Random and nonrandom integration of a polyomavirus DNA molecule containing highly repetitive cellular sequences.

RmI is a circular DNA molecule that consists of a complete polyomavirus genome with an insertion (Ins) of mouse cellular DNA. This polyomavirus genome carries a mutation which renders its replication, but not its transforming ability, temperature sensitive. Ins contains both unique and repetitive cellular DNA sequences. We transfected RmI into rat cells at the permissive and nonpermissive temperatures for replication and isolated clones that had integrated RmI in their genomes. In this paper, we describe detailed mapping of the integrated RmI sequences present in 37 different cell clones. Our results indicated that transfection at the permissive temperature resulted in a random integration pattern, whereas transfection at the nonpermissive temperature resulted in a nonrandom integration pattern. The nonrandom insertions had a preferential length and preferential endpoints. We argue from these results that the nonrandom integration pattern is related to the presence of Ins and that the switch between nonrandom integration and random integration reflects a modification of the integrating substrate. When both are active, the random mechanism dominates the nonrandom mechanism.     

Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S-25S and 5S rDNA, telomeres) and genus-specific satellite repeats (VicTR-A and VicTR-B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR-A and -B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied.     

Structural analysis of a cDNA clone from Xenopus laevis containing a repetitive sequence element

Total polysomal RNA from Xenopus laevis stage 40 embryos was probed for the presence of repetitive sequences by Northern blot analysis with a genomic DNA fragment which had previously been shown to contain several repetitive sequence elements (Spohr et al., 1981). The analysis revealed that various presumptive mRNAs contain sequences complementary to the repetitive probe. Consequently, a cDNA library was constructed and screened with the same probe. Forty-eight positive recombinants containing eucaryotic inserts of 300–700 base pairs were isolated and one such clone was characterized in detail. Analysis of its nucleotide sequence revealed the presence of an open reading frame for 118 amino acids. Comparison of nucleotide sequences located 3′ to this presumptive protein coding region with the sequence of the genomic DNA fragment used as a probe clearly identifies and allows one to define the exact location of the repetitive element in the cloned cDNA. This analysis shows furthermore that one portion of the repeated sequence is highly conserved in the two members of this repetitive sequence family, whereas the other part is more divergent. In this area blocks of oligonucleotides are scattered between nonhomologous DNA stretches. The occurrence frequency of the presumptive mRNAs which carry repetitive elements homologous to the used repetitive probe is suggested to be close to that of rare mRNAs.     

Characterization of a highly repetitive family of DNA sequences in the mouse.

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.     

Analysis of repetitive sequence elements containing tRNA-like sequences.

Several repetitive sequence elements from diverse species share extensive sequence homology with tRNA molecules. Analysis of the tRNA-like sequences within these elements suggest that they have originated from authentic tRNA sequences. Elements containing tRNA-like sequences can be divided into three distinct groups whose members share extensive sequence homology, have similar sequence organization and have unique species distribution. We suggest that these three groups represent independent examples of retroposon families that have originated from tRNAs.     

Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities.

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.     

Plasmodium berghei, P. chabaudi, and P. falciparum: similarities in phosphoproteins and protein kinase activities and their stage specific expression

Phosphoproteins from Plasmodium berghei, P. chabaudi, and P. falciparum are compared. A major phosphoprotein of 46 kDa is found in all three species. Peptide mapping indicates that this protein is indeed the same in all three cases and is phosphorylated at similar sites in all three species. Monoclonal antibodies were raised against three other P. berghei phosphoproteins. All three monoclonal antibodies recognize both P. berghei and P. chabaudi proteins. Only one of the monoclonal antibodies crossreacts with a P. falciparum protein of 36 kDa, whereas the equivalent P. berghei and P. chabaudi proteins are 34 and 32 kDa, respectively. The highest rate of synthesis of the phosphoproteins is observed during the early trophozoite stage, whereas the highest rate of phosphorylation is observed during the late trophozoite stage.     

cDNA and deduced primary structure of rat protein B23, a nucleolar protein containing highly conserved sequences

