FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells
© Nalbant et al; licensee BioMed Central Ltd. 2005
Received: 19 August 2004
Accepted: 27 January 2005
Published: 27 January 2005
Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.
One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.
The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.
Hematopoietic differentiation is a complex process whereby multiple functionally and morphologically distinct cell types arise from a population of pluripotent hematopoietic stem cells (PHSCs) . The accurate and efficient regulation of hematopoietic development is controlled by a large number of regulatory proteins that have been identified over the past few decades. These regulatory molecules include the hematopoietic growth factors (HGFs), soluble proteins that recognize specific receptors on the surface of sub-populations of hematopoietic cells, thereby initiating signal transduction pathways that modulate the differentiation, proliferation, and/or survival of target cells . The identification of regulators of hematopoiesis has been an ongoing effort for many years and has benefited from the existence of accessible cell line models as well as the characterization of genes affected by somatic mutations associated with specific human leukemias .
We have used a pair of factor-dependent murine cell lines to identify novel genes expressed within distinct hematopoietic lineages as an approach to the identification of novel candidate genes for development of diagnostic and therapeutic approaches to leukemia. The EML and MPRO cell lines were both established by infecting murine bone marrow cells with a retrovirus expressing a dominant negative retinoic acid receptor α (RARα) protein [4, 5]. The infected cells were selected in the presence of either stem cell factor (SCF) or granulocyte/macrophage colony stimulating factor (GM-CSF). EML are SCF-dependent and resemble uncommitted hematopoietic progenitor cells. They can be induced to differentiate to the promyelocyte stage of granulopoiesis in the presence of interleukin-3 (IL-3) and high doses of all trans retinoic acid (atRA) [4, 6]. Terminal neutrophil differentiation of EML cells can be induced by replacement of IL-3 and SCF with GM-CSF. MPRO cells are GM-CSF-dependent and can be induced to differentiate to neutrophils by adding high doses of atRA to the culture medium. The expression patterns of a number of genes expressed during hematopoiesis have been examined in EML and MPRO cells and generally agree with the patterns observed in other cell systems and in primary hematopoietic cells. Thus, EML and MPRO provide a powerful system for the identification and characterization of novel genes expressed within the hematopoietic lineage.
We have employed the representation difference analysis technique  to identify cDNAs representing genes expressed at higher levels in EML cells 72 hours after induction of differentiation than in uninduced cells. We describe the identification of a clone derived from an uncharacterized putative secreted protein. We have performed a comparative genomics analysis and determined that this protein is the founding member of an extended family of highly related proteins. This family contains three members in mammalian species, one or two members in invertebrate or simple vertebrate species and five or six members in fish. We have determined that one family member is a secreted glycoprotein and describe the expression pattern of the human and mouse genes in tissues and during hematopoietic differentiation.
Identification of differentially expressed genes by representational difference analysis (RDA)
Total RNA was prepared from EML cells grown in the presence of SCF alone (0 hour) or in medium supplemented with IL-3 and atRA for 72 hours. The RNA was converted to cDNA and subjected to three rounds of RDA as previously described . Six differentially-expressed clones were identified [6, 8]. Clone number 1623 was chosen for further analysis and the differential expression of this gene was confirmed by Northern blot analysis. 1623 mRNA was essentially undetectable in the 0 hour sample but readily detectable in the 72 hour sample (data not shown and see figure 10).
Sequence analysis of clone 1623
Identification of a family of related genes
Accession numbers for vertebrate FAM20 family members1
Accession numbers for invertebrate FAM20 family members1
Assignment of subfamily relationships
Features of FAM20 proteins
Fam20a is a secreted protein
Fam20a secretion requires a functional signal sequence
Expression patterns of FAM20 genes during myeloid differentiation
We originally identified Fam20a as a differentially expressed mRNA in EML cells induced to differentiate along the myeloid lineage. To determine whether Fam20b and Fam20c are also expressed during hematopoiesis, we performed RT-PCR analysis of cDNAs prepared at various times during experimentally-induced differentiation of EML and MPRO cells using primers specific to each family member. Fam20a mRNA levels were low in uninduced EML cells maintained in the presence of SCF and increased during the subsequent 72 hours of incubation in atRA and IL-3 (Figure 10A). EML cells mature to the promyelocyte stage of neutrophil differentiation under these conditions and can subsequently be differentiated into neutrophils by adding GM-CSF in place of SCF and IL-3. Fam20a mRNA levels decreased during terminal neutrophil differentiation in EML cells and also in MPRO cells induced to undergo the same differentiation process in the presence of atRA (Figure 10A and 10B). Fam20b and Fam20c mRNAs were readily detected in both cell lines and their levels did not vary dramatically during the differentiation process in either cell line (Figure 10A and 10B).
