Dieter Ammermann

The development of the macronucleus in the ciliated protozoan Stylonychia mytilus

Ciliated protozoa are characterized by generative micronuclei and vegetative polyploid macronuclei. Micronuclei of Stylonychia mytilus contain 1 600 times as much DNA per haploid genome as E. coli. Most of this DNA is shown to be repetitive. The development of the macronucleus involves, as demonstrated by cytology, only 1/3 of the chromosomes which in a first replication phase are polytenized in probably 5 replication steps and appear as giant chromosomes. At this developmental stage considerable amounts of repetitive DNA are still present in the chromosomes. During the subsequent disintegration phase more than 90% of the DNA are eliminated from the macronucleus anlage. The remainder is further replicated five times and composes the final macronucleus. Since this DNA reassociates with a reaction rate almost identical to an ideal second order reaction its kinetic complexity can be determined by comparison with the kinetic complexity of E. coli DNA. Macronuclear DNA reassociates with a kinetic complexity of 26 times the kinetic complexity of E. coli DNA (corrected for GC content) which indicates that macronuclear DNA sequences exist at a ploidy level of 4 096 C. We assume that macronuclear DNA may be present only once per haploid genome. In this case it represents only 1.6% of the DNA in micronuclei or 10% of the DNA in the giant chromosome stage.

Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus

Some stages of macronuclear anlagen development, known from earlier investigations (see Fig. 1), were studied in detail. The results are: a) The giant chromosomes of Stylonychia mytilus are not somatically paired, but are connected end-to-end to form one or a few composite chromosomes. When they later disintegrate, the bands become isolated granules. b) Spectrophotometric measurements show that during the DNA-poor stage which follows the disintegration of the chromosomes, the macronuclear anlagen of Euplotes have a DNA content of 21 c, while the syncaryotic (deriving from syncarya) and hemicaryotic (deriving from haploid hemicarya) anlagen of Stylonychia have the DNA content of diploid micronuclei (2c). Nevertheless the syncaryotic anlagen of Stylonychia and Euplotes initially develop two nucleoli at the end of this stage, the hemicaryotic anlagen of Stylonychia only one. From this it is concluded that the genes of one giant chromosome band stay together in one granule, c) Labeled DNA from the giant chromosomes which remains in the anlagen during the DNA-poor stage is distributed approximately equally to the daughter nuclei during the first few fissions of the exconjugants.-Autoradiographic experiments showed that the DNA of the macronuclei of Stylonychia that is duplicated at one time in a replication band is not duplicated simultaneously during the next DNA-duplication. The DNA duplications during the second polyploidization stage of the macronuclear anlagen development are exceptions, because the mixing of the macronuclear DNA which occurs before every fission does not occur during the second polyploidization stage.—The pseudomicronuclei which sometimes are formed from the macronuclei in emicronucleated strains of Stylonychia contain numerous elements which are much smaller than the chromosomes.—The macronucleus of Stylonychia is very insensitive to irradiation with X-rays.—The results lead to the following hypothesis: The macronuclei of the two hypotrich ciliates contain unconnected chromomeres or small aggregates which are distributed at random to the two daughter nuclei during the divisions.

Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate stylonychia mytilus

Using indirect immunofluorescence, we studied the reaction of antibodies specific for left-handed Z DNA with the nuclei of the hypotrichous ciliate Stylonychia mytilus. In the vegetative cell, the macronucleus reacts strongly with these antibodies, but no reaction can be detected with micronuclei. However, an antibody that binds to denatured and right-handed B DNA reacts with both types of nuclei. No reaction of the anti-Z DNA antibody is seen in the macronuclear replication band. Digestion of macronuclei with DNAase I leads to a decrease in the anti-Z DNA antibody reaction. Some stages of the developing macronucleus were also investigated. No reaction is seen at the polytene chromosome stage, but following DNA elimination the nucleus is seen to react with the antibody.

Characterization of macronuclear DNA in five species of ciliates

Macronuclear DNA of four hypotrichous and one holotrichous ciliate species was characterized by biochemical techniques. The renaturation kinetics of the macronuclear DNAs of all five species were similar. Repetitive sequences occur only in an amount below 2%. Although the DNA content of the macronuclei of the species differs considerably, the kinetic complexity of the macronuclear DNA is rather uniform (around 3 × 1010 daltons, i.e., 4–11 x the E. coli genome). Only in the macronuclei of the hypotrichous species the DNA exists as gene-sized fragments.

ON THE STRUCTURE OF OXYBLEPHARISMIN AND ITS FORMATION FROM BLEPHARISMIN

Abstract The blepharismins ( 1 ) from Blepharisma japonicum give the corresponding oxyblepharismins ( 2 ) on irradiation in vitro and in vivo . The chemical structures of these compounds are elucidated and a mechanism is given for this unusual transformation.

Synthese und Abbau der Nucleinsäuren während der Entwicklung des Makronukleus von Stylonychia mytilus (Protozoa, Ciliata)

