DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er

Carotovoricin Er is a phage-tail-like bacteriocin produced by Erwinia carotovora subsp. carotovora strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5alpha when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.     

Regulation of 3' splice site selection in the 844ins68 polymorphism of the cystathionine Beta -synthase gene

844ins68 is a frequent polymorphism of the cystathionine beta-synthase gene (CBS) that consists of a 68-bp insertion duplicating the 3' splice site of intron 7 and the 5'-end of exon 8. The presence of two identical 3' splice sites spaced by 68 bp should lead to either a selection of the proximal site or to at least two alternatively spliced CBS mRNA variants. Instead, an accurate selection of the distal 3' splice site is observed in the 844ins68 carriers. The duplication has generated a gene re-arrangement at the 3' splice site where two GGGG runs have been brought close to each other. Using a minigene system, we have investigated the effect this peculiar configuration might have on the selection of the 3' splice site of intron 7 in the CBS gene. Minimal disruption of the G runs resulted in a dramatic shift toward the proximal 3' splice site selection with inclusion of the 68-bp insertion and a consequent change of the reading frame. The insertional event created this peculiar configuration of two G repeats close to each other that subsequently acquired the ability to strongly bind heterogeneous nuclear ribonucleoprotein (hnRNP) H1, a specific trans-acting factor. The interaction of hnRNP H1 with G runs within the 844ins68 context might interfere with the recruitment of splicing factors to the proximal 3' splice site thus favoring the selection of the distal 3' splice site. Our results therefore suggest the possibility that the insertion was an evolutionary event that allowed the rescue of the wild-type sequence, so preserving protein function.     

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.     

Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements

An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR, was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.     

Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements

An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.     

Complete sequence and genomic analysis of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gammaHV68) infects mice, thus providing a tractable small-animal model for analysis of the acute and chronic pathogenesis of gammaherpesviruses. To facilitate molecular analysis of gammaHV68 pathogenesis, we have sequenced the gammaHV68 genome. The genome contains 118,237 bp of unique sequence flanked by multiple copies of a 1,213-bp terminal repeat. The GC content of the unique portion of the genome is 46%, while the GC content of the terminal repeat is 78%. The unique portion of the genome is estimated to encode at least 80 genes and is largely colinear with the genomes of Kaposi's sarcoma herpesvirus (KSHV; also known as human herpesvirus 8), herpesvirus saimiri (HVS), and Epstein-Barr virus (EBV). We detected 63 open reading frames (ORFs) homologous to HVS and KSHV ORFs and used the HVS/KSHV numbering system to designate these ORFs. gammaHV68 shares with HVS and KSHV ORFs homologous to a complement regulatory protein (ORF 4), a D-type cyclin (ORF 72), and a G-protein-coupled receptor with close homology to the interleukin-8 receptor (ORF 74). One ORF (K3) was identified in gammaHV68 as homologous to both ORFs K3 and K5 of KSHV and contains a domain found in a bovine herpesvirus 4 major immediate-early protein. We also detected 16 methionine-initiated ORFs predicted to encode proteins at least 100 amino acids in length that are unique to gammaHV68 (ORFs M1 to 14). ORF M1 has striking homology to poxvirus serpins, while ORF M11 encodes a potential homolog of Bcl-2-like molecules encoded by other gammaherpesviruses (gene 16 of HVS and KSHV and the BHRF1 gene of EBV). In addition, clustered at the left end of the unique region are eight sequences with significant homology to bacterial tRNAs. The unique region of the genome contains two internal repeats: a 40-bp repeat located between bp 26778 and 28191 in the genome and a 100-bp repeat located between bp 98981 and 101170. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs. Interestingly, genes associated with EBV cell tropism, latency, and transformation are all contained within these regions encoding virus-specific genes. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. The availability of the gammaHV68 genomic sequence will facilitate analysis of critical issues in gammaherpesvirus biology via integration of molecular and pathogenetic studies in a small-animal model.     

High prevalence of a mutation in the cystathionine beta-synthase gene.

We found that a mutation previously described by Sebastio et al., involving a 68-bp insertion in the coding region of exon 8 of the cystathionine-beta-synthase (CBS) gene in a single patient with homocystinuria, is highly prevalent. In our control population, 11.7% (9/77) of the individuals were heterozygous carriers of this mutation. In contrast to the previous report, which assumed that the 68-bp insertion introduced a premature-termination codon and resulted in a nonfunctional CBS enzyme, we found that the presence of this mutation is not associated with hyperhomocysteinemia. Assay of CBS activity in transformed lymphocytes from individuals who were heterozygous or homozygous for this mutation showed normal activity. Furthermore, reverse-transcripion-PCR showed that individuals carrying this mutation have normal size mRNA. Our results suggest that the insertion creates an alternate splicing site, which eliminates not only the inserted intronic sequences but also the T833C mutation associated with this insertion. The net result is the generation of both quantitatively and qualitatively normal mRNA and CBS enzyme. Although the mutation does not seem to affect the activity of the CBS enzyme, the prevalence is somewhat increased in patients with premature coronary-artery disease, although the difference is not statistically significant.     

Isolation and genetic characterization of a novel filamentous bacteriophage,a deleted form of phage f237, from a pandemic Vibrio parahaemolyticus O4:K68 strain

We isolated a filamentous bacteriophage, VfO4K68, from the pandemic Vibrio parahaemolyticus strain belonging to 04:K68 serovar. The VfO4K68 DNA lacked a 1,893-bp fragment present in that of the distinctive region of f237, a filamentous phage isolated from a pandemic 03:K6 strain (Nasu, H. et al., J. Clin. Microbiol., 38, 2156-2161, 2000). The deletion resulted in the formation of a novel open reading frame (ORF) that possesses homology to the ORF 27 of ETA phage and staphylococcal enterotoxin E (SEE) of Staphylococcus aureus. VfO4K68 was able to infect the recipient 03:K6 serovar strains. These results suggest that VfO4K68 might act as a genetic transmitter and play some roles in the pandemic V. parahaemolyticus infection.     

Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication.

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization of the human myoglobin gene.

Cross-hybridization of the grey seal myoglobin gene to human DNA detected a single human myoglobin gene plus an extensive family of sequences apparently related to the central exon of this gene. The functional human gene is 10.4 kb long and has a haemoglobin-like three exon/two intron structure with long non-coding regions similar to its seal homologue. At least 300 bp of 5'-flanking region are closely homologous between the two genes, with the exception of a divergent purine-rich region 68-114 bp upstream of the cap site. A diverged tandem repetitive sequence based on (GGAT)165 is located 1100-1750 bp upstream from the gene; internal homology units within this sequence suggest sequence homogenization by gene microconversions. A second 33-bp tandem repeat element in the first intron is flanked by a 9-bp direct repeat, shares homology with other tandem repetitive elements in the human genome and may represent a novel form of transposable element.