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Cell Growth & Differentiation Vol. 11, 11-17, January 2000
© 2000 American Association for Cancer Research


Articles

Molecular Cloning of a Novel Retinoic Acid-responsive Gene, HA1R-62, Which Is also Up-Regulated in Hoxa-1-overexpressing Cells1

Jian Shen and Lorraine J. Gudas2

Department of Pharmacology, Cornell University Medical College, New York, New York 10021

Abstract

Using a PCR-based cDNA subtractive hybridization method (L. Diatchenko et al., Proc. Natl. Acad. Sci. USA, 93: 6025–6030, 1996), we cloned a cDNA fragment of a novel gene that is highly expressed in F9-10; F9-10 is an F9 teratocarcinoma stem cell line that expresses high levels of exogenous Hoxa-1 mRNA and protein in comparison to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of retinoic acid (RA). Rapid amplification of cDNA ends was used to clone the full-length cDNA of this gene, designated HA1R-62 (Hoxa1 regulated-62). We have shown that HA1R-62 is also a RA-responsive gene and that it is expressed (mRNA size, ~4.3 kb) in adult mouse thymus, lung, kidney, and ovary as well as in 12.5-day mouse embryos. DNA sequence analysis and in vitro translation experiments have shown that HA1R-62 encodes a protein with a molecular mass of approximately 26 kDa. Elucidation of the function of the HA1R-62 gene product will provide new insights into the functions of RA and homeobox genes.

Introduction

RA,3 a member of the vitamin A family of signaling molecules called retinoids, influences the proliferation and cellular differentiation of a wide variety of cell types and plays an important role in vertebrate development (1 , 2) . Most of the effects of RA are elicited through its ability to regulate gene expression via the action of RARs. The RARs (RAR{alpha}, RARß, RAR{gamma}, and their isoforms) are activated by both all-trans-RA and 9-cis-RA, whereas the RXRs (RXR{alpha}, RXRß, RXR{gamma}, and their isoforms) are activated only by 9-cis-RA (3) . These receptors bind preferentially as RAR/RXR heterodimers to RA-responsive elements located in the regulatory regions of target genes. The many diverse effects of RA are associated with the transcriptional regulation of target genes, which include transcription factors, extracellular matrix proteins, enzymes, growth factor receptors, and so forth (4 , 5) .

F9 murine teratocarcinoma stem cells (F9 cells) resemble the pluripotent inner cell mass cells or ES cells of mouse blastocysts and thus provide an important model system in which to study critical early events in mouse development. F9 cells differentiate into primitive endoderm in response to RA (2 , 6) . The homeobox gene Hoxa-1 is transcriptionally regulated by RA and encodes a transcription factor that has been shown to play important roles in cell differentiation and embryogenesis (7, 8, 9, 10, 11, 12, 13) .

The actions of homeobox genes in embryogenesis and tumorigenesis are complicated. It follows that the identification of the gene targets of this particular homeobox gene, Hoxa-1, is crucial to the elucidation of the molecular mechanisms involved in cellular differentiation. To dissect these actions and identify the key components, it is advantageous to use a model system. By using both differential hybridization and a cDNA subtractive hybridization approach, we were able to identify genes that were differentially expressed in F9-10, a murine F9 teratocarcinoma stem cell line (14) that expresses high levels of exogenous Hoxa-1 as compared to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of RA. The F9-10 cell line was produced in this laboratory by stable transfection of F9 wild-type cells with a cDNA containing the complete coding sequence of the Hoxa-1 protein under the control of mouse metallothionein promoter. On ZnCl2 treatment, Hoxa-1 is ectopically overexpressed in the absence of RA, as assessed by both Northern and Western blot analyses (14) .

Our data have shown that the expression of a wide variety of genes is affected by the level of Hoxa-1 protein, and it is likely that these genes are either direct or indirect target genes of Hoxa-1.4 Here we report the molecular cloning and characterization of one of the putative Hoxa-1 downstream target genes, HA1R-62.

