Search

Octanoic acid is an industrially relevant compound with applications in antimicrobials or as a precursor for biofuels. Microbial biosynthesis through yeast is a promising alternative to current unsustainable production methods. To increase octanoic acid titers in Saccharomyces cerevisiae, we use a previously developed biosensor that is based on the octanoic acid responsive pPDR12 promotor coupled to GFP. We establish a biosensor strain amenable for high-throughput screening of an octanoic acid producer strain library. Through development, optimization, and execution of a high-throughput screening approach, we were able to detect two new genetic targets, KCS1 and FSH2, which increased octanoic acid titers through combined overexpression by about 55% compared to the parental strain. Neither target has yet been reported to be involved in fatty acid biosynthesis. The presented methodology can be employed to screen any genetic library and thereby more genes involved in improving octanoic acid production can be detected in the future.

Ginkgo biloba breeding commonly concentrates on the selection of superior trees that have high flavonoid contents. The flavonoids present in Ginkgo leaves have strong medicinal implications, including anti-dengue, anti-HIV, anticancer, antioxidant and anti-inflammatory properties. Flavonoids play important roles in plant immune responses; however, the molecular mechanisms underlying these interesting responses remain unclear. To obtain a comprehensive understanding, we performed the transcriptome sequencing of Ginkgo with different flavonoid contents. Using an Illumina sequencing platform, we obtained approximately 533,952,528 clean reads. After the sequences were filtered and assembled, the transcriptome data generated 37,625 unigenes, of which 21,472 (57.07%) were successfully annotated in five public databases. Among those genes, many candidates were involved in flavonoid biosynthesis, transport and regulation. Expression profiles were generated, and 457 genes were found to be significantly differentially expressed between the Sample_GB_FH1/2/3 (FH) and Sample_GB_FL1/2/3 (FL) libraries; 246 (53.83%) genes were up-regulated, and 211 (46.17%) were down-regulated. These genes included 14 genes that were enriched in flavonoid transport, 1 MYB gene that encoded a putative transcription factor (TF), and 1 dihydroflavonol reductase (DFR) gene that was involved in the flavonoid pathway. Our results provide comprehensive gene expression information about the Ginkgo transcriptome and can facilitate our understanding of the molecular mechanisms of flavonoid development in Ginkgo. Furthermore, our results markedly expand both the available Ginkgo genetic library and analyses of the species and provide valuable information to the Ginkgo-related pharmaceutical industry.

Background With about 1,000 species in the Neotropics, the Eumaeini (Theclinae) are one of the most diverse butterfly tribes. Correct morphology-based identifications are challenging in many genera due to relatively little interspecific differences in wing patterns. Geographic infraspecific variation is sometimes more substantial than variation between species. In this paper we present a large DNA barcode dataset of South American Lycaenidae. We analyze how well DNA barcode BINs match morphologically delimited species. Methods We compare morphology-based species identifications with the clustering of molecular operational taxonomic units (MOTUs) delimitated by the RESL algorithm in BOLD, which assigns Barcode Index Numbers (BINs). We examine intra- and interspecific divergences for genera represented by at least four morphospecies. We discuss the existence of local barcode gaps in a genus by genus analysis. We also note differences in the percentage of species with barcode gaps in groups of lowland and high mountain genera. Results We identified 2,213 specimens and obtained 1,839 sequences of 512 species in 90 genera. Overall, the mean intraspecific divergence value of CO1 sequences was 1.20%, while the mean interspecific divergence between nearest congeneric neighbors was 4.89%, demonstrating the presence of a barcode gap. However, the gap seemed to disappear from the entire set when comparing the maximum intraspecific distance (8.40%) with the minimum interspecific distance (0.40%). Clear barcode gaps are present in many genera but absent in others. From the set of specimens that yielded COI fragment lengths of at least 650 bp, 75% of the a priori morphology-based identifications were unambiguously assigned to a single Barcode Index Number (BIN). However, after a taxonomic a posteriori review, the percentage of matched identifications rose to 85%. BIN splitting was observed for 17% of the species and BIN sharing for 9%. We found that genera that contain primarily lowland species show higher percentages of local barcode gaps and congruence between BINs and morphology than genera that contain exclusively high montane species. The divergence values to the nearest neighbors were significantly lower in high Andean species while the intra-specific divergence values were significantly lower in the lowland species. These results raise questions regarding the causes of observed low inter and high intraspecific genetic variation. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the montane species concerned are relatively young and hybridization is probable. The release of our data set represents an essential baseline for a reference library for biological assessment studies of butterflies in mega diverse countries using modern high-throughput technologies an highlights the necessity of taxonomic revisions for various genera combining both molecular and morphological data.

