Structural annotation of equine protein-coding genes determined by mRNA sequencing.

The horse, like the majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for the majority of genes defined in the equine genome are predictions based on ab initio sequence analysis or the projection of gene structures from other mammalian species. The current study used Illumina-based sequencing of messenger RNA (RNA-seq) to help refine structural annotation of equine protein-coding genes and for a preliminary assessment of gene expression patterns. Sequencing of mRNA from eight equine tissues generated 293,758105 sequence tags of 35 bases each, equalling 10.28 gbp of total sequence data. The tag alignments represent approximately 207 × coverage of the equine mRNA transcriptome and confirmed transcriptional activity for roughly 90% of the protein-coding gene structures predicted by Ensembl and NCBI. Tag coverage was sufficient to refine the structural annotation for 11,356 of these predicted genes, while also identifying an additional 456 transcripts with exon/intron features that are not listed by either Ensembl or NCBI. Genomic locus data and intervals for the protein-coding genes predicted by the Ensembl and NCBI annotation pipelines were combined with 75,116 RNA-seq-derived transcriptional units to generate a consensus equine protein-coding gene set of 20,302 defined loci. Gene ontology annotation was used to compare the functional and structural categories of genes expressed in either a tissue-restricted pattern or broadly across all tissue samples.

Alternative processing of the human and mouse raly genes(1).

A human homolog(RALY) of the mouse Raly gene was isolated and sequenced, and shown to encode a novel protein isoform containing a 16 amino acid in-frame insert in the variable region of the protein. Analysis of the corresponding region of the mouse Raly gene demonstrated that this novel protein isoform is also present in the mouse. Comparative analysis of RALY cDNA and EST sequences suggests the presence of additional alternatively processed RALY transcripts. As in the mouse, the human RALY gene is widely expressed as a 1.7-kb transcript.

Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Ak molecules. Ak beta TM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Ak conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Ak beta chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Ak beta chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to assess a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the Ak alpha Cy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.

Class II cytoplasmic and transmembrane domains are not required for class II-mediated B cell spreading.

B cells cultured on immobilized anti-class II monoclonal antibody (mAb) change from round to flattened cells, with lamellipodia and filopodia. This change in cell morphology, termed 'spiders', occurs within 30 min upon culture and is mediated through either I-A or I-E molecules. Class II molecules that are defective in mediating protein kinase C (PKC) due to the deletions of both alpha and beta chain's cytoplasmic (Cy) domain sequences can induce spider formation. B-cell transfectants that express chimeric MHC class II/class I molecules, where the ectodomains are class II sequences and the transmembrane and Cy domains are class I sequences also form spiders when cultured on anti-class II mAb. The spider morphology is not induced by either anti-immunoglobulin (Ig) or anti-MHC class I mAb. Treatment of B cells to increase intracellular cAMP, a component of the class II signaling pathway also results in spider formation with the same kinetics and percent change in the responding population as that induced by anti-class II mAb. Cytochalasin A treatment which disrupts cytoskeletal actin filaments and the tyrosine kinase inhibitor, genistein, both inhibit spider formation. Actin redistributes from a concentric ring in round cells to the ends of the filopodia in the spiders. The mechanism of spider induction whether resultant from second messengers following class II signaling or from non-signaling-induced physical interactions of class II with intracellular cytoskeletal components only requires the extracellular domains of class II. The biologic relevance of B-cell spiders is currently not known but has been reported to be associated with class II signal transduction and efficient Ag presentation.

Participation of membrane skeleton proteins in aggregation of epidermal growth factor receptors in A431 cells.

Polyclonal antibody against alpha-spectrin of chicken erythrocytes was prepared. This antibody as well as anti-vinculin and anti-annexin I and II, were used for localization of the antigens in A431 cells during translocation of epidermal growth factor receptors (EGF-Rs) on cell surface. During aggregation of EGF-Rs only spectrin and actin aggregates colocalized with the "capped" receptors in adherent as well as in suspended cells. Physiological implication of spectrin involvement in EGF-Rs redistribution in A431 cells is discussed.

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation.

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m2) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.

Utilization of microhomologous recombination in yeast to generate targeting constructs for mammalian genes.

We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice. This procedure is rapid and efficient, and should be directly applicable to all mammalian genes. Moreover, only minimal information about the locus being targeted is required. The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene.

Heat shock proteins in Amoeba: I. Effect of high temperature on Amoeba proteus and Amoeba borokensis.

A sudden increase of temperature of surrounding medium, even in the sublethal range, induced in Amoeba proteus (strain War) and Amoeba borockensis the synthesis of a new class of proteins, which might be classified as the heat shock proteins (hsp). The new pattern of protein synthesis, determined by incorporation of (35)S-methionine, was detectable within 30 min after beginning of the exposure of the cells to the elevated temperature. Amoeba proteus (strain War) and Amoeba borokensis differ distinctly in thermosensitivity, however, no distinct difference in the rate of the heat shock proteins' synthesis between the studied species was found.

