bluuebunny/crossref_metadata_2025 huggingface dataset:
The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.
Authors: Faure M , Franke J , Hall A L , Podgorski G J , Kessin R H
Published: October 2015

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.
Identification of positive and negative regulatory elements governing cell-type-specific expression of the neural cell adhesion molecule gene.
Authors: Hirsch M R , Gaugler L , Deagostini-Bazin H , Bally-Cuif L , Goridis C
Published: October 2015

The neural cell adhesion molecule (NCAM) is one of the most prevalent cell adhesion molecules in vertebrates. Its expression is subject to complex cell-type- and developmental-stage-dependent regulation. To study this regulation at the level of transcription, we analyzed the promoter region of the mouse NCAM gene. The NCAM promoter did not contain a typical TATA box. Transcription started at several sites that were used indiscriminately by different cell types, implying that the different NCAM isoforms are expressed from a single promoter. Sequences responsible for both promotion and inhibition of transcription resided within 840 base pairs upstream of the main transcriptional start site. The sequence from positions -645 to -37 relative to the translation initiation site directed high levels of expression in NCAM-expressing N2A cells. The same fragment was six times less active but still significantly active in L cells, but this activity was repressed by inclusion of an additional upstream segment. We mapped eight domains of interactions with nuclear proteins within the 840-base-pair region. The segment with maximum promoter activity contained two adjacent footprints, the occupation of which appeared to be mutually exclusive. One of them corresponded to an Sp1-factor-binding consensus site, the other one bound a factor with nuclear factor I activity. The single protected domain in the fragment harboring a repressor activity consisted of a GGA repeat resembling negative regulatory elements in other promoters. Three adjacent binding sites occupied an A + T-rich segment and contained ATTA motifs also found in the recognition elements of homeodomain proteins. These results show that negative and positive elements interact to regulate the tissue-specific patterns of expression of the NCAM gene and indicate that a factor related to nuclear factor I is involved in its transcriptional control.
Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.
Authors: Ng D T , Hiebert S W , Lamb R A
Published: October 2015

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.
Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.
Authors: Nishizawa M , Nagata S
Published: October 2015

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.
N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.
Authors: Akeson R , Bernards R
Published: October 2015

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.
Organization and expression of the chicken N-myc gene.
Authors: Sawai S , Kato K , Wakamatsu Y , Kondoh H
Published: October 2015

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.
Characterization of the rat transforming growth factor alpha gene and identification of promoter sequences.
Authors: Blasband A J , Rogers K T , Chen X R , Azizkhan J C , Lee D C
Published: October 2015

We have determined the complete nucleotide sequence of rat transforming growth factor alpha (TGF alpha) mRNA and characterized the six exons that encode this transcript. These six exons span approximately 85 kilobases of genomic DNA, with exons 1 to 3 separated by particularly large introns. What had previously been thought to represent a species-specific difference in the size of the TGF alpha precursor (proTGF alpha) is now shown to be due to microheterogeneity in the splicing of exons 2 and 3. This results from a tandem duplication of the acceptor CAG and gives rise to two alternate forms (159 and 160 amino acids) of the integral membrane precursor. Exon 6, which encodes the 3' untranslated region of TGF alpha mRNA, also encodes, on the opposite strand, a small (approximately 200-nucleotide) transcript whose sequence predicts an open reading frame of 51 amino acids. Expression of this latter transcript does not appear to be coregulated with that of TGF alpha mRNA. Primer extension and S1 nuclease analyses of authentic TGF alpha transcripts revealed two major and multiple minor 5' ends which span more than 200 base pairs of DNA in a G + C-rich region that lacks canonical CCAAT or TATA sequences. The 5' ends of six independently derived cDNAs localized to five different sites in this same region. Restriction fragments that overlap these transcription start sites and extend approximately 300 base pairs in the 5' direction faithfully promote transcription in vitro with HeLa cell nuclear extracts. In addition, they direct the expression of the bacterial chloramphenicol acetyltransferase gene in transient-transfection assays.
Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells.
Authors: Biemann H P , Erikson R L
Published: October 2015

