bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of yeast histone genes by stimulation of stationary-phase cells.
Authors: Drebot M A , Veinot-Drebot L M , Singer R A , Johnston G C
Published: October 2015

In the cell cycle of the budding yeast Saccharomyces cerevisiae, expression of the histone genes H2A and H2B of the TRT1 and TRT2 loci is regulated by the performance of "start," the step that also regulates the cell cycle. Here we show that histone production is also subject to an additional form of regulation that is unrelated to the mitotic cell cycle. Expression of histone genes, as assessed by Northern (RNA) analysis, was shown to increase promptly after the stimulation, brought about by fresh medium, that activates stationary-phase cells to reenter the mitotic cell cycle. The use of a yeast mutant that is conditionally blocked in the resumption of proliferation at a step that is not part of the mitotic cell cycle (M.A. Drebot, G.C. Johnston, and R.A. Singer, Proc. Natl. Acad. Sci. 84:7948, 1987) showed that this increased gene expression that occurs upon stimulation of stationary-phase cells took place in the absence of DNA synthesis and without the performance of start. This stimulation-specific gene expression was blocked by the mating pheromone alpha-factor, indicating that alpha-factor directly inhibits expression of these histone genes, independently of start.
Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene.
Authors: Saatcioglu F , Perry D J , Pasco D S , Fagan J B
Published: October 2015

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.
Authors: Lees-Miller S P , Chen Y R , Anderson C W
Published: October 2015

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.
Positive and negative transcriptional elements of the human type IV collagenase gene.
Authors: Frisch S M , Morisaki J H
Published: October 2015

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.
Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.
Authors: Hobson G M , Molloy G R , Benfield P A
Published: October 2015

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.
Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.
Authors: All-Robyn J A , Brown N , Otaka E , Liebman S W
Published: October 2015

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.
Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.
Authors: Shaulsky G , Goldfinger N , Ben-Ze'ev A , Rotter V
Published: October 2015

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.
Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line.
Authors: Chavrier P , Vingron M , Sander C , Simons K , Zerial M
Published: October 2015

Molecular analysis of Saccharomyces cerevisiae secretion mutants has led to the identification of two Ras-like GTP-binding proteins, Ypt1p and Sec4p, which are essential for transport along the exocytic route. To study the regulation of membrane traffic in epithelial cells, a set of 11 clones encoding proteins similar to the YPT1/SEC4 products were isolated from an MDCK (Madin-Darby canine kidney) cell cDNA library. Four of these proteins, Rab8, -9, -10, and -11, are novel members of this subfamily of Ras-like proteins, and two of them are closely related to Ypt1p and Sec4p. The ratio of the number of clones isolated over the total number screened reveals a high level of complexity for this subfamily of GTP-binding proteins. This diversity supports their proposed function in controlling different steps in membrane traffic.
Functional expression of the genomic DNA sequences encoding mouse Na,K-ATPase alpha 1 gene by cotransfection of overlapping genomic DNA segments.
Authors: Tam S Y , Geissler E N , Graw S L , Housman D E
Published: October 2015

The entire 33-kb coding region of the mouse Na,K-ATPase alpha 1 subunit gene was cloned in two overlapping cosmids which contain inserts of 40 kb. To assess the functional expression of the mouse alpha 1 gene, the two cosmids were cotransfected into ouabain-sensitive CV-1 monkey cells yielding an average of 64 resistant colonies per 10(6) cells per microgram of DNA. Analysis of the DNA transferred to the ouabain-resistant transformants by the two cosmids suggests that the generation of a functional gene can occur by homologous recombination between the two introduced segments, as demonstrated by generation of a novel diagnostic restriction fragment. The ability to reconstruct the intact mouse alpha 1 gene in a heterologous host cell and to monitor its functional expression with a selection protocol permits direct identification and isolation of regulatory sequences for the gene.
Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.
Authors: Blake M C , Jambou R C , Swick A G , Kahn J W , Azizkhan J C
Published: October 2015

Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.
Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity.
Authors: Yu C L , Tsai M H , Stacey D W
Published: October 2015

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced. One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.
Regulation of N-myc gene expression: use of an adenovirus vector to demonstrate posttranscriptional control.
Authors: Babiss L E , Friedman J M
Published: October 2015