Results

The Identification of Genes That Are Differentially Expressed in the F9–10 Cell Line versus the F9 Wild-Type Cell Line.
We sought to isolate genes that were differentially expressed in the F9 Hoxa-1-overexpressing cell lines versus wild-type F9 cells, which do not express Hoxa-1 mRNA in the absence of RA. The F9-10 cell line expresses exogenous Hoxa-1 mRNA and protein after zinc addition at about the same level as that of the endogenous Hoxa-1 mRNA in F9 wild-type cells treated with RA (14) . Differential hybridization and a subtractive hybridization method were used to compare gene expression in F9 wild-type cells and F9-10 cells after both cell lines were cultured in the presence of ZnCl2 for 24 h to induce the expression of exogenous Hoxa-1 cDNA in the F9-10 cell line. Using both methods, 169 candidate clones were picked and then assayed by Northern blot analysis of RNA from F9 wild-type cells cultured for 24 h in the presence of 100 µM ZnCl2 versus RNA from F9-10 cells cultured for 24 h in the presence of 100 µM ZnCl2. Twenty-six of the clones were differentially expressed in F9-10 cells as compared to F9 wild-type cells.4 The characteristics and expression pattern of one of the putative Hoxa-1 target genes, HA1R-62 (Clone-62), are reported here.

Time Course of HA1R-62 (Clone-62) mRNA Induction by RA.
Wild-type F9 cells were examined for expression of both Hoxa-1 and HA1R-62 mRNAs. As shown by Northern blot analysis (Fig. 1)Citation , HA1R-62 mRNA was significantly increased in F9 wild-type cells 24 h after the addition of 1.0 µM RA. The level of HA1R-62 mRNA was further increased 48 and 72 h after RA treatment. The same blot was hybridized to the Hoxa-1 cDNA probe to show the induction of endogenous Hoxa-1 mRNA by RA in F9 wild-type cells (7) .



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Fig. 1. Time course of HA1R-62 mRNA induction by RA in F9 wild-type cells and in Hoxa-1-overexpressing F9-10 cells after ZnCl2 treatment. A, total RNA was prepared from F9 wild-type cells and F9-10 cells cultured for various lengths of time (0–72 h) and under different conditions (RA, 1 µM RA; Zn, 100 µM ZnCl2). Total RNA (12.5 µg) from different samples was loaded in each lane, and Northern blots were hybridized to random prime-labeled mouse HA1R-62, Hoxa-1, and GAPDH cDNA probes. B, quantitation of the Northern analyses (shown in A) of the HA1R-62 and Hoxa-1 mRNA levels by PhosphorImager analysis, normalization to GAPDH mRNA levels, and expression of the data as a percentage of the most abundant message level of each gene. This experiment was repeated twice with similar results. C, total RNA was prepared from F9 wild-type stem cells and F9-10 cells, both of which were cultured in the presence of 100 µM ZnCl2, and from F9 wild-type cells cultured in the presence of 1 µM RA. Total RNA (12.5 µg) from different samples was loaded in each lane, and Northern blots were hybridized to random prime-labeled cDNA probes for HA1R-62. A GAPDH cDNA probe was used as a loading control for RNA, and the Hoxa-1 cDNA probe was included to show Hoxa-1 expression.

 
Total RNA from the Hoxa-1-overexpressing cell line, F9-10, is also shown on this Northern blot. The F9-10 cell line was created by stable transfection of F9 cells with a Hoxa-1 expression vector driven by a mouse metallothionein promoter (14) . On ZnCl2 treatment, ectopic Hoxa-1 mRNA is expressed (Fig. 1, A and B)Citation . The difference between the endogenous Hoxa-1 mRNA size in F9 wild-type cells (2.2 kb) and the exogenous Hoxa-1 mRNA in F9-10 cells (1.5 kb) results from the fact that the exogenous Hoxa-1 mRNA is encoded by a cDNA missing a portion of its 3' untranslated region (14) . In F9-10 cells, even without ZnCl2 treatment, some exogenous Hoxa-1 mRNA expression is seen (Lane 7); this results from the leakiness of the metallothionein promoter. As shown in Fig. 1, A and BCitation , the level of HA1R-62 mRNA was increased in the Hoxa-1-overexpressing F9-10 cells after ZnCl2 treatment. In contrast, F9 wild-type cells treated with 100 µM ZnCl2 do not exhibit expression of the HA1R-62 gene, indicating that HA1R-62 mRNA expression is not caused by ZnCl2 treatment (Fig. 1C)Citation . These data indicate that there is a correlation between expression of the Hoxa-1 protein and HA1R-62 mRNA expression in both wild-type F9 cells after RA addition and in the F9-10 line after ZnCl2 addition.

