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Introduction: Salmonella enterica is a major cause of foodborne illness in the United States. A multi-drug resistant (MDR) emergent Salmonella Infantis (ESI) with a megaplasmid (pESI) was first identified in Israel and Italy and subsequently reported worldwide. The ESI clone carrying an extended spectrum β-lactamase blaCTX-M-65 on a pESI-like plasmid and a mutation in the gyrA gene has recently been found in the United States in poultry meat. Methods: We analyzed the phenotypic and genotypic antimicrobial resistance, genomics and phylogeny of 200 S. infantis isolates from animal diagnostic samples. Results: Of these, 33.5% were resistant to at least one antimicrobial and 19.5% were multi-drug resistant (MDR). Eleven isolates from different animal sources were phenotypically and genetically similar to the ESI clone. These isolates had a D87Y mutation in the gyrA gene conferring reduced susceptibility to ciprofloxacin and harbored a combination of 6-10 resistance genes: blaCTX-M-65, aac(3)-IVa, aadA1, aph(4)-Ia, aph(3')-Ia, floR, sul1, dfrA14, tetA, and fosA. These 11 isolates carried class I and class II integrons and three virulence genes: sinH, involved in adhesion and invasion, ybtQ and ybtP, associated with iron transport. These isolates were also closely related to each other (separated by 7 to 27 SNPs) and phylogenetically related to the ESI clone recently found in the U.S. Discussion: This dataset captured the emergence of the MDR ESI clone in multiple animal species and the first report of a pESI-like plasmid in isolates from horses in the U.S. [ABSTRACT FROM AUTHOR]

Tall fescue (Lolium arundinaceum) is a highly adaptable forage, pasture and turf grass that is grown on over 14 M ha in the eastern half of the United States and in other temperate regions of the world. A significant factor in adaptability, productivity and stand persistence is in part due to the presence of an intercellular, seed-transmissible, endophytic fungus, Epichloë coenophiala. Epichloë endophytes have been shown to produce a number of alkaloid compounds only in planta, some that are beneficial in repelling insects, while others are toxic to animals. The goal of this work was to monitor the level of the ergot and loline (classified as pyrrolizidine) alkaloid accumulation in individual plants to determine the plant genotype contribution to alkaloid concentrations. The experimental design consisted of sixteen tall fescue KY31 clones in a space-planted, replicated trial over three years. Our results demonstrated that while changes in the alkaloid concentrations for each plant/endophyte genotype were observed over the three years, the overall alkaloid levels remained relatively constant when compared to other plant/endophyte genotypes combinations in the field. Additionally, overall levels of the ergot and loline alkaloid accumulation did not vary in the same way over the three years. Since the E. coenophiala endophyte genotype was the same across all clones, our results indicate that it is the plant genotype that is responsible for determining alkaloid levels in each plant, and suggest that the signal(s) from the plant to the endophyte may not be the same for ergot and loline alkaloid production. [ABSTRACT FROM AUTHOR]

RNA interference is a powerful tool that post-transcriptionally silences target genes. However, silencing efficacy varies greatly among different insect species. Recently, we attempted to knock down some housekeeping genes in the tawny crazy ant (Nylanderia fulva), a relatively new invasive species in the southern United States, but only achieved relatively low silencing efficiency when dsRNA was orally administered. Here, we detected divalent cation-dependent, dsRNA-degrading activity in the midgut fluid of worker ants in ex vivo assays. To determine whether dsRNA degradation could contribute to low effectiveness of oral RNAi in N. fulva , we cloned its sole dsRNase gene (NfdsRNase). The deduced amino acid sequence contained a signal peptide and an endonuclease domain. Sequence alignment indicated a high degree of similarity with well-characterized dsRNases, particularly the six key residues at active sites. We also identified dsRNase homologs from five other ant species and found a tight phylogenetic relationship among ant dsRNases. NfdsRNase is expressed predominantly in the abdomen of worker ants. Oral delivery of dsRNA of NfdsRNase significantly reduced the expression of NfdsRNase transcripts, and substantially suppressed dsRNA-degrading activity of worker ants' midgut fluids as well. Our data suggest that dsRNA stability in the alimentary tract is an important factor for gene silencing efficiency in N. fulva , and that blocking NfdsRNase in gut lumen could potentially improve RNAi, a novel pest management tactic in control of N. fulva and other ant species. [ABSTRACT FROM AUTHOR]

