Partial Denaturation Mapping Research Articles

Characterization of Vibrio cholerae O1 ElTor typing phage S5

S5 (ATCC No. 51352-B2), a Vibrio cholerae O1 ElTor typing phage was characterized. The growth characteristics and inactivation kinetics (thermal, UV and pH) of this lytic phage were investigated. Phage morphology was examined by electron microscopy and was classified as belonging to the family Podoviridae. The S5 phage genome is shown to be a linear double-stranded 39-kb-long DNA as determined by electron microscopy and restriction digestion. Partial denaturation maps were constructed and were used to show that the DNA is non-permuted and terminally redundant. The replication origin of this T7-like phage was visualized by electron microscopy. The polarity of packaging of S5 DNA in the phage head was determined. SDS-PAGE of phage S5 shows two major structural polypeptides of 50 and 42 kDa. A 3D structure of the phage head was reconstructed at a resolution of 37 A using Cryo-EM and a single-particle reconstruction technique.

Physicochemical characterization of vibriophage N5

Phage N5 is one of the phages of Vibrio cholerae serovar O1 biotype El Tor (Ghosh, A. N., Ansari, M. Q., and Dutta, G. C. Isolation and morphological characterization of El Tor cholera phages. J. Gen. Virol. 70: 2241–2243, 1989). In the present communication the growth curve, molecular weight and confirmation of the genome, partial denaturation map and restriction endonuclease digestion pattern have been determined. Partial denaturation map indicates that the genome has non-permuted / invariant sequence. Presence of cohesive ends has also been documented.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis.

PSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3'-protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules. Fifty-seven open reading frames were identified on the PSA genome, which are apparently organized into three major transcriptional units, in a life cycle-specific order. Functional assignments could be made to 33 gene products, including structural proteins, lysis components, DNA packaging proteins, lysogeny control functions and replication proteins. Bioinformatics demonstrated relatedness of PSA to phages infecting lactic acid bacteria and other low G + C Gram-positives, but revealed only few similarities to Listeria phage A118. Virion proteins were analysed by amino acid sequencing and mass spectrometry, which enabled identification of major capsid and tail proteins, a tape measure and a putative portal. These analyses also revealed an unusual form of translational frameshifting, which occurs during decoding of the mRNAs specifying the two major structural proteins. Frameshifting yields different length forms of Cps (gp5) and Tsh (gp10), featuring identical N-termini but different C-termini. Matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) of tryptic peptide fragments was used to identify the modified C-termini of the longer protein species, by demonstration of specific sequences resulting from + 1 programmed translational frameshifting. A slippery sequence with overlapping proline codons near the 3' ends of both genes apparently redirects the ribosomes and initiates the recoding event. Two different cis-acting factors, a shifty stop and a pseudoknot, presumably stimulate frameshifting efficiency. PSA represents the first case of + 1 frameshifting among dsDNA phages, and appears to be the first example of a virus utilizing a 3' pseudoknot to stimulate such an event.

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution.

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross-linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy-two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle-specific manner into at least three major transcriptional units. N-terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control-associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site-specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

Detection of Partial Denaturation in AT-Rich DNA Fragments by Ion-Pair Reversed-Phase Chromatography

Temperature-dependent denaturation of DNA restriction fragments from the pBR322 plasmid ranging in length from 46 to 910 base pairs was detected by ion-pair reversed-phase high-performance liquid chromatography using columns packed with alkylated nonporous poly(styrene/divinylbenzene) particles. The presence of acetonitrile in the mobile phase was found to decrease the melting temperatures of DNA fragments by 1.5-2 °C/% of acetonitrile in the eluent. Small fragments (<120 bp) were completely denatured between 53.6 and 63.5 °C, depending on their total GC content. Whereas retention times of completely helical DNA fragments increased gradually with increasing temperature, partial denaturation of larger DNA fragments (>150 bp) was found to reduce retention at temperatures above 53.6 °C. Therefore, micropreparative fractionation and rechromatography, together with DNA restriction analysis, were applied to identify the correct elution order of completely helical and partially denatured fragments. Inspection of the DNA sequences of partially denatured fragments revealed domains with repeating AT base pairs. Positions of partial denaturation within the pBR322 plasmid detected by chromatographic analysis were in good agreement with partial denaturation maps obtained by electron microscopy desrcibed in the literature.

Vibriophage N4 DNA is nonpermuted and terminally redundant

Phage N4 is one of the five newly isolated phages used in a new phage typing scheme for Vibrio cholerae O1 biovar EITor strains and belongs to the Podoviridae family. Electron microscopic studies showed that the phage N4 has a DNA of 40.4 ± 0.1 kb. A partial denaturation map of the N4 DNA has been constructed. It has been shown with the help of this partial denaturation map that phage N4 genome is nonpermuted. Circularization of the phage genome on treatment with exonuclease III, followed by incubation in 50% formamide, indicates a terminal redundancy.Key words: vibriophage N4, DNA, Vibrio cholerae, denaturation mapping.