Expression of HA1R-62 in HW-F9-pTA-N-Hoxa1-8 Cells.
Additional Hoxa-1 stably transfected F9 cell lines were created more recently in this laboratory using the Tet-off gene expression system, and in one of these cell lines, HW-F9-pTA-N-Hoxa1-8, exogenous Hoxa-1 mRNA expression was activated by the removal of tetracycline from the medium (Fig. 2Citation , Lane 4). It is also the case that in this tetracycline-regulated expression vector, the exogenous Hoxa-1 mRNA (1.5 kb) is not full length, although the Hoxa-1 protein encoded by this vector is full length (14) . This HW-F9-pTA-N-Hoxa1-8 cell line was used to examine the expression patterns of HA1R-62 mRNAs in the presence and absence of tetracycline. In the presence of tetracycline, HA1R-62 mRNA was not expressed in the HW-F9-pTA-N-Hoxa1-8 cells (Fig. 2Citation , Lane 3). HA1R-62 mRNA expression was up-regulated by the removal of tetracycline from the medium (Fig. 2Citation , Lane 4). This again suggests that the expression of HA1R-62 is under the control of Hoxa-1 homeodomain protein in F9 cells. We included RNA samples from the F9 wild-type cells treated with 1 µM RA for 48 h on the same blot. The HA1R-62 mRNA was induced by RA treatment (Fig. 2Citation , Lane 2), as expected.



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Fig. 2. HA1R-62 mRNA expression in HW-F9-pTA-N-Hoxa1-8 cells. Northern blot analysis of total RNA isolated from F9 wild-type and HW-F9-pTA-N-Hoxa1-8 cells cultured under different conditions: Lane 1, F9 wild-type stem cells; Lane 2, F9 wild-type cells treated with 1 µM RA for 48 h; Lane 3, HW-F9-pTA-N-Hoxa1-8 cells in the presence of 1 µg/ml tetracycline; Lane 4, in the absence of tetracycline for 72 h. Actin mRNA is shown as a loading control. This experiment was performed two times with similar results. Arrow, size of the endogenous Hoxa-1 mRNA (~2.2 kb).

 
One interesting point with respect to the endogenous Hoxa-1 gene can be seen in Fig. 2Citation . Expression of the exogenous Hoxa-1 mRNA in response to tetracycline removal also resulted in increased expression of the endogenous Hoxa-1 mRNA (Fig. 2Citation , Lane 4, and arrow at right). These data suggest that the Hoxa-1 gene contains an autoregulatory element to which the Hoxa-1 protein can bind, similar to the regulation of Hoxb-1 (15) .

Expression of HA1R-62 in Hoxa-1 3' Enhancer (-/-) CCE Cells.
One method for generating a cell line with reduced amounts of Hoxa-1 was to remove both copies of the Hoxa-1 3' RA-inducible enhancer (RAIDR5) by two rounds of homologous recombination (16 , 17) . In these CCE-B30 ES cells, the coding regions of the two alleles of the Hoxa-1 gene are intact, but the RA-inducible 3' enhancer is deleted from both alleles. This prevents the induction of Hoxa-1 mRNA by RA (16 , 17) .5 Northern blot analyses of total RNA from a mouse ES cell line, CCE wild-type cells, and from a RAIDR5-/- CCE cell line, CCE-B30, after a 48-h treatment with different concentrations of RA, are shown in Fig. 3Citation . In the Hoxa-1 3' enhancer-/- CCE-B30 cells, RA induction of Hoxa-1 gene expression is almost completely eliminated (Fig. 3)Citation . The same blot was also hybridized to a HA1R-62 cDNA probe; in the CCE-B30 cells, expression of HA1R-62 mRNA is reduced as compared to that in CCE wild-type cells treated with the same concentrations of RA. These data again show a correlation between the levels of Hoxa-1 mRNA induced by RA and the levels of HA1R-62 mRNA.



