Genomic representation of the Hind II 1.9 kb repeated DNA.

The genomic representation and organization of sequences homologous to a cloned Hind III 1.9 kb repeated DNA fragment were studied. Approximately 80% of homologous repeated DNA was contained in a genomic Hind III cleavage band of 1.9 kb. Double digestion studies indicated that the genomic family, in the majority, followed the arrangement of the sequenced clone, with minor restriction cleavage variations compatible with a few base changes. Common restriction sites external to the 1.9 kb sequence were mapped, and hybridization of segments of the cloned sequence indicated the 1.9 kb DNA was itself not tandemly repeated. Kpn I bands which were homologous to the sequence contained specific regions of the repeat, and the molecular weight of these larger fragments could be simply explained. Mapping of common external restriction sites indicated that in some but not all cases the repeat could be organized in larger defined blocks of greater than or equal to 5.5 kb. In some instances, flanking regions adjacent to the repeat may contain common DNA elements such as other repeated DNA sequences, or possibly rearranged segments of the 1.9 kb sequence. It is suggested that although the 1.9 kb sequence is not strictly contiguous, at least some of these repeated sequences in the human genome are arranged in clustered or intercalary arrays. A region of the 1.9 kb sequence hybridized to a mouse repeated DNA, indicating homology beyond the primates.     

Identification and isolation of transcribed human X chromosome DNA sequences

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.     

Repetitive sequences and their organization on genomic clones of Zea mays

Fourteen recombinant clones from Zea mays were studied with regard to their composition of unique and repetitive sequences. Southern hybridization experiments were used to classify restriction fragments of the clones into a unique, middle or highly repetitive class of reiteration frequency. All three classes were often found on the same genomic clone. Crosshybridization studies between clones showed that a given repeat might be present on several clones, and thus four families of highly repetitive elements were established. Heteroduplex analysis was used to show the arrangement and size of repeats common between several clones. A short interspersion pattern of unique, middle and highly repetitive DNA was found. The dispersed repetitive elements were 300-1300 bp in length. Analysis of the pattern produced by a given repeat in genomic Southern experiments suggests that some small dispersed repeats may also exist as part of a larger repeating unit elsewhere in the genome.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum.

Sau3 A and Hind III restriction fragments of Clostridium cylindrosporum genomic DNA were used to isolate clones containing 80% of the N10-H4folate synthetase gene in a 5' fragment and the remaining 20% of the gene in the 3' fragment. These fragments were joined at a common SnaB I restriction site and expressed in Escherichia coli at a level equivalent to what is normally found in C. cylindrosporum. Sequence comparisons show a large degree of homology with genes from two other clostridial species, including a thermophile. Certain conserved sequences found in the three clostridial proteins and in the N10-H4folate synthetase portion of eukaryotic C1-H4folate synthases may represent consensus sequences for nucleotide and H4folate binding.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

Genomic arrangement of repeated PS700 elements in the nematode Panagrellus silusiae

When genomic DNA from the free-living nematode Panagrellus silusiae is digested with the restriction endonuclease BamHI and separated by electrophoresis, a band in the 700 base pair size range is evident after ethidium bromide staining. One of the 0.7-kilobase fragments (PS700-1) was characterized and found to be a member of a moderately repetitive DNA family (T. Warren and J.J. Pasternak. 1988. Nucleic Acids Res. 16: 10,833-10,847). In the current study, DNA sequence analyses of three independently isolated copies of the PS700 DNA family showed the same nucleotide sequence and greater than 98% similarity to PS700-1. Four EMBL-4 bacteriophage clones were isolated from a Panagrellus genomic DNA library with PS700-1 as the probe and were analyzed by restriction endonuclease site mapping and Southern blot DNA hybridization. These clones contain 31 copies of the PS700 DNA family. In each case, the units are arranged in head-to-tail arrays. One of the EMBL-4 clones contains copies of a novel variant of the PS700 elements. The maintenance of both nucleotide sequence and restriction endonuclease restriction site homogeneity among members of the dispersed PS700 DNA family may denote a functional role for these sequences.     

