Workshop on biological applications of X-ray microscopy and imaging. Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA, 28-29 April 2003.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
T Paunesku
Publications and source records attributed to T Paunesku.
Report on the Workshop of Biological Applications of X-ray Microbeams.
Explore the source record for details and available documents.
Databases as Topic↗
Proliferating cell nuclear antigen (PCNA): ringmaster of the genome.
Proliferating cell nuclear antigen (PCNA) protein is one of the central molecules responsible for decisions of life and death of the cell. The PCNA gene is induced by p53, while PCNA protein interacts with p53-controlled proteins Gadd45, MyD118, CR6 and, most importantly, p21, in the process of deciding cell fate. If PCNA protein is present in abundance in the cell in the absence of p53, DNA replication occurs. On the other hand, if PCNA protein levels are high in the cell in the presence of p53, DNA repair takes place. If PCNA is rendered non-functional or is absent or present in low quantities in the cell, apoptosis occurs. The evolution from prokaryotes to eukaryotes involved a change of function of PCNA from a 'simple' sliding clamp protein of the DNA polymerase complex to an executive molecule controlling critical cellular decision pathways. The evolution of multicellular organisms led to the development of multicellular processes such as differentiation, senescence and apoptosis. PCNA, already an essential molecule in the life of single cellular organisms, then became a protein critical for the survival of multicellular organisms.
Amino Acid Sequence↗
p53 gene deletions in paraffin-preserved lymphoid tumors from irradiated mice.
Experiments were performed to measure deletions in the p53 gene in paraffin-embedded tissues (tumors and control) derived from mice exposed to gamma-rays or neutrons up to 28 years ago. Deletions in exons 1, 3, 4, 5, 6, 7 and 9 were monitored by PCR and Southern blotting techniques. The results of these experiments demonstrated p53 deletions in only 1/6 spontaneous tumors but in 5/6 gamma-ray-induced and 5/6 neutron-induced tumors. Exons deleted in tumors from gamma-ray exposed mice were similar to those deleted in tumors from neutron-exposed mice. They document differences in spectra of p53 deletions in comparing spontaneous radiation-induced tumors.
Animals↗
Identification of genes regulated by UV/salicylic acid.
PURPOSE: Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells. The presumed effector molecule responsible for this modulation is NF-kappaB. In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid. MATERIALS AND METHODS: Differential-display RT-PCR was used to identify differentially expressed genes. RESULTS: Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24 kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156). CONCLUSIONS: Sequences of four of these genes contained NF-kappaB DNA binding sites of the type that may attract transrepressor p55/p55 NF-kappaB homodimers. Down-regulation of these genes upon UV irradiation may contribute to increased cell survival via suppression of p53 independent apoptosis.
Adenosine Triphosphatases↗
Mechanisms of radiation-induced gene responses.
While identifying genes differentially expressed in cells exposed to ultraviolet radiation, we identified a transcript with a 25-nucleotide region that is highly conserved among a variety of species, including Bacillus circulans, pumpkin, yeast, Drosophila, mouse, and man. In the 5' untranslated region of a gene, the sequence is predominantly in a +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3' untranslated region, the sequence is most frequently in a -/+ orientation. The element is found in many different genes that have diverse functions. Gel mobility shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to double-stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated an additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. This element may bind to protein(s) that maintain DNA in a single-stranded orientation for transcription, or be important in the transcription-coupled DNA repair process.
Animals↗
DNA sequence recognition by hybridization to short oligomers: experimental verification of the method on the E. coli genome.
A newly developed method for sequence recognition by hybridization to short oligomers is verified for the first time in genome-scale experiments. The experiments involved hybridization of 15,328 randomly selected 2-kb genomic clones of Escherichia coli with 997 short oligomer probes to detect complementary oligomers within the clones. Lists of oligomers detected within individual clones were compiled into a database. The database was then searched using known E. coli sequences as queries. The goal was to recognize the clones that are identical or similar to the query sequences. A total of 76 putative recognitions were tested in two separate but complementary recognition experiments. The results indicate high specificity of recognition. Current and prospective applications of this novel method are discussed.
