bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple mechanisms provide rapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae.
Authors: Johnston M , Flick J S , Pexton T
Published: October 2015

Expression of the GAL genes of Saccharomyces cerevisiae is induced during growth on galactose by a well-characterized regulatory mechanism that relieves Gal80p inhibition of the Gal4p transcriptional activator. Growth on glucose overrides induction by galactose. Glucose repression acts at three levels to reduce GAL1 expression: (i) it reduces the level of functional inducer in the cell; (ii) it lowers cellular levels of Gal4p by repressing GAL4 transcription; and (iii) it inhibits Gal4p function through a repression element in the GAL1 promoter. We quantified the amount of repression provided by each mechanism by assaying strains with none, one, two, or all three of the repression mechanisms intact. In a strain lacking all three repression mechanisms, there was almost no glucose repression of GAL1 expression, suggesting that these are the major, possibly the only, mechanisms of glucose repression acting upon the GAL genes. The mechanism of repression that acts to reduce Gal4p levels in the cell is established slowly (hours after glucose addition), probably because Gal4p is stable. By contrast, the repression acting through the upstream repression sequence element in the GAL1 promoter is established rapidly (within minutes of glucose addition). Thus, these three mechanisms of repression collaborate to repress GAL1 expression rapidly and stringently. The Mig1p repressor is responsible for most (possibly all) of these repression mechanisms. We show that for GAL1 expression, mig1 mutations are epistatic to snf1 mutations, indicating that Mig1p acts after the Snf1p protein kinase in the glucose repression pathway, which suggests that Snf1p is an inhibitor of Mig1p.
Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.
Authors: Cheng C , Kacherovsky N , Dombek K M , Camier S , Thukral S K , Rhim E , Young E T
Published: October 2015

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.
scid cells efficiently integrate hairpin and linear DNA substrates.
Authors: Staunton J E , Weaver D T
Published: October 2015

The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.
Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3.
Authors: Kjaerulff S , Davey J , Nielsen O
Published: October 2015

We previously identified two genes, mfm1 and mfm2, with the potential to encode the M-factor mating pheromone of the fission yeast Schizosaccharomyces pombe (J. Davey, EMBO J. 11:951-960, 1992), but further analysis revealed that a mutant strain lacking both genes still produced active M-factor. Here we describe the isolation and characterization of a third M-factor gene, mfm3. A mutant lacking all three genes fails to produce M-factor, indicating that all functional M-factor genes now have been identified. The triple mutant exhibits an absolute mating defect in M cells, a defect that is not rescued by addition of exogenous M-factor. A mutational analysis reveals that all three mfm genes contribute to the production of M-factor. Their transcription is limited to M cells and requires the mat1-Mc and ste11 gene products. Each gene is induced when the cells are starved of nitrogen and further induced by a pheromone signal. Additionally, the signal transduction machinery associated with the pheromone response is required for transcription of the mfm genes in both stimulated and unstimulated cells.
BCL3 encodes a nuclear protein which can alter the subcellular location of NF-kappa B proteins.
Authors: Zhang Q , Didonato J A , Karin M , McKeithan T W
Published: October 2015

BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.
A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.
Authors: Lu Y , Alarcon C M , Hall T , Reddy L V , Donelson J E
Published: October 2015

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.
Human replication protein A binds single-stranded DNA in two distinct complexes.
Authors: Blackwell L J , Borowiec J A
Published: October 2015

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.
Repression of cyclin D1: a novel function of MYC.
Authors: Philipp A , Schneider A , Väsrik I , Finke K , Xiong Y , Beach D , Alitalo K , Eilers M
Published: October 2015

