Empirical relationships between protein structure and carboxyl pKa values in proteins

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pK(a) values are known. Mean pK(a) values for aspartates and glutamates are < or = 3.4 (+/-1.0) and 4.1 (+/-0.8), respectively. For aspartates, mean pK(a) values are 3.9 (+/-1.0) and 3.1 (+/-0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pK(a) values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pK(a) values: pK(a) values for solvent-exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pK(a) values and these correlations are not improved by including simple surface-area-based terms to account for the effects of desolvation. Mean aspartate pK(a) values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pK(a) values are > 5.5 and most are found in active sites or ligand-binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N-termini of helices have mean pK(a) values of 2.8 (+/-0.5) and 3.4 (+/-0.6), respectively, about 0.6 units less than the overall mean values.     

Revision of Tipula (Yamatotipula) stackelbergi Alexander (Diptera, Tipulidae), and a short discussion on subspecies among crane flies

All available type material of Tipula stackelbergi Alexander, Tipula usuriensis Alexander and Tipula subpruinosa Mannheims were examined. Tipula (Yamatotipula) stackelbergistat. rev. is elevated from a subspecies of Tipula (Yamatotipula) pruinosa Wiedemann to a valid species. Two new synonyms are proposed: Tipula usuriensissyn. n. proved to be a junior synonym of. Tipula (Yamatotipula) pruinosa and Tipula subpruinosasyn. n. a junior synonym of Tipula (Yamatotipula) freyana Lackschewitz. Tipula (Yamatotipula) stackelbergi is redescribed, male and female terminalia of Tipula (Yamatotipula) pruinosa are illustrated and discussed. Female terminalia of Tipula (Yamatotipula) freyana are described and illustrated for the first time. A key to both sexes of Tipula (Yamatotipula) stackelbergi and Tipula (Yamatotipula) pruinosa, and a key to females of Tipula (Yamatotipula) chonsaniana, Tipula (Yamatotipula) freyana and Tipula (Yamatotipula) moesta are provided. Subspecies are not uncommon among crane flies, but their ranges and traits are poorly known. An interdisciplinary approach (genetics, ecology, taxonomy) is suggested if subspecific ranks are to be used in tipuloid systematics.     

A Transmission/Disequilibrium Test That Allows for Genotyping Errors in the Analysis of Single-Nucleotide Polymorphism Data

The present study assesses the effects of genotyping errors on the type I error rate of a particular transmission/disequilibrium test (TDT(std)), which assumes that data are errorless, and introduces a new transmission/disequilibrium test (TDT(ae)) that allows for random genotyping errors. We evaluate the type I error rate and power of the TDT(ae) under a variety of simulations and perform a power comparison between the TDT(std) and the TDT(ae), for errorless data. Both the TDT(std) and the TDT(ae) statistics are computed as two times a log-likelihood difference, and both are asymptotically distributed as chi(2) with 1 df. Genotype data for trios are simulated under a null hypothesis and under an alternative (power) hypothesis. For each simulation, errors are introduced randomly via a computer algorithm with different probabilities (called "allelic error rates"). The TDT(std) statistic is computed on all trios that show Mendelian consistency, whereas the TDT(ae) statistic is computed on all trios. The results indicate that TDT(std) shows a significant increase in type I error when applied to data in which inconsistent trios are removed. This type I error increases both with an increase in sample size and with an increase in the allelic error rates. TDT(ae) always maintains correct type I error rates for the simulations considered. Factors affecting the power of the TDT(ae) are discussed. Finally, the power of TDT(std) is at least that of TDT(ae) for simulations with errorless data. Because data are rarely error free, we recommend that researchers use methods, such as the TDT(ae), that allow for errors in genotype data.     

山东植物区系中的特有现象

对山东植物区系中的特有现象进行了初步研究。全省共有５３个特有种,可分为４种分布式样即全省布型、鲁中南－山东半岛间断分布型、鲁中南山地分布和山东半岛分布型;提出了山东植物区的两个特有现象中心,即崂山昆嵛山中心和泰山蒙山中心,并初步探讨了其形成原因。     

The impact of copper, nitrate and carbon status on the emission of nitrous oxide by two species of bacteria with biochemically distinct denitrification pathways

