A web-based platform for rice microarray annotation and data analysis

Rice (Oryza sativa) feeds over half of the global population. A web-based integrated platform for rice microarray annotation and data analysis in various biological contexts is presented, which provides a convenient query for comprehensive annotation compared with similar databases. Coupled with existing rice microarray data, it provides online analysis methods from the perspective of bioinformatics. This comprehensive bioinformatics analysis platform is composed of five modules, including data retrieval, microarray annotation, sequence analysis, results visualization and data analysis. The BioChip module facilitates the retrieval of microarray data information via identifiers of “Probe Set ID”, “Locus ID” and “Analysis Name”. The BioAnno module is used to annotate the gene or probe set based on the gene function, the domain information, the KEGG biochemical and regulatory pathways and the potential microRNA which regulates the genes. The BioSeq module lists all of the related sequence information by a microarray probe set. The BioView module provides various visual results for the microarray data. The BioAnaly module is used to analyze the rice microarray’s data set.     

The random versus fragile breakage models of chromosome evolution: a matter of resolution

Conserved synteny––the sharing of at least one orthologous gene by a pair of chromosomes from two species––can, in the strictest sense, be viewed as sequence conservation between chromosomes of two related species, irrespective of whether coding or non-coding sequence is examined. The recent sequencing of multiple vertebrate genomes indicates that certain chromosomal segments of considerable size are conserved in gene order as well as underlying non-coding sequence across all vertebrates. Some of these segments lost genes or non-coding sequence and/or underwent breakage only in teleost genomes, presumably because evolutionary pressure acting on these regions to remain intact were relaxed after an additional round of whole genome duplication. Random reporter insertions into zebrafish chromosomes combined with computational genome-wide analysis indicate that large chromosomal areas of multiple genes contain long-range regulatory elements, which act on their target genes from several gene distances away. In addition, computational breakpoint analyses suggest that recurrent evolutionary breaks are found in “fragile regions” or “hotspots”, outside of the conserved blocks of synteny. These findings cannot be accommodated by the random breakage model and suggest that this view of genome and chromosomal evolution requires substantial reassessment.     

A new male-specific gene “<Emphasis Type="Italic">OTOKOGI</Emphasis>” in <Emphasis Type="Italic">Pleodorina starrii</Emphasis> (Volvocaceae,Chlorophyta) unveils the origin of male and female

Eukaryotic sex was initially isogametic and it is assumed that anisogamy/oogamy evolved independently in many lineages including animals, land plants and volvocine green algae. The exact evolutionary mechanisms that were responsible for the evolution of oogamy from isogamy were poorly understood until Nozaki et al. (2006) introduced the use of molecular-genetic data in elucidating the evolutionary origin of oogamy from isogamy in the colonial volvocacean Pleodorina starrii. In the close relative Chlamydomonas reinhardtii, sexual reproduction is isogametic with mating-types plus and minus. Mating type minus represents a “dominant sex” because the MID (“minus-dominance”) gene of C. reinhardtii is both necessary and sufficient to cause the cells to differentiate as isogametes of the minus mating type. No sex-specific genes had been identified in the volvocine green algae until Nozaki et al. (2006a) successfully cloned the MID gene of P. starrii. This “OTOKOGI” (PlestMID) gene is present only in the male genome, and encodes a protein localized abundantly in the nuclei of mature sperm. Thus, P. starrii maleness evolved from the dominant sex (mating type minus) of its isogamous ancestor. This breakthrough provides an opportunity to address various extremely interesting questions regarding the evolution of oogamy and the male-female dichotomy. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Multimodality molecular imaging of disease progression in living subjects

The enormous advances in our understanding of the progression of diseases at the molecular level have been supplemented by the new field of ‘molecular imaging’, which provides for in vivo visualization of molecular events at the cellular level in living organisms. Molecular imaging is a noninvasive assessment of gene and protein function, protein–protein interaction and/or signal transduction pathways in animal models of human disease and in patients to provide insights into molecular pathogenesis. Five major imaging techniques are currently available to assess the structural and functional alterations in vivo in small animals. These are (i) optical bioluminescence and fluorescence imaging techniques, (ii) radionuclide-based positron emission tomography (PET) and single photon emitted computed tomography (SPECT), (iii) X-ray-based computed tomography (CT), (iv) magnetic resonance imaging (MRI) and (v) ultrasound imaging (US). Functional molecular imaging requires an imaging probe that is specific for a given molecular event. In preclinical imaging, involving small animal models, the imaging probe could be an element of a direct (‘direct imaging’) or an indirect (‘indirect imaging’) event. Reporter genes are essential for indirect imaging and provide a general integrated platform for many different applications. Applications of multimodality imaging using combinations of bioluminescent, fluorescent and PET reporter genes in unified fusion vectors developed by us for recording events from single live cells to whole animals with high sensitivity and accurate quantification are discussed. Such approaches have immense potential to track progression of metastasis, immune cell trafficking, stem cell therapy, transgenic animals and even molecular interactions in living subjects.     

