G4 DNA replication: I. Origin of synthesis of the viral and complementary DNA strands

The break in the complementary DNA strand of early G4 replicative form II DNA (RFII) and in the viral DNA strand of late RFII DNA was located using two single cleavage restriction enzymes (EcoRI and PstI) and by limited nick translation of the break using DNA polymerase I and ³²P-labelled deoxyribonucleotides followed by digestion with the restriction enzymes HaeIII and HindII. The break in the complementary DNA strand was unique and in HaeIII Z5 close to the EcoRI cleavage site whereas the break in the viral DNA strand was on the other side of the molecule in HaeIII Z2 approxiately 50% away from the EcoRI cleavage site. Distribution of a short ³H pulse in early G4 replicating intermediates that were synthesising both DNA strands at the same time showed that synthesis of the strands started on opposite sides of the molecule and proceeded in opposite convergent directions, suggesting that initiation of synthesis of the two strands was independent and not unified in a single growing fork.     

Fine structure of polyoma virus DNA.

A fine structure map of polyoma DNA has been made based on cleavage with a number of restriction endonucleases (including HaeII and III, BamI, HindII and III, BumI, HpaII, and in part, HphI) and depurination of wild-type DNA, the eight HpaII restriction fragments and some HaeIII fragments. This analysis has made possible some correlation with simian virus 40 DNA, and has facilitated detailed examination of various polyoma strains and variants. Sequences from the region of the origin of DNA replication have been examined.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

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.     

Molecular and physical organization of highly repetitive,undermethylated DNA from Pennisetum glaucum

A HaeIIl monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (Msp/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5′-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.     

Genomic Relatedness within Five Common Finnish Campylobacter jejuni Pulsed-Field Gel Electrophoresis Genotypes Studied by Amplified Fragment Length Polymorphism Analysis, Ribotyping, and Serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.     

Different regions of a complex statellite DNA vary in size and sequence of the repeating unit.

The 1.688 g/cm³ satellite DNA of Drosophila melanogaster is composed primarily of 359 base-pair units repeated in tandem. Most of these units contain a single cleavage site for both HaeIII and HinfI restriction endonucleases; however, some units lack one or both sites. Previously we had shown that the distribution of HaeIII and HinfI endonuclease sites varies widely between different regions of 1.688 g/cm³ satellite DNA; for example, some regions contain HaeIII sites in every unit and other regions (>10,000 base-pairs) contain no HaeIII sites (Carlson &; Brutlag, 1977). We have now cloned molecules of 1.688 g/cm³ satellite DNA which lack HaeIII sites and have shown that the absence of sites is caused by sequence variation rather than base modification. This result indicates that regions of 1.688 g/cm³ satellite DNA with different distributions of restriction sites differ in the sequence of their repeating units. We also show that a large fraction of the satellite DNA which is not cleaved by HaeIII endonuclease still contains HinfI endonuclease sites (and AluI sites) spaced about 359 base-pairs apart. However, one cloned segment lacking HaeIII sites was found to contain 33 tandem copies of a novel 254 base-pair unit. Sequence analysis showed that this 254 base-pair unit is homologous to the 359 repeat except for a 98 base-pair deletion. These data suggest that both units have evolved from a common ancestor and that each has subsequently become amplified into separate tandem arrays.     

Location of the origin-terminus of the viral strand of the duplex replicative form of bacteriophage G4 DNA

One of the products of bacteriophage G4 DNA replication late in the infectious process is an open-circular, duplex replicative form DNA, RFII. These molecules contain a single discontinuity located at a specific site in the viral strand. Limited enzymatic repair of such RFII molecules with ³²P-labeled deoxyribonucleoside triphosphates specifically labels restriction fragments HpaII A, HaeIII Z₂, Hind(II and III) A and Hind(II and III) D₂ and places the 3′OH terminus of the viral strand at a point approximately half-way round the genome from the single EcoRI site.These results taken together with the in vitro localization of the origin of the complementary strand at a point close to the EcoRI site (Zechel et al., 1975) suggest that G4 replicates by a mechanism involving distinct and widely separated origins of the individual strands (e.g., a displacement-loop mechanism).     

