bluuebunny/crossref_metadata_2025 huggingface dataset:
The human homolog of the JE gene encodes a monocyte secretory protein.
Authors: Rollins B J , Stier P , Ernst T , Wong G G
Published: October 2015

The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein.
Expression of the MyoD1 muscle determination gene defines differentiation capability but not tumorigenicity of human rhabdomyosarcomas.
Authors: Hiti A L , Bogenmann E , Gonzales F , Jones P A
Published: October 2015

Several human rhabdomyosarcoma cell lines, cultured primary tumor explants, and biopsies of tumor and normal skeletal muscle tissue expressed a 2.0-kilobase transcript that hybridized to the mouse muscle determination gene MyoD1. This transcript was found in tumor cell lines and primary explants that developed multinucleated myotubes but was absent in Wilms' tumors or cell lines and primary explants that developed multinucleated myotubes but was absent in Wilms' tumors or cell lines derived from other mesenchymal tumor cell types. Expression of the human homolog of MyoD1 therefore can define a tumor as a rhabdomyosarcoma. Transfection of the mouse MyoD1 gene into the human rhabdomyosarcoma cell line RD increased the ability of the tumor cells to differentiate into multinucleated myotubes and enhanced myosin heavy-chain gene expression but did not decrease tumorigenicity in nude mice.
Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.
Authors: Baer M , Sancar G B
Published: October 2015

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.
Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes.
Authors: Maryon E , Carroll D
Published: October 2015

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.
A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.
Authors: Query C C , Bentley R C , Keene J D
Published: October 2015

We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.
Developmental appearance of transcription factors that regulate liver-specific expression of the aldolase B gene.
Authors: Tsutsumi K , Ito K , Ishikawa K
Published: October 2015

The region from -202 to -1 of the rat aldolase B (AldB) promoter directs tissue-specific transcription in vitro. Deletion of the half of this promoter distal to its origin reduced its ability for tissue-specific expression. Two protein-binding sites in this distal element have been identified and characterized. One bound a factor named AIF-A, which is probably identical to previously characterized liver-specific factors HNF-1 and APF. The other, containing a CCAAT motif, bound a factor named AIF-B. These two factors were considerably enriched in liver cells compared with other cells not expressing AldB. In the liver, these two factors increased prior to the activation of the AldB gene during development. Liver-specific in vitro transcription assays indicated that the binding of both factors to their target sequences was required for AldB transcription. These results suggest that AIF-A and AIF-B are positively acting transcription factors that regulate tissue-specific and development stage-specific activation of the AldB gene.
Human transcription factor TFIIIC2 specifically interacts with a unique sequence in the Xenopus laevis 5S rRNA gene.
Authors: Fradkin L G , Yoshinaga S K , Berk A J , Dasgupta A
Published: October 2015

Transcription factor TFIIIC2 derived from human cells is required for tRNA-type gene transcription and binds with high affinity to the essential B-box promoter element of tRNA-type genes. Although 5S rRNA genes contain no homology with the tRNA-type gene B box, we show that TFIIIC2 is also required for Xenopus laevis 5S rRNA gene transcription. TFIIIC2 protected an approximately 30-base-pair (-10 to +18) region of a Xenopus 5S rRNA gene from DNase I digestion. This region, which spanned the transcription start site, included sequences that are highly conserved among eucaryotic 5S rRNA genes and have no homology with the B-box sequence of tRNA genes. Mutation of the TFIIIC2-binding site reduced transcription of the 5S rRNA gene by a factor of 10 in HeLa cell extracts. Methylation of C residues within the TFIIIC2-binding site interfered with binding of TFIIIC2. These results suggest a role of the TFIIIC2-binding sequence in 5S rRNA gene transcription. In addition, the 5S rRNA gene binding site and the tRNA-type gene B-box sequence did not compete with each other for binding to TFIIIC2 any better than did an unrelated DNA sequence, indicating that TFIIIC2 interacts with 5S rRNA genes and tRNA-type genes through separate DNA-binding domains or polypeptides.
Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.
Authors: Allison D S , Young E T
Published: October 2015

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.
Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.
Authors: Kozak M
Published: October 2015

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.
Two chicken erythrocyte band 3 mRNAs are generated by alternative transcriptional initiation and differential RNA splicing.
Authors: Kim H R , Kennedy B S , Engel J D
Published: October 2015