Single-enzyme amplified fragment length polymorphism and its applicability for Salmonella epidemiology

Amplified fragment length polymorphism (AFLP) is a PCR-based DNA fingerprinting technique whereby restriction fragments may be visualized without prior knowledge of nucleotide sequences. In AFLP analysis, bacterial genomic DNA is digested with a restriction enzyme and ligated to adapter oligonucleotides. A subset of DNA fragments are then amplified using primers which contain adapter-defined sequences. Selective amplification is achieved by the use of primers containing adapter-defined sequences with one additional arbitrary nucleotide. We used four primers complementary to the adapter sequence, but each differing in the final 3' base that extended into the fragment DNA. The usefulness of these primers for fingerprinting Salmonella enterica was assessed in a hierarchical manner. Using a single-enzyme approach (SAFLP) we have used this method to fingerprint 30 strains of S. enterica, belonging to 14 different serotypes. SAFLP profiles derived from Hind III fragments differentiated between the serotypes. In addition, SAFLP profiles for each serotype differentiated between the phage types and individual strains. The technique is significantly faster to perform than other DNA-based methods and has given reproducible and discriminatory results. This hierarchical SAFLP technique may provide a valuable addition to existing methods for the DNA fingerprinting of S. enterica for epidemiological studies.     

Analysis of foldback sequences and repetitive sequences in cloned segments of nuclear DNA from Physarum polycephalum

The properties of DNA segments containing foldback elements were studied after their selection from a ‘random’ recombinant library of Physarum polycephalum nuclear DNA sequences, cloned using the plasmid vector pBR322. Hybridisation of in vitro labelled recombinant plasmids to Southern blots of genomic restriction fragments demonstrated that each cloned segment contained repetitive elements located at several hundred sites in the genome. Two of the DNA clones generated hybridisation patterns which suggested that they contain repetitive elements with internal cleavage sites for the restriction endonuclease HaeIII. Cross-hybridisation of all combinations of the cloned sequences showed that most contain different arrangements of repetitive elements derived from different sequence families. The results are consistent with a model proposed previously on the basis of studies on total nuclear DNA, for the organisation of sequences closely associated with foldback elements in the Physarum genome     

Chromosome-specific subfamilies within human alphoid repetitive DNA

Nucleotide sequence data of about 20 X 10(3) base-pairs of the human tandemly repeated alphoid DNA are presented. The DNA sequences were determined from 45 clones containing EcoRI fragments of alphoid DNA isolated from total genomic DNA. Thirty of the clones contained a complete 340 base-pair dimer unit of the repeat. The remaining clones contained alphoid DNA with fragment lengths of 311, 296, 232, 170 and 108 base-pairs. The sequences obtained were compared with an average alphoid DNA sequence determined by Wu & Manuelidis (1980). The divergences ranged from 0.6 to 24.6% nucleotide changes for the first monomer and from 0 to 17.8% for the second monomer of the repeat. On the basis of identical nucleotide changes at corresponding positions, the individual repeat units could be shown to belong to one of several distinct subfamilies. The number of nucleotide changes defining a subfamily generally constitutes the majority of nucleotide changes found in a member of that subfamily. From an evaluation of the proportion of the total amount of alphoid DNA, which is represented by the clones studied, it is estimated that the number of subfamilies of this repeat may be equal to or exceed the number of chromosomes. The expected presence of only one or a few distinct subfamilies on individual chromosomes is supported by the study, also presented, of the nucleotide sequence of 17 cloned fragments of alphoid repetitive DNA from chromosome 7. These chromosome-specific repeats all contain the characteristic pattern of 36 common nucleotide changes that defines one of the subfamilies described. A unique restriction endonuclease (NlaIII) cleavage site present in this subfamily may be useful as a genetic marker of this chromosome. A family member of the interspersed Alu repetitive DNA was also isolated and sequenced. This Alu repeat has been inserted into the human alphoid repetitive DNA, in the same way as the insertion of an Alu repeat into the African green monkey alphoid DNA.     