Vibriophage D10 contains non-permuted DNA with cohesive ends

Phage D10, a Vibrio cholerae O-1 El Tor group X phage, is one of the five newly isolated phages used in the phage typing scheme developed for V. cholerae O-1 biotype El Tor and belongs to the Myoviridae family. From electron microscopic studies it is shown that phage D10 has a DNA genome of 32 +/- 0.2 kb. This is the first report where it has been shown by the construction of a partial denaturation map that this vibriophage genome is nonpermuted and has cohesive ends. The location of the ends of the DNA in the phage head has also been inferred.

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867–876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be Θ-shaped structures in which the φX174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933–1937].

Two deletions in the Epstein-Barr virus genome of the Burkitt lymphoma nonproducer line Raji

The Epstein-Barr virus genome carried in the Burkitt lymphoma nonproducer cell line Raji was characterized by partial denaturation mapping and by hybridization of cloned viral fragments to filters containing separated Raji DNA fragments. Partial denaturation mapping revealed that the EBV DNA population of Raji cells is homogenous and that two deletions are observed in distant parts of the genome compared to linear DNA isolated from virus particles of different strains. These deletions were characterized by blot analysis. One deletion of 3.15 kb lies within HindIII-E; the second is 2.4 kb and is located close to the right terminus of linear viral DNA. The two deletions were observed in several cell lines derived from the Raji line. These deletions might contribute to the inability of Raji cells to produce EBV either spontaneously or upon induction.

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S-16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3'-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.

Structure of partially denatured Escherichia coli 23 S ribosomal RNA determined by electron microscopy

The secondary structure of 23 S ribosomal RNA was analyzed by electron microscopy after partial denaturation. A reproducible pattern of loops was seen when molecules were spread for electron microscopy in 50% formamide solutions containing various concentrations of Mg2+ and Na+. Some loops were stabilized more than others by Na+ or by Mg2+; but in general, small amounts of Mg2+ (0.5 to 1.0 mM) markedly stabilized all the major loops, as did much greater amounts of Na+ (100 mM). However, at all levels of Mg2+ examined, increasing levels of Na+ destabilized loop structures. These data are consistent with the known salt dependence of double-stranded DNA and transfer RNA structure. The four most frequently observed loops correspond, within the limits of measurement error, to the major loops in the secondary structure models of Noller et al. (1981) and Glotz et al. (1981). These four loops are, in length and position of their midpoints along the 23 S rRNA molecule: 490 +/- 50 at 250 +/- 40; 350 +/- 50 at 1860 +/- 80; 400 +/- 70 at 2330 +/- 150; and 570 +/- 100 at 2350 +/- 100. Three of the four have base-paired stems with delta G0 values among the lowest of all the loops in the two indirect models. At least two are also among the most stable loops found in computer searches of the 23 S rRNA sequence for dyad symmetry. These results demonstrate that partial denaturation mapping can both identify prominent features of secondary structure in rRNA and estimate their relative stability.

Bacteriophage P2 DNA Replication: Characterization of the requirement of the gene B protein in vivo

Replicative intermediates isolated from Escherichia coli cells infected with P2 gene B mutants were circular DNA molecules with single-stranded DNA tails, as opposed to the double-stranded DNA tails of wild-type replicative intermediates. The results show that the mutant replicative intermediates arose from aberrant DNA replication, aberrant due to a lack of lagging strand DNA synthesis, but with normal leading strand synthesis, so that only one circular duplex daughter DNA molecule was made from each duplex parent molecule. The single-stranded tails were shown to correspond to the nicked (and therefore displaced) parental DNA "l" strands. By partial denaturation mapping, the ends of the single-stranded tails tended to map close to the replication origin, but not all at a unique position, probably due to partial degradation or breakage in vivo, or during cell lysis or DNA isolation. By hybridization to separated strands of P2 DNA on nitrocellulose filters, DNA synthesis was shown to be asymmetric, and consistent with more leading strand than lagging strand synthesis having occurred. We concluded that the gene B protein is required for lagging strand DNA synthesis, but not for initiation, elongation or termination of the leading strand.

A chloroplast DNA of Euglena gracilis with five complete rRNA operons and two extra 16S rRNA genes

DNA was isolated from chloroplasts of Euglena gracilis var. bacillaris (ATCC No. 10616). The structure of the rDNA was studied using partial denaturation mapping and heteroduplex analysis. Seven GC-rich stretches representing the rDNA were apparent in the partial denaturation pattern. To analyse the structure of the rDNA in detail, heteroduplexes with the E. coli rrnD operon cloned in the plasmid pBK8 were prepared. Five complete rRNA operons were found. In addition one extra 16S rRNA gene was located between the second and third complete operons and another extra 16S rRNA gene was upstream of all five operons. Each one was associated with a small inverted repeat structure.

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.

