bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.
Authors: Semba K , Kawai S , Matsuzawa Y , Yamanashi Y , Nishizawa M , Toyoshima K
Published: October 2015

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.
Alterations of G-protein coupling function in phosphoinositide signaling pathways of cells transformed by ras and other membrane-associated and cytoplasmic oncogenes.
Authors: Alonso T , Srivastava S , Santos E
Published: October 2015

We showed previously that transformation by cytoplasmic and membrane-associated oncogenes including ras results in uncoupling between surface stimulation by platelet-derived growth factor, bombesin, and serum and activation of intracellular phospholipase C (PLC); this uncoupling does not involve alterations at the receptor or effector enzyme levels (T. Alonso, R. O. Morgan, J. C. Marvizon, H. Zarbl, and E. Santos, Proc. Natl. Acad. Sci. USA 85:4271-4275, 1988). In this study, we stimulated normal and oncogene-transformed NIH 3T3 cells with fluoroaluminate (AIF4-), thus directly activating PLC-associated G protein(s) and bypassing the receptor step. A1F4(-)-elicited PLC responses were significantly impaired in transformed cells when compared with those in their normal counterparts, suggesting that the uncoupling of PLC is the result, at least in part, of functional impairment at the G-protein level. Transformation by ras oncogenes has also been reported to result in enhanced PLC response to bradykinin resulting from increased receptor numbers (G. Parries, R. Hoebel, and E. Racker, Proc. Natl. Acad. Sci. USA 84:2648-2652, 1987; J. Downward, J. de Gunzburg, R. Riehl, and R. Weinberg, Proc. Natl. Acad. Sci. USA 85:5774-5778, 1988). We demonstrate here that transformation by other membrane-associated and cytoplasmic oncogenes also results in increased responsiveness to bradykinin ("supercoupling") and enhanced receptor numbers. However, there is no direct correlation between the number of receptors and the enhancement in responsiveness, suggesting that other factors besides receptor number are also involved in the enhanced responses. We propose that a common effect of transformation by cytoplasmic and membrane-associated oncogenes is functional alteration of coupling G proteins and that a similar modification of different kinds of G proteins may account for the pleiotropic alterations of signal transduction (uncoupling and supercoupling) observed.
Maximal stress-induced transcription from the human HSP70 promoter requires interactions with the basal promoter elements independent of rotational alignment.
Authors: Williams G T , Morimoto R I
Published: October 2015

Transcription of the human HSP70 gene is regulated by a complex array of cis-acting promoter elements that respond to conditions that include normal conditions of cell growth and induction following physiological stress. We have examined the requirements of the basal and inducible promoter elements by using promoter mutations and a transient transfection assay. Multiple forms of stress-induced transcription, including heat shock and incubation with heavy metals or amino acid analogs, are mediated by a single heat shock element (HSE) between -105 and -91 consisting of three contiguous 5-base-pair units, NGAAN, that are inverted relative to adjacent units. Maximal inducible expression requires a fully functional basal promoter. Spacing mutations which alter the relative helical orientation of adjacent genetic elements have only minimal effects on basal and stress-inducible expression and show no effects of periodicity. In addition, placement of the HSE adjacent to the basal promoter removes the requirements for a fully functional basal promoter for maximal stress-inducible expression. These results suggest that factors bound at the HSE and the basal promoter can function through multiple interactions.
Characterization of chicken c-ski oncogene products expressed by retrovirus vectors.
Authors: Sutrave P , Copeland T D , Showalter S D , Hughes S H
Published: October 2015

We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.
Involvement of a second lymphoid-specific enhancer element in the regulation of immunoglobulin heavy-chain gene expression.
Authors: Libermann T A , Lenardo M , Baltimore D
Published: October 2015