Denitrifying bacteria convert nitrate (NO(3) (-) ) to dinitrogen (N(2) ) gas through an anaerobic respiratory process in which the potent greenhouse gas nitrous oxide (N(2) O) is a free intermediate. These bacteria can be grouped into classes that synthesize a nitrite (NO(2) (-) ) reductase (Nir) that is solely dependent on haem-iron as a cofactor (e.g. Paracoccus denitrificans) or a Nir that is solely dependent on copper (Cu) as a cofactor (e.g. Achromobacter xylosoxidans). Regardless of which form of Nir these groups synthesize, they are both dependent on a Cu-containing nitrous oxide reductase (NosZ) for the conversion of N(2) O to N(2) . Agriculture makes a major contribution to N(2) O release and it is recognized that a number of agricultural lands are becoming Cu-limited but are N-rich because of fertilizer addition. Here we utilize continuous cultures to explore the denitrification phenotypes of P.?denitrificans and A.?xylosoxidans at a range of extracellular NO(3) (-) , organic carbon and Cu concentrations. Quite distinct phenotypes are observed between the two species. Notably, P.?denitrificans emits approximately 40% of NO(3) (-) consumed as N(2) O under NO(3) (-) -rich Cu-deficient conditions, while under the same conditions A.?xylosoxidans releases approximately 40% of the NO(3) (-) consumed as NO(2) (-) . However, the denitrification phenotypes are very similar under NO(3) (-) -limited conditions where denitrification intermediates do not accumulate significantly. The results have potential implications for understanding denitrification flux in a range of agricultural environments.     

A Qualitative and Quantitative Study of a Sensory Epithelium in the Inner Ear of a Fish (Colisa labiosa; Anabantidae)

The macula lagenae of the anabantide fish Colisa labiosa was studied with light and transmission electron microscopy. (1) The sensory area is naturally divided in a central area (A) surrounded by a peripheral part (B). (2) Generally the central hair cells are separated by supporting cells, while the peripheral hair cells are found in groups. The cells of a group are not separated by supporting cells. (3) Tubuli-like structures, hexagonal in cross section, are found in all cells. In peripheral hair cells the longitudinally oriented tubuli-like structures are aggregated in thick bundles. (4) Variation in shape, electron density, stereocilia arrangement and size of mitochondria was found in different hair cells. (5) The central hair cells contain large accumulations of presynaptic bodies (10–44). Contrarily, the peripheral hair cells contain only a few pre-synaptic bodies (1–3). (6) The central hair cells are innervated by thick afferent (6–15 μm) and fine presumed efferent (less than 1 μm nerve fibres, while the peripheral hair cells are innervated by thin (1–6 μm) afferent nerve fibres only.     

Fluctuation analysis of nonideal shot noise. Application to the neuromuscular junction

Procedures are described for analyzing shot noise and determining the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of the shots under a variety of nonideal conditions that include: (a) slow, spurious changes in the mean, (b) nonstationary shot rates, (c) nonuniform distribution of shot amplitudes, and (d) nonlinear summation of the shots. The procedures are based upon Rice's (1944. Bell Telephone System Journal. 23: 282-332) extension of Campbell's theorem to the second (variance), lambda 2, third (skew), lambda 3, and fourth, lambda 4, semi-invariants (cumulants) of the noise. It is shown that the spectra of lambda 2 and lambda 3 of nonstationary shot noise contain a set of components that are proportional to (r) and arise from w(t), and a set of components that are independent of (r) and arise from the temporal variations in r(t). Since the latter components are additive and are limited by the bandwidth of r(t), they can be removed by appropriate filters; then (r) and (h) can be determined from the lambda 2 and lambda 3 of the filtered noise. We also show that a factor related to the ratio (lambda 3)2/(lambda 2)(lambda 4) monitors the spread in the distribution of shot amplitudes and can be used to correct the estimates of (r) and (h) for the effects of that spread, if the shape of the distribution is known and if r(t) is stationary. The accuracy of the measurements of lambda 4 is assessed and corrections for the effects of nonlinear summation of lambda 2, lambda 3, and lambda 4 are derived. The procedures give valid results when they are used to analyze shot noise produced by the (linear) summation of simulated miniature endplate potentials, which are generated either at nonstationary rates or with a distribution of amplitudes.     

Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo

In 1990 we discovered the formation of prostaglandin F(2)-like compounds, F(2)-isoprostanes (F(2)-IsoPs), in vivo by nonenzymatic free radical-induced peroxidation of arachidonic acid. F(2)-IsoPs are initially formed esterified to phospholipids and then released in free form. There are several favorable attributes that make measurement of F(2)-IsoPs attractive as a reliable indicator of oxidative stress in vivo: (i) F(2)-IsoPs are specific products of lipid peroxidation; (ii) they are stable compounds; (iii) levels are present in detectable quantities in all normal biological fluids and tissues, allowing the definition of a normal range; (iv) their formation increases dramatically in vivo in a number of animal models of oxidant injury; (v) their formation is modulated by antioxidant status; and (vi) their levels are not effected by lipid content of the diet. Measurement of F(2)-IsoPs in plasma can be utilized to assess total endogenous production of F(2)-IsoPs whereas measurement of levels esterified in phospholipids can be used to determine the extent of lipid peroxidation in target sites of interest. Recently, we developed an assay for a urinary metabolite of F(2)-IsoPs, which should provide a valuable noninvasive integrated approach to assess total endogenous production of F(2)-IsoPs in large clinical studies.     

Descriptions of six new species of Caryospora (Apicomplexa: Eimeriidae) from Guatemalan snakes (Serpentes: Colubridae and Viperidae)

One hundred and seventy snakes were collected in Guatemala and examined for coccidia. Of these, 8 individuals representing 6 host species were positive for Caryospora spp., 6 of which are described as new species. Sporulated oocysts of Caryospora bothriechis n. sp. from Bothriechis aurifer are spheroidal to subspheroidal, 12.7 x 12.5 (12-14 x 12-13) microm, with a length/width (L/W) ratio of 1.0; they lack a micropyle (M) or oocyst residuum (OR), but 1 large polar granule (PG) is usually present. Sporocysts are ovoidal, 9.0-7.5 (8-10 x 7-8) microm, and have a L/W ratio of 1.2, and a Stieda body (SB) and sporocyst residuum (SR). Oocysts of Caryospora coniophanis n. sp. from Coniophanes imperialis are spheroidal to subspheroidal, 18.8 x 18.1 (17-20.5 x 16-20) microm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 13.2 x 9.4 (12-15 x 8-10) microm with a L/W ratio of 1.4, and a SB, substieda body (SSB), and SR. Oocysts of Caryospora conophae n. sp. from Conophis lineatus are spheroid to subspheroidal, 20.4 x 19.5 (17-26 x 17-25) microm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 13.1 x 9.8 (11-15 x 8-11) microm with a L/W ratio of 1.3 and a SB, SSB, and SR. Oocysts of Caryospora guatemalensis n. sp. from Lampropeltis triangulum are spheroidal to subspheroidal, 23.9 x 23.2 (20-27 x 20-26) microm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 14.4 x 10.6 (13-18 x 9-13) microm, with a L/W ratio of 1.4 and a SB, SSB, and SR. Oocysts of Caryospora mayorum n. sp. from Conophis lineatus are spheroidal to subspheroidal, 25.6 x 24.4 (24-27 x 24-25) microm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 16.3 x 11.9 (16-18 x 11-13) microm, with a L/W ratio of 1.4 and a SB, SSB, and SR. Oocysts of Caryospora zacapensis n. sp. from Masticophis mentovarius are spheroidal to subspheroidal, 22.5 x 21.8 (19-25 x 18-25) microm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 14.6 x 11.4 (11-16 x 10-13) microm, with a L/W ratio of 1.3 and a SB, SSB, and SR.     

Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.     

Six new species of coccidia (Apicomplexa: Eimeriidae) from East African chameleons (Sauria: Chamaeleonidae)

Coprological examination of 83 East African chameleon specimens revealed 32.5% prevalence of coccidian parasites. Six species are described as new: Eimeria tilburyi n. sp. from Chamaeleo jacksonii has cylindrical oocysts, 28.9 (26-33) x 16.0 (14-18) microm and occasionally a small polar granule. Sporocysts are oval to ellipsoidal, 10.6 (9-12) x 7.2 (6-8) microm, without Stieda and substieda bodies; endogenous stages were found in the gall bladder. Oocysts of Eimeria largeni n. sp. from Chamaeleo gracilis are broadly cylindrical, 31.2 (29.5-34) x 19.3 (18.5-20) microm, with 1-3 polar granules. Sporocysts are oval, 10.2 (10-11) x 7.6 (7-8.5) microm, without Stieda and substieda bodies. Eimeria bohemii n. sp. from Chamaeleo melleri has cylindrical oocysts, 25.0 (24-26) x 14.0 (13-15) microm, without a polar granule. Sporocysts are broadly oval, 9.4 (9-10) x 6.5 (6-7) microm, without Stieda and substieda bodies. Isospora wildi n. sp. from Chamaeleo dilepis has subspherical to broadly oval oocysts, 25 (22-28) x 21.4 (18-24) microm, with a smooth wall 1 microm thick. Sporocysts are broadly oval to ellipsoidal, 12.3 (12-13) x 9.7 (9-10) microm, with Stieda and substieda bodies. Oocysts of Isospora necasi n. sp. from C. melleri are subspherical to broadly oval, 26.6 (21-30) x 24.3 (20-27) microm, with a velvetlike wall 2 microm thick. Sporocysts are broadly ellipsoidal, 12.8 (12-14) x 9.8 (9-10) microm, with slightly pointed end and with Stieda and substieda bodies. Oocysts of Isospora munriyu n. sp. from C. jacksonii are spherical to subspherical, 23.6 (21.5-25) x 21.9 (21-23) microm, with a finely granulated wall 1.5 microm thick. Sporocysts are broadly ellipsoidal, 12.4 (12-13) X 8.7 (8-10) microm, with Stieda and substieda bodies.     