Expression patterns Of FAM20 genes in human tissues
Several classes of secreted proteins, including the colony stimulating factors or hematopoietins, are important regulators of hematopoietic differentiation and function . These molecules are of clinical significance due to their use in stimulating hematopoiesis in patients with neutropenias and other hematological disorders . Consequently, the identification of novel secreted proteins that display specific spatiotemporal expression patterns in hematopoietic cells is of great interest. In this report, we describe the identification and initial characterization of a new family of secreted glycoproteins expressed within the hematopoietic lineage as well as several other cell types and tissues. The family has been named FAM20 to indicate the fact that the members are related by sequence similarity rather than a specific shared function and contains three members in mammals. We anticipate that the members will acquire new names as their specific functions are determined. The family contains three separate subfamilies which are referred to as FAM20A, FAM20B and FAM20C in humans.
FAM20 proteins: features and potential functions
At the present time, the specific function(s) of the FAM20 proteins is unknown. Analysis of the sequences of the proteins from various species failed to reveal obvious similarities to known functional domains except for an N-terminal signal sequence. Expression studies clearly demonstrated that the mouse Fam20a protein is a secreted protein and that disruption of the signal sequence prevented the detection of the glycosylated form of the protein in cell media. Surprisingly, disruption of the signal sequence resulted in redistribution of intracellular Fam20a from a cytoplasmic compartment that is likely to be the ER to the nucleus. It is unclear whether this redistribution is functionally significant and is presumably due to the presence of a cryptic nuclear localization signal (NLS) in the protein. NLSs are generally comprised of stretches of basic residues  and several candidate regions rich in basic amino acids are present in Fam20a that could direct the mislocalized protein to the nucleus.
Overall, the FAM20 proteins vary in length between 400–670 amino acids with the FAM20B family members being the shortest and the FAM20C members generally being the longest. The variation in length is due to differences in the length of the less conserved N-terminal region. The FAM20 proteins are further characterized by a highly conserved C-terminal region of approximately 350 amino acids that we named the conserved C-terminal domain (CCD). We noted the presence of three regions that are more highly conserved amongst all family members, with the most invariant extended sequence being the DRHHYE heptapeptide within conserved region 2. The function of the peptide is as yet unknown but three possible functions can be proposed. First, the histidines within this sequence could be involved in the coordination of metal ions that may be required for FAM20 protein function. Second, FAM20 proteins may be enzymes and this sequence may be a component of the active site of the enzyme. Interestingly, a weak match was detected between this region of certain FAM20 family members and a conserved domain within the phosphatidylinositol 3- and 4-kinases ; however additional experiments must be performed to address the relevance of this similarity. Third, the highly charged nature of this peptide suggests that it may be located on the surface of FAM20 proteins, where it may participate in protein:protein interactions. Although we have been unable to identify any other proteins in public databases that contain the exact sequence, the sequence RHHYE is found between amino acids 41–45 in the N-terminal region of the viral infectivity protein (Vif) from human immunodeficiency virus 1 (Accession number AAQ09611) . Vif enhances HIV-1 infectivity by blocking the antiviral activity of the nucleotide editing enzyme APOBEC3G . Vif exerts its inhibitory effect by binding to and inducing the degradation of APOGEC3G and also by blocking its translation [16, 17]. The APOBEC3G interacting domain is located within the N-terminal region that contains the FAM20-related pentapeptide although the specific involvement of this sequence has not yet been investigated . Thus, this sequence within conserved region 2 may be a site for protein:protein interaction. However, APOBEC3G or related proteins are unlikely candidate binding partners as they are intracellular proteins. We also noted that one of the Drosophila FAM20 proteins (NM_170079; CG31145) was identified as a protein that interacted with the Dynein light chain protein Dlc90f . Dyneins are motor proteins involved in intracellular transport . It appears unusual that a secreted protein would directly interact with a motor protein; therefore, this may represent a specific interaction of this particular family member in fruitfly.
