Α-tubulin Gene Research Articles

Premature Termination of Transcription Can Be Induced on an Injected α-Tubulin Gene in Xenopus Oocytes

The Xenopus laevis alpha-tubulin gene X alpha T14, which is highly expressed during oogenesis, exhibits accurate and efficient transcription initiation when microinjected into X. laevis oocytes. However, we found previously in nuclease protection assays of transcripts from injected X alpha T14 that many protected fragments that were shorter than expected could be produced. We show here by exonuclease VII mapping, Northern (RNA) blotting, and gel fractionation of RNA that these fragments were caused by truncated transcripts that share the same initiation sites as mature transcripts but whose 3' ends are located in the 5' leader just 45 to 72 nucleotides downstream. We present evidence from the analysis of in vitro pulse-labeled RNA that these truncated transcripts are formed by premature transcription termination rather than by RNA processing. At low template levels, very little premature termination occurred, but as more DNA was injected, the proportion of transcripts that were prematurely terminated increased steadily, even at template levels at which the initiation machinery was unsaturated. At high template levels, most transcripts were prematurely terminated. These results suggest that some sort of saturable antitermination function operates in oocytes in a manner that is dependent on the number of appropriate templates available rather than on the number of polymerases that initiate transcription. They also suggest that measures of initiation frequency may not always be a reliable means of assessing the amount of transcription of injected genes in oocytes.

Differential effects of N-methyl- N′-nitro- N-nitrosoguanidine on constitutive and hormone-inducible gene expression in rat hepatoma cells

Previous studies have shown that treatment of rat hepatoma cells (the Fao clone of Reuber H-35 cells) with 500 ng/ml of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) causes a 78% decrease in dexamethasone (DEX)-induced tyrosine aminotransferase (TAT) enzyme activity and a concurrent 75% decline in total steroid-induced TAT steady-state RNA levels. To determine if this inhibition was a specific or more general effect on inducible-gene expression, the effects of MNNG on other genes were examined. MNNG had little effect on total DEX or Cd-induced metallothionein (MT) RNA levels when the cells were treated with 1 μM DEX or 3 μM CdCl 2 for 4 h. In addition, the carcinogen had no effect on the basal level of MT-specific total RNA, nor did it alter the total RNA levels of the α-tubulin gene. Although attempts were made to measure the levels of the glucocorticoid receptor by both biochemical and molecular methods, receptor levels were too low to quantitate accurately. However, the lack of effect of MNNG on steroid-induced MT RNA levels suggests that the inhibitory effect of the carcinogen was not mediated through alterations in glucocorticoid receptor function. MNNG had no effect on cell number or viability, nor did the carcinogen alter the methylation pattern of the TAT gene as determined from MspI/ HpaII digests. The results suggest that MNNG mediates its inhibitory effects by a specific interaction with either the TAT gene itself or some other regulatory factor(s) involved in TAT RNA transcription or stability. This effect was relatively gene specific, since expression of the inducible, specialized liver function TAT gene was inhibited by MNNG but expression of the more ubiquitous and inducible MT gene and the constitutively expressed α-tubulin gene were not.

Short interspersed repeats fromXenopusthat contain multiple octamer motifs are related to known transposable elements

We have identified in an intron of an X. laevis alpha-tubulin gene a member of a novel family of short (226-431 bp) interspersed repetitive elements. We have isolated other members of this family, which we term Ocr, from ovary cDNA and genome libraries and have identified another two in the published sequences of an H1B histone gene cluster and an actin gene intron. The termini of the Ocr elements are formed by a 19 bp inverted repeat that has clear sequence homologies to those of certain large transposable elements, such as 1723 (Xenopus) and Ac (maize). However, the Ocr elements do not appear to be deletion derivatives of larger transposons. The internal regions of the Ocr elements contain multiple copies of the octamer motif (ATTTGCAT) arranged as divergently-orientated dyads. We have shown by a gel mobility shift assay that these octamer dyads specifically bind what is presumably an OTF-type activator protein in oocyte nuclear extracts. We speculate that short interspersed repetitive families of this type may be generated by a mechanism of replicative transposition that uses a DNA intermediate and involves the interaction of DNA-binding proteins also utilised in other cellular processes.