The majority of the parasite assessments of New World primates have been conducted through the identification of the eggs found in faeces, though many species of parasites have very similar eggs, leaving uncertainty in the diagnosis. Here, we present the results of a parasite survey of the three species of primates distributed in Mexico, combining non-invasive sampling with molecular techniques via DNA extraction of the eggs found in the faeces. Mitochondrial and ribosomal DNA were employed for species identification and Bayesian phylogenetic analysis. Nine parasite taxa were found in the three primate species: the nematodes Trypanoxyuris minutus, T. multilabiatus, T. pigrae, T. atelis, T. atelophora, Strongyloides sp., unidentified Ancylostomatid, unidentified Ascarid, and the trematode Controrchis biliophilus. We were able to extract and amplify DNA from the eggs of the five species of Trypanoxyuris reported for Mexican primates, two morphologically different trematode eggs, and Strongyloides sp. Phylogenetic analysis confirmed that the two types of trematode eggs belong to Controrchis biliophilus, a member of the family Dicrocoeliidae. For Strongyloides sp., phylogenetic analysis and genetic divergence showed an association between our samples and S. fuelleborni; however, no species could be established due to the lack of more DNA sequences from Strongyloides sp. occurring in Neotropical primates. The use of molecular and phylogenetic methods could help to overcome the limitations imposed by traditional non-invasive sampling because eggs are primarily obtained from the faeces; however, its utility relies on the extant genetic library and the contributions that expand such library. The information presented here could serve as a basis for future research on primate parasitology, allowing a more accurate parasite diagnosis and a more precise evaluation of their zoonotic potential., Graphical abstract Image 1, Highlights • Molecular diagnosis of parasites from Mexican primates through non-invasive sampling. • Phylogenetic analysis identified seven parasite species. • 28S a suitable marker for parasite DNA diagnosis. • Accurate parasite diagnosis crucial in wildlife conservation and management programs.

The Russian Far East (RFE) is an important hotspot of biodiversity whose insect fauna remains understudied, particularly its Microlepidoptera. Here we explore the diversity of leaf-mining micromoths of the family Gracillariidae, their distribution and host plant associations in RFE using a combination of field observations and sampling, DNA barcoding, morphological analysis and literature review. We collected 91 gracillariid specimens (45 larvae, 9 pupae and 37 adults) in 12 localities across RFE and identified 34 species using a combination of DNA barcoding and morphology. We provide a genetic library of 57 DNA barcodes belonging to 37 Barcode Index Numbers (BINs), including four BINs that could potentially represent species new to science. Leaf mines and leaf shelters are described and illustrated for 32 studied species, male or female genitalia as well as forewing patterns of adults are shown, especially for those species identified based on morphology. Three species, Micrurapteryx caraganella (Hering), Callisto insperatella (Nickerl), and Phyllonorycter junoniella (Zeller) are newly recorded from RFE. Five species previously known from some regions of RFE, were found for the first time in Amurskaya Oblast: Phyllonorycter populifoliella (Treitschke), Primorskii Krai: Ph. sorbicola Kumata and Sahkalin Island: Caloptilia heringi Kumata, Ph. ermani (Kumata) and Ph. ulmifoliella (Hübner). Eight gracillariid–plant associations are novel to science: Caloptilia gloriosa Kumata on Acer pseudosieboldianum, Cameraria niphonica Kumata on A. caudatum subsp. ukurundense, Parornix ermolaevi Kuznetzov on Corylus sieboldiana, Phyllonorycter ermani (Kumata) on Betula platyphylla, Ph. nipponicella (Issiki) on Quercus mongolica, Ph. orientalis (Kumata) and Ph. pseudojezoniella Noreika on Acer saccharum, Ph. sorbicola on Prunus maakii. For the first time we documented the “green island” phenotype on Phyllonorycter cavella (Zeller) mines on Betula platyphylla. Two pestiferous species have been recorded during our surveys: Micrurapteryx caraganella on ornamental Caragana arborescens in urban plantations in Amurskaya Oblast, and the lime leafminer Phyllonorycter issikii (Kumata), a species known to be native to RFE and invasive elsewhere in Russia and in European countries. A revised checklist of RFE gracillariids has been compiled. It accounts for 135 species among which 17 species (13%) are only known to occur in RFE. The gracillariid fauna of RFE is more similar to the Japanese fauna (49%), than to the fauna of the rest of Russia (i.e European part and Siberia) (32%).

