bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.
Authors: Rushforth A M , Saari B , Anderson P
Published: October 2015

We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.
Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.
Authors: Gruda M C , Zabolotny J M , Xiao J H , Davidson I , Alwine J C
Published: October 2015

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS)
Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.
Authors: Lommel L , Hanawalt P C
Published: October 2015

A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80% of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.
Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.
Authors: Murnane J P , Yu L C
Published: October 2015

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.
Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms.
Authors: Barbas J A , Galceran J , Torroja L , Prado A , Ferrús A
Published: October 2015

The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6a1, 6a2, 6b1, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6b1, resulting in the failure to produce all exon 6b1-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.
Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.
Authors: Bagrodia S , Taylor S J , Shalloway D
Published: October 2015

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.
p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.
Authors: Krook A , Rapoport M J , Anderson S , Pross H , Zhou Y C , Denhardt D T , Delovitch T L , Haliotis T
Published: October 2015

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.
Retroposition in a family of carcinoma-associated antigen genes.
Authors: Linnenbach A J , Seng B A , Wu S , Robbins S , Scollon M , Pyrc J J , Druck T , Huebner K
Published: October 2015

The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q.
Hibernation-associated gene regulation of plasma proteins with a collagen-like domain in mammalian hibernators.
Authors: Takamatsu N , Ohba K , Kondo J , Kondo N , Shiba T
Published: October 2015

In mammals, hibernation is expressed by only a limited number of species, and the molecular mechanisms underlying hibernation are not well understood. Recently, we have found plasma proteins which disappear from blood specifically during hibernation in a mammalian hibernator, the chipmunk. Here, we report the cDNA cloning of these chipmunk hibernation-related proteins, HP-20, -25, and -27, and analyses of their expression. All three proteins contain a collagen-like domain near the N terminus and are highly homologous to each other. Their mRNAs were detected only in liver in nonhibernating chipmunks, and in hibernating chipmunks, the amounts were reduced to less than 1/10 of those in nonhibernating chipmunks, indicating that HP-20, -25, and -27 mRNA expression is regulated similarly in association with hibernation. Southern blot analyses of the squirrel family with each of chipmunk HP-20, -25, and -27 cDNA revealed that a nonhibernating species (tree squirrel) as well as another hibernating species (ground squirrel) retained the corresponding genes. However, their transcripts were detected only with the hibernating species, and in hibernating ground squirrels, their levels were greatly reduced compared with those in nonhibernating animals, as were the cases with the chipmunk. These observations are the first line of evidence for occurrence of hibernation-associated gene regulation. The results would indicate the commitment of HP-20, -25, and -27 to hibernation and support the idea that genetic controls are involved in mammalian hibernation.
Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.
Authors: Ash J , Ke Y , Korb M , Johnson L F
Published: October 2015

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.
Functional analysis of developmentally regulated chromatin-hypersensitive domains carrying the alpha 1-fetoprotein gene promoter and the albumin/alpha 1-fetoprotein intergenic enhancer.
Authors: Bernier D , Thomassin H , Allard D , Guertin M , Hamel D , Blaquière M , Beauchemin M , LaRue H , Estable-Puig M , Bélanger L
Published: October 2015

During liver development, the tandem alpha 1-fetoprotein (AFP)/albumin locus is triggered at the AFP end and then asymmetrically enhanced; this is followed by autonomous repression of the AFP-encoding gene. To understand this regulation better, we characterized the two early developmental stage-specific DNase I-hypersensitive (DH) sites so far identified in rat liver AFP/albumin chromatin: an intergenic DH-enhancer site and the AFP DH-promoter site. Mutation-transfection analyses circumscribed the DH-enhancer domain to a 200-bp DNA segment stringently conserved among species. Targeted mutations, DNA-protein-binding assays, and coexpression experiments pinpointed C/EBP as the major activatory component of the intergenic enhancer. Structure-function relationships at the AFP DH-promoter site defined a discrete glucocorticoid-regulated domain activated cooperatively by HNF1 and a highly specific AFP transcription factor, FTF, which binds to a steroid receptor recognition motif. The HNF1/FTF/DNA complex is deactivated by glucocorticoid receptors or by the ubiquitous factor NF1, which eliminates HNF1 by competition at an overlapping, high-affinity binding site. We propose that the HNF1-NF1 site might serve as a developmental switch to direct autonomous AFP gene repression in late liver development. We also conclude that the intergenic enhancer is driven by C/EBP alpha primarily to fulfill albumin gene activation functions at early developmental stages. Factor FTF seems to be the key regulator of AFP gene-specific functions in carcinoembryonic states.
Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases.
Authors: Hurley T R , Hyman R , Sefton B M
Published: October 2015