Molecular-genetics analysis of natural and stimulated ovulation impairment

The influence of FMR1, INHα1, NAT2, GSTT1 and GSTM1 genes mutations on ovarian function and their association with POF and “poor response” to exogenous GT after ovulation stimulation were investigated. The carriers of Ala257Thr transition predominated in the studied “poor responders” group. In 1.6% POF patients and 2.5% persons from “poor responders” group, but nobody from control group this transition combined with intermediate alleles of FMR1 gene was observed. The frequency of deletion in GSTM1 gene in “poor responders” group was significantly higher (p = 0.01) than in normal ovulatory control group. The frequency of Ser680Ser-Ala307Ala polymorphic genotype (22.2%) in “poor responders” group was significantly higher (p = 0.028) than in normal-ovulatory control group (7.7%). The daily dosage of GT in intermediate alleles of FMR1 gene carriers as well in patients with “slow acetylation” NAT2 genotype was significantly higher in comparison to patients without intermediate alleles and patients with “quick acetylation” NAT2 genotype. Quantity of oocytes after stimulation ovulation in women with INHα1 gene Ala257Thr transition were significantly decreased in comparison to patients without such mutation. Further investigations of these genes can play a major role in POF studying and modulation of ovarian response to exogenous GT. Published in Ukrainian in Tsitologiya i Genetika, 2008, Vol. 42, No. 2, pp. 63–69. The text was translated by the authors.     

Multimodality in vivo molecular-genetic imaging

Multimodality imaging is increasingly being used in molecular-genetic studies in small animals. The coupling of nuclear and optical reporter genes represents the beginning of a far wider application of this technology. Optical imaging and optical reporter systems are cost-effective and time-efficient, they require less resources and space than PET or MRI, and they are particularly well suited for small animal imaging and for in vitro assays to validate different reporter systems. However, optical imaging techniques are limited by depth of light penetration and scatter and do not yet provide optimal quantitative or tomographic information. These issues are not limiting for PET- or MRI-based reporter systems, and PET- and MRI-based animal studies are more easily generalized to human applications. Many of the shortcomings of each modality alone can be overcome by the use of dual- or triple-modality reporter constructs that incorporate the opportunity for PET, fluorescence and bioluminescence imaging. We optimistically expect that some form of tomographic, small animal optical imaging capability will be developed soon, and that this will provide the opportunity for the colocalization of optical signals to anatomical structures provided by tomographic CT and MR imaging.     

Abstract mathematical molecular biology: II some relational consequences of the “one gene-one enzyme” hypothesis

Following the program outlined in a previous paper (Bull. Math. Biophysics,23, 237–260, 1961), a further abstract study is made of some simple relational systems which possess some properties of living organisms. It is shown that the “one gene-one enzyme” hypothesis leads to the conclusion that either all genes are built of the same chemical building blocks, or that at least all genes have a number of building blocks in common. A consistent relational application of the “one gene-one enzyme” hypothesis leads moreover to the conclusion that replication is not an inherent property of a gene. Rather there must be a set of enzymes which “copy” the genes. The number of enzymes in this set must be less than the number of genes and therefore the activity of those “copying” enzymes cannot be absolutely specific.     

Molecular-genetic imaging: a nuclear medicine-based perspective

Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology. Noninvasive in vivo molecular-genetic imaging developed more recently and is based on nuclear (positron emission tomography [PET], gamma camera, autoradiography) imaging as well as magnetic resonance (MR) and in vivo optical imaging. Molecular-genetic imaging has its roots in both molecular biology and cell biology, as well as in new imaging technologies. The focus of this presentation will be nuclear-based molecular-genetic imaging, but it will comment on the value and utility of combining different imaging modalities. Nuclear-based molecular imaging can be viewed in terms of three different imaging strategies: (1) "indirect" reporter gene imaging; (2) "direct" imaging of endogenous molecules; or (3) "surrogate" or "bio-marker" imaging. Examples of each imaging strategy will be presented and discussed. The rapid growth of in vivo molecular imaging is due to the established base of in vivo imaging technologies, the established programs in molecular and cell biology, and the convergence of these disciplines. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases and disease processes.     

Development of Novel Types of Plastid Transformation Vectors and Evaluation of Factors Controlling Expression

Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named “operon-extension” vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5′-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5′-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named “split” plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.     

