bluuebunny/crossref_metadata_2025 huggingface dataset:
The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae.
Authors: Rosenblum-Vos L S , Rhodes L , Evangelista C C , Boayke K A , Zitomer R S
Published: October 2015

The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae. The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog. The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein. This protein is essential; deletion of the coding sequence was lethal. The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function. Mutations in the rox3 gene showed an interesting pattern of intragenic complementation. A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation. These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7. Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression. The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.
A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.
Authors: McNally F J , Rine J
Published: October 2015

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.
Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum.
Authors: d'Enfert C , Barlowe C , Nishikawa S , Nakano A , Schekman R
Published: October 2015

Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae. Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein. Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential. Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature. This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport. Sar1p membrane association was enhanced by elevated levels of Sec12p. These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.
Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection.
Authors: Hekmatpanah D S , Young R A
Published: October 2015

A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.
Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides.
Authors: Hu Q J , Bautista C , Edwards G M , Defeo-Jones D , Jones R E , Harlow E
Published: October 2015

Even though the retinoblastoma gene is one of the best-studied tumor suppressor genes, little is known about its functional role. Like all tumor suppressor gene products, the retinoblastoma protein (pRB) is thought to inhibit some aspect of cell proliferation. It also appears to be a cellular target of several DNA tumor virus-transforming proteins, such as adenovirus E1A, human papillomavirus E7, or simian virus 40 large T antigen. To help in the analysis of pRB, we have prepared a new set of anti-human pRB monoclonal antibodies. In addition to being useful reagents for the study of human pRB, these antibodies display several unexpected properties. They can be used to distinguish different subsets of the pRBs on the basis of their phosphorylation states. Some are able to recognize pRB homologs in other species, including mice, chickens, and members of the genus Xenopus. In addition, some of these antibodies can bind directly to other cellular proteins that, like pRB, were originally identified through their association with adenovirus E1A. These immunologically cross-reactive proteins include the p107 and p300 proteins, and their recognition by antibodies raised against pRB suggests that several members of the E1A-targeted cellular proteins form a structurally and functionally related family.
Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.
Authors: Preston R A , Manolson M F , Becherer K , Weidenhammer E , Kirkpatrick D , Wright R , Jones E W
Published: October 2015

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.
The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.
Authors: Gibbs V C , Williams S R , Gray P W , Schreiber R D , Pennica D , Rice G , Goeddel D V
Published: October 2015

At least two species-specific gene products are required for signal transduction by interferon gamma (IFN-gamma). The first is the IFN-gamma receptor, which binds ligand with high affinity in a species-specific manner. The second is an undetermined species-specific signal transducer(s). To determine whether the human IFN-gamma receptor (hIFN-gamma R) interacts directly with this signal transducer(s) and, if so, with what functional domain(s), we constructed expression vectors for the hIFN-gamma R and three hybrid human-murine IFN-gamma receptors. The hybrid receptors contained the extracellular, human IFN-gamma (hIFN-gamma) binding domain of the hIFN-gamma R, either the human or murine transmembrane domain, and either the human or murine intracellular domain. The vectors encoding these receptors were stably transfected into two mouse cell lines, one of which (SCC-16-5) contains a single copy of human chromosome 21. The resulting cell lines were treated with hIFN-gamma, and murine major histocompatibility complex class I antigen expression was analyzed by immunofluorescence flow cytometry. All transfected cell lines lacking human chromosome 21 remained insensitive to hIFN-gamma. However, all four of the IFN-gamma receptors were able to signal when expressed in the cell line containing human chromosome 21. We conclude that the extracellular domain of the IFN-gamma receptor is involved not only in the species specificity of IFN-gamma binding but also in signalling through interaction with an as yet unidentified species-specific factor(s) encoded by a gene(s) on human chromosome 21.
A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic retention of p68c-rel.
Authors: Mosialos G , Hamer P , Capobianco A J , Laursen R A , Gilmore T D
Published: October 2015