Determining what role newly discovered genes play in the body is an important part of genetics. This task requires a lot of extra information about each gene, such as the specific cells where the gene is active, or what happens when the gene is deleted. To answer these questions, researchers need tools and methods to manipulate genes within a living organism. The fruit fly Drosophila is useful for such experiments because a toolbox of genetic techniques is already available. Gene editing in fruit flies allows small pieces of genetic information to be removed from or added to anywhere in the animal’s DNA. Another tool, known as GAL4-UAS, is a two-part system used to study gene activity. The GAL4 component is a protein that switches on genes. GAL4 alone does very little in Drosophila cells because it only recognizes a DNA sequence called UAS. However, if a GAL4-producing cell is also engineered to contain a UAS-controlled gene, GAL4 will switch the gene on. Lee et al. used gene editing to insert a small piece of DNA, containing the GAL4 sequence followed by a ‘stop’ signal, into many different fly genes. The insertion made the cells where each gene was normally active produce GAL4, but – thanks to the stop signal – rendered the rest of the original gene non-functional. This effectively deleted the proteins encoded by each gene, giving information about the biological processes they normally control. Lee et al. went on to use their insertion approach to make a Drosophila genetic library. This is a collection of around 1,000 different strains of fly, each carrying the GAL4/stop combination in a single gene. The library allows any gene in the collection to be studied in detail simply by combining the GAL4 with different UAS-controlled genetic tools. For example, introducing a UAS-controlled marker would pinpoint where in the body the original gene was active. Alternatively, adding UAS-controlled human versions of the gene would create humanized flies, which are a valuable tool to study potential disease-causing genes in humans. This Drosophila library is a resource that contributes new experimental tools to fly genetics. Insights gained from flies can also be applied to more complex animals like humans, especially since around 65% of genes are similar across humans and Drosophila. As such, Lee et al. hope that this resource will help other researchers shed new light on the role of many different genes in health and disease.

The genus Neoechinorhynchus represents a hyper-diverse group of acanthocephalans, parasites of fresh and brackish water fish and freshwater turtles, with approximately 116 species described worldwide. Forty-nine species have been recorded in the Americas, nine of them in Middle America. Even though species delimitation methods using DNA sequences have been rarely used for parasitic helminths, the genetic library for species of Neoechinorhynchus has grown in the past few years, enhancing the possibility of using these methods for inferring evolutionary relationships and for establishing more robust species boundaries. In this study, we used non-tree-based and tree-based methods through a coalescent approach to explore the species limits of specimens of Neoechinorhynchus collected in 57 localities across Middle America. We sequenced a large number of individuals to build a comprehensive dataset for three genes: the mitochondrial cytochrome c oxidase subunit I (352 individuals), the internal transcribed spacers (330 individuals), and the D2 + D3 domains of the large subunit (278 individuals). Several species delimitation methods were implemented, i.e., Automatic Barcode Gap Discovery (ABGD), General Mixed Yule-Coalescent Model (GMYC), Bayesian species delimitation (BPP) and species tree (∗BEAST). Additionally, we conducted a detailed morphological study of the diagnostic traits associated with the proboscis of 184 males and 169 females. Overall, our analyses allowed us to validate nine nominal species of Neoechinorhynchus and to identify 10 additional genetic lineages herein regarded as candidate species. This unexpected genetic diversity and the lack of reliable morphological traits show that the genus Neoechinorhynchus includes a group of cryptic species, at least in Middle America.