To determine whether enhancer elements in addition to the highly conserved octamer (OCTA)-nucleotide motif are important for lymphoid-specific expression of the immunoglobulin heavy-chain (IgH) gene, we have investigated the effect of mutating the binding site for a putative additional lymphoid-specific transcription factor, designated NF-microB, in the murine IgH enhancer. We demonstrate that the NF-microB-binding site plays a critical role in the IgH enhancer, because mutation of the microB DNA motif decreased transcriptional activity of the IgH enhancer in cells of the B-cell lineage but not in nonlymphoid cells. This effect was comparable to or even stronger than the effect of a mutation in the OCTA site. Moreover, combined mutation of both microB and OCTA sites further reduced enhancer activity in lymphoid cells. Interestingly, alteration of either the microB or E3 site in a 70-base-pair fragment of the IgH enhancer that lacks the binding site for OCTA abolished enhancer activity in lymphoid cells completely. Nevertheless, a multimer of the microB motif alone showed no enhancer activity. DNase footprinting analysis corroborated the functional data showing that a lymphoid-specific protein binds to the microB DNA motif. Our results suggest that the microB element is a new crucial element important for lymphoid-specific expression of the IgH gene but that interaction with another enhancer element is essential for its activity.
Identification of Xenopus S6 protein kinase homologs (pp90rsk) in somatic cells: phosphorylation and activation during initiation of cell proliferation.
Authors: Chen R H , Blenis J
Published: October 2015

We have identified human, mouse, and chicken homologs to Xenopus S6 protein kinase II (S6KII). In quiescent cells, the apparent molecular mass of the Xenopus homologs (referred to as pp90rsk) increased from a range of 81 to 91 to a range of 85 to 92 kilodaltons following serum addition, which is consistent with an increase in protein phosphorylation. Indeed, serum growth factors stimulated pp90rsk phosphorylation at multiple serine and threonine residues. Furthermore, pp90rsk activity was stimulated within seconds of serum addition. Distinct molecular sizes, chromatographic properties, phosphopeptide maps, and kinetics of activation, the lack of immunological cross-reactivity, and analysis of S6 kinase activities in cells that overexpressed pp90rsk suggest that pp90rsk and pp70-S6 protein kinase, a previously identified mitogen- and oncogene-regulated S6 kinase in cultured cells, are distinct and differentially regulated. The notion that both enzymes are regulated by protein phosphorylation was supported by the ability to inactivate their S6 phosphotransferase activities with potato acid phosphatase. These data demonstrate that homologs to the Xenopus S6 protein kinases are produced and regulated by protein phosphorylation in somatic cells and that, in addition to a proposed role in Xenopus oocyte maturation, these homologs may participate in the initiation of animal cell proliferation.
The upstream sequence -537 to -278 is necessary for transcription of the human nucleolar antigen p120 gene.
Authors: Haidar M A , Henning D , Busch H
Published: October 2015

Two cis-acting elements in the p120 gene play important roles in transcription; the region from -537 to -278 is necessary for initiation of transcription, and the region from -1426 to -1223 is necessary for efficient transcription. The distal element(s) which lies upstream of -278 is required for initiation of transcription.
Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae.
Authors: Boorstein W R , Craig E A
Published: October 2015

The SSA3 gene of Saccharomyces cerevisiae, a member of the HSP70 multigene family, is expressed at low levels under optimal growth conditions and is dramatically induced in response to heat shock. Sequences coinciding with two overlapping heat shock elements, located 156 base pairs upstream of the transcribed region, were necessary and sufficient for regulation of heat induction. The SSA3 promoter was also activated in an ssa1ssa2 double-mutant strain. This increase in the expression of SSA3 was mediated via the same upstream activating sequences that activated transcription in response to heat shock.
Closely related DNA sequences specify distinct patterns of developmental expression in Drosophila melanogaster.
Authors: Xiao H , Lis J T
Published: October 2015