Cytoskeletal protein synthesis and regulation of nerve regeneration in PNS and CNS neurons of the rat

Following injury to the facial nerve, facial motoneurons respond with a rapid suppression of neurofilament synthesis, and an increase in actin and tubulin synthesis. In situ hybridization studies show that these changes are the result of alterations in levels of the corresponding mRNAs. The increased ratio of neurofilament to tubulin expression is also characteristic of developing neurons, and direct evidence for the idea that axon injury provokes a partial return to a developmental state is provided by the finding that there is rapid re-expression of a developmentally regulated α-tubulin gene. In magnocellular rubrospinal neurons, whose axons do not regenerate after injury, the same initial changes in gene expression occurred, but they were not sustained. We do not know whether this is cause or effect of the failure to regenerate, but we can conclude that injured CNS neurons have the potential to respond to injury by initiating a 'regeneration programme'. Failure to sustain this programme is presumably due to differences in the trophic environment of central and peripheral axons.

Structure and expression of an α‐tubulin gene of Physarum polycephalum

Fragments of Physarum polycephalum DNA generated by partial digestion with Sau3A were cloned into phage-lambda EMBL4. A recombinant (phage-lambda E alpha Tu) containing an alpha-tubulin (E alpha-tubulin) gene was isolated. The E alpha-tubulin gene is part of the alt B locus. The gene was sequenced and was found to contain seven intervening sequences. The alpha-tubulin isotype (E alpha-tubulin) encoded by the gene has a methionine residue at the C-terminus. The E alpha-tubulin gene has much in common with the N alpha-tubulin gene cloned into phage-lambda NM1149 [M. J. Monteiro & R. A. Cox (1987) J. Mol. Biol. 193, 427-438]. However, the gene products E alpha-tubulin and N alpha-tubulin differ in amino acid sequence near to the C-terminus. E-peptide (corresponding to amino acids 440-448 of E alpha-tubulin) and N-peptide (corresponding to amino acids 437-445 of N alpha-tubulin) were synthesised and used to raise antibodies (E-antibodies and N-antibodies). The antibodies were used to show that on two-dimensional gel electrophoresis N alpha-tubulin travels to the alpha 1 position and E alpha-tubulin moves to the alpha 2 position. Gene-specific DNA probes were used to show that transcripts of the E alpha-tubulin gene were present in the plasmodial but not in the amoebal phase of the life cycle. The E- and N-antibodies detected E alpha- and N alpha-tubulins in plasmodia but not in amoebae, confirming that the expression of the E alpha- and N alpha-tubulin genes is regulated during development. E alpha- and N alpha-tubulin were shown to be components of spindle microtubules by indirect immunofluorescence microscopy.

Developmental distribution of RNA and protein products of the Drosophila α-tubulin gene family

The developmental pattern of gene expression of the Drosophila melanogaster α-tubulin family has been examined in detail at both the mRNA and protein levels. Northern data from 16 stages of development have been quantified to produce estimates of pool sizes of each of the α-tubulin transcripts through development. The in situ pattern of α67C RNA localization in developing oocytes and early embryos has also been determined. At the protein level, two of the three previously unidentified products of α-tubulin genes (α67C and α85E) have been identified. Evidence that protein from the fourth gene comigrates with the ubiquitously expressed α84B is presented. In addition to the primary translational products of the α-tubulin genes, an elaborate series of post-translationally modified α-tubulins has been resolved. The developmental profiles of both synthesis and accumulation of these α-tubulin proteins are described.

Identification of EcoRV fragments spanning the Nα-tubulin gene of Physarum

The Nα-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of Physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another α-tubulin gene. Probes derived from either 5′- or 3′-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5′-fragment) and 6.2 kb (3′-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M 3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5′-end) and 5.5 kb (3′-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.

