The nucleotide sequence of a geminivirus from Digitaria sanguinalis

The encapsidated single-stranded circular DNA of a geminivirus isolated from Digitaria sanguinalis has been sequenced. The data obtained are consistent with there being one DNA circle of 2701 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV) and wheat dwarf virus showed 64 and 47% DNA homology, respectively. The sequence has four potential coding regions for proteins of greater than 10 kDa, two in the viral (+) sense and two in the complementary (-) sense. Each of these potential coding regions has a highly homologous counterpart among the seven open reading frames previously described for MSV. Virion DNA contained, in addition to the circular single-stranded DNA, a population of small DNA molecules similar to those associated with MSV particles. A comparison with MSV DNA of the region complementary to these small DNA molecules revealed conserved sequences, which may have a role in defining the limits of these primer-like molecules.     

Encapsidation of minute virus of mice DNA: aspects of the translocation mechanism revealed by the structure of partially packaged genomes

Minute virus of mice (MVM) packages a single, negative-sense copy of its linear single-stranded DNA genome, but a chimeric virus, MML, in which >95% MVM sequence was fused to the right-hand terminus of LuIII, packages >40% positive-sense DNA. While encapsidation of both MML strands begins efficiently, genome translocation frequently stalls at specific sites in positive-sense DNA. Internalized sequences, derived from the 3' end of the strand, ranged from 1 to 5 kb in length, with species of around 2 kb predominating. When nuclease activity during isolation was minimized, these truncated species were found to be part of pre-excised 5 kb single-strands. Similarly, some partially encapsidated negative-sense DNAs were observed, forming a continuum of protected 3' sequences between 1 and 3 kb in length, but these were less abundant and more uniformly distributed than their positive-sense counterparts, indicating that the negative strand has evolved for efficient internalization. The paucity of protected DNAs shorter than 1-2 kb suggests that translocation is biphasic, proceeding efficiently through the first (3') third of the genome, but prone to stall thereafter. Sequences with conspicuous secondary structure, including stem-loop and guanidine rich regions, were found to interrupt packaging, especially when positioned near the 5' end of the strand. Since VP2 amino-terminal peptides were exposed at the particle surface in all packaging intermediates, extrusion of this peptide precedes translocation of the full-length strand.     

Mutational analysis of the small intergenic region of maize streak virus.

Maize streak virus (MSV) is a leafhopper-transmitted geminivirus containing one molecule of circular single-stranded DNA of about 2.7 kb. We have tested the infectivity of MSV mutants. A deletion of 29 bases in the small intergenic region (SIR) did not affect the infectivity of MSV. Mutants containing insertions of oligonucleotides of up to 32 bases at the AsnI site in SIR were also infectious. However, the infection efficiency of the insertion mutants decreased as the size of the oligonucleotides increased. This reduced virulence may be due to a decreased efficiency of 3'-end formation of the complementary sense mRNA(s). The stability of MSV mutants in infected maize plants was analyzed by polymerase chain reaction (PCR). Some oligonucleotide-insertion mutants were completely stable while others obtained deletions. Infectivity of the mutants, however, did not require deletion formation. Deletion rearrangement was shown to be dependent on the sequence of the inserted oligonucleotide. The AsnI site is the only site known to permit insertion of nonviral DNA without abolishing MSV infectivity. The system described may allow the use of MSV as a delivery vector to introduce at least small segments of DNA into maize.     

Novel virus-like particles containing circular single-stranded DNAs associated with subterranean clover stunt disease

Novel virus-like particles, 17-19 nm in diameter, have been isolated from subterranean clover and pea plants infected with the pathogen of subterranean clover stunt disease (SCSD). The structure and genetic organization of these particles suggest that the pathogen of SCSD is representative of a new group of plant DNA viruses. SCS virus-like particles (SCSV) are isometric and band as a single component with buoyant densities of 1.24 g/ml in Cs2SO4 and 1.34 g/ml in CsCl. The A260 nm/A280 nm is about 1.35, which is consistent with an estimated nucleic acid content of 17%. Molecular calculations suggest that the particles have a T = 1 capsid structure containing 60 polypeptide subunits each with Mr of 19,000. Nucleic acid analysis including restriction enzyme digestions of double-stranded cDNAs suggests that SCSV have a divided genome composed of multiple species of circular, single-stranded DNA molecules each of approximately 850-880 nucleotides and that each is encapsidated in a separate particle. Linear and aggregated forms of these DNAs are also detected by gel electrophoresis. Evidence suggests that these virus-like particles are the pathogen of SCSD.     

