Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

Genetic Characterization of the Saccharomyces Cerevisiae Translational Initiation Suppressors Sui1, Sui2 and Sui3 and Their Effects on His4 Expression

Saccharomyces cerevisiae strains containing mutations of the HIS4 translation initiation AUG codon were studied by reversion analysis in an attempt to identify components of the translation initiation complex that might participate in initiation site selection during the scanning process. The genetic characterization of these revertants identified three unlinked suppressor loci: SUI1, SUI2 and sui3, which when mutated restored the expression of the HIS4 allele despite the absence of the AUG initiator codon. Both sui1 and sui2 are recessive and cause temperature-sensitive growth on enriched medium. The temperature-sensitive phenotype and the ability to restore HIS4 expression associated with either sui1 or sui2 mutations cosegregate in crosses. SUI3 mutations are dominant and do not alter the thermal profile for growth. None of the mutations at the three loci suppresses known frameshift, missense or nonsense mutations. Each is capable of suppressing the nine different point mutations of the initiator codon at HIS4 or HIS4-lacZ as well as a two base change (ACC) and a three base deletion of the AUG codon, suggesting that the site of suppression resides outside the normal initiator region. sui1 and sui2 suppressor mutations were mapped to chromosomes XIV and X, respectively. Suppression by sui1, sui2 and SUI3 mutations results in 14-, 11- and 47-fold increases, respectively, relative to isogenic parent strains, in the expression of a HIS4 allele lacking the initiator AUG codon. Part of this increase in the HIS4 expression by sui2 and SUI3 can be attributed to increases of HIS4 mRNA levels, presumably mediated by perturbation of the general amino acid control system of yeast.     

The thermal denaturation of nonpolymerizable alpha alpha-tropomyosin and its segments as a function of ionic strength

Nonpolymerizable tropomyosin (NPTm) is found to unfold thermally at high ionic strength almost exactly as the parent protein, but it does not aggregate at low ionic strength. Thus, NPTm can be used as a tropomyosin surrogate whose coiled-coil structural stability can be probed by varying the ionic strength. Studies of NPTm by CD show that increasing ionic strength stabilizes the coiled-coil structure. CD spectra over a wide range of helix content, obtained by varying either temperature or ionic strength, show an isodichroic point at 203 nm, suggesting a local, residue-level, two-state model. At given temperature, such a local helix in equilibrium random equilibrium suggests ln [phi h/(1-phi h)] = A1 + A2In, wherein phi h is the fraction helix, and A1, A2, and n are constants. In the low ionic strength region, theoretical limiting laws for ionic strength mediated charge-charge, dipole-dipole, and apolar-apolar (salting out) interactions give, respectively, n = 0.5, 1.0, and 1.0. Our experimental values for 40 degrees C, where the data span a wide range of helix content, show n = 1.0, suggesting that ionic strength stabilizes either by reducing dipole-dipole repulsions or by enhancing hydrophobic interactions, both probably interhelix in nature. Two segments of tropomyosin, 11Tm127 and 142Tm281, neither of which aggregate at low ionic strength, give results similar to those for NPTm, i.e., n = 0.96 and 0.84, respectively.     

Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.     

Analysis of N-acetoxy-N-2-acetylaminofluorene-induced frame-shifts in pBR322

We present a simple system which can be used to study directly directly the sequence change and the cellular repair functions involved in frame-shift mutagenesis by a covalently reactive mutagen. Positive (+S) and negative (?S) alterations in the number of base pairs of the Tc gene of pBR322 were generated and particular clones with Ap^RTc^S phenotypes were selected for mutagenesis experiments. Exposure of these frame-shifted plasmid DNAs to a potent carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), in vitro, caused covalent alterations to DNA sequence and resulted in a number of revertants (Ap^RTc^R) not observed in the untreated controls. The dose curve indicated an exponential response suggesting single-hit kinetics. Differential inactivation of the Ap gene was observed among various E. coli strains. The wild-type AB1157 and AB2463 (yrecA) showed a similar dose curve while AB1886 (uvrA) showed a marked decrease in Ap^R clones at the same dose. Both addition (+S) and deletion (?S) plasmids exhibited similar dose curves on inactivation of Ap gene. The reversion frequency, however, of ?S plasmid was a factor of 10 times higher than +S plasmid. The reversion frequency also increase markedly with uvrA host but not with recA host. 2 types of deletion revertants of the +S plasmid were found. 1 revertant has a single GC base-pair deletion in GC-rich region which is likely to be a target for AAAF reaction. The other showed a deletion of 4 base pairs (TCGA) at the tandem repeating sequence TCGATCGA which may represent a hot spot for frame-shift mutation.     

