bluuebunny/crossref_metadata_2025 huggingface dataset:
An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice.
Authors: Johnston L A , Kotarski M A , Jerry D J , Kozak L P
Published: October 2015

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.
High-level recombination specific to polyomavirus genomes targeted to the integration-transformation pathway.
Authors: Hacker D , Fluck M M
Published: October 2015

An unusually high incidence of interviral recombination was found in the process of integration of the polyomavirus genome concomitant with neoplastic transformation of nonpermissive cells. Transformants were isolated after mixed infections of Fischer rat cells with two mutants lacking restriction endonuclease sites and were analyzed for the presence of unselected integrated recombinant restriction fragments. A large fraction of the transformants isolated (38% of the 64 transformed cell lines studied) contained recombinant viral genomes that had undergone recombination in a 1.3-, 1.7-, or 3.6-kilobase-pair interval. More than 90% of these recombinant transformants showed evidence of crossovers in multiple intervals. To our knowledge, the recombination frequencies observed in these experiments represent the highest frequencies of homologous recombination reported for a mitotic mammalian system that does not involve transfection. In contrast to the elevated level of recombination in the integrated viral genomes, no evidence of recombination was obtained among the replicated unintegrated pool of viral genomes isolated from the same population of infected cells from which the recombinant transformants were derived. Either of two hypotheses can provide an explanation for the segregated recombination: either recombination occurs at elevated levels in a small, recombination-prone fraction of the population destined to become transformed, or recombination occurs only among those viral genomes which are engaged in the process of integration and thus interact with a recombinogenic host machinery (for example, the host scaffold). We favor the latter hypothesis.
Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.
Authors: Terns M P , Jacob S T
Published: October 2015

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.
Identification and characterization of PF4varl, a human gene variant of platelet factor 4.
Authors: Green C J , Charles R S , Edwards B F , Johnson P H
Published: October 2015

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.
Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.
Authors: Zhu H , Conrad-Webb H , Liao X S , Perlman P S , Butow R A
Published: October 2015

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.
An RNA polymerase I promoter located in the CHO and mouse ribosomal DNA spacers: functional analysis and factor and sequence requirements.
Authors: Tower J , Henderson S L , Dougherty K M , Wejksnora P J , Sollner-Webb B
Published: October 2015

We report results of experiments in which we demonstrated the existence of a polymerase I promoter within the ribosomal DNA spacer upstream from the rRNA initiation site in Chinese hamsters and mice. Transcription of the CHO spacer promoter was achieved by the same protein factors, C and D, that catalyzed transcription of the gene promoter, and these factors bound stably to the CHO spacer promoter in a preinitiation complex, just as they did to the gene promoter. In contrast to the CHO spacer promoter, which was transcribed in vitro nearly as efficiently as the gene promoter, the mouse spacer promoter was far less active; this low activity was attributable to the fact that the mouse spacer promoter bound factor D inefficiently. It is striking that the active CHO spacer promoter violated the otherwise universal rule that metazoan RNA polymerase I promoters all have a G residue at position -16. Sequence comparisons also revealed a great similarity between the CHO and mouse spacer promoter regions, yet there was much less similarity between the flanking sequences. There was also only limited homology between the spacer and gene promoter regions, but despite this the two kinds of initiation regions were organized similarly, both consisting of an essential core promoter domain and a stimulatory domain that extended upstream to approximately residue -135. Evolutionary considerations argue strongly that the presence of ribosomal DNA spacer promoters offers a significant selective advantage.
Continued withdrawal from the cell cycle and regulation of cellular genes in mouse erythroleukemia cells blocked in differentiation by the c-myc oncogene.
Authors: Coppola J A , Parker J M , Schuler G D , Cole M D
Published: October 2015

Constitutive expression of the c-myc oncogene blocks dimethyl sulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (MEL) cells. During the first 12 h of treatment with DMSO, MEL cells undergo a temporary decrease in the level of c-myc mRNA, followed by a temporary withdrawal from the cell cycle. We found the same shutoff of DNA synthesis during the first 12 to 30 h after DMSO induction in normal MEL cells (which differentiate) and in c-myc-transfected MEL cells (which do not differentiate). We also examined whether deregulated c-myc expression grossly interfered with the regulation of gene expression during MEL cell differentiation. We used run-on transcription assays to monitor the rate of transcription of four oncogenes (c-myc, c-myb, c-fos, and c-K-ras); all except c-K-ras showed a rapid but temporary decrease in transcription after induction in both c-myc-transfected and control cells. Finally, we found the same regulation of cytoplasmic mRNA expression in both types of cells for four oncogenes and three housekeeping genes associated with growth. We conclude that in the MEL cell system, the effects of deregulated c-myc expression do not occur through a disruption of cell cycle control early in induction, nor do they occur through gross deregulation of gene expression.
Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.
Authors: Lim R W , Varnum B C , O'Brien T G , Herschman H R
Published: October 2015