Sequence of one α- and two β-tubulin genes of Tetrahymena pyriformis: Structural and functional relationships with other eukaryotic tubulin genes

Macronuclear DNA of the ciliate Tetrahymena pyriformis contains only one size class of fragments coding for α-tubulin, αTT. We have isolated αTT from a partial plasmid library, using Chlamydomonas reinhardtii α-tubulin gene as a probe. This gene as well as the two β-tubulin genes, βTT1 and βTT2, have been sequenced. None of these genes contains introns and all use TGA as the stop codon. In the coding region of the two β-tubulin genes, there are several TAA and TAG stop codons that probably code for glutamine. The codon usage is very biased. Regions flanking the tubulin coding sequences are A + T-rich (75%) and quite different among themselves. In these regions there are several putative transcription-regulatory sequences. Nuclear transcripts begin and terminate at multiple sites. The β-tubulin proteins differ only in two amino acid residues. Primary structure of Tetrahymena tubulins as well as their hydropathy indexes show a high degree of homology with tubulins from other organisms. Two-dimensional electrophoretic analysis of the ciliary tubulins shows the presence of eight α-tubulins and four β-tubulins. The α-tubulins migrate faster than the β-tubulins, in contrast with what happens with brain tubulins. We suggest that there are several α- and β-tubulin isoforms and the migratory inversion observed may be due to post-translational modifications.

The α- and β-tubulins of Toxoplasma gondii are encoded by single copy genes containing multiple introns

We have studied the fundamentals of gene expression in the protozoan parasite Toxoplasma gondii by analyzing, in detail, the genes encoding α- and β-tubulin. Southern analysis and quantitation studies reveal that, unlike in other organisms studied thus far, both these genes are present as single copies in the haploid Toxoplasma genome. Sequencing of these genes indicates that they both contain multiple introns with conserved 5′ and 3′ splice site signals. We have found that HeLa cell nuclear extracts are able to accurately splice a Toxoplasma pre-mRNA construct. We have mapped, for the α-tubulin gene, the exact site of transcription initiation and the approximate site of poly A addition by primer extension and RNase protection assays. Trans-splicing, as demonstrated in the Kinetoplastida, is not involved in the formation of the mature α-tubulin transcript in T. gondii.

Increased tubulin messenger RNA in the frog retina after optic nerve transection

The optic nerve of the bullfrog (Rana catesbeiana) was transected and the regeneration process was investigated. The nerve regeneration was confirmed by observing the extension of the axon and by examining the recovery of axonal transport. Following optic nerve transection, the rapid and transient increase of messenger RNA of alpha-tubulin gene was observed in the retina, but no specific change of actin messenger RNA was observed.

The complete sequence of a frog α-tubulin gene and its regulated expression in mouse L-cells

A frog alpha-tubulin cDNA and a frog alpha-tubulin gene, closely related to the cDNA, were cloned and sequenced and the structure of the gene deduced. The gene was introduced into mouse L-cells in order to investigate the transcription and regulation of the gene. The gene was transcribed and there was processing of the transcripts. Furthermore, the gene displayed the correct autoregulatory feedback control.

The ?1-tubulin gene of Arabidopsis thaliana: primary structure and preferential expression in flowers

The primary structure of the α1-tubulin gene of Arabidopsis thaliana was determined and the 5' and 3' ends of its transcript were identified by S1 nuclease mapping experiments. The information obtained was used to (i) predict the amino acid sequence of the α1-tubulin, (ii) deduce the positions of introns within the α1-tubulin gene, and (iii) construct 3' noncoding gene-specific hybridization probes with which to study the pattern of α1-tubulin transcript accumulation in different tissues and at different stages of development. The predicted amino acid sequence of the α1-tubulin has 92% identity with the predicted product of the previously characterized A. thaliana α3-tubulin gene. The coding sequence of the α1-tubulin gene is interrupted by four introns located at positions identical to those of the four introns in the α3 gene. RNA blot hybridization studies carried out with an α1-tubulin gene-specific probe showed that the α1 gene transcript accumulates primarily in flowers, with little transcript present in RNA isolated from roots or leaves. In order to investigate the pattern of α-tubulin gene expression in developing flowers, RNA was isolated from flowers at five different stages of development: flower buds, unopened flowers with pollen, open flowers, flowers with elongating carpels, and green seed pods. RNA blot hybridizations performed with 3' noncoding gene-specific probes showed that the α3 tubulin gene transcript is present in flowers at all stages of development, whereas the α1-tubulin gene transcript could only be detected in RNA from unopened flowers with pollen, open flowers, and flowers with elongating carpels.

