by using the 3Ј-RACE Milan, Italy. System (Life Technologies, Rockville, MD) and 0.5 ␮g of poly(A)ϩ §To whom reprint requests should be addressed at: Building 36, Room 1C-06 National ͞ Institutes of Health, 36 Convent Drive, MSC 4036, Bethesda, MD 20892-4036. E-mail: RNA from brains of adult BALB c mice. A cDNA fragment then [email protected]. was amplified by two sequential PCRs with nested primer pairs (for ϩ Ј The publication costs of this article were defrayed in part by page charge payment. This the first PCR, P was 5 -TCCGGATCCGAGGAAGAGGAC- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. CACTTTCAGTGT-3Ј, and ϪP was 5Ј-GGCCACGCGTCGA- §1734 solely to indicate this fact. 8904–8909 ͉ PNAS ͉ August 1, 2000 ͉ vol. 97 ͉ no. 16 Downloaded by guest on October 2, 2021 DNA Sequencing. Cloned DNA fragments were sequenced with a Perkin–Elmer͞Applied Biosystems 373A DNA sequencer by using fluorescent dideoxynucleotides. Sequence analysis and assembly of contigs were performed by using the WISCONSIN SEQUENCE ANALYSIS PACKAGE (Genetics Computer Group, Madison, WI). Sebox Chromosomal Mapping. Sebox was mapped by analysis of the cross (NFS͞N ϫ Mus spretus) ϫ M. spretus or C58͞J (6). RNA Isolation and Northern Blot Hybridization Analysis. Total RNA was isolated by a modified version of the Chomczynski and Sacchi method (7) (Totally RNA system, Ambion, Austin, TX), and poly(A)ϩ RNA was purified by chromatography on oligo-dT cellulose (5 Prime33 Prime). Northern blots containing 2 ␮gof poly(A)ϩ RNA per lane were hybridized with a double-stranded DNA probe spanning the coding region downstream of the homeobox (nucleotide residues 991-1410 in Fig. 2) labeled with [␣-32P]dCTP (Random Primed DNA labeling kit, Stratagene). Blots were washed at high stringency (50°C, 0.1 ϫ SSC͞0.1% SDS), and autoradiograms were developed after 10–15 days. In Situ Hybridization. Superovulation was induced in 4-week-old BALB͞c mice by injection of pregnant mare serum (PMS, Sigma), 5 units i.p., at 2:00 p.m., followed by human chorionic gonadotrophin (hCG, Sigma), 5 units, 46–48 h later (8). Super- ovulating mice then were either killed 21 h after hCG to obtain oviducts containing ovulated eggs or mated and killed 24 h after hCG to obtain fertilized eggs. For the analysis of two- and Fig. 1. Northern analysis of Sebox mRNA in mouse tissues. Blots (2 ␮gof four-cell embryos, oviducts of superovulated or mated females poly(A)ϩ RNA͞lane) were hybridized with a cDNA probe labeled with were collected 48 and 68 h after hCG injection, respectively. [␣-32P]dCTP (specific activity of 1.0 ϫ 109 cpm͞␮g). A 1.45-kb RNA transcript Ovaries and fallopian tubes containing either unfertilized eggs or was detected in adult ovary, skin, and liver (A and B). In addition, RNA of the one-, two-, or four-cell embryos were fixed in 2% paraformal- same length was observed during late fetal development (D). Two diffuse dehyde (PFA) in PBS for 7–8 h at 4°C, infiltrated with 20% bands of RNA, approximately 1.5 and 1.8 kb in length, were detected in sucrose overnight at 4°C, and frozen in M-1 embedding matrix postnatal brain starting from the fifth postnatal day, which increased in (Lipshaw Manufacturing, Detroit) over a mixture of 2-methyl- abundance thereafter (C). butane͞dry ice. Other tissues used for in situ hybridization were frozen immediately after dissection without fixation. Sections ␮ development. At 17.5 days, the hybridization signal is a small (12 m) were cut with a cryostat, fixed in 4% PFA, treated with fraction of the signal detected in the body and head (devoid of 0.25% acetic anhydride, and dehydrated by passage through a the brain) of newborn mice (P0). Expression of Sebox RNA in graded ethanol͞water series. Unless otherwise stated, a 12.5-day-old embryos also was detected by reverse transcription– [33P]UTP-labeled RNA probe corresponding to the portion of PCR (data not shown).
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