bluuebunny/crossref_metadata_2025 huggingface dataset:
Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.
Authors: Tajima M , Nogi Y , Fukasawa T
Published: September 2015

We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.
Structure, organization, and regulation of human metallothionein IF gene: differential and cell-type-specific expression in response to heavy metals and glucocorticoids.
Authors: Varshney U , Jahroudi N , Foster R , Gedamu L
Published: September 2015

We describe a human genomic clone containing the metallothionein (MT) IF and MT IG genes. Southern blot analysis and partial DNA sequence determinations show that these genes are organized in a head-to-head fashion and are located approximately 7.0 kilobases apart from each other. Sequence analysis shows that the MT IF gene contains three exons separated by two introns. All of the intron-exon junctions are defined by the GT-AG rule. The 5' flanking region shows the presence of a duplicated metal regulatory element (TGCGC CCGGCCC) important in heavy-metal induction of this gene and a sequence for its basal level expression (GCGGGGCGGGTGCAAAG). The 5' flanking region is also highly G + C rich (approximately 75%) and contains several GC boxes (GGGCGG), probably important in the binding of transcription factors. The TATAA box and the AATAAA sequence are represented by their variants, the TATCAA box and the AATTAA sequence, respectively. This gene is functional and inducible by heavy metals but not by dexamethasone in mouse LMTK- cells after its transfer on a plasmid containing the herpes simplex virus thymidine kinase gene. Further studies on various human cell lines show that this gene is not expressed in a splenic lymphoblastoid cell line (WI-L2) but is expressed in two hepatoma cell lines (Hep 3B2 and Hep G2) in response to cadmium, zinc, and copper. Dexamethasone appears to have no significant effect on its expression. The studies suggest that the MT IF gene shows cell-type-specific expression and is differentially regulated by heavy metals and glucocorticoids.
DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.
Authors: Upton C , McFadden G
Published: September 2015

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.
Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene.
Authors: Seliger B , Kollek R , Stocking C , Franz T , Ostertag W
Published: September 2015

A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.
Expression of viral p21ras during acquisition of a transformed phenotype by rat adrenal cortex cells infected with Kirsten murine sarcoma virus.
Authors: MacAuley A , Auersperg N , Pawson T
Published: September 2015

Rat adrenal cortex cells infected with Kirsten murine sarcoma virus acquire a transformed phenotype in a progressive fashion. The expression of the viral p21ras does not appear to correlate with the degree of transformation of the adrenocortical cells but rather is produced at similar levels as the culture becomes transformed. This indicates that the expression of an oncogenic form of p21ras is not of itself sufficient to completely transform rat adrenal cortex cells.
Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.
Authors: McLeod M , Craft S , Broach J R
Published: September 2015

The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid. In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo. We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it. This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski, Cell 40:795-803, 1985; J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA 82:7270-7274, 1985). Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines. However, mutations outside the core are not similarly suppressed. We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate. Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency. The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.
Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.
Authors: Thelander L , Berg P
Published: September 2015

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.
Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.
Authors: Moran E , Zerler B , Harrison T M , Mathews M B
Published: September 2015

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.
Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae.
Authors: Valerie K , Fronko G , Henderson E E , de Riel J K
Published: September 2015

A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity.
Activation of the adenovirus and BK virus late promoters: effects of the BK virus enhancer and trans-acting viral early proteins.
Authors: Grinnell B W , Berg D T , Walls J
Published: September 2015

We have examined the activation of the adenovirus major late promoter (MLP) by the cis-acting enhancer element of the human polyomavirus BK and by the trans-acting simian virus 40 (SV40) T antigen and adenovirus E1A proteins. By using chloramphenicol acetyltransferase expression vectors, we found that the MLP (pLP-CAT) was trans-activated in human and monkey kidney cells expressing the SV40 T antigen. In addition, the MLP could be cis-activated by the BK virus enhancer in both human and monkey kidney cells; approximately 20 times more chloramphenicol acetyltransferase was produced from expression vectors containing a hybrid promoter (BL), in which the BK enhancer was upstream of the MLP, than from expression vectors containing the MLP alone. This same level of enhancement of the MLP by the BK enhancer was observed in cells expressing the T antigen of SV40. However, in the 293 cell line, greater enhancement of MLP activity (70-fold) was observed with the BK enhancer sequence. In contrast, MLP activity in the 293 cell line was unchanged by the SV40 enhancer. In cotransfection experiments, MLP activity, augmented by the BK enhancer, could be further stimulated with a plasmid coding for the E1A gene products. By creating deletion mutants, we determined that the high-level activation of the hybrid BL transcriptional unit by the E1A proteins requires both MLP sequences and an intact BK virus enhancer. On the other hand, activation of the BL transcriptional unit by the T antigen did not require an intact enhancer sequence. Our results suggest that the SV40 T antigen and E1A proteins trans-activate the BL promoter by different mechanisms. We also demonstrate in cotransfection experiments that the BK late promoter is activated 45-fold by the SV40 T antigen.
Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.
Authors: Klobutcher L A , Vailonis-Walsh A M , Cahill K , Ribas-Aparicio R M
Published: September 2015