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.
Cellular factors that interact with the hepatitis B virus enhancer.
Authors: Ben-Levy R , Faktor O , Berger I , Shaul Y
Published: October 2015

An 83-base-pair-long hepatitis B virus DNA fragment efficiently activates the transcription of the heterologous globin gene promoter. This fragment contains binding sites for at least four distinct cellular factors termed E, TGT3, EP, and NF-I. E is a positively acting factor, responsive to phorbol ester. EP is apparently identical to the factor EF-C that binds to the polyomavirus enhancer. The conservation of the binding site sequences for most of these factors in the genomes of other members of the hepadnavirus family suggests that these viruses share common enhancer elements.
A novel pathway for transversion mutation induced by dCTP misincorporation in a mutator strain of CHO cells.
Authors: Phear G , Meuth M
Published: October 2015

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.
Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines.
Authors: Kontis K J , Arfin S M
Published: October 2015

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.
Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.
Authors: Friesel R , Burgess W H , Maciag T
Published: October 2015

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.
Stable maintenance of a 35-base-pair yeast mitochondrial genome.
Authors: Fangman W L , Henly J W , Churchill G , Brewer B J
Published: October 2015

Small deletion variants ([rho-] mutants) derived from the wild-type ([ rho+]) Saccharomyces cerevisiae mitochondrial genome were isolated and characterized. The mutant mitochondrial DNAs (mtDNAs) examined retained as little as 35 base pairs of one section of intergenic DNA, were composed entirely of A.T base pairs, and were stably maintained. These simple mtDNAs existed in tandemly repeated arrays at an amplified level that made up approximately 15% of the total cellular DNA and, as judged by fluorescence microscopy, had a nearly normal mitochondrial arrangement throughout the cell cytoplasm. The simple nature of these [rho-] genomes indicates that the sequences required to maintain mtDNA must be extremely simple.
Purification and properties of the Rous sarcoma virus internal enhancer binding factor.
Authors: Karnitz L , Poon D , Weil P A , Chalkley R
Published: October 2015

The internal enhancer binding factor (IBF) that specifically binds sequences within the gag gene internal enhancer of Rous sarcoma virus Schmidt-Ruppin A was purified to near homogeneity from BHK cells. The polypeptides that constituted IBF DNA-binding activity were identified by sodium dodecyl sulfate-polyacrylamide gel analysis. As isolated from BHK cells, IBF consisted of two different but related polypeptides. One (IBF alpha) had a molecular weight of 40,000; the other (IBF beta) had a molecular weight of 20,000 and appeared to be a proteolytic product of IBF alpha. The site within the gag gene to which IBF bounds in vitro (internal enhancer site 2; nucleotides 856 to 878 of the Rous sarcoma virus genome) were demonstrated to function as a cis-acting transcriptional stimulatory element both in vivo and in vitro. By using HeLa cell nuclear transcription extracts, purified IBF was found to function as a trans-acting transcription factor that stimulated transcription in vitro. Purified IBF was also demonstrated to be very similar to EBP20 (K. Carlberg, T. A. Ryden, and K. Beemon, J. Virol. 62:1617-1624, 1988), and it may well belong to the same family of DNA-binding proteins.
Inverse regulation of the yeast COX5 genes by oxygen and heme.
Authors: Hodge M R , Kim G , Singh K , Cumsky M G
Published: October 2015