Evolution of the FAM20 gene family
Tremendous progress has been made over the past decade in the sequencing of genomes from species at different positions on the evolutionary tree [20–25]. This vast amount of information can be used for comparative studies to elucidate the evolutionary origin of members of gene families. We have reported here the identification of orthologues of the FAM20 members in two insect species (D. melanogaster and A. gambiae), a simple chordate (C. intestinalis), three mammals (H. sapiens, M. musculus and R. norvegicus) and two fish (F. rubripes and D. rerio). In each case, the identification of these genes as bona fide transcription units is supported either by direct experimental evidence for the human and mouse genes, or by the existence of multiple EST sequences in public databases. A single FAM20 gene was identified in C. intestinalis, which is considered to be a representative of a basal chordate related to the common ancestor of humans and other higher chordates . The C. intestinalis genome encodes approximately 16,000 genes and generally contains single representatives of superfamilies in higher vertebrates, thereby permitting the elucidation of likely evolutionary origins of genes within these families . The C. intestinalis FAM20 protein clustered with the FAM20B subfamily members, as did the family members identified in invertebrate species. Therefore, we propose that the FAM20B subfamily contains the direct descendents of the ancestral FAM20 gene and that the FAM20A and FAM20C subfamilies result from duplication and subsequent evolution of this ancestral gene . This pattern is consistent with the 2R hypothesis of genome evolution proposed by Ohno in 1970 . In the FAM20 case, the loss of one gene at some stage of higher vertebrate evolution would need to be hypothesized to explain the final paralogue number of three in mammals. A single round of gene duplication appears to have occurred subsequent to the divergence of the nematode and insect lineages, giving rise to the two paralogues in fruit fly and mosquito that both cluster within the FAM20B subfamily.
A further round of gene duplication has been proposed to have occurred in fish  and the existence of five to six FAM20 genes in pufferfish and zebrafish is consistent with this hypothesis. However, it is interesting that the expansion appears to have occurred exclusively in the FAM20C subfamily. This pattern could be explained either by two successive rounds of gene duplication of a small genomic region that included the original FAM20C representative, or by two rounds of duplication of larger genomic segments followed by gene conversion of FAM20A and FAM20B descendents (or parents) to yield four FAM20C members. Analysis of the genomic regions surrounding each of the FAM20C genes in fish will be necessary to distinguish between these two possibilities. Nevertheless, the expansion of the FAM20C subfamily in fish suggests that these proteins have acquired specific functions required within these species.
FAM20 expression patterns in mammalian cells and tissues
Expression analysis of the three FAM20 family members in mammalian tissues and hematopoietic cells showed that FAM20A is expressed in a much more restricted pattern that the other two members. Importantly, Fam20a was also the only member to display obvious differential expression in hematopoietic cells undergoing myeloid differentiation. Fam20a mRNA levels were highest during intermediate stages of differentiation of EML cells, at a time when many cells are becoming committed to the myeloid lineages and undergoing extensive proliferation [4, 8, 29]. EML cell cultures also give rise to a small number of cells from B cell and erythroid lineages under these conditions but these cells do not survive after SCF and IL-3 is removed from the medium and replaced with GM-CSF. Thus, the decrease in Fam20a mRNA levels after the 72 hour timepoint could be interpreted in two ways. First, Fam20a may be expressed in one of the lineages that cannot survive in GM-CSF. Second, Fam20a may be expressed specifically in cells committed to the myeloid lineage and its expression may decrease during terminal granulocytic differentiation. The similarities in Fam20a expression patterns in EML cells after the 72 hour timepoint and in MPRO cells suggests that the second explanation is most likely. Therefore, we propose that Fam20a is primarily expressed in cells committed to the granulocytic lineage and presumably plays a role in either lineage commitment of cell proliferation. The hypothesis is currently under investigation using gene disruption techniques and through further characterization of the Fam20a protein.
