Mrvil, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1

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Abstract

Ecotropic MuLVs induce myeloid leukemia in BXH2 mice by insertional mutagenesis of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by viral tagging as common sites of viral integration in BXH2 leukemias. Previous studies showed that a frequent common integration site in BXH2 leukemias is the Nf1 tumor suppressor gene. Unexpectedly, about half of the viral integrations at Nf1 represented a previously undiscovered defective nonecotropic virus, termed MRV. Because other common integration sites in BXH2 leukemias encoding proto-oncogenes contain ecotropic rather than MRV viruses, it has been speculated that MRV viruses may selectively target tumor suppressor genes. To determine if this were the case, 21 MRV-positive BXH2 leukemias were screened for new MRV common integration sites. One new site, Mrvi1 was identified that was disrupted by MRV in two of the leukemias. Ecotropic virus did not disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, suggesting that Mrvi1 is specifically targeted by MRV. Mrvi1 encodes a novel protein with homology to Jaw1, a lymphoid restricted type II membrane protein that localizes to the endoplasmic reticulum. MRV integration occurs at the 5′ end of the gene between two differentially used promoters. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias. Like Jaw1, which is downregulated during lymphoid differentiation, Mrv1 is downregulated during monocytic differentiation of BXH2 leukemias. Taken together, these data suggest that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of a gene important for myeloid cell growth and/or differentiation. Experiments are in progress to test whether Mrvi1 is a tumor suppressor gene.

Introduction

BXH2 mice represent an important model for the identification of myeloid leukemia disease genes. Not only do these mice have the highest spontaneous incidence of myeloid leukemia of any known inbred mouse strain, but the leukemias in these mice are retrovirally-induced and the proviruses in the tumors can thus be used as insertional tags to identify the disease genes. BXH2 is one of 12 recombinant inbred (RI) BXH strains derived from crossing C57BL/6J and C3H/HeJ mice. Unlike the parental strains, or the 11 other BXH R1 strains, BXH2 mice spontaneously express a B-ecotropic murine leukemia virus (MuLV) beginning early in life and >95% of these mice die of myeloid leukemia by 1 year of age (Bedigian et al., 1981, 1984). The BXH2 B-ecotropic virus is not transmitted through the germline (Jenkins et al., 1982Jenkins et al., 1982), rather the virus is horizontally transmitted via transplacental infection of implantation stage embryos (Bedigian et al., 1993). The BXH2 B-ecotropic virus is thought to have been generated by a rare recombination event between two defective endogenous ecotropic proviruses, Emv1 and Emv2 inherited from the C3H/HeJ and C57BL/6J parents, respectively (Jenkins et al., 1982Jenkins et al., 1982). The susceptibility of BXH2 mice to myeloid leukemia is genetically determined as infection of other BXH RI strains with the BXH2 B-ecotropic virus produces either no leukemias or primarily B-cell leukemias (Bedigian et al., 1993).

Several disease genes have been identified by proviral tagging in BXH2 mice. The genes include a tumor suppressor gene (Nf1) (Buchberg et al., 1990; Largaespada et al., 1995), the Myb proto-oncogene (Buchberg et al., 1990; our unpublished results), two class I homeobox genes (Hox7 and Hox9) (Nakamura et al., 1996), and a Pbx1-related homeobox gene, Mesi1 (Moskow et al., 1995; Nakamura et al., 1996a). Two of these genes, Nf1 and Hoxa9 are also involved in human myeloid leukemia (Shannon et al., 1994; Nakamura et al., 1996b; Borrow et al., 1996). As expected, nearly all of the viral integrations at Myb, Hoxa7, Hoxa9, and Meis1 loci are B-ecotropic viruses. This is not the case, however, for the viruses located at Nf1. In this case, approximately half of the viral insertions represent a novel defective non-ecotropic virus (Cho et al., 1995). Sequence analysis showed that the defective virus carries two large deletions, a 1.8 kb deletion in pol and a 1.6 kb deletion in env. Similar deletions have been identified in another murine retrovirus, the murine AIDS (MAIDS) virus (Aziz et al., 1989; Chattopadhyay et al., 1989). Because of this structural similarity, this new virus has been designated MRV (MAIDS-related virus) (Cho et al., 1995).

Unblot analysis (hybridization to dried agarose gels; Tsao et al., 1983) with oligonucleotide probes that span the MRV env and pol deletions showed that most inbred mouse strains carry endogenous MRV-related proviruses (Cho et al., 1995). BXH2 mice carry three endogenous MRV-related proviruses. One provirus, Mrv5 maps to the Y chromosome and is carried by most inbred strain males. The other two proviruses, Mrv1 and Mrv4, are autosomal. Sequence analysis indicated that Mrv1 is the source of the virus present at Nf1 (Cho et al., 1995). Transmission of the Mrv1-encoded virus to myeloid tumor cells likely involves rescue of Mrv1-encoded virus by a B-ecotropic virus helper, which is predicted to occur only rarely. This may explain why only 12% of BXH2 leukemias harbor MRV proviruses (Cho et al., 1995).

A number of models could account for the selection of somatic MRV proviruses at Nf1. One model that is particularly intriguing suggests that this selection stems from the fact that Nf1 is a tumor suppressor gene and both alleles of Nf1 need to be inactivated for leukemia to occur. If the first Nf1 allele is inactivated by the integration of a non-defective B-ecotropic virus then this would establish viral interference and make it difficult for a second viral infection of the same leukemic cell to occur. However, if the first integration were an MRV provirus then viral interference would not occur. Consistent with this hypothesis, all BXH2 leukemias characterized to date that have two viral integrations at Nf1 contain an MRV provirus integrated in one allele and a B-ecotropic virus integrated in the second allele (Cho et al., 1995).

To determine whether there are other MRV common integration sites in BXH2 tumors that may harbor tumor suppressor genes, we screened more than 200 BXH2 tumors for new common sites of MRV integration. These studies identified one new common MRV integration site, Mrvi1 that was targeted by MRV in two BXH2 leukemias.

Subsequent studies showed that Mrvi1 encodes a novel protein with limited homology to the lymphoid restricted protein, Jaw1 and that like Jaw1, whose expression is downregulated during lymphoid differentiation, Mrvi1 expression is downregulated during myeloid differentiation. Mrvi1 represents a potentially new tumor suppressor gene involved in mouse and human myeloid leukemia.

Results

Identification of Mrvi1, a common site of MRV integration in BXH2 tumors

A 26 bp MRV-specific probe (Δenv26) was used to screen 205 EcoRI-digested BXH2 tumor DNAs by unblot analysis in order to determine if there were other common sites of MRV integration in BXH2 leukemias besides Nf1. Since EcoRI does not cleave the MRV genome, integration of MRV into the same EcoRI fragment in different tumor DNAs would produce comigrating MRV-cellular DNA fragments. Among the 205 BXH2 tumors analysed, 21 (10%) contained MRV proviruses (data not shown) and three of the 21 MRV-positive tumors, 87-15, 87-260 and 19 contained comigrating EcoRI fragments (Figure 1, left panel). The EcoRI fragment from tumor 87-15 was subsequently cloned and a unique sequence probe, p15.8.1.1. isolated from the cellular flank (Figure 2). Hybridization of p15.8.1.1. to Southern blots of EcoRI-digested DNA from tumor 87-260 and 19 indicated that tumor 19, but not tumor 87-260, had a viral integration in the same EcoRI fragment as tumor 87-15 (Figure 1, right panel). The EcoRI fragment from tumor 19 was also cloned and restriction enzyme maps of this clone, as well as clone 87-15, showed that the two MRV proviruses were integrated 3 kb apart and in the opposite transcriptional orientation (Figure 2). This common site of MRV integration has been designated Mrvi1 (MRV integration site 1). B-ecotropic virus did not target Mrvi1 in the 205 B-ecotropic virus-positive BXH2 leukemias analysed (data not shown), suggesting that Mrvi1 is specific for MRV.

