A family of repeated sequences in Drosophila genome with telomeric localization: properties and polymorphism

The previously cloned Drosophila genome fragment Dm665 (2.4 kb) hybridizing with telomers on polytene chromosomes is a representative of the family of repeats, a part of which being organized in tandem clusters. The repeats are not transcribed in cell culture, are species-specific and represented in 200-250 copies per haploid genome. In natural and laboratory Drosophila lines polymorphism has been revealed with regard to homology with Dm665 in the telomeres.     

Long poly(A) tracts in the human genome are associated with the Alu family of repeated elements

Long poly(dA).poly(dT) tracts (poly(A) tracts), regions of DNA containing at least 20 contiguous dA residues on one strand and dT residues on the complementary strand, are found in about 2 X 10(4) copies interspersed throughout the human genome. Using poly(dA).poly(dA) as a hybridization probe, we identified recombinant lambda phage that contained inserts of human DNA with poly(A) tracts. Three such tracts have been characterized by restriction mapping and sequence analysis. One major poly(A) tract is present within each insert and is composed of from 28 to 35 A residues. In each case, the poly(A) tract directly abuts the 3' end of the human Alu element, indicating that the major class of poly(A) tracts in the human genome is associated with this family of repeats. The poly(A) tracts are also adjacent to A-rich sequences and, in one case, to a polypurine tract, having the structure GA3-GA3-GA4-GA6-GA5-GA4. We suggest that repetitive cycles of unequal crossing over may give rise to both the long poly(A) and polypurine tracts observed in this study.     

Complete genome sequence of Neisseria gonorrhoeae NCCP11945

Neisseria gonorrhoeae is an obligate human pathogen that is the etiological agent of gonorrhea. We explored variations in the genes of a multidrug-resistant N. gonorrhoeae isolate from a Korean patient in an effort to understand the prevalence, antibiotic resistance, and importance of horizontal gene transfer within this important, naturally competent organism. Here, we report the complete annotated genome sequence of N. gonorrhoeae strain NCCP11945.     

Biochemical properties of Neisseria gonorrhoeae LgtE

A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by PCR and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties were determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations. LgtE was only able to mediate the addition of UDP-galactose into neisserial lipooligosaccharides (LOSs). We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity. LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity. Furthermore, it was able to add multiple galactose residues onto LOS. We also determined that this enzyme was capable of adding galactose onto both the alpha and beta chains of neisserial LOS.     

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.     

A ubiquitous family of repeated DNA sequences in the human genome

Renatured DNA from human and many other eukaryotes is known to contain 300-nucleotide duplex regions formed from renatured repeated sequences. These short repeated DNA sequences are widely believed to be interspersed with single copy DNA sequences. In this work we show that at least half of these 300-nucleotide duplexes share a cleavage site for the restriction enzyme AluI. This site is located 170 nucleotides from one end. This Alu family of repeated sequences makes up at least 3% of the genome and is present in several hundred thousand copies.Inverted repeated sequences are also known to contain a short 300-nucleotide duplex region. We find that at least half of the 300-nucleotide duplex regions in inverted repeated sequences also have an AluI restriction site located 170 nucleotides from one end.By driven renaturation techniques, the Alu family is shown to be distributed over a minimum range of 30% to 60% of the genome. (The breadth of this range reflects the presence of inverted repeated sequences which, in part, include the Alu family.) These findings imply that the interspersion pattern of repeated and single copy sequences in human DNA is largely dominated by one family of repeated sequences.     

Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae

The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative DnaA and IHF binding sites, various open reading frames that are thought to specify functional proteins, and some important DNA sequences involved with conjugative transfer of gonococcal beta-lactamase plasmids. The deletion in the Africa-type plasmid is 1827 bp and one of the DR30 repeats is also missing. The deletion in the Rio-type plasmid and several Toronto-type plasmids was determined to be 2273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. The &Ncirc;imes-type plasmid is an Africa-type plasmid and also contains an IS5 insertion sequence. Since IS5 has not been identified in gonococcal isolates, we suggest that this sequence may have been inserted after the original gonococcal plasmid was transformed into Escherichia coli. The New Zealand plasmid is an Asia-type plasmid that contains an endogenous tandem duplication of 1883 bp and the direct DR2 is implicated in this duplication. The nature of the defined truncation of Tn2 present in the various plasmids is also discussed.     

Mobile dispersed repeated DNA elements in the Drosophila genome

The molecular and cytogenetic organizations of 19 nonhomologous dispersed repeated sequence families were studied in 15 different laboratory strains of Drosophila melanogaster. Elements from each of the families appear to undergo transposition within the Drosophila genome, because there were striking differences in both the number and chromosomal locations of these elements between strains. A significant fraction (greater than 1%) of Drosophila DNA therefore has an unstable genomic organization. Each middle repetitive family exhibited similar variations in the chromosomal distribution of elements between the strains. Although the movements of these elements are not limited to a small number of genomic sites, there are chromosomal regions where elements from the different dispersed repeated DNA families appear to be clustered. The locations of such preferred integration sites are different in each of the D. melanogaster strains examined.     