Cell culture and Representational Difference Analysis (RDA)
EML and MPRO cells were cultured as described previously [6, 30]. Briefly, EML cells were cultured in Iscove's modified Dulbecco's medium containing 20% horse serum (Atlanta Biochemical, Norcross, GA) supplemented with 10% BHK-MKL conditioned medium (CM) as a source of stem cell factor (SCF). The cells were differentiated by adding 10% of WEHI-3 CM as a source of IL-3 and all trans retinoic acid (atRA, 1 × 10-5 M, Sigma) for 72 hours. Terminal granulocytic differentiation was induced by removing IL-3 and SCF and adding 10% BHK-HM5 CM as a source of granulocyte/macrophage-colony stimulating factor (GM-CSF). MPRO cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and 10% BHK-HM5 CM. Terminal differentiation was induced by adding 1 – 10-5 M atRA to the culture medium. For collection of RNA samples at different timepoints, 10 cm dishes were seeded with 150,000 cells and cultured under identical conditions. Cells were harvested from individual wells at 24 hour intervals for RNA preparation.
Total RNA was prepared from isolated cells using a modified guanidium isothiocyanite/phenol extraction procedure as described previously . Poly A+ RNA prepared from EML cells at zero hours (stem cell stage) and 72 hours (promyelocyte stage) was converted to double stranded cDNA and three rounds of RDA was performed as described previously . cDNA clones representing six putative differentially expressed genes were identified and named according to clone number. Initial characterization of these clones was described earlier [6, 8] and clone number 1623 was examined further in this study.
Cos-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, BioWhittaker, Walkersville, MD) containing 10% FBS and Penicillin/Streptomycin (BioWhittaker) under standard cell culture conditions. For transfections, cells were plated at a density of 2.5 × 104 per well in six well plates and transfected using the Effectene Transfection Reagent (Qiagen, Valencia, CA) under conditions recommended by the manufacturer.
Isolation of a full length 1623 cDNA
Analysis of the sequence of the original 1623 cDNA isolated from the RDA experiments revealed that it contained the coding sequence from the C-terminus of an uncharacterized protein. 5' and 3' rapid amplification of cDNA ends (RACE) was performed using Marathon-Ready cDNA from mouse spleen using the Advantage cDNA PCR kit (both from Clontech) as described previously . The RACE reactions were performed using gene specific primers designed based on the original 1623 sequence or on sequences identified in early RACE reactions and adaptor primers provided with the cDNA. The 5' RACE products were compared to sequences in public databases and identified a mouse cDNA (NM_017565; aka DKFZp434F2322) that was identical to the extended 1623 sequence. PCR primers were designed that overlapped the putative initiation and stop codons of the ORF encoded by NM_017565 and used to PCR amplify products from cDNAs prepared from undifferentiated MPRO cells. The product of this PCR reaction was subcloned and sequenced in its entirety. The sequence was identical to the original clone (which was isolated from mammary tissue) indicating that the same mRNA is expressed in both tissues. The full length clone (now named Fam20a) encoded a 541 amino acid protein.
All basic sequence manipulations were performed using the VectorNTI suite of sequence analysis programs (Informax/Invitrogen Corp., Carlsbad, CA). Identification of FAM20 members from different species was initially achieved using standard BLAST searches at NCBI http://www.ncbi.nlm.nih.gov/BLAST/. Genome specific searches were performed either through NCBI or Ensembl http://www.ensembl.org/ or through genome specific websites (Ciona intestinalis: http://genome.jgi-psf.org/ciona4/ciona4.home.html; Fugu rubripes: http://aluminum.jgi-psf.org/prod/bin/blast.fugu3.cgi). Searches were performed either as nucleotide-nucleotide (blastn) searches or as protein-translated nucleotide (tblastn) searches. In general, these searches were sufficient to identify gene regions encoding the most highly conserved regions of the proteins, particularly in the fish and Ciona genomes. To assemble complete genes, genomic regions containing the identified regions of similarity were downloaded into VectorNTI and searches were performed for individual exons, initially derived from mammalian genes but subsequently, from potential orthologues in fish or lower vertebrates, depending on the sequence being examined. Putative exons were examined for the presence of consensus splice donor and acceptor sites and complete sets of exons were assembled into cDNAs for translation and further comparisons. These results were compared against genes assembled by two gene prediction programs, FGENESH: http://www.softberry.com/berry.phtml and GENSCAN: http://genes.mit.edu/GENSCAN.html, however, these programs often made incorrect assignments that required manual assessment of the results. The protein sequences derived from each of the examined species were primarily used in the comparisons to define subfamily assignments and alignments were performed in the AlignX program in VectorNTI using the blosum62 scoring matrix. Protein domain searches were performed using the Simple Modular Architecture Research Tool (SMART) http://smart.embl-heidelberg.de/ and/or Profilescan http://hits.isb-sib.ch/cgi-bin/PFSCAN. Signal sequence searches were performed using the SignalP analysis program http://www.cbs.dtu.dk/services/SignalP/. Sequences derived by annotation of genome sequences in this study have been submitted to the Third Party Annotation database at NCBI under accession numbers BK001515 to BK001522. The FAM20 family name has been approved by the Human Genome Organization nomenclature committee.