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.
In vitro posttranslational modification of lamin B cloned from a human T-cell line.
Authors: Pollard K M , Chan E K , Grant B J , Sullivan K F , Tan E M , Glass C A
Published: October 2015

Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.
Courtship in Saccharomyces cerevisiae: an early cell-cell interaction during mating.
Authors: Jackson C L , Hartwell L H
Published: October 2015

During conjugation in Saccharomyces cerevisiae, two cells of opposite mating type (MATa and MAT alpha) fuse to form a diploid zygote. Conjugation requires that each cell locate an appropriate mating partner. To investigate how yeast cells select a mating partner, we developed a competition mating assay in which wild-type MAT alpha cells have a choice of two MATa cell mating partners. We first demonstrated that sterile MAT alpha 1 cells (expressing no a- or alpha-specific gene products) do not compete with fertile MATa cells in the assay; hence, wild-type MATa and MAT alpha cells can efficiently locate an appropriate mating partner. Second, we showed that a MATa strain need not be fertile to compete with a fertile MATa strain in the assay. This result defines an early step in conjugation, which we term courtship. We showed that the ability to agglutinate is not necessary in MATa cells for courtship but that production of a-pheromone and response to alpha-pheromone are necessary. Thus, MATa cells must not only transmit but must also receive and then respond to information for effective courtship; hence, there is a "conversation" between the courting cells. We showed that the only alpha-pheromone-induced response necessary in MATa cells for courtship is production of a-pheromone. In all cases tested, a strain producing a higher level of a-pheromone was more proficient in courtship than one producing a lower level. We propose that during courtship, a MAT alpha cell selects the adjacent MATa cell producing the highest level of a-pheromone.
Isolation and characterization of the alpha platelet-derived growth factor receptor from rat olfactory epithelium.
Authors: Lee K H , Bowen-Pope D F , Reed R R
Published: October 2015

We have cloned and characterized a new member of the receptor tyrosine kinase family. The cDNA clone, isolated from a rat olfactory cDNA library, has considerable homology to the family of receptors that includes the colony-stimulating factor 1 receptor, the c-kit proto-oncogene, and the platelet-derived growth factor (PDGF) receptors. Analysis of DNA sequence homology, ligand-binding, and ligand-stimulated phosphorylation data suggests that this clone encodes the rat PDGF-A/B or alpha-receptor. Comparison of its sequence to those of other receptors allows us to postulate a mechanism for receptor dimerization and activation. The expression of the rat alpha-PDGF receptor in nonneuronal cells of the olfactory epithelium and in the olfactory bulb is consistent with a role for PDGF in glial cell generation.
Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.
Authors: Morrison D K , Kaplan D R , Rhee S G , Williams L T
Published: October 2015

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.
Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts.
Authors: Sweet L J , Alcorta D A , Jones S W , Erikson E , Erikson R L
Published: October 2015

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.
Organization of subtelomeric repeats in Plasmodium berghei.
Authors: Dore E , Pace T , Ponzi M , Picci L , Frontali C
Published: October 2015

Several (but not all) Plasmodium berghei chromosomes bear in the subtelomeric position a cluster of 2.3-kilobase (kb) tandem repeats. The 2.3-kb unit contains 160 base pairs of telomeric sequence. The resulting subtelomeric structure is one in which stretches of telomeric sequences are periodically spaced by a 2.1-kb reiterated sequence. This periodic organization of internal telomeric sequences might be related to chromosome-size polymorphisms involving the loss or addition of subtelomeric 2.3-kb units.
RNA editing in the cytochrome b locus of the higher plant Oenothera berteriana includes a U-to-C transition.
Authors: Schuster W , Hiesel R , Wissinger B , Brennicke A
Published: October 2015