Expression of the CD45 tyrosine protein phosphatase is required for the response of functional lymphocytes to stimulation through the antigen receptor. One or more of its substrates may therefore be essential for signal transduction during lymphocyte activation. We have studied the phosphorylation of the closely related lck, fyn, and c-src tyrosine protein kinases in leukemic murine T-cell lines that have lost the expression of CD45. The phosphorylation of the lck kinase at an inhibitory site of tyrosine phosphorylation, Tyr-505, was increased by two-, six-, and eightfold in three different cell lines. Phosphorylation of the fyn kinase at the homologous site, Tyr-531, was unaltered in one of these cell lines, but increased by 2.5-fold in the two others. The phosphorylation of p60c-src at the homologous tyrosine was essentially unchanged in the one CD45-negative cell line in which it was examined. The expression of CD45 therefore regulates the phosphorylation and potentially the activity of the lck and fyn tyrosine protein kinases, but the effect on the lck kinase is much greater than on the fyn kinase. This finding and the observation that CD45 had no effect on the phosphorylation of p60c-src suggest that CD45 exhibits polypeptide substrate specificity in vivo. Additionally, these findings are consistent with the hypothesis that the unresponsiveness of CD45-negative lymphoid cells to antigenic stimulation is due largely to hyperphosphorylation of the lck kinase.
Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.
Authors: Moore P A , Ruben S M , Rosen C A
Published: October 2015

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.
A complex promoter element mediates transactivation of the human proliferating cell nuclear antigen promoter by the 243-residue adenovirus E1A oncoprotein.
Authors: Labrie C , Morris G F , Mathews M B
Published: October 2015

The adenovirus E1A oncoproteins interfere with the normal regulation of cellular proliferation through interactions with cell cycle regulatory proteins. In view of the essential role of proliferating-cell nuclear antigen (PCNA) in DNA replication, we performed a mutational analysis of the minimal human PCNA promoter (nucleotides -87 to +62) to define sequence elements which mediate transactivation by the 243-residue E1A protein (E1A 243R). Linker-scanning and site-directed mutants were examined for basal and E1A-induced expression of chloramphenicol acetyltransferase (CAT) from PCNA promoter-CAT reporter constructs transiently expressed in HeLa cells. The results define the cis-acting element required for induction of PCNA by E1A 243R as a region between -59 and -45 relative to the transcription initiation site. This PCNA E1A-responsive element (PERE), which is protected from DNase I digestion by nuclear extracts from 293 cells, includes the sequence AGCGTGG immediately upstream of the ATF binding site previously shown to be important for activation of PCNA by E1A 243R (G. F. Morris and M. B. Mathews, J. Virol. 65:6397-6406, 1991). Mutation of either the upstream component or the ATF site within the PERE diminishes basal promoter activity and abrogates transactivation by E1A 243R. This novel cis-acting element is also essential for both basal and E1A-induced expression in the context of the full-length PCNA promoter.
SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence.
Authors: Pendergast A M , Gishizky M L , Havlik M H , Witte O N
Published: October 2015

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.
A dosage-dependent suppressor of a temperature-sensitive calmodulin mutant encodes a protein related to the fork head family of DNA-binding proteins.
Authors: Zhu G , Muller E G , Amacher S L , Northrop J L , Davis T N
Published: October 2015