The erythrocyte anion transport protein (band 3) mediates two distinct cellular functions: it provides plasma membrane attachment sites for the erythroid cytoskeletal network, and it also functions as the anion transporter between the erythrocyte cytoplasm and extracellular milieu. We previously showed that two chicken band 3 polypeptides are encoded by two different mRNAs with different translation initiation sites. Here we show that these two band 3 mRNAs are transcribed from two separate promoters within a single gene. In addition, the two pre-mRNAs are differentially spliced, leading to fusion with coding exons used in common in the two mRNAs. The chicken erythrocyte band 3 gene is therefore the first example of a gene that has two promoters within a single locus which function equally efficiently in one cell type at the same developmental stage.
Evidence for multiple major histocompatibility class II X-box binding proteins.
Authors: Celada A , Maki R
Published: October 2015

The X box is a loosely conserved DNA sequence that is located upstream of all major histocompatibility class II genes and is one of the cis-acting regulatory elements. Despite the similarity between all X-box sequences, each promoter-proximal X box in the mouse appears to bind a separate nuclear factor.
Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.
Authors: Azzaria M , Schurr E , Gros P
Published: October 2015

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.
Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae.
Authors: Trawick J D , Rogness C , Poyton R O
Published: October 2015

Transcription of Saccharomyces cerevisiae COX6, the nuclear gene for subunit VI of cytochrome c oxidase, is activated in heme-proficient cells, requires the HAP2 gene, and is subject to glucose repression. In this study, by deletion mutagenesis of the COX6 promoter, we identified two regions that are important for transcription. The first was an upstream activation site, UAS6. It was found to be contained within an 84-base-pair (bp) sequence, between bp -256 and -340 of the COX6 translational initiation codon, and to contain sequences required for activation by heme and HAP2 and for release from glucose repression. When located upstream of a CYC1-lacZ fusion gene, deleted for both of its UASs, this segment functioned as a UAS element. Although UAS6 could promote expression in either orientation, it showed a marked orientation dependence in its response to HAP2 and the carbon source. The second region lay between bp -255 and -91. It contained two of the three major 5' termini of COX6 mRNAs and a putative TATA box. Deletion analysis of this region demonstrated that the putative TATA box is not required for transcription and that this region is separable into two redundant domains.
Purification of a protein binding to the CDEI subregion of Saccharomyces cerevisiae centromere DNA.
Authors: Jiang W D , Philippsen P
Published: October 2015

The DNA subregions CDEI and CDEIII of Saccharomyces cerevisiae centromeres are highly conserved, and both are binding sites for proteins. We describe here the purfication of a CDEI-specific binding protein using biotin-labeled synthetic CDEI DNA coupled to streptavidin agarose. The binding properties of this 64-kilodalton (kDa) protein were characterized by competition assays and by methylation interference assays. DNA fragments with single base-pair changes at positions 7 and 8 of CDEI were less efficient competitors than fragments with nonmutated CDEI. Mutations at these positions have previously been shown to decrease centromere activity in vivo. Methylation of guanosines at either side of the 8-base-pair CDEI sequence did not interfere with binding, whereas methylation of any of the four guanosines within CDEI prevented binding. A smaller CDEI-specific binding protein of 37 kDa was also purified and characterized. It is most likely a degradation product of the 64-kDa protein.
The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription.
Authors: Fassler J S , Winston F
Published: October 2015

To understand the function of SPT13, a gene encoding a trans-acting factor involved in regulation of Ty-mediated gene expression, we have cloned and sequenced the gene. Our analysis revealed that SPT13 is the same gene as GAL11, a gene in which mutations cause reduced expression of some GAL4-regulated genes. Further analysis of spt13/gal11 mutants suggested that the SPT13 gene also affects transcription of genes involved in mating-type specialization. We show here that SPT13 has both positive and negative regulatory roles in transcription.
T-kininogen gene expression is induced during aging.
Authors: Sierra F , Fey G H , Guigoz Y
Published: October 2015

We have constructed a cDNA library from senescent (24-month-old) rat liver mRNA and, by differential screening, have selected clones corresponding to mRNA species with increased abundance in aging rats. Direct sequencing of the inserts indicated that most of the clones (9 of 10) contained sequences coding for T-kininogen, also called major acute-phase protein, cysteine protease inhibitor, or thiostatin. Nuclear elongation experiments showed that the increase in mRNA concentration was controlled at the transcriptional level. RNase mapping and S1 analysis indicated that the age-dependent induction operated preferentially at one of the three transcriptional start sites of the gene(s). The acute-phase reaction (inflammation) is known to also induce these genes at the level of transcription; however, two of the three start sites are induced by inflammation. Transcription from one of these sites was induced by both phenomena, aging and inflammation.
Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product.
Authors: Fukui Y , Miyake S , Satoh M , Yamamoto M
Published: October 2015

