SEQUENCE AND POLYMORPHISM OF RHESUS MACAQUE MHC

点击翻译按钮获取中文摘要

The increased utility of the rhesus macaque animal model in both HIV vaccine development and pathogenesis studies necessitates the development of a solid genetics and immunogenetics data infrastructure for this species. Relative to human data, there is a paucity of information about the larger-scale gene content of the macaque MHC and very little polymorphism data from outside of the macaque class I and II genes. As a pointed example, indirect evidence exists that there may be as many as 3 class I B loci and 2 class I A loci on certain macaque haplotypes, but this has not been confirmed through direct genomic analysis. Thus the number of actively expressed class I genes in individual experimental macaque may be unknown. An essentially similar story und4rlies some class II loci, and very little structural data exists about the hundreds of other genes present in the macaque MHC, more than a third of which are also directly involved in the immune response. These deficiencies provide substantial motivation to better understand the genetics and immunogenetics of rhesus macaques that could be useful in the design and conduct of AIDS vaccine experiments in the similar immunodeficiency (SIV) and chimeric SIV-HIV (SHIV) animal models using this non-human primate species. Toward this end, we proposed to develop a DNA sequence of a macaque major histocompatibility complex (MHC) of finished high quality data. We further propose to annotate this data string with extensive polymorphism data (SNP) and larger-scale haplotypic variation, and with respect to the complete gene content of the region. One fundamental priority will be to effective organize and disseminate this data to the interested scientific community To develop this resource, we propose to accomplish the following specific aims: 1) To derive a complete and high quality genome sequence of a major histocompatibility complex (MHC) from the rhesus macaque. 2) To develop and define a set of SNPs and larger scale haplotypes structures useful as mapping tools in the association with MHC-linked phenotypes. 3) To identify and annotate genomes and polymorphisms in the macaque MHC and make this resource available in a useful and palatable form to the scientific community. We will accomplish these aims in part by combining resources and expertise in macaque genetics, genomics, and functions from three accomplished laboratories. There is a critical need for analysis and monitoring of cellular and sub- cellular biological functions, especially for understanding pharmacological and disease processes. Optical microscopy has been previously used for biological sample characterization but has a diffraction limited resolution of lambda 2. However, scanning near-field optical microscopy can be used to achieve nanoscale resolution on the order of lambda30 and better. The technique combines the properties of atomic force microscopy in the non-contact mode with optical microscopy, by utilizing a fiber optic probe tip that scans over a sample surface and simultaneously acquires shear force and optical signals to produce topographical and optical images, respectively. Optical fiber microtips fabricated from silica have been used for this technique in the visible wavelength region with at least 100 nm resolution. However, these fibers cannot be used in the infrared because they do not transmit at wavelengths longer than 2 microm. We propose to develop the technique using infrared-transmitting optical fiber microtips capable of accessing the "fingerprint" region from 2-10 microm, where molecular species can be readily identified by their characteristic vibrational absorption bands/ The goals of this program are: Develop the new technique of Scanning Near Field Infrared Microscopy using state-of-the-art low optical loss IR-transmitting optical fibers which NRL has developed for a variety of applications. NRL will fabricate NR-transmitting microtips capable of probing the 2-10 microm infrared wavelength region that has previously been unavailable. NRL will collaborate with Vanderbilt scientists in the implementation of the microtips with a Near Field Microscope. Demonstrate nanoscale optical resolution at least 100 nm in the infrared with the SNIM system using the IR fiber microtips. Utilizing the scanning near-field microscopy (SNIM) in the 2-10 microm wavelength region to investigate spectroscopic imaging of biological structures such as living cells at less than or equal too 100 nm resolution in an aqueous environment. The optical and topographical resolution as well as spectroscopic imaging capability of the probes will be evaluated in a biological cellular environment, to analyze cellular functions such as membrane transport and molecular recognition processes in biological samples. This new capability will be critical for the understanding of a wide variety of pharmacological and disease processes.