The grey hamster has been used in biomedical research for decades. However, effective molecular methods for evaluating the genetic structure of this species are lacking, which hinders its wider usage. In this study, we employed cross-amplification of microsatellite loci of species within the same genus by polymerase chain reaction. Loci screened included 107 from the Mongolian gerbil (MG) and 60 from the Chinese hamster (CH); of these, 15 polymorphic loci were identified for the grey hamster. Of the 167 loci screened, 95 (56.9%) with clear bands on agarose gel were initially identified. After sequencing, 74 (77.9%) of these matched the criteria for microsatellite characteristics, including 41 from MG and 33 from CH. Lastly, 15 (20.3%) loci with more than two alleles for each locus were identified through capillary electrophoresis scanning. To justify the applicability of the 15 grey hamster loci, genetic indexes of grey hamsters were evaluated using 46 generations of outbred stock, established 20 years ago, from Xinjiang, China. Mean effective allele numbers and expected heterozygosity of stock were as low as, respectively, 1.2 and 0.14; these were 2.8 and 4.0 times inferior, respectively, to wild grey hamsters. This finding suggests that the genetic structure of the stock-bred population is too weak to resist artificial and natural selection, mutation and genetic drifting. In conclusion, we have developed de novo microsatellite markers for genetic analysis of the grey hamster, providing data and methodology for the enrichment of a genetic library for this species.

The in vitro DNA binding profile of Aspergillus nidulans transcription factor AmyR was analyzed by a novel approach employing a genetic library of beads and flow cytometry analysis. An artificial library with 22 randomized nucleotides was constructed and subjected to a protein-DNA binding reaction with MalE-tagged AmyR. DNA fragments with potential AmyR-binding sites were labeled with fluorescence-conjugated antibody to be enriched by flow cytometry through 5 rounds of successive selection. Finally, a binding motif with a single CGG triplet was obtained from DNA fragments showing weak AmyR binding, while another motif with dual CGG triplets was discovered with stronger binding fragments. An informative motif, CGGNNNTTTNTCGG, was found to exist only in the promoter region of highly AmyR-dependent genes. These results suggest that this system is a powerful tool for the rapid and comprehensive analysis of the binding preferences of transcription factors.

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthalene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and growth. A genetic library of EPA505 was constructed using mini-Tn5 promoter reporter genes encoding for tetracycline resistance (tc(p-)) or luminescence (luxAB(p-)). Out of 2250 Tn5 mutants, ten were deficient in utilization of FLA, NAP, and/or PHE as sole carbon sources. Three classes of Tn5 mutants were defined: classI (nap(-)phe(-)fla(-)), classII (nap(-)phe(-)), and classIII (fla(-)). Four of five mutants in classI did not express dioxygenase function, whereas one classI mutant and all classII and classIII mutants retained dioxygenase activity. In Tn5 tc(p-) classI mutants 200 and 394 (dioxygenase negative) and classII mutant 132 (dioxygenase positive), promoter reporter was expressed when induced with FLA, NAP, PHE, other polycyclic aromatic hydrocarbons (PAHs), and several proposed PAH-derived catabolites. The Tn5 tc(p-) derived classIII mutant 104 was induced only with PAHs and not with PAH-derived catabolites. DNA sequence analysis of cloned regions of classI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DNA sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is designated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase designated pbhB. The Tn5 tc(p-) in classII mutant 132 occurred within sequences that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hydratase-aldolase gene (pbhC). Sequence analysis of the region proximal to this gene revealed a putative promoter that contained a binding site for a LysR transcriptional activator. In classIII mutant 104, the Tn5 tc(p-) resided within a region that shared 94% nucleotide homology to that of a pyruvate phosphate dikinase gene known to be involved in cellular uptake of glucose. The FLA-specific catabolic gene disrupted in mutant 104 was designated phbD. Functional and sequence analyses of promoter probe mutants allowed identification of four genes necessary for the utilization of PAHs that are controlled by at least two promoters that are affected by a wide range of aromatic compounds.