Mutations in the Schizosaccharomyces pombe ral2 gene cause a phenotype indistinguishable from that of the ras1-defective mutant. Using cloned ral2 DNA, we disrupted the chromosomal gene. The disruptants showed the same phenotype as the original ral2 isolates, i.e., they had spherical cells, had no detectable mating activity, and exhibited no response to the mating pheromone, but their vegetative growth was apparently normal. Sequence analysis of the ral2 gene suggests that it encodes a polypeptide of 611 amino acid residues whose predicted amino acid sequence shows no strong homology to any known protein. Either multiple copies or even a single copy of the ras1Val-17 allele, which is an activated form of ras1, restored rodlike cell morphology and ability to respond to the mating factor to ral2 mutants. These results suggest that the ral2 and ras1 gene products interact intimately and that the ral2 gene product is involved in activation of the ras1 protein in S. pombe.
The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.
Authors: Ma H , Bloom L M , Walsh C T , Botstein D
Published: October 2015

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.
Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.
Authors: Thorsness M , Schafer W , D'Ari L , Rine J
Published: October 2015

Responses of the yeast genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMG1 and HMG2, to in vivo changes in heme concentrations were investigated. Expression of the genes was determined by direct measurement of the mRNA transcribed from each gene, by direct assay of the enzyme activity encoded by each gene, and by measurement of the expression of lacZ fusions to the control regions of each gene. These studies indicated that expression of HMG1 was stimulated by heme, whereas expression of HMG2 was repressed by heme. The effect of heme on HMG1 expression was mediated by the HAP1 transcriptional regulator and was independent of HAP2. Thus, the genes encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase isozymes join a growing list of gene pairs that are regulated by heme in opposite ways.
Definition of an Ets1 protein domain required for nuclear localization in cells and DNA-binding activity in vitro.
Authors: Boulukos K E , Pognonec P , Rabault B , Begue A , Ghysdael J
Published: October 2015

Ets1 and Ets2 are nuclear phosphoproteins which bind to DNA in vitro and share two domains of strong identity. Deletion analyses of each of these conserved regions in Ets1 demonstrated that integrity of the carboxy-terminal domain, also conserved in the more distantly related elk and erg gene products, is essential for both nuclear targeting and DNA-binding activity in vitro.
The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.
Authors: Pawson T , Letwin K , Lee T , Hao Q L , Heisterkamp N , Groffen J
Published: October 2015

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.
Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.
Authors: Laker C , Kluge N , Stocking C , Just U , Franz M J , Ostertag W , DeLamarter J F , Dexter M , Spooncer E
Published: October 2015

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.
Many yeast chromosomes lack the telomere-specific Y' sequence.
Authors: Jäger D , Philippsen P
Published: October 2015

Chromosomal DNAs of 26 different strains representing Saccharomyces species were analyzed by pulsed-field gel electrophoresis and subsequent hybridization to Y' telomere DNA. Hybridization to Y' was found exclusively in Saccharomyces cerevisiae strains, and among these strains, Y' sequences were found to be lacking in small, middle-sized, and large chromosomes.
Participation of multiple factors, including proliferin, in the inhibition of myogenic differentiation.
Authors: Wilder E L , Linzer D I
Published: October 2015

Proliferin (PLF) is a secreted glycoprotein in the prolactin-growth hormone family in mice. PLF expression was detected in C3H 10T1/2 fibroblasts, but not in two 10T1/2-derived myogenic cell lines, and was restored in two nondifferentiating variants of one of these myogenic cell lines. Transient expression of one form of PLF (PLF1) inhibited expression from a muscle-specific gene promoter; a second form of PLF, which differed at three amino acid residues, displayed no activity in this transient assay. Introduction of a PLF1 expression construct into both muscle- and 10T1/2-derived myoblasts resulted in cell lines that were no longer myogenic or that differentiated only partially. Analysis of these cell lines revealed that differentiation could be obstructed at several steps and by one or more factors in addition to PLF. Although expected to function in vivo as an extracellular hormone, PLF did not appear to be acting through a cell surface receptor to inhibit differentiation in these cultured myoblasts.
Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae.
Authors: Nishizawa M , Araki R , Teranishi Y
Published: October 2015

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.
The major low-molecular-weight heat shock protein in chloroplasts shows antigenic conservation among diverse higher plant species.
Authors: Vierling E , Harris L M , Chen Q
Published: October 2015