We present evidence that differences in the levels of N-myc mRNA among different cell types are the result of posttranscriptional control. First, we noted that while steady-state mouse N-myc mRNA could be detected only in fetal mouse brain, it was transcribed at an equivalent rate in adult brain, liver, spleen, and placenta and in fetal brain. Similarly, the human N-myc gene was transcribed at an equivalent rate in HeLa cells, which do not accumulate this RNA in the cytoplasm, and cell lines G401 (a Wilms tumor-derived cell line) and SKNMc (established from a primitive neuroepithelioma), which do express N-myc RNA. As expected, the N-myc promoter functioned at equivalent rates, as demonstrated by the level of a reporter gene, when introduced into these cell types by using a recombinant adenovirus vector. The suggestion that posttranscriptional mechanisms control the level of this RNA was supported by the observation that sequences in the N-myc third exon specifically decreased the level of E1A mRNA when these sequences were placed downstream of the E1A promoter in a recombinant adenovirus. Finally, we further localized these sequences to a 600-bp fragment of the third exon by introducing various subclones of this sequence downstream of the E1A promoter in both viral and plasmid vectors.
A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter.
Authors: Lum L S , Sultzman L A , Kaufman R J , Linzer D I , Wu B J
Published: October 2015

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.
Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.
Authors: Silver G , Krauter K S
Published: October 2015

We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.
Regulated expression of the small nuclear ribonucleoprotein particle protein SmN in embryonic stem cell differentiation.
Authors: Sharpe N G , Williams D G , Latchman D S
Published: October 2015

The SmN protein is a component of small nuclear ribonucleoprotein particles and is closely related to the ubiquitous SmB and B' splicing proteins. It is expressed in a limited range of tissues and cell types, including several undifferentiated embryonal carcinoma cell lines and undifferentiated embryonic stem cells. The protein declines to undetectable levels when embryonal carcinoma or embryonic stem cells are induced to differentiate, producing primitive endoderm or parietal endoderm or yielding embryonal bodies. This decline is due to a corresponding decrease in the level of the SmN mRNA. The potential role of SmN in the regulation of alternative splicing in embryonic cell lines and early embryos is discussed.
Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.
Authors: Wilson C , Gilmore R , Morrison T
Published: October 2015

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a type II glycoprotein oriented in the plasma membrane with its amino terminus in the cytoplasm and its carboxy terminus external to the cell. We have previously shown that the membrane insertion of HN protein requires signal recognition particle SRP, occurs cotranslationally, and utilizes the same GTP-dependent step that has been described for secretory proteins, type I proteins, and multispanning proteins (C. Wilson, R. Gilmore, and T. Morrison, Mol. Cell. Biol. 7:1386-1392, 1987; C. Wilson, T. Connolly, T. Morrison, and R. Gilmore, J. Cell Biol. 107:69-77, 1988). The role of the amino-terminal cytoplasmic domain in the faithful membrane insertion of this type II protein was explored by characterizing the membrane integration of a mutant lacking 23 of the 26 amino acids of the cytoplasmic domain. The mutant protein was able to interact with SRP, resulting in translation inhibition, membrane targeting, and membrane translocation, but the efficiency of translocation was considerably lower than for the wild-type HN protein. In addition, a significant proportion of the mutant protein synthesized in the presence of SRP and microsomal membranes was associated with the membrane in an EDTA- and alkali-insensitive manner yet integrated into membranes with its carboxy-terminal domain on the cytoplasmic side of membrane vesicles. Membrane-integrated molecules with this reverse orientation were not detected when the mutant protein was synthesized in the absence of SRP or a functional SRP receptor. Truncated mRNAs encoding amino-terminal segments of the wild-type and mutant proteins were translated to prepare ribosomes bearing arrested nascent chains. The arrested mutant nascent chain, in contrast to the wild-type nascent chain, was also able to insert into membranes in a GTP- and SRP-independent manner. Results suggest that the cytoplasmic domain plays a role in the proper membrane insertion of this type II glycoprotein.
Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.
Authors: Tonks N K , Cicirelli M F , Diltz C D , Krebs E G , Fischer E H
Published: October 2015

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.
In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene.
Authors: Klemm D J , Roesler W J , Liu J S , Park E A , Hanson R W
Published: October 2015

