Forces controlling the rate of DNA ejection from phage lambda

The goal of this work was to investigate how internal and external forces acting on DNA affect the rate of genome ejection from bacteriophage lambda after the ejection is triggered in vitro by a lambda receptor. The rate of ejection was measured with time-resolved static and dynamic light scattering, while varying such parameters as temperature and packaged DNA length, as well as adding DNA-binding proteins to the host solution. We found that temperature has a strong effect on the ejection rate, with an exponential increase of the initial ejection rate as a function of temperature. This can possibly be explained by the temperature-induced conformational changes in the tail pore-forming proteins where the "open" conformation dominates over "closed", at elevated temperatures. The DNA length also had an effect on initial ejection rate, with a nearly linear dependence comparing the three different genomes (37.7, 45.7 and 48.5 kb DNA), with faster ejection rate for longer genomes. Since the initial rate of ejection increases in an almost direct relationship with the length of the genome, the total time needed to eject DNA completely appeared to be nearly constant for all three DNA length phage mutants. The increased initial rate of ejection with increasing DNA length is due to the increased DNA bending and inter-strand repulsion forces for the longer DNA chains. Finally, we also show that addition of non-specific DNA-binding proteins (HU and DNase I) increases the rate of ejection by exerting additional "pulling" forces on the DNA that is being ejected.     

The structure of the variable gene for the kappa chains of antibodies produced by hybridoma PTF-02

The genome of hybridoma PTF-02 has two genes for the kappa chains, and only one of these codes for the synthesis of the antibody light chains. The nucleotide sequences corresponding to the leader peptide and to the variable region of this gene were determined. An amino acid sequence corresponding to exons has been proposed on the basis of the nucleotide sequence. A nucleotide sequence adjacent to the gene at the 5'-end has also been determined, in particular, the precise localization of TATA- and CAT-boxes as well as those of the conservative deca (dc) and pentadeca (pd) sequences. The structure of the regulatory region in the gene is similar to that in the myeloma genomes. However, the 5'-region differs in its nucleotide composition and in the frequency of dc sequences from the DNA sequences adjacent to the 5'-end of eukaryotic genes which do not belong to the immunoglobulin family.     

Analyses of bifidobacterial prophage-like sequences

The genomes of 22 putative prophages (bifidoprophages), previously identified in bifidobacterial genomes, were analyzed to detect the presence and organization of functional modules. Bifidoprophages were shown to display a classical modular genomic organization in which the DNA lysogeny module and the DNA packaging regions are the most highly conserved. Furthermore, single phage gene as well as multiple phage gene-based phylogenetic analyses clearly revealed the chimeric make-up of the genomes of bifidoprophages.     

The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector

We have employed repeat units of herpes simplex virus (HSV) defective genomes to derive a cloning-amplifying vector (amplicon) that can replicate in eucaryotic cells in the presence of standard HSV helper virus. The design of the HSV amplicon system is based on the previous observation that cotransfection of cells with helper virus DNA and seed monomeric repeat units of HSV defective genomes results in the regeneration of concatemeric defective genomes composed of multiple reiterations of the seed repeats. Cotransfection of cells with helper virus DNA and chimeric repeat units containing bacterial plasmid pKC7 DNA resulted in the generation of defective genomes composed of reiterations of the seed HSV-pKC7 repeats. These chimeric defective genomes were packaged into virus particles and could be propagated in virus stocks, with the most enriched passages containing more than 90% chimeric defective genomes. Furthermore, monomeric chimeric repeat units could be transferred back and forth between bacteria and eucaryotic cells. A derivative vector constructed so as to contain several unique restriction enzyme sites could be potentially employed in the introduction of additional viral or eucaryotic DNA sequences into eucaryotic cells.     

On the concept and elucidation of endogenous retroviruses

Endogenous retrovirus (ERV) genomes integrated into the chromosomal DNA of the host were first detected in chickens and mice as Mendelian determinants of Gag and Env proteins and of the release of infectious virus particles. The presence of ERV was confirmed by DNA hybridization. With complete host genomes available for analysis, we can now see the great extent of viral invasion into the genomes of numerous vertebrate species, including humans. ERVs are found at many loci in host DNA and also in the genomes of large DNA viruses, such as herpesviruses and poxviruses. The evolution of xenotropism and cross-species infection is discussed in the light of the dynamic relationship between exogenous and endogenous retroviruses.     