Different Strategies for Molecular Differentiation of Mycobacterium bovis Strains Isolated in Sardinia, Italy

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)₅ oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed.     

DNA bending induced by DNA (cytosine-5) methyltransferases

DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay. The following bend angles were obtained: M.BspRI (GG^m5CC), 46–50°; M.HaeIII (GG^m5CC), 40–43°; M.SinI (GGW^m5CC), 34–37°; M.Sau96I (GGN^m5CC), 52–57°; M.HpaII (C^m5CGG), 30°; and M.HhaI (G^m5CGC), 13°. M.HaeIII was also tested with fragments carrying a methylated binding site, and it was found to induce a 32° bend. A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HaeIII and M.BspRI bend DNA toward the minor groove. The DNA curvature induced by M.HaeIII contrasts with the lack of DNA bend observed for a covalent M.HaeIII–DNA complex in an earlier X-ray study. Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3′ to the target cytosine tend to induce greater bends than enzymes with guanine in this position. We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.     

Restriction site variation,length polymorphism and changes in gene order in the mitochondrial DNA of the yeast Kluyveromyces lactics

The purpose of this work was to compare mitochondrial DNA restriction endonuclease patterns in strains of the yeast Kluyveromyces lactis, from different sources, to see how conserved is the organization of this organellar genome. The mitochondrial DNA of five independently-isolated strains and one of unknown origin were compared. Strains NRRL Y-1205, NRRL Y-8279 and NRRL Y-1140 gave identical patterns. Strain NRRL Y-1564 showed an insertion, with respect to the other three, of approximately 1250 bp. Strain W600B had also an insertion with extra restriction sites for EcoRI, HpaI, HaeIII, HincII and XbaI. On the other hand, strain Y-123 showed a restriction pattern quite different from the others.Sequences putatively encoding apocytochrome b, ATPase subunit 9 and ribosomal RNA large subunit, were localized on the physical maps of three strains. Results demonstrated that the order of these three genes shows a common feature in strains W600B and WM37 (auxotroph of Y-1140) but a different distribution in WM27 (auxotroph derived from Y-123). All these facts explain the extensive intraspecific polymorphism observed in the mtDNA of this yeast.     

The mitochondrial genome of wild-type yeast cells. VII. Recombination in crosses.

Two Saccharomyces cerevisiae wild-type strains were crossed, and 26 diploid clones were obtained from (1) mass mating; (2) individual buds in zygote lineages; (3) individual zygotes. The mitochondrial DNAs from these diploids were investigated in their recombination and segregation by analyzing their restriction fragment patterns.Recombinant mitochondrial genomes were present in 75% of the diploid clones. Such recombinant genomes had unit sizes different from, yet within ± 5% of, the parental ones and showed EcoRI and HindII + III fragment patterns of parental types, two strong indications that both the gene complement and the gene order were very largely preserved in the progeny.Fragment patterns produced by HpaII and HaeIII were characterized by (1) fragments originating from the DNAs of both parents; and (2) new fragments, namely fragments absent in either parent. The new fragments appear to arise from unequal crossing-over events occurring in the spacers of allelic parental genetic units and usually have preferential localizations in the genome.These results provide the first evidence for physical recombinations of mitochondrial DNA in crosses of wild-type yeast cells, indicate that recombination is very frequent in crosses, and shed some light on mitochondrial segregation. They also have interesting implications for recombination phenomena in interspersed systems of unique and repetitive nucleotide sequences.     

Unusual behaviour of SPO1 DNA with respect to restriction and modification enzymes recognizing the sequence 5''-G-G-C-C

Summary SPO1 DNA contains only 5 cleavage sites for restriction enzymes which recognize and cleave the sequence 5-G-G-C-C (HaeIII or BsuR). Fragments of SPO1 DNA cloned in E. coli to substitute 5-hydroxymethyluracil (HMU) by thymine (T) remain resistant to HaeIII indicating that this unexpectedly small number of cleavages by HaeIII is not correlated with the presence of HMU in the normal phage DNA. It was previously shown that SPO1 is neither subject to B. subtilis R restriction (Trautner et al., 1974) nor modification in vivo (Günthert et al., 1975). We now show that SPO1 DNA can however be restricted and modified in vitro.     