Three new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the lesser seed-finch, Oryzoborus angolensis (Passeriformes: Emberizidae) from Brazil

Three new coccidian (Apicomplexa: Eimeriidae) species are reported from the lesser seed-finch, Oryzoborus angolensis from Brazil. Sporulated oocysts of Isospora curio n. sp. are spherical to subspherical; 24.6 x 23.6 (22-26 x 22-25) microm, shape-index (SI, length/width) of 1.04 (1.00-1.15). Oocyst wall is bilayerd, approximately 1.5 microm thick, smooth and colourless. Micropyle and oocyst residuum are absent. The sporocysts are ovoid, 13.2 x 10.9 (15-17 x 10-13) microm, SI = 1.56 (1.42-1.71), with a small Stieda body and residuum composed of numerous granules scattered among the sporozoites. Sporozoites are elongated and posses a smooth surface and two distinct refractile bodies. Oocysts of Isospora braziliensis n. sp. are spherical to subspherical, 17.8 x 16.9 (16-19 x 16-18) microm, with a shape-index of 1.06 (1.00-1.12) and a smooth, single-layered wall approximately 1 microm thick. A micropyle, oocyst residuum and polar granules are absent. Sporocysts are ellipsoid and slightly asymmetric, 13.2 x 10.8 (12-14 x 9-12) microm, SI = 1.48 (1.34-1.61). Each sporocyst contains a barely visible Stieda body and a residuum composed numerous of granules. Sporozoites are elongated and each of them contains two distinct refractile bodies. Oocysts of Isospora paranaensis n. sp. are subspherical to broadly ellipsoid 24.3 x 19.8 (22-26 x 18-22) microm, SI = 1.22 (1.15-1.38) with smooth single-layered wall approximately 1.5 microm thick. A micropyle and oocyst residuum are absent, but one distinct ellipsoid polar granule (2.5-3.5 x 1.5-2.5 microm) is present. Sporocyst are ovoid, 15.7 x 10.1 (14-18 x 8-12) microm, SI = 1.46 (1.31-1.72), with distinct Stieda and sub-Stieda bodies. Each sporocyst contains a spherical sporocyst residuum, 4 microm in diameter All described isosporan species represent a possible cause of acute coccidiosis for O. angolensis in captivity.     

Two novel Drosophila TAF(II)s have homology with human TAF(II)30 and are differentially regulated during development

TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAF(II)s). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAF(II)s. Biochemical studies suggested that the Drosophila (d) TFIID complexes contain only eight TAF(II)s, leaving a number of yeast and human TAF(II)s (e.g., hTAF(II)55, hTAF(II)30, and hTAF(II)18) without known Drosophila homologues. We demonstrate that Drosophila has not one but two hTAF(II)30 homologues, dTAF(II)16 and dTAF(II)24, which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5' extremities overlap. We show that these novel dTAF(II)s are expressed and that they are both associated with TBP and other bona fide dTAF(II)s in dTFIID complexes. dTAF(II)24, but not dTAF(II)16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAF(II)16 and dTAF(II)24 are functional homologues of hTAF(II)30, and this is the first demonstration that a TAF(II)-GCN5-HAT complex exists in Drosophila. The two dTAF(II)s are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAF(II)16 and dTAF(II)24 may have similar but not identical functions.     

