bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels.
Authors: Zhao Y , Uyttendaele H , Krueger J G , Sudol M , Hanafusa H
Published: October 2015

We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.
The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.
Authors: Udvardy A , Schedl P
Published: October 2015

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.
A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.
Authors: Walden P D , Cowan N J
Published: October 2015

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.
AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster.
Authors: McCullough A J , Schuler M A
Published: October 2015

cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco.
Functional characterization of the L-type pyruvate kinase gene glucose response complex.
Authors: Diaz Guerra M J , Bergot M O , Martinez A , Cuif M H , Kahn A , Raymondjean M
Published: October 2015

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.
BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation.
Authors: Kihara-Negishi F , Tsujita R , Negishi Y , Ariga H
Published: October 2015

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA, preferentially in undifferentiated EC cells versus differentiated cells. Hence, BOX DNA is probably a novel transcriptional element related to EC cell differentiation.
A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.
Authors: Abeliovich H , Tzfati Y , Shlomai J
Published: October 2015

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.
Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene.
Authors: Zhang L , Churchill A C , Kazmierczak P , Kim D H , Van Alfen N K
Published: October 2015

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.
Cell cycle- and terminal differentiation-associated regulation of the mouse mRNA encoding a conserved mitotic protein kinase.
Authors: Lake R J , Jelinek W R
Published: October 2015

We determined the nucleotide sequence of a mouse and a human cDNA, which we designate STPK13, that encodes an apparent protein kinase related to that encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene. The polo and CDC5 gene products are required for normal mitosis. The STPK13 mRNA is regulated during terminal erythrodifferentiation and during the cell cycle. Within the precommitment period of murine erythroleukemia cell terminal differentiation, most of the poly(A) tail is lost from the STPK13 mRNA, but the body of the mRNA remains unchanged in abundance; this poly(A) loss does not occur in mutant erythroleukemia cells that fail to commit to terminal differentiation. During the cell cycle, the abundance of the body of the STPK13 mRNA fluctuates. The mRNA is present in growing but not in nongrowing cells. It reaches a maximum abundance during G2/M phase, is absent or present at only low levels during G1 phase, and begins to reaccumulate at approximately the middle of S phase. The cell cycle-associated accumulation and loss of the STPK13 mRNA could cause a similar fluctuation in abundance of its encoded protein kinase, thereby providing a maximum amount during M phase, when the kinase is thought to function, and little or none at other times of the cell cycle. Posttranscriptional regulation must be responsible for the cell cycle-associated fluctuations because transcription rates are relatively constant during different times of the cell cycle when there are large differences in mRNA abundance.
Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae.
Authors: Russo P , Li W Z , Guo Z , Sherman F
Published: October 2015

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.
The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.
Authors: Pesce S , Benezra R
Published: October 2015

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.
The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.
Authors: Geiser J R , Sundberg H A , Chang B H , Muller E G , Davis T N
Published: October 2015

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.
Premature termination of tubulin gene transcription in Xenopus oocytes is due to promoter-dependent disruption of elongation.
Authors: Hair A , Morgan G T
Published: October 2015

We have shown previously that the Xenopus alpha-tubulin gene, X alpha T14, exhibits premature termination of transcription when injected into oocyte nuclei. The 3' ends of prematurely terminated transcripts are formed immediately downstream of a stem-loop sequence found in the first 41 bp of the 5' leader. We show here, using deleted constructs, that premature termination requires the presence only of sequences from -200 to +19 relative to the initiation site. Deletion of the stem-loop does not increase the production of extended transcripts, and premature termination apparently continues at nonspecific sites. This finding indicates that disruption of the elongation phase of transcription rather than abrogation of a specific antitermination mechanism is the cause of premature termination in X alpha T14. We also found that disruption of elongation on a reporter gene could be induced specifically by competition with X alpha T14 promoters. To identify which elements of the promoter might interact with elongation determinants to cause this competition, we constructed a series of internal promoter mutants. Most mutations in the -200 to -60 region of the promoter had some effect on initiation frequency but did not cause any significant change in levels of premature termination. However, mutations in the core promoter that removed the TATA box consensus causes major change in initiation and resulted in a marked decrease in the production of prematurely terminated transcripts relative to extended transcripts. We discuss why such promoters can apparently escape the disruption of elongation that leads to premature termination.
TATA-binding protein and associated factors in polymerase II and polymerase III transcription.
Authors: Meyers R E , Sharp P A
Published: October 2015