Sequence, higher order repeat structure, and long-range organization of alpha satellite DNA specific to human chromosome 8.

We have characterized alphoid repeat clones derived from a chromosome 8 library. These clones are specific for human chromosome 8, as demonstrated by use of a somatic cell hybrid mapping panel and by in situ hybridization. Hybridization of the clones to HindIII digests of human genomic DNA reveals a complex pattern of fragments ranging in size from 1.3 to greater than 20 kb. One clone, which corresponds in size to the most prevalent genomic HindIII fragment, appears to represent a major higher order repeat in the chromosome 8 centromere. The DNA sequence of this clone reveals a dimeric organization of alphoid monomers. Restriction analysis of two other clones indicates that they are derivatives of this same repeat unit. The chromosome 8 alphoid clones hybridize to EcoRI fragments of genomic DNA ranging up to 1000 kb in length and reveal a high degree of polymorphism between chromosomes. Distribution of higher order repeat units across the centromere was examined by two-dimensional gel electrophoresis. Repeat units of the same size class tended to cluster together in restricted regions of centromeric DNA.     

Analysis of genes for 5S rRNA from the cricket, Acheta domesticus: two classes of repeating units

To examine the modulation of 5S rRNA gene activity during development in the cricket, Acheta domesticus, 5S X DNA was isolated from a lambda Charon 4 genomic library and characterized. Southern blot analysis of cloned A. domesticus genomic DNA revealed that restriction fragments of 3.0 and 2.1 kb represent two size classes of 5S X DNA repeating units; over 90% of the repeats measure 3.0 kb. Restriction analysis of two 5S X DNA clones suggests that the 2.1-kb repeats are not randomly interspersed within clusters of the larger 3.0-kb repeating units. Heteroduplex and restriction mapping of several clones indicate that the spacers of both repeating units account for their unusual length. The major difference between the two classes of repeats may lie in 0.9-kb spacer sequences to the 3.0-kb repeats.     

Mapping of mitochondrial DNA of individual sheep and goats: Rapid evolution in the D loop region

Mitochondrial DNA (mtDNA) from sheep and goat was compared by restriction endonuclease analysis and heteroduplex mapping in the electron microscope. The fragment patterns produced by endonuclease Hae III from three individual sheep and two goat mtDNAs all differed from each other. The three sheep mtDNAs had identical Eco RI and Hind III fragments, but the two goat mtDNA patterns differed from each other as well as from sheep mtDNA. We estimate that each sheep mtDNA differs from each other by 0.5–1% of its nucleotide sequences, the two goat mtDNAs by 1–2%, and there is a 6–11% sequence difference between sheep and goat mtDNAs. We have mapped the Eco RI and Hind III sites of goat and sheep mtDNA and determined the positions of the D loop, which marks the replication origin, relative to the restriction map. The D loops are at homologous positions on the mtDNAs from both species, but the goat D loop is only 75% as long as the sheep D loop. Regions with a high degree of sequence divergence occur at both ends of the D loop. We suggest that a duplication of about 150 base pairs has occurred in the region where the sheep and goat D loops differ in length. We discuss mtDNA evolution in terms of divergence of isolated “mitochondrial DNA clones.”     

(TG)n uncovers a sex-specific hybridization pattern in cattle

Screening of a bovine genomic library with the human minisatellite 33.6 probe uncovered a family of clones that, when used to probe Southern blots of bovine genomic DNA digested with the restriction enzyme HaeIII or MboI, revealed sexually dimorphic, but otherwise virtually monomorphic, patterns among the larger DNA fragments to which they hybridized. Characterization of one of these clones revealed that it contains different minisatellite sequences. The sexual dimorphism hybridization pattern observed with this clone was found to be due to multiple copies of two tandemly interspersed repeats: the simple sequence (TG)n and a previously undescribed 29-bp sequence. Both repeats appear to share many genomic loci including autosomal loci. In contrast, Southern analysis of AluI- or HinfI-digested bovine DNA with the (TG)n repeat used as a probe yielded substantial polymorphism. These results show that (i) different minisatellites can be found in a cluster, (ii) both simple and more complex repeated sequences other than the simple quaternary (GATA)n repeat can be sexually dimorphic, and (iii) simple repeats can reveal substantial polymorphism.     