RNA editing in the cytochrome b locus of Oenothera berteriana mitochondria modified a number of cytidine nucleotides to uridines, mostly altering codon identities. One nucleotide alteration involved a reverse modification changing a genomic thymidine to a cytidine in the cDNA sequence. The enzymatic editing activity in higher-plant mitochondria thus appears to be able to catalyze the interconversion of pyrimidines in both directions at specific nucleotides in the mRNA template.
Identification of positive and negative regulatory regions controlling expression of the cartilage matrix protein gene.
Authors: Kiss I , Bösze Z , Szabó P , Altanchimeg R , Barta E , Deák F
Published: October 2015

A complex pattern of regulation of the cartilage matrix protein gene was revealed by transient expression experiments. A minimal promoter from positions -15 to +64 functioned in chondrocytes and fibroblasts. An enhancer located in the first intron exerted chondrocyte-specific stimulation on the minimal promoter activity. The same fragment, however, had a negative effect in fibroblasts. Between -334 and -15, a silencer was found which inhibited the gene expression driven from its homologous as well as heterologous promoters both in chondrocytes and fibroblasts. Additional positive and negative control regions were mapped further upstream of the promoter.
Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either.
Authors: Pine R , Decker T , Kessler D S , Levy D E , Darnell J E
Published: October 2015

Interferon-stimulated gene factor 2 (ISGF2) was purified from HeLa cells treated with alpha interferon. The factor, a single polypeptide of 56 kilodaltons (kDa), bound both to the central 9 base pairs of the 15-base-pair interferon-stimulated response element (ISRE) that is required for transcriptional activation of interferon-stimulated genes and to the PRD-I regulatory element of the beta interferon gene. ISGF2 was a phosphoprotein, and dephosphorylation in vitro reduced its DNA-binding activity. However, conditions that changed the amount of ISGF2 did not change the phosphorylated isoforms in vivo. ISGF2 in unstimulated cells existed in trace amounts and was induced by both alpha interferon and gamma interferon as well as by virus infection. Plasmid-bearing Escherichia coli clones encoding ISGF2 were selected with antibody against purified ISGF2. Sequence analysis revealed that the ISGF2 protein was the same as that encoded by the cDNA clone IRF-1, which has been claimed to activate transcription of interferon genes. We show that transcription of the ISGF2 gene was induced by alpha interferon, gamma interferon, and double-stranded RNA. However, ISGF2 was neither necessary nor sufficient for induced transcription of the beta interferon gene, while the factor NF kappa B was clearly involved.
Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1.
Authors: Baker R E , Masison D C
Published: October 2015

CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved DNA element I (CDEI) of yeast centromeres. We cloned and sequenced the gene encoding CP1. The gene codes for a protein of molecular weight 39,400. When expressed in Escherichia coli, the CP1 gene directed the synthesis of a CDEI-binding protein having the same gel mobility as purified yeast CP1. We have given the CP1 gene the genetic designation CEP1 (centromere protein 1). CEP1 was mapped and found to reside on chromosome X, 2.0 centimorgans from SUP4. Strains were constructed in which most of CEP1 was deleted. Such strains lacked detectable CP1 activity and were viable; however, CEP1 gene disruption resulted in a 35% increase in cell doubling time and a ninefold increase in the rate of mitotic chromosome loss. An unexpected consequence of CP1 gene disruption was methionine auxotrophy genetically linked to cep1. This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (D. Thomas, H. Cherest, and Y. Surdin-Kerjan, J. Mol. Biol. 9:3292-3298, 1989) suggest that CP1 is both a kinetochore protein and a transcription factor.
Expression of the K-fgf proto-oncogene is controlled by 3' regulatory elements which are specific for embryonal carcinoma cells.
Authors: Curatola A M , Basilico C
Published: October 2015

Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.
Macrophage colony-stimulating factor-induced tyrosine phosphorylation of c-fms proteins expressed in FDC-P1 and BALB/c 3T3 cells.
Authors: Tapley P , Kazlauskas A , Cooper J A , Rohrschneider L R
Published: October 2015

