K. Zimmerman

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus‐transformed pre‐B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site‐specific join between the open reading frame of a LINE‐1 repetitive element and a remaining JH segment. We discuss this event in the context of the ‘accessibility’ model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre‐B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination‐deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre‐B cell lines and early in normal development. We propose that the predisposition of pre‐B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased ‘accessibility’ of the germline gamma 2b locus to switch‐recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.

IgH enhancer-mediated deregulation of N-myc gene expression in transgenic mice: generation of lymphoid neoplasias that lack c-myc expression

We have generated transgenic mouse lines that carry one of three different constructs in which the murine N‐myc gene is expressed under the control of the immunoglobulin heavy chain transcriptional enhancer element (E mu‐N‐myc genes). High‐level expression of the E mu‐N‐myc transgenes occurred in lymphoid tissues; correspondingly, many of these E mu‐N‐myc lines reproducibly developed pre‐B‐ and B‐lymphoid malignancies. The E mu‐N‐myc transgene also appeared to participate in the generation of a T cell malignancy that developed in one E mu‐N‐myc mouse. These tumors and cell lines adapted from them expressed exceptionally high levels of the E mu‐N‐myc transgene; the levels were comparable to those observed in human neuroblastomas with highly amplified N‐myc genes. In contrast, all of the E mu‐N‐myc cell lines had exceptionally low or undetectable levels of the c‐myc RNA sequences, consistent with the possibility that high‐level N‐myc expression can participate in the negative ‘cross‐regulation’ of c‐myc gene expression. Our findings demonstrate that deregulated expression of the N‐myc gene has potent oncogenic potential within the B‐lymphoid lineage despite the fact that the N‐myc gene has never been implicated in naturally occurring B‐lymphoid malignancies. Our results also are discussed in the context of differential myc gene activity in normal and transformed cells.

Structure and expression of the murine L-myc gene.

We have isolated a 12 kb clone from the murine genome which we show by DNA transfection studies to contain an entire functional L‐myc gene and the transcriptional promoter sequences necessary for its expression. We have also isolated a 3.1 kb cDNA sequence from a murine brain cDNA library which corresponds to most of the L‐myc mRNA. We have identified the L‐myc coding region within the genomic clone by a combination of S1 nuclease analyses. Northern blotting analyses and comparative nucleotide sequence analyses with the cDNA clone. The L‐myc gene appears to be organized similarly to the other well‐characterized myc‐family genes, c‐myc and N‐myc. The predicted amino acid coding sequence of the L‐myc gene indicates that the L‐myc protein is significantly smaller than c‐ and N‐myc, but is highly related. In particular, comparison of the N‐ and c‐myc protein sequences reveals seven relatively conserved regions interspersed among non‐conserved regions; the L‐myc gene retains five of these conserved regions but lacks two others. In addition, a portion of one highly conserved region is encoded within a different region of the L‐myc gene but, due to changes in the size of L‐myc exons relative to those of N‐ and c‐myc, maintains its overall position in the peptide backbone with respect to other conserved regions. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc‐family proteins.

Differential regulation of the N-myc gene in transfected cells and transgenic mice.

The N-myc gene is expressed specifically in the early developmental stages of numerous cell lineages. To assay for sequences that could potentially regulate N-myc expression, we transfected constructs that contained murine N-myc genomic sequences linked to a reporter gene and genomic clones that contained the complete human or murine N-myc genes into cell lines that either express or do not express the endogenous N-myc gene. Following either transient or stable transfection, the introduced N-myc sequences were expressed regardless of the expression status of the endogenous gene. In contrast, when the clones containing the complete human N-myc gene were introduced into the germline of transgenic mice, expression in some transgenic lines paralleled the tissue- and stage-specific expression of the endogenous murine gene. These findings demonstrate differences in the regulation of N-myc genes in recipient cells following in vitro versus in vivo introduction, suggesting that early developmental events may play a role in the regulation of N-myc expression.

