Aya Leder

Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells

Butyric acid is an unusually potent inducer of erythroid differentiation in cultured erythroleukemic cells. It is effective at one hundredth the concentration required of dimethylsulfoxide, a most effective inducing agent. Studies using a variety of analogues and metabolites suggest that the structural features of butyric acid are rather stringently required for induction. This effect is considered in view of the fact that butyric acid is a naturally occurring fatty acid, is effective in relatively low concentrations, and is widely used to form derivatives of cAMP.

Consequences of widespread deregulation of the c-myc gene in transgenic mice: Multiple neoplasms and normal development

We have constructed a transgenic mouse strain in which a mammary tumor virus LTR/c-myc fusion gene is anomalously expressed in a wide variety of tissues. The deregulated c-myc transgene, now glucocorticoid inducible, contributes to an increased incidence of a variety of tumors, including those of testicular, breast, lymphocytic (B cell and T cell), and mast cell origin. The deregulated gene does not, however, otherwise disturb cell proliferation, nor does it interfere with normal development in these animals. Moreover, since not all tissues that express the transgene develop neoplasms, these results begin to define the transforming spectrum of the c-myc oncogene. They also extend to several organ systems the notion that elements in addition to an activated c-myc gene are required to induce malignancy in the living organism.

A comparison of two cloned mouse β-globin genes and their surrounding and intervening sequences

The BALC/c mouse has two nonallelic beta-globin genes that appear to reside on two different Eco R1 fragments of genomic DNA. We have already cloned one of these fragments and shown that the gene encoded within it is interrupted by at least one large intervening sequence of DNA. We have now cloned and characterized the second beta-globin gene-containing fragment. The coding sequence of its gene is also interrupted by an intervening sequence of DNA that occurs in about the same position, relative to the coding sequence, as does the first. Because some shared features of the structure of these two genes might be responsible for their coordinate expression and the elimination of their intervening sequences, we have compared their surrounding, coding and intervening sequences by restriction endonuclease analysis and by visualization of the heteroduplex structures formed between them. Of the 7000 bp of sequence compared in this way, we find only a few hundred base pairs of homology in addition to the coding sequence. These shared sequences flank the coding sequence and appear to include only those portions of the intervening sequence immediately adjacent to the interrupted structural gene.

Differentiation of erythroleukemic cells in the presence of inhibitors of DNA synthesis

Erythroid differentiation can be readily induced by butyric acid in cultured erythroleukemic cells in the presence of inhibitors of DNA synthesis and in the absence of cell division. This result appears to rule out more complex models for globin gene expression which require gene replication or cell division (or both).

Perinatal lethality (ple): a mutation caused by integration of a transgene into distal mouse chromosome 15

We have used cytogenetic and recombinational analysis to determine the position of a transgene integrated into the mouse genome. The transgene maps to band F on the physical map of mouse chromosome 15 by in situ analysis and is tightly linked genetically to a cluster of loci that include the mutations caracul (Ca) and microcytic anemia (mk). Genetic analysis of the offspring of noninbred animals carrying the transgene and marker loci demonstrates a significant deficiency of homozygous progeny at weaning. When inbred mice heterozygous for the transgene are mated, about one-quarter of their offspring are homozygous; none of these animals survives more than 1 day after birth. It appears likely that a recessive insertional mutation has occurred as a result of transgene integration into a locus required for postnatal viability. We call this mutation transgenic perinatal lethality (Tg.ple).

Genetic interaction between the unstable v-Ha-RAS transgene (Tg.AC) and the murine Werner syndrome gene: transgene instability and tumorigenesis

Tg.AC transgenic mice provide a sensitive assay for oncogenic agents and a convenient alternative to the two-stage initiation/promoter model of skin tumorigenesis. Although extensively used, this model has remained in part an enigma since mice that carry the Tg.AC transgene (consisting of v-Ha-Ras driven by an embryonic ζ-globin promoter) would not ordinarily be expected to develop skin and other adult tumors. Cloning and characterizing the inserted transgene has provided an insight into the Tg.AC phenotype. We find that the transgene is inserted into a Line-1 element in such a way as to create extended inverted repeats consisting of both transgene and Line-1 sequences. Such structures would be expected to contribute to the instability of the Tg.AC locus and we suggest that this instability is critical to the Tg.AC phenotype. Further, we strengthen this notion by introducing an inactivating mutation in the murine Wrn gene (a gene important in maintenance of genome stability) and showing that bigenic Tg.AC/WrnΔhel/Δhel mice experience an eightfold increase in inactivating germline mutations at the Tg.AC locus. Similarly, Tg.AC/WrnΔhel/Δhel mice that retain an intact and thus active Tg.AC locus experience a sharp increase in papillomas as compared to Tg.AC/Wrn+/+ mice. This work demonstrates a genetic interaction between the instability of the multicopy transgene and the Werner Syndrome gene. From this, we conclude that genetic instability remains a key element in this tumor promoter model.