Biology of the temperate Streptococcus thermophilus bacteriophage TP-J34 and physical characterization of the phage genome

The temperate Streptococcus thermophilus bacteriophage TP-J34 was identified in the lysogenic host strain J34. The majority of phage particles produced upon induction was defective and noninfectious, consisting of DNA-filled heads lacking tails. A physical map (45.6 kb) was established. Analysis of minor restriction bands of the DNA isolated from phage particles as well as the analysis of the protein pattern indicated that phage TP-J34 is a pac-type phage. This was confirmed by immunoelectron microscopy using antisera raised against virulent cos- and pac-type S. thermophilus phages. The lysogenic host J34 but not its noninducible derivate J34-12 contained phage DNA in the nonintegrated state and exhibited autolysis at elevated temperatures. Prophage-carrying strains grew homogeneously while 16 of 20 prophage-cured derivatives aggregated and sedimented rapidly. When phage TP-J34 was propagated lytically on a prophage-cured host strain, a 2.7-kb site-specific deletion occurred in the phage genome. This deletion was also identified in the prophage DNAs of relysogenized strains.     

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking thehprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and includinghprt. We used bubble PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream ofhprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream ofhprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay,hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.     

Recombinant DNA related to hepatitis B and human immunodeficiency viruses in mononuclear cells of patients with AIDS

Sixty-eight of 73 patients with human immunodeficiency virus (HIV) infection were positive when tested for the presence of hepatitis B virus (HBV)-related DNA sequences in peripheral blood mononuclear cells (PBMCs) by the dot blot method. Twenty-two of the positive DNAs were examined by Southern hybridization and all exhibited a 3.2 kb extrachromosomal DNA fragment that hybridized to HBV DNA. This DNA was isolated from agarose gels and cloned into the EcoRI site of pBR322 DNA. The cloned DNA (pHBI) hybridized to both HBV DNA and HIV cDNA; HBV DNA did not hybridize to HIV cDNA under the same conditions. The results of restriction enzyme analyses indicated that pHBI contains: 1) a large deletion of HBV sequences spanning the 3' end of the HBV surface antigen gene; 2) a small deletion near the 5' end of the HBV core antigen gene; and 3) a region of homology to a one kb central section of the HIV pol gene. These data suggest that the 3.2 kb DNA found in the PBMCs is a natural recombinant between HBV and HIV DNAs raising the possibility not only that this DNA plays a role in the pathogenesis of AIDS but also that other viral recombinant DNAs may be pathogenic in human disease.     

Hairpin and dimer structures of linear plasmid-like DNAs in mitochondria of Paramecium caudatum

The molecular structure of plasmid-like DNAs (designated type-II) which were isolated from mitochondria in the ciliated protozoan Paramecium caudatum was characterized. These type-II DNAs are always detected as a set of four kinds with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The DNAs of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomer molecules, respectively. Electron microscopic observations indicated configurations of a hairpin structure that had a protruding end of single-stranded DNA in one terminus and a loop in the other terminus. The monomers stick together with base-pairing in opposite directions at the protruding end to form the dimers, suggesting the presence of inverted repeats. These unusual dimers may have a role in replication of the DNAs in which the monomers can serve as a primer for each other.     

Large accumulations of maize streak virus in the filter chamber and midgut cells of the leafhopper vector <Emphasis Type="Italic">Cicadulina mbila</Emphasis>

Maize streak virus (MSV, Mastrevirus, Geminiviridae) is persistently transmitted by Cicadulina mbila, apparently without propagation in its leafhopper vector. MSV was shown earlier by quantitative PCR to accumulate in the alimentary canal of C. mbila. We examined the alimentary canals of C. mbila leafhoppers that acquired MSV from diseased plants for various acquisition access periods (AAP) by immunofluorescence confocal laser scanning microscopy (iCLSM) and by immunogold labelling transmission electron microscopy (iTEM). Following a 7-day AAP and a 7-day inoculation period (IP) on healthy seedlings, MSV was detected by iCLSM mainly in the filter chamber and anterior midgut. Using iTEM, large accumulations of MSV particles, usually enclosed in membranous vesicles, were detected only in cells of the midgut, inside and outside the filter chamber, following 14- or 30-day AAPs, and also following 7-day AAP and 7-day IP on healthy plants. No virus was detected in the control non-vector species C. chinaï. Coated pits or vesicles, typical of clathrin-mediated endocytosis, were not observed. We discuss an alternative endocytosis pathway and suggest that the MSV accumulations are stored in endosomes in the midgut epithelial cells.     

