Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.

Enhancers for RNA polymerase I in mouse ribosomal DNA.

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

A comparison of men and women with occlusive sleep apnea syndrome.

Among 118 patients with occlusive sleep apnea syndrome (OSA), defined as daytime hypersomnolence and an apnea hypopnea index (AHI) greater than ten events/h, 41 women were compared with 77 men. Body mass index, spirometric study, PaO2, PaCO2, and results from nocturnal polysomnography were examined in a two-way analysis of variance (ANOVA) for the effects of sex, age group, and a sex-age group interaction. The age groups examined were above and below 42 years, the breakpoint for menopause in the women. Younger persons tended to be more obese and to have a higher AHI. Both sexes had similar pulmonary function, AHI, and nocturnal desaturation, but women experienced significantly fewer completely occluded breathing events and had apneas of shorter mean and maximum duration than men of similar ages. No effect of menopausal status per se was observed. In OSA patients, differences in upper airway occlusion and apnea duration suggest differences between the sexes in upper airway physiology or respiratory control.

A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin.

Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.

In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta-tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

RNA polymerase specificity of mRNA production and enhancer action.

To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific.

Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses.

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.

Four unique genes required for beta tubulin expression in vertebrates.

We have isolated the four separate segments of chicken DNA which contain sequence homology to beta tubulin. With the exception of a fifth region of DNA which appears to contain only a 5' fragment of a beta gene, these four cloned sequences represent all of the beta tubulin encoding DNA in the chicken. Each gene is very similar in structure, containing three or four small intervening sequences clustered in the 5' portion of the coding region. Using RNAs prepared from a variety of cell lines and tissues, we have found five different mRNAs which carry beta tubulin sequences, two of which are encoded by the same gene. Three of these mRNAs are unexpectedly long (between 3500 and 4000 bases). However, these large mRNAs do give authentic beta tubulin translation products. Overall, we conclude that each of the four beta tubulin genes is a functional gene which is expressed in a specific program during differentiation. These data strongly suggest that four beta tubulins are necessary for proper microtubule function in vertebrates.

Unpolymerized tubulin modulates the level of tubulin mRNAs.

Although numerous studies have suggested ways in which the assembly of cytoskeletal proteins can be regulated physiologically, less information has been generated on the regulation of the synthesis of these proteins. Ben-Ze'ev et al. recently suggested that the synthesis of tubulin in mouse 3T6 cells is affected by the state of assembly of microtubules. We have investigated the level at which this apparent modulation of tubulin synthesis takes place, using cloned cDNA probes for alpha- and beta-tubulin mRNAs to measure the amounts of tubulin mRNAs combined with immunoprecipitation of tubulin to monitor the rate of protein synthesis. We have found that in many, but not all, cell types tubulin synthesis decreases very rapidly in response to microtubule inhibitors that increase the monomer pool. This decline in synthesis is associated with decline in the amounts of both alpha- and beta-tubulin mRNAs. Kinetic studies of tubulin protein synthesis and RNA levels suggest that the tubulin monomer may regulate the rate of tubulin mRNA transcription. It is likely that tubulin synthesis can be shut off quickly in the cell as the result of short half-lives of the tubulin mRNAs, which may be as short as 1--2 hr. These data suggest that the cell exploits the instability of the tubulin mRNAs as a means to regulate precise levels of the monomer-tubulin pool.

Multiple sites for the initiation of microtubule assembly in mammalian cells.

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.

Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.