The Rel family of proteins includes a number of proteins involved in transcriptional control, such as the retroviral oncoprotein v-Rel, c-Rel, the Drosophila melanogaster developmental protein Dorsal, and subunits of the transcription factor NF-kappa B. These proteins are related through a highly conserved domain of approximately 300 amino acids, called the Rel homology domain, that contains dimerization, DNA binding, and nuclear targeting functions. Also within the Rel homology domain, there is a conserved consensus sequence (Arg-Arg-Pro-Ser) for phosphorylation by cyclic AMP-dependent protein kinase (PKA). We used linker insertion mutagenesis and site-directed mutagenesis to determine the importance of this sequence for the transformation of avian spleen cells by v-Rel and the subcellular localization of c-Rel in chicken embryo fibroblasts (CEF). The insertion of 2 amino acids (Pro-Trp) within this sequence completely abolished transformation and transcriptional repression by v-Rel and resulted in a shift in the localization of c-Rel from cytoplasmic to nuclear in CEF. When the conserved Ser within the PKA recognition sequence was replaced by Ala, there was no significant effect on transformation and transcriptional repression by v-Rel or on cytoplasmic retention of c-Rel. However, when this Ser was changed to Asp or Glu, transformation and transcriptional repression by v-Rel were significantly inhibited and c-Rel showed a diffuse nuclear and cytoplasmic localization in CEF. Although a peptide containing the recognition sequence from v-Rel can be phosphorylated by PKA in vitro, this site is not constitutively phosphorylated to a high degree in vivo in transformed spleen cells incubated with okadaic acid. Our results indicate that the transforming and transcriptional repressing activities of v-Rel and the cytoplasmic retention of c-Rel are dependent on the structure of the conserved PKA recognition motif. In addition, they suggest that phosphorylation at the conserved PKA site could have a negative effect on transformation and transcriptional repression by v-Rel and induce the nuclear localization of c-Rel.
Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation.
Authors: Adler B K , Harris M E , Bertrand K I , Hajduk S L
Published: October 2015

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.
A human alpha-fetoprotein enhancer-binding protein, ATBF1, contains four homeodomains and seventeen zinc fingers.
Authors: Morinaga T , Yasuda H , Hashimoto T , Higashio K , Tamaoki T
Published: October 2015

We have isolated a full-length cDNA encoding a protein (ATBF1) that binds to an AT-rich motif in the human alpha-fetoprotein gene enhancer. The amino acid sequence deduced from the cDNA revealed that this is the largest DNA-binding protein (306 kDa) known to date, containing four homeodomains, 17 zinc finger motifs, and a number of segments potentially involved in transcriptional regulation. Although the exact function of this protein has not been determined, these structural features suggest that ATBF1 plays a transcriptional regulatory role.
Evidence that interaction of hepatocytes with the collecting (hepatic) veins triggers position-specific transcription of the glutamine synthetase and ornithine aminotransferase genes in the mouse liver.
Authors: Kuo F C , Darnell J E
Published: October 2015

We previously demonstrated that glutamine synthetase (GS) and ornithine aminotransferase (OAT) mRNAs are expressed in the mouse liver acinus preferentially in pericentral hepatocytes, that is, those immediately surrounding terminal central veins (A.L. Bennett, K.E. Paulson, R.E. Miller, and J.E. Darnell, Jr., J. Cell Biol. 105:1073-1085, 1987, and F.C. Kuo, W.L. Hwu, D. Valle, and J.E. Darnell, Jr., Proc. Natl. Acad. Sci. USA, in press). We now show that hepatocytes surrounding large collecting hepatic veins but not portal veins also express these two mRNAs. The pericentral hepatocytes are the most distal hepatocytes with respect to acinar blood flow, whereas this is not necessarily the case for hepatocytes next to the large collecting hepatic veins. This result implies that it is contact with some hepatic venous element which signals positional expression. In an effort to induce conditions that change relationships between hepatocytes and blood vessels, regenerating liver was studied. After surgical removal of two-thirds or more of the liver, there was no noticeable change in GS or OAT expression in the remaining liver tissue during regeneration. However, treatment with carbon tetrachloride (CCl4), which specifically kills pericentral hepatocytes, completely removed GS- and OAT-containing cells and promptly halted hepatic transcription of GS. Repair of CCl4 damage is associated with invasion of inflammatory and scavenging cells, which remove dead hepatocytes to allow regrowth. Only when hepatocytes resumed contact with pericentral veins were the pretreatment levels of OAT and GS mRNA and high levels of GS transcription restored.
Regulation of asparagine synthetase gene expression by amino acid starvation.
Authors: Gong S S , Guerrini L , Basilico C
Published: October 2015

