Glycolysis and methylaminoisobutyrate uptake in rat-1 cells transfected with ras or myc oncogenes.

A high rate of aerobic glycolysis was catalyzed by rat-1 cells transfected with a ras oncogene (ras cells); rat-1 cells and rat-1 cells transfected with myc oncogene (myc cells) showed a low rate of glycolysis that was increased after exposure of the cells to type B transforming growth factor (TGF-beta). The uptake of radioactive methylaminoisobutyric acid or L-methionine via system A of amino acid transport also was accelerated after exposure of these cells to TGF-beta, with the myc cells being most sensitive and the ras cells least sensitive. Methionine was found to be a potent inhibitor of glycolysis in ras cells as well as in rat-1 or myc cells that were exposed to TGF-beta. We propose a relationship between the product of the ras oncogene (p21) and the protein(s) induced by exposure to TGF-beta.     

src- and fps-containing avian sarcoma viruses transform chicken erythroid cells.

We report here that several oncogene-transducing avian sarcoma virus strains, namely Rous sarcoma virus (src), Fujinami sarcoma virus (fps), and PRCII (fps), transform avian erythroid cells in vitro and in vivo. The src- and fps-transformed erythroblasts grow in vitro for 20-30 generations, require special growth conditions, and tend to differentiate spontaneously. In these properties, they resemble erythroid cells transformed with the erbB-containing H strain of avian erythroblastosis virus (AEV-H) but differ from those transformed with AEV-ES4 (erbA, erbB), which grow under standard culture conditions and rarely differentiate spontaneously. Erythroblasts transformed with viruses carrying temperature-sensitive mutations in the src or fps oncogene and then shifted to the nonpermissive temperature in the presence of anemic serum (as a source of an erythropoietin-like factor) differentiate terminally into erythrocytes. These results demonstrate that several members of the src gene family other than erbB have the capacity to transform erythroid cells.     

Heat shock proteins are methylated in avian and mammalian cells.

Exposure of chicken cells grown in tissue culture to heat shock or sodium arsenite results in a dramatic increase in the synthesis of three major polypeptides with molecular weights of 83,000 (HSP 83), 68,000 (HSP 68; referred to here as "thermin"), and 25,000 (HSP 25). Incubation of BHK-21 or HeLa cells under the same conditions results in induction of HSP 68 and a 66,000-dalton polypeptide (HSP 66). Chicken thermin is resolved by isoelectric focusing into a major acidic and a more-basic component; mammalian thermin is resolved only into one major acidic component. HSP 83 and the acidic form of thermin are highly conserved in all avian and mammalian cells examined as judged by their electrophoretic mobilities, isoelectric points, and one-dimensional peptide maps. In addition, the acidic form of thermin is indistinguishable from a protein that copurifies with brain microtubules and that remains associated with the intermediate filament-enriched Triton/KCl cytoskeletons of cells grown in tissue culture. Thermin is also a component of skeletal myofibrils. HSP 83 and thermin are methylated in cells cultured under normal growth conditions. Induction of heat shock proteins by incubation of cells in the presence of sodium arsenite results in a marked methylation of the newly synthesized thermin. Under the same experimental conditions, no significant increase in methylation of the HSP 83 is observed. HSP 25 is not methylated in untreated cells or in cells treated with sodium arsenite. These results suggest that methylation of heat shock proteins may have an important role in regulating their function.     

Activation changes the spectrum but not the diversity of genes expressed by T cells.

During activation T cells are thought to change their patterns of gene expression dramatically. To find out whether this is true for T cells activated in animals, the patterns of genes expressed in resting T cells and T cells 8 and 48 hr after activation were examined by using Affymetrix gene arrays. Gene arrays gave accurate comparisons of gene expression in the different cell types because the expression of genes known to vary during activation changed as expected. Of the approximately 6,300 genes assessed by the arrays, about one-third were expressed to appreciable extents in any of the T cells tested. Thus, resting T cells express a surprisingly large diversity of genes. The patterns of gene expression changed considerably within 8 hr of T cell activation but returned to a disposition more like that of resting T cells within 48 hr of exposure to antigen. Not unexpectedly, the activated T cells expressed genes associated with cell division at higher levels than resting T cells. The resting T cells expressed a number of cytokine receptor genes and some genes thought to suppress cell division, suggesting that the state of resting T cells is not a passive failure to respond to extant external stimuli.     

