Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection

Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5 h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments.     

Development of the Fifth Leaf is Indicative for Whole Plant Performance at Low Temperature in Tomato

In the search for early-detectable selection criteria for growthat low temperature conditions in tomato, first the initiationand growth of individual leaves was analysed. Scanning electronmicroscopy revealed that the first four primordia had alreadydeveloped during the germination period at 25°C. The primordiumof the fifth leaf, however, was initiated after the transferof seedlings to the experimental conditions. The increase inlength of the first three leaves, and to a lesser extent ofthe fourth leaf, was considerably smaller in comparison withthat of later formed leaves. Moreover, the morphology of thefirst three to four leaves was deviant, whereas the others showedthe normal compound leaf architecture. All these results indicatedthat the fifth leaf was the earliest formed leaf with growthcharacteristics that might reflect the growth potential of thewhole plant. Development of the fifth leaf was tested as a marker for wholeplant growth. At three temperature, 18, 15 and 12°C, growthresponses of the fifth leaf were similar to that of whole plantsin four tomato genotypes: Line A, Line B, Premier and MXXIV-13.Significant differences in relative growth rate of dry weightof whole plants and fifth leaves (RGR_W)and of leaf area of thefifth leaves (RGR_LA between two fast growing and two slow growinggenotypes were found. No genotype by temperature interactionfor RGR_W and RGR_LA was found, indicating that the effect oftemperature decrease was similar for the four genotypes. The structure of the mature fifth leaf of one fast and one slowgrowing genotype, Line A and MXXIV-13, was analysed. For bothgenotypes, leaves were small and thick at low temperature, 12°C.The total number of epidermis and palisade parenchyma cellsper leaf was smaller but the size of the cells developed at12°C was larger than at 18°C. Consequently, the slowgrowth at 12°C was due to a low rate of cell division. Atboth temperatures, the fifth leaf to MXXIV-13 was smaller comparedto that of line A. Since the size of the cells were similar,the smaller leaf size was due to lower number of leaf cells. The results confirm the suitability of the growth, especiallyexpressed as RGR_LA , of the fifth leaf as a nondestructive marketfor vegetative development of tomato at low temperature. Growthdifferences between genotypes were mainly reflected by differencesin cell number of leaves, which might be correlated with geneticallydetermined differences in cell number of leaf primordia.Copyright1993, 1999 Academic Press Lycopersicon esculentum Mill. genotypes, plant growth, selection criteria, low temperature, leaf initiation, leaf development, RGR, leaf structure, cell expansion     

Structural color production by constructive reflection from ordered collagen arrays in a bird (Philepitta castanea: Eurylaimidae)

Ordered hexagonal arrays of parallel collagen fibers produce the brilliant green structural color of the fleshy, supraorbital caruncles of male Velvet Asity (Philepitta castanea; Aves: Eurylaimidae). The collagen arrays are organized in larger macrofibrils that are packed irregularly within cone-shaped papillae that cover the surface of the caruncle. The color of the caruncle conforms closely to the wavelengths predicted by applying Bragg's Law of constructive reflection to measurements of the size and spatial organization of the collagen arrays. These observations constitute a novel mechanism of structural color production in animals. These collagen arrays are convergently similar to the smaller, highly structured collagen arrays in the mammalian cornea, which exploit the same physical mechanism to produce optical transparency. © 1994 Wiley-Liss, Inc.     

Sequence variation in the monoclonal-antibody-U36-defined CD44v6 epitope

 Monoclonal antibody (mAb) U36 was developed for the treatment of minimal residual disease of head and neck squamous cell carcinoma (HNSCC). The mAb-U36-defined antigen was characterized by cDNA cloning, and was shown to be identical to the keratinocyte-specific CD44 splice variant epican. The epitope recognized by mAb U36 was shown to be located in the v6 domain. Two amino acids within the epitope appeared to differ from the sequences that have been described in literature. The sequence of the epitope appeared to contain glutamic acid at position 367 and lysine at position 374, while valine and arginine respectively have been described before. Interestingly, another anti-CD44v6 antibody with possible clinical application, VFF18, recognizes an epitope in the same area. With respect to the applicability of these antibodies for tumor targeting, this variation might have an influence on antibody-antigen interaction and mAb accumulation in the tumor. Furthermore, this observation raised the question whether the different epitopes are related to the malignant behavior of tumor cells. In this paper we determine the relative affinity of mAb U36 for the variant epitope sequences by tumor cell binding assays using synthetic peptides for competition. The presence of glutamic acid instead of valine at position 367 caused strong competition. Further evaluation showed that the published valine variant does not exist in vivo, and is the result of a sequencing artefact. The effect of substitution of lysine for arginine at position 374 had no effect on the binding of mAb U36 to the cells. This amino acid variation was shown to be due to allelic polymorphism. There was no trend towards allelic imbalance in tumor cells as compared to normal cells. Received: 5 June 1997 / Accepted: 14 August 1997