The COX5a and COX5b genes encode divergent forms of yeast cytochrome c oxidase subunit V. Although the polypeptide products of the two genes are functionally interchangeable, it is the Va subunit that is normally found in preparations of yeast mitochondria and cytochrome c oxidase. We show here that the predominance of subunit Va stems in part from the differential response of the two genes to the presence of molecular oxygen. Our results indicate that during aerobic growth, COX5a levels were high, while COX5b levels were low. Anaerobically, the pattern was reversed; COX5a levels dropped sevenfold, while those of COX5b were elevated sevenfold. Oxygen appeared to act at the level of transcription through heme, since the addition of heme restored an aerobic pattern of transcription to anaerobically grown cells and the effect of anaerobiosis on COX5 transcription was reproduced in strains containing a mutation in the heme-biosynthetic pathway (hem1). In conjunction with the oxygen-heme response, we determined that the product of the ROX1 gene, a trans-acting regulator of several yeast genes controlled by oxygen, is also involved in COX5 expression. These results, as well as our observation that COX5b expression varied significantly in certain yeast strains, indicate that the COX5 genes undergo a complex pattern of regulation. This regulation, especially the increase in COX5b levels anaerobically, may reflect an attempt to modulate the activity of a key respiratory enzyme in response to varying environmental conditions. The results presented here, as well as those from other laboratories, suggest that the induction or derepression of certain metabolic enzymes during anaerobiosis may be a common and important physiological response in yeast cells.
Differential expression of a zinc finger-encoding gene in response to positive versus negative signaling through receptor immunoglobulin in murine B lymphocytes.
Authors: Seyfert V L , Sukhatme V P , Monroe J G
Published: October 2015

Egr-1 is a murine early growth factor-inducible gene that encodes a protein with zinc fingers and that is believed to be involved in transcriptional regulation. Expression of this gene was investigated in murine B lymphocytes stimulated through their receptor for antigen (surface immunoglobulin [sIg]) with antireceptor antibodies (anti-sIg). Rapid (by 15 min) up-regulation of Egr-1 mRNA expression was observed after sIg cross-linking at a dose of anti-sIg sufficient to drive the majority of cells into cell cycle. Interestingly, signaling through sIg on the murine B-lymphoma cell line WEHI-231 did not up-regulate Egr-1 expression even though similar signaling pathways, including up-regulation of c-fos expression, are associated with this receptor in these cells. Importantly, cell growth and proliferation of WEHI-231 cells were inhibited by anti-sIg stimulation, which suggested a relationship between Egr-1 expression and differential processing of receptor immunoglobulin signals with respect to cellular growth responses. This notion was further supported by the finding that murine B lymphomas whose proliferation was not inhibited by anti-sIg showed receptor immunoglobulin-coupled Egr-1 expression. In further support of this association are results showing that under conditions in which Egr-1 expression was induced in WEHI-231 cells in response to stimulation by anti-sIg, a concomitant change was observed in the growth response of these cells. These results, then, indicate a potential role for Egr-1 expression in the translation of receptor-generated signals into cellular activation or induced unresponsiveness.
Alternative processing of mRNAs encoding mammalian chromosomal high-mobility-group proteins HMG-I and HMG-Y.
Authors: Johnson K R , Lehn D A , Reeves R
Published: October 2015

The high-mobility-group protein HMG-I is a well-characterized nonhistone chromosomal protein that is preferentially expressed in rapidly dividing cells, binds to A. T-rich regions of DNA in vitro, and has been localized to particular regions of mammalian metaphase chromosomes. We isolated eight cDNA clones encoding HMG-I and its isoform HMG-Y from a human Raji cell cDNA library and detected blocks of nucleotide sequence rearrangements in the 5'-untranslated regions of these clones. In addition to this leader sequence variation, five of the eight cDNA clones had either a 33- or 36-base-pair in-frame deletion in their open reading frame (ORF); we found that this shortened ORF encodes the HMG-Y protein isoform. We present evidence that the 5'-untranslated-region and ORF heterogeneity of the cDNA clones is the result of alternative processing of RNA transcripts from a single functional gene. Several additional but probably nonfunctional HMG-I or HMG-Y gene copies exist in the human genome; we isolated and partially sequenced one of these pseudogenes and found that it is a processed HMG-Y retropseudogene.
The t(11;14)(p15;q11) in a T-cell acute lymphoblastic leukemia cell line activates multiple transcripts, including Ttg-1, a gene encoding a potential zinc finger protein.
Authors: McGuire E A , Hockett R D , Pollock K M , Bartholdi M F , O'Brien S J , Korsmeyer S J
Published: October 2015

