Germline Nuclei Research Articles

The DNA of ciliated protozoa

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.

Cell-cell interactions prevent a potential inductive interaction between soma and germline in C. elegans

In each gonadal arm of wild-type C. elegans hermaphrodites, the somatic distal tip cell (DTC) maintains distal germline nuclei in mitosis, while proximal nuclei enter meiosis. We have identified two conditions under which a proximal somatic cell, the anchor cell (AC), inappropriately maintains proximal germline nuclei in mitosis: when defined somatic gonadal cells have been ablated in wild type, and in lin-12 null mutants. Laser ablations and mosaic analysis indicate that somatic gonadal cells neighboring the AC normally require lin-12 activity to prevent the inappropriate AC-germline interaction. The AC-germline interaction, like the DTC-germline interaction, requires glp-1 activity. In one model, we propose that the AC sends an intercellular signal intended to interact with the lin-12 product in somatic gonadal cells; when lin-12 activity is absent, the signal interacts instead with the related glp-1 product in germline. Our data illustrate the importance of mechanisms that prevent inappropriate interactions during development.

Nonrandom chromosome arrangements in germ line nuclei of Sciara coprophila males: the basis for nonrandom chromosome segregation on the meiosis I spindle.

Meiosis I in males of the Dipteran Sciara coprophila results in the nonrandom distribution of maternally and paternally derived chromosome sets to the two division products. Based on an earlier study (Kubai, D.F. 1982. J. Cell Biol. 93:655-669), I suggested that the meiosis I spindle does not play a direct role in the nonrandom sorting of chromosomes but that, instead, haploid sets are already separated in prophase nuclei well before the onset of spindle formation. Here I report more direct evidence that this hypothesis is true; this evidence was gained from ultrastructural reconstruction analyses of the arrangement of chromosomes in germ line nuclei (prophase nuclei in spermatogonia and spermatocytes) of males heterozygous for an X-autosome chromosome translocation. Because of this translocation, the maternal and paternal chromosome sets are distinguishable, so it is possible to demonstrate that (a) the two haploid chromosome sets occupy distinct maternal and paternal nuclear compartments and that (b) nuclei are oriented so that the two haploid chromosome sets have consistent relationships to a well-defined cellular axis. The consequences of such nonrandom aspects of nuclear structure for chromosome behavior on premeiotic and meiotic spindles are discussed.

Giant chromosomes in ciliates.

Ciliates are characterized by the occurrence of two different cell nuclei. The DNA-rich macronucleus is the “somatic nucleus” of the cell and is responsible for almost the entire cellular RNA synthesis. However, during sexual reproduction, or conjugation, it is resorbed. The diploid micronuclei are the “germ line nuclei” of the cells. They are, at least in many species, dispensable during most of the cells’ life cycle, which is characterized by asexual reproduction (division). During conjugation they undergo meiosis and form a synkaryon in each cell. One of its derivatives develops into a new macronucleus in the animals which, after conjugation, are now called exconjugants. During the development of the macronucleus anlage into a new macronucleus giant chromosomes are found in several species.KeywordsPolytene ChromosomeCiliated ProtozoanSomatic NucleusInter BandReplication BandThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Tissue-specific schedule of selective replication in Drosophila nasutoides.

Dramatic changes in the DNA composition of post-mitotic versus mitotic and germ line nuclei occur during development in different organisms. Drosophila nasutoides possesses n=4 chromosomes which were quantified with a microphotometer in females. The diploid (2 C) DNA content was 0.79 pg or 7.7×108 nucleotide pairs, calculated from brain metaphases and calibrated with hen erythrocyte nuclei. The individual elements comprised X=9%, 2=16%, 3=13%, and 4=62% of the total complement. In polytene nuclei of larval salivary glands which had undergone 11 endoreplication cycles, chromosome 4 contained only 1.55% of total Feulgen DNA. Thus, in contrast with other Drosophila genomes, where under-replicating material is dispersed to all elements, a huge quantity of non-endoreplicating DNA is restricted to a single chromosome. This permits accurate determination of the timing of under-replication in the single cell. The data presented here suggest that the schedule is tissue-specific. Larval hind gut and salivary duct nuclei begin under-replication during the first endocycle, whereas adult and larval salivary glands mainly begin during the second cycle. In Malpighian tubules the onset of selective DNA syntheses occurs during either the first or the second endocycle.

