Structural analysis of both products of a reciprocal translocation between c-myc and immunoglobulin loci in Burkitt lymphoma.

The balanced translocations that occur between the c-myc and immunoglobulin loci in Burkitt lymphoma provide an unusual opportunity to analyze both products of a reciprocal recombination. Accordingly, we have determined the structure of the two reciprocal products of a translocation that joins the 5' portion of the c-myc gene on chromosome 8 to the immunoglobulin mu switch recombination signal on chromosome 14. By determining the nucleotide sequences at the translocation crossover points of both product chromosomes, we precisely locate these points with respect to nearby genes. This determination allows us to conclude that translocation involves nonhomologous recombination, is highly conservative of c-myc sequences (deleting only 16 bp at the crossover point), but deletes over 2 Kb of immunoglobulin sequences from the mu switch signal. The mu constant and c-myc genes are joined head-to-head about 3 Kb apart, while the IgH enhancer and an aberrantly rearranged D/J region are linked to sequences 5' of c-myc on the reciprocal product.

Activation and somatic mutation of the translocated c-myc gene in burkitt lymphoma cells.

In contrast to other human tumors in which the c-myc gene and its transcript are greatly amplified, careful analysis of t(8;14) Burkitt cell lines indicates that the c-myc transcript is marginally, and in some cases not at all, increased by comparison to control lymphoblastoid cell lines. Instead, there is a more subtle alteration in the expression of the translocated c-myc gene characterized by a shift in promoter utilization and an apparent insensitivity to the regulation that inactivates the normal c-myc allele within these same cells. In some Burkitt cell lines, such deregulation might be because of the loss of a putative control region through removal of the large dual promoter/leader segment of the c-myc gene. In other cell lines, however, this deregulation may be explained by somatic mutations that occur within the putative control region even though it is located many hundreds of bases from the translocation breakpoint.

The effect of translocations on the cellular myc gene in Burkitt lymphomas.

Chromosomal translocations are found to be a characteristic feature of Burkitt lymphomas. Similar translocations are found in mouse plasmacytomas and both diseases involve interchanges between one of the immunoglobulin loci and DNA in the vicinity of the myc gene. The structure of the myc gene has been elucidated from studies on translocated versions of the gene. Activation of the myc gene may play a role in transformation by promoting growth of the cells bearing the rearranged chromosomes.

Translocations among antibody genes in human cancer.

The characteristic chromosomal translocations that occur in certain human malignancies offer opportunities to understand how two gene systems can affect one another when they are accidentally juxtaposed. In the case of Burkitt lymphoma, such a translocation joins the cellular oncogene, c-myc, to a region encoding one of the immunoglobulin genes. In at least one example, the coding sequence of the rearranged c-myc gene is identical to that of the normal gene, implying that the gene must be quantitatively, rather than qualitatively, altered in its expression if it is to play a role in transformation. One might expect to find the rearranged c-myc gene in a configuration that would allow it to take advantage of one of the known immunoglobulin promoters or enhancer elements. However, the rearranged c-myc gene is often placed so that it can utilize neither of these structures. Since the level of c-myc messenger RNA is often elevated in Burkitt cells, the translocation may lead to a deregulation of the c-myc gene. Further, since the normal allele in a Burkitt cell is often transcriptionally silent in the presence of a rearranged allele, a model for c-myc regulation is suggested that involves a trans-acting negative control element that might use as its target a highly conserved portion of the c-myc gene encoding two discrete transcriptional promoters.

The human c-myc oncogene: structural consequences of translocation into the IgH locus in Burkitt lymphoma.

We have determined the sequence of the normal human c-myc gene and compared it to portions of a c-myc gene that has been translocated into the immunoglobulin heavy chain locus in a Burkitt lymphoma cell. The normal c-myc gene is encoded in three discrete exons divided by two large intervening sequences. Its mRNA is transcribed from two active promoters located about 150 nucleotides from one another. Each promoter initiates transcription of a long (approximately 550 bp) untranslatable leader sequence encoding the entire first exon. This exon and additional 5' flanking sequences are tightly conserved between mouse and man. In the Burkitt cell BL22, the rearranged c-myc gene retains both promoters and is unchanged in its amino acid coding domains. Translocation of this gene joins it to the immunoglobulin heavy chain switch region at a point approximately 1000 bp 5' to the dual c-myc promoters. These genes are joined in opposite transcriptional orientation. The structure of the translocated gene and the nature of its linkage to the immunoglobulin locus and the presence of two c-myc promoters and consequently two long leader sequences raise novel possibilities for the activation of an oncogene.

Cell surface marker proteins during mouse spermatogenesis: two-dimensional electrophoretic analysis.

Purified plasma membranes isolated from separated highly homogenous populations of mouse pachytene spermatocytes, round spermatids (step I-8), and residual bodies have been compared using 2-dimensional polyacrylamide gel electrophoresis. Two polypeptides apparently specific to pachytene spermatocytes have been identified. Component Pa has a molecular weight of 90 k daltons (K) and pI of 5.6. Component Pb has a molecular weight of 56.5 K and a pI of 6.0. Four polypeptides detected only in plasma membranes of round spermatids have been identified as follows: RSa, 90-95 K and pI 5.9; RSb, also 90-95 K and pI 5.9; RSc, approximately 88 K and pI 5.5; RSd, 58 K and pI 6.0-6.3. No polypeptides unique to residual body membranes were identified. Short-term culture experiments have established that separated adult mouse spermatogenic cells survive short-term culture in vitro. These cells actively synthesize numerous cellular proteins as determined by the incorporation of [3H]leucine. Investigations concerning the effect of the cell separation procedure on mouse spermatogenic cell membranes indicate that only 7 of 110-120 total plasma membrane constituents are degraded enzymically during cell purification. Only one of these constituents may correspond to the presumptive cell differentiation markers described for pachytene spermatocytes and round spermatids. These results indicate, therefore, that plasma membranes obtained immediately after cell separation are suitable for the detailed biochemical analysis of the most integral surface proteins during spermatogenesis in the mouse.

Isolation of plasma membranes from purified mouse spermatogenic cells.

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5'-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.