Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

Replication of bacteriophage M13: XIII. Structure and replication of cloned M13 miniphage

Restriction analysis of the duplex replicative forms of four cloned M13 miniphage indicates that all species examined contain a single copy of the intergenic space between genes II and IV plus one or more copies of a portion of the genome extending from within gene IV to a site in the HaeIII G fragment within the intergenic space. Both the viral and the complementary strand origins of replication have been localized previously within the 160 base-pair HaeIII G fragment. Since reiteration of a portion of the HaeIII G fragment could possibly lead to phages having multiple copies of the origin of replication, we have determined the location of the viral strand origin-terminus in M13 miniphage by mapping the position of the discontinuity(ies) in mini-RFII³ molecules isolated during asymmetric viral strand synthesis. Limited repair of late life-cycle mini-RFII molecules with DNA polymerase I in the presence of labeled deoxynucleoside triphosphates followed by restriction analysis demonstrates that the discontinuity in the RFII is contained at a unique site within the single HaeIII G fragment. The absence of a discontinuity in the reiterated DNA sequence containing only a portion of the HaeIII G fragment indicates that the reiterations of the origin region do not include the entire sequence specifying the viral strand origin-terminus.     

Replication origin of bacteriophage f1: Two signals required for its function

Plus strand synthesis in bacteriophage f1 initiates in a region of dyad symmetry at a specific site (plus origin) recognized and nicked by the viral gene II protein. In this paper we describe several small deletions on the 5′ side of the f1 plus origin which disrupt the region of dyad symmetry and extend up to only four nucleotides from the gene II protein nicking site. These deletions do not impair the ability of gene II protein in vitro to nick this site. However, they do inhibit in vivo plus strand synthesis. We conclude that the nucleotide sequence at the f1 plus origin contains at least two specific signals that are required for efficient plus strand synthesis.     

Analysis of the recombination event generating a vesicular stomatitis virus deletion defective interfering particle.

cDNA clones of different portions of the L cistron and 5'-terminal region of the vesicular stomatitis virus genome have been prepared and used to identify the exact site of the deletion in the defective interfering particle, DI-LT. The deletion extends from nucleotide 251 from the beginning of the L gene to a position 342 nucleotides from the end of the genome. The nucleotide sequences flanking the deletion site, as well as those at the ends of the deleted segment, did not contain any obvious vesicular stomatitis virus initiation or termination signals as had been found near the recombination sites in other defective interfering particle RNAs. The results best fit a model for the origin of this type of defective interfering particle in which the polymerase interrupts its synthesis and moves with its nascent daughter strand to a new position on the template and resumes synthesis there, further extending the nascent strand. Neither the interruption nor the resumption of synthesis appears to be in response to the template nucleotide sequence. The sequences of two partial L cistron clones also reveal open reading frames that code for amino acid sequences likely to be the amino and carboxy termini of the L protein.     

Cloning of a functional replication origin of phage G4 into the genome of phage M13.

A chimeric single-stranded DNA phage, M13Gori1, has been formed as a result of the in vitro insertion of a 2216 base-pair HaeII fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dnaG-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13Gori1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13Gori1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13Gori1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dnaG-primed G4 origin. The M13Gori1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.     

Duplications between direct repeats stabilized by DNA secondary structure occur preferentially in the leading strand during DNA replication

To ascertain a leading or lagging strand preference for duplication mutations, several short DNA sequences, i.e. mutation inserts, were designed that should demonstrate an asymmetric propensity for duplication mutations in the two complementary DNA strands during replication. The design of the mutation insert involved a 7-bp quasi inverted repeat that forms a remarkably stable hairpin in one DNA strand, but not the other. The inverted repeat is asymmetrically placed between flanking direct repeats. This sequence was cloned into a modified chloramphenicol acetyltransferase (CAT) gene containing a −1 frameshift mutation. Duplication of the mutation insert restores the reading frame of the CAT gene resulting in a chloramphenicol resistant phenotype. The mutation insert showed greater than a 200-fold preference for duplication mutations during leading strand, compared with lagging strand, replication. This result suggests that misalignment stabilized by DNA secondary structure, leading to duplication between direct repeats, occurred preferentially during leading strand synthesis.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Replication of a plasmid containing two origins of bacteriophage f1

A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.     

A role for single-strand breaks in bacteriophage phi-X174 genetic recombination

Formation of genetic recombinants in bacteriophage φX174 is stimulated up to 50-fold in host cells carrying the recA⁺ allele by subjecting the virus particles to ultraviolet irradiation before infection, or by starving the host cell for thymine during infection; in recA host strains no such increases are observed.φX174 replicative form DNA molecules formed in vivo from ultraviolet-irradiated bacteriophage consist of an intact, circular full-length viral (+) strand and a partially complete complementary (?) strand extending from the point of origin of complementary strand DNA synthesis to an ultraviolet lesion. φX174 replicative form DNA molecules formed in thymine-deficient host strains during thymine starvation have nearly complete circular viral (+) and complementary (?) strands, which contain random single-strand nicks or gaps.Correlation of these structures with the observed increases in recombination suggests that single-strand “breaks” are aggressive intermediate structures in the formation of φX174 genetic recombinants mediated by the host recA⁺ gene product.     

Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

Segregation of Replicative DNA Polymerases during S Phase: DNA POLYMERASE ?, BUT NOT DNA POLYMERASES α/δ, ARE ASSOCIATED WITH LAMINS THROUGHOUT S PHASE IN HUMAN CELLS*

DNA polymerases (Pol) α, δ, and ϵ replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ϵ being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G₁/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ϵ were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G₁/S arrest and early in S phase, Pol α, δ, and ϵ were associated with the same nucleoprotein complexes, whereas in late S phase Pol ϵ and Pol α/δ were largely associated with distinct complexes. At G₁/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ϵ, not Pol α/δ, remained associated with lamins. Consistently, Pol ϵ, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ϵ and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ϵ, to post-replicative processes such as translesion synthesis or post-replicative repair.     

A minor pathway leading to plaque-forming particles in bacteriophage lambda. Studies on the function of gene D

Lysates of bacteriophage λ, mutant in the head gene D, contain a minor amount of defective particles which can be isolated and complemented to infective particles by adding purified gene D product. The defective particles contain DNA with a specific infectivity in the helper assay of about 10% of phage DNA. This DNA is firmly held in the capsid and a tail is attached. Although the particles adsorb to sensitive bacteria, the DNA is not injected. The complemented, infectious particles differ from normal phage by having a lower density. After growing in a permissive host, phage particles of normal density are produced. The implications of the ability of gene D protein to bind to otherwise complete particles as a last step are discussed.     

The single calmodulin gene of the cephalochordate Branchiostoma

The cDNA and gene for calmodulin (CaM) from the cephalochordate Branchiostoma were isolated and characterized. The nucleotide sequence of the Branchiostoma CaM cDNA is about 80% identical to the CaM of Drosophila and Aplysia. However, all nucleotide substitutions are silent, therefore the amino acid sequences of all these CaMs are identical. Branchiostoma and Aplysia CaM genes have the same exon/intron organization. PCR, Northern and genomic Southern analyses showed that Branchiostoma CaM is encoded by a single copy gene, while fish are known to have at least four CaM genes. These results fit the hypothesis that major gene duplication events occurred close to the origin of vertebrates, i.e., after the divergence of the cephalochordate lineage.     

Comparative analysis of the constituents of two cultivars of Dioscorea dumetorum (Kunth) Pax. and their molecular barcoding

The edible and wild cultivars of Dioscorea dumetorum (Kunth) Pax. (Family: Dioscoreaceae) are commonly used as close substitutes in herbal markets due to their edibility and medicinal importance. Comparative study of the cultivars was done using chemical analysis of their methanol tuber extracts and by utilizing the DNA barcode regions, ribulose-1, 5-bisphosphate carboxylase subunits (rbcL) and the non-coding intergenic spacer gene (trnH-psbA) for nucleotide sequences comparison of cultivars. Of the 54 compounds identified from the methanol extract of the edible and wild cultivars using gas chromatography-mass spectrometry (GC-MS), 12 are similar, while 42 are remarkably different. In particular n-hexadecanoic (palmitic) acid had a higher percentage area in the edible cultivar (31.16%) compared to the wild cultivar (0.26%). The cultivars were successfully amplified using the universal primers gene rbcL and trnH-psbA. Their nucleotide sequences showed a slight variation when aligned with CodonCode Aligner V.9.0.1. They were identified by the Basic Local Alignment Search Tool (BLAST) through comparison with Genbank data, which shows close similarities with submitted data. The use of molecular barcoding in identifying the cultivars of Dioscorea dumetorum as well as the chemical composition of their tubers may form an important marker in the compilation of future herbal pharmacopoeia for its proper identification.     

Evolutionary divergence of ribosomal internal transcribed spacer 2 in lizards

Internal transcribed spacers 1 and 2 (ITS1 and ITS2) are known to play an important role in rRNA maturation, yet the mechanism of their action is still not completely understood. Comparison of the ITS1 and ITS2 nucleotide sequences for various organisms reveals conserved regions, which are potentially involved in rRNA biogenesis, and yields new information about the evolutionary divergence of the corresponding region of the genome. The rDNA fragments containing ITS2 were amplified, cloned, and sequenced for three lizard species: Darevskia armeniaca, Lacerta strigata (Lacertidae), and Agama caucasia (Agamidae). The lizard ITS2 sequences were compared with their counterparts from other organisms and proved to contain not only universally conserved elements characteristic of the consensus secondary structure of vertebrate ITS2, but also lizard-specific regions. Comparison of the ITS2 size and the distribution of homologous regions for the two lizard families made it possible to assume that evolution of the modern species involved duplication of ITS2 in the genome of their common ancestor.     

Polyoma virus defective DNAs. II. Physical map of a molecule with rearranged and reiterated sequences (D74).

The structure of the polyoma virus defective species D74 (74% the size of full-length polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24, 24 and 27% in size. The two 24% segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units.Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units are linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin &; Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.     

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso''s in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.