The lived experience of visual creative expression for young adult cancer survivors.

Engaging in visual creative expression individually and in a therapeutic setting can be a beneficial experience for cancer survivors; however, most research in this field has been conducted with older adults. The current study aimed to address this gap by utilising van Manen's hermeneutic phenomenology to answer the following question: 'What is the lived experience and meaning of visual creative expression for young adult cancer survivors?' Seven young adults, diagnosed with cancer between the ages of 18 and 35, were interviewed about creative expression experiences, which they engaged in individually and/or in a therapeutic setting. Data analysis included a thematic reflection, guided existential reflection, and a process of writing and rewriting. Two superordinate themes were identified: increased self-understanding and a healing experience. Seven subthemes were also identified and included the following: being in the flow, allowing the body to express itself, renegotiating control, changing one's environment, being seen, respect for art as a separate entity and giving back. Findings suggest that visual creative expression can be a meaningful experience for young adult cancer survivors, and that this experience espouses both similarities and differences from experiences of older adult survivors. Recommendations are made for future research, in addition to implications for practitioners.

In vivo cytotoxic T lymphocyte elicitation by mycobacterial heat shock protein 70 fusion proteins maps to a discrete domain and is CD4(+) T cell independent.

To gain insights into the mechanisms by which soluble heat shock protein (hsp) fusions can elicit CD8(+) cytotoxic T lymphocytes (CTLs) against the fusion partner, mycobacterial (Mycobacterium tuberculosis) hsp70 was dissected to ascertain whether a particular hsp domain is necessary, and knockout mice were used to determine whether the fusion protein's immunogenicity is dependent on CD4(+) T lymphocytes. We found that the ability to elicit CD8(+) CTLs depends on a discrete 200-amino acid protein domain, indicating that the fusion protein's immunogenicity for CD8(+) T cells does not require coupled chaperone function or peptide binding. Further, we found that ovalbumin (OVA).hsp70 fusion protein elicited anti-OVA CD8(+) CTLs about equally well in CD4 knockout and wild-type C57BL/6 mice, and also when the hsp70 was of murine (self) origin. The ability of hsp70 fusion proteins to elicit CD4-independent CTL responses suggests that hsp70 fusion proteins may be useful for immunological prophylaxis and therapy against disease in CD4(+) T cell-deficient individuals.

Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.

We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2-restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV-infected HLA-A2+ cell lines were specifically lysed by both A2-restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells.

Yeast RNA polymerase II genes: isolation with antibody probes.

Genes encoding yeast RNA polymerase II subunits were cloned. Efficient isolation of these genes was accomplished by probing a phage lambda gt11 recombinant DNA expression library with polyvalent antibodies directed against purified yeast RNA polymerase II. The identity of genes that specify the largest RNA polymerase II subunits, the 220,000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay. Both of these genes exist in single copy in the yeast Saccharomyces cerevisiae.

Monoclinic hydroxyapatite.

The existence of a monoclinic phase of hydroxyapatite, Ca(2)(PO(4))(4)OH, has been confirmed, by single-crystal structure analysis (weighted "reliability" factor = 3.9 percent on |F|(2)). The structure has space group P21/b, a = 9.4214(8) angstroms, b = 2a, c = 6.8814(7) angstroms, and gamma = 120 degrees , and is analogous to that of chlorapatite. The distortions from the hexagonal structure with which the monoclinic structure is pseudosymmetric are similar to those in chlorapatite, including enlargement of that triangular array of oxygen atoms in which the chlorine ion or, in hydroxyapatite, the hydroxyl hydrogen ion is approximately centered. The hydroxyapatite specimen was prepared by the conversion of a single crystal of chlorapatite in steam at 1200 degrees C, was mimetically twinned, and was approximately 37 percent monoclinic.

Dispersal pathways for particle-associated pollutants.

Particle-associated pollutants (totaling 10(7) metric tons per year) are introduced into the New York Bight by ocean dumping, estuarine discharge, sewage outfalls, eolian transport, and shipping waste and spillage. Oceanic and estuarine circulation processes dilute and transport the particles by a natural dispersal system that also tends to be highly distributive; particle-associated pollutants apparently seek the same sinks in the Hudson River shelf valley and intracoastal weltlands, regardless of their point of introduction.

The plasticity of dendritic cell responses to pathogens and their components.

Dendritic cells are involved in the initiation of both innate and adaptive immunity. To systematically explore how dendritic cells modulate the immune system in response to different pathogens, we used oligonucleotide microarrays to measure gene expression profiles of dendritic cells in response to Escherichia coli, Candida albicans, and influenza virus as well as to their molecular components. Both a shared core response and pathogen-specific programs of gene expression were observed upon exposure to each of these pathogens. These results reveal that dendritic cells sense diverse pathogens and elicit tailored pathogen-specific immune responses.

Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins.

DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes. Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication. Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome. Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function. A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats). These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

Genome-wide location and function of DNA binding proteins.

Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.

RNA polymerase II: subunit structure and function.

RNA polymerase II is the core of the complex apparatus that is responsible for the regulated synthesis of mRNA. A comprehensive knowledge of RNA polymerase II is essential to our understanding of the molecular mechanisms through which a variety of transcription factors regulate eukaryotic gene expression. The recent cloning of genes for all ten subunits of yeast RNA polymerase II has revealed intriguing similarities and differences between the eukaryotic RNA polymerase and its simpler prokaryotic counterpart. Epitope tagging and other experiments made possible by the cloning of these genes have provided a clearer picture of RNA polymerase II subunit composition, stoichiometry and function, and set the stage for further investigating the dialogue between RNA polymerase II and transcription factors.

The RNA polymerase II holoenzyme and its implications for gene regulation.

The RNA polymerase II (Pol II) transcription initiation apparatus consists of several multisubunit complexes, including Pol II, general transcription factors and suppressor of RNA polymerase B (SRB) proteins. Recent evidence indicates that many of these components assemble into a large complex, called the RNA polymerase holoenzyme, the SRB components of which participate in the response to transcriptional regulators. We discuss these results and their implications for the regulation of gene expression.

Regulatory targets in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme is the form of polymerase recruited to promoters for protein-coding genes. Several targets of mammalian activators, previously called coactivators, turn out to be subunits of the holoenzyme which activators use to recruit and regulate the holoenzyme. Several of these newly identified holoenzyme components have been implicated in human disease.

Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction.

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.

To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III.

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth.

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.

RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.

RNA polymerase II mutants defective in transcription of a subset of genes.

Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species.

Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection.

A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.

A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival.

Cells respond to stress by altering gene expression, and these adjustments facilitate stress tolerance. Although transcriptional changes are integral to most stress responses, little is known about the mechanisms that permit the transcription apparatus itself to tolerate stress. Here we report that a major role of the RNA polymerase II subunit RPB4 is to permit appropriate transcriptional responses during stress. Yeast cells lacking RPB4 have essentially wild-type growth rates at moderate temperatures (18 to 22 degrees C), but their growth rates are substantially reduced at temperatures outside this range. When subjected to a heat shock, cells lacking RPB4 rapidly lose the ability to transcribe genes and experience a dramatic loss in viability. When cells lacking RPB4 are subjected to the nutrient stress that accompanies entry into stationary phase, they also exhibit a substantial decline in mRNA synthesis and in viability relative to wild-type cells. Interestingly, the portion of RNA polymerase II molecules that contain RPB4 is small in log phase but increases substantially as cells enter stationary phase. We propose that the association of RPB4 with the other RNA polymerase II subunits increases the tolerance of the enzyme to stress.