Porcine circovirus 3 (PCV3) was first discovered in 2016, almost concomitantly by two groups of researchers in the United States. The novel case was reported in a group of sows with chronic reproductive problems with clinical presentation alike porcine dermatitis and nephropathy syndrome (PDNS), where metagenomic sequencing revealed a genetically divergent porcine circovirus designated PCV3. The discovery of PCV3 in a PDNS case, which used to be considered as part of PCVAD attributed to PCV2 (porcine circovirus 2), has garnered attention and effort in further research of the novel virus. Just when an infectious molecular DNA clone of PCV3 has been developed and successfully used in an in vivo pathogenicity study, yet another novel PCV strain surfaced, designated PCV4 (porcine circovirus 4). So far, PCV3 has been reported in domestic swine population globally at low to moderate prevalence, from almost all sample types including organ tissues, faecal, semen and colostrum samples. PCV3 has been associated with a myriad of clinical presentations, from PDNS to porcine respiratory disease complex (PRDC). This review paper summarizes the studies on PCV3 to date, with focus on diagnosis. [ABSTRACT FROM AUTHOR]

Recently, there have been reports worldwide of a multidrug-resistant, emergent Salmonella Infantis (ESI) clone with a large megaplasmid (pESI), often containing the extended-spectrum beta-lactamase gene blaCTX-M-65. This clone also has a gyrA mutation conferring fluoroquinolone resistance, further limiting treatment options. In the United States, this clone has also been found in poultry sources, indicating a likely source of human illnesses. We conducted short-read sequencing of Salmonella enterica isolated from retail meats as part of routine surveillance by the National Antimicrobial Resistance Monitoring System (NARMS). We analyzed the resulting data temporally and geographically to determine when and where the ESI clone has spread in the United States. We found the ESI clone was first found in retail meats in Tennessee in 2014, but by 2019 was throughout the United States and comprised 29% of all Salmonella isolated from retail chickens, and 7% from retail turkey. Of these isolates, 85.0% were within 20 single nucleotide polymorphisms (SNPs) of those causing human illnesses. Long-read sequencing data indicated substantial recombination in the pESI plasmid resulting in the presence of 0-10 resistance genes, despite all their chromosomes being within 31 SNPs of one another. This work demonstrates the rapid spread of this clone of Salmonella Infantis in poultry in the United States, with the potential for increased burden of human illness attributed to this multidrug-resistant pathogen. [ABSTRACT FROM AUTHOR]

Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by gene cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaqTM, proofreading Pfu polymerase, and PicoMaxxTM buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the occurrence, the genetic environment and the functionality of novel variants of the MDR gene cfr(C) in Campylobacter from China.Methods: A total of 370 Campylobacter isolates of porcine and chicken origin collected from three regions of China in 2015 were screened for cfr(C) by PCR. The phenotypes and genotypes of cfr(C)-positive isolates were investigated by antimicrobial susceptibility testing, PFGE, MLST, S1-PFGE, Southern blotting and WGS. Quantitative RT-PCR was used to compare the expression levels of the cfr(C) variants in their original isolate and clone constructs in Campylobacter jejuni NCTC 11168.Results: Four (1.1%) porcine Campylobacter coli isolates were positive for cfr(C). They failed to show elevated MICs of phenicols. The deduced Cfr(C) sequences identified exhibited 2-6 amino acid changes compared with the original Cfr(C) reported in the USA. Cloning of the cfr(C) variant genes into C. jejuni NCTC 11168 resulted in ≥32-fold increases in the MICs of phenicols, indicating that the cfr(C) variant genes are functional. The cfr(C)-carrying isolates belonged to three genotypes and WGS analysis revealed the cfr(C) genes were chromosomally located in MDR genomic islands, which contained multiple antibiotic resistance genes of Gram-positive origin.Conclusions: This study identified chromosomal cfr(C) genes in C. coli isolates from China. They appeared functionally dormant in the original isolates but were fully functional when cloned and expressed in C. jejuni. The cfr(C) genes were co-transferred with other antibiotic resistance genes, possibly from Gram-positive bacteria. These findings reveal new insights into the function and transmission of cfr(C) in Campylobacter. [ABSTRACT FROM AUTHOR]