Figure 1
figure1

Identification of a common site of MRV integration. The left panel shows an unblot of four BXH2 tumor DNAs and normal C57BL/6 liver DNA digested with EcoRI and hybridized with an MRV-specific probe (Δenv26). Somatically-acquired MRV proviruses are seen as weakly hybridizing fragments in tumor DNAs. The right panel shows a Southern blot of the same DNAs digested wtih EcoRI and hybridized with p15.8.1.1, a unique sequence cellular DNA probe flanking the MRV integration in tumor 87-15. The probe detects a rearrangement in tumor 19 in addition to tumor 87-15. Molecular weight standards in kilobase pairs are shown to the left of the figure

Figure 2
figure2

Location of viral integrations at Mrvi1. A partial long-range restriction map of the Mrvi1 locus is shown at the top. Also shown are the cosmid and P1 clones used for exon trapping. A SalI site located several kb to the left of the viral integrations at Mrvi1 was identified during sequence analysis and was used as an anchor for pulsed-field mapping. This SalI site is boxed. The location of two exons identified by exon trapping, COSET.29 and P1ET.PPI, are also shown as are the locations of three exons from the Mrvi1 gene. A partial short-range restriction map of the Mrvi-1 locus in normal C57BL/6J and two BHX2 tumor DNAs is shown at the bottom. The location of the Δenv 26 and p15.8.1.1 probes are also shown. The MRV proviruses are represented as lines connected by filled rectangles, which represent viral LTRs. The transcriptional orientation of the proviruses is also indicated. Restriction enzymes: B, BamHI; Bs, BssHII, Bg, BglI; E, EagI; N, NaeI; R, EcoRI; RV, EcoRV; S, SalI, X, XhoI; Xb, XbaI

Mrvi1 maps to chromosome 7

The murine chromosomal location of Mrvi1 was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J x Mus spretus)F1 X C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2700 loci that are well distributed among all the autosomes as well as the X chromosome (Copeland et al., 1993). C57BL/6J and M. Spretus DNAs were digested with several restriction enzymes and analysed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using probe p15.8.1.1. The presence or absence of one of these polymorphisms was then followed in backcross mice in order to determine the map location of Mrvi1. The mapping results indicated that Mrvi1 is located in the distal region of mouse chromosome 7 in a region of human chromosome 11p15 homology (Figure 3). No other common sites of viral integration have been mapped to this region, indicating that Mrvi1 is a new common integration site in the mouse.

Figure 3
figure3

Mrvi1 maps to mouse chromosome 7. (a) The chromosomal location of Mrvi1 was determined by interspecific backcross analysis. The segregation patterns of Mrvi1 and flanking genes are shown at the top. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J X Mus spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J allele while the white boxes represent the presence of a Mus spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. For individual pairs of loci more animals than are shown in the haplotype analysis were often typed. These additional animals were used in calculating the recombination distances. A partial chromosome linkage map showing the location of Mrvi1 in relation to linked genes is shown at the bottom of the figure. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. No double or multiple recombination events were observed. The number of recombinant N2 animals over the total number of N2 animals typed plus the recombination frequencies, expressed as genetic distances in centiMorgans (±one standard error), is shown for each pair of loci to the left of the map. The position of loci in human chromosomes are shown to the right of the map. References to map positions for most human loci can be obtained from the GDB (Genome Data Base), a computerized database of human linkage information maintained by the William H Welch Medical Library of the Johns Hopkins University (Baltimore, MD, USA)

A novel gene is interrupted by viral integration at Mrvi1

To determine if a gene(s) is interrupted or activated by integration at Mrvi1 a cosmid and P1 contig was created that surrounds the Mrvi1 locus (Figure 2). Cosmid and P1 clones were positioned with respect to the viral integration sites by a combination of restriction enzyme digestion and hybridization analysis (data not shown). These clones were then used in exon trapping experiments. Two exons, 101 bp (P1ET.PPI) and 149 bp (COSET.29) in size, were trapped in these experiments and they mapped 25 kb and 3 kb to the left, respectively of the viral integration site in tumor 19 (Figure 2). Subsequent Northern blot analysis of poly(A)+ mouse mRNAs using these two exons as probes indicated that they were derived from the same gene (data not shown). This hypothesis was later confirmed by reverse transcriptase-polymerase chain reaction (RT – PCR) analysis, which also indicated that the gene was transcribed from right to left (Figure 2).

Several oligo-dT-primed cDNA libraries were screened for full-length Mrvi1 cDNA clones. All attempts failed, suggesting that these two exons might be located at the extreme 5′ end of the gene. To determine if this were the case, we attempted to isolate Mrvi1 cDNA clones by rapid amplification of cDNA 5′ ends (5′ RACE). The 5′ RACE reactions, primed at the 3′ end of COSET.29, consistently generated fragments of 615 bp and 300 bp. Subsequent analysis showed that the 615 bp fragment contained the 149 bp (COSET.29) exon (now called exon 1b) and a new exon, exon 1a (Figure 2). Additional 5′ RACE reactions using primers from exon 1a consistently produced fragments that ended in exon 1a, suggesting that this exon is located at the 5′ end of the message. Physical mapping showed that exon 1a lies 30 kb 5′ of exon 1b and that the viral integrations at Mrvi1 are located within the first intron of the gene (Figure 2).

Further analysis of the 300 bp 5′ RACE product indicated that it contains the 101 bp (P1ET.PPI) exon (now called exon 2) as well as 200 bp of upstream genomic DNA. The two different 5′ RACE products therefore appear to be derived from mRNAs that initiate from different promoters. To confirm this result and prove that the clones were not simply derived from genomic DNA contamination or from pre-spliced hnRNA, we performed RT – PCR using a primer derived from the genomic sequence located just upstream of exon 1b and a primer from exon 2. This experiment produced a fragment of the expected size whereas no product was observed in the RT(−) control or from genomic DNA (data not shown). These data confirm that Mrvi1 has two separate promoters; the P1 promoter located upstream of exon 1a and P2 promoter located upstream of exon 1b. Exon 1b is used in both transcripts; as the first exon in P2-initiated transcripts and as exon 2 in P1-initiated transcripts.

To identify sequences from the 3′ end of the Mrvi1 gene, nested oligonucelotide primers were designed corresponding to the coding strand of exons 1a and 1b and 3′ RACE was performed using mouse heart marathon ready cDNA as the template. Multiple PCR fragments 5 – 6 kb in size were identified, cloned and sequenced. All 3′ RACE products began with exon 1a or exon 1b and ended in poly(A) tracts located downstream of a consensus polyadenylation signal sequence (UUUAUUUU) that was identical in all clones. Subcloning of the Mrvi1 5′ RACE products indicated that the P1 transcripts were more abundant than P2 transcripts. This was also true for the full-length P1- and P2-initiated transcripts. This phenomenon was not primer dependent as multiple primer combinations produced similar results. This suggested that the two promoters were differentially active in heart tissue, the mRNA source for the 5′ RACE analysis. To determine if this were also true for other types of cells, semi-quantitative RT – PCR analysis was done using poly(A)+ mRNA from normal mouse brain, spleen, and heart as well as two BXH2 tumor cell lines, B140 and B112. This analysis showed that the P1 promoter is approximately 4 – 5 times as active as the P2 promoter in all tissues and tumor cell lines tested (data not shown).