Base Sequence↗
Regulation of thymus PCNA expression is altered in radiation-sensitive wasted mice.
Mice bearing the autosomal recessive mutation 'wasted' (wst/wst) express a disease syndrome characterized by neurologic dysfunction, immunodeficiency, and increased sensitivity to the killing effects of ionizing radiation relative to normal littermates (wst/-) and to parental control mice (BCF1, BALB/c, and C57BL/6). Many of these abnormalities, evident as early as 21 days of age, have been localized to thymic tissues and T-lymphocyte populations. Comparison of two-dimensional gel electrophoresis patterns of proteins from wst/wst and control mouse thymus revealed that an acidic protein with a molecular mass of approximately 30 kDa was consistently expressed at lower levels in wasted mice than in controls. Microsequencing of this protein revealed a sequence of 19 N-terminal amino acids identical to the sequence of murine proliferating cell nuclear antigen (PCNA). Northern blot analyses of PCNA expression in thymus and spleen demonstrated lower accumulation of PCNA-specific transcripts in wasted mice compared with that in controls. Because PCNA expression is associated with cell cycle progression, the percentages of thymic and splenic cells in each stage of the cell cycle were examined; there were no differences in the cell stage distribution of lymphocytes freshly isolated from wasted mice compared with littermate or parental controls. After activation with concanavalin A, however, splenocytes from wst/wst mice showed a lower percentage of cells in S phase compared with that in controls. Southern blots with PCNA probes showed that the PCNA loci from the wasted mice and their normal littermates have the same restriction maps. While differences in polymerase chain reaction (PCR) priming were obtained, these could be attributed to strain-specific differences in mouse PCNA pseudogenes. These results suggest the presence of an alteration in the pathway leading to PCNA expression in radiation-sensitive tissues of wasted mice.
Animals↗
Discovering distinct genes represented in 29,570 clones from infant brain cDNA libraries by applying sequencing by hybridization methodology.
To discover all distinct human genes and to determine their patterns of expression across different cell types, developmental stages, and physiological conditions, a procedure is needed for fast, mutual comparison of hundreds of thousands (and perhaps millions) of clones from cDNA libraries, as well as their comparison against data bases of sequenced DNA. In a pilot study, 29,570 clones in duplicate from both original and normalized, directional, infant brain cDNA libraries were hybridized with 107-215 heptamer oligonucleotide probes to obtain oligonucleotide sequence signatures (OSSs). The OSSs were compared and clustered based on mutual similarity into 16,741 clusters, each corresponding to a distinct cDNA. A number of distinct cDNAs were successfully recognized by matching their 107-probe OSSs against GenBank entries, indicating the possibility of sequence recognition with only a few hundred randomly chosen oligomers.
Animals↗
Expression of thymidine kinase messenger RNA and a related transcript is modulated by radioprotector WR1065.
Previous studies have shown that the radioprotector WR1065 protects against mutagenesis across a wide concentration range (i.e., 40 microM to 4 mM) but protects against cell killing by ionizing radiation at concentrations greater than 1 mM. Other work has demonstrated that many genes are induced or repressed after exposure of cells in culture to ionizing radiation, but the actual inducing agents for this gene modulation response are unknown. In these experiments, we set out to identify genes that would be modulated in response to two different concentrations of WR1065 (i.e., a lower dose that is incapable of protecting against cell killing but effective in protecting against mutation induction, and a high dose that is effective in protecting against both end points). Using differential display reverse transcription-PCR, we compared genes expressed in untreated cells to those expressed in cells treated with different concentrations of WR1065 (4 mM or 40 microM) with or without radiation exposure (7.5 Gy). One band, which showed a differential response, was sequenced and found to have homology in the 3'-untranslated region of the mouse thymidine kinase (tk) gene but not identity to the Chinese hamster ovary tk gene. Dot blot and Northern blot analyses confirmed the differential display results and also determined that regulation of the tk-like gene is similar to that of tk itself. These experiments established that in Chinese hamster ovary cells, radiation causes a repression in accumulation of tk mRNA and a related tk-like transcript. This repression is made less dramatic by the presence of 40 microM WR1065, and, in fact, expression becomes enhanced when cells are pretreated with 4 mM WR1065. This suggests a role for regulation of tk and its related gene in the survival response of cells after exposure to ionizing radiation.
