Nuclei purified from cauliflower mosaic virus-infected turnip leaves contain subgenomic, covalently closed circular cauliflower mosaic virus DNAs

Nuclei isolated from cauliflower mosaic virus (CaMV) infected turnip leaves contain subgenomic CaMV DNA species in addition to the genome length CaMV DNA. These subgenomic CaMV DNA species are present as covalently closed circles (form I), relaxed circles (form II) and linear (form III) molecules. The subgenomic form I DNA species range in size from about 10% of genome length to nearly genome length. These subgenomic DNA species appear in tissue infected with cloned CaMV DNA, indicating that they arise rapidly and have not accumulated in the virus population from serial propagation of CaMV. No specific region of the CaMV genome appears to be preferentially deleted to form the subgenomic CaMV DNA species. At least three distinct subgenomic species appear to accumulate preferentially in nuclei isolated from infected tissue. Two of these abundant subgenomic CaMV DNA species are form I and the other one is form III. Some of the subgenomic CaMV DNA species appear to be minichromosomes.     

Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (-) strand viral DNA and reverse transcription of primed RNA templates

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (-) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (-) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (-) strand DNA. These are discussed in relation to their role as possible replicative intermediates.     

Construction of an EcoRI restriction map of Mycoplasma pneumoniae and localization of selected genes.

A restriction map of the genome of Mycoplasma pneumoniae, a small human pathogenic bacterium, was constructed by means of an ordered cosmid library which spans the complete bacterial chromosome. The positions of 143 endonuclease EcoRI restriction fragments were determined and aligned with the physical map. In addition, restriction sites for the rare-cutting enzymes XhoI (25 sites), ApaI (13 sites), NotI (2 sites), and SfiI (2 sites) were included. The resulting map consists of 185 restriction sites, has a mean resolution of 4.4 kbp, and predicts a genome size of 809 kbp. In addition, several genes were identified and mapped to their respective genomic EcoRI restriction fragments.     

Studies on the single-stranded discontinuities of the cauliflower mosaic virus genome.

The Cauliflower Mosaic Virus (CaMV) genome is a double-stranded DNA molecule of about 5 million daltons. Native DNA molecules appear heterogeneous when analysed by gel electrophoresis. We have examined the nature of this apparent heterogeneity. Besides, this genome is shown here to contain three single-stranded breaks, as revealed by different denaturation experiments: heating at 75 degrees C, treatment with NaOH or dimethyl sulfoxide (DMSO). Labelling with terminal transferase proves that the 3' ends at these interruptions all have free hydroxyl groups. Electron microscopy and alkaline gel electrophoresis indicate that these three discontinuities are shared by both strands, and that they are not randomly located. S1 nuclease is active on CaMV DNA and generates three fragments. The comparison between the sizes of these fragments and of the products of denaturation leads us to consider that S1 acts at the level of the interruptions. We have determined that two of them, distant by one third genome unit, are in the same strand; the other is in the opposite strand, distant by one sixth genome unit from the nearest other one. The combined use of restriction enzymes and S1 nuclease has enabled us to locate these three discontinuities on the restriction map of the CaMV genome that we have otherwise established.     

The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses

Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin.     

Alignment of Sfi I sites with the Not I restriction map of Schizosaccharomyces pombe genome.

A Sfi I restriction map of the fission yeast Schizosaccharomyces pombe genome was aligned with the Not I restriction map. There are 16 Sfi I sites in the S. pombe genome. Three Sfi I sites are on chromosome III which is devoid of Not I sites. The sizes of the entire genome and individual chromosomes, calculated from the Sfi I fragment sizes, are consistent with that calculated from the Not I fragment sizes. The Sfi I map provides greater physical characterization of the S. pombe genome and further validates the use of S. pombe chromosomal DNA as size standard. These maps have allowed detection of polymorphism on all three chromosomes.     

Physical Map of the Saccharomyces Cerevisiae Genome at 110-Kilobase Resolution

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.     

Complete maps of IS1, IS2, IS3, IS4, IS5, IS30 and IS150 locations in Escherichia coli K12

Summary In this paper complete distribution maps are presented of the seven IS elements 1, 2, 3, 4, 5, 30 and 150. These maps were obtained during the construction of an almost complete restriction map of the Escherichia coli genome of K12 strain BHB2600. The positions of IS elements were correlated to this map. The distribution of integration sites of all IS types is nonrandom. Besides a large gap from 79 min to 96 min, there is a pronounced IS cluster at 6 min and another at 97 min, map locations that have low gene incidences on the classical map. One cluster coincides with a region of IS induced rearrangements. The IS distribution pattern was compared to patterns of strains W3110 and HB101.     

