Tatjana Paunesku

Variant chromosomal arrangement of adult β-globin genes in rat

Abstract The genomic organization of three haplotypes of β-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 βmajor type and the remaining four are of βminir type. Partial sequencing of six out of 13 genes shows that duplications of βminor genes are causing polymorphism in a number of genes. Also, in haplotype b two βminor genes have a 6.5-kb intron 2, while in haplotype c only one βminor 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 β-globin genes found in mammals so far.

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.

A presumed B6 strain-specific p53 polymorphism is confined to a B6 cell line and is likely to represent a facilitating mutation

Sequencing by hybridization techniques (Drmanac et al. 1990) is being used to analyze the incidence of specific p53 mutations associated with radiation-induced and spontaneous lymphosarcomas in mice. One sequence difference noted as being a mouse strain-specific polymorphism has been identified through these experiments as being a mutational, rather than a polymorphic, site. Southern blots of various p53 exon DNAs produced by PCR were hybridized to [732p]ATP-labeled oligonucleotides. Table 1 contains the PCR amplification sequences, oligonucleotide probes, and other details. The reaction conditions (see Drmanac et al. 1990; 10 ng probe/1 ml hybridization buffer [0.5 M Na2HPO 4, 7% wt/vol sodium lauroyl sarcosine] at 12~ for 16 h, followed by a 40rain wash at 12~ in 4• standard saline citrate) were designed to enable oligomer probes to hybridize with fullmatch target sequences severalfold more efficiently than with mismatched targets. We designed a set of oligonucleotide probes that could be used to detect variant sequences in mutational hotspots that have been shown to be associated with p53 oncogenic changes in different tumor systems (Hollstein et al. 1991). As a model for optimizing the method, we used p53 exon VI sequences of C57BL/6 and BALB/c mouse strains. Bienz and coworkers (1984) had reported a strain-specific nucleotide polymorphism that occurs as a Met codon at amino acid position 234 in the p53 protein of the BALB/c mouse and as an Ile codon in p53 from the C57BL/6 mouse strain. This was described as one of several mouse strain-specific polymorphisms in the p53 gene. According to these reported polymorphisms (Bienz et al. 1984), probes

Changes in gene expression following EMF exposure

1 and A1 (see Table 1) would hybridize only to BALB/c exon V1 p53 sequence, whereas probes

Deficient PCNA expression and radiation sensitivity

2 and A2 would have a full-match target in the C57BL/6 exon VI p53 DNA and in one or two additional RAG-1 control DNA sequences (see sequences in Table 1). As shown in Fig. 1, however, both BALB/c and C57BL/6 mice have a sequence coding for a Met codon for amino acid position

Proliferating cell nuclear antigen (PCNA) : ringmaster of the genome.

Experiments were designed to examine the effects of electromagnetic field (EMF) exposure on specific gene expression, an effect that can be deleterious, beneficial, or neutral, depending on the long-term consequences; however, the proof of a reproducible, quantitative biological effect (such as change in gene expression) will lead to latter experiments aimed at determining the relative contribution of these changes to cellular consequences. Past work by ourselves and by others has shown that measures of gene expression are extremely sensitive indicators of the cellular and biological effects of ionizing radiation, with transcriptional changes being detected by exposure of cells to doses of {gamma}-rays as low as 0.01 cGy that have no pronounced cellular consequences. On the basis of this work, the authors hypothesized that measures of gene expression will be equally sensitive to EMF effects on cells.

DNA sequencing by hybridization: 100 bases read by a non-gel-based method

Abstract Wasted mice express radiosensitivity in lymphoid and nervous system tissues, displaying increased apoptosis, increased transcriptional activation, and altered gene induction in response to radiation when compared to control littermates. In order to evaluate altered gene induction linked with radiosensitivity in spinal cord tissue of the wasted mice, we isolated total RNA from the spinal cords of radiation-exposed and unexposed wasted and control mice and hybridized it to a gene expression microarray. Previous studies by our group had demonstrated that lymphoid tissues of wasted mice lack proliferating cell nuclear antigen (PCNA) expression. This protein is involved in almost every aspect of DNA replication and repair, and radiosensitivity in lymphoid tissues of wasted mouse can easily be linked to proliferating cell nuclear antigen (PCNA) deficiency. In total spinal cord tissue, however, PCNA mRNA expression was not altered in wasted mice relative to controls. Nevertheless, mRNAs for many proteins that were known to interact with PCNA were modulated in wasted spinal cords compared to the tissues from BCF1 controls.