We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.
Analysis of p53 mutants for transcriptional activity.
Authors: Raycroft L , Schmidt J R , Yoas K , Hao M M , Lozano G
Published: October 2015

The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.
Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure.
Authors: Pabón-Peña L M , Zhang Y , Epstein L M
Published: October 2015

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.
A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi.
Authors: Villanueva M S , Williams S P , Beard C B , Richards F F , Aksoy S
Published: October 2015

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.
Repression of immunoglobulin enhancers by the helix-loop-helix protein Id: implications for B-lymphoid-cell development.
Authors: Wilson R B , Kiledjian M , Shen C P , Benezra R , Zwollo P , Dymecki S M , Desiderio S V , Kadesch T
Published: October 2015

It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.
Oocyte-specific factors bind a conserved upstream sequence required for mouse zona pellucida promoter activity.
Authors: Millar S E , Lader E , Liang L F , Dean J
Published: October 2015

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high levels of reporter gene activity, suggesting that the element is not only necessary but also sufficient for expression from zona pellucida promoters. Oligonucleotides containing the conserved element from either Zp-2 or Zp-3 form DNA-protein complexes of identical mobility in gel retardation assays using extracts of oocytes but not other tissues. These data are consistent with the hypothesis that common factors binding to conserved element IV are involved in coordinate expression of the oocyte-specific Zp-2 and Zp-3 zona pellucida genes.
Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.
Authors: Miller S M , Magasanik B
Published: October 2015

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.
The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.
Authors: Li Y , Li D , Osborn K , Johnson L F
Published: October 2015

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.
Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest.
Authors: Marcus S , Xue C B , Naider F , Becker J M
Published: October 2015

Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized. We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity. Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone. a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor. Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.
Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Authors: Lemaigre F P , Durviaux S M , Rousseau G G
Published: October 2015

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.
Different forms of Go alpha mRNA arise by alternative splicing of transcripts from a single gene on human chromosome 16.
Authors: Murtagh J J , Eddy R , Shows T B , Moss J , Vaughan M
Published: October 2015

Go alpha, (gene symbol GNA01), a member of the signal-transducing guanine nucleotide-binding (G) protein family, has been implicated in ion channel regulation. Some tissues contain multiple Go alpha mRNAs of different sizes that differ in the 3' untranslated regions (UTRs). Using sequence-specific 48-base oligonucleotides, two complementary to the different 3' UTRs and one complementary to the coding region, we investigated the origin of the multiple Go alpha transcripts, the organization of the Go alpha gene, the interspecies conservation of 3' UTRs, and the chromosomal localization of Go alpha. Oligonucleotides labeled to high specific activity by using terminal deoxynucleotidyltransferase each hybridized with a single band of restriction enzyme-digested mouse and human DNAs. In three of four digests of human DNA, the two probes specific for the different 3' UTRs hybridized with the same restriction fragment. Thus, these nucleotide sequences are in close proximity in the human genome. The order of the UTRs in the bovine, human, and mouse genomes was confirmed directly by polymerase chain reaction (PCR) amplification and sequencing. Hybridization of bovine oligonucleotide sequence with mouse and human genomic DNA indicated a high degree of interspecies sequence conservation: conservation was confirmed by PCR amplification and sequencing. Bands detected by both UTR probes, as well as the predominant bands detected by a bovine Go alpha cDNA, segregated with human chromosome 16 on Southern blot analysis of human-mouse somatic cell hybrids. We conclude that Go alpha mRNAs with different 3' UTRs arise by alternative splicing of transcripts from a single gene. The UTRs, which exhibit a high degree of interspecies conservation, may play a role in regulation of Go alpha expression during differentiation or in specific tissues. The use of oligonucleotide probes of the type described here represents a new strategy, potentially widely applicable for mapping and elucidating structural features of genes.
A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes.
Authors: Hinkley C , Perry M
Published: October 2015

