Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes

Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination

Discrimination of wheat seed varieties on the basis of geometrical characteristics

Celem pracy było poszukiwanie takich wyróżników geometrii 16 odmian ziarna pszenicy, które pozwolą na ich dyskryminacje. Do identyfikacji właściwości geometrycznych wykorzystano stanowisko do komputerowej analizy obrazu, oparte na pozyskiwania obrazu ziarniaków za pomocą aparatu fotograficznego. Każdy z ziarniaków został opisany przez 66 zmiennych geometrycznych. Analiza statystyczna wyników przebiegała dwuetapowo. W pierwszym etapie przeprowadzono redukcję zmiennych do najlepiej dyskryminujących, natomiast w drugim etapie przeprowadzono analizę dyskryminacyjną. Błąd klasyfikacji odmian jarych wyniósł 10,55%, natomiast odmian ozimych 4,58%.The purpose of the work was to try to find these geometry characteristics for 16 wheat seed varieties, which will allow their discriminations. Workstation for computer image analysis, based on acquiring seed image using a camera, was used for identifying geometrical properties. Each seed was described by 66 geometrical variables. Statistical analysis of results proceeded in two stages. The first stage involved reduction of variables to those discriminating best, whereas discriminant analysis was made in the second stage. Classification error for spring varieties was 10.55%, and 4.58% for winter varieties