Cloning and characterization of the mouse ret finger protein (rfp), a B box zinc finger protein

An important question in biology is to understand the role of specific gene products in regulating embryogenesis and cellular differentiation. Many of the regulatory proteins possess specific motifs, such as the homeodomain, basic helix-loop-helix structure, zinc finger, and leucine zipper. These sequence motifs participate in specific protein-DNA, protein-RNA, and protein-protein interactions, and are important for the function of these regulatory proteins.^ The human rfp (ret finger protein) belongs to a novel zinc finger protein family, the B box zinc finger family. Most of the B box proteins, including rfp, have a conserved tripartite motif, consisting of two novel zinc fingers (the RING finger and the B box) and a coiled-coil domain. Interestingly, a fusion protein between the tripartite motif of rfp and the tyrosine kinase domain of c-ret has transforming activity. In this study, we examined the expression of rfp during mouse development, and characterized the role of the tripartite motif in rfp function.^ We cloned the mouse rfp cDNA, which shares a 98.4% homology with the human sequence at amino acid level. Such strikingly high degree of homology indicates the high evolutionary pressure on the conservation of the sequence, suggesting that rfp may have an important function. Using the somatic cell hybrid system, we assigned the rfp gene to mouse chromosome 13 and human chromosome 6. Rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos; however, they were restricted in adult mice, with the highest level of expression in the testis. Rfp expression in the testis is detected only in late pachytene spermatocytes and round spermatids. In both embryos and spermatogenic cells, rfp protein was distributed within cell nuclei in a punctate pattern, similar to the PODs (PML oncogenic domains) observed with another B box protein, PML. In cultured mammalian cells, we found that rfp was indeed co-localized to the PODs with PML. Using the yeast two-hybrid system, we showed that the rfp could specifically interact with PML, and that the interaction was dependent on the distal portion of the rfp coiled-coil domain.^ We also showed that rfp could form homodimers, and both the B box and coiled-coil domain were required for proper dimerization. It seems that the proximal portion of the coiled-coil domain provides the interacting interface, while the B box zinc finger orients the coil and maintains the correct structure of the whole molecule. Our data are consistent with the zinc-binding property and structural analysis of the B box. The RING finger seems to be involved in rfp nuclear localization through interaction with other proteins. We believe that homodimerization and interaction with PML are important for the normal interaction of rfp during development and differentiation. In addition, rfp homodimerization may also be essential for the oncogenic activation of the rfp-ret fusion protein. ^

Eric D. Perl, Theophany. The Neoplatonic Philosophy of Dionysius the Areopagite, State University of New York Press (SUNY series in ancient Greek philosophy), 2007. 163 p., ISBN-13: 978-0-7914-7111-1

Fil: Caram, Gabriela de los Ángeles.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Reactores Químicos. Problema 3: RFP isotermo con múltiples reacciones (curso 2011-2012)

Enunciado problema 3: RFP isotermo con múltiples reacciones.

Going live: a comparative analysis of the suitability of the RFP derivatives RedStar, mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.

Determining the appropriate proportion of owner-provided design in design-build contracts - a content analysis approach

The Request For Proposal (RFP) with the design‐build (DB) procurement arrangement is a document in which an owner develops his requirements and conveys the project scope to DB contractors. Owners should provide an appropriate level of design in DB RFPs to adequately describe their requirements without compromising the prospects for innovation. This paper examines and compares the different levels of owner‐provided design in DB RFPs by the content analysis of 84 requests for RFPs for public DB projects advertised between 2000 and 2010 with an aggregate contract value of over $5.4 billion. A statistical analysis was also conducted in order to explore the relationship between the proportion of owner‐provided design and other project information, including project type, advertisement time, project size, contractor selection method, procurement process and contract type. The results show that the majority (64.8%) of the RFPs provide less than 10% of the owner‐provided design. The owner‐provided design proportion has a significant association with project type, project size, contractor selection method and contract type. In addition, owners are generally providing less design in recent years than hitherto. The research findings also provide owners with perspectives to determine the appropriate level of owner‐provided design in DB RFPs.

Analysis of selection criteria for design-builders through the analysis of Request for Proposals (RFPs)

Selecting an appropriate design-builder is critical to the success of DB projects. The objective of this study is to identify selection criteria for design-builders and compare their relative importance by means of a robust content analysis of 94 Request For Proposals (RFPs) for public DB projects. These DB projects had an aggregate contract value of over US$3.5 billion and were advertised between 2000 and 2010. This study summarized twenty-six selection criteria and classified into ten categories, i.e.: price, experience, technical approach, management approach, qualification, schedule, past performance, financial capability, responsiveness to the RFP, and legal status in descending order of their relative importance. The results showed that even though price still remains as the most important selection category, its relative importance declines significantly in the last decade. The categories of qualification, experience, past performance, by contrast, have been becoming more important to DB owners for selecting design-builders. Finally, it is found that the importance weighting of price in large projects is significantly higher than that in small projects. This study provides a useful reference for owners in selecting their preferred design-builders.

Institute for Community College Development Addresses Leadership Crisis

[Excerpt] It is projected that between 2001 and 2007, 47 percent of community college presidents will have left their positions. At a time when challenges are growing more complex, the senior administrators who typically moved into presidencies are also "aging out," leaving fewer qualified individuals in the pipeline. The Institute for Community College Development (ICCD), a partnership between the State University of New York (SUNY) and Cornell, was founded by a group of community college presidents to respond to this leadership crisis. ICCD has been part of ILR since 2001.

Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase

We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment.