Jan M. Norrander

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.

Rib72, a Conserved Protein Associated with the Ribbon Compartment of Flagellar A-microtubules and Potentially Involved in the Linkage between Outer Doublet Microtubules

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized “ribbon” of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an ∼96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.

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.

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) andDrosophila melanogaster, to Mus musculusand 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.

Manipulation and expression of the maize zein storage proteins in Escherichia coli

Abstract The cDNA sequence for the mature form of the zein protein A20 was inserted into the lac cloning region of two different M13mp phage vectors. Translation of these recombinant phage in E. coli cells produced a β-galactosidase—zein fusion protein. Another zein clone was constructed in which the entire coding sequence, including that of the signal peptide, was cloned into a M13mp vector. This clone was designed to produce a pre-zein protein which did not contain any β-galactosidase amino acids. Expression of these phage-coded proteins in E. coli cells was detected on Western blots using zein antibodies. Expression of the β-galactosidase—zein fusion protein was extremely low, comprising less than 1% of the total E. coli proteins. Levels of expression were increased slightly when this same sequence was cloned into a pUC-derived expression plasmid containing the highly efficient trp—lac promoter.

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

SummaryWe have investigated the fate of the yeast 2 μm 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.