Attempt to calculate the allele frequency distribution of a TaqI RFLP detected by the highly polymorphic probe YNH24

Summary To improve the analysis of parentage testing with the additional technique of DNA polymorphisms, the usefulness of probe YNH24 was studied. The allele frequency distribution of restriction fragments detected by probe YNH24 on TaqI-digested genomic DNA from 100 unrelated individuals was determined. For this purpose, the size of the fragments was calculated by making use of HindIII-digested lambda DNA as an internal marker and of a digitizing tablet coupled to a computer. The size of the fragments ranged from 2.53 kb to 5.89kb. The mean standard deviation was 0.05kb. The differences between the fragment sizes appeared to be smaller than the standard deviation. For this reason, it was not possible to calculate the allele frequency distribution of this highly polymorphic genetic system.     

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

Summary A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.     

Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus

DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments were fractionated according to size by agarose gel electrophoresis, the fragments transferred to nitrocellulose filters using the technique of Southern, and the filter-bound DNA hybridized with labeled cloned histone genes of the sea urchin, Psammechinus miliaris. Results indicate, first, that sequences homologous to the five individual histone gene probes are present in discrete macronuclear fragments which appear as bands in the gel hybridization assay. Secondly, for each of the five individual histone gene probes the homologous DNA fragments are several in number, ranging in size from 7.6 Kb (Kilo base pairs) to 0.73 Kb. For example, the largest of six detected fragments hybridizing to the H3 gene probe contains approximately 10 times the amount of DNA required to code for a Stylonychia H3 histone. The smallest detected fragment hybridizing to the H3 probe contains enough DNA to code for approximately two copies of the histone. Finally, in general, no two histone gene probes hybridized to the same macronuclear DNA fragment. This result indicates that genes coding for the five histones in Stylonychia are not located together on the same macronuclear DNA fragments and implies that the five functionally related genes would not be transcribed together as a polycistronic unit.     

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca

Summary The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda⁺ isolates) hybridized to PdaT9, while only one Pda^– isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda^– phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6–2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.     

Isolation and some properties of a mammalian ribosomal DNA

The DNA coding for 28 S and 18 S ribosomal RNA, including the spacer regions, has been isolated from calf (Bos taurus) thymus gland. The method used included shearing of the total DNA to a highly homogeneous size population, selective heat denaturation and S 1 nuclease treatment to remove single stranded DNA. Repeated centrifugation on density gradients yields a 140-fold purified rDNA fraction with a GC content of 61.2%. Eco RI nuclease cleaves this DNA into two fragments of 16.4 and 4.9×10⁶ daltons. Hybridization of these fragments with 28 S and 18 S rRNA shows that the 28 S coding sequence is located mostly on the 4.9×10⁶ dalton fragment, while both the 16.4 and 4.9×10⁶ dalton fragments contain the 18 S sequence. The data indicate that the ribosomal RNA gene has a repeat unit of 21.3×10⁶ daltons which includes a nontranscribed spacer of about 12.5×10⁶ daltons.     

Human cellular sequences detectable with adenovirus probes

Previous studies suggesting homology between human cellular DNA and the DNAs from adenovirus types 2 and 5 are extended in the present paper. A clone (ChAd^h), isolated from a human genomic DNA library using an adenovirus probe, hybridized to discrete regions of adenovirus 2 DNA, including part of the transforming genes E1a and E1b, as well as to repeated sequences within human DNA. The E1a and E1b genes both hybridize to the same 300 base pair Sau3AI fragment within ChAd^h although there is no obvious homology between E1a and E1b. The Ad 2 E1a gene was also used as a probe to screen other cellular DNAs to determine whether repeated sequences detectable with Ad 2 DNA probes were conserved over long evolutionary periods. Hybridization was detected to the genomes of man, rat, mouse and fruit fly, but not to those of yeast and bacteria. In addition to a smear hybridization, discrete fragments were detected in both rodent and fruit fly DNAs. The experiments reported suggest the existence of two different types of cellular sequences detected by Ad 2 DNA: (1) repeated sequences conserved in a variety of eukaryote genomes and (2) a possible unique sequence detected with an E1a probe different from that responsible for hybridization to repeated sequences. This unique sequence was detected as an EcoRI fragment in mouse DNA and had a molecular size of about 8.8 kb.     

Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 pat−1

A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 ^pat-1 conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 ^pat-1 locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700–800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster.Abbreviations YAC Yeast artificial chromosome - BCN beet cyst nematode - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism     

Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.     

Determination and in vivo characterization of the basic replicon of natural plasmids of Methylomonas clara

The basic replicon of the endogenous Methylomonas clara plasmid pBE-2 and its derivatives was defined to a region of 2.7 kb by in vivo deletions and conjugative transfer experiments using Escherichia coli-M. clara hybrid plasmids. Origin activity was found to be confined to a maximal length of 1.3 kb. The origin consists of two fragments which can be separated more than 4 kb by the integration of foreign DNA fragments without loss of function. A fragment having a maximum size of 2.1 kb supports in trans replication initiation at the origin. In addition, two incompatibility determinants were revealed, one localized in the origin fragment and the other outside the origin. Incompatibility between two basic replicons of the natural M. clara plasmids can be overcome by the integration of one of them in the compatible IncP plasmid R68-Km^s. No homology was found between the plasmid basic replicon and the chromosomal DNA of M. clara.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> ISP5230

Consensus amino acid sequences of FADH₂-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a β–α–β fold present in FAD-binding sites of certain monooxygenases. When used to probe Southern blots of restriction-enzyme-digested DNA, the [α-³²P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes. Journal of Industrial Microbiology & Biotechnology (2002) 29, 1–5 doi:10.1038/sj.jim.7000263 Received 25 June 2001/ Accepted in revised form 02 April 2002     

Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains showed disappearance of a 70- or 80-kb fragment in their patterns associated with loss of Shiga toxin genes during maintenance or subcultivation. Hybridization experiments with a DNA probe complementary to Shiga toxin sequences revealed that the Shiga toxin genes in the parental strain were located on fragments the same size as the lost fragments from the toxin-negative derivatives. The evidence indicates that PFGE pattern of EHEC O157:H7 may change due to loss of Shiga toxin genes, which is likely to be associated with curing of Shiga toxin gene carrying phages in vitro. Received: 4 May 1998 / Accepted: 19 August 1998     

Plasmid from photosynthetic bacterium Ectothiorhodospira sp. carries a transposable streptomycin resistance gene

Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp. resolved a single extrachromosomal element, plasmid pDG1. Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping. Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3. The resistance to streptomycin and mercury found in Ectothiorhodospira sp. was transferred to E. coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3. The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E. coli HB101, using a kanamycin resistance reporter gene. High stringency molecular hybridization with ³²P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences. In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK 100 used as a target. For this test, we regrouped in the same cells of E. coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tet^r, km^r). We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E. coli DH1. The transfer frequency of streptomycin resistance into pVK100 was 10⁻⁵, compatible with a transposition event. In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

Chloroplast DNA diversity in the genusRubus (Rosaceae) revealed by Southern hybridization

The variability in chloroplast DNA type of 20Rubus genotypes was examined by Southern hybridization. DNA extracted from theRubus accessions was digested with two restriction enzymes (EcoRI and EcoRV) and heterologous chloroplast DNA sequences from barley and pea were used as probes to detectRubus chloroplast DNA sequences on Southern blots ofRubus total DNA. Restriction fragment length polymorphism was detected and a total of 92 restriction fragments were generated by the probe/enzyme combinations examined. Cladistic principles based on the parsimony assumption were used to assemble a phylogenetic tree based on chloroplast restriction fragment length data. The phylogenetic tree grouped the taxonomically defined species and is in general agreement with information based on morphological criteria. However, the Japanese red raspberryR. illecebrosus was shown to have diverged considerably in terms of evolutionary time from other species in subg.Idaeobatus. Furthermore, the molecular approach provides a quantitative estimate of the relationship between species that is difficult to obtain from morphological data. In order to complement the chloroplast DNA information a ribosomal DNA probe was also included in the analysis and provided further information on the phylogenetic relationships withinRubus.     

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×10⁶ to 14×10⁶ dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l0⁶ dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100 kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT¹¹⁰ sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT¹¹⁰ locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT¹¹⁰ and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.