Search for specific DNAs in Creutzfeldt-Jakob infectious brain fractions using "nick translation"

Scrapie agent has been reported to contain an “essential DNA” component in the viroid size range (R. F. Marsh, T. B. Malone, J. S. Semancik, W. D. Lancaster, and R. P. Hanson, Nature (London) 275, 146–147, 1978). The human transmissible encephalopathy, Creutzfeldt-Jakob disease (CJ) was searched for unique CJ DNA components in highly infectious brain fractions containing negligible amounts of nuclear DNA. DNA was quantitatively extracted from these fractions and labeled to high specific activity by “nick translation” with ³²P-nucleotides under conditions where the labeled DNA maintained its original length. Labeled DNAs were analyzed by gel electrophoresis with special emphasis on molecules in the viroid size range. Reconstruction experiments showed that ?0.1 pg DNA could be detected with these procedures. No reproducible CJ specific DNA bands were detectable when compared to normal brain fractions. Restriction enzyme analysis of these DNAs also showed no CJ specific bands integrated into larger molecular weight DNA. Use of Brij 58 to release more infective physical particles into the rough endoplasmic reticulum also did not show CJ specific DNAs. Comparison of the amounts of known infective units by animal assay, assuming a 400-base DNA sequence, indicated that CJ specific DNAs should be detectable in these fractions with the methods used. These results appear to rule out usual forms of double-stranded or partially double-stranded DNAs as components of transmissible encephalopathy agents. A systematic analysis of other specific nucleic acid components of these agents is feasible using the highly sensitive in vitro labeling approach employed here.     

Cloning of a functional human adenine phosphoribosyltransferase (APRT) gene: Identification of a restriction fragment length polymorphism and preliminary analysis of DNAs from APRT-deficient families and cell mutants

A complete human APRT gene has been isolated from a lambda phage genomic library using cloned mouse APRT DNA as a probe. The human gene, contained in a recombinant lambda phage designated Huap15, is functional by virtue of its capacity to transfer human APRT activity to Aprt^– mouse recipient cells after phage-mediated transfection. Digestion of Huap15 DNA with BamH1 generated a 2.2-kb fragment that is the only fragment of eight produced to hybridize with the mouse APRT gene. This 2.2-kb BamH1 fragment is a unique, single copy sequence, and has been used to identify a restriction fragment length polymorphism (RFLP) associated with the APRT locus. Taq1 digestion and Southern blot analysis of DNAs from 49 unrelated individuals produced three different patterns. DNAs of 30 individuals produced a restriction pattern of three labeled fragments about 500 bp, 600 bp, and 2.1 kb in size, which is characteristic for individuals homozygous for the more common allele. Two individuals homozygous for the less frequent allele displayed labeled fragments of 500 bp and 2.7 kb. The remaining 17 DNA samples produced all four labeled bands as expected for heterozygous individuals. The frequency of heterozygotes in the population is about 35%, while the frequency of the less common allele is about 0.21. Restriction enzyme analysis of DNAs from two APRT-deficient brothers and from an unrelated heterozygote revealed no gross deletions or rearrangements, nor the Taq1 polymorphism.     

Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules

Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HM-1:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb +/- 0.5 kb in size and contain structural features predicted by the restriction map of the extrachromosomal ribosomal DNA of E. histolytica.     

Structure,restriction map and infectivity of the genomic and replicative forms ofAaPV DNA

Summary We have characterized the genomic and replicative form (RF) DNA of theAedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6/36 clone ofAedes albopictus cell line [22]. The genome ofAaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated from infected C6/36 cells was established. Among the 23 restriction enzymes tested, 14 cleaved theAaPV RF DNA and 30 restriction sites were mapped and oriented with respect to the viral genomic DNA. Both viral and RF DNAs were found infectious when transfected to virus-free C6/36 cells. The asymmetrical encapsidation of the viral genome is a property common to most vertebrate autonomous parvoviruses but rather unusual among densoviruses. Both by its small size, the asymmetrical mode of encapsidation and the restriction map, theAaPV genome resembles that of theAedes Densonucleosis virus [1].     

Chloroplast DNA evolution among legumes: Loss of a large inverted repeat occurred prior to other sequence rearrangements

Summary We have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions.     

Restriction fragment length polymorphisms of the CYP11B1 gene in the Japanese population

Summary Restriction fragment length polymorphisms (RFLPs) of the CYP11B1 gene were studied in Japanese using cDNA clone P450c11 as a probe. Genomic DNAs from 60 unrelated Japanese individuals were digested with 8 different restriction enzymes and analyzed by Southern blot hybridization. Two RFLPs were detected inMspI digests of the DNA. One(A) was characterized by polymorphic bands at 3.4 and 2.5 kilobasepairs (kb) and the other (B) by polymorphic bands at 1.7 and 1.2 kb. The third RFLP was observed inPvuII-digested samples and was polymorphic at 5.8 and 4.0 kb bands. Two of the three RFLPs found, RFLP (A) and (C), have not been described in the only previous report which was based on Caucasian samples. We also examined the RFLPs of a 3 generation family of 11-hydroxylase deficiency caused by an abnormality of the CYP11B1 gene. All the family members were homozygous in all three RFLPs and was thus not informative.