Microinjection of the ras oncogene protein into nonestablished rat embryo fibroblasts.

Early (twice) and late (eighth) passage nonestablished rat embryo fibroblasts exhibit dramatically different responses to microinjection of the oncogenic form (T24) of the ras protein. Late passage cells respond like established cultures in that they both synthesize DNA and divide in response to the oncogenic protein. However, early passage cells are shown to be much less responsive to the introduction of this protein in terms of the threshold concentration required to elicit a mitogenic response. Furthermore, conditioned medium from late passage cells can both potentiate the effect of ras in early passage cells and stimulate colony formation in soft agar. In addition, we show that human cells can respond to microinjection of the ras oncogene protein by synthesizing DNA.

Elevation of c-myc protein by DNA strand breakage.

The intracellular level of the c-myc protein is elevated in a dose and time dependent manner by DNA strand breakage. Lesions introduced by either alkylating agents or gamma irradiation are capable of stimulating increased production of c-myc protein. In addition, inhibition of DNA strand break rejoining maintains the level of the c-myc protein in an elevated state, whilst inhibition of DNA replication does not cause an increase in c-myc protein. We conclude that chromatin perturbation via DNA strand breakage both increases the endogenous amounts of the c-myc protein and maintains its elevated level.

Reduced protein kinase C activity in a ras-resistant cell line derived from Ki-MSV transformed cells.

We have examined phosphatidylinositol turnover and C-kinase distribution in a flat cellular ras-resistant cell line (C11) derived from Kirsten murine sarcoma virus (Ki-MSV) transformed NIH/3T3 cells (DT). This cell type has been shown to express high levels of the p21 Ki-ras gene product yet is resistant to the transforming effects of this protein. Our data indicate that C11 cells have reduced levels of total C-kinase activity when compared to NIH/3T3 cells and do not retain the ability to phosphorylate the growth associated 80-kDa C-kinase substrate either in vivo or in vitro. Furthermore, whilst the steady state levels of diacylglycerol and the sum of inositol phosphates are elevated in DT cells, in C11 cells these levels are reduced to an amount equivalent to that seen in NIH/3T3 cells. These data indicate a correlation between a protein kinase C dependent pathway and resistance to transformation by ras.

Translational upregulation of the c-myc oncogene in Bloom's syndrome cell lines.

Previous studies have shown a constitutive increase in the levels of c-myc protein in cell lines derived from patients with the cancer-prone disorder Bloom's Syndrome (BS). We report here that this overexpression results from a specific increase in the translation of the c-myc mRNA and is not the result of either a chromosomal translocation involving the c-myc locus or an amplification of this gene. We also did not detect any increase in the stability of the c-myc protein or any significant increases in the levels of c-myc mRNA expressed in BS cells compared to control cell lines. Overall, there is a 39-80% increase in the association of the c-myc mRNA with polysomes in BS cell lines. Since, in some cases, overexpression of the c-myc protein has been shown to increase levels of the translation initiation factors eIF-4E and eIF-2 alpha, which may themselves play a role in malignant conversion, we have also examined the levels of these proteins in BS cells and found them to be either comparable or lower than those in control cell lines. These data suggest that if c-myc does contribute to the cancer predisposition phenotype in BS then it does not appear to act via an eIF-4E and eIF-2 alpha mediated pathway.

High levels of the c-myc protein in cell lines of Bloom's syndrome origin.

Lymphoblastoid cell lines derived from cancer prone Bloom's Syndrome patients differ from cell lines representative of several other disorders by exhibiting a constitutive elevation in the level of the c-myc protein. This may be a contributing factor in the strong predisposition to malignancy observed in Bloom's syndrome.

The v- and c-myc oncogene proteins colocalize in situ with small nuclear ribonucleoprotein particles.

The intranuclear distribution of the v-myc and c-myc oncogene proteins were studied by immunofluorescence and immunoelectron microscopy. The nuclear distribution pattern of these proteins is shown to be identical to the distribution of small nuclear ribonucleoprotein particles (snRNPs). Colocalization was observed in cells expressing either the v- or c-myc proteins or in cells microinjected with the recombinant human c-myc protein. Immunolocalization studies revealed the v-myc protein and snRNPs to be concentrated within a nuclear network which excludes the nucleolus, nuclear pore-lamina complex, and portions of the nucleoplasm which contain the bulk of DNA. These results identify a nuclear region enriched in the myc-oncogene protein and snRNPs and raise the possibility that these nuclear constituents may function in related processes.

Aberrant translational control of the c-myc gene in multiple myeloma.

