Molecular characterization of the gyrB region of the oral spirochete, Treponema denticola

The nucleotide (nt) sequence of the Treponema denticola (Td) DNA gyrase beta-subunit gene (gyrB) has been determined. Southern blot analysis of Td chromosomal DNA indicated that gyrB is present as a single copy. Approximately 3.2kb of the nt sequence 5' and 0.7kb of nucleotide sequence 3' of gyrB were obtained. Analysis of the deduced amino acid (aa) sequence revealed two complete open reading frames (ORFs) (ORF1 and ORF3) and a truncated ORF (ORF4'). ORF1 has no homology to sequences in the databases, whereas ORF3 and ORF4' have significant homology to several bacterial DnaA (replication initiator) and DnaE (DNA polymerase III) proteins respectively. RT-PCR data showed that orf1-gyrB are co-transcribed, while dnaA-dnaE are co-transcribed but in the opposite direction. These data indicated that the gene organization of the Td gyrB region is unique compared with that of other bacteria. Eighteen putative DnaA boxes with several AT-rich regions were identified in the dnaA-dnaE intergenic region, and three putative DnaA boxes were identified in the gyrB-dnaA intergenic region. Spontaneous coumermycin A(1)-resistant Td mutants were isolated and characterized. The mutants have a >20-fold higher resistance to coumermycin A(1) than wild-type Td. A single point mutation in gyrB that changed GyrB Lys(136) to Glu or Thr appears to be responsible for the coumermycin A(1) resistance.     

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.     

Chromosome end formation in phage lambda, catalyzed by terminase, is controlled by two DNA elements of cos, cosN and R3, and by ATP.

The terminase enzyme of phage lambda is a site-specific endonuclease that nicks DNA concatemers to regenerate the 12 nucleotide cohesive ends of the mature chromosome. The enzyme's DNA target, cos, consists of a nicking domain, cosN, and a binding domain, cosB. cosB, situated to the right of cosN, comprises three 16 bp repeat sequences, R1, R2 and R3. A similar sequence, R4, is present to the left of cosN. It is shown here that terminase has an intrinsic specificity for cosN which is independent of the R sites. The interaction with cosN is mediated by binding to target sites that include 12 bp on the 5', and 2-7 bp on the 3' side of the nick. Of the four R sites, only R3 is required for the proper formation of ends. When R3 is present, an ATP-charged terminase system correctly catalyzes the production of staggered nicks in cosN, at sites N1 and N2 on the bottom and top strands, respectively. When ATP is omitted, the bottom strand is nicked incorrectly, at the site Nx, 8 bp to the left of N1. If R3 is removed or disabled by a point mutation, nicking in cosN becomes dependent upon ATP but, even in the presence of ATP, bottom strand nicking is divided between sites N1, the correct site, and Nx, the incorrect one. Thus, R3 is an important regulatory element and must reside in cis in respect to cosN. Furthermore, cosN substrates bearing point mutations at N1 and N2 are nicked at sites Nx and Ny, 8 bp to the left of N1 and N2, respectively. When R3 is present and ATP is added, nicking is redirected to the N1 and N2 positions despite the mutations present. Thus, terminase binding to R3, on one side of cosN, regulates the rotationally symmetric nicking reactions on the bottom and top strands within cosN.     

Small single-copy region of plastid DNA in the non-photosynthetic angiosperm Epifagus virginiana contains only two genes. Differences among dicots, monocots and bryophytes in gene organization at a non-bioenergetic locus.

We have determined the nucleotide sequence of a 7 kb (1 kb = 10(3) base-pairs) region that includes the entire small single-copy region (SSC) of the plastid genome of Epifagus virginiana, a non-photosynthetic, parasitic flowering plant. The SSC (4.8 kb) is considerably smaller than those of photosynthetic plants due to the complete deletion of all photosynthetic, chlororespiratory and ribosomal protein genes. This leaves only two genes: a protein gene of 1738 codons whose product is unlikely to be involved in bioenergetic processes and a leucine tRNA gene (trn(LUAG)). Both genes span junctions between the inverted repeat and the SSC, with the consequence that the terminal 20 base-pairs of the repeat is transcribed in both directions and functions both as the 3' end of the tRNA gene and as an internal segment of orf1738. We find that the region of tobacco plastid DNA homologous to Epifagus orf1738 contains a single open reading frame (ORF) of 1901 codons rather than the three ORFs of 1244, 273 and 228 codons originally reported. However, we confirm that the equivalent region of the bryophyte Marchantia contains two genes (1068 and 464 codons) corresponding to the N and C-terminal portions of the dicot protein. In contrast, rice plastid DNA contains a severely truncated pseudogene at this locus.     