Constitutive expression of human MYC represses mRNA levels of cyclin D1 in proliferating BALB/c-3T3 fibroblasts. We expressed a series of mutant alleles of MYC and found that downregulation of cyclin D1 is distinct from previously described properties of MYC. In particular, we found that association with Max is not required for repression of cyclin D1 by MYC in vivo. Conversely, the integrity of a small amino-terminal region (amino acids 92 to 106) of MYC is critical for repression of cyclin D1 but dispensable for transformation of established RAT1A cells. Runoff transcription assays showed that repression occurs at the level of transcription initiation. We cloned the promoter of the gene for human cyclin D1 and found that it lacks a canonical TATA element. Transcription starts at an initiator element similar to that of the adenovirus major late promoter; this element can be directly bound by USF in vitro. Expression of MYC represses the cyclin D1 promoter via core promoter elements and antagonizes USF-mediated transactivation. Taken together, our data define a new pathway for gene regulation by MYC and show that the cyclin D1 gene is a target gene for repression by MYC.
Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.
Authors: Jacobs Y , Xin X Q , Dorshkind K , Nelson C
Published: October 2015

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.
Differential binding of c-Myc and Max to nucleosomal DNA.
Authors: Wechsler D S , Papoulas O , Dang C V , Kingston R E
Published: October 2015

The ability of a transcription factor to function in vivo must be determined in part by its ability to bind to its recognition site in chromatin. We have used Max and derivatives of c-Myc to characterize the effect of changes of dimerization partner on binding to nucleosomal DNA templates. We find that homo- and heterodimeric complexes of these proteins bind to the CACGTG sequence in free DNA with similar affinities. Although Max homodimers bind to nucleosomes, truncated c-Myc homodimers do not. Surprisingly, modifying the c-Myc dimerization interface or changing its dimerization partner to Max enables nucleosomal DNA binding. Thus, changes in dimer structure or dimerization efficiency can have significant effects on nucleosome binding that are not predicted from their affinity for free DNA. We conclude that domains other than the basic region per se influence the ability of a transcription factor to bind to nucleosomal DNA and that changes of dimerization partner can directly affect the ability of a factor to occupy nucleosomal binding sites.
GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.
Authors: Albertyn J , Hohmann S , Thevelein J M , Prior B A
Published: October 2015

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.
Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.
Authors: McKune K , Woychik N A
Published: October 2015

We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6.
m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain.
Authors: Plessel G , Fischer U , Lührmann R
Published: October 2015

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.
An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.
Authors: Sweeney R , Chen L , Yao M C
Published: October 2015

Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.
Myogenic differentiation triggered by antisense acidic fibroblast growth factor RNA.
Authors: Fox J C , Hsu A Y , Swain J L
Published: October 2015

Acidic fibroblast growth factor (FGF) and related family members regulate differentiation in organisms as diverse as Xenopus laevis and mammals. We utilized a well-characterized model of myogenic development to directly assess the importance of endogenously produced FGF in controlling differentiation. A role for endogenous FGF is suggested by the previous finding that acidic and basic FGF abundance in cultured myocytes decreases during differentiation. In this study we inhibited the endogenous production of FGF in murine Sol 8 myoblasts by using antisense RNA and observed precocious myogenic differentiation. Exogenously supplied acidic FGF rescues this phenotype. Further results suggest that the effect of FGF on myogenic differentiation is mediated in part through inhibition of myogenin expression. These results demonstrate a direct role for endogenously synthesized growth factors in regulating myogenesis and provide support for a general role for related proteins in mammalian development.
The human cut homeodomain protein represses transcription from the c-myc promoter.
Authors: Dufort D , Nepveu A
Published: October 2015

Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene.
TATA-dependent enhancer stimulation of promoter activity in mice is developmentally acquired.
Authors: Majumder S , DePamphilis M L
Published: October 2015

