Integration of cytogenetic landmarks into the draft sequence of the human genome.

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.

A murine model of inflammatory bone disease.

We have recently reported the identification of a new recessive mutation on murine chromosome 18 that results in tail kinks and deformity in the lower extremities of mice. Preliminary examination of the bones of these mice showed that there are abnormalities present that resembled chronic recurrent multifocal osteomyelitis. Accordingly, this new mutation was named "CMO." In this report, we describe the histology of bones in CMO mice, as well as the capacity of the bone marrow cells from these animals to form osteoclasts (OCLs). In addition, we tested conditioned media from non-adherent marrow cells and total marrow cells from CMO mice for their capacity to induce OCL formation in normal murine marrow cultures. These studies demonstrated that the bone disease in these animals is inflammatory in nature, and a soluble factor(s) that is not IL-1alpha, IL-6 or TNF-alpha is released by marrow cells from CMO animals and enhances OCL formation in normal murine marrow cultures.

Chronic multifocal osteomyelitis, a new recessive mutation on chromosome 18 of the mouse.

Mice with tail kinks and deformities in their lower extremities were observed in a litter of C.D2-Qa-2+N6F15 mice. A mutant line that exhibits this phenotype in 100% of its offspring was established by subsequent breeding. The abnormalities resembled to some degree those found in a human syndrome termed chronic recurrent multifocal osteomyelitis (CRMO). Accordingly, we name the new mutation chronic multifocal osteomyelitis (cmo). Breeding analysis showed that the defect was determined by a single autosomal recessive gene. Restriction fragment length polymorphism (RFLP) analysis of progeny from a backcross between Mus musculus domesticus (CLA) and C.D2-Qa-(2+)-cmo/cmo indicated that the cmo gene resides on mouse Chromosome 18.

Alternative splicing of RNAs transcribed from the human c-myb gene.

An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-myb homology is included as an additional 363-nucleotide coding exon (termed E6A or coding exon 9A) has been described for normal and tumor murine cells that express myb. We show here that this alternative splicing event is conserved in human c-myb transcripts. In addition, another novel exon (termed E7A or coding exon 10A) is identified in human c-myb mRNAs expressed in normal and tumor cells. Although the myb protein isoform encoded by murine E6A-containing mRNA is larger than the major c-myb protein, the predicted products of both forms of human alternatively spliced myb transcripts are 3'-truncated myb proteins that terminate in the alternative exons. These proteins are predicted to lack the same carboxy-terminal domains as the viral myb proteins encoded by avian myeloblastosis virus and E26 virus. The junction sequences that flank these exons closely resemble the consensus splice donor and splice acceptor sequences, yet the alternative transcripts are less abundant than is the major form of c-myb transcripts. The contribution that alternative splicing events in c-myb expression may make on c-myb function remains to be elucidated.

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

A chronic inflammatory response. Its role in supporting the development of c-myb and c-myc related promonocytic and monocytic tumors in BALB/c mice.

This study demonstrates that an inflammatory response caused by the injection of pristane into the peritoneal cavity of mice provides a useful system for rapid induction of myeloid tumors by retroviruses. Two such tumors, which developed in the peritoneal cavity with average latencies of 68 to 71 d and incidences of greater than 50%, are 1) the McML, mature monocyte-macrophage tumors induced by retroviral constructs containing exons 2 and 3 of c-myc cDNA, and 2) the MML, promonocytic tumors induced by Moloney murine leukemia virus infection and its integration into the c-myb locus. Development of both neoplasms is clearly dependent on the intense i.p. inflammatory response, inasmuch as mice given the viruses and not pristane fail to develop these tumors. Although both types of tumors appear in the peritoneal cavity, the MML tumors that arise by i.v. injection of Moloney murine leukemia virus may actually originate via infection, and perhaps transformation, of precursor hemopoietic cells outside the peritoneal cavity, followed by migration of the cells to the peritoneal cavity. This is suggested by the fact that i.v.v but not i.p. injection of virus is an efficient method of producing these particular myeloid tumors. Although both McML and MML tumors require the inflammatory environment for their development, treatment of mice with a nonsteroid anti-inflammatory drug, indomethacin, has no effect on McML monocyte/macrophage tumors but completely prevents the development of the MML promonocyte tumors.

Alternative internal splicing in c-myb RNAs occurs commonly in normal and tumor cells.