Animals↗
Clone clustering by hybridization.
DNA sequencing by hybridization (SBH) Format 1 technique is based on experiments in which thousands of short oligomers are consecutively hybridized with dense arrays of clones. In this paper we present the description of a method for obtaining hybridization signatures for individual clones that guarantees reproducibility despite a wide range of variations in experimental circumstances, a sensitive method for signature comparison at prespecified significance levels, and a clustering algorithm that correctly identifies clusters of significantly similar signatures. The methods and the algorithm have been verified experimentally on a control set of 422 signatures that originate from 9 distinct clones of known sequence. Experiments indicate that only 30 to 50 oligomer probes suffice for correct clustering. This information about the identity of clones can be used to guide both genomic and cDNA sequencing by SBH or by standard gel-based methods.
Algorithms↗
A presumed B6 strain-specific p53 polymorphism is confined to a B6 cell line and is likely to represent a facilitating mutation.
Explore the source record for details and available documents.
Animals↗
DNA sequence determination by hybridization: a strategy for efficient large-scale sequencing.
The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.
Animals↗
DNA sequencing by hybridization: 100 bases read by a non-gel-based method.
Determination of the sequences of human and other complex genomes requires much faster and less expensive sequencing processes than the methods in use today. Sequencing by hybridization is potentially such a process. In this paper we present hybridization data sufficient to accurately read a known sequence of 100 base pairs. In independent reactions, octamer and nonamer oligonucleotides derived from the sequence hybridized more strongly to this DNA than to controls. The 93 consecutive overlapping probes were derived from a 100-base-pair segment of test DNA and additional probes were generated by incorporation of a noncomplementary base at one of the ends of 12 of the basic probes. These 12 additional probes also had a full-match target in one of the control DNAs. The test and one of five control DNAs spotted on nylon filters were hybridized with 83 octamers and 22 nonamers under low-temperature conditions. A stronger signal in DNA containing a full-match target compared to DNA with only mismatched targets was obtained with all 105 probes. In 3 cases (2.9%), the difference of signals was not significant (less than 2-fold) due to inefficient hybridization and the consequently higher influence of background. The hybridization pattern obtained enabled us to resequence the 100 base pairs by applying an algorithm that tolerates an error rate much higher than was observed in the experiment. With this result, the technological components of large-scale DNA sequencing using the sequencing by hybridization method are in place.
Base Sequence↗
Origin of rat beta-globin haplotypes containing three and five genes.
We have reported in rat three adult beta-gene haplotypes containing either five or three genes. Detailed sequence analysis reveals that the leftmost gene is the major gene and that at the opposite end downstream lies the minor gene. All of the genes lying between them are minor-major hybrids indicating their origin by unequal crossing-over. In two haplotypes beta-globin genes were found with an L1(1) element inserted directly into IVS2. The described results allow the formulation of a pathway of mutational events leading from the ancient two-beta-gene rodent ancestor through a three-gene haplotype to five-gene haplotypes, one of which is postulated to have arisen in common laboratory strains since their capture in the wild.
Animals↗
Variant chromosomal arrangement of adult beta-globin genes in rat.
The genomic organization of three haplotypes of beta-globin genes was determined to resolve the question of the number of those genes in rat. Haplotype a, found in inbred strain DA, has three genes or pseudogenes, while haplotypes b, found in AO, Y5 and Wistar strains, and c, found in Wistar strain, have five genes or pseudogenes each. In haplotypes b and c, the first gene is of beta major type and the remaining four are of beta minor type. Partial sequencing of six out of 13 genes shows that duplications of beta minor genes are causing polymorphism in a number of genes. Also, in haplotype b two beta minor genes have a 6.5-kb intron 2, while in haplotype c only one beta minor gene contains such a large intron 2. The three structurally different haplotypes described are not interconvertible by single recombination events. The results indicate that the rat has the highest number of adult beta-globin genes found in mammals so far.
Amino Acid Sequence↗