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Fig. 3. Expression of HA1R-62 and Hoxa-1 in CCE wild-type and Hoxa-1 3' enhancer (-/-) CCE cells. Total RNA was prepared from CCE wild-type ES cells and Hoxa-1 3' enhancer (-/-) CCE ES cells (CCE-B30) cultured for 48 h in the presence of different RA concentrations (0–1.0 µM). Total RNA (12.5 µg) from different samples was loaded in each lane, and Northern blots were hybridized to random prime-labeled mouse HA1R-62, Hoxa-1, and GAPDH cDNA probes. B, quantitation of the Northern blot analyses of the HA1R-62 and Hoxa-1 mRNA levels by PhosphorImager analysis, normalization to GAPDH mRNA levels, and expression of the data as a percentage of the most abundant message level of each gene. This experiment was done twice with similar results.

 
Tissue Distribution of HA1R-62 mRNA.
The expression of HA1R-62 mRNA is evident in adult mouse thymus, lung, kidney, and ovary as well as in 12.5-day mouse embryo RNAs. (Fig. 4)Citation . The thymus and ovary showed the highest levels of expression of HA1R-62 mRNA (size, ~4.3 kb).



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Fig. 4. Tissue distribution of HA1R-62 mRNA. Total RNA (15 µg) from mouse embryo (12.5-days postcoitus) and different adult mouse organs was loaded in each lane, and Northern blots were hybridized to random prime-labeled HA1R-62 cDNA probes. Bottom panel, the ethidium bromide staining of the total RNA gel of this tissue blot as a loading control.

 
Determination of the Full-Length Nucleotide Sequence of HA1R-62 cDNA.
The entire DNA sequence of HA1R-62 cDNA is shown in Fig. 5Citation . As illustrated at the top of the Fig. 5ACitation , the original cDNA isolated from the cDNA subtraction screening was about 120 bp long. This cDNA fragment was used to screen an F9 RACT cDNA library, and a 2.8-kb clone with the same 3'-end was isolated. To isolate the full-length cDNA, 5'-RACE was used (primer design was as indicated in Fig. 5Citation ), and a product was obtained that extended 760 bp from the 5'-end of the clone isolated from the cDNA library. Alignment of the sequences enabled us to delineate the entire cDNA sequence, and we found a single long ORF starting with a methionine codon at nucleotide 505 and ending with a TGA stop codon at nucleotide 1260. Four in-frame stop codons were found upstream from the putative initiation site at positions 49, 169, 250, and 286. A putative polyadenylation signal, AATATA, was found at nucleotide position 3528–3533.



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Fig. 5. Nucleotide sequence of HA1R-62 cDNA. A, partial cDNA fragments of HA1R-62 obtained from different cloning techniques are shown, as well as the 5'-RACE strategy and RACE PCR primers (AS1 and AS2). B, full-length cDNA sequence of HA1R-62 and peptide sequence encoded by the ORF. The initiation site, stop codons, and putative polyadenylation signal are indicated with bold letters. The GenBank accession number for this sequence is AF197105. The RACE experiments were performed twice with identical results.

 
A homology search (18) using the HA1R-62 cDNA sequence revealed that there are two related sequences. Both of them are short sequences reported as ESTs. The 3'-end (nucleotide position 3183–3559) of HA1R-62 cDNA is 97% homologous to an EST (National Center for Biotechnology Information accession number AU043143) from mouse embryo. Another EST (National Center for Biotechnology Information accession number 043914), also from mouse embryo, is 96% homologous to the 3'-end (nucleotide position 3208–3559) of HA1R-62 cDNA.

A protein homology (Swissprot database) search of the peptide encoded by the HA1R-62 ORF revealed that a HA1R-62 protein segment (amino acids 51–83) is 51% homologous to one transmembrane region (amino acids 674–706) of Bride of Sevenless Protein, a Drosophila protein that is a transmembrane ligand of the sevenless transmembrane tyrosine kinase receptor (19) .

