Hans-Martin Seyfert

Molecular polymorphism of ciliary proteins from different species of the ciliate Tetrahymena.

Ciliary proteins from five different Tetrahymena species were analyzed by means of SDS-polyacrylamide gel electrophoresis. Of at least 32 different polypeptides, only 2 were found to have identical molecular weights in all species. In any comparison of 2 species, a maximum of 60% and at least 20% of the proteins had indistinguishable molecular weights, depending on the combination examined. Extensive codominance of ciliary proteins was found in a F1 hybrid. No interspecific cross-reactions occurred in double-diffusion tests involving the cilia from different species and antisera produced against them in rabbits.

An estimation of the soluble tubulin content in Tetrahymena cells of normal and of size-altered phenotype.

The amount of soluble tubulin in a temperature-sensitive (ts) size mutant of the ciliate Tetrahymena was measured in a variety of physiological conditions. For this purpose a competitive ELISA assay for tubulin was set up. The assay is based on an antiserum against Tetrahymena axonemal tubulin. Characterization of the antiserum shows its mono-specificity towards tubulin as well as its potential to recognize tubulin from a wide variety of cellular sources and organisms. After fractionation of the cells into soluble material, cold-labile and cold-resistant structures, we found very little tubulin soluble (less than 20% of the total), while most of the tubulin is polymerized, especially into cortical structures. Prolonged starvation does not alter the tubulin content. During the culture growth cycle the percentage of the soluble tubulin increases. Growing the ts mutant at high temperature to a large cell size will also increase the pool of soluble tubulin to a large extent. Only under this condition is the amount of soluble tubulin about equal to that fixed in cilia. The tubulins in the three different compartments are polymorphic and have a different metabolism. This is indicated by the much higher specific activity of soluble tubulin compared with the structurally bound material. In agreement, the half-life of the soluble tubulin is shorter than that of the cortical tubulin.

Induced tubulin synthesis is caused by induced gene transcription in Tetrahymena

Tubulin synthesis and tubulin m RNA concentrations increase to variable extents during ciliary regeneration in the ciliate Tetrahymena. Experiments described here were carried out to determine whether the increased tubulin mRNA concentrations are due to induced transcription of tubulin genes or to stabilization of tubulin mRNA. In vivo labeling experiments with [3H]uridine and in vitro transcription assays suggest that under conditions of increased protein and tubulin synthesis the rate of transcription is enhanced. Hybridization assays of in vitro transcribed RNA also demonstrate qualitatively that the tubulin genes are transcribed at higher rates when tubulin synthesis is stimulated during ciliary regeneration. This observation is supported by measurements of the half-life of tubulin mRNA molecules in nondeciliated cells: This is approximately 2 h. Since the concentration of tubulin mRNA in cells engaged in cilia regeneration increases from 5 to 19-fold during the first hour of the regeneration period, even a complete stabilization of the tubulin mRNA molecules could not account for an increase in tubulin mRNA concentration of this magnitude.

Properties of total and poly(A)+ RNA from exponentially growing and from resting cultures of Tetrahymena thermophila

Abstract Total RNA was extracted from exponentially growing and resting cultures of Tetrahymena thermophila. Poly(A)-containing RNA was separated by oligo(dT) affinity chromatography. The following characteristics of both preparations were studied: the changes in sedimentation profiles of newly made RNAs as a function of time, the length of the poly(A) segment, and the capacity of polyadenylated mRNA to code for proteins in vitro. The time-dependent sedimentation profiles of both kinds of RNA changed strikingly with the modes of growth: poly(A)+ RNA from heterodisperse in log phase into uniformly and slowly sedimenting in stationary phase, and total RNA from typical ribosomal into heterodisperse with a maximum in the pre-rRNA region. As revealed by the temperature regime developed by Ihle et al. [1] about 80% of all poly(A) RNA molecules carried a poly(A) stretch of less than 50 nucleotides. There was a tendency of the class 0–20 nucleotides to become more frequent in the stationary phase. The polyadenylated mRNAs were translated in the reticulocyte in vitro system. At least one protein of about 26 000 D was translated only in presence of mRNA of growing cells and not with that from resting cells. Another of 3 500 D was found only with mRNA from resting cultures. Three other proteins were translated with different rates according to the culture growth rate. The results demonstrate that the RNA isolated from different phases of culture growth have different dynamic as well as coding properties related to rate of cell multiplication.

A short G1 period is correlated with low macronuclear DNA contents in Tetrahymena.

Summary Macronuclear DNA content is variable in Tetrahymena . The cells which are the first to initiate macronuclear replication are those having a low DNA content. This conclusion is based on the decrease of the coefficient of variation of macronuclear DNA contents at the beginning of S, and is directly shown by correlating the time of onset of S to the macronuclear DNA content. This result is compatible with the assumption that the transcription rate is one of the factors releasing the onset of replication in the cell cycle.

Temperature dependent changes of cell growth parameters in Tetrahymena.

Abstract Cytophotometric studies revealed gross amounts of RNA and protein to be negatively correlated with incubation temperature. The rate of [3H]-uridine incorporation into TCA-insoluble material of synchronously growing cells followed a cyclic pattern during the cell cycle and was correlated to the event of the S phase.

Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.

Variants and Biotechnological Use of the Bovine Lactoferrin-Encoding Gene

Owing to its outstanding high and avid Fe3+ ion-binding capacity, the secreted 80-kDa glycoprotein lactoferrin (Lf) is thought to act as an antimicrobial agent against a broad spectrum of microbial species. To examine the proposed role of the bovine Lf-encoding gene as a candidate gene for conferring resistance against microbial infections, we systematically searched for natural variants of the gene and its promoter. Based on our previous characterization of the bovine Lf-encoding gene and published cDNA sequences, we identified 36 potentially polymorphic sites within the coding region of the gene, 15 of them altering the protein sequence. Experimental analysis confirmed two of these polymorphisms from among 11 sites checked in a variety of bovine breeds. These exchanges are tightly linked on two alleles. Comparative sequencing in dairy and beef breeds revealed 17 polymorphic sites within approx 700 bp of the proximal promoter region analyzed. One of these was proven to result in a polymorphic SP1-box. The promoter analysis within these breeds and in related Bovidae species suggests that the relatively low expression of the bovine Lf-gene is not a consequence of domestication.