A tributyltin chloride (TBTCl)-resistant bacterium, Alteromonas sp. M-1, was isolated from coastal seawater. This bacterium grew in medium containing 125 microM TBTCl. TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system. A genetic library was constructed using plasmid vector pUC 19. Three positive clones were obtained, by which E. coli was transformed to TBTCl resistance. Of the three clones, the shortest fragment from HindIII-library was analyzed. This fragment was 1.8 kb long and contained one complete open reading frame. The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage and E. coli. TBTCl-tolerant marine bacteria other than Alteromonas sp. M-1 were obtained from natural seawater to which TBTCl was added. DNA-DNA hybridization was performed between the three cloned fragments from Alteromonas sp. M-1 and chromosomal DNA of the TBTCl-tolerant bacteria. Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance.

A genetic library of tributyltin chloride (TBTC1)-resistant marine bacterium, Alteromonas sp. M-1, was constructed using plasmid vector pUC 19. Three positive clones were obtained from E. coli JM 109 transformed with the plasmids by the method of replica plating to LB medium containing 1 mM TBTC1. These clones could grow in LB liquid medium containing 100 microM TBTC1. Plasmids harbouring genes of Alteromonas sp. M-1 were designated pTBT1, pTBT2 and pTBT3 which contain 1.8 kb Hind III-fragment, 4.8 kb Pst I-fragment and 7.8 kb Pst I-fragment, respectively. Nucleotide sequence of the shortest fragment, 1.8 kb Hind III-fragment was determined, revealing an open reading frame (ORF) was contained in the fragment. The ORF was 324 bp (108 amino acids). The 48.5% of the amino acids encoded was hydrophobic, suggesting that the product relating to TBTC1 resistance might be membrane related protein. Homology search in amino acids alignment indicated that the product has homology with transport proteins.

To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.

Made available in DSpace on 2013-08-12T19:03:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-01 Made available in DSpace on 2013-09-30T17:49:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:13:14Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:13:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-01 Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI. UNESP São Paulo State Univ, FCAV, Dept Biol Appl Agr & Anim Husb Dev, BR-14884900 Jaboticabal, SP, Brazil Univ São Paulo, BR-05508 São Paulo, Brazil Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Soybean, BR-86001970 Londrina, Parana, Brazil UNESP São Paulo State Univ, FCAV, Dept Biol Appl Agr & Anim Husb Dev, BR-14884900 Jaboticabal, SP, Brazil

Water contamination by nitrate is a wideworld extended phenomena. Biological autotrophic denitrification has a real potential to face this problem and presents less drawbacks than the most extended heterotrophic denitrification. Three bench-scale UASB reactors were operated under autotrophic (R1, H2S as electron donor), mixotrophic (R2, H2S plus p-cresol as electron donors) and heterotrophic (R3, p-cresol as electron donor) conditions using nitrate as terminal electron acceptor. 16S rDNA genetic libraries were built up to compare their microbial biodiversity. Six different bacteria phyla and three archaeal classes were observed. Proteobacteria was the main phyla in all reactors standing out the presence of denitrifiers. Microorganisms similar to Thiobacillus denitrificans and Acidovorax sp. performed the autotrophic denitification. These OTUs were displaced by chemoheterotrophic denitrifiers, especially by Limnobacter-like and Ottowia-like OTUs. Other phyla were Bacteroidetes, Chloroflexi, Firmicutes and Actinobacteria that - as well as Archaea members - were implicated in the degradation of organic matter, as substrate added as coming from endogenous sludge decay under autotrophic conditions. Archaea diversity remained low in all the reactors being Methanosaeta concilii the most abundant one.

The Tasmanian devil (Sarcophilus harrisii) is currently threatened by an emerging wildlife disease, devil facial tumour disease. The disease is decreasing devil numbers dramatically and may lead to the extinction of the species. At present, nothing is known about the immune genes or basic immunology of the devil. In this study, we report the construction of the first genetic library for the Tasmanian devil, a spleen cDNA library, and the isolation of full-length MHC Class I and Class II genes. We describe six unique Class II beta chain sequences from at least three loci, which belong to the marsupial Class II DA gene family. We have isolated 13 unique devil Class I sequences, representing at least seven Class I loci, two of which are most likely non-classical genes. The MHC Class I sequences from the devil have little heterogeneity, indicating recent divergence. The MHC genes described here are most likely involved in antigen presentation and are an important first step for studying MHC diversity and immune response in the devil.