Several plant species are known to synthesize low-molecular-weight nucleus-encoded heat shock proteins (HSPs) which localize to chloroplasts. DNA sequence analysis of chloroplast HSP cDNAs from pea (Pisum sativum) and soybean (Glycine max) has shown that the carboxyl-terminal halves of these proteins are homologous to low-molecular-weight HSPs from a wide range of eucaryotes (E. Vierling, R. T. Nagao, A. E. DeRocher, and L. M. Harris, EMBO J. 7:575-581, 1988). We used a pea cDNA to construct fusion proteins containing either the carboxyl-terminal heat shock domain or the amino-terminal domain of the chloroplast HSP. The fusion proteins were overexpressed in Escherichia coli and used to produce choloroplast HSP-specific polyclonal antibodies. The carboxyl-terminal antibodies recognized chloroplast HSP precursor proteins from pea and from three divergent plant species, Arabidopsis thaliana, petunia (Petunia hybrida), and maize (Zea mays). The amino-terminal antibodies recognized effectively only the pea precursor. When intact plants of each species were subjected to a heat stress regime mimicking field growth conditions, significant levels of the mature forms of the chloroplast HSPs accumulated in pea, A. thaliana, and maize. The levels of accumulated HSPs remained unchanged for 12 h following the stress treatment. We conclude that the synthesis of chloroplast-localized HSPs is an important component of the stree response in all higher plants and that chloroplast HSPs from dicotyledonous and monocotyledonous plants have a conserved carboxyl-terminal domain.
The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor.
Authors: Boxer L M , Prywes R , Roeder R G , Kedes L
Published: October 2015

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.
Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.
Authors: Lee K F , Atiee S H , Rosen J M
Published: October 2015

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.
Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.
Authors: Reynolds A B , Roesel D J , Kanner S B , Parsons J T
Published: October 2015

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.
Molecular isolation and analysis of the erect wing locus in Drosophila melanogaster.
Authors: Fleming R J , DeSimone S M , White K
Published: October 2015

The molecular study of the erect wing (ewg) locus was initiated by isolating DNA in the 1A8-1B1 interval of the X chromosome. Previous developmental genetic analyses of the mutant alleles at the ewg locus have demonstrated that the wild-type ewg product is essential during embryogenesis and is required postembryonically at least for the development of the indirect flight muscle. To define the ewg-encoding DNA, chromosomal breakpoints that genetically flank the ewg locus were used. P-element-mediated transformation followed by subsequent rescue of the ewg-lethal alleles has defined a 11.5-kilobase genomic fragment as encoding the ewg locus. Northern blot analysis of transcription from this DNA has revealed a complex pattern of transcription with respect to both size and developmental profile. Tissue distribution of putative ewg transcription was examined by in situ hybridization to 6- to 14-h-old embryonic sections. These sections revealed that the expression of putative ewg messages is limited to the central nervous system-derived structures and not observed within the mesoderm during this developmental stage.
A yeast protein possesses the DNA-binding properties of the adenovirus major late transcription factor.
Authors: Chodosh L A , Buratowski S , Sharp P A
Published: October 2015

The adenovirus major late transcription factor (MLTF), or upstream stimulatory factor, is a human promoter-specific transcription factor which recognizes the near-palindromic sequence GGCCACGTGACC (R. W. Carthew, L. A. Chodosh, and P. A. Sharp, Cell 43:439-448, 1985; L. A. Chodosh, R. W. Carthew, and P. A. Sharp, Mol. Cell. Biol. 6:4723-4733, 1986; M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985). We describe here a protein found in the yeast Saccharomyces cerevisiae which possesses DNA-binding properties that are virtually identical to those of human MLTF. These two proteins recognize the same DNA-binding site, make the same purine nucleotide contacts, and are affected in the same manner by mutations in the MLTF-binding site.
Methylation of replicating and nonreplicating DNA in the ciliate Tetrahymena thermophila.
Authors: Harrison G S , Karrer K M
Published: October 2015

Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cells was demonstrated.
cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element.
Authors: Fu Y H , Paietta J V , Mannix D G , Marzluf G A
Published: October 2015

The sulfur-regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. The positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. Expression of the cys-3 regulatory gene was found to be controlled by scon and by sulfur availability. The nucleotide sequence of the cys-3 gene was determined and can be translated to yield a protein of molecular weight 25,892 which displays significant homology with the oncogene protein Fos, yeast GCN4 protein, and sea urchin histone H1. Moreover, the putative cys-3 protein has a well-defined leucine zipper element plus an adjacent charged region which together may make up a DNA-binding site. A cys-3 mutant and a cys-3 temperature-sensitive mutant lead to substitutions of glutamine for basic amino acids within the charged region and thus may alter DNA-binding properties of the cys-3 protein.
The PVT gene frequently amplifies with MYC in tumor cells.
Authors: Shtivelman E , Bishop J M
Published: October 2015