Nuclear division and migration during early embryogenesis of Bradysia tritici coquillet (syn. Sciara ocellaris) (diptera : Sciaridae)

Nuclear division and migration of cleavage nuclei in the embryos of Bradysia tritici (Diptera : Sciaridae) have been studied by light microscopy and nuclear staining. There are 8 cleavage cycles up to the syncytial blastoderm stage (4.5 hr), and during the 11th cycle cellularization begins (6.5 hr). The first 3 divisions take about 30 min each. During the 5th and 6th cycles, the maximum rate of division is reached (12 min/cycle at 22°C). After pole cell formation, the duration of the following mitotic cycles increases progressively. During nuclear migration, the presumptive germ line nuclei reach the egg cortex first, followed by anterior somatic nuclei and finally, posterior somatic nuclei reach the egg cortex. Possibly as a result of this region-specific nuclear migration, nuclear divisions become parasynchronous after 3 hr of embryogenesis (4th cycle). Several mitotic cycles later, between the 8th and 10th cycle in different embryos, X-chromosome elimination in somatic nuclei begins at the anterior egg pole and progresses in anteroposterior direction. Our observations suggest that the observed region-specific differences may be due to the activity of localized factors in the egg that control migration and nuclear cycle of the somatic nuclei.

Viscoelastic studies on Tetrahymena macronuclear DNA.

We have used viscoelastometry in an attempt to understand the physical organization of genetic material in Tetrahymena nuclei. The micronucleus or germ line nucleus is diploid. It divides mitotically during vegetative growth, and five pairs of chromosomes are seen in meiosis. The macronucleus, or somatic nucleus, is approximately 45-ploid, divides amitotically, and has no visible chromosomes at any stage. Viscoelastic analysis of Tetrahymena macronuclei reveals DNA Molecules of 2-3 X 10(10) daltons accounting for much, if not all, of the macronuclear DNA. Since the average chromosome in the micronucleus contains 2.4-2.7 X 10(10) daltons of DNA, we deduce that the macronucleus of Tetrahymena contains chromosome-sized DNA molecules.

Nuclear lamellae in the germ-line cells of gall midges (Cecidomyiidae, Diptera)

Lamellar structures in oogonial and spermatogonial cells of gall midges were found to form a complicated system of intranuclear compartments inside which all heteropycnotic chromosomes present in the germ-line cells of these insects are located. In this way, the heteropycnotic S-chromosomes are separated by the nuclear lamellae from the remaining, decondensed chromosomes (E-chromosomes) of the interphase germ-line nucleus.

Positive selection for mating with functional heterokaryons in Tetrahymena pyriformis.

Allelic assortment of Tetrahymena pyriformis makes possible the isolation of cells which contain both a heterozygous germ line nucleus and a somatic nucleus which expresses phenotypes of only one member of allelic pairs. Cycloheximide-sensitive segregants have been isolated from the vegetative progeny of cells heterozygous for a dominant mutation conferring resistance to cycloheximide; such segregants are called functional heterokaryons. Since progeny from crosses of these cells are cycloheximide-resistant, addition of the drug provides positive selection for successful exconjugants. The timing for expression of the new phenotype during conjugation is presented and used to identify what appear to be immature progeny of round one mating in genomic exclusion. Usefulness of functional heterokaryons in a number of genetic and developmental studies is discussed.

The cytology of paedogenesis in the gall midge Mycophila speyeri.

In paedogenetically developing female eggs of the gall midgeMycophila speyeri only one equational meiotic division occurs. The primary cleavage nucleus contains 29 chromosomes. In the fourth cleavage division 23 chromosomes are eliminated from the future somatic nuclei while the primordial germ-line nucleus keeps the high chromosome number.—The paedogenetic development of male eggs begins with two meiotic divisions. The egg nucleus with 14 or 15 chromosomes fuses with two, sometimes only one, somatic nuclei (2n=6) of maternal origin (regulation). Thus the primary cleavage nucleus contains 26 or 27 chromosomes, sometimes only 20 or 21. Elimination in cleavage divisions V and VI leeds to somatic nuclei with 3 chromosomes while the primordial germ-line nucleus keeps the high chromosome number.—Differences between male and female eggs and the evolution of regulation in gall midges are discussed.

