We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.

Based upon an intermediate morphology, Apocynum x-floribundum Greene has long been considered a hybrid involving A. androsaemifolium and A. cannabinum. The floral morphology in this genus, however, appears to prohibit both import and export of pollen, and observations of numerous insect visitors reveal that pollen is not routinely carried from flower to flower. Furthermore, reproductive success as measured by fruit set is very low in most populations. Hybridization was thus called into question, with allozyme evidence used to test the hypothesis of a hybrid origin for A. x-floribundum. Six diagnostic loci, as well as two loci exhibiting highly disparate allele frequencies, were resolved for each parent. All examined populations of A. x-floribundum were heterozygous at these loci, thus supporting the hypothesis of hybridization. Evidence from additional loci indicated that all populations tend to be strongly clonal. Observed heterozygosity was very low in the parental species, suggesting a history of inbreeding or a severe bottleneck. There was no support for earlier assumptions that some intermediates are derived from backcrosses or "secondary hybrids." Statistical analyses of plant height, leaf shape, petal length, sepal length, follicle length, seed length, and seed number per follicle supported these conclusions. The persistence and vegetative spread of hybrid clones may contribute to the illusion that hybridization is common.

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Accelerated tool wear and tool breakage are significant problems in micro-machining processes such as micro-milling. Traditional flood cooling processes are unsuitable for micro-milling due to the excessive collision force between the fluid stream and the tool being large enough to affect the accuracy of the cutting process. In this research an atomization-based cooling and lubrication system is presented that delivers atomized cutting fluids to a micro-milling tool through the use of an original nozzle design based on two orthogonally-directed streams. The system and nozzle is used to investigate the relative importance of cooling and lubrication on micro-milling of 6061 T6 cold-rolled aluminum with a 0.508 mm diameter two-fluted end mill. Six cutting conditions are experimentally evaluated based on cutting forces and tool life. Lubrication is investigated through two concentrations (10% and 25%) of a semi-synthetic cutting fluid. Cooling is investigated through the use of atomized deionized water as well as dry cutting with cooling provided by a Ranque-Hilsch vortex tube. Dry cutting was used as a control. Statistical testing revealed the importance of lubrication relative to cooling when machining on the micro-scale as deionized water performed the worst of all tests conducted. Based on the experimental results, recommendations are made for the design of future micro-machining cooling and lubrication systems.