Protein B23 (Mr/pI = 38,000/5.1) is a major RNA-associated nucleolar phosphoprotein which contains highly acidic segments and has a high affinity for silver ions. Using synthetic oligonucleotides as probes cloned cDNAs encoding protein B23 were isolated and characterized. One of the cDNAs, obtained from a rat brain library, contained an insert of 1232 base pairs of DNA encoding a polypeptide of 292 amino acid residues. Segments of the protein sequence were confirmed by partial sequencing of CNBr fragments from rat hepatoma protein B23. The protein contains a methionine-rich amino-terminal sequence and two highly acidic segments in the center of the sequence. The first acidic segment, in which 11 of the 13 residues are acidic, begins at residue 120 and contains a major phosphorylation site. In the second segment (residues 159-187) there are four copies of the sequence Asp-Asp-Glu, and all but two of the 29 residues have acidic side chains. When the sequence of the rat protein was compared with available sequences from other species a high degree of conservation was found; the 77-residue carboxyl-terminal sequence is identical with that of human protein B23 (Chan, P. K., Chan, W.-Y., Yung, B. Y. M., Cook, R. G., Aldrich, M. B., Ku, D., Goldknopf, I. L., and Busch, H. (1986) J. Biol. Chem. 261, 14335-24341), and about 63% of the residues are identical when the rat B23 sequence is compared with protein N038 from Xenopus laevis (Schmidt-Zachmann, M. S., Hügle-D?rr, B., and Franke, W. (1987) EMBO J. 6, 1881-1890). Except for the presence of highly acidic regions no significant similarities were found with protein C23 (nucleolin), the other major nucleolar protein.     

Comparative genomics of elastin: Sequence analysis of a highly repetitive protein.

Due to the low complexity associated with their sequences, uncovering the evolutionary and functional relationships in highly repetitive proteins such as elastin, spider silks, resilin and abductin represents a significant challenge. Using the polymeric extracellular protein elastin as a model system, we present a novel computational approach to the study of sequence, function and evolutionary relationships in repetitive proteins. To address the absence of accurate sequence annotation for repetitive proteins such as elastin, we have constructed a new database repository, ElastoDB (http://theileria.ccb.sickkids.ca/elastin), dedicated to the storage and retrieval of elastin sequence- and meta-data. To analyse their sequence relationships we have devised an innovative new method, based on the identification of overrepresented 'fuzzy' motifs. Applying this method to elastin sequences derived from mammals, chicken, Xenopus and zebrafish resulted in the identification of both highly conserved, and taxon and species specific motifs that likely represent important functional and/or structural elements. The relative spacing and organization of these elements suggest that exon duplication events have played an important role in the evolution of elastin. Clustering of similarity profiles generated for sets of exons and introns, revealed a pattern of putative duplication events involving exons 15-30 in mammalian and chicken elastins, exons 20-31 in both zebrafish elastins, exons 15-20 in fugu elastin and exons 35-50 in Xenopus elastin 1. The success of this approach for elastin offers a promising route to the elucidation of sequence, structure, function and evolutionary relationships for many other proteins with sequences of low complexity.     

Organization of a family of highly repetitive sequences within the human genome

Recombinant clones containing the highly repetitive human DNA sequence approximately 340 base-pairs in length obtained after EcoRI digestion (αRI-DNA) were cloned in plasmid pAT153. Two clones contained a single copy of the αRI-DNA sequence, and the third had an insert with two copies of the sequence in tandem. When radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI, a series of multiple bands was obtained up to 22 repeats in length, demonstrating that the αRI-DNA sequences occur in tandem arrays in the genomic DNA. A reassociation analysis using isolated insert DNA from one of the recombinant clones showed that the family of sequences is repeated 22,000 times in the human genome. Clones containing the αRI-DNA sequence were also isolated from a library of human genomic DNA in bacteriophage λ. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by DNA less abundant than the αRI sequence.     

