Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing

A novel series of charge-modified, dye-labeled 2′,3′-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase™ II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.     

Heterochromatin: new possibilities for the inheritance of structure

Significant portions of the eukaryotic genome are heterochromatic, made up largely of repetitious sequences and possessing a distinctive chromatin structure associated with gene silencing. New insights into the form of packaging, the associated histone modifications, and the associated nonhistone chromosomal proteins of heterochromatin have suggested a mechanism for providing an epigenetic mark that allows this distinctive chromatin structure to be maintained following replication and to spread within a given domain.     

DNA diversity among the free-living amoeba, Naegleria fowleri, detected by the random amplified polymorphic DNA method

Thirty-one Naegleria fowleri strains from different continents were studied by using the Random Amplified Polymorphic DNA (RAPD) method. Five primers among the 40 examined generated eight RAPD variants corresponding partially to their geographic origin. Four characteristic variants are detected in Oceania. The four remaining ones are present in Europe, two of which are observed on other continents. One European variant is found to exhibit similarities with strains from Oceania. The genetic diversity found in Europe and Oceania as well as the existence of ubiquitous RAPD variants bring new insights about the dispersion of N. fowleri throughout the world.     

Regulation of heterochromatin by histone methylation and small RNAs

Heterochromatin mediates various nuclear processes including centromere function, gene silencing and nuclear organization. Although it was discovered nearly 75 years ago, the pathways involved in heterochromatin establishment, assembly and epigenetic maintenance have been elusive. Recent reports have demonstrated that distinct and novel chromatin-associated factors, including DNA, RNA and histone modifications, are involved in each of these events. These new findings define a novel conserved mechanism of heterochromatin formation that is likely to have an impact on all eukaryotic silencing pathways.     

Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.     

Electromyographic (EMG) Biofeedback in the Comprehensive Treatment of Central Pain and Ataxic Tremor Following Thalamic Stroke

Peripheral pain and ataxic tremor can appear suddenly following thalamic stroke and can significantly alter a patient's psychological, social, and physical functioning. The present paper reports the case of a 70-year-old Caucasian female who sustained an acute left posterior cerebral artery infarction involving the thalamus and left mesiotemporal regions. She subsequently developed Central Poststroke Pain and ataxic movement of her right arm and hand in addition to a significant right-side claudication. She was treated over 16 weeks (6 weeks of EMG biofeedback and 10 weeks of psychotherapy) with a combination of EMG biofeedback, progressive muscle relaxation, behavioral pain coping skills training, Forced Use Therapy, and Cognitive Behavioral Therap 7 years after her initial cerebral accident. The case demonstrates the utility of biofeedback when combined as part of a comprehensive treatment program to address the multiple complications associated with thalamic stroke.     

The CD9 tetraspanin is not required for the development of peripheral B cells or for humoral immunity

The CD9 tetraspanin is known to be expressed at high levels on marginal zone (MZ) B cells, B-1 B cells, and plasma cells, and its expression is believed to be dependent on signals derived via Btk. In CD9 null mice, however, the development and survival of MZ B cells, B-1 B cells, and plasma cells all appear to be unaffected, and humoral immune responses to T-dependent and T-independent Ags are similar to those seen in wild-type littermate controls. In wild-type mice, CD9 levels may serve to distinguish between the presumed MZ precursor B cell population in the spleen and other IgD-expressing transitional B cells that express lower levels of CD21 and CD1d. These results suggest that CD9 is dispensable for B cell development and humoral immunity, but that this protein may serve as an additional marker for the presumed MZ precursor population of splenic B cells.     

Synthesis of novel piperidinyl linker based energy transfer terminators and their potential use in DNA sequencing

Synthesis of novel piperidinyl linker based ET cassettes and terminators is described These novel terminators are evaluated in the DNA sequencing experiments using thermostable DNA polymerase.