Genetic improvement of crop plants is dependent on the availability of genetic variation, which can be present naturally or can be created using mutations. Ethyl methanesulfonate (EMS) mutagenesis provides an attractive tool to create variant alleles of genes of interest in any plant. In the present paper, we report development of an EMS‐mutagenized population in a hard red winter wheat (Triticum aestivum L.) variety Jagger. Jagger has been a leading variety across the United States and has been grown internationally as well, for over two decades now. It possesses many useful traits related to disease resistance, cold tolerance, and milling and baking qualities. Because it is a landmark variety, the genome of Jagger is being sequenced by an international collaborative team, which will speed up gene mapping and cloning efforts in it. The Jagger Targeting Induced Local Lesions In Genome (TILLING) resource developed in this work will be useful for functional validation of these genes. The mutagenized population was developed using EMS and was characterized phenotypically and genotypically. Gene‐wise mutation screening was performed for three genes located on different chromosomes across all the three genomes. The population has an overall mutation frequency of 1/40.7 kb, and we were able to find multiple knockout, missense, and missplicing mutations for each targeted gene. The population will be useful as a forward and reverse genetic screen for a number of valuable traits. [ABSTRACT FROM AUTHOR]

Cytauxzoon felis is a tick-borne hemoparasite that causes cytauxzoonosis in domestic cats in the United States. Historically, feline cytauxzoonosis was reported to be nearly always fatal. However, increasing evidence of cats surviving acute infection and/or harboring a chronic, subclinical infection has suggested the existence of different C. felis strains that may vary in pathogenicity. In this study, the intraspecific variation of the C. felis first and second ribosomal RNA internal transcribed spacer (ITS1, ITS2) regions was assessed for any clinical outcome or geographic associations. Sequence data were obtained for 122 C. felis ITS1 and ITS2 clones from 41 domestic cat blood samples from Arkansas, Kansas, Missouri, Oklahoma, and Texas. Seven previously reported ITS1 region sequences were found, and a previously undescribed 23-bp insert was detected in cloned ITS1 sequences from a domestic cat in Missouri and two cats in Oklahoma. Four previously reported ITS2 region sequences were identified, and a 40-bp insert similar to that previously reported in C. felis of a domestic cat from Arkansas and pumas was detected in 18 cloned C. felis sequences from 12 domestic cats. One clone contained both the 23-bp insert and 40-bp insert within the ITS1 and ITS2 regions, respectively. Combined ITS1 and ITS2 sequence genotypes revealed that C. felis sequences from 27 cats (72/122 clones) corresponded to four previously described genotypes, ITSa, ITSc, ITSd, and ITSn. Five clones with the novel 23-bp insert from three cat isolates represented two new genotypes, ITSaa and ITSbb. Genotypes ITScc, ITSdd, ITSee, ITSff, ITSgg, and ITShh denoted 13 clones that matched prior sequences but had no previously assigned genotype. Genotypes ITSii through ITStt comprised 32 clones that were similar to, but did not exactly match, previously described genotypes. Twenty-five cats had C. felis infections with multiple ITS genotypes. Considerable C. felis genetic diversity was revealed with no significant geographic or clinical outcome associations. [ABSTRACT FROM AUTHOR]

In the United States, allyl isothiocyanate (AITC) has been registered as an insecticide, bactericide, and nematicide. And it has been confirmed that AITC has significant insecticidal activities against four stored product pests including Sitophilus zeamais Mostchulky (Coleoptera: Curculionidae). This study aimed to verify the mechanism of action of AITC on cytochrome c oxidase core subunits II in S. zeamais. Enzyme - catalyzed reactions and Fourier transform infrared spectrometer (FTIR) analysis revealed that the expressed COX II proteins could competitively bind and inhibit the activity of COX II. Furthermore, molecular docking results showed that a sulfur atom of AITC could form a 2.9 Å hydrogen bond with Ile-30, having a binding energy of −2.46 kcal/mol. [Display omitted] • A Yeast expression system for producing cytochrome c oxidase subunit II is employed. • Yeast expression system produces much higher protein content than prokaryotic expression. • The expressed COXII protein has catalytic oxidation activity. • The expressed COXII protein shows strong binding ability to AITC. • The binding site of COXII to AITC is predicted by molecular docking. [ABSTRACT FROM AUTHOR]

The article features scientists Frederick M. Ausubel, recipient of the Genetics Society of America's 2014 Thomas Hunt Morgan Medal. Particular focus is given to Ausubel's contributions to the field of genetics research. Topics discussed include his educational background, factors that influenced his career choices, and the nif gene-cloning project.

