Comparative analysis of the genetic structure and chromosomal location of the murine MyD118 (Gadd45beta) gene.

The MyD118 (Gadd45beta) protein is a member of a family of structurally related proteins, including Gadd45 (Gadd45alpha) and CR6 (Gadd45gamma), that have critical roles in regulating growth arrest and apoptosis. The MyD118 and other members of its family display distinct patterns of expression in response to stimuli that induce differentiation, growth arrest, or apoptosis. Species-blot analysis showed that MyD118 is an evolutionarily conserved gene, and comparative sequence analysis showed that MyD118 has a gene structure similar to that of other members of its gene family. Comparison of putative transcription factor-binding sites found in sequences of this gene family provides evidence that p53 is involved in regulating the expression of MyD118 and that NF-kappaB may play a role in differential expression of MyD118 and Gadd45(Gadd45alpha). Fluorescence in situ hybridization localized the MyD118 gene to mouse chromosome band 10B5.3, correcting a previous assignment to mouse chromosome 9.

Expression of myc-family, myc-interacting, and myc-target genes during preimplantation mouse development.

Previous studies indicated that members of the myc gene family may be essential for preimplantation development. Other studies revealed that preimplantation embryos lacking c-myc, N-myc, or L-myc are viable, indicating that these genes are either not essential for preimplantation development or can be substituted for functionally by other myc gene family members. To investigate the possible role of these genes during preimplantation development, we determined the temporal patterns of expression of four members of the myc gene family, genes encoding myc-associated proteins, and four putative MYC target genes. We observed a sequential pattern of myc gene expression, with the L-myc mRNA expressed as a maternal transcript, the c-myc mRNA expressed during the 4-cell through morula stages, and the B-myc mRNA expressed highly at the morula and blastocysts stages. B-myc was the predominant family member expressed during preimplantation development. The mxi mRNA was not detectable and the mad mRNA was detectable only as a maternal transcript. The max mRNA, however, was expressed both as a maternal mRNA and as an embryonic message throughout most of preimplantation development. Three putative MYC target genes (Odc, cyclin E, and prothymosin-alpha) were transcriptionally induced during the 2-cell stage, and their mRNAs increased sharply in abundance during development to the morula and blastocyst stages. Another putative MYC target gene, cyclin A, was expressed both as a maternal mRNA and as an embryonic transcript. These data support the view that the expression of myc target genes may be supported initially through the expression of maternally inherited MYC proteins and corresponding mRNAs and that subsequent stage-specific changes in expression of myc genes, myc-associated genes, and myc target genes may control early differentiative events around the time of implantation.

Arrest of spermatogenesis and defective breast development in mice lacking A-myb.

The Myb gene family currently consists of three members, named A-, B- and c-myb. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb-/- males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.

Expression and activity of L-Myc in normal mouse development.

To determine the role of L-Myc in normal mammalian development and its functional relationship to other members of the Myc family, we determined the normal patterns of L-myc gene expression in the developing mouse by RNA in situ hybridization and assessed the phenotypic impact of L-Myc deficiency produced through standard gene targeting methodology. L-myc transcripts were detected in the developing kidney and lung as well as in both the proliferative and the differentiative zones of the brain and neural tube. Despite significant expression of L-myc in developing mouse tissue, homozygous null L-myc mice were found to be viable, reproductively competent, and represented in expected frequencies from heterozygous matings. A detailed histological survey of embryonic and adult tissues, characterization of an embryonic neuronal marker, and measurement of cellular proliferation in situ did not reveal any congenital abnormalities. The lack of an apparent phenotype associated with L-Myc deficiency indicates that L-Myc is dispensable for gross morphological development and argues against a unique role for L-Myc in early central nervous system development as had been previously suggested. Although overlapping expression patterns among myc family members raise the possibility of complementation of L-Myc deficiency by other Myc oncoproteins, compensatory changes in the levels of c- and/or N-myc transcripts were not detected in homozygous null L-myc mice.

The mouse immunoglobulin kappa light-chain genes are located in early- and late-replicating regions of chromosome 6.