Differential expression of an α-tubulin gene during the development of Physarum polycephalum

The expression of an α-tubulin gene ( altB1 (NαTu) [(1987) J. Mol. Biol. 193, 427-438]) of Physarum polycephalum (strain CLdAXE) was found to be governed by a developmental switch since mRNA transcripts were detected, by S 1 nuclease analysis, in the plasmodial but not the amoebal phase of the life-cycle. The conclusion that the altB1 (NαTu) allele codes for a plasmodial specific α-tubulin isotype is supported by recent amino acid sequence data.

Primary structure of an α-tubulin gene of Physarum polycephalum

An α-tubulin gene of Physarum was isolated as a phage-λ NM1149 recombinant (designated phage-λ NαTu). Phage-λ NαTu contained a 4700 base-pair HindIII nuclear DNA fragment of an allele of the altB locus of Physarum (one of four unlinked α-tubulin gene loci). Subfragments of the 4700 base-pair insert of phage-λ NαTu were cloned into phage M13 and the nucleotide sequence was determined by the dideoxy chain termination method. The start point of transcription was identified by primer extension and a putative polyadenylation site was located by S 1 nuclease analysis. The 4650 base-pair HindIII insert into phage-λ NαTu spans the complete gene; sequences upstream from the 5′ end contain the RNA transcription promoter elements (the TATA and CCAAT boxes). The nucleotide sequence encoding α-tubulin contains seven intervening sequences, ranging from 63 to 222 nucleotides in size. The exons have a sequence that is identical with a Physarum α-tubulin cDNA clone, except for three base changes, one leading to a Val codon in place of a Met codon, another leading to a Glu codon in place of an Asp codon, and the third change is silent. The genomic clone provides the nucleotide sequence coding for the last 26 amino acid residues missing from the cDNA clone. The new sequence data indicate that the α-tubulin gene has a C-terminal methionine codon and not a tyrosine codon, which has been found in all α-tubulin genes sequenced to date.

Developmental consequences of mutations in the 84B α-tubulin gene of Drosophila melanogaster

Developmental effects of six mutations in the gene encoding the majority of α-tubulin in all tissues at all stages of Drosophila melanogaster development have been examined. All six alleles produce at least partially stable α84B protein. In genetic assays, two of these alleles approximate the null condition. The other four alleles appear to form a graded series of hypomorphs. The two most severe alleles produce a semidominant maternal-effect polyphasic lethality, plus a predominantly larval recessive zygotic lethality. Clonal analysis of one of these alleles suggests it is a cell lethal. Worsening of the lethal phenotype (negative complementation) occurs in most interallelic heterozygotes involving these two mutations. As hemizygotes, the other four alleles are predominantly larval/pupal lethals. Partial complementation is achieved by most interallelic heterozygotes involving these four alleles. Phenotypic defects associated with the six tubulin mutations include disrupted embryos, pseudopupae, pharate adults with defects in various cuticular pattern elements, pharate adults with retarded head development, adults with leg tremors and extremely short life spans, and viable but sterile adults with bristle defects.

