The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme.

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis. Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tektl staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.

The Rib43a protein is associated with forming the specialized protofilament ribbons of flagellar microtubules in Chlamydomonas.

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins as tektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1. 7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an approximately 4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43a gene on linkage group III. Sequence analysis of the RIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens (genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.

Expression of ciliary tektins in brain and sensory development.

Many types of neural tissues and sensory cells possess either motile or primary cilia. We report the first mammalian (murine testis) cDNA for tektin, a protein unique to cilia, flagella, and centrioles, which we have used to identify related proteins and genes in sensory tissues. Comparison with the sequence database reveals that tektins are a gene family, spanning evolution from Caenorhabditis elegans (in which they correlate with touch receptor cilia) and Drosophila melanogaster, to Mus musculus and Homo sapiens (in which they are found in brain, retina, melanocytes, and at least 13 other tissues). The peptide sequence RPNVELCRD, or a variant of it, is a prominent feature of tektins and is likely to form a functionally important protein domain. Using the cDNA as a probe, we determined the onset, relative levels, and locations of tektin expression in mouse for several adult tissues and embryonic stages by Northern blot analysis and in situ hybridization. Tektin expression is significant in adult brain and in the choroid plexus, the forming retina (primitive ependymal zone corresponding to early differentiating photoreceptor cells), and olfactory receptor neurons of stage embryonic day 14 embryos. There is a striking correlation of tektin expression with the known presence of either motile or primary cilia. The evolutionary conservation of tektins and their association with tubulin in cilia and centriole formation make them important and useful molecular targets for the study of neural development.

Structural comparison of tektins and evidence for their determination of complex spacings in flagellar microtubules.

Recent structural studies indicate that a tektin heteropolymer forms a unique protofilament of flagellar microtubules. We report here the sequence of tektin C (approximately 47 kDa), predicted from its cDNA (GenBank U38523), compared to tektins A (approximately 53 kDa) and B (approximately 51 kDa) from sea urchin (Strongylocentrotus purpuratus) sperm flagellar microtubules, and compared to partial sequences reported from mouse and human. We are now able to make several observations concerning the tektin family: (1) their common structural features, (2) a comparison of their structure to intermediate filament proteins, and (3) their possible organization in the tektin filament polymer. The predicted amino acid sequence identities/similarities are: for tektins A and C, 42/54%, for tektins A and B, 34/51%; for tektins B and C, 29/42%; for tektin C and a partial cDNA clone from mouse testis, 55/65%; and for tektin B and a partial cDNA clone from the human brain, 45/47%. The three tektins (and the human clone) possess the exact sequence repeat RPNVELCRD. The structural pattern of all three tektin polypeptides is similar to intermediate filament proteins. Tektins are predicted to form extended rods composed of two alpha-helical segments (approximately 180 residues long) capable of forming coiled coils, which are interrupted by short non-helical linkers. The two segments are homologous in sequence and secondary structure, indicating a gene duplication event prior to the divergence of the three tektins. Along each tektin rod cysteine residues occur with a periodicity of approximately 8 nm, coincident with the axial repeat of tubulin dimers in microtubules. From EM data and calculations of secondary structure, the segment length of tektin AB heterodimers is likely to be 16 nm. Both segments of tektin C may be 24 nm long, but one may be 16 nm. On the basis of the available evidence, we propose that coassembly of tektin AB heterodimers with tektin C dimers produces filaments with overall repeats of 8, 16, 24, 32, 40, 48 and 96 nm, generating the basis for the complex spatial arrangements of axonemal components.

Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis.