The c-fms protein is a receptor for macrophage colony-stimulating factor (M-CSF) with intrinsic protein-tyrosine kinase activity. We investigated the tyrosine phosphorylation of murine c-fms proteins expressed from a retroviral vector in factor-dependent myeloid FDC-P1 cells and in BALB/c 3T3 fibroblasts transformed by the expression of the c-fms gene. FDC-P1 cells expressing c-fms were able to grow and differentiate in response to M-CSF. Their c-fms proteins were normally phosphorylated on serine and became phosphorylated on tyrosine residues contained in five tryptic peptides when the cells were exposed to M-CSF. A subset of these peptides was constitutively phosphorylated in BALB/c cells expressing c-fms, consistent with the production of M-CSF by these cells. All the peptides detected in vivo were also phosphorylated in vitro. These peptides were analyzed by susceptibility to proteases, comparison with synthetic peptides, and site-directed mutagenesis. The identities of four of the tryptic peptides were determined; they arise from three unique tyrosine phosphorylation sites. One major site of tyrosine phosphorylation at residue 697 accounted for two of the tryptic peptides. A second major site was identified at tyrosine residue 706. These two tyrosine phosphorylation sites are located within the tyrosine kinase insert region. Tyrosine 807, which has homology to the major autophosphorylation site of the p60v-src tyrosine kinase, is a minor autophosphorylation site. Possible functional roles for these phosphorylations of the c-fms protein include interactions with substrate proteins, catalytic activity, and ligand-induced degradation.
Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase.
Authors: Colicelli J , Field J , Ballester R , Chester N , Young D , Wigler M
Published: October 2015

Large deletion and small insertion mutations in the adenylyl cyclase gene of Saccharomyces cerevisiae were used to map regions required for activation by RAS protein in vitro. The amino-terminal 605 amino acids were found to be dispensable for responsiveness to RAS protein. All other deletions in adenylyl cyclase destroyed its ability to respond to RAS. Small insertion mutations within the leucine-rich repeat region also prevented RAS responsiveness, while other insertions did not.
Negative and positive cis-acting elements control the expression of murine alpha 1-protease inhibitor genes.
Authors: Montgomery K T , Tardiff J , Reid L M , Krauter K S
Published: October 2015

The alpha 1-protease inhibitor (alpha 1-PI) proteins of mice are encoded by a group of genes whose members are expressed coordinately in a liver-abundant pattern and are regulated primarily at the transcriptional level. To better understand the developmental and tissue-specific regulation of this gene family, one member that is analogous to the human alpha 1-antitrypsin gene was chosen for study. Deletional analysis of the upstream regulatory region of this gene was performed, spanning from -10 kilobases to -80 base pairs relative to the transcriptional start site. Two functional positive cis-acting elements within the 522 bases immediately upstream of the start site for transcription were shown to modulate the level of expression from this promoter when introduced into human or mouse hepatoma cells, and a third region acted as a negative regulatory element in that its deletion resulted in a two- to sixfold increase of expression of a transfected minigene construct. Sequence comparison between the regulatory domains of two mouse alpha 1-PI genes and the human alpha 1-antitrypsin gene showed that the mouse gene contains a novel positive cis-acting element which is absent in human gene and that a specific eight-base-pair difference between species results in a strong positive cis-acting element in the human gene acting as a negative element in the mouse gene. An enhancer located approximately 3,000 base pairs upstream of the major start site for transcription was also identified. This element is position and orientation independent. Several different DNA-protein binding assays were used to demonstrate that each DNA segment with functional significance in transfection assays interacts specifically with proteins found in adult mouse liver nuclei. The major positive-acting element appeared to be specifically recognized by nuclear proteins found only in tissues that express alpha 1-PI, while the negative element binding proteins were ubiquitous. Thus, the distal regulatory domain including bases -3500 to -133 of this murine alpha 1-PI gene family member is more complex than was previously demonstrated. It is composed of a set of at least three additional functional cis-acting regulatory elements besides those which have been mapped by others and has a far upstream enhancer.
Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.
Authors: Xu H , Boeke J D
Published: October 2015