Cellular differentiation is an essential characteristic of cells required for the formation of multicellular organisms. However, many unicellular organisms, such as yeast, also differentiate in order to survive in hostile environments. Recent studies suggest that the mechanisms and factors involved in differentiation are not very cell type specific, let alone organism specific (Yamamoto et al. 1999; Hiroi et al. 2002). Schizosaccharomyces pombe, a fission yeast, resembles higher eukaryotes in several ways including genetic structure, cell cycle control, and various signaling pathways. It also undergoes cellular differentiation during sexual development involving conjugation, meiosis, and sporulation. Sexual differentiation is controlled primarily through two signals: nutrient starvation and the presence of mating pheromone. Transcriptional factor Ste11 is required and activates several genes involved in conjugation and meiosis (Kelly et al. 1988; Watanabe et al. 1988; Hughes et al. 1990; Sugimoto et al. 1991; Willer et al. 1995). Included are the mei2, rep1, and ste genes (including ste11), and other mating-type genes (Kelly et al. 1988; Watanabe et al. 1988; Hughes et al. 1990; Sugimoto et al. 1991; Miyamoto et al. 1994; Sugiyama et al. 1994; Willer et al. 1995). Nitrogen starvation as well as carbon starvation are two major nutrient-depletion signals triggering sexual development in budding yeast. Pat1/Ran1 kinase plays a unique role in the regulation of sexual development, primarily blocking the onset of meiosis until conjugation occurs by inactivating a key triggering factor, Mei2 (Beach et al. 1985; Iino and Yamamoto 1985a,b; Watanabe et al. 1997). Inactivation of Pat1 in haploid cells induces lethal meiosis that can be suppressed through inactivation of the mei2 gene. As Ste11 is required for the expression of mei2 (Sugimoto et al. 1991), factors inhibiting ste11 expression or function would rescue the pat1 inactivated cells. Rcd1 (required for cell differentiation) was identified as a differentiation-controlling factor crucial for nitrogen starvation-induced sexual development in S. pombe by screening a genetic library for suppressors of pat1 lethality (Okazaki et al. 1998). Surprisingly, cells with nonfunctional rcd1 were also shown to be sterile, despite initial assumptions that these strains would have been highly proficient for sexual development. Rcd-1 is essential for nitrogen starvation-invoked induction of ste11 (Okazaki et al. 1998). Paradoxically, overexpression of Rcd-1 rescued pat1 inactivated cells, supposedly through the inhibiting activity of a dominant active mutant of Mei2 (Y. Watanabe and M. Yamamoto, unpubl.). Homologs of the rcd1 gene from S. pombe are quite conserved throughout eukaryotic genomes (Fig. 1A; Okazaki et al. 1998). One homolog, CAF40, was identified as a component of the CCR4–NOT transcription complex in Saccharomyces cerevisiae (Chen et al. 2001). This complex is evolutionarily conserved in eukaryotes and is involved in initiation (Denis and Malvar 1990; Sakai et al. 1992; Collart and Struhl 1993), elongation (Denis et al. 2001), degradation (Tucker et al. 2001), and deadenylation (Tucker et al. 2001) of mRNA, as well as regulation of TFIID activity (Chen et al. 2002). In the CCR4–NOT complex, only NOT1 is essential (Collart and Struhl 1993; Maillet et al. 2000), although it has no identifiable functional domains, apparently acting as a scaffold. CAF40 associates with NOT1 through the latter's N-terminal 1100 residues, although its presence was not required for CCR4–NOT complex formation (Chen et al. 2001). As CAF40 has also been shown to interact with human NOT1, this suggests that hRcd-1 would be a part of the human CCR4–NOT complex (Chen et al. 2001). Figure 1. Rcd-1 sequences. (A) Alignment of the sequences of Rcd-1 from various organisms as indicated (GenBank accession nos.: Homo sapiens, {"type":"entrez-protein","attrs":{"text":"NP_005435","term_id":"4885579"}}NP_005435; S. pombe, {"type":"entrez-protein","attrs":{"text":"NP_594984","term_id":"19115896"}} ... Mammalian Rcd-1 was investigated for its functional similarity to the yeast homologs using F9 mouse teratocarcinoma cell differentiation, which is regulated by retinoic acid (RA) (Hiroi et al. 2002). F9 cells differentiate into primitive endoderm upon treatment with RA (Hogan et al. 1981; Edwards and McBurney 1983). Inhibition of mouse Rcd-1 (mRcd-1) production through the use of antisense oligonucleotides effectively blocked RA-induced differentiation (Hiroi et al. 2002). As well, overexpression of mRcd-1-induced spontaneous differentiation, apparently sensitizing the F9 cells to rather low levels of RA (Hiroi et al. 2002). Rcd-1 is highly expressed in various mammalian tissues. Antibodies against Rcd-1 were used to probe various tissues from adult and embryonic rat, showing the highest levels of expression in the lung, spleen, testes, and thymus of adult rats, while expressed in virtually all tissues of the early rat embryo (Hiroi et al. 2002). Northern blots using human Rcd-1 as a probe showed significant expression in human testes, ovaries, and thymus (Okazaki et al. 1998). mrcd1 was also isolated as an erythropoietin-responsive gene potentially involved in hematopoetic cell development (Gregory et al. 2000). A recent finding reports that mRcd-1 interacts with the c-Myb protein (Haas et al. 2004), which, in turn, is required for the generation of monocytes from a myeloid progenitor (for review, see Friedman 2002). These results are consistent with Rcd-1’s proposed involvement in differentiation control. Despite the increasing knowledge about the role Rcd-1 plays in cellular differentiation, little is known about the mechanisms by which Rcd-1 may exert its influence. Structural information could provide more insight regarding its actual functioning within cells. We now report the crystal structure of the highly conserved region of human Rcd-1 (a region spanning 90% of the full-length protein). The armadillo-repeat-like structure in conjunction with interesting surface features and charge clustering suggest possible means by which Rcd-1 can mediate its effects on cellular differentiation-related gene control.