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.
Human homologs of TU transposon sequences: polypurine/polypyrimidine sequence elements that can alter DNA conformation in vitro and in vivo.
Authors: Hoffman-Liebermann B , Liebermann D , Troutt A , Kedes L H , Cohen S N
Published: September 2015

We previously have shown that homologs of the outer domain segment of the inverted repeat termini (IVR-OD) of the sea urchin TU transposons are conserved among multiple eucaryotic species, including humans. We report here that two cloned human DNA IVR-OD homologs, Hut2 and Hut17, consist of a series of tandem repeats of the trimer AGG/TCC, forming segments (313 and 221 base pairs in length, respectively) of polypurine/polypyrimidine (pPu/pPy or "Puppy") asymmetry in the two DNA strands; these are punctuated at certain sites with variant trimers, which are different for the two clones. Sequences homologous to the Hut2 pPu/pPy tract exist at multiple sites in the DNA of a wide variety of eucaryotes. Hybridization of human DNA with a Hut2 probe or with a previously described chicken DNA pPu/pPy sequence indicates that pPu/pPy sequences can be grouped into families distinguishable by the extent of their homology with each probe at different hybridization stringencies. Moreover, particular pPu/pPy tracts show species-specific differences in their distribution. Both the Hut2 and Hut17 pPu/pPy tracts are cleaved by S1 nuclease when tested on supercoiled plasmids. Most if not all of the 313-base-pair Hut2 pPu/pPy tract is also sensitive to S1 in its native location in HeLa cell chromatin, indicating that the sequence contains conformational information that can be expressed in vivo. This view is supported by evidence that exogenously derived Hut2 pPu/pPy tracts introduced into mouse L cells and integrated in chromatin can assume an S1-sensitive conformation.
Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae.
Authors: Abrams E , Neigeborn L , Carlson M
Published: September 2015

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles. We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus. Both genes in multiple copy complemented only the cognate snf mutation. The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA. Both RNAs contained poly(A) and were present in low abundance. Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa. Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation. Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression. The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.
Transcriptional control signals of a herpes simplex virus type 1 late (gamma 2) gene lie within bases -34 to +124 relative to the 5' terminus of the mRNA.
Authors: Homa F L , Otal T M , Glorioso J C , Levine M
Published: September 2015

The cis-acting DNA sequences required for regulated expression of a herpes simplex virus type 1 (HSV-1) late (gamma 2) gene were studied by using viruses containing specific deletions in the 5' transcribed noncoding and upstream regions of the HSV-1 glycoprotein C (gC) gene, a model gamma 2 gene. Nine mutant viruses which had variable 5' and 3' deletions within bases -569 to +124 relative to the 5' terminus of the gC mRNA were isolated. The mutants were isolated by a simple in situ hybridization screening procedure not requiring any prior selective pressure for or against expression of the gC gene. Analysis of RNA extracted from cells infected with individual mutants showed that the DNA sequences required for regulated expression of this gamma 2 gene lay within bases -34 to +124. This 158-base-pair fragment was sufficient to confer accurate and quantitative expression of gC mRNA and to maintain the stringent requirement on viral DNA replication for expression of this gene. Moreover, it was found that sequences located between -34 and +14 contained signals essential for expression of gC. To determine whether the -34 to +124 sequences would function as a gamma 2 promoter when moved to another region of the HSV-1 genome, the 158-base-pair fragment was substituted for the normal thymidine kinase promoter-regulatory sequences in the thymidine-kinase gene locus. Transcription of this chimeric gene was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis. The only recognizable consensus sequence upstream of the transcription initiation site for this gene was the TATAAA sequence at -30.
Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.
Authors: Firak T A , Subramanian K N
Published: September 2015

