We have isolated clones which complement the temperature sensitivity and abnormal rRNA processing pattern of the rrp2-2 mutant of Saccharomyces cerevisiae we previously described. DNA sequencing and restriction analysis demonstrated that all clones contain the NMEI gene encoding the RNA of the ribonucleoprotein particle RNase MRP. Deletion analysis showed that the NMEI gene is responsible for the complementation of the rrp2-2 phenotype. A single base change was identified in the nmel gene in the rrp2 mutant, confirming that the RRP2 and NMEI genes are identical. Our experiments therefore indicate that RNase MRP, in addition to its previously reported role in formation of RNA primers for mitochondrial DNA replication [Clayton, D. A. (1991) Trends Biochem. Sci. 16, 107-111], is involved in rRNA processing.

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA-and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three-to fourfold slowing of both DNA 3-end-directed and RNA 5-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803-5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3-end-and RNA 5-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.Infection with human immunodeficiency virus (HIV) is the cause of AIDS and affects over 30 million people worldwide (64). The primary targets of therapy for HIV infection include the viral protease and reverse transcriptase (RT). HIV type 1 (HIV-1) RT is a heterodimer consisting of 66-and 51-kDa subunits (p66 and p51, respectively) (3). p66 contains both the polymerase and the RNase H active sites of the enzyme (34, 37, 39). The RNase H domain is present in the carboxy-terminal third of p66. Although p51 is derived from p66 by proteolytic cleavage, it assumes a very different tertiary structure and does not contain a catalytic site (37, 39). The function of p51 is not known, but it may play a role in binding the tRNA 3Lys -template complex (3, 39) and in maintaining the structural integrity of the heterodimer (1).RNase H cleavage is essential for HIV-1 replication (61; for a review see reference 11). Two modes of RNase H cleavage have been described (Fig. 1). "Polymerase-dependent" cleavage is thought to occur in concert with DNA polymerization to degrade the genomic RNA during minus strand DNA synthesis (26,46). The position of the primary DNA 3Ј-end-directed cleavage occurs 15 to 18 nucleotides (nt) from the recessed 3Ј end of the DNA (26, 33); we have referred to this mode of cleavage as DNA 3Ј-end-directed RNase H cleavage. A second mode of RNase H cleavage occurs independently of DNA polymerization. The position ...

We have identified from a collection of temperature sensitive yeast mutants strains which fail to process rRNA normally. Characterization of one such mutant is reported here. This strain accumulates increased amounts of the 35S primary transcript, '24S' molecules extending from the transcription start site to the 5.8S region, and two classes of 5.8S rRNA with 5' extensions of 7 and 149 bases, respectively. We show that this pleiotropic change in the rRNA processing pattern is due to a single mutation. Possible models for the function of the mutated gene are discussed.

Additive manufacturing (AM) is rapidly evolving as the most comprehensive tool to manufacture products ranging from prototypes to various end-user applications. Fused filament fabrication (FFF) is the most widely used AM technique due to its ability to manufacture complex and relatively high strength parts from many low-cost materials. Generally, the high strength of the printed parts in FFF is attributed to the research in materials and respective process factors (process variables, physical setup, and ambient temperature). However, these factors have not been rigorously reviewed for analyzing their effects on the strength and ductility of different classes of materials. This review systematically elaborates the relationship between materials and the corresponding process factors. The main focus is on the strength and ductility. A hierarchical approach is used to analyze the materials, process parameters, and void control before identifying existing research gaps and future research directions.

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