Effects of mammalian and avian gonadotropins on in vitro progesterone production by avian ovarian granulosa cells

Chicken ovarian granulosa cells were incubated in vitro following their preparation by collagenase treatment. The secretion of progesterone by the cell suspension was increased (between 2.3- and 3.8-fold) by the addition of either avian or mammalian luteinizing hormone (LH). The effectiveness of chicken gonadotropin fractions in stimulating progesterone release agrees well with their known LH contents. The addition of an antiserum against avian LH, to the incubation, blocked the effect of chicken LH on progesterone release. Mammalian FSH induced small (20 to 70%) increases in progesterone secretion.     

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.     

Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

DNA from six avian sarcoma virus (ASV)-transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from all of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 10(6) daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 10(6) daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 10(6) daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 10(6) daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 10(6)-dalton EcoRI fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines.     

Nucleotide sequence of two overlapping myc-related genes in avian carcinoma virus OK10 and their relation to the myc genes of other viruses and the cell.

Avian carcinoma virus OK10 has the genetic structure gag-delta pol-myc-delta env. It shares the transformation-specific myc sequence with three other avian carcinoma viruses (MC29, MH2, CMII) and also with a normal chicken gene proto-myc and the gag, pol, and env elements with non-transforming retroviruses. Unlike the other myc-containing viruses, which synthesize singular myc proteins, OK10 synthesizes two different myc-related proteins of 200 and 57 kDa. Here we have sequenced the myc region of an infectious OK10 provirus to investigate how OK10 synthesizes two different proteins from the same myc domain and to identify characteristic differences between the normal proto-myc gene and the myc-related viral transforming genes. It was found that the 1.6-kilobase myc domain of OK10 is colinear and coterminal with the myc domains of MC29, MH2, and the terminal two exons of proto-myc. It is preceded by the same splice acceptor as the myc sequence of MH2 and as the second proto-myc exon. From this and the known structure of retroviruses, it follows that the OK10 gene encoding the 57-kDa protein is discontinuous with a small 5' exon that includes six gag codons and a large 3' myc exon (delta gag-myc). This gene and the delta gag-myc gene of MH2 are isogenic. The proto-myc-derived intron preceding the myc domain of OK10 is in the same reading frame as the adjacent delta pol and myc domains and, hence, is part of the gag-delta pol-myc gene encoding the 200-kDa protein. Sequence comparisons with proto-myc and MC29 and MH2 indicate that there are no characteristic mutations that set apart the viral myc domains from proto-myc. It is concluded that transforming function of viral myc-related genes correlates with the lack of a viral equivalent of the first proto-myc exon(s) and conjugation of the viral myc domains with large or small retroviral genetic elements rather than with specific point mutations. Because OK10 and MH2 each contain two genes with potential transforming function (namely, delta gag-myc and gag-delta pol-myc or delta gag-mht, respectively), it remains to be determined whether the delta gag-myc genes have transforming function on their own or need helper genes. The possible helper requirement cannot be very specific because the two potential helper genes are very different.     

Distinctive effects of the viral oncogenes myc, erb, fps, and src on the differentiation program of quail myogenic cells.

The relationship between susceptibility to transformation in vitro by different oncogenes and terminal differentiation was analyzed in embryonic quail myogenic cells. Infection with Rous sarcoma virus (RSV), Fujinami sarcoma virus (FSV), avian erythroblastosis virus (AEV), and the avian myelocytomatosis virus MC29 led to rapid and massive transformation. Transformed cells had distinctive morphological alterations, increased proliferation rates, and the ability to grow in agar suspension. Furthermore, homogeneously transformed cultures failed to fuse into multinucleated myotubes and to express muscle-specific genes. However, cloned populations of RSV-, FSV-, and AEV-transformed myogenic cells could, under appropriate culture conditions, partially differentiate into atypical "revertant" myotubes. In contrast, competence for terminal differentiation was completely and irreversibly suppressed on transformation by MC29. The specificity of action of a given oncogenic sequence on the inhibition of differentiation was further studied by using conditional and nonconditional transformation mutants. Myogenic cells infected with temperature-sensitive (ts) mutants of RSV and FSV exhibited a fully reversible block of differentiation after shift to restrictive temperature, while cells infected with ts34 AEV were not temperature sensitive for differentiation. Cultures infected with the partially transformation-defective mutant of MC29 td10H were morphologically transformed and acquired anchorage independence for proliferation but maintained a residual competence for terminal differentiation.     

Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds.

To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution.     

Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis.

DNA topoisomerase II (EC 5.99.1.3) is necessary for chromosome condensation and disjunction in yeast but not for other functions. In mammalian cells, it has been reported to be necessary for progression toward mitosis but not for transit through mitosis. We have found, on the contrary, that specific inhibition of topoisomerase II (but not of topoisomerase I) interferes with mammalian mitotic progression. Metaphase is prolonged, and anaphase separation of chromatids is completely inhibited, in cells given high concentrations of topoisomerase II inhibitors; nevertheless these cells attempt cleavage, sometimes generating nucleate and anucleate daughters. Lower concentrations of inhibitors interfere with anaphase and produce abnormalities of segregation. DNA topoisomerase II activity is therefore necessary for mammalian chromatid separation, but it is not tightly coupled to the control of other mitotic events.     

Expression of delta-aminolevulinate synthase in avian cells: separate genes encode erythroid-specific and nonspecific isozymes.

A controversy has existed in the literature for the past several years regarding the number of vertebrate genes encoding the mitochondrial protein that initiates the first step in heme biosynthesis, delta-aminolevulinate synthase [ALAS; succinyl-CoA: glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37]. By analysis of chicken ALAS cDNA clones isolated from both liver and erythroid cells, we show that at least two separate genes encode ALAS mRNAs. These experiments show that (i) two different genes encode the ALAS isozymes found in erythroid and in liver tissues, and (ii) while the product of the erythroid gene (ALASE) is expressed exclusively in erythroid cells, the hepatic form of the enzyme is expressed ubiquitously, suggesting that this is the nonspecific form (ALASN) found in all chicken tissues.     

Identification of a Rous sarcoma virus transformation-related protein in normal avian and mammalian cells.

Avian sarcoma viruses (ASV) contain a gene (src) whose protein product mediates sarcomagenic transformation. This product is a 60,000-Mr phosphoprotein designated pp60src. We have found that normal uninfected frog, chicken, rat, and human cells contain a 60,000-Mr phosphoprotein related to the product of the ASV src gene and have designated that protein pp60. A phosphoprotein of similar size was not detectable in Drosophila cells. The pp60 proteins were detected by immunoprecipitation with rabbit antitumor serum containing broad spectrum antibodies to pp60src. Peptide maps of [35S]methionine-labeled pp60 and pp60src indicated major similarities as well as some differences in amino acid composition. Peptide maps of the 32P-labeled proteins demonstrated that the phosphopeptides of all endogenous pp60 molecules tested were identical. However, some differences were noted between the phosphopeptide patterns of pp60 and viral pp60src. The kinase activity associated with pp60src was measured in the immunocomplex and resulted in the transfer of radioactive phosphorus from [gamma-32P]ATP to the immunoglobulin heavy chain as well as to an 80,000-Mr phosphoprotein. The pp60 of chicken, rat, and human origin also contained an associated kinase activity. These results are consistent with the notion that the pp60 molecules are the protein products of endogenous sarc sequences found in vertebrate cells.     

Direct transfer of cloned genes from bacteria to mammalian cells.