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca2+ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A2 (PLA2) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs. [ABSTRACT FROM AUTHOR]

Brevundimonas diminuta is the test organism specified in the United States Environmental Protection Agency's (USEPA) reverse osmosis (RO) treatment device verification protocol. As nonselective growth medium is employed, enumeration of B. diminuta may be impaired due to interference by indigenous heterotrophic bacteria. Thus the microbial removal capability of the filtration system may be incorrectly assessed. As these treatment devices are used in emergency situations, the health of the public could be compromised. The objective of this study was to develop selective approaches for enumerating viable B. diminuta in test water. Two molecular approaches were investigated: expression of a kanamycin resistance gene and expression of a fluorescent protein gene. The USEPA protocol specifies a 0.3 µm cell size, so the expression of the selective markers were assessed following growth on media designed to induce this small cell diameter. The kanR strain was demonstrated to be equivalent to the wild type in cell dimension and survival following exposure to the test water. The kanR strain showed equivalent performance to the wild type in the RO protocol indicating that it is a viable alternative surrogate. By utilizing this strain, a more accurate validation of the RO system can be achieved. [ABSTRACT FROM AUTHOR]

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65-70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90. [ABSTRACT FROM AUTHOR]

Many plant ESTs have been sequenced as an alternative to whole genome sequences, including peanut because of the genome size and complexity. The US peanut research community had the historic 2004 Atlanta Genomics Workshop and named the EST project as a main priority. As of August 2011, the peanut research community had deposited 252,832 ESTs in the public NCBI EST database, and this resource has been providing the community valuable tools and core foundations for various genome-scale experiments before the whole genome sequencing project. These EST resources have been used for marker development, gene cloning,microarray gene expression and genetic map construction. Certainly, the peanut EST sequence resources have been shown to have a wide range of applications and accomplished its essential role at the time of need. Then the EST project contributes to the second historic event, the Peanut Genome Project 2010 Inaugural Meeting also held in Atlanta where it was decided to sequence the entire peanut genome. After the completion of peanut whole genome sequencing, ESTs or transcriptome will continue to play an important role to fill in knowledge gaps, to identify particular genes and to explore gene function. [ABSTRACT FROM AUTHOR]

Abstract: Salmonella Cerro prevalence in US dairy cattle has increased significantly during the past decade. Comparison of 237 Salmonella isolates collected from various human and animal sources between 1986 and 2009 using pulsed-field gel electrophoresis, antimicrobial resistance typing, and spvA screening, showed very limited genetic diversity, indicating clonality of this serotype. Improved subtyping methods are clearly needed to analyze the potential emergence of this serotype. Our results thus emphasize the critical importance of population-based pathogen surveillance for the detection and characterization of potentially emerging pathogens, and caution to critically evaluate the adequacy of diagnostic tests for a given study population and diagnostic application. [Copyright &y& Elsevier]

Cotton (Gossypium hirsutum L.) monoculturc under conventional tillage has been the predominant cropping system in the Southern High Plains region of the United States since the 1940s. This study evaluated other cropping systems and land uses for their potential to increase soil quality and enhance soil functioning compared with continuous cotton (Ct-Ct), including a mixture of grasses in the Conservation Reserve Program (CRP), a pasture monoculture [Bothriochloa bladhii (Retz) S.T. Blake] and a cotton-winter wheat ( Triticum aestivum L.)corn (Zea mays L.) rotation (Ct-W-Cr). Soil microbial communities were evaluated according to microbial biomass C (MBC) and N (MBN), fatty acid methyl ester (FAME) profiling, and molecular cloning techniques. Soil MBC was higher under the alternative systems at 0 to 5 cm (CRP > pasture = Ct-W-Cr > Ct-Ct), 5 to 10 cm (CRP = Ct-W-Cr> pasture> Ct-Ct), and 10 to 20cm (CRP = pasture = Ct-W-Cr> Ct-Ct). Soil DNA concentration was correlated with key soil quality parameters such as microbial biomass (r>0.52, P <0.05), total C (r = 0.372, P <0.1), and total N (r = 0.449, P <0.05). The 16S rRNA gene banding patterns (0-5 cm) of undisturbed systems (CRP and pasture) were more similar to each other than to Ct-Ct and Ct-W-Cr. Fungal/bacterial FAME ratios were higher under CRP and pasture than under Ct-Ct at 0 to 5 and 5 to 10 cm. This study found increases in sensitive soil quality parameters under alternative management compared with cotton monoculture. [ABSTRACT FROM AUTHOR]