Three short synthetic DNA sequences, which are closely related to one another, confer three distinct patterns of developmental expression on the heat shock hsp70 gene in transgenic Drosophila melanogaster lines. These results show that small variations or even single base pair changes in a repeated element of a regulatory sequence can create promoters that display new specificities of tissue and developmental regulation. Interestingly, the three patterns of developmental expression conferred by the synthetic DNAs resemble in part those of the known developmental genes: glucose dehydrogenase (Gld), Dopa decarboxylase (Ddc), and salivary gland secretory proteins (Sgs), respectively. In each case, the defined regulatory region of the known developmental gene contains multiple sequences that are similar or identical to the synthetic sequence that confers a similar pattern of developmental expression on the hsp70 gene. Thus, these results are congruent with the view that short sequence elements in multiple copies can confer either simple or relatively complex patterns of developmental expression on a receptive promoter like that of hsp70. Furthermore, the fact that the three variants tested produced three distinct patterns of expression in transgenic animals suggests that the number of different elements is large.
Most of the homeobox-containing Xhox 36 transcripts in early Xenopus embryos cannot encode a homeodomain protein.
Authors: Condie B G , Brivanlou A H , Harland R M
Published: October 2015

Multiple Xhox 36 transcripts accumulate in Xenopus embryos from gastrula to early tadpole stages. The transcripts were characterized by sequencing cDNA clones and by S1 protection and Northern (RNA) blotting of embryonic RNA with probes derived from the cDNAs. The Xhox 36 RNAs included unspliced precursor transcripts that accumulated in the embryonic nuclei, spliced transcripts that contained multiple stop codons in frame with the homeobox, and less abundant coding mRNAs. These transcripts were generated either by alternative splicing or multiple initiations from a single Xhox 36 gene. The sequence of a cDNA clone of the unspliced transcript showed that the intron contained a noncanonical 3' splice site. However, the intron was spliced efficiently when expressed from a plasmid injected into Xenopus embryos, suggesting that the inefficient splicing of the endogenous RNA is not due to the unusual 3' splice site. The accumulation of noncoding and unspliced transcripts suggests multiple levels of regulation in the embryonic expression of the Xhox 36 gene.
BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex.
Authors: Newman A P , Shim J , Ferro-Novick S
Published: October 2015

A subset of the genes required for transport from the endoplasmic reticulum (ER) to the Golgi complex in Saccharomyces cerevisiae was found to interact genetically. While screening a yeast genomic library for genes complementing the ER-accumulating mutant bet1 (A. Newman and S. Ferro-Novick, J. Cell Biol. 105: 1587-1594, 1987), we isolated BET1 and BOS1 (bet one suppressor). BOS1 suppresses bet1-1 in a gene dosage-dependent manner, providing greater suppression when it is introduced on a multicopy vector than when one additional copy is present. The BET1 and BOS1 genes are not functionally equivalent; overproduction of BOS1 does not alleviate the lethality associated with disruption of BET1. We also identified a pattern of genetic interactions among these genes and another gene implicated in transport from the ER to the Golgi complex: SEC22. Overproduction of either BET1 or BOS1 suppresses the growth and secretory defects of the sec22-3 mutant over a wide range of temperatures. Further evidence for genetic interaction was provided by the finding that a bet1 sec22 double mutant is inviable. Another mutant which is blocked in transport from the ER to the Golgi complex, sec21-1, demonstrates a more limited ability to be suppressed by the BET1 gene. The interactions we observed are specific for genes required for transport from the ER to the Golgi complex. The products of the genes involved are likely to have a direct role in transport, as bet1-1 and sec22-3 begin to display their mutant phenotypes within 5 min of a shift to the restrictive temperature.
Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein.
Authors: Price C M
Published: October 2015