The length of chromatin loops in meiotic prophase I of warm-blooded vertebrates depends on the DNA compositional organization

In meiotic prophase I, chromatin fibrils attached to the lateral elements of the synaptonemal complexes (SC) form loops. SCAR DNA (synaptonemal complex associated regions of DNA) is a family of genomic DNA tightly associated with the SC and located at the chromatin loop basements. Using the hybridization technique, it was demonstrated that localization of SCAR DNA was evolutionarily conserved in the isochore compositional fractions of the three examined genomes of warm-blooded vertebrates—human, chicken, and golden hamster. The introduction of the concept of the comparative loops (CL) of DNA that form of chromatin attach to SC in the isochore compositional fractions provided the calculation of their length. An inverse proportional relationship between the length of CL DNA and the GC level in the isochore compartments of the studied warm-blooded vertebrate genomes was revealed. An exception was the GCpoorest L1 isochore family. For different compositional isochores of the human and chicken genomes, the number of genes in the CL DNA was evaluated. A model of the formation of GC-rich isochores in vertebrate genomes, according to which there was not only an increase in the GC level but also the elimination of functionally insignificant noncoding DNA regions, as well as joining of isochores decreasing in size, was suggested.     

Comparative analysis of the human type 1a and bovine type 1 papillomavirus genomes.

The DNA sequences of the genomes of the bovine type 1 and human type 1a papillomaviruses were compared. The overall organization of both genomes is very similar. Three areas of maximal homology were found in the L1 and E1/E2 genes, and at the beginning of L2. The conservation of homologous amino acid sequences encoded in the open reading frames argues that these segments represent real genes or exons. Within these segments, however, only certain domains of the putative proteins are preferentially conserved. Two polypeptide chains show homologous arrangement of the cysteine residue clusters Cys-X-X-Cys, despite a lack of conservation of the rest of the amino acid sequence. A significant sequence divergence in a region where the three reading frames are open suggests that papillomavirus genomes have evolved not solely by accumulation of point mutations. Conserved sequences were also found in the noncoding region, and their possible involvement in regulation of viral gene expression is discussed.     

Methylation of genomes and genes at the invertebrate-vertebrate boundary.

Patterns of DNA methylation in animal genomes are known to vary from an apparent absence of modified bases, via methylation of a minor fraction of the genome, to genome-wide methylation. Representative genomes from 10 invertebrate phyla comprise predominantly nonmethylated DNA and (usually but not always) a minor fraction of methylated DNA. In contrast, all 27 vertebrate genomes that have been examined display genome-wide methylation. Our studies of chordate genomes suggest that the transition from fractional to global methylation occurred close to the origin of vertebrates, as amphioxus has a typically invertebrate methylation pattern whereas primitive vertebrates (hagfish and lamprey) have patterns that are typical of vertebrates. Surprisingly, methylation of genes preceded this transition, as many invertebrate genes have turned out to be heavily methylated. Methylation does not preferentially affect genes whose expression is highly regulated, as several housekeeping genes are found in the heavily methylated fraction whereas several genes expressed in a tissue-specific manner are in the nonmethylated fraction.     

Development of a cellular assay system to study the genome replication of high- and low-risk mucosal and cutaneous human papillomaviruses

We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3- to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono- and oligomeric HPV genomes in these confluent cell cultures. Amplification occurred as a result of the initiation of semiconservative two-dimensional replication from one active origin in the HPV oligomer. Our data suggest that the system described here might be a valuable, cost-effective, and efficient tool for use in HPV DNA replication studies, as well as for the design of cell-based assays to identify potential inhibitors of all stages of HPV genome replication.     

A test of Chargaff's second rule

In 1968, Chargaff and his colleagues discovered a rule in Bacillus subtilis: in single stranded DNA, A=T and C=G. This rule has since been confirmed many times in other bacterial and eukaryotic genomes. To the best of our knowledge, this rule has not been tested before in either single stranded DNA or RNA genomes. Over 3400 genomic sequences were examined here and included for the first time both double and single stranded DNA and RNA genomes. We found that: (1) with the exception of the organellar DNA, this parity rule holds for all types of double stranded DNA genomes and (2) that this rule fails to hold for other types of genomes. The parity rule appears to be a selective force on genome evolution and codon use.     