The synthesis of nucleic acids during the development of the macronuclear anlage from a diploid nucleus to the polyploid macronucleus has been studied. The nucleic acids were labeled with 3H-thymidine and 3H-uridine. The following results have been obtained: 1. In the first polyploidization stage the anlage incorporates 3H-thymidine into its DNA. At the end of this stage the anlage is polyploid and strongly labeled. Some hours later, during the DNA-poor stage, most of the labeling has disappeared (autoradiographs). This indicates that most of the anlagen-DNA is decomposed. Scintillation counter measurements showed that only about 9 per cent of the DNA stays within the exconjugants. These data agree with earlier spectrophotometric results (Ammermann, 1965). 2. The DNA-poor stage is followed by a second polyploidization stage in which the definitive polyploid macronucleus develops. Completely new thymidine is incorporated during this stage. 3. RNA synthesis does not occur neither in the giant chromosome anlage (end of the first polyploidization stage), nor in the DNA-poor stage, nor in the fragments of the old macronucleus. RNA synthesis starts at the beginning of the second polyploidization stage (autoradiographs).ZusammenfassungEs wurde die Nukleinsäuresynthese während der Entwicklung der Makronukleusanlage vom diploiden Kern zum polyploiden Makronukleus untersucht. Die Nucleinsäuren wurden mit 3H-Thymidin und 3H-Uridin markiert. Dabei ergab sich:1.Während einer ersten Polyploidisierungsphase baut die Anlage 3H-Thymidin in ihre DNS ein. Am Ende dieser Phase ist die nunmehr polyploide Anlage stark markiert. Einige Stunden später jedoch, während der DNS-armen Phase, ist der größte Teil der Markierung verschwunden (Autoradiographien). Daraus wird gefolgert, daß die meiste DNS aus der Anlage verschwindet. Scintillationsspektrometrische Messungen zeigten, daß die DNS-Menge der Exkonjuganten um mehr als 90% reduziert wird. Diese Ergebnisse stimmen mit spektrophotometrischen Befunden (Ammermann, 1965) überein. 2. Auf das DNS-arme Stadium folgt eine zweite Polyploidisierungsphase, in welcher der endgültige polyploide Makronukleus heranwächst. Dabei wird neu aufgenommenes Thymidin eingebaut. 3. Weder in der Anlage mit Riesenchromosomen (am Ende der ersten Polyploidisierungsphase) noch in der DNS-armen Anlage noch in den Fragmenten des alten Makronukleus konnte eine RNS-Synthese nachgewiesen werden. Sie setzt erst mit dem Beginn der zweiten Polyploidisierungsphase ein (Autoradiographien).

Die Cytologie der Parthenogenese bei dem Tardigraden Hypsibius dujardini

Hypsibius dujardiniDoy. (Articulata, Tardigrada) shows obligatory parthenogenesis under given cultivating conditions. Males were never found. The first meiotic division reduces the number of chromosomes; the (2n=10) chromosomes are divided between a small polar body and the egg nucleus. Prior to the second division the dyads divide, thus restoring the diploid number. A diploid polar body is formed subsequent to the second division. After the egg nucleus has moved toward the center of the egg, the cleavage divisions begin. — During meiosis II and the first cleavage divisions the chromosomes can develop into “large chromosomes” which presumably consist mostly of RNA. No “large chromosomes” are found after the seventh cleavage division. Sometimes a plate of coloured material (“elimination chromatin”) can be observed between the anaphase daughter plates of the first cleavage divisions. In this case the chromosomes are always small.Zusammenfassung1.Hypsibius dujardini (Articulata, Tardigrada) führt eine diploide, unter den gegebenen Kulturbedingungen obligatorische Parthenogenese durch. Männchen wurden nicht gefunden.2.Die erste Reifeteilung führt zur Reduktion der Chromosomenzahl. Die 5 Bivalente werden geteilt und anschließend auf einen kleinen Richtungskörper und den Eikern verteilt. Anschließend, ohne ein Interphasestadium, spalten sich die 5 im Eikern verbliebenen Dyaden in 10 Elemente auf, wodurch die diploide Chromosomenzahl wiederhergestellt wird.3.Die zweite Reifeteilung ist eine normale Mitose. Nach der Abschnürung des relativ großen Richtungskörpers mit 10 Chromosomen rückt der ebenfalls die diploide Zahl Chromosomen enthaltende Eikern ins Eiinnere, und die Furchungsteilungen beginnen.4.Während der zweiten Reifeteilung und während der ersten Furchungsteilungen sind die daran beteiligten Chromosomen häufig besonders groß. Diese „großen Chromosomen“ können einige Teilungen überdauern. Sie werden aber spätestens nach der 5. Furchung wieder klein. Ihre Größe wird offenbar vorwiegend durch angelagerte RNS verursacht.5.Zwischen den Tochterplatten der Anaphasen der ersten Furchungen kann manchmal eine Schicht anfärbbaren Materials beobachtet werden. Die Chromosomen sind in diesem Fall stets klein. Vielleicht handelt es sich hierbei um nicht-DNS-haltiges „Eliminationschromatin“, das bei der Verkleinerung der „großen Chromosomen“ frei wird.

The Histones of the Ciliated Protozoan Stylonychia mytilus

Histones were extracted from pure macronuclei, micronuclei and macronucleus anlagen and from chromatin which was isolated from these different nuclear fractions. Analysis of these preparations by polyacrylamide gel electrophoresis showed differences in electrophoretic patterns between the histones of these nuclei.

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences

Abstract. The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3′ overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3′ telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified α1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, α1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the α1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.

Germ line specific DNA and chromosomes of the ciliate Stylonychia lemnae

The variation in DNA content of the micronucleus (germinal nucleus) of Stylonychia lemnae and its relation to the number of chromosomes was examined. Different populations possess similar amounts of micronuclear DNA but there are differences of ±30% between clones of the same population. However, the DNA content varies by about 100% in the micronuclei during the lifetime of a clone. The haploid micronucleus contains 35 or 36 chromosomes which persist in the developing macronucleus anlagen and grow to giant chromosomes. Besides this remaining subset, the micronucleus contains a variable number of germ line restricted chromosomes (mean about 140; range between 100 and 180). The somatic macronucleus eliminates these elements early in its development. The varying number of the germ line restricted chromosomes is responsible for the variation in the micronuclear DNA content.