Transcription by RNA polymerase I (pol I), pol II, and pol III requires the TATA-binding protein (TBP). This protein functions in association with distinct TBP-associated factors (TAFs) which may specify the nature of the polymerase selected for initiation at a promoter site. In the pol III transcription system, the TBP-TAF complex is a component of the TFIIIB factor. This factor has been resolved into a TBP-TAF complex and another component, both of which are required for reconstitution of transcription by pol III. Neither the TBP-TAF complexes B-TFIID and D-TFIID, which were previously characterized as active for pol II transcription, nor TBP alone can complement pol III transcription reactions that are dependent upon the TBP-TAF subcomponent of TFIIIB. Surprisingly, the TBP-TAF subcomponent of TFIIIB is active in reconstitution of pol II transcription.
Isolation of cDNAs encoding the Drosophila GAGA transcription factor.
Authors: Soeller W C , Oh C E , Kornberg T B
Published: October 2015

To investigate the mechanisms involved in expression of the Drosophila melanogaster engrailed gene, we purified GAGA protein, one of several putative transcriptional activator proteins that binds to the proximal region of the engrailed promoter. Antibodies raised against GAGA protein were used to demonstrate that the protein is present in all nuclei of young embryos. We isolated cDNA clones encoding GAGA protein in which a putative 519-codon open reading frame contains general sequence motifs characteristic of other transcription factors. These include stretches of polyglutamine, a 60-amino-acid region with 18 (30%) lysine or arginine residues, and a single putative zinc finger motif. In addition, a 120-residue N-terminal region shares significant sequence homology with several other known Drosophila transcription factors, including those encoded by Broad Complex and tramtrack. Up to 35-fold GAGA protein-dependent stimulation of transcription in Schneider line 2 tissue culture cells was observed after transfection of GAGA protein-encoding sequences. The GAGA gene is present in one copy in the Drosophila genome, at cytological location 70EF, and it encodes RNAs which vary in size between 2.4 and 4.4 kb.
The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.
Authors: Whyatt L M , Düwel A , Smith A G , Rathjen P D
Published: October 2015

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.
Species-specific signals for the splicing of a short Drosophila intron in vitro.
Authors: Guo M , Lo P C , Mount S M
Published: October 2015

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.
U2 small nuclear RNA 3' end formation is directed by a critical internal structure distinct from the processing site.
Authors: Jacobson M R , Rhoadhouse M , Pederson T
Published: October 2015

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.
B-myc inhibits neoplastic transformation and transcriptional activation by c-myc.
Authors: Resar L M , Dolde C , Barrett J F , Dang C V
Published: October 2015

B-myc is a recently described myc gene whose product has not been functionally characterized. The predicted product of B-myc is a 168-amino-acid protein with extensive homology to the c-Myc amino-terminal region, previously shown to contain a transcriptional activation domain. We hypothesized that B-Myc might also function in transcriptional regulation, although its role in regulating gene expression is predicted to be unique, because B-Myc lacks the specific DNA-binding motif found in other Myc proteins. To determine whether B-Myc could interact with the transcriptional machinery, we studied the transcriptional activation properties of a chimeric protein containing B-Myc sequences fused to the DNA-binding domain of the yeast transcriptional activator GAL4 (GAL4-B-Myc). We found that GAL4-B-Myc strongly activated expression of a GAL4-regulated reporter gene in mammalian cells. In addition, full-length B-Myc was able to inhibit or squelch reporter gene activation by a GAL4 chimeric protein containing the c-Myc transcriptional activation domain. We also observed that B-Myc dramatically inhibited the neoplastic cotransforming activity of c-Myc and activated Ras in rat embryo cells. Because B-Myc inhibits both neoplastic transformation and transcriptional activation by c-Myc, we suggest that the transforming activity of c-Myc is related to its ability to regulate transcription. Whether B-Myc functions biologically to squelch transcription and/or to regulate transcription through a specific DNA-binding protein remains unestablished.
Biochemical analysis of torso and D-raf during Drosophila embryogenesis: implications for terminal signal transduction.
Authors: Sprenger F , Trosclair M M , Morrison D K
Published: October 2015