We demonstrate a 10- to 25-fold increase in the amount of c-myc protein in several independent cell lines derived from patients with multiple myeloma (MM). This is not accompanied by a corresponding increase in the overall level of the c-myc mRNA. There is, however, a 3.4-fold increase in the amount of c-myc mRNA associated with the polysomes in these cell lines without any detectable change in either the polysome size or the rate of translation elongation, thus suggesting that there is an increase in the extent of mobilisation of c-myc mRNA to the polysomes in MM. Analysis of the 5' untranslated region of c-myc has revealed the presence of a mutation, in all of the MM cell lines examined, in a region which has been implicated previously in the translational control of this mRNA species. These data suggest aberrant translational control of the c-myc gene in cell lines derived from patients with MM, which may contribute towards pathogenesis of the disease.

[Activity of c-myc protein is necessary in the first 6 hours of the prereplicative period for 3T3 Swiss cells]. / Aktivnost' belka c-myc neobkhodima v pervye 6 ch prokhozhdeniia prereplikativnogo perioda kletkami 3T3 Swiss.

It was shown what microinjection of polyclonal antibodies to the myc protein specifically inhibits DNA synthesis in serum-stimulated 3T3 Swiss cells during the first 6 h of the prereplicative period. The effect depends on the concentration of antibodies. Microinjections of polyclonal antibodies against the whole protein were more effective when microinjections of antibodies against parts of the protein. Microinjections of five kinds of monoclonal antibodies and their mixture were in effective. It was also shown what induction of expression of the antisense myc sequence in 3T3 Swiss cells leads to potent inhibition of DNA synthesis during the first 6 h of the prereplicative period. Thus it is clear what the myc protein participates in the early stages of preparation to replication, i.e., transition of cells from G0 to G1.

Transcriptional organization within an Escherichia coli cell division gene cluster: direction of transcription of the cell separation gene envA.

A cluster of at least 14 genes, each concerned with some aspect of cell envelope growth, morphogenesis, or function, is located at 2 min on the genetic map of Escherichia coli. We located the envA cell division gene and its promoter within the cluster and determined the direction of transcription of the gene by constructing fusions between its promoter and the galK coding sequence. In addition, we identified the promoter of a possible new gene lying between envA and the secA gene. We also present evidence from gene fusion studies which shows the direction of transcription of the ftsZ(sulB) division gene. The direction of transcription is the same for all three promoters and is the same as that of all other cluster genes for which this is known. We discuss the significance of this observation, together with the fact that every gene examined in sufficient detail within the cluster appears to have its own promoter and to be able to be expressed from isolated cloned fragments. Using a novel variable-copy plasmid vector, we demonstrate that the DNA fragment containing the envA gene is not stably maintained in multiple copies. The construction of two independent, nonoverlapping deletions allows us to conclude that the envA product itself is responsible for this effect.

Overlapping functional units in a cell division gene cluster in Escherichia coli.

The ftsZ ( sulB ) coding sequence is preceded by two promoters, at least one of which lies within the coding sequence of the neighboring gene, ftsA . This region of the ftsA gene is required for full biological activity of ftsZ .

Cancer predisposition in Bloom's syndrome.

This article focuses upon defining those factors which may contribute to the pathogenesis of cancer. The molecular basis of tumour etiology is discussed with reference to cancer predisposing syndromes, and in particular to the human inherited disease, Bloom's syndrome. In Bloom's syndrome, patients are predisposed to a wide variety of malignant disease. We propose a model in which overexpression of the ubiquitous c-myc proto-oncogene contributes to this process.

DNA sequence and transcriptional organization of essential cell division genes ftsQ and ftsA of Escherichia coli: evidence for overlapping transcriptional units.

The DNA sequence of a cloned segment of the Escherichia coli chromosome containing ftsQ, ftsA, and part of the ftsZ gene was determined and interpreted for genetic complementation and promoter fusion data for the region. The contiguous genes ftsQ, ftsA, and ftsZ were transcribed in the same direction (clockwise on the genetic map) and each had at least one associated promoter which allowed it to be transcribed independently of neighboring genes. ftsA and ftsZ possessed promoters within the coding sequences of the juxtaposed upstream structural genes, and a promoter element for ftsA was surrounded by a region of twofold symmetry which corresponded closely to a symmetrical element in the region of a putative ftsZ promoter. The structural gene of ftsQ consisted of 838 nucleotides, encoding a 276-residue amino acid polypeptide of molecular weight 31,400; the structural gene of ftsA consisted of 1,260 nucleotides, encoding a 420-residue amino acid polypeptide of molecular weight 45,400. The observation that the termination codon of ftsQ overlaps with a potential initiation codon for ftsA suggested that these two genes may be translationally coupled when transcription is initiated upstream of the ftsQ coding sequence.

Initial culture behaviour of rat blastocysts on selected feeder cell lines.

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examined the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3-6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat.