Isolation and identification of Triticeae chromosome 1 receptor-like kinase genes (Lrk10) from diploid, tetraploid, and hexaploid species of the genus Avena.

The DNA sequence of an extracellular (EXC) domain of an oat (Avena sativa L.) receptor-like kinase (ALrk10) gene was amplified from 23 accessions of 15 Avena species (6 diploid, 6 tetraploid, and 3 hexaploid). Primers were designed from one partial oat ALrk10 clone that had been used to map the gene in hexaploid oat to linkage groups syntenic to Triticeae chromosome 1 and 3. Cluster (phylogenetic) analyses showed that all of the oat DNA sequences amplified with these primers are orthologous to the wheat and barley sequences that are located on chromosome 1 of the Triticeae species. Triticeae chromosome 3 Lrk10 sequences were not amplified using these primers. Cluster analyses provided evidence for multiple copies at a locus. The analysis divided the ALrk EXC sequences into two groups, one of which included AA and AABB genome species and the other CC, AACC, and CCCC genome species. Both groups of sequences were found in hexaploid AACCDD genome species, but not in all accessions. The C genome group was divided into 3 subgroups: (i) the CC diploids and the perennial autotetraploid, Avena macrostachya (this supports other evidence for the presence of the C in this autotetraploid species); (ii) a sequence from Avena maroccana and Avena murphyi and several sequences from different accessions of A. sativa; and (iii) A. murphyi and sequences from A. sativa and Avena sterilis. This suggests a possible polyphyletic origin for A. sativa from the AACC progenitor tetraploids or an origin from a progenitor of the AACC tetraploids. The sequences of the A genome group were not as clearly divided into subgroups. Although a group of sequences from the accession 'SunII' and a sequence from line Pg3, are clearly different from the others, the A genome diploid sequences were interspersed with tetraploid and hexaploid sequences.

A putative beta2-adrenoceptor from the rainbow trout (Oncorhynuchus mykiss). Molecular characterization and pharmacology.

Extensive molecular characterization of mammalian beta-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout beta2-adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout beta2-adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate beta2-adrenoceptors. The conclusion that this sequence is a rainbow trout beta2-adrenoceptor is further supported by phylogenetic analysis of vertebrate beta-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout beta2-adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein-binding domain of the rainbow trout beta2-adrenoceptor sequence suggests that the in vivo control of trout beta2-adrenoceptor signaling differs substantially from that of mammals.

Molecular evolution of leptin.

Leptin, a hormone produced mainly by adipocytes, is involved in the regulation of food intake, metabolism, and reproduction. The objective of this study was to determine the evolutionary relationships of leptin genes. Partial nucleotide sequences of leptin were cloned and sequenced from six mammalian species: large hairy armadillo (Chaetophractus villosus), rabbit (Oryctolagus cuniculus), big brown bat (Eptesicus fuscus) [corrected], striped skunk (Mephitis mephitis), raccoon (Procyon lotor), and beluga whale (Delphinapterus leucas). The PUZZLE program was used to construct maximum-likelihood trees. Our phylogenetic analysis shows that the grouping of these new mammalian sequences with those currently available in GenBank respect the evolutionary relationships generally accepted for mammals. However, when leptin sequences for chicken and turkey are included in the analysis, these are found to group with mouse and rat leptins. Chicken and mouse leptins are 95% identical. However, when mouse is compared with closer relatives, such as rabbit or bat, identities are approximately 80%. A comparison of extant and ancestral leptin sequences suggests that convergent or parallel evolution is the most plausible hypothesis to explain the similarity between bird and rodent leptins.

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines. Theoretical model.

