Murine 86-kDa heat shock protein gene and promoter.

The class of 90 kDa heat shock proteins (Hsp90) is among the most abundant heat shock proteins (Hsps) in eukaryotic cells. In vertebrates, Hsp90 is encoded by two distinct gene families giving rise to products of 84 and 86 kDa. In mice the expression of these two genes, hsp84 and hsp86, vary with respect to each other in responses to stress, and also in response to signals for growth and development. Therefore, as a step towards understanding the molecular basis for the differential regulation of these two genes, we have isolated and characterized genomic clones of the murine hsp86 gene and its 5' flanking region. The gene is composed of eleven exons interrupted by 10 introns. The 5' region contains consensus TATA, several stimulatory protein-1 binding site (SP1) elements as well as six consensus heat shock elements (HSE) 5' of the transcription start site. An 806 bp fragment of the 5' promoter region conferred constitutive expression upon a reporter gene and this expression was increased upon heat shock.

Cloning and characterization of the promoter for murine 84-kDa heat-shock protein.

The 90-kDa heat-shock (HS) proteins (HSP90) are members of the HSP family. Their synthesis is inducible by HS and a variety of stress signals. HSP90 is also abundant under normal physiological conditions and its synthesis can be regulated during growth and differentiation. Therefore, HSP90 is speculated to have important biological functions, in addition to its role in mediating stress responses. However, the mechanism(s) regulating hsp90 gene expression in nonstressed cells is poorly understood. As a prerequisite towards understanding the basis for hsp90 regulation, we have cloned and characterized the 5' flanking region of murine hsp84, one of two genes which code for HSP90 proteins. Full basal promoter activity of hsp84 was found to be associated with a 627-bp region immediately upstream from the transcription start point (tsp). Sequence analysis revealed several putative regulatory elements, including a HS element (HSE), an AP1-binding site (AP1), a cyclic AMP response element (CRE), and four stimulatory protein-1-binding sites (SP1). HS inducibility required the HSE which was bound by HS transcription factor-1(HSF-1) present in extracts prepared from cells exposed to HS. The HSE was not required for basal (non-HS) expression, but, interestingly, two protein-HSE complexes, devoid of HSF-1 and HSF-2, were formed under these conditions. The potential significance of these findings to the expression of hsp84 under normal physiological conditions is discussed.

Estrogen dependent expression of heat shock transcription factor: implications for uterine synthesis of heat shock proteins.

Transcriptional induction of heat shock protein genes is generally mediated by binding of heat shock transcription factor(s) to the heat shock element present in the promoters of heat shock genes. Although the steady-state levels of heat shock factor mRNAs vary among different tissues, at present virtually nothing is known regarding the cellular signals responsible for their synthesis and hence the observed variations. In this report we demonstrate that the heat shock transcription factor (HSTF or HSF) is under positive regulation by estrogen. The effect of estrogen was observed with both types of heat shock factors (HSF-1 and HSF-2) and occurred at both the mRNA and protein level. Immunolocalization studies emphasized the potential biological importance of these observations whereby the increase in uterine HSF-1 and HSF-2 due to estrogen was found to be associated with the endometrium, the primary tissue component which is targeted for estrogen action. This is the first demonstration of a cellular factor which can regulate HSF-1 and HSF-2 gene expression. The implications of these findings to uterine heat shock protein gene expression are discussed.

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome.

The bacteriophage P1 Cre-lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre-lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.

Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system.

The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.

Gene transfer with subsequent removal of the selection gene from the host genome.

A general method of gene transfer that does not leave behind a selectable marker in the host genome is described. A luciferase gene was introduced into the tobacco genome by using the hygromycin phosphotransferase gene (hpt) as a linked selectable marker. Flanked by recombination sites from the bacteriophage P1 Cre/lox recombination system, the hpt gene was subsequently excised from the plant genome by the Cre recombinase. The Cre-catalyzed excision event in the plant genome was precise and conservative--i.e., without loss or alteration of nucleotides in the recombinant site. After removal of the Cre-encoding locus by genetic segregation, plants were obtained that had incorporated only the desired transgene. Gene transfer without the incorporation of antibiotic-resistance markers in the host genome should ease public concerns over the field release of transgenic organisms expressing such traits. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host.

Mutational analysis of a bacteriophage P4 late promoter.

Transcription from the late Psid promoter of satellite bacteriophage P4 is dependent on the bacterial RNA polymerase carrying the sigma 70 subunit and is positively regulated by the product of the P4 delta gene or the ogr gene of helper bacteriophage P2. Through deletion and mutational analyses of the Psid promoter, we identified mutations in the -10 region and in a region of hyphenated dyad symmetry centered around position -55 that inactivate Psid. Most of these mutations alter base pairs that are highly conserved in the five other delta-activated P4 and P2 late promoters. We propose that the P4 delta and P2 ogr gene products bind the -55 region of the P4 and P2 late promoters.

Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase.

A site-specific recombination system has many potential uses for rearranging genetic material in higher eukaryotic cells: for example, the control of gene expression by deletion or inversion of DNA segments, the clustering of transgenic constructs via site-specific integration, and the generation of chromosomal translocations. In this report, we describe a first step towards the application of a site-specific recombination system in plant cells. By use of a transient assay, we demonstrate that the bacteriophage P1 cre gene can be expressed as a functional recombinase in tobacco cells. Upon expression in tobacco protoplasts, Cre recognizes its target sites, lox, and mediates reciprocal genetic crossovers at these sites. When the lox sites are present in cis to one another, and arranged in either direct or inverted orientations, we detect Cre/lox-specific deletion and inversion events, respectively. The placement of lox sites in trans resulted in the co-integration of the substrates by Cre-mediated intermolecular recombination. These results indicate that the Cre/lox site-specific recombination system might be further developed as an additional tool for manipulating DNA in plant cells. Applications relevant to the genetic engineering of higher plants are discussed.

In vitro transcription from the late promoter of bacteriophage P4.

The late genes of satellite bacteriophage P4 are cotranscribed from a single promoter which shares little homology with known classes of Escherichia coli promoters (E. Dale, G. Christie, and R. Calendar, J. Mol. Biol. 192:793-803, 1986). In a coupled transcription-translation system, the P4 late gene promoter was activated by either the delta protein of P4 or the ogr protein of helper phage P2 in the absence of any other phage-encoded factor. delta-dependent transcription was inhibited by antibodies to the sigma 70 subunit of E. coli RNA polymerase but was restored by purified sigma 70, indicating that activation of transcription by the delta protein of P4 is dependent on the sigma 70 holoenzyme.

Organization and expression of the satellite bacteriophage P4 late gene cluster.

The satellite bacteriophage P4 genes for capsid size determination (sid), transactivation (delta), and polarity suppression (psu) are cotranscribed at late times after infection from a single P4 late promoter (Psid) that lies to the left of the sid gene. While the -10 region of this promoter is similar to the consensus sequence for Escherichia coli RNA polymerase, the -35 region shares no homology with known classes of E. coli promoters. The -10 and -35 regions of Psid share no homology with the late gene promoters of helper phage P2. Nonetheless, P4 late transcription is stimulated by coinfecting P2, as well as by P2 prophage. This stimulation depends on the P2 encoded transcription factor ogr; transcription from Psid is stimulated following the induction of the P2 ogr gene carried on a plasmid. P4 late transcription in the absence of P2 requires the P4 delta product, which is partially homologous to the P2 ogr gene product. DNA sequence analysis shows that the psu gene codes for a protein of Mr = 21,314 that is unrelated to the antitermination gene products of the lambdoid phages.