Immunoelectrophoretic analysis of erythrocytic stages of Plasmodium yoelii and P. chabaudi

Triton X-100 extracts of erythrocytes infected with Plasmodium chabaudi and P. yoelii were analysed in crossed immunoelectrophoresis with rabbit antisera. The parasite origin of the antigens detected was assessed by metabolic radiolabelling of the parasites with 35S-methionine. About 12 immunoprecipitates were obtained with both extracts and their homologous antiserum. Cross-tests showed that the two parasite strains were very similar antigenically. Species-specific antigens could, however, also be demonstrated. Two antigens, present on both P. yoelii- and P. chabaudi-infected erythrocytes, were located on the surface of the host cell membrane as judged from 125I-labellings with lactoperoxidase. Experiments with phenyl-Sepharose showed that most of the antigens detected were hydrophilic and none of them reacted with concanavalin A.     

Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events

There are several methods of modifying bovine genomes. Pronuclear microinjection is more widely used but it is still to be improved. Searches for alternatives have lead to the development of new methods including SMGT (Sperm Mediated Gene Transfer), in which live spermatozoa are used as vehicles for DNA delivery during in vitro fertilization. In previous studies, we presented evidence that a highly repetitive Alu-like repeat favours transgenesis by homologous recombination (HR). Up to 60% integration via HR was obtained following pronuclear microinjection of a Pst1 beta-actin GFP DNA construction. In the present study, we show that HR-mediated integration is also possible using SMGT, since bovine spermatozoa electroporated with the same DNA construct are able to transfer it to a high proportion of embryos obtained by in vitro fertilization. Swim-up selected bovine spermatozoa were mixed with the Pst1 beta-actin GFP construct (6 x 10(6) spermatozoids were incubated with 600 ng of muDNA), submitted or not to electroporation (300 V, 25 F) and treated or not with DNase I. The process of electroporation itself did not affect in vitro embryonic development. However, oocytes fertilized with electroporated DNA-treated spermatozoa developed beyond the 16-cell stage in proportions that were significantly lower (27% with Pst1 beta-actin GFP and 34% with beta-actin GFP) compared to the control without DNA (44%). On the other side, the use of electroporation significantly increased the uptake of DNA. The number of homologous recombination events detected by PCR went from 3.5% without electroporation to 46.5% after electroporation. In conclusion, our results confirm that spermatozoa electroporation combined with homologous recombination in a highly repetitive Pst1 sequence is a feasible method to obtain transgenic bovine embryos.     

Simple repetitive DNA sequences from primates: Compilation and analysis

Simple repeats composed of tandemly repeated units 1–6 nucleotides (nt) long have been extracted from a selected set of primate genomic DNA sequences. Of the 501 theoretically possible, different types of repeats only 67 were present in the analyzed database in at least two different size ranges over 12 nt. They include all simple repeats known to be polymorphic in the primate genome. A list of moderately expanding and nonexpanding oligonucleotide patterns has also been included. Furthermore, we have compiled statistical data with emphasis on the overall variability of the most abundant 67 types of repeats. We have demonstrated that the expandability of at least some simple repeats may be affected by the overall base composition and by flanking sequences. In particular, the occurrence of tandemly repeated CAG and GCC triplets in exons positively correlates with their G+C content. We also noted that in the vicinity of Alu sequences tetrameric repeats are more abundant than in the total genomic DNA. This paper can be used as a comprehensive guide in identification of the most abundant and potentially polymorphic simple repeats. It is also of broader significance as a step toward understanding the contribution of flanking sequences and the overall sequence composition to variability of simple repeats. Correspondence to: J. Jurka     

The occurrence of families of repetitive sequences in a library of cloned cDNA from human lymphocytes

A library of cloned cDNAs representative of lymphocyte total poly(A)+ RNA was screened with total DNA probes at high clone density. 10% of the recombinants showed the presence of sequences which are repeated in the genome. Further analysis of six such isolated cDNA clones indicated that they contain different families of repetitive sequences with reiteration frequencies of between 150 and 45,000 copies per haploid genomes. Five of the six clones were found to contain single copy sequences as well as a repetitive sequence. cDNA clones containing repetitive sequences have been found to be derived from high, intermediate and low abundance classes of lymphocyte poly(A)+ RNA.