70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences.

RTG-2 cells, a line of fibroblasts from rainbow trout (Salmo gairdnerii), are induced to synthesize a distinct set of heat-shock polypeptides after exposure to elevated temperature or to low concentrations of sodium arsenite. We isolated and characterized two cDNA sequences, THS70.7 and THS70.14, encoding partial information for two distinct species of 70-kilodalton heat shock polypeptide (hsp70) from these cells. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with derived amino acid sequences for hsp70 polypeptides from Drosophila melanogaster and Saccharomyces cerevisiae. Northern blot analysis of RNA from arsenite-induced RTG-2 cells, with the trout hsp70 cDNAs as probes, revealed the presence of three hsp70 mRNA species. Southern blot analysis of trout testis DNA cleaved with various restriction endonucleases revealed a small number of bands hybridizing to the hsp70 cDNAs, suggesting the existence of a small family of hsp70 genes in this species. Finally, trout hsp70 cDNA sequences cross-hybridized with restriction fragments in genomic DNA from HeLa cells, bovine liver, Caenorhabditis elegans, and D. melanogaster.

Induction of a novel set of polypeptides by heat shock or sodium arsenite in cultured cells of rainbow trout, Salmo gairdnerii.

The heat-shock response has been characterized in cultured fibroblasts of the rainbow trout, Salmo gairdnerii. The response has been elicited by two different stress situations; cells were either subjected to higher temperatures than normal (27 to 29 degrees C as opposed to 22 degrees C) or were incubated in medium containing sodium arsenite (15 to 100 microM final concentration). The response of the cells to these conditions is to synthesize a set of new polypeptides, the "heat-shock polypeptides" (hsps), that are not present or present in extremely low amounts in noninduced cells. Furthermore, during prolonged arsenite induction, the synthesis of normal cellular proteins is repressed. In trout fibroblasts, at least six hsps are detectable. These range from 30 000 to 87 000 in molecular weight and are referred to as hsp30, hsp32, hsp42, hsp62, hsp70, and hsp87. The hsp30 and hsp70 components are the most abundant and can be visualized by Coomassie blue staining of gels after prolonged induction. The heat-shock response is a reversible process in trout cells. Results of in vitro translation of mRNA from induced cells indicate that the control of hsp induction may be at the transcriptional level. Hsp70 from trout comigrates with the major hsp from Drosophila melanogaster on sodium dodecyl sulfate - polyacrylamide gels, suggesting that this protein may be highly conserved in evolution.

IS186: an Escherichia coli insertion element isolated from a cDNA library.

Escherichia coli IS186 was isolated from cDNA libraries made from rainbow trout RNA and maintained in E. coli RR1. The element was 1,347 base pairs in length, had a perfect inverted repeat of 25 base pairs, and had an open reading frame of 375 amino acids. The hypothetical protein sequence of IS186 had limited homology to the E. coli IS4 hypothetical protein I sequence. There were three copies of IS186 in E. coli RR1.

Inducibility of heat shock polypeptides in cells containing hyperacetylated histones.

We have examined the effect of sodium butyrate on the levels of histone acetylation, the pattern of protein synthesis and the inducibility of heat shock polypeptides (hsps) in cultured trout fibroblasts. Maximal levels of histone acetylation are achieved upon treatment of these cells with 5 mM butyrate for 24 h. No significant changes in the pattern of protein synthesis, as detected by two-dimensional gel electrophoresis, are apparent under these conditions, although changes in the levels of three polypeptides are seen at shorter times of exposure to butyrate. Heat shock polypeptides are inducible at normal levels in butyrate-treated cells. This is in contrast to the ability of butyrate to inhibit the activation of steroid-inducible genes in some systems.

Factors affecting cellular mosaicism in the expression of a lacZ transgene in two-cell stage mouse embryos.

In the present study, we have analysed the expression pattern of a lacZ transgene (CMZ12) in preimplantation stage mouse embryos. The transgene is expressed at the two-cell stage, where it shows cellular mosaicism due to variable expressivity. The variable gene expression indicates a partial penetrance of the transgene. The extent of variation in expression is influenced by the genetic background of the oocyte. DBA/2 and CFLP genetic backgrounds promote high expression of the transgene, while Balb/c, C57BL/6, DDK, and F1 (C57BL/6 x CBA) genetic backgrounds give none or very little lacZ activity. In vitro culture of one-cell embryos to the two-cell stage induces the expression of lacZ in all strain backgrounds tested. The variation in CMZ12 expression is a transient phenomenon and does not affect later stage activity of the transgene. Nuclear transfer experiments and DNA methylation analysis suggests that a heritable modification of the transgene locus has not occurred.