Review of genes in development: re-reading the molecular paradigm

Genes in Development is a collection of 13 stimulating essays on "post genomic" approaches to the concept of the gene. At the risk of caricaturing some complex balances, the contributors tend to be skeptical about genetic determinism, the central dogma of molecular biology, reductionism, genes as programs and the concept of the gene as a DNA sequence. They tend to like emergent properties, complexity theory, the parity thesis for developmental resources, developmental systems theory, and membranes. But within this broad weltanschauung the essays in Genes in Development vary widely in their interests and emphases––from the history of twentieth century genetics to the social and ethical issues raised by contemporary genetics––which makes for an attractive and valuable collection.     

Transfer of megaplasmid pKB1 from the rubber-degrading bacterium <Emphasis Type="Italic">Gordonia westfalica</Emphasis> strain Kb1 to related bacteria and its modification

Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 × 10⁻⁸ events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 × 10³, 1.9 × 10³, and 8.3 × 10² transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.     

Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis>

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T₁ progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.     

Over-expression of the apple <Emphasis Type="Italic">spermidine synthase</Emphasis> gene in pear confers multiple abiotic stress tolerance by altering polyamine titers

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. ‘Ballad’) plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 μM CuSO₄) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments. Xiao-Peng Wen and Xiao-Ming Pang contributed equally to this work.     

Cranial shape and correlated characters in crocodilian evolution

Crocodilians show a high degree of cranial variation and convergence throughout their 80 million-year fossil record that complicates their phylogenetic reconstruction. Conflicting phylogenetic results from different data partitions and character homoplasies typify crocodilian phylogeny, and differences between molecular and morphological phylogenetic hypotheses are believed to be associated with the slender-snout skull shape of Gavialis gangeticus and Tomistoma schlegelii. Slender-snout skulls are one of five identified eusuchian cranial ecomorph shape categories (ESCs) thought to reflect functional or ecological specialization. This paper tested the effect of transitions among general, blunt and slender ESCs on cranial character-state distributions in phylogeny using the concentrated changes test. In addition, 'tree-free' character compatibility analysis of character independence was conducted on the morphological character matrix to determine if character correlations are observed independent of specific tree topologies. Results suggest cranial ESCs do affect cranial character-state gains in phylogeny. Concentrated changes identify a broad suite of character-state changes that significantly correlate with transitions to slender, general and blunt ESCs on morphological, molecular and combined-data tree topologies, but numbers of correlated characters for each category differ according to topology. Character compatibility analysis results do not mirror the concentrated changes test results and reflect hierarchically distributed support throughout the data. As cranial ESCs affect character-state transitions, it is possible that nonphylogenetic variables could affect inferences of crocodilian phylogeny by affecting cranial morphology.     

Male sand crickets trade-off flight capability for reproductive potential

In this paper, we test the hypothesis that male sand crickets, Gryllus firmus, experience a trade-off between flight capability and reproductive potential expressed as reduced testis weight in flight-capable morphs. We used a half-sib design with 130 sires, three dams per sire and an average of 5.66 males per dam family, for a total of 2206 F1 offspring. Traits measured were head width, somatic dry weight, testis weight, wing morph (micropterous/macropterous), weight of the dorso-longitudinal flight muscles (DLM) and the functional status of these muscles. Heritabilities of all traits were significant and ranged from 0.14 to 0.43. All traits were positively correlated with body size, but removal of this covariance revealed a highly significant trade-off, both phenotypically and genetically, between testes size and flight capability as measured by wing morph, DLM size or DLM status. The possible implications of this for morph-specific reproductive tactics are discussed.     

Haplotype analysis revealed a genetic influence of osteopontin on large artery atherosclerosis

Osteopontin (OPN) is a cytokine that involves in vascular remodeling processes in cerebrovascular diseases. The association of its gene with ischemic stroke was investigated in a Korean population. Representative sequence variants covering the entire OPN gene were genotyped in 455 controls and 271 patients with ischemic stroke including large artery atherosclerosis (LAA), small vessel occlusion, and cardioembolism. Analysis with the individual tagging variants and their haplotypes revealed an evidence of association only with LAA. Significances were shown with the haplotypes, especially with the TCA at the loci C2140T, C5891T, and A7385G conferring a risk of 2.09 for LAA (P < 0.05). The CG at the loci C1013T and A7385G was the most protective haplotype (OR = 0.66, P < 0.05). Our findings suggested that several haplotypes of OPN gene contributed to determining risk factors as well as protective factors of LAA.     

Identification of mitochondrial disease genes through integrative analysis of multiple datasets

Determining the genetic factors in a disease is crucial to elucidating its molecular basis. This task is challenging due to a lack of information on gene function. The integration of large-scale functional genomics data has proven to be an effective strategy to prioritize candidate disease genes. Mitochondrial disorders are a prevalent and heterogeneous class of diseases that are particularly amenable to this approach. Here we explain the application of integrative approaches to the identification of mitochondrial disease genes. We first examine various datasets that can be used to evaluate the involvement of each gene in mitochondrial function. The data integration methodology is then described, accompanied by examples of common implementations. Finally, we discuss how gene networks are constructed using integrative techniques and applied to candidate gene prioritization. Relevant public data resources are indicated. This report highlights the success and potential of data integration as well as its applicability to the search for mitochondrial disease genes.     

Population structure and genetic diversity in two species of Hawaiian picture-winged Drosophila

Over the last several decades many picture-winged Drosophila have become less common in both geographical distribution and local population size (pers. obs., Foote pers. comm., Montgomerey pers. comm.). Here we report on a study of two Hawaiian Drosophila species, D. engyochracea, and D. hawaiiensis, to determine the impact that changes in population sizes over the past thirty years have had on the genetic diversity of these species. D. engyochracea is known from only two locations on the Island of Hawai'i (Kipuka Ki and Kipuka Pua'ulu), while D. hawaiiensis is currently more wide spread across Hawai'i Island. We collected 65 D. hawaiiensis and 66 D. engyochracea from two forest patches (kipuka) isolated by a 400 year old volcanic ash deposit. DNA sequence data for 515 bases of the mitochondrial gene COII was analyzed for both species to estimate relative total genetic diversity as well as inter-kipuka gene flow. The more wide spread species, D. hawaiiensis, has more genetic diversity (23 vs. 11 unique haplotypes) than the rarer species, D. engyochracea. The distribution of haplotypes in the kipuka is consistent with more gene flow in D. engyochracea than in D. hawaiiensis. Phylogenetic analysis indicates a small number of individuals morphologically identified as one species but have DNA sequence diagnostic for the other species. These results are consistent with these individuals being descendant from hybrids between species.     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.