Mrvi1 transcripts are differentially spliced

Multiple independent Mrvi1 cDNA clones were sequenced and the consensus is shown in Figure 4. Sequencing of promoter P1-derived cDNA clones indicated that complex alternative splicing occurs at the 5′ end of the gene. Five unique splicing variants were identified. No single splice variant appeared to predominate as all clones were equally represented. Two exons, 1a′ and 2′, appeared to result from the alternative use of splice donor and acceptor sites, respectively Exon 1a′ is 116 nt shorter than exon 1a and exon 2′ is 78 nt shorter than exon 2. Two forms of the gene also lack exon 1b. The sequence of the longest P1 transcript (exons 1a, 1b and 2) was 6226 nucleotides in length. The most likely initiation codon for P1-derived transcripts is the CTG codon present in exon 2. This CTG is located within the context of a very good consensus start site (Kozak, 1986). The nucleotide sequence surrounding this codon is also highly conserved between mouse and human and the amino acid homology between the predicted mouse and human polypeptides starts at this leucine. If translation starts at this CTG, then alternative splicing of the P1 transcripts would affect only the 5′ untranslated region (UTR) of the gene. The P1-derived protein is predicted to be 821 amino acids (aa) in length and end at an in frame TAG stop codon at nt 3109 (Figure 4). The 3′ UTR is fairly large (3117 nt) and contains three copies of the mRNA destabilizing sequence AUUUA (Shaw and Kamen, 1986), suggesting that the Mrvi1 mRNA may be tightly regulated.

Figure 4
figure4

Nucleotide and predicted amino acid sequence of Mrvi1 and structure of splice variants. The nucleotide and predicted amino acid sequence of P1- and P2-initiated transcripts and proteins are shown at the top. The predicted CTG start codon of the P1 transcript is shown in bold as are three mRNA destabilizing sequences and an AATAA polyadenylation signal. Nucleotide and amino acid numbers are indicated on the left and right, respectively. A diagram of mouse and human Mrvi1 splice variants is shown at the bottom. The size of the human introns is not known and the figure is not drawn to scale

No evidence of alternative splicing was observed for P2-derived transcripts. The P2 transcript if 6064 nt in length and is predicted to contain a 143 nt 5′ UTR (Figure 4). An ATG codon at nt 144, located within the context of an excellent Kozak consensus, is the predicted initiation codon. An open reading frame of 905 aa follows the start site and ends with an in frame TAG codon at nucleotide 2858. The 3′ UTR, polyadenylation site, and mRNA destabilizing sequences are identical to the P1-derived transcript. The P1-derived protein will subsequently be referred to as Mrvi1b and the P2-derived protein Mrvi1a. The major difference between the P1 and P2 transcripts is alternative splicing in P1 transcripts and the length of the 5′ UTRs (646 nt in P1 transcripts and 143 nt in P2 transcripts). Additionally, the predicted start codon of the two transcripts is different. If the initiation codon predictions are correct, Mrvi1a would contain 84 amino acids at its amino terminus not present in Mrvi1a (Figure 4).

Two different Mrvi1 proteins

To confirm that two different Mrvi1 proteins are made, P1- and P2-initiated cDNAs were translated in vitro and analysed by SDS – PAGE (Figure 5). As predicted, these two transcripts produced different-sized proteins. P1-initiated cDNA produced a protein (Mrvi1b) that migrated with an apparent molecular weight of 115 kilodaltons (kd) while P2-initiated cDNA produced a protein (Mrvi1a) that migrated at 140 kd. An additional weaker band of approximately 123 kd was also created by the P2-initiated cDNA and likely represents a product synthesized from an internal, in-frame initiation codon within a Kozak concensus site located at nt 291. The proteins are larger than predicted (90 kd for Mvri1a, 98 kd for Mrvilb), which can be attributed to the high proline content and/or N-linked glycosylation of the two proteins).

Figure 5
figure5

In vitro translated Mrvi1 proteins analysed by SDS – PAGE. P1- and P2-initiated Mrvi1 transcripts, Mrvi1b and Mrvi1a, respectively, were transcribed and translated in vitro and analysed by SDS – PAGE. The size of protein molecular weight standards is shown in kDa at the left

Mrvi1 is predicted to be a transmembrane protein associated with the endoplasmic reticulum

The 84 aa extension present in Mrvi1a is hydrophobic and computer modeling predicts that it could represent a transmembrane domain (Figure 6). Besides this extension, the two proteins are identical. Both proteins contain an α-helical coiled-coil domain (Lupas et al., 1991), which may facilitate protein-protein interactions. The proteins also contain a carboxy terminal hydrophobic region that may function as a membrane anchor domain; in fact, the PSORT program predicts that both proteins are type II membrane proteins associated with the endoplasmic reticulum (ER). Several potential N-linked glycosylation sites, various phosphorylation sites, and an ATP-binding site were also identified as well as a protein degradation (PEST) signal sequence at the carboxy terminus.

Figure 6
figure6

Alignment of mouse and human Mrvi1 proteins and functional domains. An alignment of the mouse Mrvi1b and human MRVI1B proteins is shown at the top. Conserved motifs are in bold. The coiled-coil domain is boxed. Note that the glutamic acid-rich carboxy-terminal region overlaps the PEST domain and the region between the amino terminus and the coiled-coil domain is proline rich. Amino acid identity is represented by a line between the two sequences. Conservative amino acid changes are represented by two dots and semi-conservative changes by a single dot. Gaps created in the sequences to optimize alignments are indicated by a dot in the sequence string. Abbreviations: N-GLY, N-linked glycosylation site; ProKinC, protein kinase C phosphorylation site; CaKinII, Ca2+/calmodulin kinase II phosphorylation site; cAMPKin, cAMP dependent kinase phosphorylation site; TyrKin, tyrosine kinase phosphorylation site. A cartoon showing the approximate location of Mrvi1a and Mrvi1b functional domains is shown at the bottom

Cloning of the human Mrvi1 gene

Database searches identified a human expressed sequence tag (EST) that was highly homologous to the mouse Mrvi1 gene. Full-length cDNA clones for this gene were subsequently obtained by 5′ and 3′ RACE and the gene sequenced. Like the mouse gene, the human gene recognizes a 6 kb transcript on Northern blots (Figure 8). It also has two different start sites of transcription and is predicted to code for two different proteins, one of 803 aa (88 kd) and one of 892 aa (96 kd). Like the mouse gene there is also evidence of complex alternative splicing of the exons comprising the 5′UTR of the P1-initiated transcripts. A computer-aided alignment of the human and mouse proteins indicates an overall identity of 83% (Figure 6). The coiled-coil and carboxy terminal transmembrane domains are highly conserved as are the proline-rich amino and glutamic acid rich carboxy terminus, many of the N-linked glycosylation and phosphorylation sites, the ATP-binding site, and the PEST motif. Overall, these results strongly suggest that this is the human homolog of the mouse Mrvi1 gene. Subsequently, we will refer to this gene as MRVI1 and the two proteins it encodes as MRVI1A and MRVI1B.

Figure 8
figure7

Northern blot analyses of Mrvi1 gene expression. Northern blots of various adult (a) human and (b) mouse tissues and (c) embryos at various stages of gestation were hybridized with a full-length human (a) or murine (b, c) Mrvi1 cDNA probe. The blots were also hybridized with a GAPDH or β-actin probe to control for RNA loading. The size in kb of molecular weight markers is shown on the left of each panel

Mrvi1 shares homology with a lymphoid restricted membrane protein, Jaw1

Comparison of the Mrvi1 amino acid sequence to other proteins in GenBank showed that Mrvi1 has significant homology to the human and mouse Jaw1 protein. Limited homology was detected throughout the length of the proteins (E value of 2.6e-29) with the highest degree of homology in the coiled-coil domain and the putative transmembrane anchor at the C-terminus of the proteins (Figure 7). Jaw1 was originally identified as a differentially regulated transcript in B and T cell cells (Behrens et al., 1994). Jaw1 is expressed exclusively in immature B- and T-cells and is down regulated in more mature cells of these lineages. As predicted for Mrvi1, Jaw1 is a class II type membrane protein anchored to the ER (Behrens et al., 1994).