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.
Structure of the protein encoded by the chicken proto-oncogene c-myb.
Authors: Gerondakis S , Bishop J M
Published: September 2015

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.
Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins.
Authors: Schatz P J , Solomon F , Botstein D
Published: September 2015

Microtubules in yeast are essential components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. The relative importance in these processes of the two divergent alpha-tubulin genes of the budding yeast Saccharomyces cerevisiae, TUB1 and TUB3, was examined through the construction of null mutations and by increasing their copy number on chromosomes and on plasmids. Experiments with null alleles of TUB3 showed that TUB3 was not essential for mitosis, meiosis, or mating. Null alleles of TUB3, however, did cause several phenotypes, including hypersensitivity to the antimicrotubule drug benomyl and poor spore viability. On the other hand, the TUB1 gene was essential for growth of normal haploid cells. Even in diploids heterozygous for a TUB1 null allele, several dominant phenotypes were evident, including slow growth and poor sporulation. This functional difference between the two genes is apparently due to different levels of expression, because extra copies of either gene could suppress the defects caused by a null mutation in the other. We conclude that in spite of the 10% divergence between the products of the two genes, there is no essential qualitative functional difference between them.
Regulation of adenovirus gene expression in human WI38 cells by an E1B-encoded tumor antigen.
Authors: White E , Faha B , Stillman B
Published: September 2015

Adenovirus mutants carrying alterations in the gene encoding the E1B 19-kilodalton tumor antigen (19K protein) cause enhanced cytopathic effect (cyt phenotype) and the degradation of host-cell chromosomal DNA (deg phenotype) upon infection of human HeLa or KB cells. Furthermore, E1B 19K gene mutant viruses are defective for cellular transformation. We report that these mutant viruses possess a host-range phenotype for growth in human cells. In human HeLa cells the mutant viruses grew to the same levels as the wild-type virus, but they were severely defective for growth in KB cells. In human WI38 cells, the E1B 19K gene mutant viruses had a substantial growth advantage over the wild-type virus, yielding 500-fold-higher titers. Viral DNA synthesis was reduced 10- to 20-fold in WI38 cells infected with the wild-type virus relative to that synthesized by the E1B mutant viruses. Viral early and late protein synthesis was similarly reduced in wild type- relative to mutant-infected cells. These reduced levels of early gene expression in wild-type virus-infected cells were paralleled by comparably reduced levels of early cytoplasmic mRNA. The primary cause of this host-range phenotype appeared at the level of early gene transcription, since transcription of viral early genes in the mutant-infected cells was substantially greater than levels found in cells infected with the wild-type virus. These results implicate the E1B 19K tumor antigen in the regulation of adenovirus early gene expression. Specifically, the E1B 19K protein directly or indirectly exerts a negative effect on early gene transcription accounting for efficient gene expression from the E1B mutant viruses in WI38 cells. Based on these findings it is probable that the cyt and deg phenotypes observed in mutant-infected HeLa and KB cells are the result of the pleiotropic effect of this altered gene regulation.
trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1.
Authors: Reisman D , Sugden B
Published: September 2015

Two regions of the Epstein-Barr virus (EBV) genome together make up an element, oriP, which acts in cis to support plasmid replication in cells that express the EBV nuclear antigen 1 (EBNA-1). The two components of oriP are a region containing a 65-base-pair (bp) dyad symmetry and a region containing 20 copies of a 30-bp direct repeat. Here we show that the 30-bp family of repeats of oriP can function as a transcriptional enhancer that is activated in trans by the EBNA-1 gene product. In either EBV-genome-positive cells or in cells that express EBNA-1, the 30-bp family of repeats, when positioned in either orientation upstream or downstream, enhances expression of the chloramphenicol acetyltransferase (CAT) gene expressed from either the simian virus 40 early promoter or the herpes simplex virus type 1 thymidine kinase promoter. The extent of transcriptional enhancement varies with the promoter and cell type. This enhanced CAT expression reflects an increased level of CAT mRNA and does not result from amplification of the plasmids expressing CAT. In addition, plasmids carrying the gene for resistance to hygromycin B and the 30-bp family of repeats yielded 10 to 100 times more hygromycin B-resistant colonies than the vector lacking the 30-bp family of repeats in both EBV-genome-positive cells and cells that express EBNA-1. EBNA-1 is known to bind to sequences within the 30-bp family of repeats (D. R. Rawlins, G. Milman, S. D. Hayward, and G. S. Hayward, Cell 42:859-868, 1985), and these trans- and cis-acting elements together have at least two functional roles: (i) they are required for DNA replication dependent upon oriP, and (ii) they can enhance expression of genes linked to the 30-bp family of repeats of oriP.
Characterization of a Tetrahymena thermophila mutant strain unable to develop normal thermotolerance.
Authors: Kraus K W , Hallberg E M , Hallberg R
Published: September 2015