Interchromosomal translocations within lymphoid neoplasms frequently involve the antigen receptor genes. We cloned the breakpoints of the t(11;14)(p15;q11) in a CD3-negative T-cell acute lymphoblastic leukemia cell line (RPMI 8402) in order to identify new genes potentially involved in T-cell neoplasia. An extensive comparison of both breakpoints and their germ line counterparts indicated that an inadvertant recombinase-mediated break at chromosome segment 11p15 recombined with the delta T-cell receptor at 14q11. The derivative 11 breakpoint resembles a coding joint in which 11p15 rather than a variable region was introduced 5' to a D delta 1 D delta 2 J delta 1 intermediate rearrangement. Conversely, the derivative 14 breakpoint corresponds to a signal joint between the 5' heptamer-spacer-nonamer recombinational signal of D delta 1 and an isolated heptamer at 11p15. Multiple, apparently distinct transcripts were found flanking both breakpoints of 8402. RNAs of 3.5, 4.4, 1.4, and 8.0 kilobases originating from either side of the derivative 14 breakpoint were highly expressed in 8402 compared with other cells. This suggests that this translocation deregulated multiple genes and provides the opportunity to assess any multifactorial contribution they may have to malignancy. We cloned and sequenced several cDNAs representing the 1.4-kilobase transcript (termed Ttg-1 [T-cell translocation gene 1]) from an 8402 library. The predicted protein of 156 amino acids contained two internal repeats which could potentially form zinc fingers.
Characterization of the human p53 gene promoter.
Authors: Tuck S P , Crawford L
Published: October 2015

Transcriptional deregulation of the p53 gene may play an important part in the genesis of some tumors. We report here an accurate determination of the transcriptional start sites of the human p53 gene and show that the majority of p53 mRNA molecules do not contain a postulated stem-loop structure at their 5' ends. Recombinant plasmids of the human p53 promoter-leader region fused to the bacterial chloramphenicol acetyltransferase gene (cat) were constructed. After transfection into rodent or human cells, a 350-base-pair fragment spanning the promoter region conferred 4% of the CAT activity mediated by the simian virus 40 early promoter/enhancer. We monitored the efficiency with which 15 3' and 5' promoter deletion constructs initiated transcription. Our results show that an 85-base-pair fragment, previously thought to have resided in exon 1, is all that is required for full promoter activity.
Cross-binding of factors to functionally different promoter elements in c-fos and skeletal actin genes.
Authors: Walsh K
Published: October 2015

A conserved 28-base-pair element in the skeletal actin promoter was sufficient to activate muscle-specific expression when placed upstream of a TATA element. This muscle regulatory element (MRE) is similar in structure to the serum response element (SRE), which is present in the promoters of the c-fos proto-oncogene and the nonmuscle actin genes. The SRE can function as a constitutive promoter element. Though the MRE and SRE differed in their tissue-specific expression properties, both elements bound to the same protein factors in vitro. These proteins are the serum response factor (SRF) and the muscle actin promoter factors 1 and 2 (MAPF1 and MAPF2). The SRF and MAPF proteins were resolved by chromatographic procedures, and they differed in their relative affinities for each element. The factors were further distinguished by their distinct, but overlapping, methylation interference footprint patterns on each element. These data indicate that the differences in tissue-specific expression may be due to a complex interaction of protein factors with these sequences.
Regulatory elements in the 5'-flanking region and the first intron contribute to transcriptional control of the mouse alpha 1 type I collagen gene.
Authors: Rippe R A , Lorenzen S I , Brenner D A , Breindl M
Published: October 2015

We have identified two blocks of regulatory sequences located in the 5'-flanking region and the first intron of the mouse alpha 1 type I collagen (COL1A1) gene. Both blocks were found to contain positive as well as negative regulatory elements. Sequences located within 222 base pairs upstream of the transcription start site showed a strong stimulatory effect on the COL1A1 promoter and were sufficient for tissue-specific regulation of the COL1A1 gene. The combined upstream and intron regulatory sequences showed a marked inhibition of COL1A1 promoter activity in fibroblasts. This finding suggests that additional, more remote regulatory sequences may be required for establishing the high level of activity of the endogenous COL1A1 gene in fibroblastoid cells.
Effects of botulinum toxin type D on secretion of tumor necrosis factor from human monocytes.
Authors: Imamura K , Spriggs D , Ohno T , Kufe D
Published: October 2015