Construction of a swine YAC library allowing an efficient recovery of unique and centromeric repeated sequences

A swine DNA genomic library was constructed in yeast artificial chromosome (YAC) using the pYAC4 vector and the AB1380 strain. The DNA prepared from two Large White males was partially digested with EcoRI and size selected after both digestion and ligation. The YAC library contained 33792 arrayed clones with an average size of 280 kb as estimated by analysis of 2% of the clones, thus representing a threefold coverage of the swine haploid genome. The library was organized in pools to facilitate the PCR screening. The complexity of the library was tested both for unique and centromeric repeated sequences. In all, 20 out of 22 primer sets allowed the characterization of one to six clones containing specific unique sequences. These sequences are known to be on Chromosomes (Chrs) 1, 2, 5, 6, 7, 8, 13, 14, 15, 17, and X. Eight additional clones carrying centromeric repeat units were also isolated with a single primer set. The sequencing of 37 distinct repeat units of about 340 bp subcloned from these eight YACs revealed high sequence diversity indicating the existence of numerous centromeric repeat unit subfamilies in swine. Furthermore, the analysis of the restriction patterns with selected enzymes suggested a higher order organization of the repeat units. According to preliminary FISH experiments on a small number of randomly chosen YACs and YACs carrying specific sequences, the chimerism appeared to be low. In addition, primed in situ labeling experiments favored the idea that the YACs with centromeric repeat sequences were derived from a subset of metacentric and submetacentric chromosomes. Received: 14 July 1996 / Accepted: 24 October 1996     

Sequence heterogeneity of murine complementary DNA clones related to the C4 and C4-Slp isoforms of the fourth complement component

Two classes of mRNA encoding the murine C4 protein were identified by sequence analysis of clones isolated from a liver complementary DNA library. The divergence found within a 357 base pair sequence available for comparison is limited to five nucleotide replacements located in the region corresponding to the carboxy-terminal end of the C4d peptide fragment. One of the nucleotide substitutions influences the presence of a site for the Hind III restriction endonuclease. That this restriction site indeed discriminates the two non-allelic genes encoding the mouse C4 and C4-Slp isoforms has been demonstrated by Southern blot analysis and nucleotide sequencing at the genomic level. Circumstantial evidence supports the identification of the gene lacking the Hind III site in the region corresponding to the carboxy-terminal end of the C4d fragment as the one encoding the C4-Slp isotype.     

东北虎粪细菌区系的16S rRNA基因序列分析

为研究东北虎粪微生物区系建立了东北虎粪细菌的16SrDNA文库。通过EcoRⅠ和HindⅢ分别对阳性克隆进行酶切分析,从东北虎的16SrDNA文库中分别获得了15个具有酶切差异的克隆。BLAST分析结果显示,在15个克隆中,10个克隆与梭菌属成员有97%以上的同源性,其中有6个序列与诺维梭菌A型(Clostridiumnovyitype A)有99%的同源性,为诺维梭菌A型;4个序列与猪粪细菌RT-18B(Swine manure bacteriumRT-18B)有97%的同源性,为消化链球菌属(Peptostreptococcus)成员。其它序列与GenBank中登录的序列同源性低于97%,为5种未培养细菌,其中4种16SrRNA基因序列分别与Clostridiumpascui、破伤风梭菌E88(ClostridiumtetaniE88)、梭菌(Clostridiumsp.)14505及产气荚膜梭菌(Clostridiumperfringens)有94%~95%的相似性。第5种与肉杆菌(Carnobacteriumsp.)R-7279株有94%的同源性。