The nucleoprotein structure of telomeres from Euplotes crassus was studied by using nuclease and chemical footprinting. The macronuclear telomeres were found to exist as DNA-protein complexes that are resistant to micrococcal nuclease digestion. Each complex encompassed 85 to 130 base pairs of macronuclear DNA and appeared to consist of two structural domains that are characterized by dissimilar DNA-protein interactions. Dimethyl sulfate footprinting demonstrated that very sequence-specific and salt-stable interactions occur in the most terminal region of each complex. DNase I footprinting indicated that DNA in the region 30 to 120 base-pairs from the 5' end lies on a protein surface; the interactions in this region of the complex are unlikely to be sequence specific. A 50-kilodalton telomere-binding protein was isolated. Binding of this protein protected telomeric DNA from BAL 31 digestion and gave rise to many of the sequence-specific DNA-protein interactions that were observed in vivo. The telomeric complexes from E. crassus were very similar in overall structure to the complexes found at Oxytricha telomeres. However, telomeric complexes from the two ciliates showed significant differences in internal organization. The telomeric DNA, the telomere-binding proteins, and the resultant DNA-protein interactions were all somewhat different. The telomere-binding proteins from the two ciliates were found to be less closely conserved than might have been expected. It appears that the proteins are tailored to match their cognate telomeric DNA.
Functional analysis of elements affecting expression of the beta-actin gene of carp.
Authors: Liu Z J , Moav B , Faras A J , Guise K S , Kapuscinski A R , Hackett P B
Published: October 2015

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.
Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon.
Authors: Nikovits W , Mar J H , Ordahl C P
Published: October 2015

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.
Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.
Authors: Abraham L J , Bradshaw A D , Shiels B R , Northemann W , Hudson G , Fey G H
Published: October 2015

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.
Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.
Authors: Hays J B , Ackerman E J , Pang Q S
Published: October 2015

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.
Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.
Authors: Michaeli J , Lebedev Y B , Richon V M , Chen Z X , Marks P A , Rifkind R A
Published: October 2015

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.
Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate.
Authors: Gangloff S P , Marguet D , Lauquin G J
Published: October 2015

We have isolated genomic clones complementing the aconitase-deficient strain (glu1-1) of Saccharomyces cerevisiae. Identification of the aconitase gene was established by enzymatic assays and molecular analyses. The corresponding mRNA has been characterized, and its direction of transcription has been determined. The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein. Disruption of the gene by deletion-insertion led to glutamate auxotrophy. Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate.
Immunoglobulin D switching can occur through homologous recombination in human B cells.
Authors: White M B , Word C J , Humphries C G , Blattner F R , Tucker P W
Published: October 2015

Prototypical class switching in mouse and human immunoglobulin heavy chains occurs through recombination of tandem blocks of short repeats located 5' to each heavy chain constant region (CH) except C delta. Deletion of C mu in immunoglobulin D (IgD)-secreting murine plasmacytomas occurs illegitimately. We demonstrate here that in human IgD-secreting myeloma cells freshly isolated from patient bone marrow and in normal peripheral blood B lymphocytes, an IgD switch can occur through homologous recombination of a direct repeat consisting of a 442-bp sequence 1.5 kbp 3' of the JH complex and a 443-bp sequence that is duplicated almost perfectly (96% similarity) 1.7 kbp 5' of the C delta gene (442/443-base-pair [bp] repeat). This homologous recombination mechanism is not exclusive for IgD switching, since C mu deletion endpoints in two established IgD-secreting myeloma cell lines fall outside the 442/443-bp repeat. The 442/443-bp mediated recombination shows cell type specificity, and we propose that it represents a unique mode for increased levels of IgD secretion in humans.
The c-myc proto-oncogene regulates cardiac development in transgenic mice.
Authors: Jackson T , Allard M F , Sreenan C M , Doss L K , Bishop S P , Swain J L
Published: October 2015

During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.
The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor.
Authors: Leiting B , Lindner I J , Noegel A A
Published: October 2015