Figure 7
figure8

Alignment of Mrvi1b and Jaw1 proteins. Conserved amino acids are boxed with amino acid identities in bold. Gaps created in the sequences to optimize alignments are represented by dashes. Amino acid positions are indicated to the right and left of the sequence. Transmembrane and coiled domains are overlined. Although the sequence identity in the transmembrane domain is low, the position of the hydrophobic domain in the two proteins is very similar (41 and 36 aa from the carboxy terminus of Mrvi1 and Jaw1, respectively)

Mrvi1 expression analysis

Northern analysis identified a 6 kb Mrvi1 transcript in all embryonic and adult murine and adult human tissues examined (Figure 8). Within hematopoietic cells, Mrvi1 expression was restricted to BXH2 myelomonocytic cell lines and to the myelocytic cell line 32D (Figure 9a). Low levels of expression were also observed in the mast cell line P815 (data not shown). No expression was detected in macrophage cell lines IC21 and WEHI3B, pre B-cell lines ABLS1 and WEHI231, plasmacytoma cell lines ABPL45 and ABPC22, T-cell line EL4, or the fibroblast cell line NIH3T3.

Figure 9
figure9

Northern blot analyses of Mrvi1 gene expression in human and murine cancer cell lines. Various mouse (a) hematopoietic and (b) BXH2 myelomonocytic cell lines and human (c, d) cancer cell lines were hybridized with mouse (a, b) and human (c, d) Mrvi1 cDNA probes. The human cancer cell lines are (c) normal placenta; G401, Wilm's tumor; RD, rhabadomyosarcoma; THP-1, acute monocytic leukemia; U937, histiocytic lymphoma; HL60, promyelocytic leukemia; HeLa, cervical carcinoma; K-562, chronic myelogenous leukemia; MOLT4, T-lymphoblastic leukemia; Raji, Burkitt's lymphoma; SW480, colon adenocarcinoma; A549, lung carcinoma; G361, melanoma; and (d) MEG01, megakaryoblastic leukemia and HEL, erythroid leukemia with megakaryocytic featuers. The blots were also hybridized with a GAPDH or β-actin probe to control for RNA loading. The size in kb of molecular weight markers is shown on the left of each panel

Northern analysis of a larger panel of BXH2 leukemia cell lines indicated that Mrvi1 expression was variable among the cell lines, with expression ranging from high levels in B132 and B106, to moderate levels in B162, B140 and B117, to little or no expression in B139, B119, B114 and B113 (Figure 9b). The expression patterns could not be correlated with the presence or absence of any particular cell surface markers, with the exception that Mac-1+ cells are negative for Mrvi1 expression while CD34+ cells are positive. (Largaespada et al., 1995).

We also analysed the expression of MRVI1 in a number of human cancer cell lines. MRVI1 was expressed at undetectable levels in all the lines tested with the exception of the HeLa cervical carcinoma cell line and the SW480 colon adenocarcinoma cell line (Figure 9c). In HeLa cells a weakly expressed 2 – 3 kb message was detected while SW480 cells expressed high levels of the normal 6 kb transcript. Interestingly, the expression of MRVI1 was undetectable in SW620 cells (data not shown), a cell line established from a lymph node metastasis from the same patient from which SW480 was derived. The high level of MRVI1 expression in SW480 appears to be unique to this colon carcinoma cell line, as several others such as COLO201, COLO205, COLO320DM, and COLO320HSR show very low to undetectable MRVI1 expression (data not shown).

Mrvi1 expression was also analysed by in situ hybridization. In bone marrow, fetal liver, and spleen high level Mrvi1 expression was observed exclusively within megakaryocytes (Figure 10, data now shown). This hybridization appeared specific for Mrvi1 as the same pattern was observed in three different tissues and in multiple experiments. Additionally, the sense control was negative in all cells tested (Figure 10, data not shown). This high level of expression is not likely to result from the high ploidy of mature megakaryocytes, as other multinucleated cells, such as osteoclasts, do not express the gene (data not shown). In non-hematopoietic cells, Mrvi1 was expressed at high levels in Paneth cells, which are located at the base of the crypts of Lieberkuhn in the small intestine (Figure 10). Paneth cells contain strongly eosinophilic granules and have the ultrastructural characteristics of exocrine protein secreting cells. High levels of Mrvi1 were also seen in the brain in the nucleus-motorius, nucleus-trigemini, and hippocampus (Figure 10).

Figure 10
figure10

;In situ hybridization analysis of Mrvi1 gene expression. Adjacent embryonic or adult tissue sections were hybridized with antisense or sense Mrvi1 probes or stained with H&E

Mrvi1 expression in megakaryocytes

To confirm and extend experiments suggesting that Mrvi1 is expressed at high levels in megakaryocytes, Mrvi1 expression was also analysed in two human cell lines that are reported to have megakaryocytic features, HEL and MEG01. Both cell lines expressed high levels of MRVI1 (Figure 9d). Megakaryocytes were also purified from normal mouse fetal liver and analysed for Mrvi1 expression. In these studies, fetal livers were dissociated and cultured in the presence of thrombopoietin and a feeder layer that supports the outgrowth of megakaryocytes. After 10 days in culture, 90 – 100% of the cells were megakaryocytes by morphology. Poly(A)+ RNA was isolted from these cells and Mrvi1 expression quantitated by RT – PCR. As shown in Figure 11, Mrvi1 was expressed in these cells. However, unlike all other cells tested, Mrvi1 transcripts appeared to originate exclusively from the P2 promoter. Consistent with these findings, MRVI1 is also expressed exclusively from the P2 promoter in MEG01 and HEL cells (Figure 11).

Figure 11
figure11

Semi-quantitative RT – PCR analysis of P1- and P2-initiated Mrvi1 transcripts in normal and transformed megakaryocytes. RNA from normal megakaryocytes is analysed on the left while RNA from two megakaryocytic cancer cell lines is analysed on the right. β-actin was used as a control for semi-quantitative RT – PCR analysis and to control for RNA integrity. H2O, no cDNA added; (+) control, 10 ng of mouse P1 and P2 full-length cDNAs added to the reaction

Mrvi1 expression is downregulated during macrophage differentiation

The homology between Mrvi1 and Jaw1, a gene that is down regulated during lymphoid differentiation, and studies reported here showing that Mrvi1 is expressed in myelomonocytic cells, but not mature macrophages, suggested that Mrvi1 might be downregulated during myeloid differentiation. To determine if this were the case, two BXH2 myelomonocytic tumor cell lines, B140 and B112, were induced to differentiate into macrophages by treatment with IL-6 (Figure 12a) and Mrvi1 expression levels measured by Northern analysis. As shown in Figure 12b, Mrvi1 expression was rapidly downregulated during macrophage differentiation. By day 2, Mrvi1 expression was only 17% of the levels seen in uninduced cells. Mrvi1 expression did not go to zero, however, even in day 4 cells. This may be explained by the fact that not all cells at day 4 had differentiated into macrophages (data not shown). To determine if this were the case, the experiment was repeated but this time at day 4 all the non-adherent undifferentiated cells were removed. As seen in Figure 12c, Mrvi1 was not expressed in day 4 adherent (i.e. differentiated) B140 and B112 cells, even by RT – PCR analysis. As expected, Mrvi1 was expressed in day 4 non-adherent cells. These data confirm that Mrvi1 expression is down regulated during macrophage differentiation in an analogous fashion to Jaw1 during lymphoid differentiation.