Determination of anterior and posterior terminal structures of Drosophila embryos requires activation of two genes encoding putative protein kinases, torso and D-raf. In this study, we demonstrate that Torso has intrinsic tyrosine kinase activity and show that it is transiently tyrosine phosphorylated (activated) at syncytial blastoderm stages. Torso proteins causing a gain-of-function phenotype are constitutively tyrosine phosphorylated, while Torso proteins causing a loss-of-function phenotype lack tyrosine kinase activity. The D-raf gene product, which is required for Torso function, is identified as a 90-kDa protein with intrinsic serine/threonine kinase activity. D-Raf is expressed throughout embryogenesis; however, the phosphorylation state of the protein changes during development. In wild-type embryos, D-Raf is hyperphosphorylated at 1 to 2 h after egg laying, and thereafter only the most highly phosphorylated form is detected. Embryos lacking Torso activity, however, show significant reductions in D-Raf protein expression rather than major alterations in the protein's phosphorylation state. This report provides the first biochemical analysis of the terminal signal transduction pathway in Drosophila embryos.
Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.
Authors: Cegielska A , Virshup D M
Published: October 2015

The initiation of simian virus 40 (SV40) DNA replication is regulated by the phosphorylation state of the viral initiator protein, large T antigen. We describe the purification from HeLa cell nuclei of a 35-kDa serine/threonine protein kinase that phosphorylates T antigen at sites that are phosphorylated in vivo and thereby inhibits its ability to initiate SV40 DNA replication. The inhibition of both origin unwinding and DNA replication by the kinase is reversed by protein phosphatase 2A. As determined by molecular weight, substrate specificity, autophosphorylation, immunoreactivity, and limited sequence analysis, this kinase appears to be identical to casein kinase I, a ubiquitous serine/threonine protein kinase that is closely related to a yeast kinase involved in DNA metabolism. The HeLa cell phosphorylation cycle that controls the initiation of SV40 DNA replication may also play a role in cellular DNA metabolism.
A new serum-responsive, cardiac tissue-specific transcription factor that recognizes the MEF-2 site in the myosin light chain-2 promoter.
Authors: Zhou M D , Goswami S K , Martin M E , Siddiqui M A
Published: October 2015

We have identified a serum-responsive, cardiac tissue-specific transcription factor, BBF-1, that recognizes an AT-rich sequence (element B), identical to the myocyte enhancer factor (MEF-2) target site, in the cardiac myosin light chain-2 (MLC-2) promoter. Deletion of the element B sequence alone from the cardiac MLC-2 promoter causes, as does that of the MEF-2 site from other promoters and the enhancer of skeletal muscle genes, a marked reduction of transcription. BBF-1 is distinguishable from cardiac MEF-2 on the basis of immunoprecipitation with an antibody which recognizes MEF-2 but not BBF-1. Unlike MEF-2, BBF-1 is present exclusively in nuclear extracts from cardiac muscle cells cultured in a medium containing a high concentration of serum. Removal of serum from culture medium abolishes BBF-1 activity selectively with a concomitant loss of the positive regulatory effect of element B on MLC-2 gene transcription, indicating that there is a correlation between the BBF-1 binding activity and the tissue-specific role of the element B (MEF-2 site) sequence. The loss of element B-mediated activation of transcription is reversed following the refeeding of cells with serum-containing medium. These data demonstrate that cardiac muscle cells contain two distinct protein factors, MEF-2 and BBF-1, which bind to the same target site but that, unlike MEF-2, BBF-1 is serum inducible and cardiac tissue specific. BBF-1 thus appears to be a crucial member of the MEF-2 family of proteins which will serve as an important tool in understanding the regulatory mechanism(s) underlying cardiogenic differentiation.
Conditional silencing: the HMRE mating-type silencer exerts a rapidly reversible position effect on the yeast HSP82 heat shock gene.
Authors: Lee S , Gross D S
Published: October 2015