Dictyostelium discoideum plasmid Ddp2 from the wild strain WS380B is a 5.8-kilobase (kb) supercoiled circle with a copy number of 300 per haploid genome. We previously described the construction of an extrachromosomally replicating transformation vector pnDeI carrying 4.7 kb of Ddp2 sequences (B. Leiting, and A. Noegel, Plasmid 20:241-248, 1988). In order to reduce the sequences required for extrachromosomal maintenance in D. discoideum, we characterized Ddp2 by sequence analysis, by deletion experiments, by transcription mapping, by electrophoretic mobility shift assays, and by expression of its single open reading frame in Escherichia coli. Two elements were involved in replication of Ddp2: a cis-acting sequence located on a 592-base-pair (bp) fragment that consisted of 220 bp of essential and 372 bp of auxiliary sequences, and a 2.7-kb open reading frame which most likely encodes a trans-acting factor. The cis- and trans-acting elements did not overlap and were shown to act independently from the location of the sequences encoding the trans-acting factor.
Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.
Authors: Ni B F , Needleman R B
Published: October 2015

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.
Developmental expression of myeloid leukemia inhibitory factor gene in preimplantation blastocysts and in extraembryonic tissue of mouse embryos.
Authors: Conquet F , Brûlet P
Published: October 2015

Murine leukemia inhibitory factor (LIF) protein is a growth factor which has the ability to maintain the developmental potential of pluripotent embryonic stem cells through a specific receptor. We have examined the expression pattern of the LIF gene from the preimplantation stage (3.5 days post coitum) to the midgestation stage (12.5 days post coitum) of the mouse embryo. LIF transcripts were detected at the preimplantation blastocyst stage, whereas no transcripts were detectable in embryonic stem cells. LIF gene transcription continued in the extraembryonic tissue of the 7.5-day and in the placenta of 9.5-, 10.5-, and 12.5-day post coitum embryos. No transcripts were detected in the embryo proper of the corresponding stages. Our results suggest that this growth factor is synthesized in the extraembryonic part of the embryo and acts on the embryonic tissues during early mouse development.
Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a kappa B-like sequence.
Authors: Zhang Y H , Lin J X , Vilcek J
Published: October 2015

Using variable-length deletion constructs of the 5'-flanking region of the human interleukin-6 (IL-6) gene linked to the chloramphenicol acetyltransferase gene, we showed that the region from positions -109 to -50 mediated the bulk of the response to tumor necrosis factor (TNF) or interleukin-1 (IL-1), while it was less responsive to forskolin. DNA mobility shift assays and DNase I footprinting analysis identified a nuclear protein from TNF- or IL-1-treated fibroblasts that bound to a region comprising a kappa B-like element located between positions -72 and -63 on the IL-6 gene. On the basis of these and other experiments, we conclude that TNF and IL-1 apparently activate IL-6 gene expression by closely related mechanisms involving activation of a NF-kappa B-like factor, whereas the pathway of IL-6 induction by forskolin is, in part, different.
Involvement of cyclic AMP-dependent protein kinases in the signal transduction pathway for interleukin-1.
Authors: Chedid M , Mizel S B
Published: October 2015

Expression of a highly specific protein inhibitor for cyclic AMP-dependent protein kinases in interleukin-1 (IL-1)-responsive cells blocked IL-1-induced gene transcription that was driven by the kappa immunoglobulin enhancer or the human immunodeficiency virus long terminal repeat. This inhibitor did not affect protein kinase C-mediated gene transcription, suggesting that cyclic AMP-dependent protein kinases are involved in the signal transduction pathway for IL-1 in a number of responsive cell types.
Transfected human beta-polymerase promoter contains a ras-responsive element.
Authors: Kedar P S , Lowy D R , Widen S G , Wilson S H
Published: October 2015

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.
Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro.
Authors: Wobbe C R , Struhl K
Published: October 2015