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.
Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.
Authors: Borzillo G V , Ashmun R A , Sherr C J
Published: October 2015

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.
The carboxy-terminal domains of erbB-2 and epidermal growth factor receptor exert different regulatory effects on intrinsic receptor tyrosine kinase function and transforming activity.
Authors: Di Fiore P P , Segatto O , Lonardo F , Fazioli F , Pierce J H , Aaronson S A
Published: October 2015

The erbB-2 gene product, gp185erbB-2, displays a potent transforming effect when overexpressed in NIH 3T3 cells. In addition, it possesses constitutively high levels of tyrosine kinase activity in the absence of exogenously added ligand. In this study, we demonstrate that its carboxy-terminal domain exerts an enhancing effect on erbB-2 kinase and transforming activities. A premature termination mutant of the erbB-2 protein, lacking the entire carboxy-terminal domain (erbB-2 delta 1050), showed a 40-fold reduction in transforming ability and a lowered in vivo kinase activity for intracellular substrates. When the carboxy-terminal domain of erbB-2 was substituted for its analogous region in the epidermal growth factor receptor (EGFR) (EGFR/erbB-2COOH chimera), it conferred erbB-2-like properties to the EGFR, including transforming ability in the absence of epidermal growth factor, elevated constitutive autokinase activity in vivo and in vitro, and constitutive ability to phosphorylate phospholipase C-gamma. Conversely, a chimeric erbB-2 molecule bearing an EGFR carboxy-terminal domain (erbB-2/EGFRCOOH chimera) showed reduced transforming and kinase activity with respect to the wild-type erbB-2 and was only slightly more efficient than the erbB-2 delta 1050 mutant. Thus, we conclude that the carboxy-terminal domains of erbB-2 and EGFR exert different regulatory effects on receptor kinase function and biological activity. The up regulation of gp185erbB-2 enzymatic activity exerted by its carboxy-terminal domain can explain, at least in part, its constitutive level of kinase activity.
Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.
Authors: Reitman M , Felsenfeld G
Published: October 2015

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.
A DNA-binding protein is required for termination of transcription by RNA polymerase I in Xenopus laevis.
Authors: McStay B , Reeder R H
Published: October 2015

We describe a partially fractionated in vitro transcription system from Xenopus laevis for the assay of transcription termination by RNA polymerase I. Termination in vitro was found to require a specific terminator sequence in the DNA and a DNA-binding protein fraction that produces a footprint over the terminator sequence.
A genomic clone encoding a novel proliferation-dependent histone H2A.1 mRNA enriched in the poly(A)+ fraction.
Authors: Fecker L , Ekblom P , Kurkinen M , Ekblom M
Published: October 2015

Replication-dependent histone mRNAs are prime examples of nonpolyadenylated mRNAs. We isolated and characterized cDNAs and a genomic clone for a replication-dependent histone H2A.1 mRNA which segregated into the poly(A)+ fraction during mRNA isolation through an oligo(dT)-cellulose column. However, the results of sequencing of the genomic clone suggested that the mRNA did not contain a poly(A) tail. Instead, the genomic sequence revealed a nonterminal oligo(A) tract directly upstream from the typical 3'-terminal hairpin loop of replication-dependent histone mRNAs. The nonterminal oligo(A) tract consisted of 14 adenylate residues interrupted by one guanylate residue (A4GA10). We concluded that this short oligo(A) stretch mediated binding of the mRNA to oligo(dT) even after stringent washes with 0.1 M NaCl, indicating that rather short oligo(A) sequences can ensure binding to oligo(dT)-cellulose. The cDNA and genomic clones contained an AAATAAG sequence at the end of the coding region. It has been suggested that this sequence contains a polyadenylation signal in some yeast and mouse transcripts, but it does not function as a polyadenylation signal in the histone transcript described in this paper.
Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.
Authors: Quilliam L A , Der C J , Clark R , O'Rourke E C , Zhang K , McCormick F , Bokoch G M
Published: October 2015