Figure 12
figure12

Mrvi1 expression is downregulated during monocyte differentiation. (a) Wright-Giemsa stained cytospin preparations of B140 BXH2 myelomonocytic leukemia cells 0 and 4 days after IL6 treatment. Note that IL6-treated cells have a marked monocytic appearance with ruffled plasma membranes and kidney shaped nuclei. (b) Northern analysis of Mrvi1 expression in B140 cells 0, 1, 2, 3, 4, and 5 days after IL6 treatment. (c) RT – PCR analysis of Mrvi1 expression in non-adherent (NA) and adherent (a) B140 and B112 cells 0 and 4 days after IL6 treatment. β-actin primers were added to each reaction to monitor RNA quality and to quantitate Mrvi1 expression levels

Discussion

In studies reported here we describe the identification of Mrvi1, a new common site of MRV viral integration in BXH2 myeloid leukemias. Viral integration at Mrvi1 occurs within the 5′ end of a novel gene that is predicted to be a type II transmembrane protein associated with the ER. Type II proteins are proteins configured to have a cytoplasmic amino terminus and a lumenal carboxy terminus. Northern analysis indicated that Mrvi1 is expressed in most adult mouse tissues while in situ studies indicated that Mrvi1 expression is restricted to megakaryocytes, Paneth cells of the small intestine, the hippocampus and specific motor nuclei in the brain. While it is not yet clear why Mrvi1 expression is so ubiquitous when assayed by Northerns but so restricted when assayed by in situ hybridization, it is possible that Mrvi1 expression is simply too low in many tissues to be detected by in situ hybridization. Alternatively, the Mrvi1 expression seen in many tissues may result from circulating megakaryocytes or some other widely distributed cell type.

Mrvi1 transcription is complex with two differentially used promoters (P1 and P2) and alternative splicing in the 5′ UTR. The transcripts initiated from the P1 and P2 promoters are predicted to code for two different proteins designated Mrvi1b and Mrvi1a, respectively. The only difference between the two proteins is that the longer Mrvi1a protein contains an 84 amino acid extension at its amino terminus not present in the Mrvi1b. The region shared by these proteins contains an α-helical coiled-coil protein interaction domain, a carboxy terminal hydrophobic region that may function as a membrane anchor, several N-linked glycosylation sites, various phosphorylation sites, an ATP-binding site, and a protein degradation (PEST) sequence. Like the mouse gene, the human MRVI1 gene also contains two different start sites of transcription and is predicted to code for two different proteins that differ only at their amino terminus, MRVI1A and MRVI1B. The human and mouse proteins are also well conserved (83% amino acid identify overall) and share most of the structural features found in the mouse protein.

The N-terminal amino acid extension in Mrvi1a/MRVI1A is predicted to encode a hydrophobic transmembrane domain that is not present in Mrvi1b/MRVI1B and may affect the subcellular localization of Mrvi1a/MRVI1A. Consistent with this hypothesis, GFP-tagged Mrvi1a protein expressed in COS-7 cells has been found to exhibit a strong perinuclear staining pattern characteristic of endoplasmic reticulum while COS-7 cells expressing a GFP-tagged Mrvi1b protein exhibit no discernible staining pattern (JD Shaughnessy Jr, unpublished results). This amino acid extension may therefore be important for localizing Mrvi1/MRVI1 to the endoplasmic reticulum.

Semi-quantitative RT – PCR analysis showed that the P1 promoter is approximately 4 – 5 times less active than the P2 promoter in all murine tissues and tumor cell lines tested, with the exception of normal murine megakaryocytes or human megakaryoblastic-like leukemia cell lines, where Mrvi1 is expressed exclusively from the P2 promoter. The viral integrations at Mrvi1 are located between the P1 and P2 promoters and these integrations are therefore likely to affect both Mrvi1 transcripts. Unfortunately, we have not been able to determine the affect of viral integration on Mrvi1 expression. Tumor DNAs are all that are available from the two BXH2 leukemias with Mrvi1 integrations at Mrvi1 and the low frequency of viral integration at this locus has so far precluded the identification of other BXH2 leukemias with viral integrations at Mrvi1.

The carboxy terminal half of Mrvi1 shows a high degree of homology to another type II protein associated with the ER, Jaw1. Northern analysis has shown that Jaw1 expression is restricted to the lymphoid lineage where it is expressed in early but not late stages of lymphoid development. While Mrvi1 does not appear to be expressed in the lymphoid lineage, Mrvi1 is expressed in several myelomonocytic BXH2 leukemia cell lines and the myelocytic cell line 32D. Mrvi1 expression is also downregulated during IL-6 induced macrophage differentiation of BXH2 myelomonocytic leukemias [but not G-CSF induced differentiation of 32D cells (JD Shaughnessy Jr, unpublished results)] like Jaw1 expression in lymphoid differentiation.

Integral membrane proteins are typically targeted to translocation-competent membranes by virtue of a signal sequence located close to the amino-terminus of the protein. Membrane anchoring is caused by the signal sequence or other hydrophobic segments located after it. However, some integral membrane proteins like Jaw1 and possibly Mrvi1 do not adhere to these rules – rather they have no signal sequence but possess a hydrophobic segment near the carboxy-terminus that orients them with their amino-termini in the cytoplasm. The mechanism by which these proteins are inserted into the membrane is not well-understood. Several tail-anchored proteins associated with the ER seem to be involved in vesicular transport. In support of a possible role in vesicle transport or processing for Mrvi1, GenBank homology searches have shown that the carboxy-terminal portion of Mrvi1 has a limited degree of homology to the rat synapsin Ia and Ib proteins (E value=0.013). These proteins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. (McCaffery and DeGennaro, 1986; Suedhof et al., 1989).

The tight control of Jaw1 and Mrvi1 expression during terminal differentiation suggests that these two proteins may be intimately involved in the control of growth and/or differentiation of hematopoietic cells. Although no function of these genes is known, it has been proposed that the sublocalization and secondary structure data for Jaw1 indicate that it may be involved in vesicle docking and transport (Berhens et al., 1994). Because Jaw1 has been shown to be exclusively expressed in lymphoid cells, it has also been suggested that Jaw1 may be involved in antigen receptor processing and presentation in both B- and T-cells (Berhens et al., 1994). The discovery of Mrvi1, a second Jaw1-related gene that is expressed in cells that lack antigen receptors, suggests an alternative function for these genes- namely, they are involved in processing lineage specific growth and differentiation factors. It is of course also possible that the function of these genes is entirely unrelated to their location in the endoplasmic reticulum, as proteins such as the cytochromes and the antiapoptosis protein, bcl-2, are anchored in the ER yet have no major ER-specific function.

The mouse and human Jaw1 proteins also contain a proline rich amino terminus with multiple PXXP motifs, which represent the core ligand for the Src homology domain 3 (SH3) (Cohen et al., 1995). The SH3 domain was first identifed as a conserved sequence in the non-catalytic part of several cytoplasmic (non-receptor) tyrosine kinases (e.g. Src, Abl, and Lck; Mayer et al., 1988) but is now known to exist in many other intracellular or membrane associated proteins (Pawson, 1995). The function of the SH3 domain is not well understood but it is generally thought to medicate assembly of specific protein complexes via binding to proline-rich peptides. It is interesting to speculate that the Mrvi1 protein is in fact a substrate for one of the oncogenic tyrosine kinases and that the proline rich amino terminus facilitates the interaction between these proteins. Given the important role of tyrosine phosphorylation in regulating cell proliferation and differentiation, and the involvement of many protein kinases in oncogenesis, future studies will be aimed at investigating whether Mrvi1 is a substrate for phosphorylation and whether Mrvi1 may be differentially phosphorylated during the cell cycle or in response to growth and/or differentiation.

Finally, Mrvi1 as well as the Jaw1 proteins also have a highly conserved coiled-coil domain that bears significant homology to the myosin heavy chains, dystrophin, intermediate filament components, and other coiled-coil proteins. Behrens et al. (1994) have demonstrated that the coiled-coil domain of Jaw1 is most closely related to myosin, yet there is no ATP-binding or actin-binding domain which suggests that Jaw1 is not a motor protein. However, a PROSITE protein motif search has shown that the Mrvi1 proteins of human and mouse do contain an ATP-binding domain, the so called `A' consensus sequence or P-loop. The significance of this ATP-binding site is not known but suggests that Mrvi1 function involves an energy dependent step.