PCR-RFLP analysis of fliC,fimH and 16S rRNA genes in Salmonella Typhimurium isolates of varied origin

Restricted fragment length polymorphism (RFLP) was used in analyses on the typing and heterogeneity, typeability and polymorphism of the 16S rRNA, fliC and fimH genes in Salmonella Typhimurium isolates of varied origin. The digestion of PCR products with restriction enzymes EcoRV, ClaI, HaeIII and ScaI (fliC genes), HincII, ClaI, EcoRV and MluI (fimH genes) and EcoRI, SmaI and HaeIII (16S rRNA genes) generated two to four bands of ranging in size from 100 to 1,104 bp. Of all the restriction profiles obtained, only the ClaI profile for fimH could be used to classify Salmonella Typhimurium isolates into different groups. According to this profile, pattern A with uncut fimH was observed in eight isolates (36.36 %) and pattern B with 755- and 253-bp bands was observed in 14 isolates (63.63 %). No pattern was allotted for a special region or source. These results demonstrate that PCR-RFLP based on these genes showed good typeability but low discriminatory power. Moreover, the highly conserved nature of fliC, fimH and 16S rRNA illustrated in our study suggests the importance of these genes as immunization and diagnostic factors in Salmonella Typhimurium. Simultaneously, our results also illustrate the potential of ClaI-based fimH analysis as a marker for the sub-serotype level differentiation of Salmonella Typhimurium isolates.     

Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tc^r) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tc^r gene also plays a role in regulating plasmid copy number.     

Molecular and physical organization of highly repetitive, undermethylated DNA from Pennisetum glaucum

A HaeIIl monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (Msp/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.     

Analysis of novel alleles of brother of tout‐velu,the drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovo^D dominant female‐sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini‐white (w ^{+ mW.hs}), or rosy (ry⁺) and neomycin (neo^R) transgenes are in common use. Parallel ovo^D lines were developed using w ^{+ mW.hs} FRT insertions on the X and chromosomes 2R and 3L, as well as ry⁺, neo^R FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry⁺, neo^R FRT background, are not amenable to germline clonal analysis without labor‐intensive recombination onto chromosome arms containing a w ^{+ mW.hs} FRT. Here we report the creation of a new ovo^D line for the ry⁺, neo^R FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal‐effect phenotypes of novel brother of tout‐velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.     

Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster

The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and ry² mutants. In vitro complementation experiments demonstrated the presence of the mal⁺ complementation factor in the oocyte, suggesting an explanation for the mal maternal effect. The ry⁺ complementation factor synthesized from paternal template was detected at gastrulation. This is the earliest detection of a paternal enzyme during nonmammalian embryonic development.     

Identification of Actinomyces,Propionibacteria, Lactobacilli and Bifidobacteria by Amplified 16S rDNA Restriction Analysis

Amplified 16S rDNA restriction analysis (ARDRA) with Hae III and Hpa II was applied to 37 reference strains, 179 human clinical and four veterinary isolates of Propionibacterium, Lactobacillus andBifidobacterium and some other anaerobic, non-sporing, Gram-positive bacilli. Results were compared with those obtained by ARDRA for reference strains (26) and clinical isolates (469) of Actinomyces spp. Reference strains were clearly differentiated to species level. Clinical isolates of Propionibacterium and Lactobacillus were identified with confidence to species level. Bifidobacterium spp. were identified in ARDRA with confidence to genus, but anomalies in species level identification of some reference strains and clinical isolates may reflect unreliable identification in conventional tests. Isolates of Arcanobacterium spp., Actinobaculum schaalii, Eggerthella lenta, some Eubacterium spp., Gardnerella vaginalis, Mobiluncus spp., Atopobium vaginae, Abiotrophia defectiva, Streptococcus mutans, Streptococcus intermedius and Clostridium sp. were clearly differentiated in ARDRA. ARDRA is a simple, rapid, and highly discriminatory method for identification of anaerobic, non-sporing, Gram-positive bacilli.