Lymphohematopoietic and Other Malignant Neoplasms Occurring Spontaneously in Transgenic Mice Carrying and Expressing MTV/myc Fusion Genes

Transgenic mice carrying and expressing exogenously introduced cellular oncogenes offer the opportunity to study oncogenesis in the context of the living organism (Stewart et al., 1984; Adams et al., 1985). To this end, we have produced various strains of transgenic mice that carry a normal mouse c-myc gene in which increasingly larger portions of the myc promoter region have been replaced by a hormonally inducible mouse mammary tumor virus promoter (Stewart et al., 1984). Two of these transgenic strains were of considerable interest, since virtually all of the available female progeny, which were in their second and third pregnancies, spontaneously developed mammary adenocarcinomas of the breast (Stewart et al., 1984). It was also of interest that the MTV/myc fusion gene was expressed both in nonneoplastic and neoplastic mammary glands, and with the exception of the normal salivary gland, was not significantly expressed in any other tissue (Davis et al., 1986). In contrast, another transgenic strain, hereafter referred to as the “K” strain, expresses MTV/myc fusion gene mRNA. in a much wider range of tissues, with the subsequent development of a high spontaneous incidence of malignant neoplasms (Leder et al., 1986). Although the largest portion of malignant neoplasms are lymphoid cell neoplasms of B cell derivation, we also have observed smaller numbers of T and non-B, non-T lymphomas as well as mast cell sarcomas, testicular neoplasms of Sertoli cell type, and mammary adenocarcinomas. We have described our experience with this strain in detail elsewhere (Leder et al., 1986).

The organization and evolution of cloned globin genes.

The globin genes represent a complex set of sequences that are expressed in a coordinate fashion during the development of red blood cells. while this complex family of genes may consist of as many as ten to fourteen members [34], three of these genes have now been cloned and their entire nucleotide sequence determined. As was initially observed in the case of beta globin major gene, all are encoded in three distinct coding blocks separated by two intervening sequences of DNA. Their intervening sequences of DNA are preserved, with respect to location, but are widely divergent, with respect to size and sequence. The divided information in each gene is edited and spliced together at the level of its initial RNA transcript which is complementary to the entire gene sequence including its intervening sequences. Structural correlation analyses have allowed us to identify sites in all three genes that might be responsible for the initiation of transcription, RNA splicing, and poly A addition. The function of these sites has been tested by cloning these genes in an animal virus vector SV40. Such animal virus hybrids have been used to infect tissue culture cells and have directed the synthesis of both alpha and beta mouse globin in cells of monkey origin. These studies indicate that such signals operate across species barriers and further indicate that the animal virus vector system will be useful in elucidating their function.

THE ARRANGEMENT, REARRANGEMENT AND ORIGIN OF IMMUNOGLOBULIN GENES

Publisher Summary This chapter analyzes the arrangement, rearrangement, and origin of immunoglobulin genes. The immune system requires sufficient genetic information to encode approximately a million different antibody molecules, each exhibiting remarkable specificity with respect to the antigens that it binds. These antibody molecules comprised of pairs of heavy and light chains display an interesting structural feature wherein their 100 or so N-terminal amino acids differ from antibody chain to antibody chain while their C-terminal portions remain identical in primary amino acid sequence. In a study described in this chapter, the tetrasegmental structure of kappa light chain genes was examined. Immunoglobulin kappa variable region genes constitute a multiple gene family that has arisen through evolution and now consists of many small subfamilies of variable region genes that are closely related both in their structure and flanking sequences. The chapter analyzes the possible role of unequal-crossing in generating immunoglobulin gene diversity. It is illustrated in the chapter that the kappa light chain genes of the mouse are encoded as four separate genetic elements. The first two comprise a large set of split variable region genes encoding a major portion of antibody diversity.

Genome-wide SNP analysis of Tg.AC transgenic mice reveals an oncogenic collaboration between v-Ha-ras and Ink4a, which is absent in p53 deficiency

Oncogenesis is a progressive process often involving collaboration between various oncogenes and tumor suppressors. To identify those genes that collaborate with oncogenic ras, we took advantage of the Tg.AC transgenic mouse, a line that harbors the v-Ha-ras transgene and spontaneously develops an array of malignant tumors. By crossing Tg.AC mice on an inbred FVB background to other inbred strains, F1 mice were created that could be analysed using genome wide, single nucleotide polymorphism (SNP) screens. Loss of heterozygosity (LOH) in tumors and tumor cell lines marked a somatic event, possibly the inactivation of tumor suppressor gene(s). LOH could also represent DNA damage, a sign of genomic instability in the pretransformed cell. Nonetheless, the screens showed no evidence of such generalized genomic instability. Instead, they revealed a single region of LOH on chromosome 4 that occurred via somatic recombination/gene conversion, generating a region of isoparental disomy. This LOH provided a clue that linked v-Ha-ras to the inactivation of the Ink4a locus in 25 of 32 tumor cell lines. This collaboration is seen regardless of tumor type or genetic background. In contrast, tumors that develop in bitransgenic mice bearing both the v-Ha-ras gene and a heterozygous mutant p53 allele tend to retain the Ink4a locus and instead lose the p53 wild-type allele. This suggests that different strategies can be selected to collaborate with v-Ha-ras in tumorigenesis.