Heat-shock-induced assembly of Hsp30 family members into high molecular weight aggregates in Xenopus laevis cultured cells.

In this study, we have examined whether members of the small heat shock protein family, hsp 30, were capable of forming heat-induced aggregates in Xenopus laevis A6 kidney epithelial cells. Rate-zonal centrifugation coupled with immunoblot analysis demonstrated the presence of hsp30 aggregates with an estimated sedimentation coefficient of 10-16S. Also, pore exclusion limit electrophoretic analysis of labeled protein from heat-shocked A6 cells revealed four heat-induced aggregates, including a prominent 510 kDa aggregate, as well as weaker 350, 290, and 240 kDa aggregates. Immunoblot analysis of the aggregates employing an hsp30C antibody suggested that the 510 and 350 kDa aggregates were comprised of hsp30 protein. One- and two-dimensional SDS-PAGE analysis of the proteins isolated from the 510 kDa region of the pore exclusion limit electrophoretic gel confirmed the presence of 30 kDa heat-induced protein. A total of eight small hsps were present in this aggregate, suggesting that virtually all of the major small hsps in Xenopus A6 cells were involved in aggregate formation. This study also detected the presence of heat-inducible hsp70 in the 510 kDa gel fraction containing the small hsps, but it could not be determined whether it was part of the multimer complex.

Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells.

In this study, we report the detection of a new group of five stress-inducible basic small heat shock proteins (BShsps) in Xenopus laevis kidney epithelial A6 cells by means of two-dimensional non-equilibrium pH gradient gel electrophoresis. These basic 30-kDa small hsps are distinct from the previously described X. laevis acidic hsp30 family on the basis of their charge and lack of cross-reactivity with an hsp30 antibody. Furthermore, at least two of the five BShsps were present constitutively, an observation that has not been made with the acidic hsp30 family. The heat inducibility of the BShsps was regulated at the level of transcription as indicated by their inhibited synthesis in the presence of the transcriptional inhibitor actinomycin D. Furthermore, the optimal temperature of BShsp induction, temporal pattern of synthesis, and induction of BShsps by other stressors such as herbimycin A and sodium arsenite were similar to those reported for the acidic hsp30 family. This study suggests that X. laevis contains at least two unique groups of small heat shock proteins that are coordinately expressed.

Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos.

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33 degrees C for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tailbud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37 degrees C for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximide-treated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development.

Reverse transcription-polymerase chain reaction: an overview of the technique and its applications.

The polymerase chain reaction (PCR) has galvanized molecular biologists by virtue of its ability to provide them with large quantities of any desired fragment (up to 11kb) of DNA. This power combined with its flexibility has also inspired many useful applications, including new methods of DNA sequencing, cloning and mutagenesis. One logical variation of PCR has been its application to the detection and analysis of messenger RNA by the addition of a reverse transcription step prior to performing PCR. Due to the exquisite sensitivity of PCR, reverse transcription-PCR (RT-PCR) has been used to characterize mRNAs previously undetectable by established methods of RNA analysis such as Northern hybridization and RNase protection assays. Furthermore, its capacity as a method of quantitative analysis is currently being developed. RT-PCR has also been used to diagnose the presence of certain diseases. Recently, RT-PCR has been employed to identify and isolate genes that are differentially expressed in different cells or environmental conditions.