Herpes simplex virus (HSV) thymidine kinase (tk) promoter activity depends on four transcription factor binding sites, one of which is a TATA box sequence, and the presence of either a cis-acting enhancer sequence or a transactivator protein. Studies presented here show that this TATA box was required for promoter activity only after cells began to differentiate and then only when promoter activity was stimulated by either an enhancer or a transactivator. When the HSV tk promoter was utilized by mouse embryos from the one-cell to eight-cell stage of development or by undifferentiated mouse embryonic stem cells, disruption of the HSV tk TATA box by site-specific mutations did not reduce promoter activity. This was true even when HSV tk promoter activity was stimulated strongly by either the embryo-responsive polyomavirus F101 enhancer or its natural transactivator, the HSV ICP4 gene product. However, stimulated expression was dependent on a distal Sp1 DNA binding site. Similarly, disruption of the TATA box did not reduce tk promoter activity in primary mouse embryonic fibroblasts or in immortalized 3T3 mouse fibroblasts; in fact, promoter activity was increased up to 2.6-fold. However, in these differentiated cells, stimulation of the HSV tk promoter by either the F101 enhancer or ICP4 protein required the TATA box. HSV tk promoter activity also was dependent on its TATA box in the mouse oocyte, a terminally differentiated cell with an endogenous transactivating activity. These results reveal that the need for a TATA box is developmentally acquired and depends on at least two parameters: the differentiated state of the cell and stimulation of the promoter by either an enhancer or a transactivator.
GATA elements are necessary for the activity and tissue specificity of the T-cell receptor beta-chain transcriptional enhancer.
Authors: Henderson A J , McDougall S , Leiden J , Calame K L
Published: October 2015

Three high-affinity binding sites for the GATA family of transcriptional regulators have been identified within the T-cell receptor beta-chain (TCR beta) transcriptional enhancer, and their functional significance has been determined in an effort to understand the T-cell specificity of the enhancer more fully. One site, TE4, is important for activity of the enhancer in T cells. Neither site TE1 nor site TE2 can functionally replace a mutated TE4 site in T cells; however, the same protein, probably GATA-3, binds all three sites, as judged by electrophoretic mobility shift, oligonucleotide competition, and proteolytic clipping assays. These data suggest that additional proteins are critical for the ability of GATA-3 to activate the TCR beta enhancer. In fibroblasts, the GATA sequence at site TE1 appears to bind a negative regulator. Since this is not true in B cells, B cells and fibroblasts appear to have different mechanisms for negative regulation of the TCR beta enhancer.
Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element.
Authors: Ueda T , Wang Z , Pham N , Messing J
Published: October 2015

By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter. The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization. A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells. In vitro binding and DNase I footprinting assays with nuclear protein prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region. The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes. A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro. It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner. Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor. The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the alpha-class zein promoter. These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes.
Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.
Authors: Li C Y , Yandell D W , Little J B
Published: October 2015

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.
The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.
Authors: Chen L I , Nishinaka T , Kwan K , Kitabayashi I , Yokoyama K , Fu Y H , Grünwald S , Chiu R
Published: October 2015

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.
Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins.
Authors: McAlear M A , Howell E A , Espenshade K K , Holm C
Published: October 2015

In addition to its role as a processivity factor in DNA replication, proliferating cell nuclear antigen (PCNA) may function in the regulation of cell cycle progression. We present genetic evidence that PCNA interacts with the gene product of CDC44, an essential nucleotide-binding protein that encodes the large subunit of yeast replication factor C (K. Fien and B. Stillman, personal communication). Mutations in POL30 (PCNA) suppress cold-sensitive alleles of cdc44 that contain mutations in or near nucleotide-binding consensus domains, but they do not suppress a null allele. Thus, it appears that PCNA interacts with Cdc44p but cannot substitute for its function. pol30 mutations suppress additional phenotypes of cdc44 mutations, including the cold sensitivity that they were selected to suppress. This observation suggests an intimate association between PCNA and Cdc44p. Each of five independent pol30 mutants contains a unique single mutation that maps to a localized region on one face of the predicted three-dimensional structure of PCNA. This face identifies a region likely to be important for functional interaction between the CDC44 and POL30 gene products.
Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.
Authors: Ramaiah K V , Davies M V , Chen J J , Kaufman R J
Published: October 2015

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.
trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.
Authors: Xu G L , Wieland B , Bindereif A
Published: October 2015