The murine immunoglobulin kappa (kappa) light-chain multigene family includes the constant region (C kappa), joining-region genes, and approximately 30 kappa-variable (V kappa) region families. The entire region occupies an estimated 1,000 to 3,000 kilobases, and some V kappa families have been linked by recombinant inbred mapping. The C kappa gene and 14 V kappa families replicated differently among cell lines of lymphoid and nonlymphoid origin. In nonlymphoid cells, the C kappa gene replicated earlier than the V kappa families. A transition from replication during the second third of S phase for the C kappa gene to later replication during S for V kappa families was observed. The V kappa family (V kappa 21) that maps closest to the C kappa gene, replicated during the first half of the S phase; most of the other V kappa families replicated during the second half of S, and some replicated during the last quarter of the S phase. In lymphoid cells, the kappa locus replicated earlier in the pre-B than in the B-cell lines. In one pre-B-cell line, 22D6, the kappa genes examined replicated at the beginning of the S phase. In the B-cell lines, the EcoRI segment containing the transcribed gene replicated near the beginning of the S phase. Other V kappa families replicated within the first two-thirds of S phase. Some linked V kappa families replicated at similar times. In the B-cell lines, a transition from replication at the beginning of S for the transcribed C kappa and V kappa genes and surrounding DNA sequences to later replication for the other V kappa families was observed. However, in contrast to the non-lymphoid cell lines, the replication of this locus occurred predominantly during the first half of S. The kappa locus contains both early- and late-replicating genes, and early replication is usually associated with transcriptional activity. The results are discussed with respect to the organization of transcriptionally active chromatin domains.

Replication program of active and inactive multigene families in mammalian cells.

In a comprehensive study, the temporal replication of tissue-specific genes and flanking sequences was compared in nine cell lines exhibiting different tissue-specific functions. Some of the rules we have determined for the replication of these tissue specific genes include the following. (i) Actively transcribed genes usually replicate during the first quarter of the S phase. (ii) Some immunoglobulin genes replicate during the first half of S phase even when no transcriptional activity is detected but appear to replicate even earlier in cell lines where they are transcribed. (iii) Nontranscribed genes can replicate during any interval of S phase. (iv) Multigene families arranged in clusters of 250 kilobases or less define a temporal compartment comprising approximately one-quarter of S phase. While these rules, and others that are discussed, apply to the tissue-specific genes studied here, all tissue-specific genes may not follow this pattern. In addition, housekeeping genes did not follow some of these rules. These results provide the first molecular evidence that the coordinate timing of replication of contiguous sequences within a multigene family is a general property of the mammalian genome. The relationship between replication very early during S phase and the transcriptional activity within a chromosomal domain is discussed.

The human myc gene family: structure and activity of L-myc and an L-myc pseudogene.

We have determined the nucleotide sequence and transforming activity of the human L-myc gene and a processed L-myc pseudogene (L-myc psi). We demonstrate by cotransformation assays that a 10.6-kb EcoRI fragment derived from a human placental library contains a complete and functional L-myc gene including transcriptional regulatory sequences sufficient for expression in rat embryo fibroblasts. Organization of the L-myc gene was determined by comparing its sequence to those of the L-myc psi gene and an L-myc cDNA clone derived from a human small cell lung carcinoma. Our results show that L-myc has a three-exon organization similar to that of the c-myc and N-myc genes. The putative L-myc gene product consists of 364 amino acids and contains five of the seven homology regions highly conserved between c-myc and N-myc. These conserved regions are located along the entire length of the putative L-myc protein and are interspersed among nonconserved regions. While the putative L-myc gene product is of a smaller size when compared to the c- and N-myc proteins, the relative positions of certain conserved residues occur in corresponding locations along the peptide backbone of the three proteins. In addition, comparison of the human and murine L-myc gene sequences indicate that the relatively large 5' and 3' untranslated regions are evolutionarily conserved, but that these sequences are totally divergent between the L-, c-, and N-myc genes. Finally, we demonstrate that, like the N- and c-myc genes, the L-myc gene can cooperate with a mutant Ha-ras gene to cause malignant transformation of rat embryo fibroblasts in culture. Our analyses clearly prove that L-myc represents a functional member of the myc oncogene family and further delineate structural features that may be important for the common and divergent functions of the members of this gene family.

Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

c-myc mRNA levels in the cell cycle change in mouse erythroleukemia cells following inducer treatment.

Several lines of evidence suggest that the c-myc protooncogene is involved in some aspect of cell division in mammalian cells. We have been investigating changes in the expression of c-myc mRNA in mouse erythroleukemia cells during chemically induced terminal erythroid differentiation. In vitro induction of erythroleukemia cell differentiation results in a switch from cells with unlimited proliferative capacity to cells that undergo a small number of terminal cell divisions. The level of c-myc mRNA changes rapidly following treatment with inducing agents. After a very rapid decline the mRNA is restored to pretreatment levels and then declines again. We have now measured the level of c-myc mRNA with respect to position in the cell cycle. Prior to inducer treatment the level of c-myc mRNA is relatively constant throughout the cell cycle. However, when the mRNA is restored following treatment with hypoxanthine or hexamethylenebisacetamide, it is found primarily in cells in the G1 phase. Thus, treatment with inducers of differentiation leads to a change in the cell cycle regulation of c-myc mRNA. This change may be involved in the altered proliferative capacity of the cells that occurs during terminal differentiation.