Nucleotide sequence of gene 31 of the T4 bacteriophage

We have determined the nucleotide sequence of a region of 656 nucleotides comprising the 31 gene of bacteriophage T4. The coding region consisted of 333 nucleotides directing the synthesis of a polypeptide of 111 amino acids, with a calculated molecular weight of 12,060. The upstream sequence contains the consensus sequences for T4 early and two middle promoters. The downstream sequence contains the consensus sequence for T4 late promoter and the inverted repeats. In addition, there are two incomplete open reading frames in the sequenced region.     

Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA).

Molecular cloning and nucleotide sequence analysis were performed for the identification of the regulator genes of methicillin resistance in the genome of a MRSA strain N315. Two open reading frames (orfs) were identified in the 5'-flanking region of the mecA gene. Predicted amino acid sequences of these orfs showed extensive homology to the co-inducer and the repressor protein of the penicillinase (PCase) production in Staphylococcus aureus as well as in Bacillus licheniformis. These orfs are considered to encode putative co-inducer and repressor proteins specific for the regulation of methicillin resistance in MRSA.     

Haloviruses HF1 and HF2: evidence for a recent and large recombination event

Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment.     

Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches

Among the total of 274 orfs within bacteriophage T4, only half have been reasonably well characterized, and the functions of the rest have remained obscure. In order to predict the molecular functions of the orfs, a position-specific iterated (PSI)-BLAST search of bacteriophage T4 against the sequence database of known 3D structures was carried out. PSI-BLAST is one of the most powerful iterative sequence search methods using multiple sequence alignment, with the ability to detect many more proteins with distant homology than standard pairwise methods. The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly homologous proteins are likely to possess highly similar 3D structures. Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise methods, were found to have significantly homologous counterparts by this method. The plausibility of the results was confirmed by checking whether important residues at substrate/ligand-binding sites were conserved. Among them, two orfs, vs.1 and e.1, which are similar to Escherichia coli lytic enzyme and MutT protein, respectively, had not been studied previously. Also, gp rIIA, a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly similar to the N-terminal region of the heat shock protein Hsp90.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Factors regulating baculovirus late and very late gene expression in transient-expression assays.

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.     

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.     

Identification of three late expression factor genes within the 33.8- to 43.4-map-unit region of Autographa californica nuclear polyhedrosis virus.

A transient transactivation assay system was used in combination with an overlapping Autographa californica nuclear polyhedrosis virus clone library to identify genes involved in late and very late baculovirus gene expression. We have identified three genes within the 33.8- to 43.4-map-unit region of the A. californica nuclear polyhedrosis virus genome which contribute to expression from promoters of the vp39 major capsid protein and polyhedrin genes. One of these three genes corresponds to the previously identified DNA polymerase gene, while the other two genes encode previously unidentified polypeptides of 59,418 and 8,706 Da. None of these genes were required for expression from the early etl promoter.     

Nucleotide sequence of the right early region of Bacillus subtilis phage PZA completes the 19366-bp sequence of PZA genome. Comparison with the homologous sequence of phage phi 29

We have sequenced the rightmost 2079 bp of the Bacillus subtilis phage PZA genome. This region encompasses the right early region. We compared it with the homologous region of phage phi 29. Six open reading frames (ORFs) were found in this region of PZA and one of them was assigned to gene 17. Analysis of putative ribosome-binding sites and comparison with phi 29 ORFs indicate that at least some of the remaining ORFs could encode proteins. Corresponding genes were not identified so far by genetic methods. Promoter candidates in the right early region of PZA were found and compared to phi 29 promoters. The sequenced region together with previously determined sequences [Paces et al., Gene 38 (1985) 45-56 and 44 (1986) 107-114] completes the entire 19,366-bp sequence of phage PZA genome.