A cell-free system for the study of transcription from the promoter of the phosphoenolpyruvate carboxykinase (GTP) gene by using nuclear extracts from rat tissues was developed. The level of basal transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter between -490 and +73 was highest when extracts from liver nuclei, rather than kidney, spleen, and HeLa nuclear extracts, were used. A series of 5' deletions and block mutations were also tested for their effects on basal transcription in vitro. The promoter truncated to -355 had the highest rate of basal transcription, while subsequent deletion to -277 markedly decreased the rate of transcription. Further deletion of the promoter to -134 resulted in a twofold increase in the basal level of transcription compared with that of the promoter deleted to -277. However, subsequent deletion of the NF-1-CCAAT-binding transcription factor binding site or the proximal cyclic AMP (cAMP) regulatory element caused a decrease in basal transcription. Block mutations were inserted into nine specific protein-binding regions of the PEPCK promoter previously shown to be of functional significance or to bind nuclear proteins. Mutation of the TATA box resulted in a 94% decrease in the level of transcription noted with the intact promoter, while sequence substitutions within the proximal cAMP regulatory element decreased the transcription rate to 25%. The addition of the catalytic subunit of cAMP-dependent protein kinase to the in vitro system stimulated transcription from the intact promoter or from a promoter deletion to -109. However, a promoter deletion to -68, which removes the proximal cAMP regulatory element, was unresponsive to added protein kinase catalytic subunit. These findings indicate that the PEPCK promoter between -490 and +73 contains sequences responsive to hormonal and tissue-specific factors in nuclei from rat tissues. The sensitivity of this in vitro transcription system closely mimics the process regulating PEPCK transcription in rat tissues and should make it ideal for testing the function of purified transcription factors.
Rapid and transient decrease of N-myc expression in retinoic acid-induced differentiation of OTF9 teratocarcinoma stem cells.
Authors: Kato K , Kanamori A , Kondoh H
Published: October 2015

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.
The Adh gene promoters of Drosophila melanogaster and Drosophila orena are functionally conserved and share features of sequence structure and nuclease-protected sites.
Authors: Moses K , Heberlein U , Ashburner M
Published: October 2015

The sibling species Drosophila melanogaster and D. orena show similar patterns of alcohol dehydrogenase expression, both spatially and temporally. These two species diverged from a common ancestor 6 million to 15 million years ago, and the DNA sequences of the promoter regions of their Adh genes show a mosaic pattern of conservation and change. By interspecific transformation of D. orena sequences into D. melanogaster, we demonstrate a functional equivalence between these sequences. Using both D. melanogaster embryo extracts and purified transcription factor Adf-1, we compare the protection of these promoter sequences from nuclease, demonstrating considerable conservation.
Schizosaccharomyces pombe ras1 and byr1 are functionally related genes of the ste family that affect starvation-induced transcription of mating-type genes.
Authors: Nadin-Davis S A , Nasim A
Published: October 2015

We have further investigated the function of the ras1 and byr1 genes, which were previously shown to be critical for sexual differentiation in fission yeast cells. Several physiological similarities between strains containing null alleles of these genes supports the idea that ras1 and byr1 are functionally closely related. Furthermore, we have found that byr1 is allelic to ste1, one of at least 10 genes which when mutated can cause sterility. Since ras1 had previously been found to be allelic to ste5, both ras and byr genes are now clearly shown to be a part of the ste gene family, thus confirming their close functional relationship. The observation that the mating-type loci could overcome the sporulation block of ras1 and byr1 mutant strains prompted investigation of the role of the ras-byr pathway in the induction of the mating-type gene transcripts upon nitrogen starvation. By Northern analysis of RNA preparations from strains carrying wild-type or mutant ras1 alleles and grown to different stages of the growth cycle, we have shown that ras1 plays an important role in inducing the Pi transcript of the mating-type loci and the mei3 gene transcript. These observations provide a molecular basis for the role of the ste gene family, including ras1 and byr1, in meiosis and indicate that further characterization of other ste genes would be very useful for elucidating the mechanism of ras1 function in fission yeast cells.
Characterization of the S-phase-specific transcription regulatory elements in a DNA replication-independent testis-specific H2B (TH2B) histone gene.
Authors: Hwang I W , Lim K , Chae C B
Published: October 2015

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes of meiotic prophase I during rat spermatogenesis. The TH2B RNA and histones are not synthesized in any other tissues, and the synthesis is independent of DNA replication. However, the cloned TH2B gene has two DNA sequence elements which stimulate transcription of the cloned gene in an S-phase-dependent manner when introduced into somatic cells. The factors interacting with the two elements, CCAAT at -127 base pairs and octamer ATTTGCAT at -93 base pairs, interact with each other to bring about a maximum stimulation of S-phase-dependent transcription. The level of CCAAT and octamer-binding proteins is unchanged during the cell cycle, and the S-phase-dependent transcription of TH2B and endogenous mouse H2B genes does not require synthesis of new proteins during the S phase. Cell cycle-specific posttranslational modification of regulatory proteins may be responsible for the S-phase-dependent transcription of H2B histone genes. The biological significance of the presence of S-phase-specific transcription regulatory elements in the DNA replication-independent and tissue-specific TH2B gene is not known.
Mouse Zfx protein is similar to Zfy-2: each contains an acidic activating domain and 13 zinc fingers.
Authors: Mardon G , Luoh S W , Simpson E M , Gill G , Brown L G , Page D C
Published: October 2015