H-Y antigen in X,i(Xq) gonadal dysgenesis: Evidence of X-linked genes in testicular differentiation

Summary Three years ago, we detected H-Y antigen in the white blood cells of a phenotypic female with several of the stigmata of Turner's syndrome, and the mosaic karyotype: 45,X/46,X,i(Xq). We surmised at the time that the isochromosome, i(Xq), may have contained occult Y-chromosome-derived material. We have now confirmed the presence of H-Y in this patient and we have obtained evidence for the presence of H-Y in four of five other similar patients, all of whom are notable for carrying at least a single cell line with the karyotype 46,X,i(Xq). Although we cannot categorically exclude the presence of Y-chromosomal genes in the cells of these patients, there is no cytogenetic evidence of structural rearrangement involving the Y in any of the cases. Expression of H-Y antigen in association with i(Xq) thus implies that H-Y structural genes are X-situated, or alternatively that they are autosomal and X-regulated. It would follow that the H-Y⁺ cellular phenotype per se is not a valid marker for the Y-chromosome, and that H-Y genes that have been mapped to the pericentric region of the Y may be regulatory.     

H-Y antigen in 46,XY gonadal dysgenesis

Summary Presence of H-Y antigen has been correlated with testicular differentiation, and absence of H-Y with failure of testicular differentiation, in a variety of mammalian species. To determine more precisely the relationship between expression of H-Y antigen and development of the testis, we studied the cells of phenotypic females with the 46,XY male karyotype. Blood leukocytes were typed H-Y⁺ in five XY females with gonadal dysgenesis, although in other studies blood leukocytes from XY females with gonadal dysgenesis were typed H-Y^-. Thus mere presence of H-Y antigen is not sufficient to guarantee normal differentiation of the testis. In the present paper we review evidence for an additional factor in gonadal organogenesis, the H-Y antigen receptor. We infer that testicular development requires engagement of H-Y and its receptor. It follows that XY gonadal dysgenesis is the consequence of functional absence of the H-Y testis inducer as in the following conditions: failure of synthesis of H-Y or failure of specific binding of H-Y.     

Evolution of Hawaiian Drosophilidae. II. Patterns and Rates of Chromosome Evolution in an Antopocerus Phylogeny

The phylogenetic relationships of seven species of the genus Antopocerus (Family Drosophilidae) have been determined by means of a study of the metaphase configurations and polytene chromosomes. Based on biogeographical, behavioral and cytogenetic information, A. longiseta from Molokai is tentatively identified as the primitive species of the genus. The metaphase karyotypes of all Antopocerus species are either five pairs of rod chromosomes and a pair of dots (5R1D), or six rods (6R). Heterochromatin additions converted the dots to rods. Chromosome breakpoints for inversions also are clustered at heterochromatic loci. The chromosome segments between heterochromatic loci may represent sets of functionally related loci, evolving as a unit. The rate of chromosomal inversion substitution is estimated in the origin of the taxon (probably a subgenus of Drosophila rather than a separate genus). It averages no greater than one substitution per 1,000 years, or one per 5,000 generations. The average genetic death rate per generation of one individual per hundred is required to achieve this substitution rate. The rate of inversion substitution during radiation of this taxon may be only 4.4 x 10^-3 times as fast as that present in forming the taxon. Alternatively, radiation may have required only 250,000 years if rates of substitution are the same as in the origination of the taxon. Average rates of substitution reflect genetic accidents, selection pressures and rates of adaptation to new niches, as well as the rate of encountering new niches. Rate of adaptation probably is much greater in this instance than rate of encountering new niches. Therefore, the average rate of evolution reflects more nearly biogeographic and ecological factors than genetic factors.