A frameshift mutation in the gamma E-crystallin gene of the Elo mouse.

The murine Elo (eye lens obsolescence) mutation confers a dominant phenotype characterized by malformation of the eye lens. The mutation maps to chromosome 1, in close proximity to the gamma E-crystallin gene which is the 3'-most member of the gamma-crystallin gene cluster. We have analysed the sequence of this gene from the Elo mouse and identified a single nucleotide deletion which destroys the fourth and last "Greek key" motif of the protein. This mutation is tightly associated with the phenotype, as no recombination was detected in 274 meioses. In addition, the mutant mRNA is present in the affected lens, providing further support for our hypothesis that the deletion is responsible for the dominant Elo phenotype.

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene.

Transgenic mice carrying the gamma 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of beta-galactosidase activity. These results suggest that gamma 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse gamma 2-crystallin gene. In a broader context, this study also demonstrates the utility of beta-galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.

Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis.

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.

Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.

The proximal promoter of the mouse arrestin gene directs gene expression in photoreceptor cells and contains an evolutionarily conserved retinal factor-binding site.

Regulatory sequences and nuclear factors governing tissue-restricted expression of the mouse arrestin gene were investigated. The results showed that while proximal promoter sequence positions -38 to +304 are sufficient to direct low levels of retina-specific gene expression, sequences extending upstream to position -209 support higher levels of expression in the retina, as well as detectable expression in the lens, pineal gland, and brain. Within the interval between positions -209 and -38, a broadly expressed nuclear factor, Bd, binds to sequences centered between positions -205 and -185, a region which contains two direct repeats of the hexamer, TGACCT. The proximal promoter binds three apparently retina-specific nuclear factors, Bp1, Bp2, and Bp3, through overlapping sequences centered between positions -25 and -15. Bp1 and Bp3 also recognize a closely related sequence found in the promoter regions of several other vertebrate photoreceptor-specific genes. Moreover, the consensus binding site for Bp1, designated PCE I, is identical to RCS I, an element known to play a critical role eliciting photoreceptor-specific gene expression in Drosophila melanogaster. The results suggest that PCE I and RCS I are functionally as well as structurally similar and that, despite marked differences in the fly and vertebrate visual systems, the transcriptional machinery involved in photoreceptor-specific gene expression has been strongly evolutionarily conserved.

Gamma-crystallins of the human eye lens: expression analysis of five members of the gene family.

While only two gamma-crystallins have been identified in the human eye lens, molecular studies indicate that the human gamma-crystallins are encoded in a multigene family comprising at least seven closely related members. Sequence analysis of five of these genes has suggested that three (gamma 1-2, G3, and G4) are potentially active, while two (G1 psi and G2 psi) correspond to closely related pseudogenes. Here we report on the detailed structure of a sixth gamma-crystallin gene, G5, and our results obtained with transient expression assays to characterize both the promoter activity and translation products of five members of the gene family. We show that 5'-flanking sequences of G1 psi and G2 psi lacked detectable promoter activity, while the corresponding sequences of G3, G4, and G5 were able to direct high levels of expression of the bacterial chloramphenicol acetyltransferase gene in primary lens epithelia, but not in cultures of nonlens origin. Detailed sequence comparisons indicated that active genes contained several conserved sequence tracts 5' of the TATA box which may constitute functional elements of a lens-specific gamma-crystallin promoter. Expression of the gamma-crystallin coding sequences from the human metallothionein IIA promoter in nonlens cells facilitated characterization of the polypeptides encoded by individual gamma-genes and, in future studies, should permit comparison of these proteins with distinct gamma-crystallins in the human lens.

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene.

