Günther Steinbrück

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.

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as l...

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.

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus.

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×106 to 14×106 dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l06 dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.

Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus

DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments were fractionated according to size by agarose gel electrophoresis, the fragments transferred to nitrocellulose filters using the technique of Southern, and the filter-bound DNA hybridized with labeled cloned histone genes of the sea urchin, Psammechinus miliaris. Results indicate, first, that sequences homologous to the five individual histone gene probes are present in discrete macronuclear fragments which appear as bands in the gel hybridization assay. Secondly, for each of the five individual histone gene probes the homologous DNA fragments are several in number, ranging in size from 7.6 Kb (Kilo base pairs) to 0.73 Kb. For example, the largest of six detected fragments hybridizing to the H3 gene probe contains approximately 10 times the amount of DNA required to code for a Stylonychia H3 histone. The smallest detected fragment hybridizing to the H3 probe contains enough DNA to code for approximately two copies of the histone. Finally, in general, no two histone gene probes hybridized to the same macronuclear DNA fragment. This result indicates that genes coding for the five histones in Stylonychia are not located together on the same macronuclear DNA fragments and implies that the five functionally related genes would not be transcribed together as a polycistronic unit.

Interspecific relationship of ten European orchid species as revealed by enzyme electrophoresis

Ten species of orchid plants belonging to the generaOrchis (7),Dactylorhiza (2), andGymnadenia (1) were analyzed by enzyme electrophoresis. Each species can be identified by a combination of enzyme bands different from those of all other species examined. The electrophoretic data were used for the construction of phenetic and phylogenetic trees with the help of computer programs. The trees were almost identical regardless which method was used. Our results differ considerably from a classification based on morphological evidence. The electrophoretic data indicate that the genusOrchis is not a monophyletic group.

Characterization of interspecific hybrids betweenOrchis mascula andO. pallens (Orchidaceae) by enzyme electrophoresis

The European orchidsOrchis mascula, O. pallens and their hybrids have been analysed by enzyme electrophoresis on starch gels. The two species differ in the electrophoretic mobilities of four out of eight enzymes tested. Three enzymes, phosphoglucomutase, phosphoglucoisomerase and “malic enzyme” exhibit typical heterozygote patterns in the hybrid plants demonstrating the presence of both differing parental alleles. Thus, species identification is easy by the electrophoretic analysis of a low number of enzyme loci, and hybrids are detectable even if morphological characters fail.

Recent advances in the study of ciliate genes

This review gives a brief report on some recent advances in the study of molecular biology of ciliate genes. The main emphasis will be put on those results which may contribute to a better understanding of how gene structure and genome organization has evolved in ciliates. New data will be discussed in relation to the contribution they might give for a general understanding of eukaryotic gene evolution. The whole topic is divided into four parts: (1) a short introduction into genome organization of ciliates and an account of new insights in this field; (2) new results about telomere formation and replication; (3) regulation of transcription with new details about the atypical usage of the genetic code; (4) some speculations on unresolved problems and their possible solution by new methods. Other important new aspects of the molecular biology of ciliates which would fit well into this context, for example RNA self-splicing and evolutionary divergence of 17 S rRNA sequences, are not included as excellent newer reviews are available [e.g., 13-15, 42].

Location of rDNA in the heteromeric macronucleus of Chilodonella steini

As it was shown previously the chromatin of the heteromeric macronucleus of Chilodonella steini consists of two specific zones, the outer one (the orthomere) made up from late replicating electron dense material, and the inner zone (the paramere) of medium electron density and early replicating, with a concentration of electron dense material called the endosome in its central part. New details regarding the reorganization of the chromatin after macronuclear division are presented here. After the division no endosome is visible. It reappears within the first two hours after the separation of sister macronuclei. With the use of in situ rDNA - DNA hybridization it was shown that the rDNA in the macronucleus of Chilodonella steini is located in the outer zone (orthomere) and the endosome. Since it has been found earlier that the DNA content of the macronucleus depends on culture conditions the authors suggest that the quantitative DNA changes are caused preferentially by differential replication of rDNA dependent on the food conditions of the cells.

Amplification of tropharium rDNA in the telotrophic ovariole of the bug, Dysdercus intermedius.

Summary In the telotrophic ovariole of the bug, Dysdercus intermedius, the highly endoploid tropharium nuclei which are functioning throughout the gonocycles were analysed with respect to rDNA amplification. The likewise polyploid follicle cell nuclei which degenerate at the end of each cycle of egg formation and diploid larval brain nuclei were used for comparison. A heavy satellite is pronounced only in the tropharium DNA. The restriction patterns after hybridization with a labelled probe of cloned rDNA indicated considerable differences in the amount of ribosomal genes present in the three types of nuclei. These results evidence a high degree of rDNA amplification in the germ-line derived tropharium nuclei, but nothing like this in the diploid or polyploid somatic cells. This is the first record of an amplification of rDNA in a telotrophic insect ovary occurring probably during early trophocyte differentiation. The developmental significance of high amounts of ribosomal genes in the long persisting tro...