Temperate Bacillus bacteriophage SP16 genome is circularly permuted and terminally redundant.

The physical nature of temperate Bacillus bacteriophage SP16 DNA was analyzed by electron microscopy, exonuclease digestion, denaturation-renaturation experiments, and restriction enzyme analysis. The SP16 genome is a linear molecule 60.0 +/- 2.0 kilobases in length without cohesive ends. Electron micrographs of denatured and renatured SP16 DNA showed that the DNA is circularly permuted. The genome possesses terminal redundancy, as demonstrated by electron microscopy of exonuclease III-digested DNA.     

Construction and behavior of circularly permuted and telocentric chromosomes in Saccharomyces cerevisiae.

We developed techniques that allow us to construct novel variants of Saccharomyces cerevisiae chromosomes. These modified chromosomes have precisely determined structures. A metacentric derivative of chromosome III which lacks the telomere-associated X and Y' elements, which are found at the telomeres of most yeast chromosomes, behaves normally in both mitosis and meiosis. We made a circularly permuted telocentric version of yeast chromosome III whose closest telomere was 33 kilobases from the centromere. This telocentric chromosome was lost at a frequency of 1.6 X 10(-5) per cell compared with a frequency of 4.0 X 10(-6) for the natural metacentric version of chromosome III. An extremely telocentric chromosome whose closet telomere was only 3.5 kilobases from the centromere was lost at a frequency of 6.0 X 10(-5). The mitotic stability of telocentric chromosomes shows that the very high frequency of nondisjunction observed for short linear artificial chromosomes is not due to inadequate centromere-telomere separation.     

Antigenic variation in visna virus.

Two antigenic variants of visna virus were isolated sequentially from a single sheep inoculated with a plaque-purified strain of virus designated 1514. The genetically stable variants, LV1-1 and LV1-4, are of two classes: LV1-1 is partially neutralized by antibody to the inoculum strain 1514, while LV1-4 is not neutralized by antibody to 1514. The genetic mechanism responsible for generating the antigenic variants was investigated by comparing the chymotryptic and tryptic maps of the envelope glycoprotein gp135 and core polypeptides (p30, p16, p14), and by comparing the pattern of large oligonucleotides produced by digestion of the RNAs by T1 ribonuclease. We show that only the peptide maps of gp135 differ among strains, that the number of peptide fragments altered is small and that gp135 is the polypeptide that elicits neutralizing antibody. The maps of the RNAs are identical. We conclude that mutation in the glycoprotein gene rather than recombination is more probably responsible for antigenic variation, and speculate on the special aspects of visna virus replication relevant to this phenomenon.     

Tomato genome is comprised largely of fast-evolving, low copy-number sequences

Summary Fifty random clones (350–2300 bp), derived from sheared, nuclear DNA, were studied via Southern analysis in order to make deductions about the organization and evolution of the tomato genome. Thirty-four of the clones were mapped genetically and determined to represent points on 11 of the 12 tomato chromosomes. Under moderate stringency conditions (80% homology required) 44% of the clones were classified as single copy. Under higher stringency, the majority of the clones (78%) behaved as single copy. Most of the remaining clones belonged to multicopy families containing 2–20 copies, while a few contained moderately or highly repeated sequences (10% at moderate stringency, 4% at high stringency). Divergence rates of sequences homologous to the 50 random genomic clones were compared with those corresponding to 20 previously described cDNA (coding sequence) clones. Rates were measured by probing each clone (random genomics and cDNAs) onto filters containing DNA from various species from the family Solanaceae (including potato, Datura, petunia and tobacco) as well as one species (watermelon) from another plant family, Cucurbitaceae. Under moderate stringency conditions, the majority of the random clones (single copy and repetitive) failed to detect homologous sequences in the more distantly related species, whereas approximately 90% of the 20 coding sequences analyzed could still be detected in all solanaceous species. The most highly repeated sequences appear to be the fastest evolving and homologous copies could be detected only in species most closely related to tomato. Dispersion of repetitive sequences, as opposed to tandem clustering, appears to be the rule for the tomato genome. None of the repetitive sequences discovered by this random sampling of the genome were tandemly arranged — a finding consistent with the notion that the tomato genome contains only a small fraction of satellite DNA. This study, along with a companion paper (Ganal et al. 1988), provides the first general sketch of the tomato genome at the molecular level and indicates that it is comprised largely of single copy sequences and these sequences, together with repetitive sequences are evolving at a rate faster than the coding portion of the genome. The small genome and paucity of highly repetitive DNA are favourable attributes with respect to the possibilities of conducting chromosome walking experiments in tomato and the fact that coding regions are well conserved among solanaceous species may be useful for distinguishing clones that contain coding regions from those that do not.     