We have analyzed the DNA sequence requirements for TATA element function by assaying the transcriptional activities of 25 promoters, including those representing each of the 18 single-point mutants of the consensus sequence TATAAA, in a reconstituted in vitro system that depends on the TATA element-binding factor TFIID. Interestingly, yeast TFIID and HeLa cell TFIID were virtually identical in terms of their relative activities on this set of promoters. Of the mutated elements, only two had undetectable activity; the rest had activities ranging from 2 to 75% of the activity of the consensus element, which was the most active. In addition, mutations of the nucleotide following the TATAAA core strongly influenced transcriptional activity, although with somewhat different effects on yeast and HeLa TFIID. The activities of all these promoters depended upon TFIID, and the level of TFIID-dependent transcription in vitro correlated strongly with their activities in yeast cells. This suggests that the in vivo activities of these elements reflect their ability to functionally interact with a single TATA-binding factor. However, some elements with similar activities in vitro supported very different levels of transcriptional activation by GAL4 protein in vivo. These results extend the degree of evolutionary conservation between yeast and mammalian TFIID and are useful for predicting the level of TATA element function from the primary sequence.
Transcriptional control of the invariant chain gene involves promoter and enhancer elements common to and distinct from major histocompatibility complex class II genes.
Authors: Zhu L , Jones P P
Published: October 2015

The invariant chain (Ii) is a glycoprotein coexpressed with the major histocompatibility complex (MHC) class II antigens. Although Ii is encoded by a single gene unlinked to the MHC gene complex, Ii and MHC class II appear to have similar patterns of tissue specific expression and generally are coordinately regulated by cytokines. Here we present evidence that transcription of the murine Ii gene is controlled by multiple cis-acting elements. The 5' regulatory region of the Ii gene appears to be combined of conserved class II regulatory elements with promoter elements commonly found in other eucaryotic genes. A region containing characteristic class II promoter elements (H box, X box, and a modified Y box) serves as an upstream enhancer in the Ii gene and might contribute to the coexpression of MHC class II and Ii genes. A series of positive control elements, the kappa B element, Sp1-binding site, and CCAAT box, are present in the Ii promoter and apparently serve distinct regulatory functions. The kappa B site in the Ii gene is a cell type-specific element, contributing to expression in a B-cell line but not in a fibroblast cell line, and the Sp1 site is required by the H-X-Y' enhancer element to stimulate promoter activity. In addition, an Ii enhancer in the first intron that specifically stimulates its own promoter has been identified. Our results suggest that a sequence match between enhancers and certain promoter elements is critical.
Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III.
Authors: Van Houten J V , Newlon C S
Published: October 2015

Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.
Enhanced translation and increased turnover of c-myc proteins occur during differentiation of murine erythroleukemia cells.
Authors: Spotts G D , Hann S R
Published: October 2015

To determine whether regulation of c-myc proteins occurs during the differentiation of murine erythroleukemia cells, we examined c-myc protein synthesis and accumulation throughout dimethyl sulfoxide (DMSO)- or hypoxanthine-induced differentiation. c-myc protein expression exhibited an overall biphasic reduction, with an initial concomitant decrease in c-myc RNA, protein synthesis, and protein accumulation early during the commitment phase. However, as the mRNA and protein levels recovered, c-myc protein synthesis levels dissociated from the levels of c-myc mRNA and protein accumulation. This dissociation appears to result from unusual translational and posttranslational regulation during differentiation. Translational enhancement was suggested by the observation that relatively high levels of c-myc proteins were synthesized from relatively moderate levels of c-myc RNA. This translational enhancement was not observed with c-myb. Under certain culture conditions, we also observed a change in the relative synthesis ratio of the two independently initiated c-myc proteins. Posttranslational regulation was evidenced by a dramatic postcommitment decrease in the accumulated c-myc protein levels despite relatively high levels of c-myc protein synthesis. This decrease corresponded with a twofold increase in the turnover of c-myc proteins. The consequence of this regulation was that the most substantial decrease in c-myc protein accumulation occurred during the postcommitment phase of differentiation. This result supports the hypothesis that the reduction in c-myc at relatively late times is most important for completion of murine erythroleukemia cell terminal differentiation.
Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.
Authors: Drawbridge J , Grainger J L , Winkler M M
Published: October 2015

Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.
Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells.
Authors: Loh T P , Sievert L L , Scott R W
Published: October 2015

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.
Transformation and pH homeostasis of fibroblasts expressing yeast H(+)-ATPase containing site-directed mutations.
Authors: Perona R , Portillo F , Giraldez F , Serrano R
Published: October 2015

Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.