Comparative analysis of the chromosomal and genomic organization of Ty1-copia-like retrotransposons in pteridophytes, gymnosperms and angiosperms.

We have investigated the physical distribution of the reverse transcriptase genes of Ty1-copia-like retrotransposable elements from 12 plant species belonging to different subdivisions by hybridization in situ on chromosome preparations. Ty1-copia-like elements showed different and non-random hybridization patterns. A dispersed distribution throughout most of the chromosomes with reduced hybridization at some regions or with some weak clustering at other regions was found in Allium cepa, Beta vulgaris, Brassica campestris, Brassica oleracea, Pennisetum glaucum, Pinus elliottii, Selaginella apoda, Vicia faba and Vicia narbonensis. Reduced hybridization occurred mainly at centromeric regions, nucleolus-organizing regions and regions known to be mainly composed of tandemly repeated sequences. In the fern Pteris cretica the retroelements showed a dispersed genomic organization with clustering at some chromosomal regions and whole chromosomes showing little signal. In Arabidopsis thaliana and Cicer arietinum Ty1-copia-like elements were found in clusters at the paracentromeric heterochromatin, a novel organization for a repetitive element in A. thaliana. New retroelement families were isolated from A. thaliana and from Beta vulgaris. Alignment of the deduced peptide sequences with Ty1-copia-like elements from other plants showed considerable divergence which was used to calculate their relationships, indicating the value of reverse transcriptase gene analysis in phylogenetic and biodiversity studies.

The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution.

Retrotransposons make up a major fraction--sometimes more than 40%--of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g., slash pine, 23,000 Mbp, or barley, 5500 Mbp) is frequent, whereas many species exclude retrotransposons from other sites of heterochromatin (e.g., intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana, widely used for plant molecular genetic studies because of its small genome (c. 100 Mbp), the Ty1-copia group reverse transcriptase gene domains are concentrated in the centromeric regions, colocalizing with the 180 bp satellite sequence pAL1. Unlike the pAL1 sequence, however, the Ty1-copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons and comparisons of the distribution between species is critical to understanding their evolution and the significance for generation of the new patterns of variability and in speciation.

The genomic and physical organization of Ty1-copia-like sequences as a component of large genomes in Pinus elliottii var. elliottii and other gymnosperms.

A DNA sequence, TPE1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliottii (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8 kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE1 represents a large subgroup of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE1 transcription could be detected, it is most likely an inactive element--at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three- and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.

Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species.

We have analysed a family of highly repetitive DNA from Arabidopsis arenosa (L.) Lawalrée [syn. Cardaminopsis arenosa (L.) Hayck] composed of AT-rich tandem repeats of 166-179 bp in head to tail organization. Sequence comparison between several repeat units revealed a high level of divergence of 4.5% to 25%. The sequence family shows more than 58% homology to satellite sequences described in Arabidopsis thaliana (L.) Heynh. but no homology to other satellite repeats in the Cruciferae. Within the genus Arabidopsis the satellite sequence was found to be present in A. thaliana and Arabidopsis suecica (Fries) Norrlin, but not in Arabidopsis griffithiana (Boiss.) N. Busch and Arabidopsis pumila (Stephan) N. Busch. In situ hybridization to metaphase chromosomes of A. arenosa (2n = 4x = 32) showed the sequence to be localized at the centromeres of all 32 chromosomes with substantial hybridization along the chromosome arms. Using Southern hybridization and in situ hybridization, we give evidence that A. suecica is a hybrid of A. thaliana and A. arenosa. A considerable reorganization of the A. thaliana satellite sequence pAL1 occurred in the hybrid genome while no molecular change of the A. arenosa repeat was observed in the hybrid. Analysis of related repeats enabled differentiation between closely related genomes and are useful for the investigation of hybrid genomes.

Molecular and physical organization of highly repetitive, undermethylated DNA from Pennisetum glaucum.

A HaeIII monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (MspI/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5'-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.

Parasuicide in psychiatric inpatients: results of a controlled investigation.

A group of 20 psychiatric inpatients who committed parasuicide during their hospital stay were compared with 58 randomly selected control inpatients and with 34 patients who were admitted following a parasuicide. The findings indicate: (1) The frequency of in-hospital parasuicide amounts to 3.2% of all admissions. (2) Apart from the more frequent past suicidal activity of the in-hospital parasuicide group, no really important differences were identified between these and control patients. The broad definition of parasuicide probably accounts for this result. (3) The patients who committed parasuicide before admission represent a diagnostically different, clinically less severely ill population. The differences between both groups of parasuicide patients support the notion of the heterogeneity of the parasuicide population.

Isolation and characterization of a tumor cell surface antigen from spontaneously transformed BALB/c mouse fibroblasts.

This work describes the detection, isolation, and partial characterization of a BALB/c mouse fibroblast cell surface antigen. This antigen migrates as a polypeptide of approximately 100,000 daltons in a discontinuous sodium dodecyl sulfate/polyacrylamide gel electrophoresis system, can be labeled by either lactoperoxidase-catalyzed cell surface 125I iodination or metabolic incorporation of [3H]glucosamine, and can be isolated by concanavalin A affinity chromatography. This cell surface glycoprotein is antigenic in BALB/c mice and has been correlated with the rejection of immunogenic tumor cells. Also, antiserum specific for Moloney leukemia virus precipitates the 100,000-dalton cell surface protein from viral and immunogenic spontaneous transformants. This virus-related antigen comigrates on sodium dodecyl sulfate gels with the major iodinated cell surface protein of these transformants. Rabbit antiserum to the purified antigen demonstrates a marked preference for the surfaces of immunogenic tumor cells as compared with normal cells and nonimmunogenic tumor cells.

Chemical and immunological studies of cell surfaces from normal and transformed cells.

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-125I or [3H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.