U6 RNA genes from the trypanosomatids Crithidia fasciculata and Leptomonas seymouri have been isolated and sequenced. As in Trypanosoma brucei, the U6 RNA genes in both C. fasciculata and L. seymouri are arranged in close linkage with upstream tRNA genes. The U6 RNA sequences from C. fasciculata and L. seymouri deviate in five and three positions, respectively, from the published T. brucei sequence. Interestingly, both C. fasciculata U6 RNA genes carry a C-->T change at the second position of the ACAGAG hexanucleotide sequence, which is important for splicing function and has been considered phylogenetically invariable. A compensatory base change of the C. fasciculata spliced leader RNA at the highly conserved 5' splice site position +5, G-->A, suggests that an interaction between the 5' splice site region and U6 RNA recently proposed for the yeast cis-splicing system may also occur in trans splicing.
A functional insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the epidermal growth factor receptor.
Authors: Coppola D , Ferber A , Miura M , Sell C , D'Ambrosio C , Rubin R , Baserga R
Published: October 2015

When wild-type mouse embryo cells are stably transfected with a plasmid constitutively overexpressing the epidermal growth factor (EGF) receptor (EGFR), the resulting cells can grow in serum-free medium supplemented solely with EGF. Supplementation with EGF also induces in these cells the transformed phenotype (growth in soft agar). However, when the same EGFR expression plasmid is introduced and overexpressed in cells derived from littermate embryos in which the insulin-like growth factor I (IGF-I) receptor genes have been disrupted by homologous recombination, the resulting cells are unable to grow or to be transformed by the addition of EGF. Reintroduction into these cells (null for the IGF-I receptor) of a wild-type (but not of a mutant) IGF-I receptor restores EGF-mediated growth and transformation. Our results indicate that at least in mouse embryo fibroblasts, the EGFR requires the presence of a functional IGF-I receptor for its mitogenic and transforming activities.
Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.
Authors: Matsumoto K , Ishimi Y
Published: October 2015

DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively.
The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain.
Authors: Romac J M , Graff D H , Keene J D
Published: October 2015

Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to U1 RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus.
Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites.
Authors: Prescott J , Falck-Pedersen E
Published: October 2015

The adenovirus major late transcription unit is a well-characterized transcription unit which relies heavily on alternative pre-mRNA processing to generate distinct populations of mRNA during the early and late stages of viral infection. In the early stage of infection, two major late transcription unit mRNA transcripts are generated through use of the first (L1) of five available poly(A) sites (L1 through L5). This contrasts with the late stage of infection when as many as 45 distinct mRNAs are generated, with each of the five poly(A) sites being used. In previous work characterizing elements involved in alternative poly(A) site use, we showed that the L1 poly(A) site is processed less efficiently than the L3 poly(A) site both in vitro and in vivo. Because of the dramatic difference in processing efficiency and the role processing efficiency plays in production of steady-state levels of mRNA, we have identified the sequence elements that account for the differences in L1 and L3 poly(A) site processing efficiency. We have found that the element most likely to be responsible for poly(A) site strength, the GU/U-rich downstream element, plays a minor role in the different processing efficiencies observed for the L1 and L3 poly(A) sites. The sequence element most responsible for inefficient processing of the L1 poly(A) site includes the L1 AAUAAA consensus sequence and those sequences which immediately surround the consensus hexanucleotide. This region of the L1 poly(A) site contributes to an inability to form a stable processing complex with the downstream GU/U-rich element. In contrast to the L1 element, the L3 poly(A) site has a consensus hexanucleotide and surrounding sequences which can form a stable processing complex in cooperation with the downstream GU/U-rich element. The L3 poly(A) site is also aided by the presence of sequences upstream of the hexanucleotide which facilitate processing efficiency. The sequence UUCUUUUU, present in the L3 upstream region, is shown to enhance processing efficiency as well as stable complex formation (shown by increased binding of the 64-kDa cleavage stimulatory factor subunit) and acts as a binding site for heterogeneous nuclear ribonucleoprotein C proteins.
Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes.
Authors: Wolf V J , Dang T , Hartl P , Gottesfeld J M
Published: October 2015

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.
N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.
Authors: Cadwallader K A , Paterson H , Macdonald S G , Hancock J F
Published: October 2015

Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.
Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes.
Authors: Firpo E J , Koff A , Solomon M J , Roberts J M
Published: October 2015

Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.