Structure of the bacteriophage lambda cohesive end site: location of the sites of terminase binding (cosB) and nicking (cosN)

The extents of the sites for nicking (cosN) and binding (cosB) of bacteriophage lambda DNA by terminase have been determined by studying cos cleavage and terminase binding in vitro. The cosN site is located in the segment from -22 to +24 bp (numbered from the center of the cohesive end sequence in the circular lambda genome). The cosB site is located in the segment from +51 to +120 (the +120 boundary determined by Miwa and Matsubara, 1983). Additional sequences are necessary for packaging into infectious phage particles, including regions to the left (Rz gene side) of cosN, and between cosN and cosB. Small deletions (7 and 11 bp) between cosN and cosB abolish packaging in vivo without affecting cos binding and cleavage in vitro, whereas a large deletion (26 bp) abolishes packaging in vivo and cleavage in vitro.     

The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB.

cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.     

The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site.

lambda terminase is an ATP-interactive, site-specific endonuclease comprising the products of lambda genes Nu1 and A. Terminase binds to cos, at the junction of two chromosomes in a concatemer, catalyzes cos cleavage and initiates the packaging of lambda DNA into proheads. cos consists of a nicking domain, cosN, where terminase cleaves to regenerate the 12 nucleotide cohesive ends of mature lambda chromosomes and a binding domain, cosB, where terminase binds to 16 bp repeat sequences called R3, R2 and R1. Evidence is presented that terminase is a single-strand endonuclease that can nick DNA by one of two mechanisms, both of which require ATP. (i) When bound to any R site, terminase nicks the strand which, within that R site, is purine-rich; the position of this nick is 47 +/- 2 nucleotides away from the mid-point of that R site, measured in the 3' direction; (ii) enzymes that are not bound to R sites nick DNA within certain specific sequences that resemble cosN half sites. These two modes of action are nicely combined for the R3-bound protomer that nicks the bottom strand at position N1 in cosN since the interval between N1 and the R3 midpoint is 47 nucleotides. Within cosN, the bottom and top strand nicks are generated by a rigid protein couple with a 2-fold rotational symmetry. The location of both of these nicks, however, is gauged asymmetrically from R3, 47 nucleotides away. Again, R1 and R2 are separated by 47 bp and orient bound protomers towards each other but, unless the DNA between these R sites is lengthened, the enzymes do not nick, indicating an inhibitory gpA-gpNu1 apposition.     

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.     

Physical map of the 3'' region of the human immunoglobulin heavy chain locus: clustering of autoantibody-related variable segments in one haplotype.

We have constructed the physical map of the 3' region of the human immunoglobulin heavy chain variable region (VH) genes. DNA segments extending to 200 kb upstream of the JH segment were isolated in two YAC clones. Five VH segments were identified in this region in the 5' to 3' order, V(II-5), V(IV-4), V(I-3), V(I-2), and V(VI-1) segments which were all structurally normal and orientated in the same direction as the JH segments. From DNA of a different cell line we have isolated a cosmid contig containing the same DNA region which has extraordinary polymorphism. The YAC and cosmid DNAs were called haplotypes A and B, respectively. Haplotype B contained an additional VH-I segment (V(I-4.1b)) between the V(II-5) and V(IV-4) segments. V(I-4.1b) segment is almost identical to a previously published VH sequence encoding a rheumatoid factor. Another VH segment in the B haplotype (V(I-3b)) corresponding to the V(I-3) segment also showed 99.7% nucleotide sequence homology with an anti-DNA autoantibody VH sequence. However, none of the VH sequences in haplotype A showed such strong homology with autoantibody VH sequences. The results suggest that VH haplotypes may have linkage with autoantibody production.     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Cloning and functional characterization of Neisseria gonorrhoeae tonB, exbB and exbD genes

Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of sources including transferrin (TF) and lactoferrin (LF). To gain insight into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild-type DNA that repaired the defect in tlu mutants, which are unable to take up Fe from either TF or LF despite exhibiting apparently normal ligand binding to the receptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged in tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26–32% identity with TonB proteins of nine other bacteria. The 663 bp ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32–38% and 24–36% identity, respectively, with ExbB and ExbD proteins of three other bacteria. Thus, the sequence comparison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of the E. coli tonB , exbB and exbD genes. An insertional mutation in the tonB homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to take up ⁵⁵Fe from TF and LF. The tonB mutation did not prevent the utilization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid-phase binding assay was largely unaffected by the tonB mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on the TonB system.     

Genetics of cosQ, the DNA-packaging termination site of phage lambda: local suppressors and methylation effects

The cos site of the bacteriophage lambda chromosome contains the sites required for DNA processing and packaging during virion assembly. cos is composed of three subsites, cosQ, cosN, and cosB. cosQ is required for the termination of chromosome packaging. Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the lambda chromosome; and by missense mutations affecting the prohead's portal protein, gpB. In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor. The 7-bp-long cosQ, with the sequence 5'-GGGTCCT-3', coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5'-PuGGNCCPy-3'. In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants. cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated. Models of cosQ suppression are presented.     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.