Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.     

High Density Molecular Linkage Maps of the Tomato and Potato Genomes

High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.     

Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression.

Published reports indicate that CD45RO-CD45RAbright T cells are native T cells, CD45RObrightCD45RA- T cells are memory T cells, and that concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status. Thus, following in vitro activation of CD45RO- CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed. Interestingly, all three of these phenotypic subsets are represented in the circulating human lymphocyte pool. We thus used dual-color flow cytometry to phenotypically characterize CD45RObrightCD45RA-, CD45ROdimCD45RAdim, and CD45RO- CD45RAbright lymphocytes. Both the CD45RObrightCD45RA- and CD45ROdimCD45RAdim subsets consisted almost entirely of T cells, whereas the CD45RO-CD45RAbright subset contained T cells plus essentially all of the B and natural killer cells. Additional studies used three-color flow cytometry to assess activation markers on T cells within the three subsets defined by CD45RO/CD45RA expression. CD25 expression increased with conversion from naive to memory status (5% of CD45RO-CD45RAbright, 24% of CD45ROdimCD45RAdim, and 42% of CD45RObrightCD45RA- T cells), whereas CD38 expression decreased during conversion (76, 53, and 27%, respectively). We also assessed the fluorescent intensities of CD11a, CD2, and CD44, shown by others to be increased on memory, compared to naive T cells. Visual inspection of fluorescence cytograms confirmed these findings, and further showed that transitional T cells express these markers at levels indistinguishable from those for naive T cells. These findings suggest that acquisition of CD25 and loss of CD38 occur relatively early in the naive-to-memory transition process, being evident in the transitional cell subset. In contrast, increased expression of CD11a, CD2, and CD44 appear to represent late events, occurring after loss of CD45RA and gain of CD45RO has been completed.     

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.     

EPR properties of the reaction center of Rhodopseudomas gelatinosa in situ and in a detergent-solubilized form

The photochemical reaction centers from a variety of purple photosynthetic bacteria are composed of a trimer of protein subunits. However, the recently isolated reaction center from Rhodopseudomonas gelatinosa appears to have only two subunits. In this paper we examine the EPR characteristics of the primary photochemical reactants in this species, and compare them with those of other species. Despite of the differences in protein composition, no dramatic differences in EPR properties are seen in vivo, although some interesting effects are seen upon solubilization of the reaction center, which may be related to the unusual lability of the isolated preparation. Perhaps the most noteworthy phenomenon seen in Rps. gelatinosa is the apparent ability of electrons on the reduced intermediary electron carrier to tunnel at low temperatures to the oxidized c-type cytochrome, which has not been seen in other species studied to date.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium.

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of < 5 μs and ≈ 50 μs). We believe that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll.We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below ?450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD⁺ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.