The HMRE silencer of Saccharomyces cerevisiae has been previously shown to transcriptionally repress class II and class III genes integrated within the HMR silent mating-type locus up to 2.6 kb away. Here we study the ability of this element to repress at an ectopic position, independent of sequences normally associated with it. When integrated 750 bp upstream of the HSP82 heat shock gene, the silencer represses basal-level transcription approximately 5-fold but has no effect on chemical- or heat-shock-induced expression. Such conditional silencing is also seen when the HMRE/HSP82 allele is carried on a centromeric episome or when the entire HMRa domain is transplaced 2.7 kb upstream of HSP82. Notably, the a1 promoter within the immigrant HMRa locus remains fully repressed at the same time HSP82 is derepressed. The position effect mediated by the E silencer is absolutely dependent on the presence of a functional SIR4 gene product, is lost within 1 min following stress induction, and is fully reestablished within 15 min following a return to nonstressful conditions. Similar kinetics of reestablishment are seen in HMRE/HSP82 and HMRa/HSP82 strains, indicating that complete repression can be mediated over thousands of base pairs within minutes. DNase I chromatin mapping reveals that the ABF1, RAP1, and autonomously replicating sequence factor binding sites within the silencer are constitutively occupied in chromatin, unaltered by heat shock or the presence of SIR4. Similarly, the heat shock factor binding site upstream of HSP82 remains occupied under such conditions, suggesting concurrent occupancy of silencer and activator binding sites. Our results are consistent with a model in which silencing at the HMRE/HSP82 allele is mediated by direct or indirect contacts between the silencer protein complex and heat shock factor.
Heat shock factor is required for growth at normal temperatures in the fission yeast Schizosaccharomyces pombe.
Authors: Gallo G J , Prentice H , Kingston R E
Published: October 2015

Schizosaccharomyces pombe is becoming an increasingly useful organism for the study of cellular processes, since in certain respects, such as the cell cycle and splicing, it is similar to metazoans. Previous biochemical studies have shown that the DNA binding ability of S. pombe heat shock factor (HSF) is fully induced only under stressed conditions, in a manner similar to that of Drosophila melanogaster and humans but differing from the constitutive binding by HSF in the budding yeasts. We report the isolation of the cDNA and gene for the HSF from S. pombe. S. pombe HSF has a domain structure that is more closely related to the structure of human and D. melanogaster HSFs than to the structure of the budding yeast HSFs, further arguing that regulation of HSF in S. pombe is likely to reflect regulation in metazoans. Surprisingly, the S. pombe HSF gene is required for growth at normal temperatures. We show that the S. pombe HSF gene can be replaced by the D. melanogaster HSF gene and that strains containing either of these genes behave similarly to transiently heat-shocked strains with respect to viability and the level of heat-induced transcripts from heat shock promoters. Strains containing the D. melanogaster HSF gene, however, have lower growth rates and show altered morphology at normal growth temperatures. These data demonstrate the functional conservation of domains of HSF that are required for response to heat shock. They further suggest a general role for HSF in growth of eukaryotic cells under normal (nonstressed) growth conditions.
Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.
Authors: Dornreiter I , Copeland W C , Wang T S
Published: October 2015

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.
Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens.
Authors: McCormack W T , Hurley E A , Thompson C B
Published: October 2015

Somatic immunoglobulin diversity is generated in avian species by sequential gene conversion of variable (V) gene segments of the immunoglobulin heavy- and light-chain loci during B-cell development. The germ line pools of donor sequence information for somatic V-region gene conversion are found in families of V pseudogenes, located 5' of the single functional V gene of each locus. The sequence relationships among the pseudogenes (psi VL) and functional VL1 gene of the chicken light-chain alleles in three inbred strains were compared to determine the extent of diversity within the germ line pseudogene cluster. Numerous differences were observed. For example, compared with the previously reported CB allele and the G4 allele, the S3 allele contains two intact pseudogenes between psi VL16 and psi VL18. These two adjacent psi VL gene segments (psi VL17a and psi VL17b) could have given rise to the psi VL17 segment of the G4 and CB alleles by homologous recombination. The majority of other sequence polymorphisms among the psi VL alleles appear to be the result of meiotic gene conversion. The incidence of untemplated mutations within psi VL segments is significantly lower than the incidence of mutation within the pseudogene flanking regions. Together with the observations that most psi VL segments have open reading frames and lack stop codons, these data support the hypothesis that the psi VL cluster resembles a functional multigene family maintained by evolutionary selection for its functional role in generating somatic antibody diversity. Meiotic gene conversion events within the psi VL cluster serve both to introduce diversity by the exchange of short segments between family members and to prevent the accumulation of random mutations.
N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65.
Authors: Toledano M B , Ghosh D , Trinh F , Leonard W J
Published: October 2015

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.
Correction of a deletion mutant by gene targeting with an adenovirus vector.
Authors: Wang Q , Taylor M W
Published: October 2015

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.
In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum.
Authors: Vallett S M , Brudnak M , Pellegrini M , Weber H W
Published: October 2015

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.