Human ornithine decarboxylase sequences map to chromosome regions 2pter----p23 and 7cen----qter but are not coamplified with the NMYC oncogene

Using a mouse cDNA probe for ornithine decarboxylase (ODC), we have identified and isolated an ODC cDNA clone from a lambda gt11 recombinant library prepared from human liver cell mRNA. The 2.0-kb insert of this clone hybridizes with several mouse genomic ODC DNA restriction fragments under conditions of low stringency, but reacts with only few human DNA fragments and a polyA+ RNA species of 2.2 kb under both nonstringent and stringent hybridization conditions. This suggests that, unlike the mouse genome, there are only few ODC genes in the human genome. The human ODC DNA fragments segregate with chromosome regions 2pter----p23 and 7cen----qter in mouse X human somatic cell hybrid clones containing normal, translocated, and deleted human chromosomes. Sequences of the short arm of chromosome 2 containing the NMYC oncogene at 2p23----p24 are often involved in DNA amplification in neuroblastomas and small-cell lung cancers. However, in at least three cases--one neuroblastoma cell line, one neuroblastoma tumor, and one lung carcinoma--the ODC sequences are not coamplified with the NMYC oncogene.     

The structure of M6, a recombinant plasmid containing Dictyostelium DNA homologous to actin messenger RNA

The recombinant plasmid M6 contains a DNA sequence from the cellular slime mold Dictyostelium discoideum which hybridizes to actin messenger RNA. The plasmid contains 6 kilobase pairs (kb) of Dictyostelium DNA inserted into a pMB9 vector. Ten cleavage sites for four different restriction enzymes have been mapped. Other work has shown that a central restriction fragment, 1.7 kb in length, contains sequences repeated about fifteen times in the genome, and that this fragment hybridizes to actin mRNA. Heteroduplexes between M6 and pDd actin 2, a chromosomal plasmid which contains two copies of the actin repeated sequence, were used to define the position of this repeat in M6. Two plasmids with inserts of cDNA made from actin mRNA were heteroduplexed to M6 to define the position and orientation of the message complementary region. This orientation was confirmed by inserting the fragment into phage λ and determining which of the separated λ strands was complementary to actin mRNA. An electron microscope technique has been developed for identifying poly(dA) sequences by hybridizing to them dBrU polymers attached to suitable markers. The mapping of the (dA) tracts that occur in the Dictyostelium insert of M6 is described here. The positions of the A:T tracts do not correlate in any simple way with the position of the actin gene sequence.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Viscosinamide-producing Pseudomonas fluorescens DR54 exerts a biocontrol effect on Pythium ultimum in sugar beet rhizosphere

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.     

DNA fingerprinting of the intestinal parasite Giardia duodenalis with the M13 phage genome

A DNA fingerprint procedure has been established for the intestinal parasite, Giardia duodenalis. This permits the identification of individual strains from both human and animal sources. Analysis of strain movement, resurgence and variation is now possible. The fingerprint probe is based on the tandem repetitive sequence found in bacteriophage M13 which hybridizes to a set of hypervariable polymorphic minisatellite sequences found in higher eukaryotes. This probe recognizes a weakly homologous set of hypervariable regions in the Giardia genome to provide a DNA fingerprint comparable to those seen in higher eukaryotes.     

稻瘟菌突变体T-DNA插入位点的精细定位和插入模式的研究

随机挑取已构建的37个稻瘟菌T-DNA突变株,利用TAIL-PCR技术扩增出T-DNA插入位点的侧翼序列,测序并进行比对分析。结果显示:成功获得扩增产物并测序的序列共有39条,T-DNA边界序列为稻瘟菌序列的有19条,其余20条为载体主干序列。在这有效扩增为稻瘟菌序列的19条中,有10条是T-DNA右侧翼序列与稻瘟菌序列,9条为左侧翼序列加稻瘟菌序列。分析T-DNA剪切位点,10条右侧翼序列中有9条的剪切位点相同,这与农杆菌介导T-DNA转化植物一样。而左边界的剪切位点就没有这种规律性。研究也精细确定了17个不同突变株的T-DNA插入位置,为后续的基因功能研究奠定基础。     

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.     

Two regions of M13 phage genome hybridizing with human DNA are similar to several keratin genes.

