The t(7;12)(q36;p13) is a recurrent abnormality in acute myeloid leukemia (AML) of childhood. The involved gene on chromosome 12 is TEL; the 7q36 partner gene has not been identified. We describe morphologic, molecular and cytogenetic characterization of two cases of 7q36/12p13-associated AML that provide important insights regarding the consequences of this rearrangement. First, the molecular organization of the breakpoint regions differ significantly: one case is a reciprocal 7;12 translocation (RTR); the other has an insertion of 7q into 12p (INS). While 12p13 breakpoints in both patients interrupt TEL intron 1, the centromere to telomere orientation of the 7q36 sequences relative to the TEL sequences are inverted in INS compared to RTR. This difference makes it difficult to postulate a mechanism whereby both patients could produce a common fusion transcript. Further, no evidence was obtained for any TEL-containing fusion transcripts. Finally, we report the first cloning of a 7;12 genomic breakpoint (from RTR) and find that it maps to a site 30 kbp proximal to the HLXB9 gene in 7q36. Together, these data suggest that, unlike most leukemia-associated chromosomal rearrangements, the important consequence of the t(7;12) is likely not the generation of a novel fusion transcript, but instead the inactivation of TEL and/or a gene at 7q36.

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rousassociated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.The endogenous viruses of chickens, which are inherited in chromosomal DNA, are closely related to the exogenous avian leukosis proviruses (for review, see 26). At least 13 distinct endogenous proviruses have been identified by DNA restriction endonuclease mapping (3, 18). Although closely related to one another, proviruses at different endogenous virus (ev) loci differ in DNA content and transcriptional activity. Thus, they may provide a set of genes for the study of elements, both viral and host, that contribute to the control of gene expression.The nondefective endogenous proviruses have a structure similar to integrated proviral DNA found after exogenous infection. That is, they retain the gene order 5'-gag-pol-env-3' of genome RNA and are flanked by two long terminal repeat (LTR) sequences, consisting of sequences derived from the 5' (U5) and 3' (U3) ends of viral RNA, as well as a sequence (R) which is itself repeated at the termini of genome RNA. Thus the overall structure is U3-R-U5-gag-pol-env-U3-R-U5 (17, 28; for review, see 6). The U3 region contains sequences important for regulation of growth rate (39) and appears to include the promoter for viral RNA synthesis (10,21,50).The ev-1 provirus is found in more than 99% of white leghorn chickens (38) and does...

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Avian retroviruses of subgroups B and D efficiently infect chicken (C/E) but not turkey (T/BD) cells. We describe here three variants of subgroup B and D viruses that infect both cell types equally well. One of these viruses, NTRE-4, was a recombinant between transformation-defective Prague (Pr) strain Rous sarcoma virus (RSV) subgroup B and the endogenous virus RAV-O; the second, SR-DE-1, was a recombinant between Schmidt-Ruppin RSV subgroup D and defective endogenous virus information. Ti oligonucleotide fingerprint analysis of the genomes of these two viruses showed only a small alteration in the portion of the env gene responsible for subgroup specificity, as indicated by the presence of a single subgroup E oligonucleotide in an otherwise purely subgroup B or D gene. The third virus, hrBOlPr-B, was a variant of Pr-RSV-B that did not appear to be a recombinant and whose altered host range we attribute to mutation. Analysis of the host range of all three viruses by infection of selectively resistant cells and by interference testing indicates that all use the subgroup B receptor on chicken ce-ls and the subgroup E receptor on turkey cells. These viruses may be analogous to the polytropic recombinant viruses recently found to be associated with leukemia in some strains of mice. Avian retroviruses can be classified into subgroups based on ability to infect various genetically defined chicken cells, resistance of infected cells to superinfection with viruses of the same subgroup, and neutralization of infectivity with specific antibodies (1). These properties are functions of the virion envelope glycoprotein, encoded by the env gene of the virus (1). In chickens, sensitivity to virus infection is controlled by at least three dominant autosomal loci: tv-a, tv-b, and tv-c (2). The tv-a and tv-c loci probably code for receptors that recognize glycoproteins of subgroups A and C, respectively. The tv-b locus, which includes four known alleles (3) showed that their novel host range was due to an alteration in a small region of the env gene. These viruses may be analogous to the "dual" host range viruses of mice (4, 5). MATERIALS AND METHODSChicken embryo fibroblasts (C/E, chf-gs-V-) were prepared from fertilized eggs purchased from SPAFAS (Norwich, CT). Cooper and recloned in this laboratory. RAV-1, RAV-2, and RAV-60 (derived from Pr-RSV-B) were gifts of H. Hanafusa. Pr-RSV-E was the recombinant between Pr-RSV-B and RAV-0, clone TRE-9, as described (7).Transforming viruses were titrated and cloned by infecting cells with serial dilutions of virus after treatment of the cells with DEAE-dextran (8). This pretreatment increased the titers approximately 100-fold but did not affect the relative infectivity on different cell types (data not shown). Cells were overlaid with agarose-containing medium immediately after infection.Three to 5 days later, foci were counted and virus was harvested from discrete foci of transformed cells from cultures that had no more than 20 foci per 60-mm culture dish.Nontransforming viruse...

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15dRBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.Avian and murine retroviruses can be expressed at different levels after integration into the host cell genome (2,14,21,26,29). In some cases, this variation is not due to genetic differences between the proviruses but instead is dependent on their site of integration (26,29). Analyses of two mouse mammary tumor virus proviruses acquired by exogenous infection in cultured cells indicated that the differential activity of these proviruses is dependent on their location in nuclease-sensitive or nuclease-resistant regions of the host cell genorne (14). An analogous relationship is not evident, however, for murine viruses introduced into the mouse germ line (Mov proviruses;27,28). In the latter case, inherited proviruses are inactive, even when located in transcriptionally active regions of chromatin (4,20). In contrast to Mov proviruses, some of the naturally occurring avian endogenous viruses (evs) are active in their inherited chromosomal location (2, 21). We were therefore interested in the relationship between provirus expression and the structure and activity of virus preintegration sites in this system.The avian endogenous viruses are highly related and stable genetic elements that were introduced into the germ line of chickens during evolution (1,7,24,25,33). Of the three viruses analyzed in this report, ev-1 and ev-2 are normally inactive in their inherited locations (producing less than 1 copy of stable RNA per cell; 2, 21), while ev-3 is active (producing 50 to 100 copies of stable mRNAs per cell; 2, 21...