Coccidia from the Tiger Salamander, Ambystoma tigrinum, in Northeastern Colorado and in Northern New Mexico

SYNOPSIS. Oocysts of Eimeria ambystomae Saxe, 1955, Eimeria microcapi sp. n., and Eimeria urodela sp. n. are described from the tiger salamander, Ambystoma tigrinum , collected in Colorado and New Mexico. The oocysts of E. ambystomae are ellipsoid, 29.8 × 17.3 (24–38 × 15–25) μm, and the sporocysts lanceolate, 22.6 × 5.4 (16–27 × 5–7) μm. Oocyst and sporocyst residua are present, but not a polar granule and a micropyle. The oocysts and sporocysts of E. microcapi are ellipsoid, measuring respectively 38.1 × 25.3 (35-41 × 23-26) μm and 18.1 × 7.4 (16-19 × 6–8) μm. Oocyst and sporocyst residua, a micropyle (mean 3 μm), and a distinct micropyle cap (2 μm high) are present, but not a polar granule. The oocysts of E. urodela are spheroid, 22.2 (14-26) μm, and the sporocysts lanceolate, 16.3 × 5.8 (12-19 × 4-7) μm. Oocyst and sporocyst residua are present, but a polar granule and a micropyle are absent.     

How many victims will a pitfall make?

A model for the trapping of animals with a circular pitfall is formulated. The model's assumptions are: (1) The animals move independently according to the same Brownian motions. (2) The boundary of the pitfall acts as an absorbing or elastic barrier. (3) Initially a fixed number of animals is independently homogeneously distributed over a finite study area (a), or the initial positions follow a homogeneous planar Poisson process (b). The model depends on three free parameters: (i) the motility of the animals, (ii) their reaction to the pitfall, (iii) the initial density.It appears that the catches in disjoint time intervals are multinomially (a) or independently Poisson (b) distributed. The parameters of these distributions are obtained by solving certain partial differential equations.Estimation and testing problems are considered, and the data of some laboratory and field experiments are analyzed. It appears that it is possible to estimate both the animals' motility and density from a pitfall experiment. However, the accuracy is very low. To solve this problem at least partially, experiments for the separate estimation of parameters other than the density are discussed.     

Structure and molecular mobility of soy glycinin in the solid state

We report a multitechnique study of structural organization and molecular mobility for soy glycinin at a low moisture content (<30% w/w) and relate these to its glass-to-rubber transition. Small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy are used to probe structure and mobility on different length and time scales. NMR (approximately 10(-6) to 10(-3) s) reveals transitions at a higher moisture content (>17%) than DSC or SAXS, which sample for much longer times (approximately 10 to 10(3) s) and where changes are detected at >13% water content at 20 degrees C. The mobility transitions are accompanied by small changes in unit-cell parameters and IR band intensities and are associated with the enhanced motion of the polypeptide backbone. This study shows how characteristic features of the ordered regions of the protein (probed by SAXS and FTIR) and mobile segments (probed by NMR and DSC) can be separately monitored and integrated within a mobility transformation framework.     

THE MAINTENANCE OF GYNODIOECY AND ANDRODIOECY IN A METAPOPULATION

Males and females are at a selective disadvantage relative to hermaphrodites (cosexuals) in species with a colonizing habit, as only cosexuals are able to establish new colonies on their own. The implications of this disadvantage are assessed by means of a computer model of metapopulation dynamics, in which individual colonies are established through different rates of immigration and suffer different rates of local extinction. Results are given for simulations of an island model, a stepping-stone model, and for a partial analysis of the island model with simplifying assumptions. It is shown that: (1) unisexual frequencies in a metapopulation can be reasonably approximated by a linear function of the logarithm of the ratio of the immigration rate to the colony extinction rate; (2) metapopulation dynamics favor the maintenance of females (gynodioecy) over males (androdioecy) with cosexuals when they would otherwise be equally likely in a panmictic situation; (3) the way in which extinction and immigration rates affect unisexual frequencies at metapopulation equilibrium interacts with whether sterility is determined by a dominant or a recessive allele; and (4) unisexual frequencies are affected in a qualitatively similar way by the dynamics of a metapopulation when cosexuals are self-incompatible to when they are self-compatible, although only in the former case are high frequencies of unisexuals maintained when extinction and colonization rates approach the threshold at which the metapopulation goes extinct. These results are discussed with reference to existing data from species with nuclear male or female sterility.     

Structural basis of the RNase H1 activity on stereo regular borano phosphonate DNA/RNA hybrids

Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN's are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.     

Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.