[The capping of receptors for the epidermal growth factor and the participation of the cytoskeleton in this process in A-431 cells]. / Képping retseptorov k épidermal'nomu faktoru rosta i uchastie tsitoskeleta v étom protsesse u kletok A-431.

Epidermal growth factor (EGF) receptor capping results from the interaction between the receptors and polyvalent ligands in A-431 cells examined in suspension at 22 degrees C. Colocalization of actin and spectrin with the ligand-receptor complexes during the redistribution was shown using double immunofluorescence. The obtained data show that the cortical microfilaments are involved in capping. EGF receptors become associated with the Triton-insoluble cytoskeleton as a consequence of ligand binding. EGF-receptor capping is not sensitive to the action of cytochalasin B. Capping in A-431 cells is discussed as a new model for studying the redistribution of the ligand-receptor complex.

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) is the key photosynthetic enzyme that catalyzes the first step of CO2 fixation. The chloroplast-localized holoenzyme of plants and green algae contains eight nuclear-encoded small subunits and eight chloroplast-encoded large subunits. Although much has been learned about the enzyme active site that resides within each large subunit, it has been difficult to assess the role of eukaryotic small subunits in holoenzyme function and expression. Small subunits are coded by a family of genes, precluding genetic screening or nuclear transformation approaches for the recovery of small-subunit mutants. In this study, the two small-subunit mutants. In this study, the two small-subunit genes of the green alga Chlamydomonas reinhardtii were eliminated during random insertional mutagenesis. The photosynthesis-deficient deletion mutant can be complemented with either of the two wild-type small-subunit genes or with a chimeric gene that contains features of both. Thus, either small subunit is sufficient for holoenzyme assembly and function. In the absence of small subunits, expression of chloroplast-encoded large subunits appears to be inhibited at the level of translation.

Actin-binding proteins involved in the capping of epidermal growth factor receptors in A431 cells.

A capping process of epidermal growth factor receptors (EGF-Rs) was used for the study of the relation between the receptors and the actin-binding proteins (spectrin, vinculin, annexin I) that may be involved in EGF-R-cytoskeleton interaction. In intact, adherent A431 cells, EGF-Rs were diffusively distributed on the cell surface. Spectrin, vinculin, and annexin I were located beneath the plasma membrane. An abundance of EGF-Rs as well as submembrane proteins was observed in regions of membrane ruffles and cell-cell contacts. Annexin I was localized also in cytoplasm being attached to filamentous structures surrounding the nucleus and extending to the cell periphery. Under polyvalent ligand treatment, EGF-Rs of adherent cells were aggregated on one side of the cell. Spectrin, vinculin, and annexin I dislocated together with EGF-Rs and were concentrated under plasma membrane at regions where cap formation took place. In suspended A431 cells only spectrin was located under the plasma membrane whereas annexin I and vinculin were diffusively distributed through the cells. During cap formation only spectrin was colocalized with EGF-Rs. The results confirmed the major role of spectrin as a receptor-microfilament linking protein.

The role of microfilaments in the capping of epidermal growth factor receptor in A431 cells.

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.

MHC class II molecules that lack cytoplasmic domains are associated with the cytoskeleton.

MHC class II molecules, composed of alpha- and beta-chain heterodimers, are required for Ag presentation. The carboxyl-terminal domains of class II molecules are believed to mediate the location of class II in the plasma membrane and are important for signal transduction and Ag presentation. These domains contain typical transmembrane sequences, and cytoplasmic sequences of 12 or 18 amino acids for the alpha- and beta-chains, respectively. We examined these domains to determine whether they linked class II molecules to the actin-based cytoskeleton. Our analyses of class II-cytoskeleton interactions, such as a colocalization with actin filaments during capping, association with the detergent-insoluble cytoskeleton, and direct binding of filamentous actin, revealed that both the cytoplasmic and transmembrane domains contributed to class II interactions with the cytoskeleton. Detergent-extracted and immunoprecipitated full-length class II molecules had quantitatively stronger interactions with the cytoskeleton than did molecules with deleted cytoplasmic domains. A secondary Ab, which was used to cross-link primary Ab bound to class II, up-regulated the class II-cytoskeletal associations. This association was efficiently inhibited by dihydrocytochalasin B, but only partially disrupted by chlorpromazine. The mechanism of interaction with actin filaments after ligation of class II occurred without a measurable increase in filamentous actin levels. This suggested that enhanced class II-cytoskeleton associations involved a rearrangement of existing actin filaments, possibly through the multiple kinases that are activated after class II transmembrane signaling.