Previous studies have shown that tektin A, one of three integral filamentous protein components of outer doublet microtubules, is synthesized in sea urchins in an amount correlating to the length of embryonic cilia initially assembled or experimentally regenerated. To investigate further the molecular mechanism for the regulation of tektin synthesis, tektin cDNA clones were used to assess mRNA levels during ciliogenesis, zinc-induced animalization, deciliation-induced regeneration and theophylline-induced elongation. Possibly involved in centriole replication, low, near-constant levels of mRNA for all three tektins are present in the unfertilized egg and during cleavage stages. Preceded by new synthesis of tektin B and C mRNAs, tektin A mRNA is up-regulated during ciliogenesis, but only tektin A mRNA levels correlate directly with ciliary length in animalized embryos; the others augment larger, non-limiting pools of tektins B and C. Tektin mRNAs decrease to steady-state levels after ciliogenesis, but are up-regulated again when the embryos are deciliated, correlating with the length of cilia to be deployed. In a species where a 3-fold ciliary length increase can be induced by theophylline treatment of zinc-arrested embryos, the mRNAs accumulate to proportionately higher levels during arrest but are not translated until induction, whereupon they decrease inversely with ciliary elongation. This suggests transcriptional control with respect to mRNA amounts but post-transcriptional control with respect to the expression of this phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of tektin A1: comparison with intermediate-filament proteins and a model for its association with tubulin.

Tektins are proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules and that have biochemical and immunological properties similar to those of intermediate-filament proteins. We report here the sequence of a cDNA for tektin A1, one of the main tektins from Strongylocentrotus purpuratus sea urchin embryos. By hybridization analysis, tektin A mRNA appears maximally at ciliogenesis. The predicted structure of tektin A1 (M(r) 52,955) is a series of alpha-helical rod segments separated by nonhelical linkers. The two halves of the rod appear homologous and are probably related by gene duplication. Comparison of tektin A1 with intermediate-filament proteins, including nuclear lamins, reveals a low amino acid homology but similar molecular motif, i.e., pattern of helical and nonhelical domains. This study indicates that tektins are unique proteins but may be evolutionarily related to intermediate-filament proteins, and suggests a structural basis for the interaction of tektins and tubulin in microtubules.

Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules.

Rabbit antibodies raised and purified against three tektins, proteins of flagellar doublet microtubules from sea-urchin sperm (Lytechinus pictus and Strongylocentrotus purpuratus), were used to study tektin biochemistry and their structural localization. Doublet microtubules were fractionated into tektin filaments and separated by SDS-PAGE into three major tektin polypeptide bands (Mr = 47, 51 and 55 (X 10(3)), which were used to immunize rabbits. Antibodies against each tektin (anti-tektins) were affinity-purified and then characterized by two-dimensional isoelectric focusing/SDS-PAGE immunoblotting and by immunofluorescence microscopy. In two-dimensional immunoblots of 0.5% Sarkosyl-resistant fractions of flagellar microtubules, the antibody against the 55 X 10(3) Mr tektin (anti-55) stained one major polypeptide of 55 X 10(3) Mr and pI 6.9, anti-51 stained two polypeptides of 51 X 10(3) Mr and pI approximately 6.15, and anti-47 stained one major polypeptide of 47 X 10(3) Mr and pI 6.15. The anti-tektins also stained several minor neighbouring polypeptides, which may be isoelectric variants, novel tektins or unrelated proteins. Furthermore, anti-47 crossreacted with the major 55 X 10(3) Mr polypeptide. By immunofluorescence microscopy all three anti-tektins stained methanol-fixed echinoderm sperm flagella and embryonic cilia. In addition, anti-47 and anti-55 stained unfixed, demembranated axonemes. Besides staining axonemes, all anti-tektins labelled the basal body region, and anti-51 labelled the sperm head envelope. These results indicate that the tektins are a complex family of proteins that are components of axonemal microtubules and possibly other cytoplasmic and nuclear structures.

Accumulation of single strand interruptions within the yeast 2 microns DNA plasmid during replication in a DNA ligase mutant.

We have investigated the fate of the yeast 2 micron DNA plasmid in strains with a temperature sensitive mutation of DNA ligase. At the restrictive temperature the plasmid DNA collects as an open circular form with single strand interruptions. Both alpha factor pheromone, which arrests cells before the start of S phase, and hydroxyurea, which blocks progression through S phase, prevent the appearance of the open circular form. Thus, interrupted plasmid DNA does not accumulate in the absence of DNA replication. On average the interrupted molecules contain four to five interruptions per newly replicated strand. Most of the interruptions are nicks (breaks in a single phosphate ester bond) rather than gaps (absence of one or more nucleotides in a strand) as judged by the in vitro conversion of the interrupted molecules into a covalently closed form by DNA ligase. Mapping of the position of the interruptions reveals no predominate sites.

Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.

The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.