Highly repetitive component α and related alphoid DNAs in man and monkeys

The genomes of Old-World, New-World, and prosimian primates contain members of a large class of highly repetitive DNAs that are related to one another and to component DNA of the African green monkey by their sequence homologies and restriction site periodicities. The members, of this class of highly repetitive DNAs are termed the alphoid DNAs, after the prototypical member, component of the African green monkey which was the first such DNA to be identified (Maio, 1971) and sequenced (Rosenberg et al., 1978). The alphoid DNAs appear to be uniquely primate sequences. — From the restriction enzyme cleavage patterns and Southern blot hybridizations under different stringency conditions, the alphoid DNAs comprise multiple sequence families exhibiting varying degrees of homology to component DNA. They also share common elements in their restriction site periodicities (172 · n base-pairs), in the long-range organization of their repeating units, and in their banding behavior in CsCl and Cs₂SO₄ buoyant density gradients, in which they band within the bulk DNA as cryptic repetitive components. — In the three species from the Family Cercopithecidae examined, the alphoid DNAs represent the most abundant, tandemly repetitive sequence components, comprising about 24% of the African green monkey genome and 8 to 10% of the Rhesus monkey and baboon genomes. In restriction digests, the bulk of the alphoid DNAs among the Cercopithecidae appeared quantitatively reduced to a simple series of arithmetic segments based on a 172 base-pair (bp) repeat. In contrast with these simple restriction patterns, complex patterns were observed when human alphoid DNAs were cleaved with restriction enzymes. Detailed analysis revealed that the human genome contains multiple alphoid sequence families which differ from one another both in their repeat sequence organization and in their degree of homology to the African green monkey component DNA. — The finding of alphoid sequences in other Old-World primate families, in a New-World monkey, and in a prosimian primate attests to the antiquity of these sequences in primate evolution and to the sequence conservatism of a large class of mammalian highly repetitive DNA. In addition, the relative conservatism exhibited by these sequences may distinguish the alphoid DNAs from more recently evolved highly repetitive components and satellite DNAs which have a more restricted taxonomical distribution.     

DNA-Mediated Prophage Induction in Bacillus subtilis Lysogenic for φ105c4

Prophage was induced when strains of Bacillus subtilis 168 lysogenic for 105c4 were grown to competence and exposed to specific bacterial DNAs. The time course of phage production was similar to that observed for mitomycin C induction of wild-type prophage. Induction was directly dependent upon DNA concentration up to levels which were saturating for the transformation of bacterial auxotrophic markers. The extent of induction varied with the source of DNA. The burst of phage induced by DNA isolated from a W23 strain of B. subtilis was fivefold less than that induced by DNA from B. subtilis 168 strains, while B. licheniformis DNA was completely inactive. This order of inducing activity was correlated with the ability of the respective DNAs to transform auxotrophic markers carried by one of the 105c4 lysogens. Differences in inducing activity also were observed for different forms of 105 DNA. The DNAs isolated from 105 phage particles and 105c4 lysogens were inactive, whereas DNA from cells lysogenized by wild-type 105 induced a burst of phage. When tested for transforming activity, however, both 105c4 and 105 lysogen DNAs were equally effective. An induction mechanism which involves recombination at the prophage insertion site is proposed to explain these differences.     

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

Summary All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and 105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo-or endonuclease activity. Only Mg²⁺ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5×10⁷) have an average molecular weight of about 3×10⁵.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships among isolates of oral haemophili as determined by DNA-DNA hybridization

In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G+C) content of DNAs from strains of Haemophilus segnis and Haemophilus para-influenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G+C content of 39.0 to 42.9% and were 49–92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70–81% homologous with the ATCC 9796 DNA probe and had a G+C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G+C content of 42.4%, and was 65–78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.     

