Comparison of three 3' non-coding regions in Schizosaccharomyces pombe expression vectors: efficiencies of transcription termination and mRNA 3'-end formation.

Analysis of an established Schizosaccharomyces pombe episomal shuttle vector suggested that inefficient transcription termination was deleterious to plasmid function. We undertook a study to determine if transcription in the presence and absence of 3'-processing within a vector could affect the ability of the plasmid to transform, transcribe and translate the RNA produced. This report provides an analysis of the effects that three S. pombe 3' non-coding regions have on the transformation and expression efficiencies of a fission yeast plasmid vector. The 3' regions from adh1, act1 and ura4 were tested for their ability to terminate and process adh1 promoter-driven transcription of a lacZ reporter gene. Differences between the 3'-processing sequences were observed, with transcription termination mediated by the ura4 3' region being more efficient than termination by the 3' regions of adh1 and act1. We show that plasmids containing inefficient transcription termination signals result in readthrough transcription and reduced transformation efficiencies. In addition, the readthrough transcripts containing 3' non-coding regions show impaired translation efficiencies. We describe an S. pombe vector (pURAS) with a high transformation efficiency that directs the production of highly translatable, discrete-sized transcripts.

Expression and analysis of the green fluorescent protein gene in the fission yeast Schizosaccharomyces pombe.

This report demonstrates that the Aequorea victoria green fluorescence protein (gfp) gene product will fluoresce in the fission yeast Schizosaccharomyces pombe when expressed from an episomal expression vector. Fluorescence was readily detectable at both the colony and single cell level. Application of fluorescence-activated cell sorting (FACS) techniques showed that gfp-expressing cells could be detected when they were as rare as 1% of a total yeast population. Quantitative analysis of gfp-expressing cells constituting as little as 5% of a total population was possible. These observations establish the suitability of the gfp gene for use in S. pombe and, in combination with FACS, offers an experimental strategy for quantitative analysis of gene expression in yeast populations.

Gene regulation by antisense RNA in the fission yeast Schizosaccharomyces pombe.

This report describes experiments designed to demonstrate the suitability of the fission yeast Schizosaccharomyces pombe as a host for antisense RNA regulation. A lacZ gene-expressing yeast strain was constructed and used as a host for the expression of a series of antisense RNAs complementary to various regions of the target lacZ mRNA. All lacZ antisense genes were placed under control of the thiamine-repressible nmt1 promoter of S. pombe and expressed from episomal plasmids. For each antisense plasmid a corresponding sense control plasmid was constructed. All lacZ antisense genes were shown to express antisense RNAs of the expected size at equivalent steady-state levels. beta-Galactosidase activity in transformed cells expressing the long, short 5' or short 3' lacZ antisense RNAs was shown to be reduced by 45%, 20%, and 10%, respectively, relative to control transformants. Further experiments indicated that antisense RNA regulation in this system was conditional and reversible, with the observed reduction of beta-galactosidase activity being dependent on the transcription of lacZ antisense RNA. Our results represent the first successful example of antisense RNA regulation of gene expression in yeast and establish S. pombe as an experimental model for the biochemical analysis of antisense RNA regulation.

The ade6 gene of the fission yeast as a target for antisense and ribozyme RNA-mediated suppression.

A genetic system for the analysis of antisense and ribozyme mechanisms is a much needed experimental tool, and yeast represent a favorable organism on which to base such a system. We have shown previously that the fission yeast Schizosaccharomyces pombe has potential to satisfy the requirements of such a system. This report describes experiments designed to determine if antisense and ribozyme RNA-mediated gene suppression will be generally applicable to other genes in S. pombe. Antisense and ribozyme RNAs designed to suppress the ade6 gene were expressed at high levels from episomal expression vectors. The ade6 gene was chosen as a target as mutations within the gene confer adenine auxotrophy and a red colony phenotype, and it was expected that antisense or ribozyme RNA-mediated mutant phenocopies would exhibit the same readily detectable phenotype. No phenotypic indication of ade6 suppression was detected in transformed yeast, and ade6 target mRNA was analyzed by primer extension and Northern analysis. Initially, conflicting results were obtained from these techniques, which were determined to be due to duplex formation between antisense and target RNA in vitro. No detectable reduction in the ade6 mRNA levels was found, and it was concluded that the gene was not suppressed by the antisense or ribozyme RNAs tested. These results confirm that in S. pombe as with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene specific and that design of antisense and ribozyme genes will be an empirical process.

