Supporting the structural basis of prion strains: induction and identification of [PSI] variants.

The [PSI] genetic element, which enhances the nonsense suppression efficiency in the yeast Saccharomyces cerevisiae, is thought to be amyloid-like aggregates of the Sup35 protein, and to self-propagate by a prion-like mechanism. Analogous to strains of the mammalian prion, variants of [PSI], with different nonsense suppression efficiencies and mitotic stabilities, can be isolated from the same yeast genetic background. In the framework of the "protein-only" hypothesis, variants of prion are assumed to be distinct conformers of the same prion polypeptide. This study aims to provide further support for the structural basis of [PSI] variation. Three variants of [PSI] were induced and distinguished by a panel of 11 single point mutations of the Sup35 protein. The variant phenotypes are intrinsically associated with [PSI] elements, presumably structurally different amyloids, rather than produced from variations in the genetic background. Differential incorporation to [PSI] variants of a Sup35 point mutation as well as N and C-terminally truncated Sup35 fragments is further demonstrated in vivo, suggesting that distinct patches of amino acid residues are involved in the assembly of [PSI] variants. These results establish a method for [PSI] variant-typing and indicate that heritable variations of amyloid structures can be derived from the same polypeptide.

Brain imaging in posttraumatic stress disorder.

This is a review article of neuroimaging studies in post-traumatic stress disorder (PTSD). Findings from structural, biochemical, and functional studies are summarized. Magnetic resonance imaging (MRI) volumetric studies have consistently reported decreased hippocampal volumes in PTSD. Proton magnetic resonance spectroscopy studies report decreased N-acetyl aspartate (NAA) ratios and absolute concentrations in the medial temporal lobe. Although still controversial, these findings from volumetric and spectroscopic studies are thought to represent decreased neuronal density of the hippocampus. Functional imaging studies document different patterns of limbic and paralimbic structure activation in PTSD compared with controls. Of theoretical importance are findings of failure to activate the anterior cingulate as well as amygdala activation during symptom provocation studies. Also, increased amygdala activation was found with a behavioral task targeted to this structure. A neurobiological model is presented that takes into account findings from neuroimaging studies in PTSD as well as animal studies of fear conditioning. This model proposes that central to symptom mediation is a dysfunction of the anterior cingulate, with a failure to inhibit amygdala activation and/or an intrinsic lower threshold of amygdala response to fearful stimuli. The model further proposes that hippocampal atrophy is a result of the chronic hyperarousal symptoms mediated by amygdala activation.

SRYand architectural gene regulation: the kinetic stability of a bent protein-DNA complex can regulate its transcriptional potency.

Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY's potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif's cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates.

The SRY cantilever motif discriminates between sequence- and structure-specific DNA recognition: alanine mutagenesis of an HMG box.

The high-mobility-group (HMG) box defines a DNA-bending motif conserved among architectural transcription factors. A "hydrophobic wedge" at the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain "cantilever." First described in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the crux of the HMG box (residues 12 and 13). Here, the role of these side chains in DNA recognition is investigated by alanine mutagenesis. F12A and I13A substitutions in the SRY HMG box each permit native folding and thermal stability (as monitored by circular dichroism and 1H-NMR) but eliminate sequence-specific DNA-binding activity (as detected by gel-mobility shift). On binding to the sharp angles of a four-way DNA junction (4WJ), however, the substitutions each promote formation of a high-molecular-weight aggregate, presumably by DNA-dependent oligomerization. The substitutions have opposite effects on initial binding to the 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enhanced by I13A. A foreshortened "alanine cantilever", not observed among specific HMG boxes, occurs in a non-specific domain (HMG-1A) and may enhance architecture-selective DNA recognition.

Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments.

The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.

Imino proton exchange provides an 1H-NMR footprint of protein-DNA interactions: general strategy and application to the SRY HMG box.

A novel 1H nuclear magnetic resonance (NMR) strategy for "footprinting" specific protein-DNA target sites is demonstrated. Relative rates of site-specifc imino-proton exchange in the free and bound DNA duplex are determined by use of laminar-shifted shaped pulses in NOESY spectra. 2D exchange crosspeaks between imino (omega2 dimension) (omega1 dimension) resonances in principle provide site-specific probes of protein binding. Chemical exchange is distinguished from nuclear Overhauser enhancements(NOEs) to bound water by use of ROESY spectroscopy. This strategy is illustrated in 1H-NMR studies of the SRY high-mobility group (HMG) box, the Y-chromosome-encoded "master switch" for testis determination in man. In a specific complex between the protein and a 15-basepair DNA site, imino-proton exchange was observed to be damped selectively within the six basepair subsite 5'-ATTGTT, previously identified by random binding-site selection as an optimal SRY target sequence. The extent of damping correlates with sites of protein-DNA contacts in the minor groove but not with the magnitude of 1H-NMR complexation shifts. SRY binding has recently been shown to introduce significant distortions in DNA structure. The DNA is sharply bent and underwound; the minor groove is widened and major groove compressed. Our results demonstrate that despite such distortions base pairing is stably maintained. Protein binding in the DNA minor groove shields DNA imino protons from exchange with solvent.