The Fam20a and Fam20aΔ23 were constructed in the pEGFP-N1 vector by PCR cloning. Xho I and Hind III restriction sites were engineered into the PCR primers (Fam20a: 5'GGCCTCGAGGCCATGCCCGGGCTGCGCAGG3', 5'GGCAAGCTTGCTCGTCAGATTAGCCTG3'; Fam20aΔ23: 5'GGCCTCGAGGCCATGTACTTCCACCTCTGGCCG3' and reverse primer was as above). Similar strategy was used to clone Fam20a and Fam20aΔ23 in the pcDNA3.1 vector (Forward primers were same as above; Fam20a and Fam20aΔ23 reverse primers: 5'GGCAAGCTTGGGCTCGTCAGATTAGCCTG3'). The Fam20a(SSmut) was generated using a PCR-based site-directed mutagenesis kit (Quickchange, Stratagene). All of the sequences were verified by sequencing using an ABI automated sequencer.
Whole cell extracts were prepared from transfected COS-1 cells by lysing directly in 2X Laemmli Sample Buffer as described previously . Purified His-tagged proteins were prepared as described below and mixed with 2X Laemmli Sample Buffer (120 mM Tris-HCl (pH 6.8), 10% Glycerol, 3.3% SDS, 0.2 M dithiothreitol, 0.004% Bromophenol Blue). Equal amounts of total cellular and purified secreted proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Micron Separations, Westborough, MA). Membranes were blocked in 5% non-fat milk in TBST (100 mM Tris-HCl (pH 7.5), 0.9% NaCl, 0.1% Tween-20) for 1 hour. Myc-tagged Fam20a proteins were detected using an anti-myc mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated donkey anti-mouse antibody (Promega, Madison, WI) was used for the secondary antibody. The immune complexes were detected using SuperSignal chemiluminescence detection kit (Pierce, Rockford, IL). In vitro transcribed and translated Fam20a was generated using the TnT T7 coupled reticulocyte lysate system (Promega) as described previously .
Purification of His-tagged proteins from cell media
COS-1 cells were transfected as described above and media were collected after 48 hours and mixed with freshly made 10X binding buffer (500 mM NaH2PO4-H2O (pH 8.0), 150 mM NaCl, 10 mM Imidazole). His-tagged proteins were purified using Ni-NTA Magnetic Agarose Beads (Qiagen, Valencia, CA) according to manufacturer's recommendations. Following the purification step they were either used directly in western blot analysis or for deglycosylation experiments. Purified proteins were subjected to enzymatic deglycosylation using the GlycoPro deglycosylation kit (ProZyme, San Leandro, CA) following the manufacturer's protocol.
COS-1 cells were plated on glass coverslips and transfected with the indicated constructs the following day. After 48 hours of incubation, the cells were fixed in 1% formaldehyde for 30 minutes at room temperature. Coverslips were incubated in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 (pH 7.3)) containing 1% Triton X-100 for 10 minutes to permeabilize the cell membranes. They were then transferred to PBST (PBS containing 0.1% Tween-20) and incubated for 30 minutes. Mouse monoclonal anti-Myc antiserum (Santa Cruz) was used at 1:2,000 dilution in PBST containing 1% bovine serum albumin (1 hour at room temperature). Coverslips were then washed with PBST three times and incubated with the secondary antibody (Alexa Fluor 488 [Molecular Probes, Eugene, OR]) for 1 hour. Coverslips were washed three times in PBST and once in water for 15 minutes each, and were mounted on glass slides. The cells were observed by epifluorescence microscopy using an Axiovert 135 TV microscope (Zeiss, Gottingen, Germany). Images were captured with a Kodak DC290 zoom camera and analyzed with MetaMorph 6.0 (Universal Imaging Corporation, Downingtown, PA).
