Ontogeny of cloned cattle to lactation

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.     

Association between a high-expressing interferon-gamma allele and a lower frequency of kidney angiomyolipomas in TSC2 patients

Tuberous sclerosis complex (TSC) is a familial hamartoma syndrome in which renal involvement is common and, at times, life threatening. We have investigated the potential effect of a non-TSC gene on renal disease in a cohort of 172 TSC patients with TSC2 mutations. Patients were genotyped for an interferon-gamma (IFN-gamma) microsatellite polymorphism, within intron 1, for which one common allele (allele 2, with 12 CA repeats) has been shown to have a higher expression of IFN-gamma. A chi(2) analysis was used to examine the association between IFN-gamma allele 2 and the development of kidney angiomyolipomas (KAMLs) in this TSC2 cohort. Because of the age-dependent development of KAMLs in TSC, we initially focused on the 127 patients who were >5 years old. Additional subgroup analyses were done to investigate the influence of age and gender. The transmission/disequilibrium test (TDT) was also performed in a subset of this cohort (46 probands) for whom parent and/or sibling samples were available for analysis. Both chi(2) analysis and TDT suggested an association between IFN-gamma allele 2 and the absence of KAMLs in patients who have known TSC2 mutations. Among the 127 patients who were >5 years old, KAMLs were present in 95 (75%) and were absent in 32 (25%). In the group with KAML present, the frequency of IFN-gamma allele 2 was 56%; in the group with KAML absent, the frequency of IFN-gamma allele 2 was significantly higher, at 78% (P=.02, by chi(2) analysis). The family-based TDT analysis gave similar results, with a TDT statistic (TDT chi2=5.45) corresponding to a P value of.02. Subgroup analyses show that both age and gender may influence the impact of this association. Although these results should be replicated in other populations with TSC, the present study suggests that modifier genes play a role in the variable expression of TSC and also suggests a potential therapy for KAMLs in patients with TSC.     

Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis

Arrestin binding to activated, phosphorylated G protein-coupled receptors (GPCRs) represents a critical step in regulation of light- and hormone-dependent signaling. Nonvisual arrestins, such as arrestin-2, interact with multiple proteins for the purpose of propagating and terminating signaling events. Using a combination of X-ray crystallography, molecular modeling, mutagenesis, and binding analysis, we reveal structural features of arrestin-2 that may enable simultaneous binding to phosphorylated receptor, SH3 domains, phosphoinositides, and beta-adaptin. The structure of full-length arrestin-2 thus provides a uniquely oriented scaffold for assembly of multiple, diverse molecules involved in GPCR signal transduction.     

PESI--an intelligent system for prediction of enzyme-substrate interactions based on experimental constraints

We present a system for predicting protein-protein modifications, and demonstrate its usefulness in the field of signal transduction research. Signal transduction is one of the most important areas of investigation in biological research. One of the major mechanisms frequently employed by cells to regulate signal transduction processes involves protein phosphorylation by various kinases. As many as 1,000 protein kinases and 500 protein phosphatases in the human genome are thought to be involved in phosphorylation processes which regulate all aspects of cell function. The complexity of such interactions stems from the enormous number of factors and interactions, which makes the identification of putative substrates for any given enzyme by straightforward experimentation increasingly difficult. We present here a data mining algorithm, based on the similarity between the modifier proteins and between the modified proteins, and on experimental constraints. The application presented here (PESI) focuses on substrate phosphorylation by various enzymes. This algorithm reduces the number of substrate candidates for experimental study by about two orders of magnitude. Moreover, this algorithm has already yielded predictions for previously unknown substrates of the enzymes PKCdelta and PKCeta, which we have confirmed experimentally.     

The chemistry and transport of mercury in a small wetland in the Adirondack region of New York, USA

The biogeochemistry of Hg was evaluated in a small wetland in the Adirondack region of New York. Concentrations of total Hg (Hg_T) in streamwater draining the wetland showed little temporal variation. The annual areal watershed flux of Hg_T (2.2 µg/m²-yr) was considerably smaller than regional inputs of atmospheric deposition of Hg_T, indicating that the terrestrial environment is a net sink for atmospheric deposition of Hg_T. Drainage inputs of Hg_T were conservatively transported through the beaver impoundment. The annual flux of total methyl mercury (CH₃Hg⁺ _T was greater than literature values of atmospheric deposition suggesting that the watershed is a net source of CH₃Hg⁺ _T . Stream concentrations of CH₃Hg⁺ _T increased during low-flow summer conditions in a riparian wetland, and particularly at the outlet of the beaver impoundment. Net production of CH₃Hg⁺ _T occurred in the beaver impoundment (0.45 µg/m²-yr). Rates of net methylation for the beaver impoundment were comparable to values reported in the literature for wetlands.     

c-Type Cytochromes and Manganese Oxidation in Pseudomonas putida MnB1

Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.     

Microbial Diversity in a Hydrocarbon- and Chlorinated-Solvent-Contaminated Aquifer Undergoing Intrinsic Bioremediation

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria- or Archaea-specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.