Molecular cloning and characterization of two distinct hsp 85 sequences from the steroid responsive fungus Achlya ambisexualis

Abstract

In Achlya ambisexualis, hsp85 is one of the characteristic mycelial heat shock proteins induced in response to a rapid elevation in temperature (Silver et al. 1983). This heat shock protein has the same electrophoretic mobility on two-dimensional gels and is antigenically related to an 85 kDa steroid hormone-regulated protein which constitutes a component of the putative Achlya steroid hormone-receptor complex. We report here the isolation of two distinct, yet highly related, hsp85 gene sequences from Achlya genomic libraries. Northern analyses, using these two Achlya genomic sequences as probes, suggest that there are two hsp85 message population in Achlya and that at least one of these is regulated by the steroid hormone antheridiol.

A Bipartite Operator Interacts with a Heat Shock Element To Mediate Early Meiotic Induction of Saccharomyces cerevisiae HSP82

“…HSP82 is an intensively studied heat shock gene that relies primarily on a single heat shock element (HSE) adjacent to the promoter to drive vegetative and heatinduced transcription (18,19,35,46,72). It is likely that Hsp82p has a phylogenetically conserved function during meiosis or early oogenesis because its homologs are similarly expressed in other fungi (9), the fruit fly (12), and mammals (22,36).…”

“…4) that is a good match to a region upstream of SPO13 (Ϫ88 to Ϫ113) (10). The SPO13 sequence is in reverse orientation to that of HSP82 and contains a perfect CAR1 URS1 motif (9). The sequence adjacent to URS82 (sequence A) bears a 6-of-7-bp identity to the SPO13 counter- The amount of RNA loaded on the gel was monitored by visualizing 18S rRNA with ethidium bromide.…”

Regulation of hsp90 and hsp70 Genes during Antheridiol-Induced Hyphal Branching in the OomyceteAchlya ambisexualis

“…The RNA isolation, Northern analyses, and probe preparation procedures were essentially as described previously (Brunt and Silver, 1991;Silver et al, 1993). The probes used in the Northern analyses were: a 1.1-kb XbaI-EcoRI insert from the hsp90 genomic clone pJS851 that represents one of two A. ambisexualis hsp90 genes (Brunt and Silver, 1991), a 3.5-kb EcoRI genomic fragment (designated C14) from the hsp70 genomic clone pSNC14 (Silver et al, 1993), a 2.9-kb EcoRI fragment (designated MB8) from the hsp70 genomic clone pMB8 (Borkar and Silver, unpublished), and a coding region probe from the Saccharomyces cerevisiae actin gene (Ng and Abelson, 1980). Hybridization signals were quantified using a scanning densitometer (Bio-Rad Model 1650) and, when required, the values normalized for loading using the actin probe.…”

“…The nucleotide sequence of 1.14 kb of the 5Ј flanking region from each genomic clone, i.e., pJS851 and pJS852 (Brunt and Silver, 1991), which represent the two A. ambisexualis hsp90 genes (designated hsp90-1 and hsp90-2, respectively), was determined using a combination of subcloning and primer walking. Subclones of pJS851 and pJS852 were constructed, and the nucleotide sequence of both strands of the double-stranded plasmid DNAs was determined by the dideoxy chain-termination method (Sanger et al, 1977), using Sequenase version 2.0 (Amersham) and T3 and T7 primers (Stratagene).…”

“…Among these are proteins that have electrophoretic behaviors similar to those of A. ambisexualis Hsp90-family and Hsp70-family heat shock proteins (Silver et al, 1983;Brunt and Silver, 1986b;Brunt et al, 1990;Silver et al, 1993). The changes in the synthesis of these proteins is accompanied by changes in the levels of hsp90 transcripts and hsp70 transcripts (Brunt and Silver, 1991;Silver et al, 1993).…”