Reverse transcription-polymerase chain reaction
Total RNA was purified as described above from EML and MPRO cells at 24 hour timepoints during the differentiation process of each cell line The primers were designed using the Vector NTI sequence analysis suite of programs (InforMax, Frederick, MD) and were follows: MmFam20a forward 5'-catagaggcccacggcgagcg-3' and reverse 5'-atggaatggggcaacag gggc-3'; Mmfam20b forward 5'-tggacaggattctgggtttc-3' and reverse 5'-ccagggatgtcgatgtttct-3'; MmFam20c forward 5'-agcagacgagagagcaggag-3' and reverse 5'-cggatctccttggtcatgtt-3'; HsFAM20a forward 5'-ctggcaggaaaagagtg-3' and 5'-cccgaacttggtgaacatct-3', HsFAM20b forward 5'-ccctgaagagacaccagaagagc-3' and reverse 5'-gaaacccagaatcctgtcca-3', HsFAM20c forward 5'-ggctcacgttccacattggt-3' and reverse 5'-aaagtcagggggtgtctcct-3'.; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5'-aatggtgaaggtcggtg tgaac-3' and reverse 5'-gaagatggtgatgggcttcc-3'; human (GAPDH) forward 5'-tgaaggtcggagtcaacggatttggt-3' and reverse 5'-catgtgggccatgaggtccaccac-3'. GAPDH was used as control for cDNA integrity. Single stranded cDNA was reverse transcribed from 2 μg of total RNA using 400 Units of Superscript RNase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA), 0.125 mM dNTPs, 10 mM DTT, and 1 μM oligo (dT)15 in a total volume of 50 μl for 1 hour at 37°C. 2 ul of the RT reaction was then mixed with 1 ul of 10X PCR Buffer (500 mM Tris-HCl (pH8.3), 2.5 mg/ml crystalline BSA and MgCl2 at 10, 20 or 30 mM (Idaho Technology Inc., Idaho Falls, ID), 0.2 mM each dNTP, 0.05 μM of each primer and 1.25 Units Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA) in a total volume of 10 μl. Reactions were loaded into a capillary tube and PCR cycles were carried out using the Rapidcycler Thermal cycler (Idaho Technology). Annealing temperatures and Mg2+ concentrations were initially optimized for each primer. A commercial set of tissue cDNAs, (Human Multiple Tissue cDNA (MTC) Panels, Clontech, Palo Alto, CA) was used for the RT-PCR analysis of human FAM20 family members.
DN isolated full length Fam20a cDNA, performed transfection experiments and drafted the manuscript. HY performed RT-PCR assays. IN assisted in construction of expression vectors. SS performed genomics analyses. EC and EGB provided reagents and assistance in performing RT-PCR assays. YD performed the original representational difference analysis and SCW directed the project and performed genomics analyses. SCW also wrote the final draft of the manuscript and all authors read and approved this version.
List of Abbreviations
family with sequence similarity 20
Erythroid, myeloid, lymphoid cell line
mouse promyelocyte cell line
granulocyte/macrophage-colony stimulating factor
pluripotent hematopoietic stem cell
hematopoietic growth factor
retinoic acid receptor
all trans retinoic acid
stem cell factor
representational difference analysis
rapid amplification of cDNA ends
conserved C-terminal domain
green fluorescent protein
This work was supported by grants from The CH Foundation and Texas Tech University Health Sciences Center. SCW is an Established Investigator of the American Heart Association. We appreciate the assistance of Susan San Francisco and Ruwantha Wettisinghe at the Center for Biotechnology and Genomics at Texas Tech University in sequencing and sequence analysis. We also thank Dan Hardy for critical reading of the manuscript and Patricia Frisbie for technical and editorial assistance.
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