AbstractAgronomical applications of cloned livestock produced by somatic cell nuclear transfer (SCNT) have been authorized in the United States and the European Food Safety Authority published that there was no evidence of risks associated with the use of cloned animal in the breeding industry. Both assessments, however, underlined that complementary data are needed to update their conclusions. SCNT is associated with a high incidence of perinatal losses. After birth, cloned cattle appear to possibly present subtle immune defects, requiring extensive studies to be properly evidenced. Twenty-five cloned Holstein heifers from five distinct genotypes and their contemporary age- and sex-matched controls were compared. An extensive survey of leukocyte subsets was performed and the humoral and T-cell immune responses to exogenous antigens were studied. Cloned cattle presented a normal representation of leukocyte subsets. Functional immunity was not modified in cloned heifers, as they were able to raise an antibody response and to develop B and T cell-specific responses against the model antigen OVA (ovalbumin) and against a rotavirus vaccine as in controls. Thus, this extensive analysis supports previous data suggesting that cloned cattle have a normal immunity. [ABSTRACT FROM AUTHOR]

AbstractIn an effort to minimize the probability of forward contamination of pristine extraterrestrial environments, the National Aeronautics and Space Administration requires that all US robotic spacecraft undergo assembly, testing and launch operations (ATLO) in controlled clean-room environments. This study examines the impact of ATLO activity on the microbial diversity and overall bioburden contained within the air of the clean-room facility in which the Mars Exploration Rovers (MERs) underwent final preparations for launch. Air samples were collected from several facility locations and traditional culture-based and molecular methodologies were used to measure microbial burden and diversity. Surprisingly, the greatest estimates of airborne bioburden, as derived from ATP content and cultivation assays, were observed prior to the commencement of MER ATLO activities. Furthermore, airborne microbial diversity gradually declined from the initiation of ATLO on through to launch. Proteobacterial sequences were common in 16S rDNA clone libraries. Conspicuously absent were members of the Firmicutes phylum, which includes the genus Bacillus. In previous studies, species of this genus were repeatedly isolated from the surfaces of spacecraft and clean-room assembly facilities. Increased cleaning and maintenance initiated immediately prior to the start of ATLO activity could explain the observed declines in both airborne bioburden and microbial diversity. [ABSTRACT FROM AUTHOR]

Stem cell research has become one of the hottest scientific topics in the past few years. By combining the ability to exactly self-duplicate and the potential to differentiate themselves to constitute any organic tissue, embryonic stem cells have presented geneticists and medical researchers with a new wealth of possibilities to treat and cure degenerative diseases such as Parkinson's and Alzheimer's. By relying heavily on discarded embryonic lines, stem cell research has also generated a new host of ethical and political controversies. The concept of media frames was employed to analyze how some of the main Brazil- and U.S.-based newspapers have framed and presented the stem cell research debate. The analysis indicates that while in Brazil the issue has been presented from a scientific (and mostly positive) point of view, in the United States the debate has been dominated by its political and ethical dimensions. [ABSTRACT FROM AUTHOR]

We have isolated and characterized 17 tetranucleotide microsatellite loci for Blanchard's cricket frog ( Acris crepitans blanchardi), an anuran common in the central USA. Sixteen loci were organized into four multiplex amplification reactions. These loci were highly polymorphic when screened in 55 individuals from two distant populations, with 11–48 alleles per locus (average = 24.8). Observed and expected heterozygosities ranged from 0.18 to 0.97 and from 0.17 to 0.96, respectively. Nine loci were also polymorphic in Acris crepitans crepitans, with seven polymorphic in Acris gryllus. Five loci amplified in all three taxa. These loci will be useful for population- and species-level investigations of this widespread group. [ABSTRACT FROM AUTHOR]

The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries. [ABSTRACT FROM AUTHOR]