In Vitro Translation of HA1R-62 mRNA.
Using the full-length cDNA clone of HA1R-62 in pBluescript plasmid vector as a template, the corresponding mRNA was transcribed by T7 RNA polymerase and translated in a rabbit reticulocyte lysate. As shown in Fig. 6Citation , the mRNA programmed the synthesis of a peptide with a molecular mass of about 26 kDa, which corresponds to the molecular mass estimated from the amino acid sequence.



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Fig. 6. In vitro translation of HA1R-62 cDNA. Full-length cDNA of HA1R-62 was cloned into pBluescript vector and used for in vitro translation. Protein markers are indicated on the left. The complete cDNA construct of murine RA hydroxylase (RAH; Ref. 27 ) was used to program the synthesis of an expected 50-kDa protein as a positive control. The - sign indicates no vector. This experiment was done twice with similar results.

 
Discussion

The cloning of a RA-responsive gene, HA1R-62, in F9 murine teratocarcinoma cells is described. Interestingly, this gene was initially cloned in our screening to identify genes that are differentially expressed in F9-10 cells. F9-10 is a murine F9 teratocarcinoma stem cell line that expresses high levels of exogenous Hoxa-1 in comparison to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of RA. The Hoxa-1 gene has been shown to be transcriptionally activated by retinoids such as RA via a RA-responsive element 3' of its coding region (13 , 16 , 17) . The loss of both copies of RAR{gamma} in F9 cells results in the loss of Hoxa-1 induction by RA and in the partial loss of ability of cells to differentiate in response to RA (20) . It is therefore likely that this RA-induced differentiation is mediated in part through the actions of homeobox genes and specifically through the action of Hoxa-1. The HA1R-62 gene shows a dramatically reduced induction by RA in Hoxa-1 3' RARE-/- CCE cells (Fig. 3)Citation . Conversely, in cell lines in which the Hoxa-1 gene is stably overexpressed, HA1R-62 mRNA is elevated (Figs. 1Citation and 2)Citation . These data strongly suggest that this novel RA-responsive gene, HA1R-62, is a target gene of the Hoxa-1 protein, which is itself a direct target of RARs (7 , 21) .

We do not know whether HA1R-62 is a direct target of the Hoxa-1 protein. It is possible that HA1R-62 is indirectly regulated by Hoxa-1 as a result of Hoxa-1 changing the differentiation state of the cell. However, this is unlikely because the F9-10 and the HW-F9-pTA-N-Hoxa1-8 cell lines were designed to conditionally overexpress only the Hoxa-1 protein, and these cell lines also overexpress HA1R-62 mRNA.

It is difficult to speculate about the function of the HA1R-62 gene product based on the DNA sequence data. However, a protein homology search of the peptide encoded by the HA1R-62 ORF revealed that a HA1R-62 protein segment (amino acids 51–83) is 51% homologous to one transmembrane region (amino acids 674–706) of the Bride of Sevenless Protein, a Drosophila protein that is a transmembrane ligand of the sevenless transmembrane tyrosine kinase receptor (19) . Thus, it is possible that HA1R-62 encodes a transmembrane protein. HA1R-62 mRNA is expressed not only in the mouse embryo, but also in adult mouse thymus, lung, kidney, and ovary, suggesting that RA is involved in regulation of its expression in these adult organs. Much research needs to be done to determine the function of the HA1R-62 protein. We are currently further investigating the function and possible regulatory relationships between Hoxa-1 and HA1R-62 in the cascade of events that takes place during the cellular differentiation induced by RA.

Very few gene targets of the Hoxa-1 protein have been identified to date. This laboratory is in the process of identifying additional putative Hoxa-1 target genes in F9 cells.4 Future research aimed at identifying more target genes of Hox proteins and analyzing precisely how target genes work together to control proper organogenesis and differentiation in a variety of species should clarify many aspects of Hox complex gene function.