Baboon endogenous virus genome. I. Restriction enzyme map of the unintegrated DNA genome of a primate retrovirus

A detailed restriction map was deduced for the genome of an endogenous retrovirus of a higher primate, that of baboon. The cleavage sites for 12 restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with baboon endogenous virus was isolated. Hybridization with a strong-stop complementary DNA probe demonstrated presence of a terminal repetition in the linear viral DNA. The positions of restriction sites for two particular enzymes, SmaI and XhoI, near each end were consistent with this result and indicated that the length of the repetition is 0.55 +/- 0.01 kilobase. The linear viral DNA had a unique restriction map indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-specific probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. We observed a minor heterogeneity in a BamHI recognition site 1.95 kilobases from the right end of the linear map.     

Genetic mapping of Ty elements in Saccharomyces cerevisiae.

We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty. The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17. Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S. cerevisiae genome. These transformants were used to map genetically sites of several Ty elements. The 24 transformants recovered at Ty sites define 19 distinct loci. Seven of these were placed on the genetic map. Two classes of Ty elements were identified in these experiments: a Ty1-17 class and Ty elements different from Ty1-17. Statistical analysis of the number of transformants at each class of Ty elements shows that there is preferential integration of the CV9 plasmid into the Ty1-17 class.     

The human mitochondrial ribosomal protein genes: mapping of 54 genes to the chromosomes and implications for human disorders.

Mitochondria possess their own translational machinery, which is composed of components distinct from their cytoplasmic counterparts. To investigate the possible involvement of mitochondrial ribosomal defects in human disease, we mapped nuclear genes that encode mitochondrial ribosomal proteins (MRPs). We generated sequence-tagged sites (STSs) of individual MRP genes that were able to be detected by PCR. They were placed on an STS content map of the human genome by typing of radiation hybrid panels. We located 54 MRP genes on the STS-content map and assigned these genes to cytogenetic bands of the human chromosomes. Although mitochondria are thought to have originated from bacteria, in which the genes encoding ribosomal proteins are clustered into operons, the mapped MRP genes are widely dispersed throughout the genome, suggesting that transfer of each MRP gene to the nuclear genome occurred individually. We compared the assigned positions with candidate regions for mendelian disorders and found certain genes that might be involved in particular diseases. This map provides a basis for studying possible roles of MRP defects in mitochondrial disorders.     

Comparison of a Brassica oleracea genetic map with the genome of Arabidopsis thaliana

Brassica oleracea is closely related to the model plant, Arabidopsis thaliana. Despite this relationship, it has been difficult to both identify the most closely related segments between the genomes and determine the degree of genome replication within B. oleracea relative to A. thaliana. These difficulties have arisen in part because both species have replicated genomes, and the criteria used to identify orthologous regions between the genomes are often ambiguous. In this report, we compare the positions of sequenced Brassica loci with a known position on a B. oleracea genetic map to the positions of their putative orthologs within the A. thaliana genome. We use explicit criteria to distinguish orthologous from paralogous loci. In addition, we develop a conservative algorithm to identify collinear loci between the genomes and a permutation test to evaluate the significance of these regions. The algorithm identified 34 significant A. thaliana regions that are collinear with >28% of the B. oleracea genetic map. These regions have a mean of 3.3 markers spanning 2.1 Mbp of the A. thaliana genome and 2.5 cM of the B. oleracea genetic map. Our findings are consistent with the hypothesis that the B. oleracea genome has been highly rearranged since divergence from A. thaliana, likely as a result of polyploidization.     

Correlation between genetic map and map of cleavage sites for sequence-specific endonucleases SalI, KpnI, BglI, and BamHI in bacteriophage T4 cytosine-containing DNA.

Cleavage sites for SalI, KpnI, BglI, and BamHI in cytosine-containing DNA from T4 alc10(alc) nd28(denA) D2a2(denB) amE51x5(56) amN55x5(42) have been mapped relative to each other, and the positions of deletions sa delta 9 (D1-stp), r1589(rII), del(39-56)12, and tk2(rI-tk) relative to these cleavage sites have been determined. Based on these analyses, a physical map of the T4 genome containing 166 kilobase pairs has been constructed.