The Zfy gene is located on the Y chromosome of placental mammals and encodes a zinc finger protein which may serve as the primary sex-determining signal. A related gene, Zfx, is similarly conserved on the X chromosome. Unlike that in most mammals, the mouse genome contains four homologous zinc finger loci: Zfy-1, Zfy-2, Zfx, and Zfa (on an autosome). We report that, in contrast to the mouse Zfy genes, Zfx is widely transcribed in embryos, newborns, and adults, both male and female. Moreover, Zfx transcripts contain long 3' untranslated sequences which are phylogenetically conserved. Zfa is a processed gene derived from Zfx. An analysis of cDNA clones demonstrated that Zfx encodes a 799-amino-acid protein that is 70% identical to the mouse Zfy-1 and Zfy-2 proteins. Zfx, Zfy-1, and Zfy-2 contain highly acidic amino-terminal domains and carboxy-terminal regions containing 13 zinc fingers. When fused to the DNA-binding domain of GAL4, the acidic domains of Zfx and Zfy-2 activated transcription in yeast cells.
DNA-mediated gene transfer into adult rat hepatocytes in primary culture.
Authors: Rippe R A , Brenner D A , Leffert H L
Published: October 2015

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.
Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.
Authors: McClinton D , Stafford J , Brents L , Bender T P , Kuehl W M
Published: October 2015

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.
Premature termination of transcription can be induced on an injected alpha-tubulin gene in Xenopus oocytes.
Authors: Middleton K M , Morgan G T
Published: October 2015

The Xenopus laevis alpha-tubulin gene X alpha T14, which is highly expressed during oogenesis, exhibits accurate and efficient transcription initiation when microinjected into X. laevis oocytes. However, we found previously in nuclease protection assays of transcripts from injected X alpha T14 that many protected fragments that were shorter than expected could be produced. We show here by exonuclease VII mapping, Northern (RNA) blotting, and gel fractionation of RNA that these fragments were caused by truncated transcripts that share the same initiation sites as mature transcripts but whose 3' ends are located in the 5' leader just 45 to 72 nucleotides downstream. We present evidence from the analysis of in vitro pulse-labeled RNA that these truncated transcripts are formed by premature transcription termination rather than by RNA processing. At low template levels, very little premature termination occurred, but as more DNA was injected, the proportion of transcripts that were prematurely terminated increased steadily, even at template levels at which the initiation machinery was unsaturated. At high template levels, most transcripts were prematurely terminated. These results suggest that some sort of saturable antitermination function operates in oocytes in a manner that is dependent on the number of appropriate templates available rather than on the number of polymerases that initiate transcription. They also suggest that measures of initiation frequency may not always be a reliable means of assessing the amount of transcription of injected genes in oocytes.
Reconstitution of an insulin signaling pathway in Xenopus laevis oocytes: coexpression of a mammalian insulin receptor and three different mammalian hexose transporters.
Authors: Vera J C , Rosen O M
Published: October 2015

We report the functional expression of the mammalian muscle-adipocyte insulin-sensitive hexose transporter in Xenopus laevis oocytes. Oocytes microinjected with RNA synthesized in vitro showed enhanced hexose transport activity compared with uninjected controls. However, like the endogenous oocyte hexose transporter, activity was stimulated only twofold by 1 microM insulin. X. laevis oocytes injected with in vitro-synthesized RNA encoding the human insulin proreceptor expressed a functionally active insulin receptor that enhanced the insulin sensitivity of injected oocytes. This increase was not observed in oocytes expressing a mutant insulin receptor that lacked protein tyrosine kinase activity. In the presence of the coexpressed human insulin receptor, insulin induced a two- to threefold increase in hexose transport. The muscle-, brain-, and liver-type hexose carriers normally expressed in tissues with different responses to insulin exhibited the same insulin sensitivity when expressed in oocytes. This was observed whether or not the insulin signal was transduced through a coexpressed human insulin receptor or the endogenous oocyte insulin-like growth factor I receptor. We conclude that the expressed human insulin receptor is able to couple efficiently with preexisting postreceptor regulatory pathways in oocytes and that the regulation of hexose transport in these cells can be mediated through the combined actions of the expressed human insulin receptor and the endogenous oocyte insulin-like growth factor I receptor.
Highly conserved upstream regions of the alpha 1-antitrypsin gene in two mouse species govern liver-specific expression by different mechanisms.
Authors: Latimer J J , Berger F G , Baumann H
Published: October 2015