Structure of mRNA encoded by tubulin genes in Leishmania enriettii

Both the α- and β-tubulin genes of Leishmania enriettii are encoded by mRNA of 2.0–2.2 kb in length. We have shown previously that the α- and β-tubulin genes are arranged in separate, tandem repeats of 2 and 4 kb, respectively, and now report the mapping of mature mRNA onto these cloned genes. Here we show that the α-tubulin gene contains a very short intergenic region (100–200 bases) whereas the larger, tandemly repeated β-tubulin gene contains a 1.8–2.0 kb region not found in mature mRNA. Comparison of S-1 mapping and primer extension results indicates that the messenger RNAs for both α- and β-tubulin contain a sequence of about 35 base pairs located at the 5′ end that is not encoded contiguously with the rest of the mRNA. This short 5′ sequence may be added to the body of the tubulin mRNAs either through splicing of a precursor RNA molecule or by a novel post-transcriptional processing reaction. The α- and β-tubulin genes are arranged identically in the two developmental stages of the parasite life cycle and are present in equal copy number.

Structural features and restricted expression of a human α-tubulin gene

The nucleotide sequence of a human alpha-tubulin gene (b alpha 1) is described. This gene is extensively homologous to a rat alpha-tubulin gene in its coding regions, 3'-untranslated region and, indeed, in segments of its largest intron. However, with the exception of three short conserved blocks of homology, the 5' flanking regions of the rat and human genes are unrelated. Hence, these genes each encoding an identical protein are transcribed under the influence of divergent promoters. Blot analyses using RNA from a variety of transformed cells derived from different tissues indicate that expression of the human alpha-tubulin gene is restricted to cells of neurological origin. Among neurological cell types b alpha 1 expression is further restricted to adherent cells that are morphologically differentiated. The data presented suggest that the b alpha 1 gene encodes a prominent neuronal and glial alpha-tubulin and that b alpha 1 expression is a function of the differentiated state of these cells.

Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense: nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins

Most tubulin genes of the African trypanosome Trypanosoma rhodesiense are contained in 3.7-kb tandemly repeating units. One member of the 3.7-kb repeat family has been isolated from a T. rhodesiense genomic library, cloned, and sequenced. The 3646-bp fragment contains a complete α-tubulin gene and portions of two β-tubulin genes. No introns are present. The genes are separated by 634- and 333-bp intergenic regions, which lack typical eukaryotic promoter and poly(A) signal sequences. However, both intergenic regions exhibit some structural similarity with sequences proposed to be involved in transcription termination and poly(A) addition in yeast. The 634-bp intergenic region shows homology to the “mini-exon” sequence associated with variable surface glycoprotein (VSG) and other trypanosome mRNAs. A comparable sequence is not found in the 333-bp intergenic region. T. rhodesiense α and β-tubulins exhibit about 84–85% amino acid (aa) sequence homology with tubulins of mammals; the genes show about 74–75% nucleotide sequence homology. The α-tubulin contains 451 aa and the β tubulin 442 aa; both have tyrosine as the C-terminal aa.

Isolation and characterization of an α-tubulin gene from Leishmania enriettii

An α-tubulin gene of Leishmania enriettii has been identified in genomic Southern blots by hybridization with a heterologous α-tubulin gene from Drosophila melanogaster. A clone containing this gene has been isolated from a plasmid library of size-selected L. enriettii DNA. It was identified by hybridization with the D. melanogaster tubulin gene. The cloned DNA fragment was characterized by restriction analysis and partial DNA sequence analysis. The cloned DNA fragment is 2 kb in length, bounded by Pst I sites, and appears to contain the entire coding region of the α-tubulin gene.

The sequences of an expressed rat alpha-tubulin gene and a pseudogene with an inserted repetitive element.

The rat genome contains two segments closely related to a rat alpha-tubulin mRNA. Both have been cloned and complete nucleotide sequences are presented. Analysis of the structure and sequence of one of these establishes it as a functional alpha-tubulin gene. The second segment is a processed alpha-tubulin pseudogene. Comparison of this pseudogene to the mRNA and gene coding for alpha-tubulin strongly suggests that a mature mRNA was involved in its origin. Features of the pseudogene and a dispersed repetitive element inserted within it possibly reflect a common RNA-mediated process of insertion.