2 types of molecular structure (composition) of a genome in one species of transposable bacteriophages of Pseudomonas aeruginosa

Comparison of heteroduplexes (HD) between DNAs of different transposable phages of Pseudomonas aeruginosa belonging to two previously described subgroups (D3112 and B3) revealed two types of structure (composition) of the bacteriophages, designated "type A" and "type B". The properties of genome structure of type A (phages of D3112 subgroup) are as follows: high level of conservation (up to 70% of genomes of different phages are represented as blocks of homologous DNA sequences); substitutions in genomes revealed as nonhomology regions in HD are, as a rule, small and located in certain sites; the distribution of the nonhomologous regions in HD of these phages is highly reproducible in independent experiments. Bacteriophages of subgroup B3 have genomes of type B: only a small part (approx. 30%) of genomes retain homology general for all of the phages; the nonhomologous regions are distributed in a large number of sites in HD; the sizes of nonhomologous regions are substantially larger than for the phages of subgroup D3112; distribution of the regions in HD is highly variable, which is characteristic of DNAs with partial homology. There is no difference between genomes of types A and B in G + C content (approx. 61-63%). Viable recombinants can be formed in crosses between phages of different genome types not only in regions with earlier revealed large DNA/DNA homology (right ends of genomes), but also in central portions of the genomes. Nevertheless, functional incompatibility of some regions of phage genomes of types A and B was demonstrated.     

Arrangement of nucleotide sequences in adeno-associated virus DNA

There are two types of adeno-associated virus virions which contain complementary single-stranded DNA genomes of about 1.4 × 10⁶ daltons. The purified complementary single polynucleotide chains anneal to form duplex linear monomers, circular monomers, and linear dimers, in addition to other less well-defined structures, as identified by sedimentation and electron microscopy. All duplex species are formed by linear single polynucleotide chains of unit length, thus duplex circles and linear dimers are assumed to be held together by relatively short overlapping hydrogen-bonded regions. The initial linear monomers present after annealing the complementary single strands do not form duplex circles or oligomers when re-exposed to annealing conditions, but DNA which sediments as linear monomers after heating linear dimers at a temperature from 7 to 25 deg. C below the T_m of duplex AAV² DNA does re-form oligomers and circles when exposed to annealing conditions. Denaturation and reannealing of any duplex species leads to the formation of all forms, indicating that the over-all single strand composition of all species is equivalent. Disruption of duplex circles by limited exonucleolytic digestion using either 3′ or 5′ exonucleases leads to the conclusion that the overlap region may have either 3′ or 5′ termini and that the overlap region represents less than 6% of the length of the genome. Exonucleolytic digestion of linear monomers to the extent of 50% leaves polynucleotide chains which cannot reanneal after denaturation, thus AAV DNA is not randomly circularly permuted. Duplex linear monomers which do not form circles when exposed to annealing conditions do form duplex circles after 1% exonuclease III digestion. A model consistent with these data is one in which the linear single polynucleotide chains present in AAV virions consist of two or more permutations. All of these chains contain terminal repetitions and their start points occur within a limited region, representing < 6% of the length of the genome. According to this interpretation AAV DNA would exist within the virion as a linear single polynucleotide chain or is cleaved at a few specific sites during extraction.     

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Abstract Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis , and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.     

Genetic Relationship and Genomic in situ Hybridization Analysis of the Three Genomes in Triticum aestivum