Botulinum toxins are potent neurotoxins which block the release of neurotransmitters. The effects of these toxins on hematopoietic cells, however, are unknown. Monocytes secrete a variety of polypeptide growth factors, including tumor necrosis factor (TNF). In the study reported here, the effects of botulinum toxin type D on the secretion of TNF from human monocytes were examined. The results demonstrate that botulinum toxin type D inhibits the release of TNF from monocytes activated by lipopolysaccharide (LPS) but not by 12-O-tetradecanoylphorbol-13-acetate. Botulinum toxin type D had no detectable effect on intracellular TNF levels in LPS-treated monocytes, indicating that the effects of this toxin involve the secretory process. This inhibitory effect of botulinum toxin type D on TNF secretion from LPS-treated monocytes was partially reversed by treatment with 12-O-tetradecanoylphorbol-13-acetate or introduction of guanosine 5'-[gamma-thio]triphosphate into these cells. The results demonstrate that TNF secretion is regulated by at least two distinct guanine nucleotide-binding proteins, one responsible for the activation of phospholipase C and another which acts as a substrate for botulinum toxin type D. ADP-ribosylation of monocyte membranes by botulinum toxin type D demonstrated the presence of three substrates with Mrs of 45,000, 21,000, and 17,000. While the role of these substrates in exocytosis is unknown, the results suggest that the Mr 21,000 substrate is involved in a process other than TNF secretion.
Rapid induction of polyadenylated H1 histone mRNAs in mouse erythroleukemia cells is regulated by c-myc.
Authors: Cheng G H , Skoultchi A I
Published: October 2015

Chemically induced differentiation of murine erythroleukemia cells is a multistep process involving a precommitment period in which exposure to inducer leads to cells that are irreversibly committed to terminal differentiation. Certain changes in the expression of cellular proto-oncogenes are an important feature of the precommitment phase. We have identified two H1 histone genes that are rapidly induced during this period. Unlike most histone genes, these two H1 genes encode polyadenylated mRNAs with long 3' untranslated regions. To investigate the relationship between induction of the H1 mRNAs and changes in proto-oncogene expression, we studied two independent series of mouse erythroleukemia cell lines that are inhibited from differentiating because of deregulated expression of transfected copies of c-myc or c-myb. The results showed that induction of the H1 mRNAs was negatively regulated by c-myc. The two H1 histone genes are among the first examples of specific cellular genes that are regulated by c-myc. The timing of their induction suggests that they may play an important role in achieving commitment to terminal differentiation.
Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.
Authors: Neuhold L A , Shirayoshi Y , Ozato K , Jones J E , Nebert D W
Published: October 2015

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.
Growth factor gene activation and clonal heterogeneity in an autostimulatory myeloid leukemia.
Authors: Leslie K B , Schrader J W
Published: October 2015

Cell lines were isolated from an in vivo-passaged myelomonocytic leukemia, WEHI-274, that arose in a mouse infected with the Abelson leukemia virus-Moloney leukemia virus complex. Clones were isolated in vitro in the presence or absence of a source of a hemopoietic growth factor, interleukin-3 (IL-3), and were divisible into three distinct classes. All three classes were leukemogenic in vivo. In vitro, the class I clone grew slowly at low cell density but responded with an increased growth rate to IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and autoconditioned medium. Supernatants of these cultures contained a factor with the biological, biochemical, and antigenic properties of IL-3. Class II clones grew better in vitro at low cell densities than did the class I clone and also responded with an increased growth rate to IL-3, GM-CSF, and autoconditional medium but produced GM-CSF rather than IL-3. In contrast, class III clones died in vitro at all cell densities unless exogenous IL-3 or GM-CSF was added. Moreover, they produced no autostimulatory factors. In the class I and class II clones, one allele of the respective IL-3 or GM-CSF gene is rearranged, and in each case, grossly abnormal RNA transcripts of the rearranged gene are present. Neither rearrangements nor abnormal RNA transcripts of the IL-3 or GM-CSF gene were detected in the class III clones. All three classes exhibited a common rearrangement of the c-myb gene, which suggested that all were derived from the one ancestral cell. These experiments demonstrate that two distinct and independent autostimulatory events were involved in the progression of a single disease.
Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.
Authors: Braun T , Tannich E , Buschhausen-Denker G , Arnold H H
Published: October 2015

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.
E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex.
Authors: Williams G T , McClanahan T K , Morimoto R I
Published: October 2015