For Tetrahymena thermophila cells to survive extended periods of time at 43 degrees C, they must continuously synthesize heat shock proteins. For its translational machinery to function at 43 degrees C, T. thermophila requires either prior nonlethal heat shock treatment or brief treatment with partially inhibiting doses of cycloheximide or emetine. We have identified and characterized a mutant strain of T. thermophila (MC-3) in which prior nonlethal heat shock does not prevent protein synthesis inactivation at 43 degrees C. In addition, treatment of MC-3 cells with either of the antibiotics that normally confer 43 degrees C thermoprotection on wild-type cells elicited no similar thermoprotective response in these cells. Despite these phenotypic characteristics, by other criteria MC-3 synthesized a normal, functional array of heat shock proteins at 40 degrees C, a nonlethal heat shock protein-inducing temperature. The mutation in MC-3 which prevents the thermostabilization of protein synthesis by nonlethal heat shock is, by genetic criteria, most likely the same one which prevents the induction of thermotolerance by drug treatments. We present evidence that this mutation may affect some ribosome-associated functions.
Structural and functional analysis of the MAL1 locus of Saccharomyces cerevisiae.
Authors: Charron M J , Dubin R A , Michels C A
Published: September 2015

We describe the isolation of a 22.6-kilobase fragment of DNA containing the MAL1 locus of Saccharomyces cerevisiae. Our results demonstrate that the MAL1 locus, like the MAL6 locus, is a complex locus containing three genes. These genes were organized similarly to their MAL6 counterparts. We refer to them as MAL11, MAL12, and MAL13 and show that they are functionally homologous to the MAL61 (encoding maltose permease), MAL62 (encoding maltase), and MAL63 (encoding the positive regulator) genes of the MAL6 locus. Transcription from each of the three genes was analyzed in a strain carrying the undisrupted MAL1 locus and in strains carrying single disruptions in each of the MAL1 genes. The MAL1 and MAL1 loci were found to be highly sequence homologous and conserved throughout the region containing these three genes. The strain used to isolate the MAL1 locus also carried the tightly linked SUC1 gene. The SUC1 gene was found to be located on the same 22.6-kilobase fragment containing the MAL1 locus and 5 kilobases from the 3' end of the MAL12 gene. The meaning of these results with regard to the mechanism of regulation of maltose fermentation is discussed.
Participation of the upstream region of the fibroin gene in the formation of transcription complex in vitro.
Authors: Tsuda M , Hirose S , Suzuki Y
Published: September 2015

The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructed by preincubation of the templates with the extracts, the inhibitory effect of histones was greatly reduced. Transcription of a fibroin gene construct, pFb5' delta-238, having the upstream region beyond the TATA box was relatively less inhibited than that of pFb5' delta-44 lacking the upstream region. This tendency toward differential inhibition was observed in the silk gland extracts but not in a HeLa cell extract and persisted even after complex formation in the silk gland extracts, suggesting a specific interaction of the upstream region with some factors in the extracts. The complexes formed on pFb5' delta-44 are probably more susceptible to the inhibitory effect of histones. On the basis of these results we propose a participation of the upstream region of the fibroin gene in the formation of stable transcription complexes at the promoter through an interaction with specific factors in the silk gland. Since the transcription-enhancing effect via the upstream region is augmented at a high histone/DNA ratio, it may mimic the in vivo situation in which the fibroin gene can be transcribed in the posterior silk gland even in the presence of excess suppressive materials.
Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.
Authors: Harashima S , Hinnebusch A G
Published: September 2015