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.
A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase.
Authors: Steinberg R A
Published: October 2015

Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.
Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.
Authors: McCusker J H , Yamagishi M , Kolb J M , Nomura M
Published: October 2015

Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.
Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.
Authors: Yano R , Nomura M
Published: October 2015

The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.
Myristylation alters DNA-binding activity and transactivation of FBR (gag-fos) protein.
Authors: Kamata N , Jotte R M , Holt J T
Published: October 2015

FBR murine sarcoma virus (gag-fos) protein, a virally transduced Fos protein, exhibits decreased gene transactivation in comparison with the cellular Fos protein. Biochemical analysis suggests that myristylation of the virally encoded N-terminal gag region results in decreased DNA binding and transcriptional activation without affecting heterodimerization with Jun protein. These findings demonstrate that protein myristylation can modulate gene regulation by a DNA-binding protein.
Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans.
Authors: Burger G , Tilburn J , Scazzocchio C
Published: October 2015

We have cloned an 11-kbp segment of the genomic DNA of Aspergillus nidulans which complements mutations in nirA, the pathway-specific regulatory gene of the nitrate assimilation pathway. Gene disruption in the corresponding region of the nuclear DNA leads to a phenotype and a gene complementation pattern indistinguishable from that observed in known noninducible nirA mutants. Transformation studies with subclones of the 11-kbp genomic segment showed that a nonreverting null mutation nirA87, maps to a 1.5-kbp stretch within that segment. These data confirm that the cloned segment contains the nirA gene. The gene is completely encompassed in the 11-kbp genomic segment, as a plasmid carrying the corresponding insert gives rise to multicopy transformants exhibiting better growth than wild type on nitrate or nitrite as the sole nitrogen source. Southern and genetic analyses of transformants obtained with various plasmid subclones established a gene size of at most 5.9 kbp. Northern (RNA) hybridization experiments revealed a 4-kb nirA transcript which is barely visible in the wild type but clearly seen in a transformant carrying about 10 gene copies. In both strains, nirA mRNA is synthesized constitutively. Upstream of nirA, a neighboring transcript about 2.8 kbp in length which is transcribed from the opposite strand with respect to nirA was localized. The transcript levels of niaD and niiA, encoding the nitrate and nitrite reductase core proteins, respectively, were investigated in nirA mutants and a nirA multicopy transformant. The results show that the nirA product regulates the transcript steady-state level of these structural genes and that it is a limiting factor for their expression.
Alpha B crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts.
Authors: Klemenz R , Fröhli E , Aoyama A , Hoffmann S , Simpson R J , Moritz R L , Schäfer R
Published: October 2015

The conditional expression of the v-mos and Ha-ras(EJ) oncogenes in NIH 3T3 cells leads to the accumulation of a 23-kDa protein (p23) (R. Klemenz, S. Hoffmann, R. Jaggi, and A.-K. Werenskiold, Oncogene 4:799-803, 1989). We purified p23 to homogeneity and determined part of the amino acid sequence. The obtained sequence is identical with that of the eye lens protein alpha B crystallin. Northern (RNA) blot and Western immunoblot experiments were performed to demonstrate that alpha B crystallin mRNA and protein do indeed accumulate as a consequence of v-mos and Ha-ras oncogene expression. Comparison of cDNA clones obtained from the mRNA of eye lenses and of oncogene-expressing fibroblasts revealed identity between them. The major transcription initiation site of the alpha B crystallin gene in our experimental system was shown by primer extension experiments to be identical with the one used in eye epithelial cells. In addition, we identified a second minor initiation site 49 nucleotides further upstream. Serum growth factors did not stimulate alpha B crystallin expression in growth-arrested cells.