Host acceptance behavior of the Japanese aquatic wasp,Agriotypus gracilis (Hymenoptera: Ichneumonidae) toward the caddisfly host,Goera japonica (Trichoptera: Limnephilidae)

The host acceptance behavior of the Japanese aquatic wasp,Agriotypus gracilis Waterston, an ectoparasitoid of the sand case building caddisfly,Goera japonica Banks was investigated in the laboratory. Female wasps were observed to enter the water by walking down a stone protruding from the water surface. Antennae were held backward and not utilized in searching for hosts under the water. Female wasps examined hosts from the outside of their cases by 2 consecutive steps, “turning” and “probing”. Turning behavior, in which female wasps move between the anterior and posterior ends of host cases, may be related to the measurement of case size. Host stages are considered to be discriminated by probing, in which females probe host cases with their sheathing ovipositors. Female wasps most frequently accepted and oviposited on pupal and prepupal hosts.     

Md-ACS1 and Md-ACO1 genotyping of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis> Borkh.) breeding parents and suitability for marker-assisted selection

Fruit ethylene production genotypes for Md-ACS1 and Md-ACO1 were determined for 60 apple cultivars and 35 advanced breeding selections. Two alleles for each gene are commonly found in cultivated apple. Earlier studies showed that genotypes homozygous for the ACS1-2 allele produce less ethylene and have firmer fruit than ACS1-1/2 and ACS1-1/1 genotypes. ACO1 plays a minor role compared to ACS1, with homozygous ACO1-1 having lower ethylene production. In this study, ACS1-2 and ACO1-1 homozygotes had firmer fruit at harvest and after 60 days of 0–1°C cold storage compared to other genotypes. These genotypes, ACS1-2/2 and ACO1-1/1, were observed for the following 8 of 95 cultivars/selections: “Delblush”, “Fuji”, “Pacific Beauty”, “Sabina” and four breeding selections. Cultivars/selections that were homozygous ACS1-2 but not ACO1-1 were: “Ambrosia”, “Aurora Golden Gala”, “CrimsonCrisp”, “Gala”, “GoldRush”, “Huaguan”, “Pacific Rose, “Pacific Queen”, “Pinova”, “Sansa”, “Sonja”, “Sundance”, “Zestar”, and 17 breeding selections. Cultivars with the heterozygous ACS1-1/2 genotype were “Arlet”, “Braeburn”, “Cameo”, “Delicious”, “Delorgue”, “Empire”, “Enterprise”, “Ginger Gold”, “Golden Delicious”, “Granny Smith”, “Honeycrisp”, “Orin”, “Pink Lady”, “Silken”, “Suncrisp”, “Sundowner”, “Sunrise” and 11 breeding selections. No cultivars were detected homozygous for both ACS1-1 and ACO1-1, or for both ACS1-2 and ACO1-2. This study is the first large-scale allelic genotyping of both ethylene synthesis genes for a comprehensive set of apple breeding parents used in an ongoing breeding project. The data reported here are important for informative selection of parent combinations and marker-assisted selection of progeny for breeding low ethylene-producing apple cultivars for better storability and improved consumer acceptance.     

Assessment of Gene Flow from Genetically Modified Anthracnose-Resistant Chili Pepper (Capsicum annuum L.) to a Conventional Crop

We conducted a 2-year field assessment of the gene flow from genetically modified (GM) chili pepper (Capsicum annuum L.), containing the PepEST (pepper esterase) gene, to a non-GM control line “WT512” and two commercial hybrid cultivars, “Manidda” and “Cheongpung Myeongwol (CM).” After seeds were collected from the pollen-recipient non-GM plants, hybrids between them and the GM peppers were screened by a hygromycin assay. PCR with the targeting hpt gene was performed to confirm the presence of transgenes in hygromycin-resistant seedlings. Out of 7,071 “WT512” seeds and 6,854 “Manidda” seeds collected in 2006, eight and 12 hybrids, respectively, were detected. In 2007, 33 hybrids from 3,456 “WT512” seeds and 50 hybrids from 3,457 “CM” seeds were found. The highest frequency of gene flow, 6.19%, was observed in that 2007 trial. These results suggest that a limited isolation distance would be sufficient to prevent gene flow from GM to conventionally bred chili peppers.     

Cancer imaging: Gene transcription-based imaging and therapeutic systems

Molecular-genetic imaging of cancer is in its infancy. Over the past decade gene reporter systems have been optimized in preclinical models and some have found their way into the clinic. The search is on to find the best combination of gene delivery vehicle and reporter imaging system that can be translated safely and quickly. The goal is to have a combination that can detect a wide variety of cancers with high sensitivity and specificity in a way that rivals the current clinical standard, positron emission tomography with [(18)F]fluorodeoxyglucose. To do so will require systemic delivery of reporter genes for the detection of micrometastases, and a nontoxic vector, whether viral or based on nanotechnology, to gain widespread acceptance by the oncology community. Merger of molecular-genetic imaging with gene therapy, a strategy that has been employed in the past, will likely be necessary for such imaging to reach widespread clinical use.     

