Organization and dynamics of satellite and telomere DNAs in Ascaris: implications for formation and programmed breakdown of compound chromosomes.

In the nematode genus Ascaris the germline genome contains considerable amounts of extra DNA, which is discarded from the somatic founder blastomeres during early cleavage. In Parascaris univalens the haploid germline genome is contained in one large compound chromosome, which consists of a euchromatic region containing the somatic genome flanked by large blocks of heterochromatin. Fluorescence in situ hybridization of fractions of the germline-limited satellite DNA revealed two highly repeated sequence families establishing the entire heterochromatin (HET blocks). The repeats, a pentanucleotide, TTGCA, and a decanucleotide, TTTGTGCGTG, constitute separate segments of the HET blocks. The blocks are polymorphic in length and, hence, in copy number of the repeats, and the arrangement of the segments. The numerous sequence variants of both repeats display a disperse distribution. The type and rate of base substitutions within both repeat units depend on position. Prior to the elimination process in presomatic cells, termed chromatin diminution, the chromosomes undergo differential mitotic condensation. Interstitial 'chromatin linkers' flanking the prospective numerous somatic chromosomes remain entirely decondensed. The somatic chromosomes are released from the plurivalent chromosomes via excision of the linkers at onset of anaphase, followed by exclusion of the akinetic linker chromatin and HET blocks from the daughter nuclei. In Ascaris suum, the germline-limited satellite, which consists of one 123 bp repeat, is scattered throughout the numerous chromosomes in small heterochromatic knobs of variable sizes, residing at chromosomal ends and/or intercalary positions. The programmed breakage, which appears to proceed in a similar manner to that in P. univalens, results in the loss of all heterochromatic knobs, accompanied by an increase in chromosome number. In both species, all germline chromosomes are capped by tracts of TTAGGC repeats. In P. univalens, such telomeric tracts also occur at the termini of the euchromatic intercalary regions. Upon diminution all telomeric tracts are discarded. De novo telomere addition occurs in all somatic cell lineages of both species. The presented data shed light on the evolutionary history of chromosome aggregation and satellite DNA formation, and putative mechanisms involved in the process of site-directed breakage to reestablish stable somatic chromosomes.

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.

The highly variable pentameric repeats of the AT-rich germline limited DNA in Parascaris univalens are the telomeric repeats of somatic chromosomes.

We have characterized the organization of the germline limited DNA of P. univalens by means of sequence analysis. The repeat unit of this satellite DNA is the pentanucleotide 5'TTGCA, although there is a high degree of sequence variation. Repeat variants are not arranged in tandem but in a disperse, nonrandom manner. In the somatic genome which arises from the germline genome through extensive genomic rearrangement early in development, copies of these pentamers represent the telomeric repeats, indicated by their sensitivity to Bal 31 and their presence in a somatic endlibrary. Unlike telomeric sequences from other species the P. univalens telomeres do not display consecutive guanines and no strand bias for that base, recently suggested as universal features of eukaryotic telomeres. Investigation of fragments that carry pentameric repeats along with sequences of different type identifies a 5 bp consensus sequence at the junction point. We suggest a model in which pentameric repeats originate via amplification by a terminal transferase (telomerase) in both the germline and the somatic genome.

Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA.

We have determined the prototype sequence of the DNA which is eliminated in the course of chromatin diminution in Ascaris suum. This DNA which is virtually absent from somatic cells but retained in the germ line consists predominantly of highly repetitive sequences which are variants of an AT rich 123 base pair repeat unit. Both major and minor variants have been sequenced. The overall structure of this germ line limited DNA corresponds to the segmental organization characteristic of satellite DNAs. Possible correlations between the mechanism of chromatin diminution and some properties of the satellite sequence are discussed.

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum.

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33 258 -- CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 x 10(5) copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a 'theme and variation' pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.