Activation of the c-myb gene by viral transduction or proviral insertional mutagenesis that is likely to result in the production of structurally altered myb proteins has been shown to be predominantly associated with myelomonocytic tumors. An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-myb homology is included as an additional 363-nucleotide coding exon has recently been identified in a mouse tumor that carries a provirus-activated myb gene. This alternative splicing was hypothesized to be a tumor-specific aberrant form of 3'-myb RNA processing as a consequence of the disruption of upstream 5'-sequences by proviral insertion. However, RNA blot analyses and RNase mapping studies presented here show that a significant portion (approximately 10%) of all myb transcripts examined, whether in normal or in clonal tumor cells, contains the additional exon. Hence the alternative splicing is a hitherto unrecognized common normal event that potentially increases the diversity of the myb proteins expressed in normal tissues including thymus and spleen, as well as in tumor cells with either normal or 5'-rearranged myb alleles. The lack of change in the ratio of the two spliced products expressed from either the normal or the 5'-rearranged myb further indicates that the insertion of the unique 121 amino acids in the larger myb transcripts is not a consequence of tumor-specific activation of the mouse myb oncogene.

Moloney murine leukemia virus-induced myeloid tumors in adult BALB/c mice: requirement of c-myb activation but lack of v-abl involvement.

BALB/c mice treated with pristane and Abelson virus have been used as an animal model system for the rapid induction of plasmacytomas. Myelomonocytic tumors with helper Moloney murine leukemia virus clonally inserted into the c-myb locus were observed in about 10% of pristane-primed BALB/c mice infected with Abelson virus. However, v-abl was absent in almost all of those tumors. Since Moloney virus is thought to induce mostly T-cell lymphomas, we have carried out studies to investigate this alteration of disease specificity and to determine whether v-abl played an obligatory role in the development of these tumors. We found that, whereas lymphomas developed late (greater than 3 months) in both pristane-primed and unprinted control mice, the myelomonocytic tumors arose at a high frequency, within 3 months, but only in pristane-treated mice. Clonal Moloney virus insertion was again found in each of the seven myelomonocytic tumors examined. Northern blot analyses and S1 mapping studies revealed the presence of virally promoted chimeric mRNAs that lack the three 5'-most myb coding exons. Hence it appears that the requirement for the v-abl gene product in tumor induction is not obligatory. Our results also indicate that tumor-specific alteration at the 5' end of the myb gene plays an important role in the development of these tumors.

Phorbol ester-induced growth arrest of murine myelomonocytic leukemic cells with virus-disrupted myb locus is not accompanied by decreased myc and myb expression.

A class of mouse neoplasms termed Abelson virus-induced plasmacytoid lymphosarcomas has previously been found to express abnormal myb transcripts due to a helper virus insertion into the 5' end of the c-myb locus. We have established clonal cell lines from these tumors and have shown that they all express markers characteristic of myelomonocytic, rather than lymphoid, cells. Treatment of the plasmacytoid lymphosarcoma lines with phorbol 12-myristate 13-acetate led to various degrees of growth arrest, presumably due to myelomonocytic differentiation. To date, characterization of myeloid cell lines with varying responses to phorbol 12-myristate 13-acetate has not been reported. The different clonal plasmacytoid lymphosarcoma lines should, therefore, provide a good model to help elucidate the role of altered myb locus and other protooncogenes in myelomonocytic leukemia. Contrary to studies on induced differentiation of myelomonocytic cells with normal myb locus, our results on plasmacytoid lymphosarcoma cells indicate that reduced myb and myc expression may not be obligatory for growth arrest to occur. The present study, however, supports the previous notion that the myb transforming ability may be restricted to cells of the myelomonocytic lineage. In addition, we found that only the more mature cells can undergo prolonged phorbol 12-myristate 13-acetate-induced growth arrest, suggesting that the maintenance of these leukemic cells in their proliferative state, presumably by the myb gene product, can be overcome with appropriate differentiation signal(s).

Two modes of c-myb activation in virus-induced mouse myeloid tumors.

Two modes of disruption of the protooncogene c-myb by viral insertional mutagenesis in mouse myeloid tumor cells are described. The first mode was found in six tumors in which a Moloney murine leukemia virus component had inserted in the same transcriptional orientation upstream of the 5'-most exon with v-myb homology (vE1). cDNA sequence data indicate the presence of a truncated c-myb mRNA that is initiated in the upstream 5' long terminal repeat of the integrated provirus and processed via a cryptic splice donor sequence in the gag region to the splice acceptor site in vE1 of the c-myb gene, thus removing the remaining downstream viral and myb intronic sequences. Unlike most gag-onc transcripts, the gag and myb sequences in the hybrid transcript were not in the same reading frame. It is presumed that the gag sequence provides a cryptic translation initiation site for the novel amino-truncated c-myb protein. The second mode of disruption was by downstream virus insertion at the 3' side of the c-myb, which results in the synthesis of a small (approximately 2 kilobase) myb transcript. The 5' long terminal repeat of the inserted provirus provides a TGA termination codon that results in the elimination of 240 normal c-myb amino acid residues from the carboxyl terminus of the tumor-specific myb protein. These results suggest that truncated myb proteins play a role in neoplastic transformation of myeloid cells.

