No abstract

Plasmid DNA directing transcription of the noncoding (anti-sense) DNA strand can specifically inhibit the expression of several test genes as well as normal, endogenous genes. The anti-sense plasmid constructions can be introduced into eukaryotic cells by transfection or microinjection and function in both transient and stable transformation assays. Anti-sense transcripts complementary to as little as 52 bases of 5' untranslated target gene mRNA specifically suppress gene activity as well as, or more efficiently than, anti-sense transcripts directed against the protein coding domain alone. Conditional anti-sense inhibition is accomplished with the use of hormone-inducible promoter sequences. Suppression of endogenous actin gene activity by anti-sense RNA is detected as a decrease in growth rate and as a reduction in the number of actin microfilament cables. These observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.

Hybridomas that secret IgG reacting specifically with the brain microtubule-associated protein MAP2 have been prepared with spleen cells from BALB/c mice hyperimmunized with high molecular weight neurotubule-associated proteins. Immunofluorescence microscopy using dual fluorochrome labeling of tubulin and MAP2 antigens revealed identical patterns of interphase fiber networks in cells from explants of newborn mouse brain. The anti-MAP2 antibody did not stain primary mouse kidney cells or CHO, 3T3, HeLa, or PtKI cell lines. Immunoprecipitation and antibody gel staining techniques failed to demonstrate any crossreacting antigen in these cells. MAP2 antigen was not seen in association with the mitotic spindle in any of the cells examined. Radioimmunoassay showed species crossreactivity of the anti-MAP2 antibody wi mammalian but not avian neural cell extracts. Glial cells and some neuroblastoma cell lines did not appear to contain MAP2. However, in the B104 rat neuroblastoma cell line the MAP2 antigen appeared to be associated with the cytoskeleton concomitant with differentiation induced by dibutyryl cyclic AMP.In disagreement with most previously published reports, our data suggest that MAP2 is found only in differentiated neuronal cells and raises the possibility that MAP2 is involved in neuronal differentiation or neuron-specific processes. Two classes of microtubule-associated proteins (MAPs) copurify with brain tubulin through cycles of assembly/disassembly: tau factor, 55,000-62,000 daltons (1, 2); and the high molecular weight protein (HMW), 250,000-350,000 daltons (3, 4). These MAPs increase rates of both nucleation and elongation during tubulin polymerization in vitro (2, 4), and it has been suggested that they play a role in the control of the in vivo utilization of microtubules during interphase and cell division. Consistent with this hypothesis, antisera prepared against MAPs stain interphase microtubule networks and mitotic spindles in a wide variety of cells (5-9). Recently, however, carefully characterized antisera to high molecular weight assembly MAPs from brain and cultured cells have been shown to have limited crossreactivity between different cell types (ref. 10; unpublished results) and species (11).The reasons for the discrepancy between the different antisera are not easily understood. Serum contains many different immunoglobulins, and it is possible that one subset of immunoglobulin is giving rise to immunofluorescent staining patterns whereas another is responsible for Ouchterlony immunoprecipitation lines. This complexity is not remedied by affinity chromatography purification of the antiserum because, during elution, partial denaturation of antibody may occur, possibly modifying the specificity of the immunoglobulins.To alleviate some of the problems inherent in the use of animal sera, we have been studying the in vvo function of HMW by using monoclonal antibody probes produced against MAPs according to the lymphocyte hybridoma method originated by Kohler and Milstein (12). In th...

Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca2+ sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas

In this report, we have compared the physical properties and actin-binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin-I1 and brush border (BB) myosin-I. All of the bacterially produced nonmuscle tropomyosins bind to F-actin with the expected stoichiometry and with affinities comparable to that of a tissue produced a-tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity for F-actin than a tissue-purified striated muscle a tropomyosin. The bacterially produced isoforms also protect F-actin from severing by villin as effectively as tissue-purified striated muscle a-tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin-activated MgATPase activity of muscle myosin S1 by -70-85%, comparable to the inhibition seen with tissue-purified striated muscle a tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin-activated MgATPase activity of muscle myosin S1 approximately two-to threefold. The in vitro sliding of actin filaments translocated by muscle myosin-11 (2.4 Wdsec at 19"C, 5.0 p d s at 24°C) increased 25-65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-IT motility. The actinactivated MgATPase activity of BB myosin-I was inhibited 75-90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin-I motility (50 nmis) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo. 0 1994 Wiley-Lisu, Inc.