Antisense gene expression in yeast.

The use of antisense and ribozyme RNA to modulate gene expression is emerging as an effective genetic technique. A compilation of successful antisense gene suppression experiments reveals the absence of reports on the use of the yeast Saccharomyces cerevisiae as a host. We examine the field of antisense and ribozyme use in S. cerevisiae and discuss that this result is not due to any lack of attempts and may reflect unique features of S. cerevisiae biology. In an attempt to learn from cellular RNA physiology we review evidence for naturally occurring antisense RNA regulation. Although there are many examples of well characterised overlapping RNA transcripts there is, as yet, no clear evidence suggesting complementary RNA-dependent gene regulation in S. cerevisiae. The application of artificial antisense and ribozyme genes is then discussed with an emphasis on the role of yeast as a model system for the systematic and genetic analysis of antisense and ribozyme RNA function. In addition, potential reasons for the lack of attempts to use antisense or ribozyme genes to create pseudogenetic mutants are considered. We conclude that the application of successful antisense and ribozyme strategies in yeast may have to address features of S. cerevisiae RNA biology and offer experimental approaches that may identify some of these features.

Differential regulation of skeletal muscle myosin-II and brush border myosin-I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms.

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-II 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 alpha-tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity for F-actin than a tissue-purified striated muscle alpha tropomyosin. The bacterially produced isoforms also protect F-actin from severing by villin as effectively as tissue-purified striated muscle alpha-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 approximately 70-85%, comparable to the inhibition seen with tissue-purified striated muscle alpha 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-II (2.4 microns/sec at 19 degrees C, 5.0 microns/s at 24 degrees C) increased 25-65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-II motility. The actin-activated 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 nm/s) 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.

Xenopus annexin II (calpactin I) heavy chain has a distinct amino terminus.

We have isolated cDNAs encoding annexin II (calpactin I) heavy chain homologues from a Xenopus oocyte cDNA library. Two of the clones are full length, while two appear to be derived from incompletely spliced mRNAs. The 1230- and 1240-base pair full length clones are 99% identical, and both have 84 bases of 5'-untranslated sequence, 1020-base open reading frames, and either 126- or 136-base 3'-untranslated domains. Northern blots show a 1.4-kilobase (kb) annexin II heavy chain transcript throughout oogenesis and in mature eggs. Xenopus annexin II mRNA levels are constant during early embryogenesis, but decrease at 8 h. After midblastula transition, the steady state level of the 1.4-kb transcript increases substantially, and a 3.3-kb transcript appears. Adult brain, heart, striated muscle, and liver contain moderate amounts of the 1.4-kb transcript, while skin has the 3.3-kb transcript and very high levels of the 1.4-kb transcript. Synthetic mRNA derived from the Xenopus annexin II cDNAs directs the synthesis of an apparent Mr = 36,500 polypeptide when microinjected into Xenopus oocytes. The predicted 339-amino acid protein products are 80% identical with murine annexin II heavy chain. Most of the differences are concentrated in the amino end from residues 15 to 24. The Xenopus annexin II heavy chain lacks the highly conserved tyrosine at position 23 which is the site of src oncogene tyrosine kinase phosphorylation in the murine protein. These results demonstrate that Xenopus oocytes contain an annexin II (calpactin I) heavy chain mRNA with a distinct amino terminus and suggest that multiple annexin II isoforms may be expressed during amphibian embryogenesis and development.

Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA.

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.

Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis.

As an alternative approach to classical genetic analysis, we are investigating the potential of anti-sense (nonsense) DNA strand transcription to inhibit gene activity. A promoter will direct transcription of the complementary nonsense DNA strand when the protein coding sequence of a cloned gene is excised and then reinserted in reverse orientation. When such flipped gene constructions of the HSV thymidine kinase (TK) gene are coinjected with the wild-type gene at a 100:1 ratio, there is a reduction of transient TK expression in TK- mouse L cells. The proportion of viable cells with demonstrable TK activity drops 4-fold as compared with neighboring cells coinjected with TK and an excess of control plasmid. Furthermore, autoradiography of the cells still expressing TK shows that 3H-thymidine incorporation is reduced. Cells contransformed with flipped TK gene constructions have a reduced capacity to express subsequently microinjected TK genes, suggesting that the anti-message phenomenon is due to a trans-inhibition of TK and is probably not an artifact of rearrangements following microinjection.