Although it is apparent that Mrvi1 expression is downregulated during macrophage differentiation, it is not yet clear whether this downregulation occurs at the level of transcription or by post-transcriptional mechanisms. It is interesting to note that the Mrvi1 3′ UTR contains three mRNA destabilizing sequences ATTTA, which could mean that Mrvi1 downregulation occurs through increased mRNA degradation. Similar destabilizing sequences are found in the mRNAs for several lymphokines, cytokines, and proto-oncogenes where they have been shown to be important for regulating the concentration of these proteins (Shaw and Kamen, 1986). Alternatively, Mrvi1 is also predicted to contain a protein degradation PEST sequence near its carboxy terminus. Mrvi1 expression may therefore also be regulated by protein degradation.

The myeloproliferative leukemia virus (MPLV) is an acute leukemogenic murine retrovirus that transduced the cellular oncogene c-mpl (Souyri et al., 1990), which is the receptor for thrombopoietin (de Sauvage et al., 1994; Wendling et al., 1994; Lok et al., 1994). In vivo infection of bone marrow cells with MPLV results in the outgrowth of a broad spectrum of MPLV-infected hematopoietic progenitor cells that have acquired factor independence for both proliferation and terminal differentiation (Wendling et al., 1986). Mrvi1 and c-mpl have very similar patterns of expression in hematopoietic tissues and cells. Both genes are detected in mouse spleen, bone marrow and fetal liver (Souyri et al., 1990) and in the HEL megakaryocytic leukemia cell line (Methia et al., 1993). c-mpl is also expressed in the CD34+ cell fraction of the bone marrow (Methia et al., 1993) while Mrvi1 is expressed in CD34+ BXH2 leukemia cell lines. Interestingly, c-mpl and Mrvi1 are not expressed in K562 cells, an erythroleukemia cell line reported to have megakaryocytic differentiation potential (Tetteroo et al., 1984; Methia et al., 1993; JD Shaughnessy Jr, unpublished results). It will be important to determine if Mrvi1 is in some way involved in the c-mpl/thrombopoietin signal transduction pathway.

As yet, we also do not know if Mrvi1 is a tumor suppressor as predicted by the model. The data on this issue are still inconclusive. In the case of the two BXH2 leukemias with MRV integrations at Mrvi1, 4MRV integration occurs only in one allele, which would seem to argue against Mrvi1 being a tumor suppressor gene. JAW1, which maps to human 12p, has recently been shown to be amplified in testicular germ cell tumors (Mostgert et al., 1998), which would argue that JAW1 is also not a tumor suppressor gene. It should be noted however, that 70% of BXH2 leukemias with viral integration at Nf1 have viral integrations only on one Nf1 allele, yet neurofibromin is still not expressed in these leukemias (Cho et al., 1995; Largaespada et al., 1995). Apparently, the second Nf1 allele contains a non-virally-induced mutation, not detectable by Southern analysis, that prevents its expression. In the absence of RNA from the two leukemias with Mrvi1 integrations at Mrvi1, it has not been possible to determine if Mrvi1 is still expressed in these leukemias.

Other data however could be used to argue that Mrvi1 is a tumor suppressor gene. For example, we have been able to show, by FISH mapping, that human MRVI1 maps to chromosome 11p15 (JD Shaughnessy Jr and J Sawyer, unpublished data) a site where several putative tumor suppressor genes have been localized (Ali et al., 1987; Bepler and Garcia-Marco., 1994; Loh et al., 1992; Baffa et al., 1996). We have also shown that MRVI1 is expressed at high levels in the colon carcinoma cell line SW480, but not in SW620, a cell line derived from a lymph node metastasis from the same patient. Expression of EIF4G2, which is physically linked to MRVI1 (Shaughnessy et al., 1997), is expressed in both cell lines. Finally, EXT1, a putative human tumor suppressor gene mutated in hereditary multiple exostoses, has recently been shown to encode a type II transmembrane protein associated with the ER (McCormick et al., 1998), providing support for the notion that this class of proteins can be tumor suppressor gene. EXT1 is required for the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). It is known that GAGs function as co-factors in several signal-transduction systems that affect cellular growth, differentiation, motility and adhesion, and may play a role in the malignant transformation of cells, tumor-cell adhesion, invasiveness, and metastasis. A similar type of function could easily be envisioned for Mrvi1. Further studies involving gene knockouts and transgenic mice should help define whether Mrvi1 is a tumor suppressor gene or proto-oncogene.

Materials and methods

Mice and cell lines

BXH2 mice were obtained from the Jackson Laboratory (Bar Harbor, Maine) and maintained at the NCI-Frederick Cancer Research and Development Center. The derivation of BXH2 leukemic cell lines has been previously described (Largaespada et al., 1995). Human tumor cell lines were purchased from the ATCC and grown in either RPMI 1640 (GIBCO – BRL) or DMEM (GIBCO – BRL) supplemented with 2 mM glutamine, 4.5 g/L glucose, Pen-Strep, and 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA, USA).

Southern and unblot analysis

High molecular weight genomic DNAs were extracted from frozen normal tissues and leukemic spleens and lymph nodes as previously described (Jenkins et al., 1982Jenkins et al., 1982). Bacteriophage and plasmid DNAs were purified using standard procedures (Sambrook et al., 1989). Restriction enzyme digestions, agarose gel electrophoresis, Southern blot transfers, hybridizations, and washes were performed as previously described (Sambrook et al., 1989). Unblots were prepared and hybridized as described (Cho et al., 1996). Briefly, 10 μg of restriction enzyme-digested DNA was electrophoresed on a 0.8% agarose gel and the gel treated with an alkaline solution to denature the DNA. The gel was then neutralized and dried down under vacuum for 1.5 h at 50°C. The dried gel was prehybridized in a solution of 5×SSCP, 0.1%SDS for 1 h at 55°C. The probe, Δenv26 was end labeled with γ32P-dATP to a high specific activity using T4 polynucleotide kinase (New England Biolabs). The prehybridization solution was removed from the blot and fresh solution containing 3×107 c.p.m./ml of probe was added. The unblot was hybridized for 4 h at 55°C. The hybridization solution was removed and the blot was washed in 5×SSCP for 15 min, 3×SSCP for 15 min, and 1×SSCP for 15 min. The unblot was wrapped in plastic wrap and autoradiographed by exposure to XAR-5 film (Kodak) using two intensifying screens. Exposures were done at −70°C for 10 days.

Gegomic cloning

Seventy μg of BXH2 tumor DNA (tumor 87-15 or 19) was digested to completion with EcoRI, electrophoresed on 1.0% Ultrapure LMP agarose gels (GIBCO – BRL), Gaithersburg, MD, USA) and fragments of the appropriate size cloned into EMBL4 lambda phage (Stratagene, La Jolla, CA, USA). Positive plaques were identified with an MRV-specific probe (Δenv42; Cho et al., 1995) and DNA from positive clones subcloned into pBluescript (Stratagene). A murine 129/SV-derived cosmid library (Stratagene) was screened with probe p15.8.1.1. and single clones purified as described in the manufacturers protocol. Murine P1 clones were purchased from Research Genetics after PCR-based screen for positive clones.