Induction of a virus infection by cloned simian virus 40 DNA was chosen as a test system to detect transfer of genes from bacteria to cultured mammalian cells. Escherichia coli cells containing a recombinant plasmid with three tandem inserts of simian virus 40 DNA were able to infect CV-1 monkey cells under various conditions. The gene transfer was resistant to DNase I and therefore seems not to occur via free DNA but most likely via uptake of whole bacteria, followed by release of plasmid DNA and generation of infectious circular simian virus 40 DNA in a recombination-excision process. Spontaneous transfer was found to be infrequent, 4 x 10(9) bacteria yielding one infection per 10(7) monkey cells. The frequency was greatly increased by adding bacteria as a calcium phosphate coprecipitate or by fusion of lysozyme-treated bacteria (protoplasts) with monkey cells in the presence of polyethylene glycol. With the latter technique, 10(4) protoplasts gave rise to one infection per 15 monkey cells. Experiments with other cell lines of human, monkey, and mouse origin, and also with bacteria harboring another recombinant plasmid, indicate that DNA transfer from bacteria to mammalian cells is a general phenomenon.     

Thyroid hormone receptors from IM-9 cells but not HeLa cells bind to promoters of triiodothyronine-responsive genes

We have used thyroid hormone receptors from two different human cell lines to investigate receptor binding to the promoters of thyroid hormone-responsive genes. Receptors extracted from IM-9 cells or HeLa cells displayed virtually identical affinity and specificity for [125I]triiodothyronine binding. The cells expressed a c-erbA alpha gene in the same relative proportions as the receptor concentrations. Both receptors were bound to DNA-cellulose and could be displaced with increasing concentrations of calf thymus DNA or pBR322 DNA. Relative to pBR322 DNA (designated as 1), binding to the hGH gene promoter was 8.1 +/- 1.1 using the IM-9 cell receptor. With the HeLa cell receptor relative binding was only 1.1 +/- 0.2. Similar relative differences were obtained with the mouse glandular kallikrein gene, mGK-6. In heat stability studies the IM-9 cell receptor was more resistant to heat inactivation than the HeLa receptor. Triiodothyronine receptors with identical hormone binding patterns may require the presence of an unidentified factor(s) which allows correct recognition of regulation sequences within responsive genes.     

Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells.

We demonstrate that an adenosine deaminase (ADA) cDNA gene can function as a dominant selectable and amplifiable marker for gene transfer experiments in mammalian cells. Cells that incorporate the gene can be selected by growth in the presence of low concentrations of the ADA inhibitor 2'-deoxycoformycin with cytotoxic concentrations of adenosine or its analogue 9-beta-D-xylofuranosyl adenine. The DNA copy number of the transfected ADA minigene in the isolated transformants of Chinese hamster ovary cells can be amplified greater than 100-fold by growth in ADA selection media and increasing concentrations of 2'-deoxycoformycin. This selection scheme may allow for the introduction and subsequent amplification of heterologous DNA in a variety of mammalian cells.     

Locus unlinked to alpha-fetoprotein under the control of the murine raf and Rif genes.

The levels of alpha-fetoprotein mRNA in mice are determined by at least two trans-acting, unlinked genes, raf and Rif. raf determines the basal levels of alpha-fetoprotein mRNA in adult mice, while Rif determines its degree of inducibility during liver regeneration. To determine whether these regulatory loci affect other structural genes, we screened a murine fetal liver cDNA library for clones containing mRNA sequences that decrease after birth. One such clone, termed pH19, was identified, and its mRNA was shown to be under the control of both raf and Rif. The single-copy gene for H19 mRNA was localized to chromosome 7, and genetic crosses established that it was unlinked to either raf or Rif. It encodes a 2.5-kilobase mRNA that was identified in those tissues that produce alpha-fetoprotein: visceral endoderm, liver, and fetal gut. The repression of H19 mRNA in neonatal liver occurs several days after the decrease in alpha-fetoprotein mRNA, whereas inductions of both mRNAs during the differentiation of F9 teratocarcinoma cells into visceral endoderm were identical. The tissue-specific expression of H19 mRNA is different from that of alpha-fetoprotein in that H19 mRNA was detected also in both cardiac and skeletal muscle where no alpha-fetoprotein mRNA is produced. Despite the fact that the levels of H19 mRNA decline to 1/10th to 1/20th in cardiac muscle after birth, the adult basal levels are not under the influence of raf. This observation argues that the raf gene is a tissue-specific regulator of mRNA levels.