A transposon-like sequence with short terminal inverted repeats in the nuclear genome of Chlamydomonas reinhardtii

A 1.2 kb DNA sequence, flanked by a potential seven base target-site duplication, was found inserted into a TOC1 transposable element from Chlamydomonas reinhardtii. The insertion sequence, named TOC2, is a member of a family of repeated DNA sequences that is present in all the C. reinhardtii strains tested. It resembles class II transposable elements: it possesses short 14 bp imperfect terminal repeats that begin AGGAGGGT, and sub-terminal direct repeats located within 250 bp of the termini. No large open reading frames were found. The terminal bases and length of target-site duplication are important in classifying transposable elements. On this basis TOC2 does not fall readily into existing families of class II transposable elements found in plants.     

Some properties of the human erythrocyte receptors for Neisseria gonorrhoeae

The nature of the receptors for T1 and T4 Neisseria gonorrhoeae on erythrocytes and other cells was investigated. In general, cells of nonprimate origin contained few receptors for gonococci. Receptors for T4 gonococci were only uncovered when host cells were pretreated with trypsin. Trypsinization, while unnecessary for T1 adherence to erythrocytes, enhanced attachment in inverse proportion to original erythrocyte sensitivity. Receptors for T1 and T4 organisms on trypsinized and trypsin-neuraminidase-treated erythrocytes were blocked by concanavalin A and peanut lectins, respectively, but a distinction could be made between them with wheat germ lectin and galactose oxidase. Of a number of sugars tested as inhibitors, only D-galactose blocked adherence of T4 but was without effect on T1. While the identity of erythrocyte receptors is uncertain, likely candidates are "band 3" protein and glycophorin, by virtue of their galactose content, lectin binding capacity, and partial exposure on the outer surface of the erythrocyte.     

A family of repeated sequences dispersed through the genome of Lilium henryi

A family of dispersed repeats longer than 7 kilobase pairs (kbp) has been identified in the very large genome of Lilium henryi, and two subregions cloned. Initially a rapidly reannealing probe (C₀t<1 M s) was prepared by hydroxyapatite chromatography. Half the copies of all sequences repeated 15000 times per genome are expected to reanneal by this C₀t value. The probe hydridized to abundant fragments of 2, 5, and 7 kbp released from genomic DNA by Bam HI digestion. Twelve 2-kb fragments and ten 5-kb sequences were cloned into pBR322. Restriction mapping of the two sets of clones showed individual members to be quite similar. Length variation was no more than 200 base pairs (bp) between repeats, and consensus sites were present on 80%–90% of occasions. In situ hybridization using representative 2-kbp and 5-kbp clones showed each sequence to be dispersed throughout all chromosomal regions. Studies on the genomic organization suggested that the 2-kbp and 5-kbp sequences are usually adjacent, and that occasional absence of the internal Bam HI site results in the release of the 7-kbP fragment. There are at least 13000 copies of the full repeat per L. henryi genome, thus accounting for approximately 0.3% of the total of 32 million kbp.     

SINEs: Short interspersed repeated elements of the eukaryotic genome

Much of the eukaryotic genome is composed of a variety of repetitive sequences. Amongst these, there are two kinds of retroposons (sequence elements derived from nonviral cellular RNA): SINEs (short interspersed elements) and LINEs (long interspersed elements). Amplification of SINEs occurs in a single germ cell, and the members of SINEs spread and become fixed in populations through genetic drift. SINEs can be regarded as phylogenetic landmarks: they are specific to one species, a few species, a genus or in some cases a family, indicating a specific time of amplification during evolution. Recent studies concerning the structure and origin of many SINEs revealed that retroposons are more widespread in animal genomes than was previously thought.     

Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae.

Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta-lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase-specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful.     

Survival of Neisseria gonorrhoeae in the Mail

Cultures of Neisseria gonorrhoeae, which used Thayer-Martin slants as transport medium, survived at least 1 day in the mail.     

A novel strategy of epitope design in Neisseria gonorrhoeae

In spite of genome sequences of both human and N. gonorrhoeae in hand, vaccine for gonorrhea is yet not available. Due to availability of several host and pathogen genomes and numerous tools for in silico prediction of effective B-cell and T-cell epitopes; recent trend of vaccine designing has been shifted to peptide or epitope based vaccines that are more specific, safe, and easy to produce. In order to design and develop such a peptide vaccine against the pathogen, we adopted a novel computational approache based on sequence, structure, QSAR, and simulation methods along with fold level analysis to predict potential antigenic B-cell epitope derived T-cell epitopes from four vaccine targets of N. gonorrhoeae previously identified by us [Barh and Kumar (2009) In Silico Biology 9, 1-7]. Four epitopes, one from each protein, have been designed in such a way that each epitope is highly likely to bind maximum number of HLA molecules (comprising of both the MHC-I and II) and interacts with most frequent HLA alleles (A*0201, A*0204, B*2705, DRB1*0101, and DRB1*0401) in human population. Therefore our selected epitopes are highly potential to induce both the B-cell and T-cell mediated immune responses. Of course, these selected epitopes require further experimental validation.     

Neisseria gonorrhoeae: evaluation of some methods used for carbohydrate utilization.

A total of 989 Neisseria gonorrhoeae cultures were examined for acid production from dextrose in seven different carbohydrate media. Four of these media were found to be quite accurate for this purpose. Some pitfalls in the preparation and usage of cystine trypticase agar carbohydrate media are discussed. The term 'fermentation' of carbohydrates by Neisseria species is inaccurate and responsible for some N. gonorrhoeae false-dextrose-negative results.