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.
Tripartite sequences within and 3' to the sea urchin H2A histone gene display properties associated with a transcriptional termination process.
Authors: Johnson M R , Norman C , Reeve M A , Scully J , Proudfoot N J
Published: September 2015

We have defined a DNA sequence that behaves as an RNA polymerase II termination signal by using the human HeLa cell transient expression system. Surprisingly, this sequence is tripartite, including part of the coding region of the sea urchin H2A histone gene together with two separate sequences in the 3' flanking region of the gene. We demonstrate that this signal functions both in its normal gene environment and also when placed within the human alpha-globin gene. However, we have failed to detect a discrete 3' terminus. Rather, our data indicate the presence of an extremely heterogeneous series of nonpolyadenylated RNAs. These heterogeneous nonpolyadenylated RNAs are stable when transcribed from the intact histone gene but are highly unstable within the human alpha-globin gene. This provides evidence for the role of poly(A) in the stability of mRNA.
Deregulation of the c-myc oncogene in virus-induced thymic lymphomas of AKR/J mice.
Authors: Reicin A , Yang J Q , Marcu K B , Fleissner E , Koehne C F , O'Donnell P V
Published: September 2015

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.
Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28.
Authors: Lörincz A T , Reed S I
Published: September 2015

Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation. The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product. The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles. When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.
Specific activation of the cellular Harvey-ras oncogene in dimethylbenzanthracene-induced mouse mammary tumors.
Authors: Dandekar S , Sukumar S , Zarbl H , Young L J , Cardiff R D
Published: September 2015

Genomic DNAs from dimethylbenzanthracene-induced BALB/c mouse mammary tumors arising from the transplantable hyperplastic outgrowth (HPO) line designated DI/UCD transformed NIH 3T3 cells upon transfection. Transforming activity was attributed to the presence of activated Harvey ras-1 oncogenes containing an A----T transversion at the middle adenosine nucleotide in codon 61. DNAs from untreated DI/UCD HPO cells and radiation-induced and spontaneous mammary tumors from the DI/UCD HPO line failed to transform NIH 3T3 cells. The results indicated that the mutation activation of Harvey ras-1 oncogenes was specific to dimethylbenzanthracene treatment in the mouse mammary tumor system.
The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts.
Authors: Dougherty J P , Augereau P , Chambon P
Published: September 2015

The effect of the cell-specific mouse immunoglobulin heavy-chain gene (IgH) enhancer on transcription from heterologous promoter elements was studied in vitro with a HeLa whole-cell extract. No stimulation of transcription could be seen under conditions in which an activation was observed with the simian virus 40 enhancer. We found, however, that a specific segment of the IgH enhancer region contains sequences which inhibit transcription in vitro.
Negative regulation of the Saccharomyces cerevisiae ANB1 gene by heme, as mediated by the ROX1 gene product.
Authors: Lowry C V , Lieber R H
Published: September 2015

In Saccharomyces cerevisiae the anaerobic (oxygen-repressed) ANB1 gene and a group of aerobic (oxygen-induced) genes are coordinately regulated by the ROX1 gene. We report here that heme, known as an inducer of aerobic genes, also causes inhibition of ANB1 expression. Thus, in combination with the ROX1 gene product heme has an opposite effect on the expression of anaerobic and aerobic genes. Accumulation of ANB1 mRNA was sharply decreased in anaerobic cells grown in the presence of heme. This effect must operate at the level of transcription since heme also inhibited accumulation of CYC1 mRNA from an ANB1-CYC1 fusion. Heme precursors did not appear to function either as inhibitors or as activators. Oxygen itself also had no effect on transcription of ANB1. Repression by heme cannot be attributed to the respiratory competence conferred by heme since both ANB1 and the aerobic genes tr-1 and CYC1 were regulated normally in [rho 0] mutants. The results are consistent with a classical allosteric coeffector function for heme, although more indirect explanations are tenable. A role for the ROX1 gene product in transcriptional regulation can be inferred from the observation that there was no inhibition of ANB1 expression by heme in rox1 mutants. Judging from this epistasis the rox1 phenotype is not due to a defect in heme production; this would indicate that the ROX1 factor functions by mediating the effect of heme on transcription.
An unusual adenine phosphoribosyltransferase pseudogene is syntenic with its functional gene and is flanked by highly polymorphic DNAs.
Authors: Dush M K , Tischfield J A , Khan S A , Feliciano E , Sikela J M , Kozak C A , Stambrook P J
Published: September 2015