DNA from two regions of the phage M13 genome hybridizes with DNA restriction fragments from genomes of various species including man [15, 20]. As the pattern of hybridization is individual-specific, this phage M13 probe can be used for DNA fingerprinting. We demonstrate here that the regions of many keratin genes coding for glycine-rich parts of C and N end domains are very similar to the phage M13 probe, and this similarity may be responsible for hybridization.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Physicochemical properties of temperate phage DNA and its possible effect on the variability of Mycobacterium lacticolum

The temperate phage 104 S was isolated from the S variant of Mycobacterium lacticolum, strain 104, and some of its characteristics were studied. The content of GC pairs in the phage DNA was 77 mole% as was calculated from the melting profile or 65 mole% as was calculated from the value of buoyant density in CsCl. The DNA was shown to be composed of 18,000 nucleotide pairs. DNA restriction fragments of M. lacticolum R, S and M variants were subjected for the first time to molecular hybridization with [32P]DNA of the temperate phage. The genome of the three M. lacticolum variants and the genome of a non-dissociating S variant clone were shown to contain sequences homologous to the DNA sequence of phage 104 S. Differences are found among the variants in the hybridizing DNA fragments. These data indicate that the phage DNA may actively be involved in the variability of the culture. Its participation can be realized by the different mode of prophage incorporation into the genome of the variants.     

The nucleotide sequence of repetitive monkey DNA found in defective simian virus 40.

DNA fragments containing monkey DNA sequences have been isolated from defective SV40 genomes that carry host sequences in place of portions of the SV40 genome. The fragments were isolated by restriction endonuclease cleavage and contain segments homologous to sequences in both the highly repetitive and unique (or less repetitive) classes of monkey DNA. The complete nucleotide sequence of one such fragment [151 base pairs (bp)] predominantly homologous to the highly reiterated class of monkey DNA was determined using both RNA and DNA sequencing methods. The nucleotide sequence of this homogeneous DNA segment does not contain discernible multiple internal repeating units but only a few short oligonucleotide repeats. The reiteration frequency of the sequence in the monkey genome is >10⁶. Digestion of total monkey DNA (from uninfected cells) with endonuclease R Hind III produces relatively large amounts of discrete DNA fragments that contain extensive regions homologous to the fragment isolated from the defective SV40 DNA.A second fragment, also containing monkey sequences, was isolated from the same defective substituted SV40 genome. The nucleotide sequence of the 33 bp of this second fragment that are contiguous to the 151 bp fragment has also been determined.The sequences in both fragments are also present in other, independently derived, defective substituted SV40 genomes.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

Conserved sequences flank variable tandem repeats in two S-antigen genes of Plasmodium falciparum

We describe the isolation of two chromosomal DNA fragments from Plasmodium falciparum. These fragments encode the antigenically distinct S antigens of two different P. falciparum isolates, namely FC27 from Papua New Guinea and NF7 from Ghana. The complete nucleotide sequences of both fragments are presented. The fragments are homologous over most of their lengths, including the entire regions flanking the protein coding sequences. Whereas the N- and C-terminal portions of sequences encoding the S antigens are homologous, major portions of the coding sequences are not. The nonhomologous regions are comprised of tandemly repeated sequences, of 33 bp in FC27 and predominantly of 24 bp in NF7. The 33 bp tandem repeats encoded by the FC27 S-antigen gene could not be detected in the NF7 genome. Conversely, the 24 bp tandem repeats encoded by the NF7 S-antigen gene could not be detected in the FC27 genome. The pattern of sequence variation within the repeats of both genes suggests a mechanism for the generation of S-antigen diversity.     

Interspersion of different repeated sequences in the wheat genome revealed by interspecies DNA/DNA hybridisation.

The repeated sequences in oats DNA have been used to study chromosomal repeated sequence organisation in wheat. Approximately 75% of the wheat genome consists of repeated sequences but only approximately 20% will form heteroduplexes with repeated sequences from oats DNA at 60 degrees C in 0.18 M Na+. The proportion of wheat DNA that forms heteroduplexes with oats DNA is shown to be independent of the wheat DNA fragment length. However, the proportion of wheat DNA that is retained with the heteroduplexes when fractionated on hydroxyapatite is very dependent upon the wheat fragment length up to 3500 nucleotides. This is because more non-renatured wheat DNA is attached to the heteroduplexes with longer fragments. The results indicate that the repeated sequences in the wheat genome homologous to repeated sequences in oats are not clustered in the chromosomes but distributed amongst other repeated and possible non-repeated sequences.