AAAS President Mary Ellen Avery reported to the Council on the AAAS Board's activities over the last year. She reported that the Board was carefully following congressional actions with regard to stem cell research and therapeutic cloning and had taken the opportunity to reissue the AAAS statement in support of therapeutic cloning. She noted that the Board had met with a variety of scientific leaders throughout the year, including the Assistant Director-General of UNESCO, the Director of the Department of Energy's Office of Science, and Congressman Rush Holt. Website, which is an online science news service that provides reporters with free and timely access to the most current science research news from around the world. INSETS: RESOLUTION ON THE OMB PROPOSED..;RESOLUTION REGARDING HUBBLE SPACE TELESCOPE..;RESOLUTION ENDORSING THE DECLARATION..;AAAS Annual Election: Preliminary Announcement

Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155T and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format. When cultured in low-nutrient seawater media, these strains copiously produced Alcian Blue-stainable viscous TEP. Growing cells were attached to each other by the TEP in a three dimensional network. Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans. The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community. In phylogenetic analyses, the strains consistently formed a new phylum-level lineage within the domain Bacteria, together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats. Five putative subgroups were delineated within this phylum-level lineage, including a marine group and an anaerobic group. The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere-shaped bacteria. The DNA G+C content of strain HTCC2155T was 48.3 mol% and the genome size was 2.9 mb. It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155T = ATCC BAA-859T = KCTC 12141T) gen. nov., sp. nov., the cultured marine representative of the Lentisphaerae phyl. nov., and the phylum be divided into two novel orders named the Lentisphaerales ord. nov. and the Victivallales ord. nov. [ABSTRACT FROM AUTHOR]

Determines the compositions of marine bacterioplankton communities in the coastal waters in California. Use of clone libraries of 16S rRNA genes and fluorescence in situ hybridization; Differences in community composition of the clone libraries; Dominance of cytophaga-flavobacter group.

Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry (Vaccinium spp.) floral buds during chill unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. Indeed, levels of the 65-, 60-, and 14-kDa dehydrins increase such that they become the most predominant proteins visible on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The peptide sequence information from the 65- and 60-kDa dehydrins was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding the dehydrins. One pair of primers amplified a 174-bp fragment. The 174-bp fragment was used to screen a cDNA library (prepared from RNA from cold-acclimated blueberry floral buds) and resulted in the isolation of a clone with a 2.0-kb insert. The cDNA was sequenced and found to be a full-length clone encoding a K5-type dehydrin (5 K boxes). Five high-confidence peptide sequences, ranging from 9 to 25 amino acids long, obtained from the 60-kDa dehydrin exactly matched sequences encoded within the cDNA clone. Furthermore, amino acid composition of the 60-kDa dehydrin agreed well with the expected amino acid composition based on the cDNA sequence. However, the DNA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of ∼40 kDa instead of 60 kDa. Experiments to determine if the dehydrins undergo post-translational modifications revealed that the 65- and 60-kDa dehydrins are glycosylated. Thus, our results indicate that the 2.0-kb dehydrin cDNA encodes the native version of the 60-kDa dehydrin. The dehydrin cDNA hybridized on RNA blots to two chilling/cold-responsive messages of 2.0 and 0.5 kb. Both the 2.0- and 0.5-kb messages increased to higher levels more quickly in the cold-hardy cultivar Bluecrop than in the less hardy cultivar Tifblue. In addition, the 0.5-kb message remained at a higher level longer in Bluecrop than in Tifblue. [ABSTRACT FROM AUTHOR]

The article examines laboratory and field enzyme-immunoassay (EIA) for diazinon and their application to residue analysis in lanolin, water and fruit juice by researchers from Australia and the U.S. In this study, the researchers describe the development of laboratory and field competitive IEA formats, based on polyclonal antibodies. These provide an alternate method of diazinon quantitation, and require little or no cleanup or sample concentration for detection of residues in well water, lanolin and fruit juice.

The article offers information on human cloning and stem cell research and the government policies on this issue in the U.S. It has been informed that U.S. president George W. Bush has always denied support to embryonic stem cell research as unethical and that he has advocated the sourcing of stem cells by methods not involving a human infant, like molecular cloning, regarding which he gave an executive order to the National Institutes of Health of the U.S.

Reports the development of a method to genetically engineer the entire influenza A virus. Difficulty in producing the virus from cloned genes; Negative-strand RNA; Details of the study conducted by Yoshihiro Kawaoka and colleagues at the University of Wisconsin, United States; Significance of the study.

Editorial. Reflects on the filing of patent applications on the majority of human genes. Generation of a collection of expressed sequence tags which identify the genes transcribed by the tissues; Use of cloned sequences to express proteins and test their biological activity; Patentability of most sequences, even those of unknown function, in the United States.