Three cloned Klebsiella pneumoniae nitrogenase gene probes were used to perform genomic Southern hybridization analysis and to screen an Anabaena variabilis-Charon 30 genetic library for Anabaena nitrogenase genes. One of ten phage clones that were isolated using a Klebsiella nifHD gene probe was studied in detail. This clone (ABN 10) contains a 9 kb Eco R1 fragment which was observed in genomic Southerns. ABN 10 does not hybridize with a Klebsiella nifK gene probe. A 2.8 kb Hind III fragment that lies within the 9 kb Eco R1 fragment contains the linked Anabaena nifH and nifD genes; the nifK gene lies at least 4 kb away. Partial sequencing of the nifH and nifD genes confirmed their identity and showed strong conservation of amino acid sequence in comparison to Anabaena 7120, Klebsiella pneumoniae, Azotobacter vinlandii, and Clostridium pasteurianum. When the cloned Anabaena nif genes were put into Klebsiella cells on plasmids, they were not expressed, suggesting that the Anabaena promoters are not recognized by Klebsiella transcription factors.

The gtfA gene of Streptococcus mutons GS-5, which encodes a 55-kDa glucosyltransferase has been isolated from a genetic library in an Escherichia coli-Bacillus subtilis shuttle vector, pMK3. The construction containing the gene enables E. coli JM83 and a sucrase-deficient mutant of B. subtilis to grow on sucrose as the sole carbohydrate source. The gene is expressed under its own control in both organisms. The level of biochemical activity detectable in B. subtilis carrying the clone is approx. 50% ofthat found in E. coli harboring the same construction. In Bacillus, the gene is expressed through exponential and stationary phases of growth with a decrease in activity as the culture enters stationary phase, corresponding to increases in intracellular protease levels. The enzyme produced in E. coli or B. subtilis harboring the cloned gene is identical to the enzyme produced by S. mutans GS-5 as determined by migration in native polyacrylamide gels.

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for α(1→6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.