We have previously generated microphthalmic mice lacking lens fiber cells by targeting the expression of the diphtheria toxin A (DT-A) gene in transgenic mice with regulatory sequences associated with the mouse gamma 2-crystallin gene. Because of the extreme toxicity of DT to animal cells and the potential leakiness of many tissue-specific regulatory regions, we investigated whether there might be an experimental advantage in using a mutant, attenuated form of the DT-A gene (tox-176) fused to gamma 2-crystallin regulatory sequences to ablate fiber cells in the ocular lens. In contrast to the microphthalmia observed in transgenic animals carrying the native DT-A gene, independent lines of mice transgenic for the gamma 2tox176 construct displayed predominantly cataracts or clinical anophthalmia. These contrasting phenotypes were transmitted within each pedigree, although for some lines some phenotypic heterogeneity among offspring was noted. The difference in phenotype between cataractous and clinically anophthalmic transgenic lines could not be ascribed to differences in the transgene copy number. Instead, the results suggest that transgene expression and hence the extent of genetic ablation are modulated by the site of chromosomal integration and, to a lesser extent, by epigenetic events. They also suggest that the attenuated gamma 2tox176 construct can integrate into chromosomal regions that are particularly favorable for expression without compromising embryological development and therefore that the tox-176 gene may be more versatile and effective than the wild-type DT-A gene for achieving genetic ablation with a broad range of cell- or tissue-specific regulatory sequences.

Interaction of a lens cell transcription factor with the proximal domain of the mouse gamma F-crystallin promoter.

The elements regulating lens-specific expression of the mouse gamma F-crystallin gene were examined. Here we show that mouse gamma F-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different gamma-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse gamma F-crystallin gene.

Lens-specific promoter activity of a mouse gamma-crystallin gene.

Crystallins are the major water-soluble proteins in vertebrate eye lenses. These lens-specific proteins are encoded by several gene families, and their expression is differentially regulated during lens cell differentiation. Here we show that a cloned mouse gamma-crystallin promoter is active in lens explants derived from 14-day-old chicken embryos but inactive in a variety of cells of non-lens origin. We also show that sequences required for proper utilization of this promoter are contained between nucleotide positions -392 and +47 relative to the transcription initiation site; deletion of sequences from positions -392 to -171 completely abolishes promoter activity. Since chickens do not have gamma-crystallin genes, the expression of a mouse gamma-crystallin promoter in chicken lens cells suggests that different classes of crystallin genes may be regulated by common lens tissue-specific mechanism(s) independent of species.

Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens.

Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.

Genetic evidence for progressive selection and overgrowth of primary tumors by metastatic cell subpopulations.

We have exploited random insertions of transfected DNA as unique clonotypic markers to follow cell lineages during primary and metastatic tumor growth of a mouse mammary adenocarcinoma, SP1. Southern analysis was undertaken of primary solid tumors and metastases obtained after injection of a pooled population of individual SP1 transfectants, or reconstituted mixtures of genetically marked metastatic and unmarked nonmetastatic cells. Here we provide evidence for the reproducible selection and eventual overgrowth of primary tumors by genotypically distinct metastatic clones, thereby illustrating that late-state, advanced primary tumors can evolve to become biologically similar, or even identical, to distant metastases. The selective growth advantage of metastatic cells within primary tumors was shown to occur despite the fact that tumors generated by both metastatic and nonmetastatic SP1 cell populations grew at comparable growth rates when injected and analyzed separately. The extent of the local growth advantage manifested by individual metastatic clones varied considerably, from 5- to 50-fold. Clonal overgrowth was also observed whether the tumor cells were injected ectopically, or orthotopically (i.e., into the mammary fat). This type of experimental approach should provide new insights into the dynamics of tumor progression and metastasis, the lineage relationship of primary tumors to metastases, the influence of clonal interactions on tumor behavior, and the physiological changes which are causative of malignant disease.

Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in v-jun transgenic murine cells.