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.
Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.
Authors: Suh E R , Waring R B
Published: October 2015

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.
A truncated, secreted form of the epidermal growth factor receptor is encoded by an alternatively spliced transcript in normal rat tissue.
Authors: Petch L A , Harris J , Raymond V W , Blasband A , Lee D C , Earp H S
Published: October 2015

Two independent cDNA clones corresponding to a 2.7-kilobase (kb) epidermal growth factor receptor (EGF-R) mRNA were isolated from a rat liver cDNA library. Sequence analysis revealed 100% homology in the external domain when compared with the full-length rat EGF-R nucleotide sequence and 80 to 90% similarity relative to the human EGF-R. However, the 3'-terminal sequence of these clones did not match EGF-R or any other known sequence(s) and was distinct from the 3' end of the 2.8-kb mRNA, which encodes a truncated EGF-R in A431 cells. The deduced amino acid sequence revealed an open reading frame which is homologous to the external domain of the EGF-R but which terminates prior to the transmembrane region. Southern blot analysis of rat genomic DNA indicated that the 3'-terminal sequence of this transcript is derived from the EGF-R gene. Analysis of a genomic clone containing the 3' end of the 2.7-kb transcript revealed that this sequence is present as a discrete exon in the mid-region of the receptor gene in proximity to the exon encoding the transmembrane domain. Introduction of an expression vector containing the truncated EGF-R cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 95-kilodalton protein which was detected in conditioned media, by using antisera directed against the EGF-R. A similarly sized protein was also detected in the media of WB cells, a continuous, nontransformed line of rat hepatic epithelial cells. Northern (RNA blot) analysis established that the truncated receptor is encoded by a 2.7-kb transcript found in normal rat liver. Furthermore, Northern analysis of rat poly(A)+ RNA showed that the 2.7-kb EGF-R transcript is expressed at differing levels in various fetal and adult tissues. These data indicate that alternative splicing of the EGF-R primary transcript yields a 2.7-kb mRNA which codes for a truncated form of the receptor. This receptor is secreted by rat hepatic epithelial cells in culture, which suggests that it may be secreted by normal rat cells or tissues and perhaps serve an as yet unknown physiological function.
Interferon-regulatory factor 1 is an immediate-early gene under transcriptional regulation by prolactin in Nb2 T cells.
Authors: Yu-Lee L Y , Hrachovy J A , Stevens A M , Schwarz L A
Published: October 2015

The pituitary peptide hormone prolactin (Prl) is a potent inducer of Nb2 T lymphoma cell proliferation. To analyze the early genetic response to the mitogenic signals of Prl, a cDNA library was constructed from Nb2 T cells stimulated for 4 h with Prl and the protein synthesis inhibitor cycloheximide. Of 26 distinct clones isolated by differential screening, one clone, designated c25, exhibited extremely rapid but transient kinetics of induction by Prl and superinduction by Prl plus cycloheximide. Run-on transcription analysis indicated that c25 gene transcription was induced greater than 20-fold within 30 to 60 min of Prl stimulation. Surprisingly, DNA sequence analysis of c25 cDNA revealed that this Prl-inducible early-response gene is the rat homolog of the mouse transcription factor interferon-regulatory factor 1 (IRF-1), sharing 91% coding sequence similarity with mouse IRF-1. At the protein level, rat IRF-1 shares 97% and 92% homology with mouse IRF-1 and human IRF-1, respectively, suggesting that this molecule has been functionally conserved throughout evolution. Our studies show that the gene for IRF-1 is an immediate-early gene in Prl-stimulated T cells, which suggests that IRF-1 is a multifunctional molecule. In addition to its role in regulating growth-inhibitory interferon genes, IRF-1 may, therefore, also play a stimulatory role in cell proliferation. The gene for IRF-1 is one of the earliest genes known to be transcriptionally regulated by Prl.