Identification of the cystic fibrosis gene: chromosome walking and jumping

An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.     

Improving potato storage and processing characteristics through all-native DNA transformation

The dominant potato (Solanum tuberosum) variety for French fry production in the United States is the 131-year-old Russet Burbank. Market penetration of the higher yielding and more uniform Ranger Russet variety is limited to about one-fifth of that of the Russet Burbank because of two storage deficits: black spot bruise sensitivity and high levels of cold-induced sweetening. Here, these trait weaknesses are turned into strengths by simultaneously lowering the expression of Ranger Russet's tuber-expressed polyphenol oxidase (Ppo), starch-associated R1, and phosphorylase-L (PhL) genes. This genetic modification was accomplished without inserting any foreign DNA into the plant genome. French fries from the intragenic potatoes also contained reduced amounts of the antinutritional compound acrylamide while, unexpectedly, displaying enhanced sensory characteristics.     

Intragenic crop improvement: combining the benefits of traditional breeding and genetic engineering

New crop varieties are developed by applying traditional breeding methods that rely on random genome modifications. These varieties combine multiple traits that support farm efficiency and acceptable yields but also contain genes associated with the production of toxins, allergens, and/or antinutritional compounds that were not considered during the selection process. Furthermore, existing cultivars frequently lack the functional genes required for specific sensory traits and the formation of health-promoting antioxidants. One new method efficiently addresses some of these issues by either silencing undesirable genes or enhancing the expression of genes that are linked to dormant beneficial traits. Rather than incorporating foreign DNA into the plant's genome, these methods transform crops with plant-derived transfer (P-) DNAs that consist of only native genetic elements. The genetic modification can be characterized molecularly so that any inadvertent transfer of undesirable DNA, as may be the case with traditional methods, is excluded. A recently developed intragenic potato plant is silenced for the polyphenol oxidase, dikinase R1, and phosphorylase-L genes in a tuber-specific manner. French fries derived from these tubers lack discolorations, display an enhanced potato flavor, and produce greatly reduced amounts of the suspected carcinogen acrylamide. It is argued that intragenic modification is unlikely to trigger phenotypic, biochemical, or physiological variation that is new to the species. Similarly, the targeted traits are similar to those that breeders select for and often have a history of domestication and reduced fitness. For these reasons, an updated regulatory system is proposed whereby intragenic crops are considered as low risk and should be cleared for commercial release in a timely and cost-effective manner. By using modern techniques to modify the same genetic material that is used by breeders, intragenic approaches may be perceived as an acceptable extension of traditional methods in crop improvement.     

Identification of the cystic fibrosis gene: genetic analysis

Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.     

Assessment of sex and evaluation of ovarian maturation of fish using ultrasonography

Ultrasonography is a non‐invasive method that can be used to assess sex and maturity of fish. This study was conducted to examine the feasibility of using ultrasound images to sex juvenile and mature halibut Hippoglossus hippoglossus (Linnaeus), mature winter Pleuronectes americanus (Walbaum) and yellowtail flounder Pleuronectes ferruginea (Storer) and mature haddock Melanogrammus aeglefinus (Linnaeus). In females, both immature and mature ovaries were relatively easy to distinguish, but in males only mature testes were consistently distinguishable. Ovarian maturation of haddock and ovulatory cycles of halibut may also be assessed using ultrasound.     

Exophiala angulospora Causes Systemic Mycosis in Atlantic Halibut: a Case Report

Filamentous black yeasts from the genus Exophiala are ubiquitous, opportunistic pathogens causing both superficial and systemic mycoses in warm- and cold-blooded animals. Infections by black yeasts have been reported relatively frequently in a variety of captive and farmed freshwater and marine fishes. In November 2012, moribund and recently dead, farm-raised Atlantic Halibut Hippoglossus hippoglossus were necropsied to determine the cause of death. Histopathology revealed that three of seven fish were affected by a combination of an ascending trans-ductual granulomatous mycotic nephritis, necrotizing histiocytic encephalitis, and in one fish the addition of a fibrogranulomatous submucosal branchitis. Microbial cultures of kidney using selective mycotic media revealed pure growth of a black-pigmenting septated agent. Application of molecular and phenotypic taxonomy methodologies determined that all three isolates were genetically consistent with Exophiala angulospora. This is the first report of E. angulospora as the causal agent of systemic mycosis in Atlantic Halibut.

Received January 27, 2014; accepted July 23, 2014     

Establishing a Holocene sediment budget for the river Dijle

A Holocene sediment budget was constructed for the 758 km² Dijle catchment in the Belgian loess belt, in order to understand long-term sediment dynamics. Hillslope sediment redistribution was calculated using soil profile information from 809 soil augerings, which was extrapolated to the entire catchment using morphometric classes. As large parts of the forests within the catchment prove to have undergone little or no erosion since medieval times, a correction was applied for the presence of forests. Total Holocene erosion amounts 817 ± 66 Mt for the catchment, of which 327 ± 34 Mt was deposited as colluvium. This corresponds with a net Holocene soil erosion rate of 10.8 ± 0.8 × 10³ Mg ha^− 1 for the entire Dijle catchment. Alluvial deposits were studied through 187 augerings spread over 17 cross-valley transects. The total alluvial sediment deposition equals 352 ± 11 Mt or 42% of total eroded sediment mass. Results indicate that at the scale of a medium-sized catchment the colluvial sediment sink is as important as the alluvial sediment sink and should not be neglected. As a result the estimation of erosion through alluvial storage and sediment export would yield large errors. Dating of sediment units show an important increase in alluvial deposition from medieval times onwards, indicating the important influence of agricultural activities that developed from that period. Mean sediment export rates from the catchment for the last 1000–1200 years range between 0.8 and 1.3 Mg ha^− 1 a^− 1 and are consistent with present suspended sediment measurements in the Dijle. Erosion for agricultural land for this period is 9.2 ± 2.2 Mg ha^− 1 a^− 1. Sediment budgets for the various tributary catchments provide an insight in the sources and sinks of sediment at different scales within the catchment.     

Genetic dissection of bacterial speck disease resistance in tomato

The bacterial speck disease of tomato has been developed as a model system to elucidate the molecular basis of specificity in plant-bacterial interactions and to study signal transduction events involved in the expression of plant disease resistance. We have employed a mutagenic approach to define the steps involved in the expression of disease resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato (Pst) Eleven disease susceptible mutants have been identified and characterized twith altered recognition of Pst strains that express the avirulence gene avrPto. Genetic analysis of these mutants has revealed that they fall into two complementation groups. Five of the mutants map at the Pto locus, while six map a new locus that we have termed Prf. Further characterization of these mutants has revealed that the mutants that map at Pto are still sensitive to the insecticide fenthion, while the prf mutants are altered in resistance and also are insensitive to fenthion. Genetic mapping has also determined that the Prf locus maps near Pto. We are currently employing a map-based cloning strategy to isolate the Prf locus.