Homology is not required for recombination mediated by DNA gyrase of Escherichia coli

Summary We have previously shown that DNA gyrase of Escherichia coli can promote recombination between heterologous DNAs in a cell-free system (Ikeda et al. 1982). In the present paper, we have studied the nucleotide sequences of several recombination junctions of -pBR322 recombinants and found that there were not more than three-basepair homologies between the parental DNAs in six combinations of the and pBR322 recombination sites. Based on this and previous results, we concluded that homology was not required for the DNA gyrase-mediated recombination. Furthermore, the structures of the recombinant DNAs we have analyzed suggest the occurrence of multiple crossovers in our in vitro system.     

The Genome of the Lepidopteran Mamestra brassicae has a Vertebrate-like Content of Methyl-cytosine

Mamestra brassicae genomic DNAs, isolated from larvae and adult tissues and from in vitro cultured CRL-8003 cells, were enzymatically hydrolysed to nucleosides that were separated by HPLC. HPLC analysis showed that 5mC content in cabbage moth larvae, adults and cultured cells was 8.9±0.5, 9.3%±0.2 and 10.2%±0.4 respectively. Cabbage moth 5mC content results the highest reported till now in insects and it is similar to the typical vertebrate one. Analysis of MspI and HpaII restriction pattern on M. brassicae DNA showed that a portion of its genome was methylated at CpG sites. Moreover, the absence of small digestion products after MspI digestion suggested that CpG are not clustered in the cabbage moth genome. Finally, methylation of repeated DNAs has been studied. Comparison of the restriction pattern of MspI and HpaII after hybridisation with the hobo, mariner, 28S and 5S rDNA probes did not evidence any difference indicating the absence of CpG methylation in all the studied repeated DNAs.     

Development of a sequence-tagged site for the centromere of chromosome 10: its use in cytogenetic and physical mapping

We sequenced the alphoid centromere probe p10RP8 (D10Z1), aligned it to three published consensus sequences, and developed a sequence-tagged site (STS), sJRH-2, based upon oligonucleotide primers having two 3 mismatches with these consensus sequences. Polymerase chain reaction (PCR) amplification using genomic DNA from a somatic cell hybrid panel representing all human chromosomes demonstrated amplification from only those cell lines containing chromosome 10. Fluorescence in situ hybridization of the amplified product demonstrated intense and specific hybridization of the PCR product to 10p11.1-q11.1. A human genomic yeast artificial chromosome (YAC) library was screened using the sJRH-2 PCR assay, and five clones were identified. Sequence analysis of one chimeric clone (consisting of DNA segments derived from chromosomes 5p and 10cen) confirmed specificity of the STS for the centromere of chromosome 10. sJRH-2 provides a convenient cytogenetic marker for chromosome 10, which will also be useful for physical mapping of the pericentromeric region of chromosome 10, a region that harbors the gene(s) for three forms of multiple endocrine neoplasia (types 2A, 2B, and familial medullary thyroid carcinoma). The GenBank accession number for the p10RP8 sequence is X63622.     

Complex and simple sequences in human repeated DNAs

Highly repeated human DNA sequences were isolated by isopycnic centrifugation, or were eluted from gels after restriction enzyme cleavage. High molecular weight DNA peaks separable from the bulk of the DNA in a variety of gradients were shown to consist of very simple sequences characteristic of simple satellite DNAs; DNA fingerprint studies indicated each of these peaks could consist of tandem repeats of a specific oligonucleotide sequence as low as 10 base pairs (bp) long. All the gradient peaks could be assigned to one of two sequence groups and several different buoyant density peaks revealed the same sequence. — Restriction fragment multimers did not share common sequences with the satellite DNAs as judged by hybridization data. They could not be separated by isopycnic centrifugation. Furthermore these highly repeated DNAs were more complex in sequence and more variable than the satellites. Even the smallest (50 bp) fragments by depurination and other direct sequencing methods were shown to be more complex than the high molecular weight satellite peaks. — The idea that subsets of repeated DNAs may be defined by sequence complexity, possibly with discrete or separable functions, is proposed.     

A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.     

Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Clostridium propionicum strain 19acry3 and further characteristics of the species

Two mixed cultures able to ferment acrylate to equimolar acetate and propionate were enriched from anaerobic sediments. From one of these mixed cultures a pure culture of a Gram-positive, obligately anaerobic bacterium was isolated. This strain, designated 19acry3 (= DSM 6251) was identified as belonging to the species Clostridium propionicum. Only a narrow range of organic compounds supported growth, including acrylate and lactate. Acrylate and lactate were fermented to acetate and propionate in a 1:2 molar ratio. When co-cultured with the non-acrylate-fermenting Campylobacter sp. strain 19gly1 (DSM 6222), the fermentation balance shifted to almost equimolar acetate and propionate. Strain 19acry3 was compared with Clostridium propionicum type strain X2 (DSM 1682). The two strains displayed similar phenotypic properties. The mol% G+C of DNA isolated from both strains was 36–37 (by thermal denaturation). Both strains displayed a characteristic fluorescence when observed by fluorescence microscopy. Cell-free extracts of both strains were examined by spectrophotofluorimetry. In both strains, two excitation peaks were observed at 378 and 470 nm. Excitation at either of these wavelengths resulted in an emission maximum at 511 nm.     

Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2→q26.3

Summary Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2 q26.3.     

Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria ofS. cerevisiae

A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria ofS. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 35 exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.Abbreviations BCIP 5 bromo 4 chloro 3 indolyl phosphate - mtDNA mitochondrial DNA - NBT Nitroblue tetrazolium - PBS phosphate buffered saline     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

The Use of Competitive PCR Mimic to Evaluate a Limulus Lambda Phage Genomic DNA Library

1. A phage genomic DNA library for Limulus (L.) polyphemus brain was constructed using the GEM-12 vector and the host strain KW251.2. The primary library contained approximately 1.275 × 10⁶ independent clones, increasing upon amplfication to 6.66 × 10⁹ pfu/ml in a total volume of 58 ml.3. A total of 28 clones was randomly chosen for a determination of the average size of inserts in the library. All clones contained inserts and the average size was 14.9 kb, ranging from 11.7 to 28.0 kb. The library provides a 10-fold equivalent of the L. polyphemus genome.4. A new approach for evaluating a genomic DNA library was developed, in which competitive PCR MIMIC was employed to determine the target gene copy number in both constructed library and brain genomic DNA. The putative protein kinase C (PKC) was selected as the target gene because its partial sequence of cDNA was recently cloned from L. polyphemus brain in our laboratory (Cao et al., 1998). A 419-bp fragment of nonhomologous sequence derived from putative PKC and a 306-bp fragment from plasmid pUC 18 were generated for use as target and competitor in PCR MIMIC, respectively.5. Within the genomic library DNA, a 0.8 value was obtained for the copy number of the putative PKC gene that was detected in 0.1 amol of one equivalent L. polyphemus genome in terms of the average recombinant molecular weight. In the genomic DNA, a single copy of putative PKC was found in 0.1 amol of one coverage for the L. polyphemus genome. Thus, it was implied that nearly 80% genetic resource was incorporated into the library. This percentage was termed the incorporation rate.6. Based on these findings, we suggest that the incorporation rate is an essential factor for evaluating genomic libraries, particularly, when using partial digestion with restriction enzymes for library construction.     

Restriction cleavage maps of the DNAs of Streptococcus pneumoniae bacteriophages containing protein covalently bound to their 5'' ends

Summary Several pneumococcal bacteriophages showing a morphology similar to that previously described for Cp-1 (Ronda et al. 1981) have been isolated and purified from throat samples taken from healthy children. Three of these phages (Cp-5, Cp-7 and Cp-9) have been studied in detail and compared to Cp-1. The four phages differed in several respects, e.g. size, structural polypeptides, restriction enzyme cleavage patterns, etc. The DNA of Cp-5, Cp-7 and Cp-9 showed protease-sensitive transfecting activity. This, together with the results obtained by electrophoretic analyses as well as by isotopic labelling of these DNAs with [-³²P] ATP and polynucleotide kinase indicated that all these new phages have a protein covalently linked to the 5 ends of their DNAs as in the case of Cp-1 (García et al. 1983). Restriction enzyme cleavage maps of Cp-1, Cp-5, Cp-7 and Cp-9 have been constructed.