We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.
Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.
Authors: Barnhart K M , Kim C G , Sheffery M
Published: October 2015

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.
Cells that overproduce protein kinase C are more susceptible to transformation by an activated H-ras oncogene.
Authors: Hsiao W L , Housey G M , Johnson M D , Weinstein I B
Published: October 2015

We recently developed rat fibroblast cell lines that stably overproduce high levels of the beta 1 form of protein kinase C (PKC). These cells display several disorders in growth control and form small microscopic colonies in agar. In the present study we demonstrate that one of these cell lines, R6-PKC3, is extremely susceptible to transformation by an activated human bladder cancer c-H-ras oncogene (T24). Compared with control cell line R6-C1, T24-transfected R6-PKC3 cells yielded a 10-fold increase in the formation of large colonies in agar. Cell lines established from these colonies displayed a highly transformed morphology, expressed the T24-encoded p21 ras protein, continued to express high levels of PKC, and were highly tumorigenic in nude mice. These results provide genetic evidence that PKC mediates some of the effects of the c-H-ras oncogene on cell transformation. Data are also presented suggesting that optimum synergistic effects between c-H-ras and PKC require critical levels of their respective activities. These findings may be relevant to the process of multistage carcinogenesis in tissues containing cells with an activated c-H-ras oncogene.
Prosomes and heat shock complexes in Drosophila melanogaster cells.
Authors: de Sa C M , Rollet E , de Sa M F , Tanguay R M , Best-Belpomme M , Scherrer K
Published: October 2015

Prosomes and heat shock protein (HSP) complexes isolated from the cytoplasm of Drosophila cells in culture were biochemically and immunologically characterized. The two complexes were found to separate on sucrose gradients, allowing the analysis of their protein constituents by two-dimensional polyacrylamide gel electrophoresis and by reaction with anti-HSP sera and prosome-specific monoclonal antibodies. All of the prosomal proteins were found to be clearly distinct from the HSP; none of the prosomal proteins was synthesized de novo in heat shock. However, an antiprosome (anti-p27K) monoclonal antibody (mouse anti-duck) recognizing the Drosophila p29K prosomal protein allowed immunoprecipitation from a heat-shocked postmitochondrial supernatant of the crude HSP complex, including the low- and the high-molecular-weight components, in particular the 70 x 10(3)-molecular weight HSP. The highly purified small 16S HSP complex still contained this preexistent p29K prosomal protein, which thus also seems to be a metabolically stable constituent of the HSP complex. The significance of this structural and possibly functional relationship between prosomes and HSP, involving the highly ubiquitous and evolutionarily conserved prosomal protein p27/29K, remains to be elucidated.
Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium.
Authors: Schalch H , Gaskell J , Smith T L , Cullen D
Published: October 2015

The genomic clones encoding lignin peroxidase isozyme H8 and two closely related genes were isolated from Phanerochaete chrysosporium BKM-1767, and their nucleotide sequences were determined. The positions and approximate lengths of introns were found to be highly conserved in all three clones. Analysis of homokaryotic derivatives indicated that the three clones are not alleles of the same gene(s).
Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.
Authors: Mroczkowski B , Reich M , Chen K , Bell G I , Cohen S
Published: October 2015

NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.
Binding of nuclear factor EF-C to a functional domain of the hepatitis B virus enhancer region.
Authors: Ostapchuk P , Scheirle G , Hearing P
Published: October 2015

Nuclear factor EF-C is present in extracts prepared from human HepG2 liver cells and from other, nonliver cell lines and binds to the hepatitis B virus and polyomavirus transcriptional enhancer regions in vitro. An inverted repeat (5'-GTTGCNNNGCAAC-3') is located within both binding regions. Diethyl pyrocarbonate interference binding assays and competition binding experiments using altered binding sites demonstrated that EF-C contacts symmetrical nucleotides within the inverted repeat. Mutations that changed the length of the spacer region between the arms of the inverted repeat were introduced in the hepatitis enhancer region. Introduction of 1 or 2 base pairs between the repeats did not affect EF-C binding, but deletion of 1 base pair or introduction of 3 to 9 base pairs reduced binding dramatically. Introduction of 10 base pairs restored partial EF-C binding ability. These and other results suggest that EF-C binding is stabilized by dimerization. In vivo assays for enhancer function using these mutants demonstrated that the EF-C binding site is a functional and important component of the hepatitis B virus enhancer region.