Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.
We have investigated the human cytomegalovirus (HCMV) US22 gene family members UL23, UL24, UL43 and US22. Specific antibodies were generated to identify pUL23 (33 kDa), pUL24 (40 kDa) and pUL43 (48 kDa), while pUS22 was identified by monoclonal antibody HWLF1. A Cterminally truncated UL43 product (pUL43t ; 21 kDa) produced by a deletion mutant was also investigated. The UL24 and UL43 genes were expressed with early-late (γ1) and true-late (γ2) kinetics, respectively. Immunoblot and immuno-EM studies demonstrated that pUL23, pUL24, pUL43 and pUS22 were virion tegument components. Immunofluorescence and immuno-EM studies showed that pUL23, pUL24, pUL43 and pUL43t were located in cytoplasmic protein aggregates, manifesting two forms : complex juxtanuclear structures and smaller, membranebound aggregates resembling dense bodies. The complex-type aggregate is a putative site of particle maturation. Because pUL43t was present in protein aggregates, but under-represented in virus particles compared to pUL43, it was concluded that N-terminal sequences target pUL43 to protein aggregates and that C-terminal sequences are important for incorporation into particles. Since three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components, at least seven of the twelve US22 family genes encode tegument proteins, suggesting that the products of the remaining five genes might be similarly located. These findings demonstrate a common biological feature among most, if not all, US22 family proteins and implicate the family in events occurring immediately after virus penetration.
Compared with the published DNA sequence (M. S. Chee, et al. Curr. Top. Microbiol. Immunol. 154:125-170, 1990), most isolates of human cytomegalovirus strain AD169 contain an additional 929 bp after nucleotide 54612. This results in a changed reading frame for the 5-terminal 50 codons of gene UL42 and expansion of gene UL43 (a US22 family member) from 187 (3-truncated) to 423 (full-length) codons. The UL42 and UL43 gene products are nonessential for growth in culture. The published DNA sequence of the AD169 strain of human cytomegalovirus (HCMV) (4) is a key resource for molecular studies with HCMV. Here, we report a 929-bp segment of AD169 DNA absent from the published sequence. This sequence is unrelated to the 15-kbp fragment of HCMV DNA present in clinical isolates and the Toledo strain of HCMV but not in AD169 (3). Unlike this 15-kbp deletion, the 929-bp fragment is present in most stocks of AD169, and an adjustment to the published DNA sequence is therefore proposed. Detection and mapping of a novel DNA sequence in the HCMV AD169 genome. The following four distinct stocks of HCMV AD169 were obtained: AD169 (ATCC) from the American Type Culture Collection (ATCC), AD169 (London) from J. Booth
We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA set to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental nonsuppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 produced fulllength UL12 protein in SupD12 cells which yielded a level of 25.9 % of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup + cells. Possible reasons are discussed.
SUMMARYAntisera to two herpes simplex virus-type 1 (HSV-1) immediate-early gene products (IE12 and IE175) have been produced using oligopeptides, constructed on the basis of proposed DNA coding sequences, as immunogens. In both cases the synthetic peptides were linked to bovine serum albumin via an N-terminal tyrosine prior to immunization. Both the IE12 and the IE175 antisera reacted with their respective HSV-1 antigens and with antigens produced by the HSV-1 immediate-early mutant tsK but not with functionally equivalent antigens induced by HSV-2, IE12 induced by tsK was found to have an altered mobility with respect to the wild-type IE12 and its precipitation was accompanied by a second, minor, component of lower mobility. Revertants of tsK gave similar results. Labelling IE12 with a variety of amino acids indicated that the first of three possible initiation codons was used in its translation from mRNA. The results imply that the other initiation codons were not used. Wildtype IE 12 is produced for at least 3 h after release of cycloheximide block and appears to be turned over rapidly.
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