A mouse adenine phosphoribosyltransferase (aprt) pseudogene that had previously been recovered from a BALB/c sperm DNA library possessed several unusual features. Its nucleotide sequence, like that of other processed pseudogenes, was colinear with its corresponding mRNA, but it was truncated at its 3' end and lacked a poly(A) tail. The pseudogene was 82% homologous with corresponding regions of the functional gene and had incurred mutations that included transitions, transversions, deletions, and a point insertion. Even though the pseudogene was truncated within the protein-coding region of the corresponding functional gene, it was flanked at both ends by 13-base-pair direct repeats. Curiously, the direct repeats exhibited homology to APRT mRNA at the site of pseudogene divergence. The pseudogene appeared to be common to BALB/c and A/J mice, but it was contained on a 3-kilobase EcoRI fragment in the former strain and a 4.5-kilobase EcoRI fragment in the latter. The BALB/c and apparently the A/J pseudogene both mapped to chromosome 8, which also contains the functional aprt gene. The DNA sequences immediately surrounding the pseudogene in the two strains appeared to be similar, suggesting that the BALB/c and A/J pseudogenes are allelic. However, DNA sequences more distal to the pseudogene in the two strains appeared to vary. Thus, the EcoRI polymorphism was not due to simple loss of an EcoRI site, but was more complex. The pattern of flanking restriction sites was different for each of several enzymes, consistent with extensive DNA rearrangement. Double digests of BALB/c and A/J genomic DNAs revealed complex polymorphisms on both sides of the pseudogene. The results were consistent with insertion, deletion, or other rearrangement of DNA sequences that flank the pseudogene and suggest that this region of mouse chromosome 8 may be a region active for mutation or recombination.
Activation of an enhancerless gene by chromosomal integration.
Authors: Hamada H
Published: September 2015

Expression of enhancerless (E-) and enhancer-containing (E+) genes that are chromosomally integrated was examined. An E- plasmid (pE-cat) containing a chloramphenicol acetyltransferase (cat) gene linked to the simian virus 40 (SV40) early promoter or its E+ counterpart plasmid (pE+-cat) containing the SV40 enhancer was cotransfected into thymidine kinase (TK)-deficient L cells with a cloned tk gene. A number of TK+ transformants were isolated, and expression of the cointegrated cat gene in these cell lines was quantitatively determined by the assay of CAT activity. The results indicated unexpectedly that the E- cat gene was as actively expressed as the E+ cat gene. Analysis of CAT mRNA by primer extension indicated that the E- cat gene, as well as the E+ cat gene, was transcribed from the "native" initiation site contained in the SV40 early promoter region. The active expression of the E- cat gene was maintained in secondary TK+ transformants that arose by transfection with genomic DNA from the primary transformant. These results suggest that expression of the integrated E- cat gene is activated by endogenous enhancer elements.
Random isolation of gene activator elements from the human genome.
Authors: Hamada H
Published: September 2015

Long-range-acting gene activator elements were randomly isolated from the human genome by functional selection. HeLa cells were transfected with an enhancer trap, a plasmid containing an enhancerless xanthine-guanosine phosphoribosyltransferase (gpt) gene transcribed from the simian virus 40 early promoter, and stably transformed GPT+ cells were selected. From several transformants, human DNA sequences flanking the enhancer trap were cloned. Two gene activators (GA1 and GA2) were found in the cloned human DNAs. GA1 and GA2 showed strong enhancer activity both in a stable transformation assay and in a transient expression assay. They had functional properties similar to those of other known enhancers: GA1 and GA2 activated the expression of a linked gene over distances of at least 5 kilobases both upstream and downstream in an orientation-independent fashion. GA1 may be required for the initial establishment of gene activation but was not essential for the maintenance of active expression. GA1 and GA2 were active not only in HeLa cells but also in other types of human cells, such as neuroblastoma cells. This indicates a limited but relatively broad cell type specificity. The HeLa genome contains multiple copies of GA1, while GA2 exists once in the genome.