The cmd1-1 mutation of calmodulin causes temperature-sensitive growth in Saccharomyces cerevisiae. We have isolated a dosage-dependent suppressor of cmd1-1, designated HCM1. Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1. Thus, overexpression of HCM1 does not bypass the requirement for calmodulin but enhances the ability of the mutant calmodulin to function. HCM1 is not essential for growth, but deletion of HCM1 exacerbates the phenotype of a strain carrying cmd1-1. HCM1 is located on chromosome III, which was recently sequenced. Our results correct errors in the published DNA sequence. The putative polypeptide encoded by HCM1 is 564 amino acids long and has a predicted molecular weight of 63,622. Antisera prepared against Hcm1p detect a protein that is overproduced in yeast strains overexpressing HCM1 and has an apparent molecular mass of 65 kDa. Eighty-six amino acid residues in the N terminus of Hcm1p show 50% identity with a DNA-binding region of the fork head family of DNA-binding proteins. When fused to the DNA-binding domain of Gal4p, residues 139 to 511 of Hcm1p can act as a strong activator of transcription. However, overexpression of HCM1 does not affect the expression of calmodulin. Furthermore, Hcm1p does not bind to calmodulin in a gel overlay assay. Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism.
Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.
Authors: Wang H , Stillman D J
Published: October 2015

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.
Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.
Authors: Aoyama A , Fröhli E , Schäfer R , Klemenz R
Published: October 2015

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.
COUP-TF acts as a competitive repressor for estrogen receptor-mediated activation of the mouse lactoferrin gene.
Authors: Liu Y , Yang N , Teng C T
Published: October 2015

We previously demonstrated that the estrogen response module (mERM) of the mouse lactoferrin gene, which contains an overlapping chicken ovalbumin upstream promoter transcription factor (COUP-TF)- and estrogen receptor-binding element, is responsible for estrogen induction. In this report we show that COUP-TF represses the mERM response to estrogen stimulation. Mutation and deletion of the COUP-TF-binding element or reduction of the endogenous COUP-TF increases mERM estrogen responsiveness. Likewise, overexpression of the COUP-TF expression vector blocked the estrogen-stimulated response of mERM in transfected cells. The molecular mechanism of this repression is due to the competition between COUP-TF and the estrogen receptor for binding at identical contact sites in the overlapping region of the mERM. Our results indicate that two members of the steroid-thyroid receptor superfamily work in concert to modulate lactoferrin gene expression.
Downregulation of Ke 6, a novel gene encoded within the major histocompatibility complex, in murine polycystic kidney disease.
Authors: Aziz N , Maxwell M M , St Jacques B , Brenner B M
Published: October 2015

Polycystic kidney disease (PKD) is characterized by progressive enlargement of the kidneys due to numerous expanding cysts ultimately leading to renal failure. We have identified a gene, Ke 6, located within the H-2K/tw5 region on mouse chromosome 17, which is downregulated in two distinct murine models of heritable PKD. Ke 6 is a member of the short-chain alcohol dehydrogenase family and possess remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function. The Ke 6 gene gives rise to two transcripts--a 1-kb Ke 6a mRNA which is abundant in kidney and liver tissue and a 1.4-kb Ke 6b mRNA which is found at a moderate level in spleen tissue. We report here the complete nucleotide sequence of Ke 6a cDNA and the expression of the Ke 6 gene in murine models of PKD. The Ke 6 gene may be intimately involved in the manifestation of these cystic kidney diseases.
PRP19: a novel spliceosomal component.
Authors: Cheng S C , Tarn W Y , Tsao T Y , Abelson J
Published: October 2015

We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae. The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base. Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif. Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction. Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components.
Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA.
Authors: Cheng J , Maquat L E
Published: October 2015