An SRY mutation causing human sex reversal resolves a general mechanism of structure-specific DNA recognition: application to the four-way DNA junction.

SRY, a genetic "master switch" for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (168T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition alpha-helix of the HMG box [Haqq, C.M., King, C.-Y., Ukiyama, E., Haqq, T.N., Falsalfi, S., Donahoe, P.K., & Weiss, M.A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided 1H-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits > 50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.

Molecular basis of mammalian sexual determination: activation of Müllerian inhibiting substance gene expression by SRY.

The pathway of male sexual development in mammals is initiated by SRY, a gene on the short arm of the Y chromosome. Its expression in the differentiating gonadal ridge directs testicular morphogenesis, characterized by elaboration of Müllerian inhibiting substance (MIS) and testosterone. SRY and MIS each belong to conserved gene families that function in the control of growth and differentiation. Structural and biochemical studies of the DNA binding domain of SRY (the HMG box) revealed a protein-DNA interaction consisting of partial side chain intercalation into a widened minor groove. Functional studies of SRY in a cell line from embryonic gonadal ridge demonstrated activation of a gene-regulatory pathway leading to expression of MIS. SRY molecules containing mutations associated with human sex reversal have altered structural interactions with DNA and failed to induce transcription of MIS.

The SRY high-mobility-group box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity.

SRY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome, regulates a genetic switch in male development. Impairment of this switch leads to intersex abnormalities of the newborn and is observed in association with mutations in the SRY DNA-binding domain [the high-mobility-group (HMG) box]. Here we show that the SRY HMG box exhibits a novel mechanism of DNA recognition: partial intercalation of a nonpolar side chain in the DNA minor groove. Base stacking (but not base pairing) is interrupted at the site of insertion. Sequence specificity reflects topological requirements of partial intercalation rather than direct readout of base-specific functional groups. Our results predict that the SRY HMG box inserts an alpha-helix into a widened minor groove at the center of a sharp DNA bend. A similar mechanism may underlie binding of SRY and homologous HMG proteins to four-way junctions (Holliday intermediates) and other noncanonical DNA structures.

SRY recognizes conserved DNA sites in sex-specific promoters.

Formation of male-specific structures and regression of female primordia are regulated in early male embryogenesis by SRY, a single-copy gene on the Y chromosome. Assignment of SRY as the testis-determining factor in eutherian mammals is supported by molecular analysis of cytogenetic sex reversal (i.e., XX males and XY females) and by complementary studies of transgenic murine models. Here we characterize the putative DNA-binding domain of SRY, which contains a conserved sequence motif shared by high-mobility group nuclear proteins and a newly recognized class of transcription factors. The SRY DNA-binding domain specifically recognizes with nanomolar affinity proximal upstream elements (designated SRYe) in the promoters of the sex-specific genes encoding P450 aromatase and Mullerian inhibiting substance (MIS). P450 aromatase catalyzes the conversion of testosterone to estradiol, and in the male embryo its expression is down-regulated. Conversely, MIS is expressed in the male embryo to induce testicular differentiation and regression of female reproductive ducts. SRYe-binding activity is observed in nuclear extracts obtained from embryonic urogenital ridge immediately preceding morphologic testicular differentiation. Our results support the hypothesis that SRY directly controls male development through sequence-specific regulation of target genes.

Analysis of the in vitro interaction between vancomycin and cholestyramine.

Vancomycin and cholestyramine have been utilized both alone and in combination for the treatment of antibiotic-associated pseudomembranous colitis. Previous work for the treatment of antibiotic-associated pseudomembranous colitis. Previous work has demonstrated significant binding of vancomycin by the anion-exchange resin. The antibacterial activity of vancomycin was markedly reduced when the suspension was centrifuged and the supernatant was tested for antibacterial activity. This study confirmed these findings but demonstrated that there was no immediate loss of antibacterial activity of bound vancomycin. The degree of inactivation appeared to be dependent upon the duration of incubation of vancomycin and cholestyramine in the testing system.