Materials and Methods

Cell Culture.
The murine F9 teratocarcinoma wild-type stem cell line and the metallothionein promoter:Hoxa-1 stably transfected derivatives JG-F9-MT-H1.6-993-10 (F9-10) were grown in DMEM supplemented with 10% heat-inactivated bovine calf serum and 2 mM glutamine, as described previously (22) . This F9-10 cell line, in response to zinc, expresses an exogenous Hoxa-1 mRNA that is about 1.5 kb because its 3'-untranslated region was truncated in this metallothionein promoter:Hoxa-1 construct; these cells also express only the longer form of the Hoxa-1 protein (331 amino acids) and not the shorter Hoxa-1 protein (133 amino acids), which lacks the homeobox domain (7 , 14 , 21) . [The Hoxa-1 gene was formerly named ERA-1 and Hox1.6 (21) .] CCE murine ES cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 µM minimal essential medium nonessential amino acids, 1 µM ß-mercaptoethanol, and 1 x 103 units/ml leukemia inhibitory factor (LIF), as described previously (23) .

cDNA Subtractive Hybridization.
Two µg of mRNA from the F9 wild-type cells and F9-10 cells, both treated with 100 µM ZnCl2 for 24 h, were used to synthesize double-stranded cDNA. A new PCR-based cDNA subtraction method, suppression subtractive hybridization (24) , was carried out using the PCR-Select kit (Clontech, Palo Alto, CA). Products from the secondary PCR reactions were inserted into the vector pCRII using a T/A cloning kit (Invitrogen, Carlsbad, CA). The insert DNA of each clone was prepared, radiolabeled, and used for Northern analysis.

Northern Analysis and DNA Sequence Analysis.
Total RNA samples were isolated from F9 wild-type and F9-10 cells and then subjected to Northern analysis as described previously (22) . DNA sequences of the candidate clones were determined at the Biotechnology Center (Cornell University, Ithaca, NY). Sequence searches of GenBank were carried out using the BLAST program (18) .

cDNA Library Screening.
Approximately 400,000 plaques of a F9 RACT 3-day cDNA library in the ZapExpress vector6 were screened with the original cDNA fragment of HA1R-62 isolated from the cDNA subtraction screening, and four positive clones were isolated and sequenced.

RACE.
RACE (25 , 26) , a procedure to amplify nucleic acid sequences from a mRNA template between a defined internal site and unknown sequences at 5'-end of mRNA, was performed by using a Marathon cDNA Amplication Kit (Clontech) according to the supplier’s protocol. The primer for reverse transcription and the two internal anchoring primers for nested PCR were 5'-TCCCCACCAAAGCCACCTCCATT-3', 5'-GAAAAAGTGCGAAGATCCAAAACGAA-3' (AS1), and 5'-GCGAAGATCCAAAACGAAATGTAAAGT-3' (AS2), respectively.

In Vitro Translation.
In vitro transcription and translation were performed by using a TNT-coupled reticulocyte lysate system (Promega, Madison, WI) with [S35]methionine radiolabeled protein labeling mix (DuPont New England Nuclear) according to the manufacturer’s instructions. The synthesized proteins were analyzed by 7.5% SDS-PAGE, followed by autoradiography.

Acknowledgments

We thank Dr. Ker Yu, Dr. Alex Langston, and Hong Wu, all of the Pharmacology Department, Cornell University Medical School, New York, NY, for Hoxa-1 genomic constructs and cell lines.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant R01CA39036 (to L. J. G.). Back

2 To whom requests for reprints should be addressed, at Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, New York 10021. Phone: (212) 746-6250; Fax: (212) 746-8858; E-mail: ljgudas{at}mail.med.cornell.edu Back

3 The abbreviations used are: RA, retinoic acid; EST, expressed sequence tag; ORF, open reading frame; RXR, retinoid X receptor; RAR, retinoic acid receptor; ES, embryonic stem; RACE, rapid amplification of cDNA ends; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

4 J. Shen and L. J. Gudas. Cloning of genes differentially expressed in cells which overexpress Hoxa-1, a homeobox gene, submitted for publication. Back

5 A. W. Langston and L. J. Gudas. Functional analysis of the retinoic acid inducible enhancer 3' of the Hoxa-1 gene, submitted for publication. Back

6 J. R. Thompson and L. J. Gudas, unpublished data. Back

Received for publication 8/17/99. Revision received 11/ 3/99. Accepted for publication 11/ 3/99.

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E. Martinez-Ceballos, P. Chambon, and L. J. Gudas
Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal
J. Biol. Chem., April 22, 2005; 280(16): 16484 - 16498.
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