IgH enhancer deregulated expression of L-myc: abnormal T lymphocyte development and T cell lymphomagenesis.

Transgenic constructs containing the murine L‐myc gene under the control of the immunoglobulin transcriptional enhancer element (Emu) are expressed at unexpectedly high levels in thymocytes and proliferating T cells compared with cells from bone marrow and proliferating B cells. In contrast, double transgenic animals bearing constructs containing the L‐ and N‐myc genes similarly linked to the Emu element maintain preferential L‐myc expression in T cells but express the N‐myc transgene preferentially in B cells. These results indicate that the L‐myc gene contains elements that act in concert with the Emu element to allow preferential expression in T lineage cells. In correspondence to the expression pattern, Emu‐L‐myc transgenic mice show expanded thymic cortices and irregularly formed splenic follicles with expanded T cell areas. Moreover, the percentage of thymocytes positive for the surface marker 1C11, which defines thymic progenitor cells, activated T cells and preleukemic T cells, is dramatically raised in transgenic mice compared with normal littermates. Emu‐L‐myc transgenic animals are predisposed to clonal lymphoid tumors, most of which are T cell lymphomas. The relative incidence, latency period, and degree of malignancy of Emu‐L‐myc tumors compared with Emu‐N‐ or c‐myc tumors is consistent with a lower oncogenic potential of the L‐myc gene. However, the Emu‐L‐myc tumors do not express detectable levels of endogenous myc family genes indicating that the L‐myc protein can substitute for c‐ or N‐myc in the generation and growth of lymphoid neoplasms.

Differential Expression of myc -family Genes During Development: Normal and Deregulated N- myc Expression in Transgenic Mice

The myc-family of cellular oncogenes is a dispersed multi-gene family that includes the c-, N- and L-myc genes (For review see Alt et al., 1986). The human and murine c-, N, and L-myc genes encode related but distinct nuclear proteins and have a similar overall organization (Kohl et al., 1986; DePinho et al., 1986, Stanton et al., 1986; DePinho et al., 1987; Legouy et al., 1987; Kay et al., 1988); all have three exons with the first encoding a potentially untranslated leader sequence. All three genes also cooperate similarly with an activated Ha-Ras oncogene to transform primary rat embryo fibroblasts (REFs) (Yancopoulos et al., 1985; DePinho et al., 1987). Despite striking similarities of regions of the myc proteins and the in vitro transforming activities, the three genes are conserved as distinct sequences throughout vertebrate species suggesting unique functional roles. This possibility is supported by findings that the genes are differentially expressed in a stage-and tissue-specific manner during human and murine differentiation (Zimmerman et al., 1986). In addition, deregulated c-myc expression has been causally implicated in the genesis of a wide variety of different tumor types and occurs by a variety of different mechanisms; deregulation of the N- and L-myc genes has been clearly implicated only in a few naturally occurring tumors (eg. human neuroblastomas and small cell lung carcinomas) and only by the mechanism of gene amplification (reviewed by Alt et al., 1986).

Gene Expression in Renal Growth and Regrowth

To elucidate the molecular events associated with postnatal and compensatory renal growth, the expression of growth/differentiation genes (c-fos, c-myc, c-H-ras, c-K-ras), a stress-related gene (HSP70) and a structural gene (collagen type IV, alpha 1 and 2) were examined. Northern analysis of messenger ribonucleic acid from the newborn mouse reveals high levels of expression of HSP70, c-H-ras, and c-K-ras during the first week of life. By day 40 HSP70-related and c-H-ras expression decreases somewhat, c-K-ras remains unchanged and collagen type IV, which encodes for the renal glomerular basement membrane, expression decreases significantly. During compensatory renal growth increased expression of HSP70, c-H-ras and c-K-ras occurs. The results seem to indicate that growth/differentiation genes may be necessary for continued cell growth (hypertrophy) in postnatal and compensatory renal growth, and that collagen type IV formation continues up through week 2 of postnatal growth consistent with the interval of glomerular basement membrane formation.