Multisequence comparisons in protein coding genes

A very powerful method for detecting functional constraints operative in biological macromolecules is presented. This method entails performing a base permanence analysis of protein coding genes at each codon position simultaneously in different species. It calculates the degree of permanence of subregions of the gene by dividing it into segments,c codons long, counting how many sites remain unchanged in each segment among all species compared. By comparing the base permanence among several sequences with the expectations based on a stochastic evolutionary process, gene regions showing different degrees of conservation can be selected. This means that wherever the permanence deviates significantly from the expected value generated by the simulation, the corresponding regions are considered “constrained” or “hypervariable”. The constrained regions are of two types: α and β. The α regions result from constraints at the amino acid level, whereas the β regions are those probably involved in “control” processing. The method has been applied to mitochondrial genes coding for subunit 6 of the ATPase and subunit 1 of the cytochrome oxidase in four mammalian species: human, rat, mouse, and cow. In the two mitochondrial genes a few regions that are highly conserved in all codon positions have been identified. Among these regions a sequence, common to both genes, that is complementary to a strongly conserved region of 12S rRNA has been found. This method can also be of great help in studying molecular evolution mechanisms.     

Grapevines engineered to express cisgenic <Emphasis Type="Italic">Vitis vinifera</Emphasis> thaumatin-like protein exhibit fungal disease resistance

Cisgenic engineering involves isolation and modification of genetic elements from the host genome, which are reinserted to develop plant varieties with improved characteristics. As a first step toward production of fungal-disease resistant cisgenic grapevines, the Vitis vinifera thaumatin-like protein (vvtl-1) gene was isolated from “Chardonnay” and reengineered for constitutive expression. Embryogenic cultures of “Thompson Seedless” were initiated from leaves and transformed with Agrobacterium to regenerate cisgenic VVTL-1 plants. Cisgene presence and copy number were confirmed by PCR and quantitative real-time PCR. Protein expression was measured using ELISA. Among the plant lines tested, two exhibited a 7–10 day delay in powdery mildew disease development during greenhouse screening and decreased severity of black rot disease in field tests. Berries exhibited a 42.5% reduction in sour-bunch rot disease incidence compared to non-transformed controls after 3 wk of storage at room temperature. Although plants recovered in this study contain viral promoters and reporter/marker genes, this is the first report of a cisgenic approach to obtain broad-spectrum fungal-disease resistance in genetically engineered grapevine.     

Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells

Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.     

Detection of gene flow from GM to non-GM watermelon in a field trial

Gene flow from genetically modified (GM) crops to conventional non-GM crops is a serious concern for protection of conventional and organic farming. Gene flow from GM watermelon developed for rootstock use, containing cucumber green mottle mosaic virus (CGMMV)-coat protein (CP) gene, to a non-GM isogenic control variety “Clhalteok” and grafted watermelon “Keumcheon” was investigated in a small scale field trial as a pilot study. Hybrids between GM and non-GM watermelons were screened from 1304 “Chalteok” seeds and 856 “Keumcheon” seeds using the duplex PCR method targeting theCGMMV- CP gene as a marker. Hybrids were found in all pollen recipient plots. The gene flow frequencies were greater for “Chaiteok” than for “KeumcheonD; with 75% outcrossing in the “Chaiteok” plot at the closest distance (0.8 m) to the GM plot. A much larger scale field trial is necessary to identify the isolation distance between GM and non-GM watermelon, as the behaviors of insect pollinators needs to be clarified in Korea.     

Terahertz vibrational properties of water nanoclusters relevant to biology

Water nanoclusters are shown from first-principles calculations to possess unique terahertz-frequency vibrational modes in the 1–6 THz range, corresponding to O–O–O “bending,” “squashing,” and “twisting” “surface” distortions of the clusters. The cluster molecular-orbital LUMOs are huge Rydberg-like “S,” “P,” “D,” and “F” orbitals that accept an extra electron via optical excitation, ionization, or electron donation from interacting biomolecules. Dynamic Jahn–Teller coupling of these “hydrated-electron” orbitals to the THz vibrations promotes such water clusters as vibronically active “structured water” essential to biomolecular function such as protein folding. In biological microtubules, confined water-cluster THz vibrations may induce their “quantum coherence” communicated by Jahn–Teller phonons via coupling of the THz electromagnetic field to the water clusters’ large electric dipole moments.