The line of human colon carcinoma cells known as COLO320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 of MYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion of PVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification of MYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC.
Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae.
Authors: Bernstein M , Kepes F , Schekman R
Published: October 2015

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.
Identification of transcriptional regulatory elements in human mitochondrial DNA by linker substitution analysis.
Authors: Topper J N , Clayton D A
Published: October 2015

Human mitochondrial DNA contains two major promoters, one for transcription of each strand of the helix. Previous mapping and mutagenesis data have localized these regulatory elements and have suggested regions important to their function. In order to define, at high resolution, the sequences critical for accurate and efficient transcriptional initiation, a linker substitution analysis of the entire promoter region was performed. Each promoter was shown to consist of approximately 50 base pairs comprising two functionally distinct elements. These and previous data strongly support a mode of transcription initiation requiring minimal sequences surrounding the initiation sites that are likely interactive with core polymerase and upstream regulatory domains capable of binding a transcription factor that modulates the efficiency of transcription initiation. Furthermore, in at least one case, this upstream regulatory domain is capable of operating bidirectionally.
Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.
Authors: Nelson F K , Frankel W , Rajan T V
Published: October 2015

Heterozygous mammalian cell lines normally express both parental alleles at most autosomal loci. However, mutants can be isolated that fail to express one of the alleles. Using a murine pre-B cell line that is heterozygous for several loci on chromosome 12, including one encoding the cell surface antigen Ly-18, we found that one of the two Ly-18 antigenic forms was lost at a rate of 1.5 x 10(-5) per cell per generation. Molecular analysis revealed that a genetic marker distal to Ly-18 became homozygous. Analysis of the genotype of the mutants at the rDNA cluster, located close to the centromere, strongly suggests that the mutants arose by mitotic recombination within this multicopy locus.
Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris.
Authors: Cregg J M , Madden K R , Barringer K J , Thill G P , Stillman C A
Published: October 2015

In Pichia pastoris, alcohol oxidase (AOX) is the first enzyme in the methanol utilization pathway and is encoded by two genes, AOX1 and AOX2. The DNA and predicted amino acid sequences of the protein-coding portions of the genes are closely homologous, whereas flanking sequences share no homology. The functional roles of AOX1 and AOX2 in the metabolism of methanol were examined. Studies of strains with disrupted AOX genes revealed that AOX1 was the major source of methanol-oxidizing activity in methanol-grown P. pastoris. The results of two types of experiments each suggested that the difference in AOX activity contributed by the two genes was a consequence of sequences located 5' of the protein-coding portions of the genes. First, the coding portion of AOX2 was able to functionally substitute for that of AOX1 when placed under the control of AOX1 regulatory sequences. Second, when labeled oligonucleotide probes specific for the 5' nontranslated region of each gene were used, it was apparent that the steady-state level of AOX1 mRNA was much higher than that of AOX2. Except for the difference in the amount of mRNA present, the two genes appeared to be regulated in the same manner. A physiological reason for the existence of AOX2 was sought but was not apparent.
An AP1-binding site in the c-fos gene can mediate induction by epidermal growth factor and 12-O-tetradecanoyl phorbol-13-acetate.
Authors: Fisch T M , Prywes R , Roeder R G
Published: October 2015

We have demonstrated that two sequence elements in the c-fos promoter can mediate the response of the gene to epidermal growth factor and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). The first is the previously described serum response element. The second is a sequence element highly homologous to the consensus binding site for the HeLa cell transcription factor AP1. Although recent reports have shown that fos protein binds to AP1-binding sites through an interaction with AP1 protein and have raised the speculation that fos protein may negatively regulate expression of the c-fos gene via this interaction, we found no role for the AP1 consensus homology in the downregulation of c-fos expression following induction by epidermal growth factor and TPA.
A plant DNA-binding protein that recognizes 5-methylcytosine residues.
Authors: Zhang D L , Ehrlich K C , Supakar P C , Ehrlich M
Published: October 2015

A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.
Transient expression of the proto-oncogene int-1 during differentiation of P19 embryonal carcinoma cells.
Authors: Schuuring E , van Deemter L , Roelink H , Nusse R
Published: October 2015

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.