A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells.

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.

The role of calcium ions during mitosis. Calcium participates in the anaphase trigger.

Calcium-containing solutions were microinjected into dividing PtK1 cells to assess the effect of calcium ion concentration on the morphology and physiology of the mitotic spindle. Solutions containing 50 microM or more CaCl2 are immediately and irreversibly toxic to PtK1 cells. Those containing 5-10 microM CaCl2 cause reversible reduction in spindle birefringence followed by normal anaphase and cytokinesis. Microinjection of 5 microM or less CaCl2 into anaphase PtK1 cells has no detectable effect on the rate or extent of chromosome movement. Metaphase cells tend to enter anaphase 4-5 min after injection with 1-10 microM CaCl2, compared with an average of 16 min after injection with calcium-free buffer. Reducing the intracellular calcium concentration by injection of EGTA-CaCl2 buffers increases the lag between injection and anaphase to 20 min or more. Microinjection of calcium solutions does not promote precocious chromatid separation in nocodazole-arrested metaphase cells, indicating that the increase in calcium concentration does not induce centromere separation directly. An increase in the concentration of free calcium ions during metaphase appears to stimulate the onset of anaphase. Such an increase, regulated by the cell itself, may contribute to the initiation of chromosome separation in mammalian cells.

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins.

Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca(2+) sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas microtubules are completely stable at Ca(2+) concentrations below 1 muM, Ca(2+) at greater than 1-4 muM induces microtubule disassembly that begins in the cell periphery and proceeds towards the cell center. At concentrations of up to 500 muM, both the pattern and time course of disassembly are not markedly altered, suggesting that, within this concentration range, Ca(2+) effects are catalytic rather than stoichiometric. Higher (millimolar) Ca(2+) concentration results in rapid destruction of microtubules. Of other divalent cations, only Sr(2+) has a slight depolymerizing effect, whereas millimolar Ba(2+), Mg(2+), or Mn(2+) is ineffective. Disassembly induced by micromolar Ca(2+) is inhibited by pharmacological agents known to bind to calmodulin and inhibit its function, suggesting that calmodulin mediates Ca(2+) effects. Both the addition of exogenous brain microtubule-associated proteins (MAPs) after lysis and the retention of endogenous cellular MAPs normally extracted during the lysis step stabilize microtubules against the depolymerizing effect of micromolar Ca(2+). The results indicate that, in this model system, microtubules are sensitive to physiological Ca(2+) concentrations and that this sensitivity may be conferred by calmodulin associated with the microtubules. MAPs appear to have a modulating effect on microtubular Ca(2+) sensitivity and thus may function as a discriminating factor in cellular functions performed by calmodulin. It is hypothesized that Ca(2+)-stimulated microtubule disassembly depends on the relative amount of MAPs.

Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons.

Hybridomas that secret IgG reacting specifically with the brain microtubule-associated protein MAP2 have been prepared with speen cells from BALB/c mice hyperimmunized with high molecular weight neurotubule-associated proteins. Immunofluorecence 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 PtK1 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 with 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.

Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis.

A two-dimensional gel electrophoresis system is used to investigate some of the properties of desmin, the major subunit of the 100-A filaments from chick muscle cells, and to compare these properties to those of the other major contractile and regulatory proteins of muscle. Desmin from embryonic and adult smooth, skeletal, and cardiac muscle cells is resolved into two isoelectric variants, alpha and beta, which possess slightly different electrophoretic mobilities in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both the alpha and the beta variants from all six preparations appear to be identical in isoelectric point and apparent molecular weight. The alpha and beta desmin are present in approximately equal amounts in all three types of muscle, suggesting that both isoelectric variants of desmin serve as the structural subunits of the 100-A filaments in chick muscle cells. Tropomyosin also can be resolved into two subunits, alpha and beta, in all three types of muscle. However, in each type of muscle both subunits differ from their counterparts in the other types of muscle, either by molecular weight or by isoelectric point. These results indicate that, with regard to apparent isoelectric point and molecular weight, desmin, a major muscle structural protein, is invariant, while tropomyosin, a major muscle regulatory protein, exhibits heterogeneity in the three types of muscle.