Activation of the c-myb locus by viral insertional mutagenesis in plasmacytoid lymphosarcomas.

Rearrangement in the c-myb locus of each of four independently derived BALB/c plasmacytoid lymphosarcoma (ABPL's) is due to the insertion of a defective Moloney murine leukemia virus (M-MuLV) into a 1.5-kilobase-pair stretch of cellular DNA at the 5' end of the v-myb-related sequences. This retroviral insertion is associated with abnormal transcription of myb sequences and probably represents a step in the neoplastic transformation of ABPL cells.

Amplification of a specific set of intracisternal A-particle genes in a mouse plasmacytoma.

Intracisternal A-particles (IAPs) derive from endogenous retrovirus-like genes that are present in ca. 1,000 copies per haploid mouse genome. A particular IAP sequence variant that represents only 2% of the endogenous genes was found to be the predominant transcript in five out of seven plasmacytoma lines examined, and these genes have been amplified two- to fourfold in MOPC 315. Restriction enzyme digests of normal and tumor DNAs revealed a set of conserved sites within the population of amplified genes which could be used to generate a consensus restriction enzyme map of the amplified IAP genome. Molecular clones of IAP genes related to those amplified in MOPC 315 were isolated from gene libraries of MOPC 315 and MOPC 104E DNAs. Each clone was characterized for the presence of the restriction enzyme sites conserved in the amplified genes. Of 26 clones isolated from MOPC 315 DNA, 14 contained all of the conserved sites, whereas none of the clones isolated from MOPC 104E DNA contained all of the sites. These results suggested that approximately one-half of the genes isolated from MOPC 315 DNA were members of the amplified IAP gene population, representing newly acquired proviruses in the tumor DNA. Unique cellular flanking sequences were isolated from four of the MOPC 315 clones and four of the MOPC 104E clones. Probes from three of the MOPC 315 clones detected restriction fragments that were unique to MOPC 315 DNA and equivalent in size to the cloned gene, demonstrating that these three IAP genes had integrated into new sites in the plasmacytoma DNA. The other five clones were found to be endogenous to the BALB/c genome. Thus, IAPs represent a significant source of insertional mutations for any cell expressing the retrovirus-like particles. Furthermore, these results suggest that only one or a small number of endogenous IAP sequences are activated to give the homogeneous population of amplified genes.

Novel myc oncogene RNA from abortive immunoglobulin-gene recombination in mouse plasmacytomas.

We have found that the myc oncogene has been modified by abortive recombination with the alpha heavy-chain immunoglobulin constant-region (C alpha) gene in five different mouse plasmacytoma lines. Recombination occurred approximately 0.8-2.0 kb to the 5' side of two distinct coding regions, defined by sequence homology between the chicken cellular and plasmacytoma myc genes. The myc and C alpha genes were always in opposite transcriptional orientation, with the recombination site within the C alpha switch region sequences. DNA recombination was found to correlate with the production of a novel 2.1 kb species of myc RNA that was 0.4 kb shorter than the normal cellular transcript. No elevated levels of myc RNA were evident, suggesting that DNA rearrangements have altered the myc oncogene product. This oncogene activation corresponds to the chromosomal translocations found in nearly all plasmacytomas.

Differing populations of intracisternal A-particle genes in myeloma tumors and mouse subspecies.

Intracisternal A-particle genes form a family of endogenous retrovirus-like genetic elements that are transcribed in mouse plasmacytomas (myeloma tumors). Two types of A-particle genes that can be differentiated by a sequence of 0.5 kilobase found in one type but not the other have been identified. Quantitative Southern blot analysis was used to measure the populations of different A-particle genes in DNAs from BALB/c mice, the Japanese subspecies Mus musculus subsp. molossinus, and myeloma tumors. The majority of the genes (715 copies per haploid genome or 76%) were found to be nearly identical except for small changes in conserved restriction enzyme sites. The second type of A-particle gene was much less abundant with 90 copies representing approximately 10%. The A-particle RNA in MOPC104E and MOPC315 was found to be colinear with a small portion of this latter type, comprising only 2% of the endogenous intracisternal A-particle sequences. Myeloma tumor DNA was found to have a two- to fourfold increase in the number of these genes, suggesting that the intracellular viruses have been activated to produce a double-stranded complementary DNA which subsequently integrated into the tumor genome. Analysis of M. musculus subsp. molossinus DNA revealed similar but shifted populations of A-particle genes, when compared with BALB/c DNA, except for the absence of a prominent EcoRI-HindIII band at 3.9 kilobases. This latter band, representing approximately 15% of the A-particle genes in BALB/c DNA, was shown to be a deletion variant of the most abundant gene family.