The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to approximately 20% of normal by frameshift and nonsense mutations that cause translation to terminate at a nonsense codon within the first three-fourths of the reading frame. Results of previous studies inhibiting RNA synthesis with actinomycin D suggested that the decrease is not attributable to an increased rate of cytoplasmic mRNA decay. However, the step in TPI RNA metabolism that is altered was not defined, and the use of actinomycin D, in affecting all polymerase II-transcribed genes, could result in artifactual conclusions. In data presented here, the nonsense codon-mediated reduction in the level of TPI mRNA is shown to be characteristic of both nuclear and cytoplasmic fractions of the cell, indicating that the altered metabolic step is nucleus associated. Neither aberrancies in gene transcription nor aberrancies in RNA splicing appear to contribute to the reduction since there were no accompanying changes in the amount of nuclear run-on transcription, the level of any of the six introns in TPI pre-mRNA, or the size of processed mRNA in the nucleus. Deletion of all splice sites that reside downstream of a nonsense codon does not abrogate the reduction, indicating that the reduction takes place independently of the splicing of a downstream intron. Experiments that placed TPI gene expression under the control of the human c-fos promoter, which can be transiently activated by the addition of serum to serum-deprived cells, verified that there is no detectable effect of a nonsense codon on the turnover of cytoplasmic mRNA.
Epidermal growth factor regulates the exchange rate of guanine nucleotides on p21ras in fibroblasts.
Authors: Buday L , Downward J
Published: October 2015

Treatment of intact Rat-1 fibroblasts with epidermal growth factor (EGF) leads to rapid activation of cellular ras-encoded proteins. By using the bacterial toxin streptolysin O to permeabilize these cells, it was shown that the low basal rate at which guanine nucleotides bind to, and dissociate from, ras-encoded protein in quiescent fibroblasts was greatly accelerated by EGF treatment. Nucleotide binding to other proteins was not affected. Stimulation of nucleotide exchange on ras-encoded protein required tyrosine kinase but not phospholipase activity. EGF had no effect on total GTPase-activating protein activity. Regulation of ras-encoded protein in Rat-1 fibroblasts is therefore mediated by stimulation, either directly or indirectly, of ras-encoded protein-specific guanine nucleotide exchange factors by the EGF receptor tyrosine kinase.
Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway.
Authors: Nakai A , Morimoto R I
Published: October 2015

We have cloned three avian heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and the avian homologs of mammalian HSF1 and HSF2. The predicted amino acid sequence of HSF3 is approximately 40% related to the sequence of HSF1 and HSF2. The sequences for all three factors exhibit extensive identify in the DNA binding motifs and the heptad repeats of hydrophobic amino acids which are common to all eukaryotic HSFs. Despite these overall similarities, each avian HSF exhibits distinct DNA binding properties. HSF2 when expressed in vitro binds constitutively to the heat shock element promoter sequence, whereas neither HSF1 nor HSF3 expressed in vitro binds to DNA. HSF1 DNA binding is induced upon heat shock or treatment with nonionic detergents, whereas the DNA binding properties of HSF3 are not induced by these conditions in vitro. These results suggest that HSF3 activation may involve an induction pathway distinct from the traditional forms of heat shock gene induction. HSF3 DNA binding activity, however, is obtained when the carboxyl-terminal region including the distal heptad repeat is deleted, indicating the presence of negative cis-regulatory sequences. The HSF3 message, like HSF1 and HSF2 messages, is coexpressed during development and in most tissues, which suggests a general role for the regulatory pathway involving HSF3.
Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.
Authors: Webster M K , Goya L , Ge Y , Maiyar A C , Firestone G L
Published: October 2015

A novel member of the serine/threonine protein kinase gene family, designated sgk, for serum and glucocorticoid-regulated kinase, was identified in a differential screen for glucocorticoid-inducible transcripts expressed in the Con8.hd6 rat mammary tumor cell line. sgk encodes a protein of 49 kDa which has significant sequence homology (45 to 55% identity) throughout its catalytic domain with rac protein kinase, the protein kinase C family, ribosomal protein S6 kinase, and cyclic AMP-dependent protein kinase. sgk mRNA is expressed in most adult rat tissues, with the highest levels in the thymus, ovary, and lung, as well as in several rodent and human cell lines. sgk mRNA was stimulated by glucocorticoids and by serum within 30 min, and both inductions were independent of de novo protein synthesis. The transcriptional regulation by glucocorticoids is a primary response, since the promoter of sgk contains a glucocorticoid response element consensus sequence 1.0 kb upstream of the start of transcription which is able to stimulate chloramphenicol acetyltransferase reporter gene activity in a dexamethasone-dependent manner. Antibodies that specifically recognize sgk-encoded protein on an immunoblot were generated. This protein was shown to increase in abundance with glucocorticoid treatment in a manner which paralleled the mRNA accumulation. This is the first report of a presumed serine/threonine protein kinase that is highly regulated at the transcriptional level by glucocorticoid hormones and suggests a novel interplay between glucocorticoid receptor signalling and a protein kinase of the second messenger family.
Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.
Authors: Shirataki H , Kaibuchi K , Sakoda T , Kishida S , Yamaguchi T , Wada K , Miyazaki M , Takai Y
Published: October 2015