alpha 1-Antitrypsin (AT), the major elastase inhibitor in mammalian serum, is produced primarily in the liver. We have characterized AT gene structure and expression in the mouse species Mus caroli, which expresses high levels of AT in the kidneys as well as in the liver. Analysis of cDNA and genomic clones showed that the AT gene in M. caroli exhibits high sequence homology (greater than 90%) to the gene in laboratory mice (M. domesticus) throughout the coding and 5'-flanking regions. Despite this extensive sequence conservation, the functional organization of cis-acting regulatory elements governing liver-specific expression is strikingly different between these species. Transient-transfection assays showed that the proximal region of the M. caroli promoter (i.e., between -120 and -2 relative to the transcriptional start site) is 10-fold more active than the analogous region of M. domesticus in driving the expression of an indicator gene in cultured liver cells. The increased activity of the proximal region of the M. caroli AT promoter appears to be the result of one or both of the two base substitutions at positions -46 and -48. The weak proximal promoter in M. domesticus is compensated for by the presence of upstream, liver-specific enhancers between -199 and -520; the analogous region in M. caroli is inactive. Thus, during the course of evolution, the modest 7% sequence divergence that has occurred between the 5'-flanking regions of the AT genes in these two species has generated distinct, yet equally effective, modes of hepatocyte-specific expression.
Phosphotyrosine-containing lactate dehydrogenase is restricted to the nuclei of PC12 pheochromocytoma cells.
Authors: Zhong X H , Howard B D
Published: October 2015

There are five lactate dehydrogenase (LDH) isoenzymes, composed of various combinations of two types of subunits. LDH-5, which contains only the LDH A subunit, is known to be present in both the cytoplasm and the nucleus, to act as a single-stranded DNA-binding protein possibly functioning in transcription and/or replication, and to undergo phosphorylation of tyrosine 238 in approximately 1% of the enzyme after cell transformation by certain tumor viruses. We have characterized LDH from wild-type PC12 pheochromocytoma cells and from a PC12 variant (MPT1) that exhibits altered lactate metabolism and altered expression of multiple genes. Wild-type and MPT1 cells contain different proportions of LDH isoenzymes, with LDH-5 being more predominant in wild-type cells than in the variant. A small fraction of LDH from PC12 cells contains phosphotyrosine. Approximately 99% of the total LDH activity is located in the cytoplasm, but all of the phosphotyrosine-containing LDH is located in the nucleus. Furthermore, essentially all of the nuclear LDH contains phosphotyrosine. These results suggest that tyrosine phosphorylation can affect its role in the nucleus.
Increased rRNA gene activity during a specific window of early pea leaf development.
Authors: Baerson S R , Kaufman L S
Published: October 2015

rRNA gene transcription rates were determined during light-mediated leaf development in Pisum sativum. The rate of transcription was observed to increase within 1 day of exposure to light and return to control levels 4 days after exposure. A striking similarity was observed between periods of elevated rRNA gene transcription and increased mitotic activity, suggesting a possible link between the two events.
Priming affects the activity of a specific region of the promoter of the human beta interferon gene.
Authors: Dron M , Lacasa M , Tovey M G
Published: October 2015

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.
The src protein contains multiple domains for specific attachment to membranes.
Authors: Kaplan J M , Varmus H E , Bishop J M
Published: October 2015

The proteins encoded by the oncogene v-src and its cellular counterpart c-src (designated generically here as pp60src) are tightly associated with both plasma membranes and intracellular membranes. This association is due in part to the amino-terminal myristylation of pp60src, but several lines of evidence suggest that amino-terminal portions of the protein itself are also involved. We now report that pp60src contains at least three domains which, in conjunction with myristylation, are capable of mediating attachment to membranes and determining subcellular localization. We identified these domains by fusing various portions of pp60src to pyruvate kinase, which is normally a cytoplasmic protein. Amino acids 1 to 14 of pp60src are sufficient to mediate both myristylation and the attachment of pyruvate kinase to cytoplasmic granules. In contrast, amino acids 38 to 111 mediate association with the plasma membrane and perinuclear membranes, whereas amino acids 204 to 259 mediate association primarily with perinuclear membranes. We conclude that pp60src contains independent domains that target the protein to distinctive subcellular locations and thus may facilitate diverse biological functions of the protein.