ABSTRACT
Annotating the druggable genome estimates the potential maximum size of the playing field for current small-molecule drug design but It does not consider biologicals or future breakthroughs in medicinal chemistry or biology.
Subject(s)
Drug Design , Proteins , Genome , Humans , Proteins/chemistry , Proteins/metabolismABSTRACT
Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.
Subject(s)
Axonal Transport , Dyneins/genetics , Dyneins/physiology , Motor Neuron Disease/genetics , Motor Neurons/physiology , Nerve Degeneration , Animals , Anterior Horn Cells/pathology , Apoptosis , Cell Differentiation , Cell Movement , Central Nervous System/embryology , Chromosome Mapping , Dimerization , Dyneins/chemistry , Female , Ganglia, Spinal/pathology , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Heterozygote , Homozygote , Lewy Bodies/pathology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Motor Neuron Disease/pathology , Motor Neuron Disease/physiopathology , Motor Neurons/ultrastructure , Mutation , Mutation, Missense , Peptide Fragments/metabolism , Phenotype , Point Mutation , Spinal Nerves/growth & development , Tetanus Toxin/metabolismABSTRACT
To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.
Subject(s)
Carrier Proteins/chemical synthesis , Carrier Proteins/metabolism , Peptide Nucleic Acids/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Antigens, Polyomavirus Transforming/chemistry , Antigens, Polyomavirus Transforming/metabolism , Base Sequence , Carrier Proteins/chemistry , Cell Nucleus/metabolism , Humans , Male , Microscopy, Confocal , Molecular Sequence Data , Nuclear Localization Signals/chemical synthesis , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/metabolism , Prostatic Neoplasms/metabolism , Rats , Spectrometry, Fluorescence , Tumor Cells, CulturedABSTRACT
Genome-wide screening for chromosomal imbalances using comparative genomic hybridization (CGH) revealed a wealth of data on previously unrecognized tumor-specific genomic alterations. CGH to microarrays of DNA, an approach termed matrix-CGH, allows detection of genomic imbalances at a much higher resolution. We show that matrix CGH is also feasible from small tissue samples requiring universal amplification of genomic DNA. Because widespread application of matrix-CGH experiments using large numbers of DNA targets demands a high degree of automation, we have developed a protocol for a fully automated procedure. The use of specialized instrumentation for the generation of DNA chips, their hybridization, scanning, and evaluation required numerous alterations and modifications of the initial protocol. We here present the elaboration and testing of automated matrix-CGH. A chip consisting of 188 different genomic DNA fragments, cloned in bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors and immobilized in replicas of 10, was used to assess the performance of the automated protocol in determining the gene dosage variations in tumor cell lines COLO320-HSR, HL60, and NGP. Although ratios of matrix-CGH were highly concordant with results of chromosomal CGH (85%), the dynamic range of the matrix-CGH ratios was highly superior. Investigation of the two amplicons on 8q24 in COLO320-HSR and HL60, containing the MYC gene, revealed a homogeneous amplicon in COLO320-HSR but a heterogeneous amplification pattern in HL60 cells. Although control clones for normalization of the signal ratios can be predicted in cases with defined chromosomal aberrations, in primary tumors such data are often not available, requiring alternative normalization algorithms. Testing such algorithms in a primary high-grade B-cell lymphoma, we show the feasibility of this approach. With the matrix-CGH protocol presented here, robust and reliable detection of genomic gains and losses is accomplished in an automated fashion, which provides the basis for widespread application in tumor and clinical genetics.