The oncogene jun encodes a transcription factor of the AP-1 family. In mice carrying viral jun (v-jun) as a transgene, wounding is a prerequisite for tumorigenesis, suggesting collaboration between the transgene and a wound-related event. To define possible candidates for this collaborative process, we examined the effect of several wound-related polypeptide growth factors on cells from transgenic mice. Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in embryo fibroblasts and tumor cell revertants from these mice. This effect was specific for the two cytokines and was restricted to cells from v-jun transgenic mice. Anchorage independence required the continued presence of the cytokines. Transfection of transgenic cells with a v-jun expression plasmid also induced anchorage independence and a tumorigenic phenotype in transgenic tumor cell revertants. However, there was no correlation between anchorage independence, expression of Jun, and AP-1 activity. These results suggest that while increased transgene expression can enhance the growth properties of v-jun transgenic cells, there exist other cytokine-dependent mechanisms that have a similar effect. Retinoic acid, dexamethasone, or forskolin inhibits induction of anchorage independence by tumor necrosis factor alpha, interleukin 1 alpha, and transfected v-jun. Although these agents affect both AP-1 transactivation potential and DNA binding in the transgenic cells, the changes are not correlated with the inhibition of growth.

Metastatic potential of SP1 mouse mammary adenocarcinoma cells is differentially induced by activated and normal forms of c-H-ras.

The mouse mammary adenocarcinoma cell line SP1 was transfected with either the activated T24-H-ras or the normal c-H-ras gene and assessed for metastatic potential in syngeneic CBA/J or nude mice. Unlike the parental control cells, which were tumorigenic but unable to metastasize from a subcutaneous site, all SP1 transfectants expressing the T24-H-ras gene were able to metastasize (predominantly to the lung). In contrast, a much smaller fraction of the clones obtained following transfection with either normal c-H-ras or pSV2neo were metastatic and, significantly, elevated expression of the c-H-ras proto-oncogene did not correlate with acquisition of metastatic potential. We conclude that activated and normal forms of the c-H-ras gene differ in their ability to confer metastatic potential to SP1 mouse mammary adenocarcinoma cells.

Two distinct target cells for v-jun mediated wound tumorigenesis.

Transgenic mice expressing v-jun under the control of the H-2K promoter develop dermal fibrosarcomas and rhabdomyosarcomas via a multistep process following wounding. To assess the relative roles that wounding and the H-2K promoter play in this process, we compared the phenotype of H-2K-v-jun mice with that of animals expressing v-jun under the control of the metallothionein I (MTI) promoter. MT-v-jun animals also develop wound-induced neoplasms by a multistage process. Both early and late features of tumorigenesis in MT-v-jun mice are different, however, from what is observed in H-2K-v-jun animals. First, the acute hyperplastic response that is characteristic of H-2K-v-jun granulation tissue is not observed in MT-v-jun wounds. Second, the myogenic components that are readily detected in the majority of late stage H-2K neoplasms are never observed in their MT counterparts. Moreover, analysis of wound tumours arising in animals expressing both MT-v-jun and H-2K-v-jun reveals that the two transgenes are not expressed in identical malignant cell populations. These results imply that mesenchymal granulation tissue is heterogeneous in composition and that the different cellular phenotypes of MT-v-jun and H-2K-v-jun malignancies result from oncogenic activation of wound-derived cells which differ in their differentiation potential. Thus, whereas the wounding component of multistage tumorigenesis is attributable to the action of v-jun, the transcriptional regulatory elements which drive its expression determine the nature of the target cells which give rise to wound-induced neoplasms.

Oncogenes conferring metastatic potential induce increased branching of Asn-linked oligosaccharides in rat2 fibroblasts.

Increased -GlcNA beta-6 Man alpha 1-6 Man beta 1- branching of Asn-linked oligosaccharides has been observed in a number of rodent and human tumor cell lines and the structures have been correlated with enhanced metastatic potential of murine tumor cells. Here we have compared the malignant potential and levels of beta 1-6-branched oligosaccharides in rat 2 fibroblast transfected with the cytoplasmic tyrosine kinase v-fps/fes, the activated GTPase T24 H-ras, and the nuclear oncogene c-myc. Rat2 cells transfected with activated c-myc were non-tumorigenic in nude mice and did not show elevated levels of beta 1-6 branched oligosaccarides, whereas transfectants carrying H-ras or v-fps were tumorigenic and generally exhibited metastatic potential which was associated with increased beta 1-6 branching. Enhanced expression of beta 1-6 branched oligosaccharides did not correlate with increased ratios of UDP-HexNAc to UDP-Hex, but was accompanied by elevated GlcNAc-transferase V activity, increased sensitivity of the cells to the toxic effects of leukoagglutinin, and an altered intracellular distribution of beta 1-6-branched oligosaccharides as visualized by fluorescent lectin staining. These results provide information on the quantitative and qualitative relationships between oncogene expression and cellular glycosylation and suggest that the ability of an oncogene to confer metastatic potential may be related to its ability to induce increased branching of Asn-linked oligosaccharides.

Expression of the bZIP transcription factor gene Nrl in the developing nervous system.

Proteins of the Maf/Nrl subfamily of bZIP transcription factors are involved in the regulation of tissue-specific gene expression. The Nrl gene, initially identified from a subtracted retinal library, is expressed in all cell layers of the adult retina, including photoreceptors. The Nrl protein has high sequence homology with Maf proteins, binds to an AP-1 like sequence element, and in photoreceptors appears to be involved in regulating the expression of rhodopsin. In the present study, we investigated the expression of Nrl in the developing and adult mouse using in situ hybridization and RT-PCR. We demonstrate that beginning at embryonic day 12.5 Nrl is expressed throughout the developing central and peripheral nervous system, with the exception of the nasal epithelium. The spatial pattern of hybridization suggests that Nrl is transcribed in post-mitotic, differentiating neurons, the developing cephalic mesenchyme and lens. Nrl expression is downregulated postnatally in the brain, and becomes restricted to neocortex and brainstem in the adult. High levels of Nrl transcripts, however, persist in the mature photoreceptors and other retinal neurons. Our studies suggest a role for the Nrl protein in neuronal differentiation and in mature neurons of the adult retina.

Skeletal muscle arises as a late event during development of wound sarcomas in v-jun transgenic mice.

Mice carrying an H-2K-v-jun transgene develop malignant sarcomas by a multistage mechanism following wounding. Here we show that these malignancies are often heterogeneous in composition, containing both undifferentiated mesenchymal cells as well as focal areas of skeletal muscle. Such myogenic areas are not detectable in premalignant precursor lesions, suggesting that cells competent for muscle differentiation arise at a late stage of tumorigenesis. Immunohistochemical staining of transgenic sarcomas reveals that levels of v-Jun correlate inversely with muscle-specific gene expression, suggesting that high levels may be inhibitory to myogenesis. Consistent with this idea, we demonstrate that whereas high levels of v-Jun are able to block MyoD-dependent gene expression in vitro, the levels of v-Jun in sarcoma-derived myogenic cells are below the threshold required to produce this effect. The cell of origin of v-jun wound sarcomas, as well as the relationship between myogenic determination and multistage tumorigenesis, are discussed in the light of these results.

tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors.

A search for protein tyrosine kinases expressed during murine cardiogenesis resulted in the isolation of a novel tyrosine kinase, designated tek, which maps to mouse chromosome 4 between the brown and pmv-23 loci. The deduced amino acid sequence of tek predicts that it encodes a putative receptor tyrosine kinase that contains a 21 amino acid kinase insert and which is most closely related in its catalytic domains to FGFR1 and the product of the ret proto-oncogene. In situ hybridization analysis of adult tissues, as well as sectioned and whole-mount embryos, showed that tek is specifically expressed in the endocardium, the leptomeninges and the endothelial lining of the vasculature from the earliest stages of their development. Moreover, examination of the morphology of tek-expressing cells, and staging of tek expression relative to that of the endothelial cell marker von Willebrand factor, revealed that tek is expressed prior to von Willebrand factor and appears to mark the embryonic progenitors of mature endothelial cells. tek encodes a novel putative receptor tyrosine kinase that may be critically involved in the determination and/or maintenance of cells of the endothelial lineage.