Biomedical subjects

K Ainger

Publications and source records attributed to K Ainger.

8 recordsLinked to original sources

PubMed1997

Transport and localization elements in myelin basic protein mRNA.

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3' UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1, 473 in the 3' UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.

Actins↗

PubMed1997

Translocation of myelin basic protein mRNA in oligodendrocytes requires microtubules and kinesin.

Myelin basic protein (MBP) mRNA is localized to the myelin membranes of oligodendrocytes. When exogenous MBP mRNA is microinjected into oligodendrocytes in culture, it is transported along the processes and localized to the myelin compartment in a multistep intracellular RNA trafficking pathway. In the work described here, oligodendrocytes were treated with agents that affect the cytoskeleton including: nocodazole, to disrupt microtubules; taxol, to stabilize microtubules; cytochalasin, to disrupt microfilaments; and kinesin anti-sense oligonucleotide, to suppress kinesin expression. Digoxigenin-labeled MBP mRNA was microinjected into the treated cells and the extent of translocation of the microinjected RNA was determined by confocal microscopy. Nocodazole, taxol, and kinesin anti-sense oligonucleotide inhibited translocation of microinjected MBP mRNA, while cytochalasin B and kinesin sense oligonucleotide did not. These results indicate that translocation of MBP mRNA in oligodendrocytes requires intact microtubules and kinesin but does not require intact microfilaments. The results are discussed in relation to the current multistep model for intracellular RNA trafficking in oligodendrocytes.

Actin Cytoskeleton↗

PubMed1995

Protein translation components are colocalized in granules in oligodendrocytes.

The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

Animals↗

PubMed1992

Molecular genetic analysis of the mldr mouse: a spontaneous revertant at the mld locus containing a recombinant myelin basic protein gene.

The mld mutation is a complex genetic lesion affecting the myelin basic protein (MBP) locus in the mouse. The mutation consists of a variety of DNA rearrangements including: tandem duplication of the MBP structural gene, partial inversion of the 3' end of the upstream gene copy, duplication of a region flanking the rearrangement junction in the upstream copy and insertion between the two gene copies of a segment of extraneous DNA not associated with the wild-type MBP locus. The net result of the mutation is a dysfunctional MBP locus. Homozygous mld/mld mice produce very little MBP and consequently very little myelin. They exhibit a clinical phenotype characteristic of hypomyelination (shaking, convulsions). We have discovered a revertant mld mouse which does not exhibit clinical symptoms of hypomyelination. Genetic analysis indicates that the reversion is allelic to mld. We have designated the revertant locus mldr. Restriction analysis of mldr genomic DNA indicates that there is a single intact MBP gene. Analysis of various junction regions using the polymerase chain reaction indicates that the single MBP gene in mldr is derived by recombination from the 5' end of the upstream gene and the 3' end of the downstream gene. Studies on MBP expression in mldr mice indicate that the developmental regulation, level of expression and pattern of post-transcriptional processing of MBP gene products in mldr are similar to wild type. These results indicate that the recombinant MBP gene in mldr is fully functional. From this we infer that the MBP-deficient phenotype of the original mld mutant is attributable to the complex rearrangements in the upstream gene copy which render the locus dysfunctional.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1989

Type VI collagen: high yields of a molecule with multiple forms of alpha 3 chain from avian and human tissues.

A differential extraction procedure followed by molecular sieve column chromatography for the isolation of large quantities of the tissue form of type VI collagen is described. Recovery of the protein was more than 60% from both chick gizzard and human placenta. On reduced NaDodSO4-gels chick type VI collagen migrated as two major bands at Mr = 140,000 and 150,000 that were present in a 1:1 ratio and five less intense bands between Mr = 230,000 and 180,000. By immunoblotting with a polyclonal antibody against the pepsinized form of chick type VI collagen, all these bands were stained. Furthermore, the amino acid composition of the five higher Mr polypeptides indicated that they all contained hydroxyproline and hydroxylysine. In the chick type VI collagen molecule the five bands of higher Mr belong to the alpha 3 chain since they were recognized by monoclonal antibodies specific for the chick Mr = 260,000 alpha 3 chain. On examination of antigenic activity by solid-phase radioimmunobinding, densitometry of stained NaDodSO4 polyacrylamide gels, and protein content type VI was found to be an abundant collagen since it accounted for up to 0.1% of the tissue wet weight. The yields per tissue wet weight and the migration pattern of human type VI collagen polypeptides were similar to those of the chick. Agarose/polyacrylamide composite gels indicated that the molecular size of the tissue form of type VI collagen molecules under non-reduced conditions corresponded to a basic type of tetrameric molecule.

Amino Acids↗

PubMed1988

Monoclonal antibodies for the different chains of chick type VI collagen.

Avian type VI collagen is composed of three subunits of Mr 140,000, 150,000 and 260,000. Monoclonal antibodies were raised against type VI collagen isolated from chick embryo gizzard, and these antibodies were used to immunoprecipitate type VI collagen from metabolically labeled embryo cells. Several antibodies appeared to react with epitopes independent of glycosylation and hydroxylation processes. The antibody-binding sites were identified on the different chains by immunoblotting of total cell extracts. In addition, antibodies that recognized different epitopes on the Mr 260,000 subunit could be grouped in at least three different clusters by competitive inhibition radioimmunobinding assays.

Animals↗

PubMed1987

Biosynthesis of chick type VI collagen. I. Intracellular assembly and molecular structure.

A monospecific rabbit antiserum to pepsin-extracted chick gizzard type VI collagen was used to characterize the intact forms of type VI collagen in tissues and cultured cells. Immunoblotting of gizzard extracts revealed polypeptides of Mr ranging from 260,000 to 140,000. Components of about Mr = 260,000, 150,000, and 140,000, each with a different peptide profile, were immunoprecipitated from labeled matrix-free chick embryo cells. Cleavage of the immunoprecipitated polypeptides with pepsin generated pepsin-resistant fragments of about Mr = 70,000, 55,000, and 45,000 that represent the alpha 1(VI), alpha 2(VI), and alpha 3 (VI) fragments. Immunoblotting with affinity-purified antibodies indicated that the Mr = 150,000 is the intact parent polypeptide of the alpha 1(VI) pepsin; the Mr = 140,000 of the alpha 2(VI) pepsin, and the Mr = 260,000 of the alpha 3(VI) pepsin. Association of the three parent chains was studied by pulse-chase experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under nonreduced conditions. A complex of Mr = 500,000 is already present intracellularly at the end of a 7-min pulse and increases considerably with time while the three unassembled chains show a comparable decrease. After 5-15 min of chase larger forms appeared along with small amounts of aggregated material that did not enter the gel. Analysis of the immunoprecipitate by diagonal electrophoresis indicated that the component of Mr = 500,000 and the larger forms dissociated into the Mr = 260,000, 150,000, and 140,000 polypeptides. Sedimentation profile of a labeled cell extract on a 5-20% sucrose gradient under nondenaturing conditions confirmed the presence of three different peptides in the complex.

Animals↗