Experiments on chromosome elimination in the gall midge, Mayetiola destructor

ABSTRACT Cleavage in Cecidomyidae (Diptera) is characterized by the elimination of chromosomes from presumptive somatic nuclei. The full chromosome complement is kept by the germ-line nuclei. The course of cleavage in Mayetiola destructor (Say) is described. After the fourth division two nuclei lie in the posterior polar-plasm and become associated with polar granules, and fourteen nuclei lie in the rest of the cytoplasm. All the nuclei possess about forty chromosomes. During the fifth division the posterior nuclei do not divide and the polar-plasm becomes constricted to form primordial germ cells (pole cells). The remaining fourteen nuclei divide and lose about thirtγ-two chromosomes so that twenty-eight nuclei are formed containing only eight chromosomes. These are the presumptive somatic nuclei. During subsequent divisions the pole cell nuclei retain the full chromosome number; these divisions occur less frequently than those of the somatic nuclei. Experiments were performed on early embryonic stages to elucidate the properties of the posterior end during the time that chromosome elimination was taking place from the presumptive somatic nuclei. Ultraviolet irradiation, constriction, and centrifugation techniques were used. The polar granules are concerned with the non-division of the germ-cell nuclei during the fifth division, since if the granules are dispersed by centrifugation, or if nuclei are prevented by constriction from coming into contact with them before the fifth division, all the nuclei divide with chromosome elimination at this division. With each technique it is possible to obtain embryos possessing germ cells with only eight chromosomes in their nuclei. Individuals possessing germ-line nuclei with only eight chromosomes were allowed to develop to maturity. Abnormalities were confined to the germ cells only and were the same regardless of which technique had been used to produce the deficient germ line. An ovary containing germ-cell nuclei with only eight chromosomes is unable to form both oocytes and nurse cells. A testis containing germ-cell nuclei with only eight chromosomes is unable to form spermatocytes but cells which come to resemble gametes are formed. Experimental males and females are both sterile. The results are discussed in relation to other experimental work on Cecidomyidae and the following main conclusions are reached: (a) the polar granules are responsible for preventing an irreversible loss of chromosomes from the germ-cell nuclei by preventing the mitosis of these nuclei during the fifth division; (b) the chromosomes normally retained in the germ line are required for gametogenesis, particularly for oogenesis. The significance of chromosome elimination is discussed.

Oogenesis in Mikiola fagi Hart. (Cecidomyiidae; Diptera).

1. Female somatic nuclei of Mikiola fagi contain only diploid sets of S-chromosomes (8 S), while germ-line nuclei have 24 chromosomes (8 S-chromosomes + 16 E-chromosomes). 2. In oogenesis only S-chromosomes form chiasmatic bivalents, while E-chromosomes occur as univalents. At prometaphase, and immediately before the first maturation division, the E-chromosomes may temporarily form associations of two, three or four, a contact which is of the somatic pairing type. 3. At metaphase, E-chromosomes are simultaneously eliminated from the spindle. This phenomenon is probably the result of a temporary inactivation of the kinetochores of E-chromosomes and may be interpreted as proof of the existence of transverse elimination forces in the spindle. 4. The E-chromosomes eliminated from the spindle aggregate as a rule in one or, less frequently, two or three groups, each of which forms then its own spindle. 5. Shortly before the first maturation division E-chromosomes rejoin the spindle with the bivalents. 6. During the first maturation division, which is a reduction division for S-chromosomes, the anaphase movement of E-chromosomes is greatly delayed. E-chromosomes, owing to the strong anaphase elongation of the spindle, are passively distributed on its surface. 7. There is no interphase between the first and second maturation division. Towards the end of anaphase I two small spindles of the second meiotic division are formed at the spindle poles, each of which contains only one haploid set of S-chromosomes. 8. Equational splitting of E-chromosomes occurs mostly when the S-chromosomes are at metaphase of the second maturation division. The anaphase movement of daughter E-chromosomes begins without prior formation of a metaphase plate. The position of E-chromosomes on the spindle surface seems to have no influence on the separation of the daughter chromosomes and their migration to the opposite poles of the first meiotic spindle. The anaphase movement of the daughter E-chromosomes, which occurs at the time when S-chromosomes undergo the second maturation division, leads to the formation of two groups each containing 16 chromosomes. In the early stages of this movement, each of the daughter E-chromosomes is oriented towards the pole not with its kinetochore but with one of its ends. Thus, the bivalent S-chromosomes undergo two maturation divisions whereby their number is reduced, while the univalent E-chromosomes undergo only one equational division. 9. It is postulated that the egg nucleus is formed as the result of a fusion between one haploid set of S-chromosomes and one set of E-chromosomes. As it is not known whether the eggs develop without fertilization, the establishment of the diploid number is open to discussion. Probably, in the Polish populations of the species with a very low percentage of males, the development starts with the fusion of the egg-nucleus with a haploid set of S-chromosomes whereby the number is raised from 20 to 24. 10. The evolution of cecidomyiid oogenesis is discussed. The modifications of oogenesis observed in the subfamily Cecidomyiinae are interpreted as mechanisms preventing the decrease of the number of univalent E-chromosomes during maturation divisions. 11. During prophase a special nuclear material accumulates around the chromosomes grouped inside the nucleus and leads to its characteristic differentiation into an interior part containing both S- and E-chromosomes and an exterior zone of nuclear sap. In prometaphase the first meiotic spindle arises from material localized in the interior part of the nucleus. The problems of the origin of the spindle-forming material as well as the formation of spindles by the E-chromosomes eliminated into the cytoplasm are discussed.