Analysis of the Spodoptera frugiperda Nuclear Polyhedrosis Virus Genome by Restriction Endonucleases and Electron Microscopy

Restriction endonuclease analysis was used to differentiate between four strains of Spodoptera frugiperda nuclear polyhedrosis virus from different geographical areas. In addition, partial denaturation was performed, and a partial denaturation map was constructed for the Ohio strain of this virus.

The Drosophila X virus contains a 1-μ M double-stranded RNA circularized by a 67-kd terminal protein: High-resolution denaturation mapping of its genome

The effect of urea on Drosophila X virus (DXV) is presented and analyzed by electron microscopy. At 4 M urea a RNA-protein complex is liberated consisting of one segment of double-stranded (ds) RNA which is maintained in a circular form inside the virion by a protein of 67 kd. The RNA-protein complex was purified by chromatography and had a tendency to form very spectacular flower-like structures. By crosslinking the RNA inside the virus with psoralen and uv radiation it is shown that the RNA was double-stranded in situ. A new method for partial denaturation mapping of double-stranded nucleic acids by electron microscopy is described and was applied to DXV RNA. Under these conditions it is shown that native and denatured regions as small as 30 RNA base pairs (bp) can be visualized. In the case of DXV RNA it was observed that the RNA possessed at one extremity a GC-rich region of 30 by followed by an AU-rich region of 160 bp. These molecules were observed in a conventional transmission electron microscope and a scanning transmission electron microscope under dark-field illumination. Absolute electron microscope length measurements and molecular weight determinations by gel migration revealed that the DXV RNA is one segment of ds-RNA of 0.97 μm in the electron microscopic conditions used in this work, giving a molecular weight of 2.2 × 10 6 d which corresponds to 3170 RNA bp. An average melting temperature of 83.3° was obtained in 0.1 SSC. Viral protein analysis was performed on the virus and on the purified protein-RNA complex. The virus is made of six major proteins and two minor proteins which were revealed after in vitro iodination.

Proteus mirabilis phage 5006M: Restriction maps of genome in relation to headful packaging

The genome of Proteus mirabilis phage 5006M was fragmented by eleven restriction enzymes: AvaI, AvaII, BamHI, BglI, BglII, BstEII, EcoRI, HindIII, SstII, XbaI, and XhoI. Variable yields obtained for fragments in restriction patterns were interpreted in terms of headful packaging of the genome. Restriction maps constructed for these enzymes were related to a partial denaturation map of the genome. The location of the pac site derived from partial denaturation experiments differs by 5% of a genome length from that obtained from restriction data but the same direction of packaging was observed.

Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.

Physical evidence for early transcription in intracellular bacteriophage P2 DNA

Escherichia coli were infected with bacteriophage P2, and the phage DNA replication was blocked by a mutation in the phage A gene. The resulting intracellular DNA molecules contained displacement loops, which were shown to contain RNA. The positions of these R loops were mapped by electron microscope partial denaturation mapping, and 90% were found to be located in the same region on the DNA, spanning less than 15% of the P2 chromosome. Also, most of the R loops have a common left end located at 77 ± 1.4% on the P2 DNA map, which corresponds well with the position of the promoter for the P2 early genes as determined by the position of several P2 virulent deletion mutations. We conclude that the observed R loops on intracellular P2 DNA represent transcription from the promoter for the early operon in P2.

Structure of frog virus 3 genome: size and arrangement of nucleotide sequences as determined by electron microscopy

The structural features of frog virus 3 (FV 3) DNA have been analyzed by electron microscopy. The FV 3 genome is a linear duplex molecule of 52.00 ± 5.60 μm in length which corresponds to 98.28 ± 10.58 X 106 daltons. To compare the structure of the genomes of FV 3, vaccinia virus, and herpes simplex virus, the three viral DNAs were denatured, quickly renatured, and examined in the electron microscope. The FV 3 DNA consisted of linear single strands while the vaccinia virus DNA contained reannealed duplexes and the herpesvirus DNA contained single-stranded circles with bushes or molecules in which two single-stranded circles were joined by a duplex region. These data suggest that the FV 3 genome contains neither crosslinked terminii nor inverted repeats. To construct a partial denaturation map of the FV 3 genome, the DNA was denatured with 85% formamide and examined in the electron microscope. The partially denatured molecules failed to match by their denatured regions when lined up end to end, suggesting that the molecules were dissimilar with respect to the arrangement of nucleotide sequences. However, a distinct denaturation map was obtained when the molecules were matched by their denatured regions rather than by the physical ends. The map revealed (1) a continuous stretch of about 40 μm (or 75.60 × 10 6 daltons) of uniquely ordered sequences common to all molecules, and (2) a distribution of the ends within 20 to 28% of the map length. These observations suggest that FV 3 DNA molecules contain various circular permutations of a common overall nucleotide sequence and that the extent of circular permutation is restricted as in bacteriophage P22 ( B. K. Tye, J. A. Huberman, and D. Botstein, 1974, J. Mot Biol. 85, 501–532).