Characterization and occurrence of two repeated palindromic DNA elements of Brucella spp.: Bru-RS1 and Bru-RS2

Two repeated DNA elements of 103 bp and 105 bp were discovered in brucellae and designated Bru-RS1 and Bru-RS2, respectively. The two elements are palindromic, are 65% similar in sequence, form two families of elements that are slightly divergent in sequence, appear to be intergenic, and are found, collectively, in more than 35 copies in brucellae. These elements are bounded by perfect or nearly perfect inverted repeats. A third copy of the terminal repeat is found within the elements and is the terminus for several truncated copies of the Bru-RS1 family. Hybridization patterns for the elements among brucellae were unique. The elements are dispersed, highly conserved among brucellae, and hot-spots for insertion by IS711.     

Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.     

Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes.     

Nucleic acid hybridization of highly repeated DNA in extracts of single Drosophila.

We have developed and characterized a method for the rapid detection and quantitation of specific DNAs in partially purified extracts of single Drosophila. While the method should be applicable to a number of repetitious DNA sequences, we have used the polypyrimidine DNA sequences (TCTCT)n to develop this technique. Using hydroxyapatite chromatography, we were able to measure the amount of nucleic acid hybrid formed and to obtain a thermal elution profile of the hybrid formed in extracts of single flies. Under a variety of conditions, purified DNA and DNA in partially purified extracts gave essentially identical results. The procedure can be used to detect the presence of rare sequences, or to measure the relative abundance of a prevalent DNA species. 40 different wild type strains of Drosophila melanogaster were examined using this technique and all contain similar amounts of the same polypyrimidine/polypurine sequence. From a small scale screening of different laboratory stocks of D. melanogaster, a variant was found which formed more DNA-DNA hybrid with labelled polypyrimidine tracts than did wild type. The additional hybrid was distinguished by a lower thermal stability than the hybrid formed in wild type.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae.

We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5' to 3', BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.     

Distribution of highly repeated DNA sequences in species of the genus Lens Miller.

Multiple-target fluorescence in situ hybridization (FISH) was applied on mitotic chromosomes of seven Lens taxa using two highly repetitive sequences (pLc30 and pLc7) isolated from the cultivated lentil and the multigene families for the 18S-5.8S-25S (pTa71) and 5S rRNA (pTa794) from wheat simultaneously as probes. The number and location of pLc30 and pLc7 sites on chromosomes varied markedly among the species, whereas the hybridization pattern of 5S rDNA and 18S-5.8S-25S rDNA was less variable. In general, each species showed a typical FISH karyotype and few differences were observed among accessions belonging to the same species, except for the accessions of Lens odemensis. The most similar FISH karyotype to the cultivated lentil is that of Lens culinaris subsp. orientalis, whereas Lens nigricans and Lens tomentosus are the two species that showed the most divergent FISH patterns compared with all taxa for number and location of pLc30 and 18S-5.8S-25S rDNA sites.     

Organization of DNA sequences highly repeated in tandem in rice genomes

Digestion of the total genomic DNA from rice Oryza sativa L. cv. C5924 with EcoRI generated an intense band of a DNA fragment of about 0.36 kb long. The DNA fragment cloned into pUC19 was used to hybridize with the total rice genomic DNA partially digested with EcoRI. A ladder of bands of DNA fragments with multiplied length of 0.36 kb was observed, demonstrating that this sequence occurs in tandem in the genome. The copy number of the sequence estimated by dot blot hybridization analysis was 2000-3000 copies per haploid genome from callus or seedling of C5924. This sequence was present in other O. sativa cultivars, such as Sasanishiki in 700-900 copies, Koshihikari in 3400-4300, and Nipponbare in 4600-6000 copies. Another rice species, O. glaberrima, also had this sequence in 540-680 copies, but four lines of foxtail millet had none. The DNA fragments containing the repeated sequences in Nipponbare were then cloned into lambda EMBL3, and sequences of nine units consecutively repeated and an AT-rich sequence connected with them in a phage clone could be determined. Each repeating unit showed sequence divergency mostly by substitution of bases in a range from 3% to 7%, when compared with a 355-bp consensus sequence. Analyses of the substituted bases indicate that these are due to spontaneous mutations which occurred at random, after reiteration of a unit sequence by unequal crossing over events. Gene conversion within the repeated sequences might have further diversified their sequences.     

Antibodies to Brucella spp. in Pacific bottlenose dolphins from the Solomon Islands

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.     

Antibodies to Brucella spp. among blue wildebeest and African buffalo in Kenya

A serologic survey of blue wildebeest (Connochaetes taurinus Burchell) and African buffalo (Syncerus caffer Sparrman) in the Masai Mara area was conducted. Antibodies to Brucella spp. were found in 18% of the blue wildebeest and 30% of the African buffalo examined. There were titers in all age groups and in both sexes. Hygromata were seen in both species. The increase in numbers of blue wildebeest and African buffalo which share grazing and watering areas with cattle of the Masai people, makes the presence of infections by Brucella spp. in wildlife an important consideration in any program for control of brucellosis.     

Differential expansion of highly repeated DNA sequences in the swine subgenomes

Differences in highly repeated DNA sequences among three swine breeds genomes were detected by means of whole‐comparative genomic hybridization (W‐CGH). The results showed that Duroc, Iberian and Landrace/Large White breeds share similar DNA sequences in their centromeric regions, but the number of copies of the highly repeated DNA sequences building the blocks of heterochromatin in the metacentric chromosomes is differentially expanded among them. That is not the case in the acrocentric subgenome where the chromosomes share similar sequence composition and number of copies among the three breeds in the centromeric regions. The highly repeated DNA sequences in the chromosome Y also displayed differences among the breeds studied. The reported results are discussed in the light of the possible evolutionary tendencies of these particular DNA sequences.     

Identification of Brucella spp. genes involved in intracellular trafficking

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago–lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn 5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis . We also examined the intracellular fate of three virB mutants ( virB2 , virB4 and virB9 ) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago–lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61β protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.     

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.