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.
Three downstream sites repress transcription of a Ty2 retrotransposon in Saccharomyces cerevisiae.
Authors: Farabaugh P J , Vimaladithan A , Türkel S , Johnson R , Zhao H
Published: October 2015

Transcription of Ty1 and Ty2 retrotransposons of the yeast Saccharomyces cerevisiae is modulated by multiple downstream regulatory sites. Both transposon families include a positively acting site within the transcribed region which resembles a higher eukaryotic enhancer. We have demonstrated the existence of a repression site distal to the enhancer of the Ty2-917 element. Here we describe experiments investigating the internal structure of this site. We show that this 200-bp region includes three distinct repression sites which we term DRSI (downstream repression site I), DRSII, and DRSIII. Individually each site causes almost twofold repression, and together the sites repress eightfold. Unexpectedly, when the entire region encompassing the DRS sites is moved outside the transcription unit, it acts as a qualitatively positively acting element. In this context the DRS sites still repress transcription, since eliminating them increases transcription further. That the region can activate transcription implies that it includes activation sites in addition to the three repression sites. The change from qualitatively negatively acting to positively acting must reflect a change in the relative effects of the multiple positive and negative sites; when moved outside the transcription unit, the activators predominate. Importantly, DRSII and DRSIII repress transcription autonomously when inserted upstream of a heterologous promoter activated by the transcriptional activator GCN4, showing that they are indeed transcriptional repression sites.
The murine myeloperoxidase promoter contains several functional elements, one of which binds a cell type-restricted transcription factor, myeloid nuclear factor 1 (MyNF1).
Authors: Suzow J , Friedman A D
Published: October 2015

The myeloperoxidase (MPO) gene is expressed specifically in myeloid cells. There is significant homology between the murine and human MPO genes in the 1.6-kb region located upstream of the murine MPO transcription initiation sites. 5',3', and internal deletions of this DNA segment localized several cis-acting DNA elements in the murine MPO promoter which are functional in 32D cl3 cells, a murine myeloblast cell line which expresses MPO. These DNA elements did not function well in mouse L-cell fibroblasts. Additional mutagenesis of the most active promoter region allowed the delimitation of a functional 20-bp segment. Mutation of the enhancer core motif within this segment was functionally deleterious, and an oligonucleotide containing these base pairs increased the activity of a minimal promoter. This same oligonucleotide, but not a mutant variant, could bind a set of nuclear proteins, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -1 beta), present in 32D cl3 cells but absent from L cells, murine erythroleukemia cells, and SP2 lymphoid cells.
GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.
Authors: Belhumeur P , Lee A , Tam R , DiPaolo T , Fortin N , Clark M W
Published: October 2015

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.
SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells.
Authors: Rabin S J , Cleghon V , Kaplan D R
Published: October 2015

To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13suc1-agarose, a reagent that binds p34cdc2/cdk2. By using this reagent, we identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in p13suc1-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc2/cdk2 complexes. In addition, SNT was associated predominantly with nuclear fractions following subcellular fractionation of NGF-treated PC12 cells. Finally, in PC12 cells, NGF-stimulated tyrosine phosphorylation of SNT was dependent on the levels of Trk tyrosine kinase activity and was constitutively induced by expression of pp60v-src. However, Ras was not required for constitutive SNT tyrosine phosphorylation, suggesting that this protein functions distally to Trk and pp60v-src but in a pathway parallel to that of Ras. SNT is the first identified specific target of differentiation factor-induced tyrosine kinase activity in neuronal cells.