We recently demonstrated that the molecular mass distribution of an uncharged polymer sample can be analyzed using free-solution capillary electrophoresis of DNA-polymer conjugates. In these conjugates, the DNA is providing the electromotive force while the uncharged polydisperse polymer chains of the sample retard the DNA engine with different amounts of hydrodynamic drag. Here we present a theoretical model of this new analytical method. We show that for the most favourable, diffusion-limited electrophoresis conditions, there is actually an optimal DNA size to achieve the separation of a given polymer sample. Moreover, we demonstrate that the effective friction coefficient of the polymer chains is related to the stiffness of the two polymers of the conjugate, thus offering a method to estimate the persistence length of the uncharged polymer through mobility measurements. Finally, we compare some of our predictions with available experimental results.

Diffusion coefficient of DNA molecules during free solution electrophoresis.

The free-draining properties of DNA normally make it impossible to separate nucleic acids by free-flow electrophoresis. However, little is known, either theoretically or experimentally, about the diffusion coefficient of DNA molecules during free-flow electrophoresis. In fact, many authors simply assume that the Nernst-Einstein relation between the mobility and the diffusion coefficient still holds under such conditions. In this paper, we present an experimental study of the diffusion coefficient of both ssDNA and dsDNA molecules during free-flow electrophoresis. Our results unequivocally show that a simplistic use of Nernst-Einstein's relation fails, and that the electric field actually has no effect on the thermal diffusion process. Finally, we compare the dependence of the diffusion coefficient upon DNA molecular size to results obtained previously by other groups and to Zimm's theory.

Vasectomy and prostate cancer in Québec, Canada.

The present investigation measured the association between vasectomy and prostate cancer (PC) in the male population of Québec, Canada. The Québec Health Insurance Board and the Québec Cancer Registry were our principal sources of information. Lung cancer cases and the male population of Québec served as controls for comparative purposes. Within a retrospective design, our preliminary results indicate an association between vasectomy and PC. Among the 1925-39 birth cohort of individuals diagnosed with PC in 1990--93, the global odds ratio was 2.6 (95% CI=1.7--4.3) while it was compared with lung cancer as the control group. This risk increased with the length of time between vasectomy and the diagnosis of cancer. An historical design indicated strong cohesion of the results. Besides, the risk does not vary when we control for the place of residence of the individuals. Vasectomy seems to increase the risk of PC at least 10 years after the operation, but we cannot exclude the impact of a possible detection bias among vasectomized individuals.

Molar mass profiling of synthetic polymers by free-solution capillary electrophoresis of DNA-polymer conjugates.

The molar mass distribution of a polymer sample is a critical determinant of its material properties and is generally analyzed by gel permeation chromatography or more recently, by MALDI-TOF mass spectrometry. We describe here a novel method for the determination of the degree of polymerization of polydisperse, uncharged, water-soluble polymers (e.g., poly(ethylene glycol) (PEG)), based upon single-monomer resolution of DNA-polymer conjugates by free-solution capillary electrophoresis. This is accomplished by end-on covalent conjugation of a polydisperse, uncharged polymer sample (PEG) to a monodisperse, fluorescently labeled DNA oligomer, followed by electrophoretic analysis. The monodisperse, charged DNA "engine" confers to each conjugate an equal amount of electromotive force, while the varying contour lengths of the uncharged, polydisperse polymers engender different amounts of hydrodynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a function of the size of the uncharged PEG tail. This provides a profile of the molar mass distribution of the original polymer sample that can be detected by laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE). Theory-based analysis of the resulting electrophoresis data allows precise calculation of the degree of polymerization of the PEG portion of each conjugate molecule. Knowledge of the molecular mass of the uncharged polymer's repeat unit allows for direct calculation of the molar mass averages as well as sample polydispersity index. The results of these analyses are strikingly reminiscent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3.4-, 5-, and 20-kDa nominal average molar mass were analyzed by FSCE and MALDI-TOF; the values of the molar mass averages, Mw and Mn, typically agree to within 5%. Measurements and molar mass calculations are performed without any internal standards or calibration. Moreover, when DNA-polymer conjugate analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min. FSCE offers an alternative to MALDI-TOF for the characterization of uncharged, water-soluble polymers that can be uniquely conjugated to DNA.

Long term follow-up of intravesical Bacillus Calmette-Guerin for the treatment of bladder transitional cell carcinoma.

OBJECTIVE: To review the long-term follow-up, in terms of recurrence and progression, of transitional cell carcinoma of the bladder treated with intravesical BCG with the following indications: CIS, Ta and T1. MATERIALS AND METHODS: Ninety-two patients who had received complete course of BCG between 1987 and 1993 were included in the study and followed for an average of 59 months (range 12 to 102). RESULTS: The recurrence and progression were looked at. Patients treated with BCG for Carcinoma in situ, 11 of 19 (53%) remained tumor-free after 1 or 2 courses of BCG for the duration of the follow-up (mean 4.9 years, range 1.5 to 8.5 years). For patients treated for recurring tumors, 17 of 50 (34%) had no recurrences after 1 or 2 courses of BCG with the same follow-up. When facing multiple tumors, 10 of 23 (43%) patients did not experience recurrences. Therefore, in the 92 patients treated, 38 presented no recurrences after 1 or 2 courses of BCG, for a success rate of 41%. In terms of progression, of the 19 patients treated with BCG for CIS, 4 (21%) went on to develop muscle invasive disease. Of the 50 patients treated for recurrent tumors, 2 (4%) eventually developed lamina propria invasion (initial lesion was a Ta tumor), 4 (8%) carcinoma in situ and 7 (14%) muscle invasive disease, for an overall progression rate of 26% in this group. Of the 25 patients treated for multiple tumors, 1 (4%) developed CIS and 3 (12%) presented with muscle invasive disease, for an overall progression rate of 16% for the duration of the follow-up. Therefore, 21 of 92 (23%) patients had progression of their disease following BCG therapy. No prognostic factors for recurrence or progression could be identified in these tumors. CONCLUSION: When indications warrant its use, BCG is effective in reducing recurrences and limiting progression in TCC of the bladder. Recurrence within 2 years of treatment is, however, a sign of poor prognosis and other therapeutic options should be sought.

Gel electrophoretic mobility of single-stranded DNA: the two reptation field-dependent factors.

The reptation model is the dominant theory in understanding the electrophoretic separation of single-stranded DNA molecules in gels or entangled polymer solutions. Recently, we showed that the Ogston and reptation regimes are separated by an entropic trapping regime at low field intensities. Here, we report the first comparison of the field-dependent part of the DNA mobility for both small and long reptating molecules. We show that both mobilities increase linearly with field intensity, with the mobility of the longer (comigrating) fragments increasing faster than that of the smaller ones. We compare our results to the predictions of the biased reptation model.

Expressed retrotransposed 5S rRNA genes in the mouse and rat genomes.

The analyses of previously described 5S rRNA gene sequences show that some of the expressed 5S rRNA genes present in the mouse and rat genomes were derived from the retrotransposition of 5S rRNA transcripts. These analyses demonstrate that new 5S rRNA gene copies can originate by retrotransposition and that some of these retrotranscribed genes are expressed.

Detecting and characterizing gene conversions between multigene family members.

We used a variety of methods to detect known gene conversions in the actin gene families of five angiosperm species, the beta-globin gene families of two primate species, and the Zfx/Zfy gene families of seven mammalian species. Our goal was to devise a working strategy which would allow the analysis of the members of a multigene family in order to determine whether there had been gene conversions between its members, identify the genes involved in the gene conversions, establish the lengths of the converted regions, and determine the polarities of the gene conversions. We show that three phylogenetic methods and the homoplasy test of Maynard Smith and Smith perform relatively poorly on our data sets because the sequences we analyzed had large levels of multiple substitutions. The method of Sawyer, the compatibility method of Jakobsen and Easteal, the partition matrix method of Jakobsen, Wilson, and Easteal, and the co-double method of Balding, Nichols, and Hunt can be used to identify the genes which have been involved in gene conversions. The co-double method is more powerful than other methods but requires orthologous sequences from related species. Compatibility, phylogenetic, and nucleotide substitution distribution statistics methods can be used to identify the location of the converted region(s). Site-by-site compatibility analyses can also be used to identify the direction of the conversion event(s). Combinations of these methods can therefore be used to establish the presence, locations, and polarities of gene conversions between multigene family members.

Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.

Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: molecular phylogeny and evidence for a new GAD paralog.

The evolution of chordate glutamic acid decarboxylase (GAD; EC 4.1.1.15), a key enzyme in the central nervous system synthesizing the neurotransmitter gamma-amino-butyric acid (GABA) from glutamate, was studied. Prior to this study, molecular data of GAD had been restricted to mammals, which express two distinct forms, GAD65 and GAD67. These are the products of separate genes and probably are derived from a common ancestral GAD following gene duplication at some point during vertebrate evolution. To enable a comprehensive phylogenetic analysis, molecular information of GAD forms in other vertebrate classes was essential. By reverse transcriptase-polymerase chain reaction (RT-PCR), partial nucleotide sequences of GAD were cloned from brains of zebra finch (Taeniopygia guttata), turtle (Trachemys scripta), goldfish (Carassius auratus), zebrafish (Danio rerio), and armoured grenadier (Coryphaenoides (Nematonurus) armatus, a deep-sea fish), and from the cerebral ganglion plus neural gland of Ciona intestinalis, a protochordate. Whereas GAD65 and GAD67 homologs were expressed in birds, reptiles, and fish, only a single GAD cDNA with equal similarities to both vertebrate GAD forms was found in the protochordate. This indicates that the duplication of the vertebrate GAD gene occurred between 400 and 560 million years ago. For both GAD65 and GAD67, the generated phylogenetic tree followed the general tree topology for the major vertebrate classes. In turtle, an alternative spliced form of GAD65, putatively encoding a truncated, nonactive GAD, was found. Furthermore, a third GAD form, which is equally divergent from both GAD65 and GAD67, is expressed in C. (N.) armatus. This third form might have originated from an ancient genome duplication specific to modern ray-finned fishes.

Recent developments in DNA electrophoretic separations.

DNA electrophoresis is now a fairly mature technology. Nevertheless, as we approach the 21st century, new ideas are frequently suggested that could lead to a revolution for DNA sequencing and mapping. Here, we review some of the novel concepts that have been studied since ca. 1990. Our review focuses on new separation mechanisms, new sieving matrices and recent conceptual advances.

The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis.

It has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris-44.5 mM boric acid-1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient.

Relationships among msx gene structure and function in zebrafish and other vertebrates.

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Computer assisted knee surgery: diagnostics and planning of knee surgery.

Cruciate ligament rupture, a common injury among young active adults, disrupts the knee's complex movement and often leads to premature degenerative arthritis of the joint. Prosthetic cruciate ligaments can be used for replacement but often fail owing to incorrect surgical placement. To aid in the planning of cruciate prosthetic substitution, a computerized system has been developed to provide the surgeon with a virtual interface allowing accurate visualization of three-dimensional (3D) bone structure and movement normally hidden beneath layers of soft tissue. Preoperatively, precise in vivo kinematics are quantified with the help of 3D medical images. Three-dimensional imagery techniques based on computed tomography (CT) have been developed to obtain accurate 3D reconstruction of knee geometry preoperatively. The system allows the surgeon to know the real-time spatial position of the patient via magnetic position and orientation sensors attached noninvasively onto the femur and tibia via a new attachment system. An interactive computer program has been developed to allow the user to simulate different prosthetic ligament insertions and compute elongation, bending, and torsion values that will be imposed on the prosthesis. Through comparison with cadaver studies and a perturbation analysis, the system is shown to be sufficiently accurate to predict certain in vivo ligament bending and torsion deformations.