Characterization of visna virus mRNA.

Visna virus is a retrovirus responsible for a classical slow infection of the central nervous system of sheep. In the present work we focused our attention on the viral mRNA's. We found that, during the acute infection in vitro, (i) viral mRNA's amount to only 0.1% of the total cytoplasmic RNA, (ii) 20% of the total cytoplasmic viral RNA is found in polyribosomes, and (iii) three viral mRNA's can be identified by sucrose gradient sedimentation or polyacrylamide gel electrophoresis. Their sedimentation coefficients are 36S, 27S, and 21S.     

Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP

Background  

The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.     

Structure and restriction enzyme maps of the circularly permuted DNA of staphylococcal bacteriophage phi 11.

One restriction enzyme map of Staphylococcus aureus bacteriophage phi 11 DNA was established by reciprocal double digestions with the enzymes EcoRI, HaeII, and KpnI. The sequential order of the EcoRI fragments was thereafter established by a novel approach involving blotting of DNA partially cleaved with EcoRI and the probing the blots with nick-translated terminal fragments. A circular map of the phi 11 DNA was established, and the phage genome was circularly permuted based on the failure to end label mature viral DNA, restriction maps of replicating DNA, and finally, homoduplex analysis in the electron microscope. A restriction enzyme map of the prophage form of phi 11 DNA was obtained by analysis of chromosomal DNA from a lysogenic strain.     

A fully active variant of dihydrofolate reductase with a circularly permuted sequence.

The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.     

Absence from the mouse genome of DNA sequences related to the globin gene family.

Radioactively labelled DNA, complementary to mouse α and β globin messenger RNA, was annealed with unlabelled mouse embryo DNA under conditions of both optimum and lowered stringency. Although an increase in saturation of unlabelled DNA fragments with complementary DNA molecules is produced by lowered stringency, the values obtained are within the range expected for the known globin chains.It is concluded that within the limits of our experimental conditions, there does not exist a large family of DNA sequences related to the globin genes.     

Small differences in electrophoretic mobility among circularly permuted sequence isomers of duck hepatitis B virus linear, single-stranded DNA

Abstract Two linear minus-strand viral DNAs, disparate in size by 10 nucleotides, were isolated from duck hepatitis B virus infected tissues and observed to migrate differently in nondenaturing agarose gels. We examined this phenomenon using both synthetic and cloned viral DNAs and discovered that distinct, circularly permuted, linear isomers of single-stranded DNA could have slightly different electrophoretic mobilities under nondenaturing conditions. This finding reveals a novel feature for consideration in assessing the conformations of native or renatured single-stranded nucleic acids. The study also suggests that the virion-derived minus-strand DNAs of the avihepadnaviridae may necessarily possess a minimal secondary structure.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

Characterization of visna virus envelope neuraminic acid.

Visna virus particles inhibit influenza virus hemagglutination in an assay for neuraminic acid-containing viruses. Pretreatment of visna virus with neuraminidase abolished hemagglutination inhibition activity but did not significantly affect attachment, infectivity, or virus-induced cell fusion in sheep choroid plexus cell monolayers.     

Clustered repeat sequences in the genome of Epstein Barr virus

The genome of Epstein-Barr virus is composed of unique DNA interspersed with repetitive sequences. This organization suggests that Epstein-Barr virus provides a useful model for studying the function(s) of repetitive sequences in eukaryotic chromosomes. The primary structure of two of the repeat sequences, the 3072 bp large internal repeat, or BamHI-W repeat, and a smaller 125 bp, G, C-rich NotI repeat, are presented here. Their structures and possible functions are discussed.     

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.     

Regulation of the visna virus long terminal repeat in macrophages involves cellular factors that bind sequences containing AP-1 sites.

Visna virus gene expression is highly restricted in monocytes but is induced by monocyte-macrophage differentiation in vivo. Deletion and linker-scanning mutants, gel shift assays, and DNase I footprinting were used to identify sequences in the visna virus long terminal repeat involved in the developmental regulation of gene expression in the U937 monocytic cell line. We found that an AP-1 and an AP-4 binding site were critical for basal activity and that the AP-1 site was required for phorbol ester-inducible gene expression. These results suggest that cellular factors that interact with AP-1 sites are involved in the developmental regulation of visna virus gene expression in macrophages.