Conserved regions at the DNA primase locus of IncP alpha and IncP beta plasmids

Genes specifying DNA primases (pri) are common in all IncP plasmids examined so far. These plasmids suppress the thermosensitive character of the Escherichia coli dnaG3 mutation. The mechanism of suppression appears to be identical to that known for RP4 and IncI alpha plasmids. The DNA primases of both these plasmid types can substitute for the dnaG protein in chromosomal DNA replication. The pri genes of the alpha and beta subgroup of IncP plasmids are related to each other as judged from Southern hybridization and immunological data. Extensive DNA and protein sequence homology has been detected although the gene products of the alpha and beta subgroups exhibit substantial differences in size. The arrangement of overlapping genes at the pri locus of IncP alpha plasmids also appears to be present in the IncP beta group.     

Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA

A method that permits the in vitro amplification and cloning of DNA dissected from specific regions of a chromosome and does not require prior knowledge of the DNA sequence is described. DNA from several different chromosomal loci in the Drosophila melanogaster genome has been isolated by this method. Although the procedure was developed to permit the isolation of DNA sequences from serial sections of a single microdissected polytene chromosome, it should be useful for obtaining DNA clones from specific regions of the nonpolytene chromosomes of other organisms as well.     

DNA repair defects associated with chromosomal translocation breaksite regions.

Using an assay that measures the removal of UV-induced pyrimidine dimers in specific DNA sequences, we have found that the Pvt-1, immunoglobulin H-C alpha (IgH-C alpha), and IgL-kappa loci are poorly repaired in normal B lymphoblasts from plasmacytoma-susceptible BALB/cAnPt mice. Breaksites in these genes are associated with the chromosomal translocations that are found in > 95% of BALB/cAnPt plasmacytomas. In contrast to those from BALB/cAnPt mice, B lymphoblasts from plasmacytoma-resistant DBA/2N mice rapidly repair Pvt-1, IgH-C alpha, and IgL-kappa. Further, (BALB/cAnPt x DBA/2N)F1 hybrids, which are resistant to plasmacytoma development, carry an efficient (DBA/2N-like) repair phenotype. Analysis of allele-specific repair in the IgH-C alpha locus indicates that efficient repair is controlled by dominant, trans-acting factors. In the F1 heterozygotes, these factors promote efficient repair of BALB/cAnPt IgH-C alpha gene sequences. The same sequences are poorly repaired in the BALB/cAnPt parental strain. Analysis of the strand specificity of repair indicates that both strand-selective and nonselective forms of repair determine repair efficiency at the gene level in nonimmortalized murine B lymphoblasts.     

Conserved pore-forming regions in polypeptide-transporting proteins

Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the beta-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organelles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the beta-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed.     

Toxicity and extrachromosomal DNA in strains of the cyanobacterium Microcystis aeruginosa

Abstract Correlations were sought between toxicity and the presence of plasmids in toxic and non-toxic strains of Microcystis aeruginosa . Plasmids were present in toxic and non-toxic cultures. Cultivation of the toxic Microcystis PCC7820 in the presence of novobiocin did not influence toxicity, although extrachromosomal DNA was no longer detected after novobiocin treatment. The data indicate that it is unlikely that plasmids are involved in the toxicity of Microcystis PCC7820.     

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.     

An extrachromosomal fragment of telomeric DNA in wheat

A procedure developed orginally for selective extraction of viral (extrachromosomal) DNA from virus-infected mammalian cells was applied to cell nuclei isolated from uninfected wheat embryos. The resulting nuclear extrachromosomal DNA (exDNA) was enriched for telomere-type sequences by isopycnic centrifugation and inserted into the Sma I site of pUC119. A cloned DNA fragment (241 bp) was found to consist primarily of tandemly repeated heptamere units of the same sequence (5-CCCTAAA-3) that is known to predominate in telomeric DNA of Arabidopsis thaliana. Hybridization experiments indicate that extrachromosomal telomeric repeats are abundant in resting embryos and disappear rapidly during germination.     

Highly variable regions of DNA flank the human alpha globin genes.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human alpha globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the alpha globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.     

Conserved and variable elements in RNA genomes of potexviruses

The nucleotide sequences of genomic RNAs and predicted amino acid sequences of two strains of potato virus X and white clover mosaic potexvirus were compared to each other, and the proteins of different plus-RNA-containing plant viruses. The predicted non-virion proteins of potexviruses have direct sequence homology and common structural peculiarities with those of several 'Sindbis-like' plant viruses. The most conserved amino acid sequences were found to be located in the polypeptide encoded by the long 5'-proximal open reading frame (ORF1). The putative polypeptide encoded by the ORF2 starting beyond the ORF1 stop codon is clearly related to the presumptive NTP-binding domain of the ORF1-coded polypeptide. These results suggest possible functions for all of the potexvirus proteins and also indicate that potexviruses have a genome organization which is considerably different from that of other plant viruses.     

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluroescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromosome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.     

Survey of extrachromosomal DNA found in the filamentous cyanobacteria.

Cleared lysates of 13 species of filamentous cyanobacteria were examined for the presence of extrachromosomal DNA by using agarose gel electrophoresis and ethidium bromide staining. Seven of the 13 species contained extrachromosomal covalently closed circular DNA, and all but 1 species contained multiple elements. There was no correlation between the presence of extrachomosomal DNA and either the range of metabolic activities found in the cyanobacteria or the differentiated cell types or structures elaborated by the morphologically complex filamentous cyanobacteria.     

Tn7 transposes proximal to DNA double-strand breaks and into regions where chromosomal DNA replication terminates

We report that the bacterial transposon Tn7 can preferentially transpose into regions where chromosomal DNA replication terminates. DNA double-strand breaks are associated with the termination of chromosomal replication; therefore, we directly tested the effect of DNA breaks on Tn7 transposition. When DNA double-strand breaks are induced at specific sites in the chromosome, Tn7 transposition is stimulated and insertions are directed proximal to the induced break. The targeting preference for the terminus of replication and DNA double-strand breaks is dependent on the Tn7-encoded protein TnsE. The results presented in this study could also explain the previous observation that Tn7 is attracted to events associated with conjugal DNA replication during plasmid DNA transfer.     

3 micron DNA - an extrachromosomal ribosomal DNA in the yeast Saccharomyces cerevisiae

A new class of extrachromosomal DNA which consists predominantly of covalently closed molecules with lengths around 3 micron, has been detected in Saccharomyces cerevisiae strain 6-1G-P188 from the Peterhof collection. Restriction analysis of the 3 micron DNA as well as of recombinant plasmids carrying HindIII fragments of the 3 micron DNA permitted construction of a physical map of the new extrachromosomal DNA species, and detection of two types differing by one EcoRI restriction site. Molecular hybridization, as well as comparison of the restriction maps, revealed the complete structural identity of the 3 micron DNA with a chromosomal repetitive unit of rDNA containing the genes for 25 S, 18 S, 5.8 S and 5 S rRNAs.     

Nuclear rDNA in Euglena gracilis: paucity of chromosomal units and replication of extrachromosomal units

Copy number of chromosomal rDNA units was investigated in two Euglena gracilis wild-type strains. It was established by dot blot analysis that these strains possess about four integrated units per haploid genome. This is the first example of a photosynthetic cell with only a few chromosomal ribosomal genes. In addition to these units, Euglena has 800 to 4000 extrachromosomal rDNA units. Electron microscopy revealed that these free rDNA circles bear a replication origin, and intermediates of replication show a D-loop structure.