Chromosome mapping

Interspecific backcross progeny were generated by mating (C57BL/6J × Mus spretus)F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 N2 mice were used to map the Mrvi1 locus (see text for details). Southern blot analysis was performed as described (Jenkins et al., 1982Jenkins et al., 1982). All blots were prepared with Hybond-N+ membrane (Amersham). The Mrvi1 probe p15.8.1.1. was labeled with α32P-dCTP using the Prime It II labeling kit (Stratagene). A major band of 6.9 kb was detected in TaqI-digested C57BL/6J DNA while a 3.5 kb fragment was detected in TaqI-digested Mus spretus DNA. Blots were washed to a final stringency of 0.5×SSCP, 0.1% SDS, 65°C. The segregation of the 3.5 kb Mus spretus-specific TaqI fragment was followed in backcross mice.

Exon trapping

The 30 kb insert in cosmid clone COS1-1 was released by NotI digestion and fractionated by size on a 1.0% Ultrapure LMP agarose (Gibco – BRL) gel in 1×TAE buffer. The 30 kb insert was then cut from the gel and purified by B-agarose treatment according to the manufacturers protocol (NEB). The COS1-1 insert and the P1 clone P114929 were digested to completion with BglII and BamHI and purified by phenol extraction and ethanol precipitation. Insert DNA was then ligated to a BamHI-digested pSPL3 vector (Gibco – BRL). DNA from the ligations we used to transform DH10B cells (Gibco – BRL). Additional steps in the experiment were performed exactly according to the manufacture protocol (Gibco – BRL). Approximately 100 individual clones from each ligation were sequenced as described below.

RNA isolation and Northern analysis

Northern blots containing 2 μg of twice selected poly(A)+ RNA from various normal mouse tissues and cell lines were purchased from Clonetech. Total mouse RNA was extracted from normal tissues, spleens and lymph nodes with leukemia cell infiltration, and cell suspensions by the RNAzol B method (Tel-Test). Poly(A)+ RNA was purified from the total RNA preps by oligo-d(T) column chromatography according to the manufacturers recommendations (Pharmacia). 2 – 5 μg of poly(A)+ RNA was fractionated by electrophoresis on 1.0% agarose gels containing formaldehyde and transferred to Hybond N+ membranes (Amersham). The membranes were prehybridized and hybridized according to the method of Church and Gilbert or using the ExpressHyb solution (Clonetech). Blots were then exposed to X-ray film at −70°C with an intensifying screen.

cDNA cloning

cDNA cloning was carried out by a combination of 5′ and 3′ rapid amplification of cDNA ends (5′ and 3′ RACE) and modified RT – PCR. The nucleotide sequence of the trapped exon COSET29 was used to design specific nested oligonucleotides: The oligonucelotides COS29-5′-RACE1-CCTGCGGAGCCCAAGCCTCTTGGAGC, COS29-5′-RACENEST - TTGGG GT TGGT ACAGCAGAGG, COS29 -3′-RAC 1 - G TC T G GGC TG CC TGT GG CT CT CC GG, and COS29 - 3′ - RACENEST - CTC CTC ATCCTCAGGAATGTGGGG were synthesized by conventional methods (Gibco – BRL). The primary 5′ and 3′ RACE reactions were performed using mouse heart Marathon Ready cDNA (Clonetech) as a template according to the manufacturers protocol using the Advantage cDNA amplification kit (Clonetech). The primary PCR was performed with the primers COS29-5′-RACE1 and COS29-3′-RACE1 in combination with the manufacturer supplied adapter primer 1 (AP1 – CCATCCT AATAC GACTC ACTA TAG GG C). A nested PCR reaction (1 cycle: 94°C 1 min; 20 cycles: 94°C, 30 s, 68°C, 5 min) was performed using a 1 : 50 dilution of the primary reaction as a template and primers COS29-5′-RACENEST and COS29-3′-RACENEST in combination with adapter primer 2, AP2-ACTCACTATAGGGCTCGAGCGGC). After determining the sequence of the 5′ and 3′ RACE products we designed 5′ primers specific for the P1 transcript (MoMrvi1P15′-3′-CATTGACCTTAAATGGTAAAAGCTCCCCAG) and the P2 transcript (MoMrvi1P25′-3′-TG GATCCT GG AAATG GAA CCCAGT TTG GG) and a common 3′ primer (MoMrvi1polyA3′-5′-TCACCACTGCCACACCTACCTGTGTGAGG) located just upstream of the polyadenylation sequence. Using the mouse heart Marathon Ready cDNA as a template and the above primers, full-length cDNA for both P1 and P2 transcripts were generated by PCR using the Advantage cDNA amplification kit (Clonetech) and a Perkin-Elmer-Cetus 480 thermocycler under the following conditions: 1 cycle: 94°C 1 min; 30 cycles: 94°C, 30 s, 68°C, 7 min. The products of the reaction were subcloned into pCR2.1 (Invitrogen) and sequenced. The murine full-length cDNA sequences of the P1 and P2 generated transcripts have been submitted to GenBank under accession numbers U63407 and U63408, respectively.

Human cDNAs covering the coding region of the human MRVI1 gene were synthesized in a similar manner as described above. Briefly, a human EST (accession number M78793) with homology to the mouse Mrvi1 cDNA was identified. Nested oligonucelotide primers used in 5′ and 3′ RACE reactions were synthesized: M787935′1°: 5-TCCTGGCGGACGGCGCCTACCACCTCAGC-3′; M787933′1°: 5′ - ACAC C C TAA GC ATCA GA CCT GG AA TT TG G -3′; M787935′NEST: 5′-GGTGCAGGACAGCGATGTCCTCCAGC-3′; M787933′NEST: 5′-CAAAGTGACTATCTTCCCATTTCCC-3′. Ten μM of M787935′1° and M787933′1°C were combined with 10 μM of AP1 and 5 μl of the human heart marathon ready cDNA and primary reactions preformed as described above. Five μl of a 1 : 50 dilution of the primary reaction was used as the template in the secondary reactions. Ten μM of M787935′NEST and M787933′NEST were combined with 10 μM of AP2 and 5 μl of the dilution and reactions performed as above. The PCR products were subcloned and sequenced. Sequence of the ends of the 5′ and 3′ RACE products showed a high degree of homology to mouse cDNA sequences. 5′ RACE products were generated that had 5′ ends that corresponded to the mouse P1 and P2 initiated transcripts. 3′ RACE products ended in poly-(A) tracts with unique sequences upstream of the poly(A)+ tract that was homologous to the corresponding mouse sequence. RT – PCR fragments of the entire human MRVI1 coding sequences were generated using the following primers: HuP15-3′: 5′-CATTGACCTTA AAT GGT AAAA GC TC CC GAG HuP25′5′-TGGCTCCGG GAA A TGGAA CC CAGCTTG GG - 3′, Hu3′UTR3′5′: 5′- ACTG AGAAC AGTGCTT GGCTC ATAG -3′. Reactions were performed exactly as for mouse full-length synthesis with the exception that the extension times were reduced to 4 min. Products were subcloned into pCR2.1 (Invigrogen) and sequenced. The nulceotide sequence of the human cDNAs for the P1 and P2 derived transcripts have been submitted to GenBank under the accession numbers AFO81249 and AFO81250.

Cell growth and differentiation

BXH2 cell lines B112 and B140 were grown as previously described (Largaespada et al., 1995). Approximately 1×105 cells were seeded in ten 20 cm dishes. 5 – 10 ng/ml of recombinant murine IL-6 was added to each plate. After 1, 2, 3, and 4 days all cells were harvested by pooling and centrifugation. In one set of day 4 plates only the adherent cells were harvested. In this case the plates were washed free of dead cells and those living cells that had not adhered to the plate bottom. The adherent cells were then scrapped from the plates and pooled by centrifugation. Cells were then pelleted and total and poly(A)+ RNA was purified as described above. Poly(A)+ RNA from day 0 (no treatment), days 1, 2, 3, and 4 was separated by gel electrophoresis and Northern blotted and hybridized as described above.