Common wheat ( Triticum　aestivum L.) is an allohexaploid, consisting of three different genomes (Au, B and D ) which are genetically closely related. Genomic DNA of the three possible genome donors, T. urartu Thum., Aegilops speltoides Tausch and Ae. tauschii Coss.,were employed as probes to hybridize with the diploid genomic DNA digested by Eco RⅠand Hin dⅢ respectively. Both the hybridization strength and band patterns among the genomes would be good indicators of genome relationships. Combining distr ibution data of some repetitive DNA sequences cloned from T. urartu in the three genomes, the authors draw a conclusion that Au and D are more closely related to each other than either one to the B genome. Genomic in situ hybridization (GISH) of T. aestivum cv. Chinese Spring with genomic DNA probes of the three diploid progenitors respectively indicated that the three genomes could be discriminated clearly via GISH. The signals on the chromosomes of Au and D genomes were even. However, when Ae. speltoides DNA was used as probe, there were very strong cross hybridization and the signals condensed on some areas of the metaphasic chromosomes. In the interphase nucleus, the chromatin of B genome dispersed on the same region and the signals on the homologous chromosomes distributed symmetrically. Rich repetitive DNA sequences in B genome, especially the tandem repetitives, perhaps take an important role for the formation of the special hybridization pattern. The main difference between B and the other two genomes probably is in the repetitive DNA sequences.     

CC1, a novel crenarchaeal DNA binding protein

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.     

A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes

Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.     

What tangled web: barriers to rampant horizontal gene transfer

Dawkins in his The Selfish Gene(1) quite aptly applies the term "selfish" to parasitic repetitive DNA sequences endemic to eukaryotic genomes, especially vertebrates. Doolittle and Sapienza(2) as well as Orgel and Crick(3) enlivened this notion of selfish DNA with the identification of such repetitive sequences as remnants of mobile elements such as transposons. In addition, Orgel and Crick(3) associated parasitic DNA with a potential to outgrow their host genomes by propagating both vertically via conventional genome replication as well as infectiously by horizontal gene transfer (HGT) to other genomes. Still later, Doolittle(4) speculated that unchecked HGT between unrelated genomes so complicates phylogeny that the conventional representation of a tree of life would have to be replaced by a thicket or a web of life.(4) In contrast, considerable data now show that reconstructions based on whole genome sequences are consistent with the conventional "tree of life".(5-10) Here, we identify natural barriers that protect modern genome populations from the inroads of rampant HGT.     

Parental genomes are separated throughout the cell cycle in a plant hybrid

The positions of the genomes originating from each parent were analysed in root-tip nuclei of the mature, sexual F₁ hybrid plant Hordeum vulgare (barley) x Secale africanum (a wild rye). The two genomes of the hybrid were identified in both spread and sectioned material by non-radioactive DNA:DNA in situ hybridization using labelled total genomic DNA from one parent as a proble and unlabelled total genomic DNA from the other parent to block non-specific hybridization. Complete three-dimensional reconstructions of sets of labelled sections enabled detailed analysis of the position of the genomes at interphase. The parental genomes lay in various non-intermixed configurations, including lateral and concentric arrangements. On spread preparations, the two parental genomes were found to be spatially separated throughout the cell cycle; the genome originating from H. vulgare tended to be located more centrally than that from S. africanum. This results show that the nucleus is spatially organized above the level of the DNA and chromosome at the genome level.by M.F. Trendelenburg     

Generation and analysis of defective genomes of Autographa californica nuclear polyhedrosis virus.

We have generated defective genomes of Autographa californica nuclear polyhedrosis virus (AcNPV) by serial, undiluted passage in IPLB-SF-21 cell culture in an attempt to identify potential cis-acting sequences important for AcNPV DNA replication. Viral DNA isolated from some of the 81 serial passages was analyzed by pulsed-field gel electrophoresis, restriction endonuclease analysis, and Southern blot hybridization. AcNPV-defective genomes appeared to be generated through a series of successively smaller and transiently stable intermediates. Although the defective genomes at passages later than passage 65 (P65) were somewhat heterogeneous in size, those of the majority of the population had a mean size estimated to be 50 kb, or 40% of that of standard virus. Defective genomic DNA at P81 hybridized strongly only to a 2.8-kb region mapping within 85.0 to 87.2 map units of AcNPV DNA (most of HindIII-K and a small part of HindIII-B), suggesting that the majority of P81-defective genomes were missing most of the 128-kb wild-type DNA sequence, except for this small 2.8-kb fragment. Furthermore, our results indicated that the defective genomes of P81 were composed largely of reiterations of this sequence. We suggest that the 2.8-kb DNA segment retained by the defective AcNPV genomes of P81 contains an important cis-acting element(s) sufficient for viral DNA replication in AcNPV-infected cells.