Semi-quantitative RT – PCR analysis of Mrvi1 expression following IL-6 treatment

One μg of total RNA derived from BXH2 cell lines B112 and B140 from day 0 and day 4 adherent cells was used as a template for first strand synthesis using SuperScript II MoLV reverse transcriptase (Gibco – BRL) according to the manufacturers protocol. Briefly, the RNA and oligo-dT primer mixture was heated to 70°C for 15 min then chilled on ice for 5 min. The reaction buffer was added to the RNA/primer mix and then incubated at 42°C for 5 min. Ten units of reverse transcriptase was added, mixed and the reaction incubated for another 50 min at 42°C. The reaction was stopped by incubation at 70°C for 15 min. The reaction was centrifuged and placed on ice. Five units of RNase H was added to the mixture and incubated for 20 min at 37°C. The 20 μl reaction was then diluted to 100 μl and frozen at −20°C until used in the PCR reaction. PCR reactions were performed using the Advantage cDNA amplification kit (Clonetech). Briefly, 10 μl of the RT reaction described above was added to 5 μl of 10× reaction buffer, 1 μl of 50×dNTPs, 1 μl of each 10 mM primer solution, 1 μl of KlenTaq enzyme and 29 μl of ddH20 to a final volume of 50 μl. For the PCR reaction the primer pair was TA4.2 REVRACE1-GTTCCACTTCATATGCCAGGGTGGAGC) and MoMrvi1polyA3′-5′. The reactions were performed in a Perkin-Elmer-Cetus 480 thermocycler under the following conditions: 1 cycle: 94°C 1 min; 30 cycles: 94°C, 30 s, 69°C, 4 min. The β-actin control primer pair consisted of BA5′-GTGACGAGGCCCAGAGCAAGAG and BA3′-AGGGGCCCGGACTCATCGTACTC. Ten μM of the control primers was added to each reaction with 18 cycles remaining. Ten μl of each reaction was analysed by gel electrophoresis.

Semi-quantitative RT – PCR analysis of Mrvi1 promoter usage

For the Mrvi1 P1-specific PCR reaction the primer combination was MoMrvi1P15′-3′ and OSW 151-CAATGGGATGGGTGGCCACATGTGCCC), for the P2 specific reaction the primer pair was MoMrvi125′-3′ and OSW 151. The reactions were performed in a Perkin-Elmer-Cetus 480 thermocycler under the following conditions: 1 cycle: 94°C 1 min; 30 cycles: 94°C, 30 s, 58°C, 4 min. The β-actin control primer pair consisted of BA5′ and BA3′. Ten μM of the control primers was added to each reaction with 18 cycles remaining. Ten μl of each reaction was analysed by gel electrophoresis. Megakaryocytes were purified as follows. Approximately 1 – 5×105 fetal liver cells from normal fetuses were plated in a methyl cellulose mix which included Thrombopoietin. At day 5 – 6, CFU-Megakaryocyte (CFU-Mk) defined as colonies with 2 – 50 cells were picked and placed in DMEM with 10% FCS. The cells were then washed once in Phosphate Buffered Saline (PBS). RNA was extracted using RNAZOL B (Tel-Test Inc) according to the manufacturer's protocol. First strand cDNA synthesis was performed using 1 μg of poly(A)+ RNA. PCR reactions on a fraction of the first strand cDNA were performed as described above. A similar strategy was used to determine the promoter usage in the human cell lines HEL and MEG01. The primers used for this analysis were the same pairs used to clone the cDNAs.

Probes

All probes were labeled with α32P-dCTP using the Prime It II labeling kit (Stratagene), unless stated otherwise. The probe p15.8.1.1. was a 1.4 kb EcoRI – PstI fragment derived from λ15.8.1.1. The probe p129.8XS was a 560 bp XbaI – SalI fragment derived from cosmid clone COS 1-1. The probe Δenv26 was a 26 nt oligonucleotide described in Cho et al. (1995). The probe GAPDH was a murine cDNA probe. The β-actin probe was 2.0 kb human cDNA (Clontech). The probes COSET.29 and P1ET.PPI were 149 bp and 101 bp fragments derived from an exon trap experiment as described above. The probe Δenv42 was a 42 bp PCR generated fragment containign 21 bp on either side of the MRV specific envelope gene deletion described by Cho et al. (1995) that was amplified from the genomic clone pMRV. The primers used for the amplification were: Δenv42-5′-CACCAGGTCTTC and Δenv42-3′-CAGTCTCTCAAACC. The fragment was labeled with α32P-dCTP by PCR using the GeneAmp PCR reagent kit. Briefly, the reaction components were as follows: 2.2 μl of 10×reaction buffer, 1 μl of Δenv-5′ and Δenv42-3′ (20 μM), 1 μl of pΔSC (100 ng of plasmid sublcone of a full length MRV provirus), 0.5 μl of each dATP, dGTP, and dTTP (10 μM), 15 μl of [α32P]dCTP (3.3 mM, 3000 Ci/mmole), and 0.5 μl of AmpliTaq (1 U/μl). The reactions were performed under the following conditions: 1 cycle: 94°C 2 min; 30 cycles: 94°C, 30 s, 37°C, 30 s, 72°C, 1 min. The labeled fragment was purified by column chromatography. Mrvi1 cDNA probes were 3.5 kb DNA fragments derived from the open reading frame of the human or murine cDNA.

DNA sequencing

DNA sequencing was performed using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin Elmer) on the ABI Model 373A DNA Sequences (Applied Biosystems). Sequence primers were either the T3, T7 sequencing primers or synthetic oligomers derived from previously determined sequence.

In situ analysis

Dissected tissue was fixed at 4°C for 96 h in 4% paraformaldehyde. Paraffin embedded tissue was cut at five microns in sagittal and horizontal planes. Probe preparation, tissue processing, and hybridization were carried out as described (Tessarollo and Parada, 1995). Slides were dipped in Kodak NBT emulsion and exposed for 9 days.

In vitro transcription and translation

cDNAs corresponding to the complete open reading frame of both the P1- and P2-derived Mrvi1 transcripts were PCR amplified from Marathon ready heart cDNA (Clontech, Palo Alto, CA, USA) using the Advantage cDNA PCR kit (Clontech). The fragments were sublconed into pCR2.1 vectors (Invitrogen) and amplified. The cDNA inserts were released from the vector by BamHI and NotI digestion and subcloned into BamHI – NotI digested pCDNA3.1 (Invitrogen). One μg of purified plasmid DNA was transcribed using T7 RNA polymerases, and in vitro translation was performed using a TnT translation kit (Promega) with [35S]methionine. Translated proteins were analysed by 10% SDS – PAGE.

DNA and protein sequence analysis

Nucleotide and protein sequence analysis was performed on a VAX 8600 using the software package of the Genetics Computer Group. Sequence homology searches were conducted at the protein level using the National Center for Biotechnology Information and the BLAST network service. The protein sequence alignment between the mouse Mrvi1 and mouse Jaw1 were performed and analysed based on the progressive sequence alignment program (Feng and Dolittle, 1987; Devereux et al., 1984). Protein subsequence motifs were identified using MacVector (Oxford Molecular Group, OR, USA), PSORT (Nakai and Kanehisa, 1992), COILS (Lupas et al., 1991) and PESTfind (Rogers et al., 1986).

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Acknowledgements

This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL. The contents of this publication do not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. We thank J Frederic Mushinski for the plasma cell and pre B-cell mRNAs. We also thank Deborah Gilbert, Linda Cleveland and Deborah Householder for expert technical assistance. Accession no. Mouse MRVi1 U63407, U63408. Human MRVi1 AFO81249, AFO81250.

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Correspondence to John D Shaughnessy Jr.

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Jr, J., Largaespada, D., Tian, E. et al. Mrvil, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1. Oncogene 18, 2069–2084 (1999) doi:10.1038/sj.onc.1202419

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Keywords

  • myeloid leukemia model
  • retroviral insertional mutagenesis
  • common sites of integration

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